First International Symposium of Veterinary Medicine – ISVM2015

Transcription

SCIENTIFIC VETERINARY INSTITUTE „NOVI SAD“
INSTITUTE OF VETERINARY MEDICINE OF SERBIA
„One Health – New Challenges“
First International Symposium of
Veterinary Medicine
(ISVM2015)
PROCEEDINGS
Hotel "Premier Aqua" - Vrdnik
May 21 – 23, 2015
Publisher
Scientific Veterinary Institute „Novi Sad“, Novi Sad, Serbia
For the Publisher
Prof Dr Miroslav Ćirković, Principal Research Felow
Editor in Chief
Dr Tamaš Petrović, Senior Research Associate
Technical Editor
Printed by
Multidizajn, Novi Sad
250 copies
Novi Sad, 2015
ISBN: 978-86-82871-36-1
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ORGANISERS
SCIENTIFIC VETERINARY INSTITUTE „NOVI SAD“
and
INSTITUTE OF VETERINARY MEDICINE OF SERBIA
CO-ORGANISERS and SUPPORTERS
MINISTRY OF EDUCATION SCIENCE AND TECHNOLOGICAL DEVELOPMENT
PROVINCIAL SECRETARIAT FOR SCIENCE AND TECHNOLOGICAL DEVELOPMENT
MINISTRY OF AGRICULTURE AND ENVIRONMENTAL PROTECTION
GENERAL SPONSOR
FISH Corp.2000 d.o.o.
SPONSORS
GLOBOS Osiguranje a.d.o.
NOACK
ALEA I.A.
SANO
ORGANIZING COMMITTEE
Dr Miroslav Ćirković, Principal Research Fellow - President
Dr Dobrila Jakić-Dimić,Principal Research Fellow
Dr Vladimir Polaček, Research Associate
Dr Branka Vidić, Principal Research Fellow
Dr Jadranka Žutić, Research Associate
Dr Sava Lazić, Principal Research Fellow
Dr Milica Živkov-Baloš, Senior Research Associate
Dr Jelena Petrović, Senior Research Associate
Dr Vladimir Radosavljević, Research Associate
Dr Dragana Ljubojević, Research Associate
Dr Sara Savić, Research Associate
Petar Bakmaz, secretary of Organizing Committee
TECHNICAL SECRETARIAT
Jelena Babić, DVM, MSc, Research Assistant
Biljana Božin, DVM, MSc, Research Trainee
Nikola Bakmaz, Secretary
Dobrisav Vuković, IT engineer
Vera Prokić, librarian
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SCIENTIFIC COMMITTEE
Dr Tamaš Petrović, Senior Research Associate – President (Serbia)
Dr Miroslav Ćirković, Principal Research Fellow (Serbia)
Dr Dubravka Milanov,Senior Research Associate(Serbia)
Dr Dobrila Jakić-Dimić,Principal Research Fellow (Serbia)
Dr Tania Hubenova, professor (Bulgaria)
Dr Theresa Bernardo, Principal Research Fellow (USA)
Dr Igor Stojanov, Senior Research Associate (Serbia)
Dr Branka Vidić, Principal Research Fellow (Serbia)
Dr Sara Savić, Research Associate (Serbia)
Dr Tamaš Bakonyi, Principal Research Fellow (Hungary)
Dr Dejan Bugarski, Research Associate (Serbia)
Dr Sava Lazić, Principal Research Fellow (Serbia)
Dr Vladimir Polaček, Research Associate (Serbia)
Dr Dušan Orlić, Principal Research Fellow (Serbia)
Dr Marjana Todorčević, Postdoc.Research Assistant(UK)
Dr Juan Carlos Saiz, Principal Research Fellow (Spain)
Dr Jadranka Žutić, Research Associate (Serbia)
Dr Ivan Pavlović, Principal Research Fellow (Serbia)
Dr Miroslav Urošević, Research Associate (Serbia)
Dr Vladimir Radosavljević, Research Associate (Serbia)
Dr Jasna Prodanov Radulović,ResearchAssociate (Serbia)
Dr Aleksandar Mašić, Adjunct professor (Canada)
Dr Petr Kralik, Research Associate (Czech Republic)
Dr Stanko Boboš, professor (Serbia)
Dr Dragana Dimitrijević , PhD (Serbia)
Dr Maja Velhner, Principal Research Fellow (Serbia)
Dr Bojana Grgić, MSc (Serbia)
Dr Aymeric Hans, Senior Research Associate (France)
Dr Milovan Jovičin, Research Associate (Serbia)
Dr Vesna Milošević, professor (Serbia)
Dr Radomir Ratajac, Research Associate (Serbia)
Dr Dušan Lalošević, professor (Serbia)
Dr Dragana Ljubojević, Research Associate (Serbia)
Dr Ivan Bogut, professor (Croatia)
Dr Živoslav Grgić, Research Associate (Serbia)
Dr Milica Živkov-Baloš,Senior ResearchAssociate(Serbia)
Dr Diana Lupulović, Research Associate (Serbia)
Dr Miloš Kapetanov, Principal Research Fellow (Serbia)
Dr Aleksandar Milovanović, Research Associate (Serbia)
Dr. Corinna Kehrenberg, professor (Germany)
Dr Željko Mihaljev, Research Associate (Serbia)
Dr Slavica Košarčić,Principal Research Fellow(Serbia)
Dr Danka Maslić-Strižak, Research Associate (Serbia)
Dr Jelena Petrović, Senior Research Associate (Serbia)
Dr Dragica Vojinović, Research Associate (Serbia)
Dr Vladimir Savić, Senior Research Associate(Croatia)
Dr Ksenija Nešić, Research Associate (Serbia)
Dr Ivana Hrnjaković-Cevtković, professor (Serbia)
Dr Živka Ilić, Research Associate (Serbia)
Dr Sandra Stefan-Mikić, professor (Serbia)
Dr Božidar Savić, Research Associate (Serbia)
Dr Snežana Ivanović, Principal Research Fellow (Serbia)
Dr Branislav Kureljušić, Research Associate (Serbia)
Dr Nataša Golić, Principal Research Fellow (Serbia)
Dr Sandra Jakšić, Research Assosiate (Serbia)
HONORARY COMMITTEE:
Dr Srđan Verbić, prof dr Snežana Bogosavljević Bošković, prof dr Zoran Mašić, Vladimir Pavlov, Branislav
Bogaroški, prof dr Dragan Glamočić, prof dr Vlada Teodorović, prof dr Radovan Pejanović, prof dr Milan
Popović, prof dr Zora Mijačević, prof dr Brana Radenković Damjanović, prof dr Nikola Grujić, prim dr
Dragan Ilić, doc dr Vladimir Petrović, prof dr Dine Mitrov, prof dr Nihad Fejzić, doc dr Boris Habrun, prof
dr Željko Cvetnić, prof dr Andrej Kirbiš, prof dr Tadej Malovrh, prof dr Boris Stegny, prof dr Paul Pirsan,
prof dr Gheorghe Darabus, dr Teodor Dujin, dr Radovan Pavlović, dr Ivana Kovinčić, dr Marijana Galić, dr
Ksenija Stevanović, dr Milan Šurjanović, dr Mira Velhner, dr Dragan Milićević, Petar Matijević, prof dr
Dragan Šefer, dr Aleksandar Lončarević, dr Milenko Žutić, dr Vojin Ivetić, dr Snežana Janković, dr Vesna
Đorđević, dr Jovanka Lević.
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PREFACE
Concept of „One Health“ is a strategy which enrolls several disciplines in all aspects of
human and animal health care and environment protection. This kind of approach to solving
different public health issues proved to be the best practice of preservation of public health on a
global level. For this reason a need for different, unique and multidisciplinary perspective has been
recognized.
In Vrdnik, from 21st to 23rd of May, 2015, First International Symposium of Veterinary
Medicine will be held, named „One Health – New challenges“. At the Symposium, the existence of
„One Health“ initiative in Serbia, our region and also broader international surrounding will be
analyzed and also current topics and different perspectives will be discussed. The main goal of
Symposium is primarily to gather all the people interested in the topic of „One Health“concept.
Also it would be essential to perceive a large number of issues of this platform, the possibilities of
improvement on national, regional and international level and to propose future activities and
recognized challenges which are possible at this moment. It is very important that the participants of
the symposium are researchers and experts of different occupations, because of the broader
initiative proposals and different perspectives for the same issues.
A successful implementation of „One Health“ concept cannot be done without cooperation
and exchange of opinions among doctors, veterinarians, agronomists, technologists, biologists, etc.
It is needed to perceive a particular problem from a perspective of different professions and
expertise, regardless if the topic is a zoonozis, emergent and infectious diseases, food safety,
antimicrobial resistance, etc. Only with a „One Health“ concept and approach there can be an
appropriate response to the challenges that come up with the aim of preservation of public health
and environment. Basically this concept represents a mutual effort of different disciplines which
operate locally on a national level, and also globally, so that optimal health of humans, animals and
ecosystems is achieved. Necessary interdisciplinary requires a continuous cooperation among
epizootiological and epidemiological service, infectologists, veterinary practitioners, doctors, food
safety experts, biologists, environmentalists and other above mentioned disciplines.
At the „One Health – New challenges“ Symposium, work of experts, researchers and their
collaborators of different occupations, profiles and research groups will be presented. The
Symposium is supported by the Ministry of education, science and technological development,
Ministry of agriculture and environment protection, Province Secretary of science and technological
development, Province Secretary of agriculture, aquaculture and forestry and our sponsors. I would
like to use this opportunity and express my great gratitude to the companies and institutions which
have sponsored our Symposium „One Health – New challenges“, or helped in any other way.
Special thanks, I owe to the stuff of Scientific Veterinary Institute „Novi Sad”, as well as the stuff
from Scientific Veterinary Institute of Serbia, for the help with organization and a successful
Symposium!
President of Organizing Committee
Prof. Dr Miroslav Ćirković
Director of Scientific Veterinary Institute „Novi Sad“
.
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Content
Page №
PROGRAM
10 - 18
Key note speach: One Health Concept – role and importance of veterinarians.
Milijan Jovanović, Miroslav Ćirković
19 - 26
27 - 104
Session 1
DISEASES OF FARM ANIMALS
Plenary lecture: EIA and EVA two equine viral diseases present in Europe
Aymeric Hans
28 - 35
Invited presentation: The voluntary programme for control and eradication of bovine viral
diarrhoea virus infections in Slovenia.
Ivan Toplak, Peter Hostnik, Danijela Rihtarič, Jože Grom
36 - 41
Invited presentation: Comparison of macroscopic and microscopic lession in mesenterial lymph
nodes in pigs naturally infected with Mycobaterium avim subsp hominissuis.
Vladimir Polaček, Jasna Prodanov-Radulović, Dejan Vidanović, Sanja Kovačević-Aleksić
42 - 49
Immune response and protective effect of vaccine against listeriosis in sheep in Serbia. Dragan
Bacić, Blagoje Dimitrijević, Mila Savić
50 - 56
Oral fluid as a potential sample for viral diseases detection in pigs. Vesna Milicević, Branislav
Kureljusić, Jelena Maksimovic-Zorić, Ljubisa Veljović, Vladimir Radosavljević, Nemanja
Jezdimirović, Bozidar Savić
57 - 63
Mycobacteria in animals in Slovenia: from cattle to aquarium fish. Mateja Pate, Darja Kušar,
Urška Zajc, Vlasta Jenčič, Diana Žele, Gorazd Vengušt, Jože Starič, Jožica Ježek, Katarina Logar,
Tina Pirš, Petra Bandelj, Brane Krt, Matjaž Ocepek
64 - 70
Sanitary and quality conditions of imported bulls semen in Serbia analyzed at "NIV-NS"
(retrospective from 2010-2014). Aleksandar Milovanović, Tomislav Barna, Milovan Jovičin, Jelena
Apić, Sava Lazić, Igor Stojanov, Miroslav Urošević
71 - 79
The health status of breeding stallions for natural breeding and artificial insemination,
regulatory compliance in European Union, South America and West Balkan. Miroslav I.
Urosević, Luis Losinno, Aleksandar Milovanović, Dragisa Trailović, Slobodanka Vakanjac, Jelena
Petrović, Natasa Filipović
80 - 86
Sperm pathological forms and acrosomal membrane integrity in boar AI dose on pig farms in
AP Vojvodina (Serbia). Jelena Apić, Aleksandar Milovanović, Tomislav Barna, Milovan Jovičin
87 - 91
Echinococcus granulosus of domestic pigs: a case control study. Doroteja Marčić, Jasna ProdanovRadulović, Igor Stojanov, Siniša Grubač, Ivan Pušić, Vladimir Polaček
92 - 96
The prevalence of aspergillosis in poultry and control measures - our experience. Miloš
Kapetanov, Dragana Ljubojević, Igor Stojanov, Milica Živkov-Baloš, Miloš Pelić, Marko Pajić
97 - 104
Session 2 - Main Hall
105 - 188
FOOD AND FEED SAFETY AND QUALITY
Invited presentation: Game meat safety – wild boars.
Jelena Petrović, Milica Živkov Baloš, Živoslav Grgić
106 - 114
Invited presentation: The determination of qualities in cereals silages supplemented with pumpkin
and carrot.
Milica Živkov-Baloš, Sandra Jakšić, Milovan Jovičin
115 - 123
5
Invited presentation: Mycotoxicological assessment of feed in Serbia in 2014 in the light of new
legislation.
Ksenija Nešić, Sandra Jakšić, Milica Živkov-Baloš, Bojana Prunić
124 – 132
Effects of fed diets with a different n-6/n-3 PUFAs ratio on oxidative stability, and physicochemical properties of chickens meat. Dragan Milićević, Dejana Trbović, Zoran Petrović, Breda
Jakovac-Strajn, Ivan Nastasijević, Nenad Perunović, Mirjana Lukić
133 – 141
Ecological safe production of smoked common carp meat. Jelena Babić, Brankica Kartalović,
Jelena Petrović, Đorđe Okanović, Biljana Božić, Miloš Pelić, Miroslav Ćirković
142 – 147
Effect of diet on improving fatty acid composition of pig meat. Radmila Marković, Milica
Todorović, Srđan Pantić, Milan Baltić, Jelena Ivanović, Dobrila Jakić-Dimić, Dragan Šefer, Branko
Petrujkić, Stamen Radulović
148 – 156
Effects of some dieatary supplementation with phytonutrients on selected biochemical
parameters and growth performance in broiler chikens. Milanka Jezdimirović, Blagoje
Dimitrijević, Saša Ivanović, Nemanja Jezdimirović, Mila Savić, Dragan Bacić, Slavoljub Jović
157 – 165
The influence of climatic factors in Serbia on mycotoxin production. Sandra Jakšić, Milica ŽivkovBaloš, Nadežda Prica, Zoran Mašić, Ksenija Nešić, Igor Jajić, Biljana Abramović
166 – 172
Radioactivity of the soil in Vojvodina (northern province of Serbia). Željko Mihaljev, Dragana
Ljubojević, Miroslav Ćirković, Milica Živkov-Baloš, Sandra Jakšić, Brankica Kartalović, Nadežda
Prica
173 – 177
Physicochemical analysis as an indicator of the quality of honey originating from Vojvodina
region. Nadežda Prica, Milica Živkov-Baloš, Sandra Jakšić, Željko Mihaljev, Dragana Ljubojević,
Branka Vidić, Sara Savić
178 – 182
Effects of dietary hot red pepper addition on productive performance and blood lipid profile of
broiler chickens. Nikola Puvača, Ljiljana Kostadinović, Dragana Ljubojević, Dragomir Lukač, Sanja
Popović, Jovanka Lević, Olivera Đuragić, Rade Jovanović
183 - 188
Session 3
189 - 241
VECTOR BORNE INFECTIONS
Plenary lecture: Mosquito-borne flaviviruses in Europe: current perspectives.
Juan Carlos Saiz
190 – 195
Invited presentation: An overview of the recent studies on tick borne pathogens in Serbia.
Sara Savić, Branka Vidić, Aleksandar Potkonjak, Željko Čonka
196 - 203
Invited presentation: WNV in Serbia: update of current knowledge.
Tamaš Petrović, Dušan Petrić, Diana Lupulović, Ivana Hrnjaković Cvetković, Vesna Milošević, Sava
Lazić, Juan Carlos Saiz
204 – 212
Pathomorphological findings in organs of sheeps died of bluetongue disease. Ivan Dobrosavljević,
Milena Živojinović, Slavonka Stokić-Nikolić, Milica Lazić, Dragan Rogožarski
213 – 219
Species diversity and life stages dominance of hard ticks (acari: ixodidae) at Vojvodina hunting
resorts. Aleksandra Petrović, Aleksandar Jurišić, Ivana Ivanović, Aleksandar Potkonjak, Vuk Vračar,
Dragana Rajković
220 – 228
Cross-reactions in serological diagnosis of flavivirus infections. Ivana Hrnjaković Cvjetković,
Dušan Petrić, Tamaš Petrović, Gordana Kovačević, Jelena Radovanov, Aleksandra Jovanović Galović,
Dejan Cvjetković, Sandra Stefan Mikić, Aleksandra Patić, Nataša Nikolić, Vesna Milošević
229 – 233
Vector-borne infection (Ehrlichia canis) – clinical case. Zlatko Dimeski, Biljana Petrovska, Zivko
Gacovski, Goce Cilev, Elena Buntevska, Bojan Stamadziovski
234 - 236
6
Leshmaniasis in 12 years old male pekingese-clinical case. Zlatko Dimeski, Biljana Petrovska,
Zivko Gacovski, Goce Cilev, Blagica Trajanoska, Martin Kamceski, Bojan Stamadziovski, Elena
Buntevska
237 – 241
Session 4
242 – 311
WILDLIFE DISEASES AND PATHOGENS IN ENVIRONMENT
Plenary lecture: Influenza in Birds and Other Animals.
Vladimir Savić
243 – 250
Invited presentation: Virus infections of the honeybee (Apis mellifera l.) in central Europe.
Tamás Bakonyi, Petra Forgách, Aleš Gregorc, Ivana Tlak Gajger, László Békési, Miklós Rusvai,
Norbert Nowotny
251 – 259
Invited presentation: The most common health disturbances detected in wild boars in enclosed
hunting grounds in Vojvodina province.
Jasna Prodanov-Radulović, Radoslav Došen, Igor Stojanov, Tamaš Petrović
260 – 270
Contamination of public places at central Belgrade municipalities with dogs parasites in period
2013-2014. Ivan Pavlović, Dubravka Jovičić, Vladimir Terzin, Dragana Petković, Dragana Terzin,
Miloš Pavlović, Zoran Tambur, Snežana Radivojević, Borivoje Savić, Slobodan Stanojević
271 – 274
Risk factors in domestic and wild cycles of Trichinella species. Milena Živojinović, Ivan
Dobrosavljević, Ljiljana Sofronić Milosavljević, Budimir Plavšić, Zoran Kulišić, Sonja Radojičić
275 – 282
Insight into the present pesticide contamination and copepods status (Crustacea: Copepoda) of
surface water in irrigation canals in Vojvodina. Vojislava Bursić, Gorica Vuković, Aleksandra
Petrović, Maja Meseldžija, Tijana Zeremski, Aleksandar Jurišić, Dragana Rajković
283 – 289
A serological survey on Aujeszky's disease in wild boars in the region of Vojvodina in Serbia.
Sava Lazić, Vesna Milićević, Gospava Lazić, Ljubiša Veljović, Diana Lupulović, Jasna ProdanovRadulović, Jelena Maksimović -Zorić, Siniša Grubač, Radoslav Došen, Tamaš Petrović
290 – 294
Viruses in environment: situation in Vojvodina province of Serbia. Gospava Lazić, Siniša Grubač,
Bugarski Dejan, Diana Lupulović, Sava Lazić, Petar Knežević, Tamaš Petrović
295 – 300
Development of a multi-residue method for the determination of insecticides in animal fat by
LC-MS/MS. Vojislava Bursić, Gorica Vuković, Tijana Zeremski, Dejan Beuković, Miloš Beuković,
Aleksandra Petrović, Magdalina Cara
301 – 305
The unusual colony losses in the region of Vojvodina. Jelena Babić, Sara Savić, Miroslav Ćirković,
Igor Stojanov, Ivan Pihler, Nada Plavša
306 - 311
Session 5
312 – 389
AQUACULTURE
Plenary lecture: Converting waste landfill in pond areas.
Miroslav Ćirković, Brankica Kartalović, Miloš Pelić, Nikolina Novakov, Dragana Ljubojević, Sanja
Jovanić, Željko Mihaljev
313 – 320
Invited presentation: Rapid detection of important carp viruses by loop-mediated isothermal
amplification (LAMP).
Vladimir Radosavljević, Dragana Ljubojević, Vesna Miličević, Miroslav Ćirković, Dobrila JakićDimić, Jelena Maksimović-Zorić, Jadranka Žutic
321 – 326
Invited presentation: Link between lipid quality of cyprinid fish species and human health.
Dragana Ljubojević, Miroslav Ćirković
327 – 339
Seasonal variations of physico chemical parameters of the pond. Brankica Kartalović, Miroslav
Ćirković, Miloš Pelić, Sanja Jovanić, Nikolina Novakov, Biljana Božić, Željko Mihaljev
340 - 345
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Nodular coccidiosis of carp fingerlings caused by Goussia subepithelialis. Nikolina Novakov,
Miroslav Ćirković, Dragana Ljubojević, Miloš Pelić, Biljana Božić, Jelena Babić, Dalibor Todorović
346 – 350
Drum-form fitoreactor in seawater recirculation system. Dmitry Dementyev
351 – 356
Body traits and chemical composition of carp grown in production systems with different level of
intensification. Liliana Hadzhinikolova, Angelina Ivanova, Tania Hubenova
357 – 361
Meat lipid quality in carps (Сyprinus carpio L) grown in production systems with different level
of intensification. Angelina Ivanova, Liliana Hadzhinikolova
362 – 368
Results of breeding of juveniles of huchen (Hucho hucho) obtained by insemination with fresh
and cryopreserved sperm in artificial conditions. Nataša Radojković, Aleksandra Milošković,
Simona Kovačević, Tijana Veličković, Snežana Simić, Miroslav Ćirković, Ákos Horváth, Vladica
Simić
369 – 373
Distribution and some ecological impacts of fluke Posthodiplostomum cuticola (Digenea,
Trematodes) in the fish assemblage of the Zapadna Morava River (Danube basin, Serbia). Goran
Marković, Nikolina Novakov
374 – 378
The effect of OTC and flumequine antibiotic in fresh feed for the control of erythrodermatitis in
common carp (Cyprinus carpio L.). Miloš Pelić, Brankica Kartalović, Dragana Ljubojević, Nikolina
Novakov, Dalibor Todorović, Biljana Božić, Miroslav Ćirković
379 – 384
Malignant anaemia of the carps. Biljana Božić, Nikolina Novakov, Miloš Pelić, Dalibor Todorović,
Miroslav Ćirković
385 - 389
Session 6
390 – 444
EMERGING AND RE-EMERGING ZOONOSES
Invited presentation: Important zoonotic viral diseases of wildlife and their impact on human
health.
Diana Lupulović, Gospava Lazić, Jasna Prodanov-Radulović, Tamas Petrović
391 – 400
Invited presentation: Influenza and international health regulations.
Dragana Dimitrijević, Slavica Rakić Adrović, Milunka Milinković, Jovanka Ćosić
401 – 404
Invited presentation: Comparation of Rhodococcus equi of human and animal origin.
Ljiljana Suvajdžić, Maja Velhner, Maja Bekut, Gordana Bojić, Tamara Krstić, Zoran Suvajdžić,
Milenko Lazić
404 – 413
The role of veterinary medicine in One Health concept. Branka Vidić, Sara Savić, Stanko Boboš,
Nadežda Prica, Miodrag Radinović
414 – 419
The concept “One World, One Health” in the Macedonian state institutions. Misho Hristovski,
Blazho Janevski, Martin Josheski
420 – 433
More than two years without detection of wild rabies virus in Slovenia. Peter Hostnik, Jedert
Maurer Werning, Breda Hrovatin, Danijela Rihtarič, Ivan Toplak
434 – 438
Epizootiological importance of Salmonella spp. isolated in different poultry farms in Southern
Bačka and Srem region. Marko Pajić, Dalibor Todorović, Maja Velhner, Dubravka Milanov,
Vladimir Polaček, Spomenka Đurić, Igor Stojanov
439 – 444
Session 7
445 - 484
ANTIBIOTIC RESISTANCE
Invited presentation: Potential spread of antimicrobial resistance via drinking water in livestock
husbandry.
Igor Stojanov, Jasna Prodanov Radulović, Miloš Kapetanov, Jelena Petrović
8
446 - 453
Invited presentation: Antimicrobial activity of the essential oils from some aromatic medicinal
plants cultivated in Serbia.
Radomir Ratajac, Marina Žekić Stošić, Aleksandar Milovanović, Igor Stojanov
454 – 461
Antimicrobial activity of blackberry juice from Serbia on animal pathogens. Tamara Krstić,
Ljiljana Suvajdžić, Srđan Stojanović, Tamaš Petrović, Maja Bekut, Nebojša Ilić, Zoran Suvajdžić
462 – 467
Antibiotic resistance to fluoroqionoles in Salmonella spp.: Recent findings in Serbia and brief
overview of resistance mechanisms and molecular typing methods. Maja Velhner, Gordana
Kozoderović, Zora Jelesić
468 – 472
Antibacterial susceptibility of mastitis pathogens to iodine-lithium complex in vitro. Marina Žekić
Stošić, Radomir Ratajac, Aleksandar Milovanović, Jelena Petrović, Igor Stojanov, Jelena Apić
473 – 478
Random amplified polymorphic DNA analysis – RAPD and resistance to antimicrobial agents in
Salmonella spp. isolated from poultry flocks in Southern Bačka and Srem region. Dalibor
Todorović, Maja Velhner, Bojana Prunić, Marko Pajić, Dubravka Milanov, Vladimir Polaček, Dejan
Bugarski
479 - 484
Supplement to Session 1: “Diseases of farm animals”
Detection of infected goats with CAE virus-control measures and prevention. Miodrag Radinovic,
Marija Pajić, Stanko Boboš, Branka Vidić, Sara Savić, Annamaria Galfi
485 – 488
Immunoglobulin G concentration and ultrasonography of the cows udder with subclinical
mastitis. Annamaria Galfi, Miodrag Radinović, Marija Pajić, Stanko Boboš, Branka Vidić, Sara Savić,
Dubravka Milanov
489 - 492
Supplement to Session 2: “Food anf feed safety and quality”
Remediation of by-products of slaughtered livestock. Djordje Okanović, Milutin Ristić
9
493 – 498
PROGRAM
Thursday, May 21, 2015
13:00 – 15:30
Registration of participants
Main Hall entrance
15:30 – 17:40
Session 1 – Main Hall
Diseases of farm animals
Chairs: Sava Lazić, Ivan Toplak, Vladimir Polaček
15:30 – 15:50
Plenary lecture: EIA and EVA two equine viral diseases present in Europe. Aymeric Hans
(ANSES - French Agency for Food, Environmental and Occupational Health & Safety)
15:50 – 16:05
Invited presentation: The voluntary programme for control and eradication of bovine viral
diarrhoea virus infections in Slovenia. Ivan Toplak, Peter Hostnik, Danijela Rihtarič, Jože Grom
(National Veterinary Institute, Veterinary Faculty, University of Ljubljana)
16:05 – 16:20
Invited presentation: Comparison of macroscopic and microscopic lession in mesenterial lymph
nodes in pigs naturally infected with Mycobaterium avim subsp hominissuis. Vladimir Polaček,
Jasna Prodanov-Radulović, Sanja Kovačević-Aleksić (Scientific Veterinary Institute Novi Sad, Novi
Sad, Serbia)
16:20 – 16:30
Immune response and protective effect of vaccine against listeriosis in sheep in Serbia. Dragan
Bacić, Blagoje Dimitrijević, Mila Savić
16:30 – 16:40
Listeriosis: a continuous challenge in veterinary practice. Majda Golob, Mateja Pate, Darja Kušar,
Matjaž Ocepek, Irena Zdovc
16:40 – 16:50
Oral fluid as a potential sample for viral diseases detection in pigs. Vesna Milicević, Branislav
Kureljusić, Jelena Maksimovic-Zorić, Ljubisa Veljović, Vladimir Radosavljević, Nemanja
Jezdimirović, Bozidar Savić
16:50 – 17:00
Mycobacteria in animals in Slovenia: from cattle to aquarium fish. Mateja Pate, Darja Kušar,
Urška Zajc, Vlasta Jenčič, Diana Žele, Gorazd Vengušt, Jože Starič, Jožica Ježek, Katarina Logar,
Tina Pirš, Petra Bandelj, Brane Krt, Matjaž Ocepek
17:00 – 17:10
Detection of infected goats with CAE virus-control measures and prevention. Miodrag
Radinovic, Marija Pajić, Stanko Boboš, Branka Vidić, Sara Savić, Annamaria Galfi
17:10 – 17:20
Sanitary and quality conditions of imported bulls semen in Serbia analyzed at "NIV-NS"
(retrospective from 2010-2014). Aleksandar Milovanović, Tomislav Barna, Milovan Jovičin, Jelena
Apić, Sava Lazić, Igor Stojanov, Miroslav Urošević
17:20 – 17:30
Discussion for all presentations and posters in Session 1
10
Posters – Hall B (Piva)
1.1 Serological survey for antibodies against morbus Aujeszky virus in pigs originating from
unvaccinated herds. Ivan Pušić, Jasna Prodanov-Radulović, Radoslav Došen, Doroteja
Marčić, Miroslav Urosević, Diana Lupolović, Igor Stojanov
1.2 Biosafety in laboratories for diagnosis of transboundary diseases. Ljubisa Veljović, Vesna
Milicević, Jelena Maksimovic-Zorić, Vladimir Radosavljević, Jadranka Žutić, Danka Maslić
Strižak, Božidar Savić
1.3 Some facts of the rdar morphotype of Salmonella spp. and Escherichia coli. Dubravka
Milanov, Bojana Prunić, Marko Pajić, Maja Velhner, Ivana Čabarkapa
1.4 The health status of breeding stallions for natural breeding and artificial insemination,
regulatory compliance in European Union, South America and West Balkan. Miroslav I.
Urosević, Luis Losinno, Aleksandar Milovanović, Dragisa Trailović, Slobodanka Vakanjac,
Jelena Petrović, Natasa Filipović
1.5 Sperm pathological forms and acrosomal membrane integrity in boar AI dose on pig
farms in AP Vojvodina (Serbia). Jelena Apić, Aleksandar Milovanović, Tomislav Barna,
Milovan Jovičin
1.6 Immunoglobulin G concentration and ultrasonography of the cows udder with subclinical
mastitis. Annamaria Galfi, Miodrag Radinović, Marija Pajić, Stanko Boboš, Branka Vidić, Sara
Savić, Dubravka Milanov
1.7 Echinococcus granulosus of domestic pigs: a case control study. Doroteja Marčić, Jasna
Prodanov-Radulović, Igor Stojanov, Siniša Grubač, Ivan Pušić, Vladimir Polaček
1.8 The prevalence of aspergillosis in poultry and control measures - our experience. Miloš
Kapetanov, Dragana Ljubojević, Igor Stojanov, Milica Živkov-Baloš, Miloš Pelić, Marko Pajić
1.9 The influence of Aspergillus fumigatus infection on some hematological parameters in
turkey’s poults. Nemanja Jezdimirović, Branislav Kureljušić, Božidar Savić, Jasna Kureljušić,
Đorđe Cvetojević, Blagoje Dimitrijević, Dobrila Jakić-Dimić, Vesna Milićević, Danka MaslićStrižak, Vojin Ivetić, Milijan Jovanović
1.10 Diseases of digestive system in suckling piglets detected on swine farms in Vojvodina
province. Jasna Prodanov-Radulović, Radoslav Došen, Igor Stojanov, Ivan Pušić, Vladimir
Polaček, Siniša Grubač, Doroteja Marčić
17:30 – 18:00
Coffe break with posters
18:00 – 20:00
Welcome, Symposium Opening Session
Main hall
 Program dedicated for 65th Anniversary of Scientific Veterinary Institute “Novi Sad” 
Symposium opening - welcome addresses:
- Welcome address: Miroslav Ćirković (President of the Organizing Committee)
- Welcome address: Tamaš Petrović (President of the Scientific Committee)
- Welcome address: Representative of local administration – Municipality Irig
- Welcome address: Dejan Bugarski (Director of Veterinary Directorate (CVO), Ministry of
Agriculture and Environmental Protection)
- Welcome address: Representative of Secretariat for Science and Technological Development,
Autonomous Province of Vojvodina
- Welcome address and opening: Representative of Ministry of Education, Science and
Technological Development, Republic of Serbia
Key note speech: One Health Concept – role and importance of veterinarians. Milijan
Jovanović, Miroslav Ćirković
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0
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Welcome Coctail and party
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Friday, May 22, 2015
Morning Sessions – Main Hall
08:30 – 13:10
Paralel Sessions
08:30 – 10:30
Session 2 - Main Hall
Food and feed safety and quality
Chairs: Dragan Milićević, Jelena Petrović, Milica Živkov Baloš
08:30 – 08:45
Invited presentation: Game meat safety – wild boars.
Jelena Petrović, Milica Živkov Baloš, Živoslav Grgić (Scientific Veterinary Institute Novi Sad,
Novi Sad, Serbia)
08:45 – 09:00
Invited presentation: The determination of qualities in cereals silages supplemented with
pumpkin and carrot. Milica Živkov-Baloš, Sandra Jakšić, Milovan Jovičin (Scientific Veterinary
Institute Novi Sad, Novi Sad, Serbia)
09:00 – 09:15
Invited presentation: Mycotoxicological assessment of feed in Serbia in 2014 in the light of new
legislation. Ksenija Nešić, Sandra Jakšić, Milica Živkov-Baloš, Bojana Prunić (Institute of
Veterinary Medicine of Serbia, Belgrade, Serbia)
09:15 – 09:25
Development of probiotic cultures for domestic animals. Nataša Golić, Katarina Veljović,
Miroslav Dinić, Nikola Popović, Marija Miljkovic, Milan Kojić, Igor Mrvaljević, Amarela TerzićVidojević
09:25 – 09:35
Effects of fed diets with a different n-6/n-3 PUFAs ratio on oxidative stability, and physicochemical properties of chickens meat. Dragan Milićević, Dejana Trbović, Zoran Petrović, Breda
Jakovac-Strajn, Ivan Nastasijević, Nenad Perunović, Mirjana Lukić
09:35 – 09:45
Remediation of by-products of slaughtered livestock. Djordje Okanović, Milutin Ristić
09:45 – 09:55
Ecological safe production of smoked common carp meat. Jelena Babić, Brankica Kartalović,
Jelena Petrović, Đorđe Okanović, Biljana Božić, Miloš Pelić, Miroslav Ćirković
09:55 – 10:05
Development of starter cultures for fermented dairy products based on autochthonous lactic
acid bacteria. Amarela Terzić-Vidojević, Katarina Veljović, Dušanka Popović, Ivana Strahinić,
Maja Tolinački, Milica Nikolić, Sanja Mihajlović, Milan Kojić
10:05 – 10:15
Effect of diet on improving fatty acid composition of pig meat. Radmila Marković, Milica
Todorović, Srđan Pantić, Milan Baltić, Jelena Ivanović, Dobrila Jakić-Dimić, Dragan Šefer,
Branko Petrujkić, Stamen Radulović
10:15 – 10:30
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2.1. Analysis of pesticide residues in vegetables on green markets in Novi Sad (Serbia). Mira
Pucarević, Nataša Stojić, Biljana Panin
2.2. Effects of some dieatary supplementation with phytonutrients on selected biochemical
parameters and growth performance in broiler chikens. Milanka Jezdimirović, Blagoje
Dimitrijević, Saša Ivanović, Nemanja Jezdimirović, Mila Savić, Dragan Bacić, Slavoljub
Jović
2.3. The influence of climatic factors in serbia on mycotoxin production. Sandra Jakšić,
Milica Živkov-Baloš, Nadežda Prica, Zoran Mašić, Ksenija Nešić, Igor Jajić, Biljana
Abramović
2.4. Occurrence of selected foodborne pathogens in foods of plant origin. Monika Moravkova,
Petra Vasickova, Michal Slany, Petr Kralik
2.5. Radioactivity of the soil in Vojvodina (northern province of Serbia). Željko Mihaljev,
Dragana Ljubojević, Miroslav Ćirković, Milica Živkov-Baloš, Sandra Jakšić, Brankica
Kartalović, Nadežda Prica
2.6. Physicochemical analysis as an indicator of the quality of honey originating from
Vojvodina region. Nadežda Prica, Milica Živkov-Baloš, Sandra Jakšić, Željko Mihaljev,
Dragana Ljubojević, Branka Vidić, Sara Savić
2.7. Some quality parameters of industrial kulen from market of Vojvodina. Natalija Džinić,
Maja Ivić, Branislav Šojić, Snežana Škaljac, Vladimir Tomović, Marija Jokanović, Predrag
Ikonić, Tatjana Tasić, Đorđe Okanović
2.8. Effects of dietary hot red pepper addition on productive performance and blood lipid
profile of broiler chickens. Nikola Puvača, Ljiljana Kostadinović, Dragana Ljubojević,
Dragomir Lukač, Sanja Popović, Jovanka Lević, Olivera Đuragić
2.9. Influence of new product fitokokci-sto in broilers nutrition on productive characteristics
and biochemical blood status. Ljiljana Kostadinović, Nikola Puvača, Dragomir Lukač,
Sanja Popović, Jovanka Lević, Olivera Đuragić, Rade Jovanović
2.10. Fatty acid composition of two skeletal muscles from native Serbian swallow-belly
mangulica pigs reared outdoors. Predrag Ikonić, Dušica Čolović, Tatjana Tasić, Đorđe
Okanović, Vladimir Tomović, Natalija Džinić, Jovanka Lević
10:30 – 11:00
11:00 – 13:10
Wildlife diseases and pathogens in environment
Chairs: Tamaš Petrović, Vladimir Savić, Tamas Bakonyi
11:00 – 11:20
Plenary lecture: Influenza in Birds and Other Animals.
Vladimir Savić (Croatian Veterinary Institute, Zagreb, Croatia)
11:20 – 11:40
Invited presentation: Virus infections of the honeybee (Apis mellifera l.) in central Europe.
Tamás Bakonyi, Petra Forgách, Aleš Gregorc, Ivana Tlak Gajger, László Békési, Miklós Rusvai,
Norbert Nowotny (Faculty of Veterinary Science, Szent István University, Budapest, Hungary)
11:40 – 12:00
Invited presentation: The most common health disturbances detected in wild boars in enclosed
hunting grounds in Vojvodina province. Jasna Prodanov-Radulović, Radoslav Došen, Igor
Stojanov, Tamaš Petrović (Scientific Veterinary Institute Novi Sad, Novi Sad, Serbia)
12:00 – 12:10
Roe deer vs. Red deer papillomavirus an odd pair in delta-papillomavirus evolution and
ecology. Károly Erdélyi, Alessandra Scagliarini
12:10 – 12:20
Contamination of public places at central Belgrade municipalities with dogs parasites in
period 2013-2014. Ivan Pavlović, Dubravka Jovičić, Vladimir Terzin, Dragana Petković, Dragana
Terzin, Miloš Pavlović, Zoran Tambur, Snežana Radivojević, Borivoje Savić, Slobodan Stanojević
12:20 – 12:30
Bacillus thuringiensis as the model for the development of reliable method for the detection
and quantification of spore of Bacillus anthracis from environmental samples. Vera
Sedlackova, Iva Kubikova, Petr Kralik
13
12:30 – 12:40
Occurrence of Toxoplasma gondii in domestic pigs and wild boars in the Czech Republic.
Michal Slany, Lea Jakubcova, Nikol Reslova, Alena Lorencova
12:40 – 12:50
Risk factors in domestic and wild cycles of Trichinella species. Milena Živojinović, Ivan
Dobrosavljević, Ljiljana Sofronić Milosavljević, Budimir Plavšić, Zoran Kulišić, Sonja Radojičić
12:50 – 13:00
Insight into the present pesticide contamination and copepods status (Crustacea: Copepoda)
of surface water in irrigation canals in Vojvodina. Vojislava Bursić, Gorica Vuković,
Aleksandra Petrović, Maja Meseldžija, Tijana Zeremski, Aleksandar Jurišić, Dragana Rajković
13:00 – 13:10
4.1 Ecto- and endoparasitic burden of apodemus mice (Rodentia: Muridae) at hunting resort
Kamarište (Serbia). Olivera Bjelić Čabrilo, Aleksandra Petrović, Aleksandar Jurišić, Borislav
Čabrilo, Dina Tenji, Ivana Ivnović
4.2 A serological survey on Aujeszky's disease in wild boars in the region of Vojvodina in
Serbia. Sava Lazić, Vesna Milićević, Gospava Lazić, Ljubiša Veljović, Diana Lupulović,
Jasna Prodanov-Radulović, Jelena Maksimović -Zorić, Siniša Grubač, Radoslav Došen, Tamaš
Petrović
4.3 Dermal fibromatosis of the roe deer- case report. Dušan Lalošević, Zoran Ristić, Sava Lazić
4.4 Viruses in environment: situation in Vojvodina province of Serbia. Gospava Lazić, Siniša
Grubač, Bugarski Dejan, Diana Lupulović, Sava Lazić, Petar Knežević, Tamaš Petrović
4.5 Development of a multi-residue method for the determination of insecticides in animal
fat by LC-MS/MS. Vojislava Bursić, Gorica Vuković, Tijana Zeremski, Dejan Beuković,
Miloš Beuković, Aleksandra Petrović, Magdalina Cara
4.6 The unusual colony losses in Vojvodina Province. Jelena Babić, Sara Savić, Miroslav
Ćirković, Igor Stojanov, Ivan Pihler, Nada Plavša
13:10 – 14:30
Lunch break and poster viewing
Morning Sessions – Hall A (Tara)
08:30 – 13:00
Paralel Sessions
08:30 – 10:30
Session 3 – Hall A (Tara)
Vector borne infections
Chairs: Tamaš Petrović, Juan Carlos Saiz, Sara Savić
08:30 – 08:50
Plenary lecture: Mosquito-borne flaviviruses in Europe: current perspectives.
Juan Carlos Saiz (INIA, Madrid, Spain)
08:50 – 09:10
Invited presentation: An overview of the recent studies on tick borne pathogens in Serbia.
Sara Savić, Branka Vidić, Aleksandar Potkonjak, Željko Čonka (Scientific Veterinary Institute
Novi Sad, Novi Sad, Serbia)
09:10 – 09:30
Invited presentation: WNV in Serbia: update of current knowledge.
Tamaš Petrović, Dušan Petrić, Diana Lupulović, Ivana Hrnjaković Cvetković, Vesna Milošević,
Sava Lazić, Juan Carlos Saiz (Scientific Veterinary Institute Novi Sad, Novi Sad, Serbia)
09:30 – 09:40
Pathomorphological findings in organs of sheeps died of bluetongue disease. Ivan
Dobrosavljević, Milena Živojinović, Slavonka Stokić-Nikolić, Milica Lazić, Dragan Rogožarski
09:40 – 09:50
Species diversity and life stages dominance of hard ticks (acari: ixodidae) at Vojvodina
hunting resorts. Aleksandra Petrović, Aleksandar Jurišić, Ivana Ivanović, Aleksandar Potkonjak,
Vuk Vračar, Dragana Rajković
14
09:50 – 10:00
Babesia spp. in ticks parasitizing wild canids in Serbia. Ratko Sukara, Salem Juwaid,
Dimosthenis Chochlakis, Sanja Ćakić, Darko Mihaljica, Duško Ćirović, Snežana Tomanović
10:00 – 10:10
Diversity and spatial distribution of sandflies (diptera: psychodidae) and possibility of reemergence of leishmaniasis in Serbia. Slavica Vaselek, Gizem Oguz, Nazli Ayhan, Sara Savić,
Trentina Di Muccio, Ozge Erisoz Kasap, Yusuf Ozbel, Vladimir Ivović, Luigi Gradoni, Bulent
Alten, Dušan Petrić
10:10 – 10:30
3.1 Cross-reactions in serological diagnosis of flavivirus infections. Ivana Hrnjaković
Cvjetković, Dušan Petrić, Tamaš Petrović, Gordana Kovačević, Jelena Radovanov, Aleksandra
Jovanović Galović, Dejan Cvjetković, Sandra Stefan Mikić, Aleksandra Patić, Nataša Nikolić,
Vesna Milošević
3.2 Epidemiology of Bluetongue disease in Southeastern Europe. Josheski Martin, Hristovski
Mišo
3.3 Vector-borne infection (Ehrlichia canis) – clinical case. Zlatko Dimeski, Biljana Petrovska,
Zivko Gacovski, Goce Cilev, Elena Buntevska, Bojan Stamadziovski
3.4 Leshmaniasis in 12 years old male pekingese-clinical case. Zlatko Dimeski, Biljana
Petrovska, Zivko Gacovski, Goce Cilev, Blagica Trajanoska, Martin Kamceski, Bojan
Stamadziovski, Elena Buntevska
10:30 – 11:00
11:00 – 13:00
Session 5 – Hall A (Tara)
Aquaculture
Chairs: Miroslav Ćirković, Vesna Đorđević, Vladimir Radosavljević
11:00 – 11:20
Plenary lecture: Converting waste landfill in pond areas. Miroslav Ćirković, Brankica
Kartalović, Miloš Pelić, Nikolina Novakov, Dragana Ljubojević, Sanja Jovanić, Željko Mihaljev
(Scientific Veterinary Institute Novi Sad, Novi Sad, Serbia)
11:20 – 11:35
Invited presentation: Rapid detection of important carp viruses by loop-mediated isothermal
amplification (LAMP). Vladimir Radosavljević, Dragana Ljubojević, Vesna Miličević, Miroslav
Ćirković, Dobrila Jakić-Dimić, Jelena Maksimović-Zorić, Jadranka Žutic (Institute of Veterinary
Medicine of Serbia, Belgrade, Serbia)
11:35 – 11:50
Invited presentation: Link between lipid quality of cyprinid fish species and human health.
Dragana Ljubojević, Miroslav Ćirković (Scientific Veterinary Institute Novi Sad, Novi Sad,
Serbia)
11:50 – 12:00
Seasonal variations of physico chemical parameters of the pond. Brankica Kartalović, Miroslav
Ćirković, Miloš Pelić, Sanja Jovanić, Nikolina Novakov, Biljana Božić, Željko Mihaljev
12:00 – 12:10
The impact of benthic and planktonic bacteria on microbiological status of the digestive tract
of carp (Cyprinus carpio). Vesna Đorđević, Miroslav Ćirković, Dragana Ljubojević, Vesna
Janković, Dejana Trbović, Radivoj Petronijević, Danka Spirić
12:10 – 12:20
A closer look at toll-like receptor 4 (TLR4) and toll-like receptor 20 (TLR20) of common carp
(Cyprinus carpio). Danilo Pietretti, Maria Forlenza and Geert Wiegertjes
12:20 – 12:30
Nodular coccidiosis of carp fingerlings caused by Goussia subepithelialis. Nikolina Novakov,
Miroslav Ćirković, Dragana Ljubojević, Miloš Pelić, Biljana Božić, Jelena Babić, Dalibor
Todorović
12:30 – 12:40
Drum-form fitoreactor in seawater recirculation system. Dmitry Dementyev
15
12:40 – 12:50
Body traits and chemical composition of carp grown in production systems with different
level of intensification. Liliana Hadzhinikolova, Angelina Ivanova, Tania Hubenova
12:50 – 13:00
Meat lipid quality in carps (Сyprinus carpio L) grown in production systems with different
level of intensification. Angelina Ivanova, Liliana Hadzhinikolova
13:00 – 13:10
5.1 Results of breeding of juveniles of huchen (Hucho hucho) obtained by insemination with
fresh and cryopreserved sperm in artificial conditions. Nataša Radojković, Aleksandra
Milošković, Simona Kovačević, Tijana Veličković, Snežana Simić, Miroslav Ćirković, Ákos
Horváth, Vladica Simić
5.2 Distribution of fluke Posthodiplostomum cuticola (Digenea, Trematodes) in the fish
assemblage of the Zapadna Morava River (Danube basin, Serbia). Goran Marković,
Nikolina Novakov
5.3 The effect of OTC and flumequine antibiotic in fresh feed for the control of
erythrodermatitis in common carp (Cyprinus carpio L.). Miloš Pelić, Brankica Kartalović,
Dragana Ljubojević, Nikolina Novakov, Dalibor Todorović, Biljana Božić, Miroslav Ćirković
5.4 Malignant anaemia of the carps. Biljana Božić, Nikolina Novakov, Miloš Pelić, Dalibor
Todorović, Miroslav Ćirković
13:10 – 14:30
Lunch break and poster viewing
Friday, May 22, 2015
Afternoon Sessions – Main Hall
14:30 – 16:35
Emerging and re-emerging zoonoses
Chairs: Branka Vidić, Dobrila Jakić Dimić, Sara Savić
14:30 – 14:45
Invited presentation: Important zoonotic viral diseases of wildlife and their impact on human
health. Diana Lupulović, Gospava Lazić, Jasna Prodanov-Radulović, Tamas Petrović (Scientific
Veterinary Institute Novi Sad, Novi Sad, Serbia)
14:45 – 15:00
Invited presentation: Influenza and international health regulations. Dragana Dimitrijević,
Slavica Rakić Adrović, Milunka Milinković, Jovanka Ćosić (Institute of Public Health of Serbia –
„Batut“, Serbia)
15:00 – 15:15
Invited presentation: Comparation of Rhodococcus equi of human and animal origin. Ljiljana
Suvajdžić, Maja Velhner, Maja Bekut, Gordana Bojić, Tamara Krstić, Zoran Suvajdžić, Milenko
Lazić (Faculty of Medicine, Department of Pharmacy, University of Novi Sad, Serbia)
15:15 – 15:25
The role of veterinary medicine in One Health concept. Branka Vidić, Sara Savić, Stanko
Boboš, Nadežda Prica, Miodrag Radinović
15:25 – 15:35
The concept “One World, One Health” in the Macedonian state institutions. Misho Hristovski,
Blazho Janevski, Martin Josheski
15:35 – 15:45
More than two years without detection of wild rabies virus in Slovenia. Peter Hostnik, Jedert
Maurer Werning, Breda Hrovatin, Danijela Rihtarič, Ivan Toplak.
15:45 – 15:55
Hepatitis E virus in the Czech Republic. Petra Vasickova, Monika Kubankova, Petr Kralik
16
15:55 – 16:05
Seroprevalence of hepatitis E virus in commercial pig farms, backyard pigs and slaughtered
pigs in the region of the city Belgrade. Branislav Kureljušić, Božidar Savić, Nemanja
Jezdimirović, Đorđe Cvetojević, Jasna Kureljušić, Dobrila Jakić-Dimić, Oliver Radanović, Vesna
Milićević, Danka Maslić-Strižak, Vojin Ivetić
16:05 – 16:15
Epizootiological importance of Salmonella spp. Isolated in different poultry farms in
Southern Bačka and Srem region. Marko Pajić, Dalibor Todorović, Maja Velhner, Dubravka
Milanov, Vladimir Polaček, Spomenka Đurić, Igor Stojanov
16:15 – 16:25
Sepsis and meningoencephalitis caused by Streptococcus suis bacteria – a case report. Vesna
Turkulov, Grozdana Čanak, Siniša Sevic, Mila Rajković, Dajana Lendak, Ljiljana Živanović
16:25 – 16:35
6.1 Listeria monocytogenes meningoencephalitis in humans. Grozdana Čanak, Vesna
Turkulov, Siniša Sević, Miloš Vujanović, Ljiljana Živanović, Vedrana Petrić
6.2 Hemorhagic fever with renal syndrome in Serbia. Vesna Kovačević-Jovanović, Jelena
Protić
6.3 Detection of L. interrogans serovar hardjo in experimentaly sub cutaneus infected rabbits
by diferent diagnostical methods. Živoslav Grgić, Branka Vidić, Sara Savić, Diana
Lupulović, Ivan Pusic
6.4 Live attenuated swine influenza viruses as vaccine candidates. Aleksandar Mašić, Yan
Zhou, Shawn Babiuk, Bojana Prunić, Tamaš Petrović
16:35 – 17:00
17:00 – 19:00
Antibiotic resistance
Chairs: Jelena Petrović, Maja Velhner, Corinna Kehrenberg
17:00 – 17:30
Plenary lecture: Mechanisms of multidrug resistance in bacteria of animal origin. Corinna
Kehrenberg (University of Veterinary Medicine, Hannover, Germany)
17:30 – 17:45
Invited presentation: Potential spread of antimicrobial resistance via drinking water in
livestock husbandry. Igor Stojanov, Jasna Prodanov Radulović, Miloš Kapetanov, Jelena
Petrovic (Scientific Veterinary Institute Novi Sad, Novi Sad, Serbia)
17:45 – 18:00
Invited presentation: Antimicrobial activity of the essential oils from some aromatic medicinal
plants cultivated in Serbia. Radomir Ratajac, Marina Žekić Stošić, Aleksandar Milovanović,
Igor Stojanov (Scientific Veterinary Institute Novi Sad, Novi Sad, Serbia)
18:00 – 18:10
Antimicrobial resistance of Enterococcus faecalis strains from pork.
Zdovc
18:10 – 18:20
Antimicrobial activity of blackberry juice from Serbia on animal pathogens. Tamara Krstić,
Ljiljana Suvajdžić, Srđan Stojanović, Tamaš Petrović, Maja Bekut, Nebojša Ilić, Zoran Suvajdžić
18:20 – 18:30
The concept of reserve antibiotic. Maja Bekut, Ljiljana Suvajdžić, Tamara Krstić
18:30 – 18:40
Antibiotic resistance to fluoroqionoles in Salmonella spp.: Recent findings in Serbia and brief
overview of resistance mechanisms and molecular typing methods. Maja Velhner, Gordana
Kozoderović, Zora Jelesić
18:40 – 18:50
Research on the antibiotic resistance APEC strains isolated from broilers, in the western part
of Romania. Iancu Ionica, Viorel Herman, Cătană Nicolae
18:50 – 19:00
17
Majda Golob, Irena
7.1
Growth competition experiments revealed a fitness cost in triclosan tolerant salmonella
enterica mutants of different serovars. Ulrike Rensch, Matthias Greiner, Günter Klein,
Corinna Kehrenberg
7.2 Bordetella bronchiseptica: proposal for a modification of the broth microdilution
susceptibility testing method. Sandra Prüller, Cornelia Frömke, Heike Kaspar, Günter Klein,
Lothar Kreienbrock, Corinna Kehrenberg
7.3 Quantification of viable campylobacter cells by qPCR after ethidium monoazide and
propidium monoazide treatments of cells. Diana Seinige, Carsten Krischek, Günter Klein,
Corinna Kehrenberg
7.4 In-vitro susceptibility to eight biocides in extended-spectrum βeta-lactamase (ESBL)
producing Escherichia coli–isolates of human and avian origin. Daniela Deus, Carsten
Krischek, Yvonne Pfeifer, Günter Klein, Felix Reich, Corinna Kehrenberg
7.5 Comparison of agar dilution and broth microdilution susceptibility testing of
Haemophilus parasuis isolates from Germany. Sandra Prüller, Conny Turni, Patrick J.
Blackall, Katrin Strutzberg-Minder, Heike Kaspar, Diana Meemken, Günter Klein, Corinna
Kehrenberg
7.6 Susceptibility of methicillin-resistant and susceptible Staphylococcus aureus isolates of
various clonal lineages to eight biocides. Isa Adriana Kernberger-Fischer, Carsten Krischek,
Birgit Strommenger, Günter Klein, Corinna Kehrenberg
7.7 Surveillance of antimicrobial resistance at the clinic of infection disease in Clinical
Centre of Vojvodina. Vedrana Petric, Sandra Stefan-Mikić, Siniša Sević, Vesna Milošević,
Ivana Hrnjaković-Cvjetković, Miloš Vujanović
7.8 Antibacterial susceptibility of mastitis pathogens to iodine-lithium complex in vitro.
Marina Žekić Stošić, Radomir Ratajac, Aleksandar Milovanović, Jelena Petrović, Igor
Stojanov, Jelena Apić
7.9 Random amplified polymorphic DNA analysis – RAPD and resistance to antimicrobial
agents in Salmonella spp. isolated from poultry flocks in Southern Bačka and Srem
region. Dalibor Todorović, Maja Velhner, Bojana Prunić, Marko Pajić, Dubravka Milanov,
Vladimir Polaček, Dejan Bugarski
7.10 Resistance of Black-Headed gull (Chroicocephalus ridibundus) and mute swan (Cygnus
olor) Escherichia coli to second and third generation of cephalosporins. Aleksandra
Petrović, Milivoje Petrušić, Verica Aleksić, Petar Knežević
Dinner and free time
1 9 : 0 0
Saturday, May 23, 2015
09:00 – 09:40
Main Hall
Symposium Conclusions and Closing Remarks
Chairs: Miroslav Ćirković, Tamaš Petrović
09:00 – 09:30
Symposium Conclusions with discussions
09:30 – 09:40
Closing Remarks
09:40 – 12:30
Tourst excursion - Visits of cultural and historical places - optional
1
End of Symposium
2
:
3
0
18
Key note speech
ONE HEALTH CONCEPT - THE ROLE AND IMPORTANCE OF VETERINARIANS
Milijan Jovanović1*, Miroslav Ćirković2
1.Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia
2. Scientific Veterinary Institute „Novi Sad“, Novi Sad, Serbia
* Corresponding author: milijan@vet.bg.ac.rs
Abstract
The One Health concept is defined as the collaborative efforts of multiple disciplines, working
locally, nationally and globally, to reach optimal health for people, animals and the environment.
One Health is a new phrase, but the concept extends back to ancient times and can be traced as far
back as to the Greek physician Hippocrates.
In the late 19th century, German physician and pathologist Rudolf Virchow (1821–1902), to
indicate the infectious disease links between animal and human health, coined the term "zoonosis",
and said "...between animal and human medicine there are no dividing lines –nor should there be".
The phrase "One Medicine" was developed and promoted by an American veterinary
epidemiologist Calvin W. Schwabe (1927–2006).
The term “One Health” is used in many different contexts. However, there appears to be some
confusion as to what the term really means. Some authors consider the terms “One Medicine”, “One
Health” and “One World-One Health-One Medicine” to be entirely synonymous.
Because in the early year of 21st century emerging zoonotic diseases created several international
crises governments and scientists worldwide recognized that greater interdisciplinary collaboration
was required to prevent and control diseases such as bovine spongiform encephalopathy (BSE),
severe acute respiratory syndrome (SARS), highly pathogenic avian influenza (HPAI H5N1) and
etc. It had highlighted the need for professional collaboration and that such collaboration should
include not only physicians and veterinarians, but also wildlife specialists, environmentalists,
anthropologists, economists and sociologists, among others. The One Health concept recognized
that the health and wellbeing of humans, animals and ecosystems are interconnected. The concept
One Health has been adopted with great enthusiasm by the veterinary profession. Veterinarians
have opportunities and responsibilities to protect the health and well being of people, animals and
ecosystems in all the areas in which they work: food security, food safety, antibiotic sensitivity
testing, research on zoonoses, emerging infectious diseases, ecosystem protection, and comparative
medical research. The tripartite group FAO/OIE/ WHO promotes and supports One Health
approach. We see One Health as a concept that would drive new research ideas and greater
collaboration.
Keywords: animals, ecosystem, humans, One Health, zoonoses
Introduction
The One Health Concept is a worldwide strategy for expanding interdisciplinary collaborations and
communications to improve all aspects of the health and welfare of humans, animals and the
environment. It could be defined as the collaborative efforts of multiple disciplines, working
locally, nationally and globally, to reach optimal health for people, animals and the environment
(Gibbs, 2014).
The term ‘One Health’ is used in many different contexts and by people with varying backgrounds.
However, there appears to be some confusion as to what the term really means, and it is used in a
19
wide range of contexts, often including or bordering concepts such as infection biology, contagious
diseases, zoonotic infections, evolutionary medicine, comparative medicine, and translational
medicine.
Some authors consider the terms “One Medicine”, “One Health” and “One World, One Health, One
Medicine” to be entirely synonymous (Gibbs and Anderson, 2009) while others point out some
differences, which are subtly nuanced (Lerner and Berg, 2015). It is certain that the term One
Health most commonly used today and has the widest and comprehensive meaning, which refers
not only to infectious diseases but also encompasses the general public health, comparative
medicine and ecology.
History
One Health is a new phrase, but the concept extends back to ancient times. The recognition that
environmental factors can impact human health can be traced as far back as to
the Greek physician Hippocrates (c. 460 BCE – c. 370 BCE) and his text "On Airs, Waters, and
Places". in which he promoted the concept that public health depended on a clean environment.
(Anon, 2015).
Also, in India on a stone edict, laid down by the roadside, written in the time of King Ashoka
(ca. 304 BC-232 BC), a common concern about the health of humans and animals:
. . . everywhere has Beloved-of-the-Gods, King Piyadasi, made provision for two types of medical
treatment: medical treatment for humans and medical treatment for animals. Wherever medical
herbs suitable for humans or animals are not available, I have had them imported and grown.
Wherever medical roots or fruits are not available I have had them imported and grown. Along
roads I have had wells dug and trees planted for the benefit of humans and animals (Lerner and
Berg 2015).
Figure 1. Rudolf Virchow (1821–1902)
20
In recent history, the great advocate of a unique approach to medicine was the founder of modern
pathology German pathologist Rudolf Virchow (Rudolf Virchow, 1821-1902) (Figure 1) which is
its position formulated in the following statement: “Between animal and human medicine there is
no dividing line-nor should there be. The object is different but the experience obtained constitutes
the basis of all medicine”. The son of a butcher, Virchow noted the link between diseases of
humans and animals and coined the term ‘zoonosis’ to indicate the infectious disease links between
animal and human health. He described the life cycle of Trichinella spiralis in swine and its
zoonotic consequences. Another pathologist, William Osler (1849-1919), a Canadian physician was
also ardent supporter of unique medicine. He briefly studied in Germany with Virchow and after he
got a lectureship in the Medical Faculty of McGill University in Montreal. He lectured to not only
medical students, but also he demonstrated anatomy and pathology on a daily basis to the veterinary
students from the Montreal Veterinary College. As an active participant in comparative pathology,
he became vice president and later president of the Veterinary Medical Association.
In the early 20th century Virchow concept is beginning to fade and lose interest in the medical
and veterinary profession, which develop as an independent discipline. In the middle of the
twentieth century Calvin Schwabe (1927-2006), a veterinary epidemiologist and parasitologist at
the University of California, Davis School of Veterinary Medicine, established a modern approach
to the role of animals in human health which developed and promoted through concept One
Medicine. His concepts of One Medicine were based on the close relationship between humans,
domestic animals and public health. Schwabe proposed a unified human and veterinary approach to
zoonoses in the 1964 edition of his monograph ‘Veterinary Medicine and Human Health’ which
has reached the third edition that appeared in 1984(Cardiff et al, 2008).
The term One World-One Health was first introduced at the meeting of the Wildlife Conservation
Society (WCS), which was held in New York in 2004. At this meeting were adopted 12
recommendations to embrace and protect both medicine and ecosystem health. This series of
recommendations became known as the Manhattan Principles, in recognition of the fact that the
meeting was hosted by Rockefeller University in New York (Gibbs, 2014).
The idea of greater collaboration between veterinary and medical profession over time more and
more developed in the USA. In 2006, the American Veterinary Medical Association (AVMA)
established the One Health Initiative Task Force, and a year later in 2007, the American Medical
Association unanimously approved a resolution calling for increased collaboration between the
human and veterinary medicine. The term ‘One Health’ had entered the medical and scientific
lexicon (Cardif et al. 2008; Gibbs, 2014; Kaplan et al.2009).
The World Veterinary Association (WVA) recognizes and supports the Tripartite Concept Note
titled “Sharing responsibilities and coordinating global activities to address health risks at the
animal-human-ecosystem interfaces” jointly developed and adopted by the World Organization for
Animal Health (OIE), World Health Organization (WHO) and the Food and Agriculture
Organization of the United Nations (FAO) in 2010 (Anon, 2014). The tripartite note clearly
recognizes that addressing health risks at the human-animal-ecosystem interface requires strong
partnerships among all stakeholders.
The goal of the concept
The One Health Concept recognizes that the health and wellbeing of humans, animals and
ecosystems are interconnected. It involves applying a coordinated, collaborative, multidisciplinary
and cross-sectorial approach to address potential or existing risks that originate at animal-humanecosystem interfaces.
21
The importance of One Health concept in the modern world is increasingly gaining in, it is not
difficult to understand because that about two-thirds (60.3 %) of emerging infectious diseases
(EID) result from zoonoses; the majority of these have their origin in wildlife (71.8%) and have
been increasing in recent years. A major finding was that there was a high correlation between EID
origins and socio-economic, environmental and ecological factors, thereby providing a mechanism
by which areas (called “emerging disease hotspots”) where EIDs are most likely to originate.
Global resources to combat the emergence of infectious disease are not well allocated as most of the
research and surveillance activities are occurring in countries that do not fit the predicted sites from
which new diseases are likely to emerge. Humans serve as a primary reservoir for only 3% of
known zoonotic pathogens (Frank, 2008).
In the early years of the 21st century with the advent of diseases such as highly pathogenic avian
influenza (HPAI H5N1) severe acute respiratory syndrome (SARS), Middle East respiratory
syndrome (MERS), Bovine spongiform encephalopathy (BSE) AIDS, Ebola, West Nile virus,
hantavirus infections and others diseases that had the potential to become pandemic, including
extensive human mortality, created several international crises (Gibbs, 2005). Governments and
scientists worldwide have found themselves facing a great challenge and recognized that greater
interdisciplinary collaboration was required to prevent and control of these diseases. Some believe
that the One Health concept was just created and developed in fear of these diseases (Gibbs,
2014).
With the development of technology, air and water transport, the world has continued to shrink.
Microorganisms can travel by plane across the world in time frames shorter than their incubation
periods. “Nowhere is remote and no one is disconnected“(Frank, 2008). Special problems are
caused by vectors transmitted diseases. Country borders do not exist for vector-borne diseases
(Valčić et al, 2015).
The population of the world is increasing from the current 7 billion, is projected to reach 9 billion
in 2050. The increase in population will require greater need for safe and quality food of animal
origin (Wall, 2014). Food safety, combating microbial resistance to antibiotics, climate change and
wildlife conservation are further mega-concerns where One Health can make contributions.
As a starting point for an explanation of "One Health concept" many authors have taken symbolic
umbrella model (Figure 2), which was developed in Sweden and from which we can see the
complexity of the concept. A number of scientific fields are present under the umbrella of One
Health (see the top row circles): biology, human medicine, veterinary medicine, public health,
environmental chemistry, and health economy, to mention some of the most important ones. The
concept of health could be defined on at least three different levels: the individual level, the group
or population level, or the ecosystem level (Lerner and Berg, 2015).
Health is a state of complete physical, mental and social well-being and not merely the absence of
disease or infirmity. It is the outcome of a complex of several inter-dependent medical, economic,
socio-cultural, environmental and ecological factors. People’s health and wellbeing, and equally
animal health and welfare are strongly interlinked. Both also influence and are impacted by the
health of the environment. Health is a precondition for wellbeing and respectively welfare.
Wellbeing and welfare reinforce health.
Animals, domesticated and wild, play a key role in the wellbeing of people and the future of our
planet. They provide working power, food, protection, companionship and enjoyment, facilitate the
advancement of biomedical research, and are a crucial part of healthy ecosystems. Keeping animals
healthy is essential for the health and well-being of people and the environment (Mills and Hall,
2014). The man and animals in a broader sense belong to the one multispecies animal world in
22
which in some cases shared a common pathogen, and also they are together exposed to harmful,
toxic agents from the environment.
Figure 2. The One Health concept presented in the form of an umbrella
In the One Health concept, plant health is often perceived as a part of ecosystem health rather than
as a separate entity and has essential impact on human and animal health
Recognizing the basic fact that all humans and animals must eat to survive, it follows that since our
food sources are all derived directly or indirectly from plants we must protect plant life on this earth
to maintain healthy humans and healthy animals. Thus, plant pathogens play a direct and indirect
role in the ecological existence of human and animal species. We need human doctors (physicians)
and animal doctors (veterinarians), as well as plant doctors (plant pathologists) – to work together
for the common good of all species. How to plant health can affect the health of animals, animal
products safety and health of people and which problems of that might arise, well you can see
from our example from 2013 regarding the appearance of larger amounts of aflatoxin in corn
grain and corn products.
Besides their role in the preservation of ecosystems and the importance as a source of food plants
will be in the future more and more used in the protection of animal health. Recent advances in
immunology and in biotechnology, demonstrated the feasibility and advantages of plant-based
production platforms for various recombinant proteins with therapeutic use, including, subunit
vaccines, complex antibodies and immunogenic virus-like particles. Plant made therapeutic
products are currently on the cusp of widely entering biotechnology markets. To date, vaccines
been made against chicken infectious bronchitis virus (IBV) and infectious bursal disease virus
(IBDV), enterotoxigenic E. coli (ETEC) and porcine transmissible gastroenteritis coronavirus
23
(TGEV), bovine herpes virus (BHV) and bovine viral diarrhea virus (BVDV) (Kolotilin et al.,
2014).
The role of the veterinarians
Veterinarians, regardless of their field of practice, all play a significant role in human health and
animal health Veterinarians in all areas of the profession have opportunities and responsibilities to
protect the health and well being of people in all the areas in which they work: food security, food
safety, antibiotic sensitivity testing, research on zoonoses, emerging infectious diseases, ecosystem
protection, comparative medical research, and human physical/mental health. The veterinarian is the
only health care professional likely to see both people and their animals, so he/she therefore has an
awareness of the potential threat of zoonotic disease and has the ability and responsibility for
detecting zoonotic/emerging diseases. Fortunately, veterinarians have considerable training in
comparative medicine, zoonoses, and public health. Physicians on the other hand do not receive
extensive training in comparative medicine and zoonoses. Therefore veterinarians are in a better
position to discover public health threats than are physicians. They are also in an ideal position for
establishing a disease surveillance system using pets as sentinels of disease exposure in the home
environment and in the wild (Frank, 2008).
There should be a good relationship and communication between public and private veterinary
sectors as well as between veterinarians and animal owners and keepers. In achieving the One
Health concept veterinary profession has a special responsibility and prominence in the
implementation of the following:

enhancing the health and welfare of animals (e.g., through treatment and prevention of
diseases and promotion of animals’ physical and mental wellbeing);

improving the health of people (e.g., through detection and prevention of zoonotic diseases);

ensuring safer animal products for human consumption (e.g., through preventing foodborne
diseases and food frauds; and overseeing best practices for use of animal medicines);

ensuring sufficient food (e.g., through technological and management advances);

protecting the environment (e.g., through conservation, prudent use of medicines, and
chemicals, and the careful disposal of waste);

competencies in the use of antimicrobials and thus insuring their effectiveness in both
animal and human medical practice; in the use of antimicrobials and thus preserve their
effectiveness in veterinary and medical practice (Anon, 2014).
Comparative medicine
Comparative medicine is the study of disease processes across species and is based on the study of
naturally occurring diseases of animals that also afflict humans Comparative medicine is, by
definition, based on the idea of comparing humans to one or more chosen animal species, or the
other way around. Hence, it has limitations from a veterinary point of view, as veterinarians are also
often interested in comparing different animal species with each other, that is, making comparisons
that include several different species but not necessarily always including Homo sapiens. Animals
suffer from many of the same chronic diseases such as heart disease, cancer, diabetes, asthma, and
arthritis as humans. Sometimes a disease entity is recognized in animals long before it is recognized
in humans. The concept of comparative medicine is very old. The ancient Greeks understood that
dissecting and studying animals could yield important clues to understanding human diseases. The
24
musculoskeletal system is particularly well-suited to comparative medicine studies since acute and
chronic disorders of bones and joints have the same counterparts in humans and animals.
Information gained from one species can be directly translated to another, thereby advancing the
diagnosis and treatment of musculoskeletal disorders. Some of the solutions in orthopedic surgery,
such as a particular method of fixation in fractures were first applied in veterinary practice (Cook
and Arnoczky, 2009).
In the United Kingdom has been established the Comparative Clinical Science Foundation to fund
comparative studies in cancer, aging and genetic disorders (http://www.onemedicine.org.uk/).
Comparative sequence analyses demonstrate extensive genetic homologies among species.
Furthermore, it has been proven that mutations of one gene in one species cause a similar disease in
other species. For example, the virus discovered to cause sarcomas in chickens by Peyton Rous and
others, 100 years ago harbours the gene associated with cancers in rats, mice and human. As the
genes and diseases are the same, the medicine will be the same: the genetic version of One
Medicine or One Health approach (Cardiff, 2008). Isolation and cloning of a gene associated with
human disease, when inserted into and expressed by the mouse genome, recapitulates the disease in
the mouse. Clearly, one gene can cause the same disease in another species. In modern biological
science is dominated by molecular biology and there is a need to learn more and more about less
and less. To enhance interdisciplinary scientific collaboration, like the existence of "Open access
publishing", there is thinking about creating "Open access bio-banks" where will be kept
biological material (be it tissue, blood, fecal samples, effluent water, or anything else possibly
relevant) that would be available to scientists who perceive certain problems from different
aspects (Lerner, 2015).
One Health in education
Given the importance of One Health to the enhancement of human, animal and environmental
health, it is essential to embed the One Health Concept into the curricula at colleges, schools and
faculties educating animal, human, and environmental health professionals. New communication
technologies provide excellent opportunities to spread information and engage people in
postgraduate education. In some parts of the world today is possible in this field to gain a degree
MSc and PhD at the University (Gibbs, 2014). To familiarize professionals with One Health,
numerous international, regional, and national conferences, symposia, and workshops have been
organized. Two international congresses have specifically addressed One Health, the first in
Australia in 2011 and the second in Thailand in 2013. Each was attended by several hundred
professionals. A third international congress is planned for 2015 in Amsterdam.
Conclusion
The advancement of the health and well-being of people and animals depends on effective and
sustained collaboration between varied professions and disciplines, both in the public and private
sectors. Observation of animal health and welfare, public health, zoonotic and environmental
problems as a separate discipline and entities would have in a long term, limited impact on
solving global problems. For continuous improvement it is necessary a broad interdisciplinary
holistic approach to develop effective action that fully emphasizes One Health philosophy. It is
time to consider whether One Health will prove to be a short-lived response to a spate of emerging
diseases that apparently threatened to the world, or a paradigm shift that will lead to a wider and
deeper commitment to interdisciplinary action addressing the protection and needs of society in the
21st century. We have an optimistic attitude, and we hope that the future will most likely bring
25
more collaborations of veterinarians from all fields with multiple professions such as public health,
human medicine, bio-engineering, animal science, environmental science, and wildlife. Together,
we are stronger to fight disease, and we are, indeed, wiser.
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by Francis Adams. 2015, http://classics.mit.edu/Hippocrates/airwatpl.html
Anon: World Veterinary Association Position on One Health Concept, 2014, www.worldvet.org.
Cardiff RD, Ward MJ, Barthold SW: ’One medicine - one pathology’: are veterinary and human
pathology prepared? Laboratory Investigation, 88: 18-26, 2008.
Cook JL, Arnoczky SP. "The One Health Concept in Comparative Orthopaedics." One Health
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49(11): 1063-1065, 2008.
Gibbs EPJ:The evolution of One Health: a decade of progress and challenges for the future, Veterinary
Record 174: 85-91, 2014.
Gibbs EPJ, Anderson TC: “One World-One Health” and the global challenge of epidemic diseases of
viral aetiology, Veterinaria Italiana, 45(1): 9-18, 2009.
Kaplan B, Kahn LH, Monath TP: The brewing storm, Veterinaria Italiana, 45(1): 9-18, 2009.
Kolotilin I, Topp E, Cox E, Davriendt B, Conrad U, Joensuu J et al.: Plant-based solutions for veterinary
immunotherapeutics and prophylactics. Veterinary Research 2014, 45: 117.
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education: what is One Health? Infection Ecology and Epidemiology 2015, 5: 25300 http://dx.doi.org./10.3402/iee.v5.25300.
Mills D, Hall S: Animal-assisted interventions: making better use of the human-animal bond Veterinary
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Osterholm MT, Moore KA, Kelley S, Brosseau LM, Wong G, Murphy FA et al.: Transmission of Ebola
viruses: what we know and what we do not know. mBio 2015, 6(2): e0013715.doi:10.1128/mBio.00137-15.
Valčić M, Radojičić S, Obrenović S, Stević N, Vasić A: Vektori kao rezervoari I prenosioci infektivnih
bolesti životinja, Zbornik predavanja XXXVI Seminara za inovacije znanja veterinara, 2015, 3-18.
Verner-Carlsson J, Lõhmus M, Sundström K, Strand TM, Verkerk M, Reusken C et al.: First evidence
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Wall P: One Health and the food chain: maintaining safety in a globalised industry, Veterinary Record,
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________________________________________________________________________
Session № 1
DISEASES OF FARM ANIMALS
Full Papers
_______________________________________________________________________
27
Plenary lecture:
EIA AND EVA TWO EQUINE VIRAL DISEASES PRESENT IN EUROPE
Aymeric Hans1
1 ANSES-Dozulé Laboratory for Equine Diseases, Virology Unit, France
* Corresponding author: Aymeric.HANS@anses.fr
Abstract
Equine infectious anemia (EIA) and Equine Viral Arteritis (EVA) are two equine diseases of
importance to international trade listed by the World Organization for Animal Heath (OIE). Equine
infectious anemia is caused by a virus (EIAV) belonging to the retroviridae family, genus
Lentivirus as the Human Immunodeficiency Virus (HIV) and has a worldwide distribution. EIAV
causes a persistant infection associated to recurrent febrile episodes. Asymptomatic infected
animals are the viral reservoir and can transmit the virus to others equids. EIAV is bloodborne and
transmitted primarily via biting flies, but also iatrogenically if non-sterile needles or surgical
equipment are used. Insects — mainly horseflies and stable flies — are mechanical vectors:
although the virus does not replicate within the insect, the infectious virus can remain in its
mouthparts for several hours after biting. Equine arteritis virus (EAV), the causative agent of EVA,
is a member of the Arteriviridae family. EAV infects equidae and may be transmitted through
respiratory and venereal routes. EAV can persist in the reproductive tract of stallions only.
Following the primary infection, up to 70% of stallions can be persistently infected and shed the
virus in their semen sometimes for life. These “shedder” stallions spread the virus in the horse
population during breeding or when their semen is used for artificial insemination. The financial
consequences of these diseases are potentially huge, since EIAV positive horses are in general
slaughtered and stallions shedding EAV in the semen lose their value. Moreover, EAV infection
may cause abortion in pregnant mares and the death of young foals. Romania and Italy are the most
European countries affected by EIA. The disease is endemic in Romania whereas only sporadic
cases have been notified in others European countries. Also, EVA cases are mainly sporadic in
European countries with very few descriptions of “pathogenic” strains leading to death or abortion
in pregnant mares. To better understand and prevent the viral spread in equine population
epidemiological surveys as well as molecular characterization of strains isolated in Europe are
investigated.
Keywords: Virus, Equine, notifiable diseases, Phylogeny
Equine Infectious Anemia Virus
Introduction
Equine infectious anaemia (EIA) is a viral disease. It is caused by Equine Infectious Anaemia virus
(EIAV) belonging to the Retroviridae family, genus Lentivirus, which also includes Human
Immunodeficiency Virus (HIV), Bovine and Feline Immunodeficiency Viruses (BIV and FIV) and
the visna-maedi virus. EIAV causes a persistent infection in equids. Clinical signs associated to the
infection appear between one to three weeks after infection and are characterized by fever, anemia,
edemas and various signs of listlessness and depend on the immune status of the host and viral load
upon infection.
28
The disease progresses and can be divided in three distinct phases. In its acute phase, the horse
shows serious clinical signs that can lead to the animal death. The chronic stage is characterized by
a recurrence of clinical phases such as pyrexia and anemia, whereas the final stage is asymptomatic
(Hammond et al., 2000; Leroux et al., 2001) with no overt signs of disease. However, these
unapparent carriers never eliminate the virus and represent an economical threat for the equine
industry. Moreover, they remain a source of infection for other horses (Issel and Adams, 1982).
Virus is transmitted mainly by infected blood through biting flies, but also by contaminated
instruments when non-sterile needles or dental equipment are used (Foil et al., 1983; Hawkins et al.,
1973). Insects, mainly horseflies and stable flies, are mechanical vectors: while the virus does not
replicate within the insect, the infectious virus remains in its mouthparts several hours after biting
(Williams et al., 1981). The virus is disseminated most effectively by this type of mechanical
vector-borne transmission when horses are gathered for equestrian events.
EIA Control is based on identification of unapparent carriers by detection of EIAV antibodies using
serological tests. The Agar Gel Immuno-Diffusion test (AGID) known as a Coggins test (Coggins et
al., 1972) is the most widely used to confirm an EIA case. Recently, some Enzyme Linked-Immuno
Sorbent Assays (ELISA) have been developed as well as immunoblots (Issel et al., 2013). EIA test
is required for horse importation/exportation and for stallions prior semen collection or in hand
breeding. Basically, leisure horses not used for breeding purposes are barely tested for EIA, thus
EIA outbreaks are often detected by a veterinarian following suspicious clinical signs in a horse
farm. Since year 2000, more and more outbreaks of EIA have been reported in Europe and USA.
Review of EIA surveillance in France
Outbreak surveillance
As EIA is a Category 1 health hazard, any clinical suspicion or confirmation by the results of
analysis by an accredited laboratory must be declared to the Directorate General for Food (DGAL)
and sample(s) must be sent to the National Reference Laboratory (NRL) for confirmation. This
clinical surveillance relies on the owners, veterinarians and the network of laboratories accredited to
perform serological analyses to detect EIA. Any equidae testing positive with an agar gel
immunodiffusion test (AGID, also known as Coggins test) is considered to be infected.
When EIA is confirmed, a declaration of infection (APDI) is issued by state veterinary services,
thus initiating health control measures. Control measures mainly consist of movement restrictions
placed on the outbreak site and decontamination by euthanasia of the infected horses, the
disinfection of facilities and materials, and monthly serological testing on the horses remaining at
the outbreak site. The outbreak is considered to have been eliminated and the restrictive measures
are lifted when the remaining horses have tested negative at least twice, three months apart.
At the same time, the horses at risk of infection (i.e. those that had been in contact with the infected
animal and those present within 500 m) are identified and placed under surveillance (restriction of
movements and regular serological testing to ensure there is no seroconversion within 90 days of
the last contact with an infected horse).
Programmed surveillance
Programmed (active) surveillance includes several different measures:
• Breeding stallions are monitored systematically:
- All stallions used in artificial insemination are tested on a regular basis. If the semen is to be
sent to another European Union country, a negative Coggins result must be produced in the
two weeks preceding the first collection.
29
-
Stallions serving naturally in certain breeds must also be tested in accordance with their stud
book recommendations. A negative Coggins result must be produced in the three months
prior to the first service then every three years.
Furhtermore, “voluntary” surveillance is recommended and a test for EIA should be performed
whenever there is a change of ownership. All exported equidae must be tested in accordance with
the health requirements of the importing country. Imported equidae must also be screened for EIA
according to the exporting country, the type of importation (temporary, definitive or readmission
after temporary export) and the type of use (slaughter or other). Screening is not mandatory for
equids transported within the EU except for those from Romania. This measure was introduced in
2010 (2010/346/EU) following several cases of EIA in the United Kingdom, Belgium and France in
2009 and 2010 among equids imported directly from Romania.
2012 outbreaks
In 2012, the French network of accredited laboratories carried out 15,691 serological analyses of
which 27 tested positive. The 27 positive analyses involved eight equines in two distinct outbreaks
in two counties: Vaucluse and Gard. These two outbreaks were discovered following the
appearance of clinical signs that were suggestive of EIA (listlessness, weight loss, anaemia, nasal
bleeding, etc.).
Vaucluse outbreak
The primary outbreak was declared on January 30th, 2012, after confirmation of the infection in a
16 year-old part-bred Arabian gelding that showed suggestive clinical signs such as fever, nasal
bleeding, swelling of the lower abdomen, hind legs and the penile sheath, and pale mucus
membranes. The APDI order for the outbreak site was issued on February 2nd, 2012, requiring
restriction of movements and testing of all horses on the site. This site held seven other horses, three
of which were also diagnosed with an EIAV infection on February 6th, 2012 and all four infected
horses were euthanised on February 9th, 2012.
The epidemiological surveys covered a six-month period from August 2011 to January 2012 and
identified (1) all horses that had been in ‘contact’ and at risk for EIA infection and (2) all horses
present within a 1 km radius of the outbreak site.
These surveys revealed that some of the four infected horses had participated in an endurance race
in September 2011 in Vaucluse. As a result, more than 180 horses were identified as being at risk of
EIA infection; no cases of infection were detected among those 180 horses. Despite the high
number of at-risk horses exposed during the endurance race in late September 2011, the virus
transmission was not demonstrated outside of the outbreak site, and there was no secondary
transmission to horses off site.
Gard outbreak
The primary EIA outbreak declared in September 2012 in Gard involved six horses of the
Camargue, Pura Raza Española (PRE) Andalusian, Barb and Merens breeds. The index case, which
exhibited fever (40°C) and low haematocrit levels (16%), was originally thought to be infected with
babesiosis (for which it tested positive), and was treated for this disease. In the absence of
subsequent improvement, the veterinarian suspected an EIA infection and, noting the poor state of
its health (massive weight loss of about 100 kg), the horse was euthanised.
The epidemiological survey was first carried out on horses present within 500 meters of the primary
outbreak site as well as on all horses that had been in contact with the infected horses during the
four months period preceding the confirmation of the outbreak. The surveys identified 378 at-risk
30
‘contact’ horses, among which only 364 could be sampled. Of these 364 horses, 3 were tested
positive for EIA.
Molecular epidemiology
To genotype the EIA strains isolated from various outbreaks, gag gene (1400 nucleotides long) was
sequenced. The 2012 EIA outbreaks led to the identification of eight seropositive horses. Virus
isolates were characterised from five of the eight horses. The phylogenetic analysis, carried out
using the MEGA 5.0 software package, was used to compare and classify the viruses isolated in
2012 with respect to those isolated previously in France and those reported in the literature (Figure
1).
Figure 1: Phylogenetic analysis of 50 EIA isolates using MEGA 5.0. Analysis performed using the full gag
gene sequence (1400bp).
The obtained phylogenetic tree shows that the isolates from the outbreak declared in Vaucluse and
Gard were not only different from each other but also from those identified previously in France, in
particular those isolated in Var in 2009 (Ponçon et al., 2011). Although there were indications from
the epidemiological field survey that there was an epidemiological connection between the
Vaucluse and Gard outbreaks via the purchase, sale or trade of horses, this hypothesis was not
confirmed by phylogenetic analysis. In contrast, data of this phylogenetic study show that Vaucluse
and Gard outbreaks had two distinct origins because the sequenced virus isolates were not identical.
Furthermore, the seropositive horses in the Vaucluse outbreak were infected by two different
isolates (Figure 1). The isolate characterised from horse 12D134 shows that it was related to isolates
sampled in 2007 on donkeys in Ardèche county (Reme et al., 2009). However, no epidemiological
31
connection could be established from the field surveys between horse 12D134 and the Ardèche
donkey euthanised in 2007. Likewise, the viral isolates characterised from horses 12D128 and
12D131 were similar to the virus isolated in 2010 in Dordogne county on a cull trotting horse. Once
again, no epidemiological connection was demonstrated between these two outbreaks. Moreover,
the isolates characterised from horses 12D899 and 12D903, found in Gard county, were similar to
viruses isolated in Italy in the past few years (Hans et al., 2012).
Review of EIA surveillance in Europe 2010-2014
EIA is a notifiable disease in Europe. Each country has to report any EIA cases, even if no clinical
signs are seen. Thus, each AGID positive test has to be declared to the national authority then to the
European authority. The vast majority of outbreaks declared in Europe are sporadic cases without
sanitary and economic consequences for horse industry. Between 2010 and 2014, no EIA outbreaks
have been reported by Baltic and Scandinavian countries (Estonia, Latvia, Lithuania, Finland
Sweden and Denmark). The western European countries (UK, Ireland, Netherlands, Belgium,
France, Spain and Portugal) have reported 16 EIA outbreaks with 35 positives horses during the last
four years. Eighty five outbreaks were reported by central European countries (Germany, Poland,
Czech Republic, Slovakia, Austria and Hungary) with a total of 105 horses tested positive for EIA
during the last four years. Finally, Italy and the Balkans countries (Croatia, Romania, Bulgaria,
Slovenia) have reported more than 7 400 EIA outbreaks between 2010 and 2014 associated to
around 10 000 positive horses for EIA. Officially, EIA outbreaks have been reported from France,
UK, Italy, Romania, Belgium, Netherlands, Germany, Croatia and Hungary during the last four
years, showing that EIA is present all around Europe with different prevalence level.
In Europe, only two countries, Italy and Romania, have implemented a national surveillance
program to test the horse population registered for EIA.
Following the 2006 EIA outbreak, due to the use of contaminated plasma, Italy has decided to
implement a surveillance program as follow: Annual test of all equids over 6 months of age kept in
regions with high prevalence (Latium, Umbria and Abruzzo) and a test every two year for equids
living in area where EIA seroprevalence is lower. Italian authority decided to test also donkeys and
mules. In Italy, around 230 000 horse sera samples, 10 000 donkeys and 1500 mules are tested each
year. Prevalence of the disease is different between horses, donkeys and mules. Indeed, prevalence
in mule is around 10%, whereas prevalence is weak in donkeys and horse around 0.08% and 0.15%,
respectively. These data, from Italy, show that the main reservoir for EIA seems to be the mules.
Since 2007, and the implementation of the surveillance program in Italy, the number of EIA
outbreaks and the number of positive horses detected has steadily decreased over the past few years.
Situation in Romania is totally different compare to Italy, since Romania has very few breeding
farms. The majority (55%) out of 700 000 equids identified in 2010 in Romania are used for work
in the fields and not for sport or leisure activity. Less than 1 %, corresponding to 4 400 equids only,
was kept in stud-farms, whereas the 99% left are owned and kept by private individual who
possessed in general only one equid. The first description of the disease dates back to the 50’s in the
Baragan region located in the South East of Romania. It is only after the integration of Romania in
European Union in 2006, that the EIA problem has been seen. Indeed, between 2000 and 2004
Romania has declared 9 953 EIA outbreaks with 30 132 positive equids. Since 2010, Romania has
implemented a surveillance program aimed at testing all equids older than 6 months of age, two
times per year for stallions and sport horses and once a year for others. Incidence is still difficult to
estimate in Romania. Nevertheless, a decrease in the number of outbreaks and of positive horses is
seen since 2010.
32
Equine arteritis Virus
Introduction
Equine arteritis virus (EAV) is one of the major viral pathogens of horses (Rola et al., 2011). It is
the causative agent of equine viral arteritis (EVA), and can infect horses, donkeys, mules, and
zebras (Balasuriya et al., 2013). EAV is an Arterivirus belonging to the Arteriviridae family in the
order Nidovirales (Cavanagh, 1997). EAV was initially isolated from an aborted foetus on a
Standardbred breeding farm near Bucyrus, Ohio, USA, in 1953 (Bryans et al., 1957a; Bryans et al.,
1957b). Based on phylogenetic analysis of a portion of ORF5 encoding viral glycoprotein 5 (GP5),
EAV isolates are classified into two groups: The North American and the European group. The
latter can be further divided into two subgroups: European subgroup 1 (EU-1) and European
subgroup 2 (EU-2) (9, 10). It has been previously reported that North American and European EAV
isolates present 85% of nucleotide identity (Vairo et al., 2012). EVA is a respiratory and
reproductive disease of horses that occurs worldwide (Glaser et al., 1996; Timoney and McCollum,
1993). The vast majority of EAV infections are subclinical, but acutely infected animals may
develop a wide range of clinical signs including pyrexia, depression, anorexia, dependent edema
(scrotum, ventral trunk, and limbs), stiffness of gait, conjunctivitis, lacrimation and swelling around
the eyes (periorbital and supraorbital edema), respiratory distress and leukopenia. The direct
consequences of EVA outbreaks are financial losses mainly due to abortions of pregnant mares and
death of young foals (Vairo et al., 2012). Following primary EAV infection, up to 70% of the
stallions will carry the virus in their reproductive tract sometimes for years (Holyoak et al., 1993a;
Holyoak et al., 1993b). Those stallions will shed the virus in their semen.
Figure 2: Phylogenetic analysis by Maximum Likelihood method. The analysis involved 87 nucleotide
sequences
33
During the summer of 2007, an outbreak of EVA occurred in Normandy, France. Only draught and
saddle horses were affected. This 2007 outbreak occurred following the used of semen
contaminated by EAV for insemination. The objectives of this study were to undertake a thorough
epidemiological investigation to identify shedders stallions following the 2007 EVA outbreak as
well as the molecular characterization of EAV isolates encountered in France.
A total of 35 positive semen samples and 5 tissues samples from aborted foetuses were analyzed in
this study. All of them have been collected between 2007 and 2010. The evolutionary history was
inferred by using the Maximum Likelihood method based on the Data specific model. Evolutionary
analyses were conducted in MEGA5 (Tamura et al., 2011). The phylogenetic tree has been
constructed using 87 sequences (figure 2). 52 of them have retrieved from Genbank database and 35
were from viruses isolated in France between 2007 and 2010. Analysis was performed using the
3kb sequences covering ORF2 to ORF7 of the EAV genome.
The phylogenetic tree constructed shows that EVA viruses circulating in France belong to 3 groups
defined as the American group (AG), the European subgroup 1 (EU1) and European subgroup 2
(EU2). Nevertheless the majority of viruses belong to the European group, since only one virus
from the American group has been isolated in France during these 3 years. Interestingly, virus
responsible of the 2007 outbreak in Normandy belongs to the European subgroup 2 and seems to be
slightly different from viruses that use to be isolated in France before the outbreak. Indeed, all viral
isolates which are related to the 2007 outbreak formed a smaller group inside the European
subgroup 2. This genetic feature might explain the severity of the clinical signs and the
pathogenicity of the virus that caused several deaths in 2007
This work gives a clear and precise view of equine arteritis viruses strains that are circulating in
France. EAV isolates belong to the 3 groups described in the literature AG, EU1 and EU2.
Nevertheless the majority of them are from the European lineage. In addition, EAV viruses isolated
during the 2007 outbreak grouped in the European subgroup 2 and are slightly divergent from the
ones seen in France the past years. This genetic diversity might explain the severity of clinical signs
and the higher mortality rate seen during this outbreak. Investigations are in progress to understand
and decipher the pathogenic features of this isolate.
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35
Invited lecture
THE VOLUNTARY PROGRAMME FOR CONTROL AND ERADICATION OF BOVINE
VIRAL DIARRHEA VIRUS INFECTIONS IN SLOVENIA
Ivan Toplak1, Peter Hostnik1, Danijela Rihtarič1, Jože Grom1
1. University of Ljubljana, Veterinary Faculty, National Veterinary Institute, Ljubljana, Slovenia
* Corresponding author: Ivan.Toplak@vf.uni-lj.si
Abstract
In Slovenia the control of bovine viral diarrhea viral (BVDV) infection was started 1994. About
half of the bovine herds were recognized with BVDV antibody positive animals. During last twenty
years only limited progress towards the eradication was achieved. With the beginning of year 2014,
according to new national rules, which prescribes the conditions for recognition, acquisition and
maintenance, a free status of BVDV for an individual herd can be achieved, if specific requirement,
based on laboratory testing, and preventive measurements are implemented in a herd. The basis of
voluntary programme is identification and certification of herds which are free of virus. For the
recognition of officially BVDV free status of herd the breeder should provide two consecutives
laboratory testing at intervals of at least six months in all animals which are during the sample
collection in the age group of 7 to 13 months, with negative results for antibodies. The farmer will
then maintained the status by the implementation of preventive measures and each year renewed
this status with one laboratory testing of all animals in the age group of 7 to 13 months. If herd is
recognized as BVDV positive, it can start with the acquisition programme through the identification
and elimination of persistently infected animals and laboratory testing all newborn calf for BVDV
during one year of period.
At the beginning of year 2015 in Slovenia already 11 bovine herds are officially free of BVDV.
First herds with officially BVDV free status confirming, that this practice is a good and cost
effective solution for our farmers. All herds with recognized BVDV free status are presented on
web site of The administration of the Republic of Slovenia for food safety, veterinary and plant
protection. We hope that voluntary programme is first step towards the eradication of BVDV at
national level.
Keywords: bovine viral diarrhoea virus, diagnostics, eradication, cattle
Introduction
Bovine viral diarrhea (BVD) is a disease of cattle that reduces productivity and may increases death
loss. It is caused by bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus of the
family Flaviviridae (Lindenbach and Rice, 2001). BVDV is distributed throughout the world, with
endemic areas detecting antibodies among 70–100 % of herds, while in some European countries
such as Sweden, Norway, Finland and Austria, the disease has already been eradicated
(Obritzhauser et al., 2005; Houe et al., 2006). BVDV is spreading by close contact (nose-to-nose)
between cattle and produce heavy economic losses in infected herds.
Virus is shedding by both acutely and persistently infected (PI) animals, but levels of shedding are
much higher in PI cattle, which are the natural reservoir for the virus. Foetuses that become infected
between 30 and 125 days of gestation and survive the infection may be born as BVDV infected
calves (Brownlie et al., 1989). The BVDV infection will persist for the life of the calf, hence the
term ''persistent infection'', or PI. The incidence of PI animals is estimated between 0.3 and 2.6 %
36
(Baker, 1995). PI animals are the main source of infection in infected herds and tend never to reach
their productive potential and growth because of reduced fertility and increased susceptibility to
other diseases (Loneragan et al., 2005). By removing the source of infection (PI animals) from the
population the disease can be controlled (Houe et al., 2006). Blood tests are the most frequently
used method to identify BVDV in live animals, but also other samples such as skin biopsies (taken
from the ear), milk or even oral swab samples can be collected for detection of virus (Kenedy et al.,
2006; Fulton et al., 2009; Toplak et al., 2015).
The monitoring of herds infected with BVDV in Slovenia started in 1994, but the disease is likely to
be present in breeding farms for centuries (Grom and Barlič-Maganja, 1999, Toplak, 2004). Since
1994, all bulls in breeding and artificial insemination centers are under supervision based on the
regular laboratory testing (Hostnik et al., 2002). Since this time, the collection centres are free of
BVDV, which are important preventive measures in a country to prevent spreading of BVDV by the
semen. During the period 1996 and 2003 from 260 to 312 breeding herds BVD were monitored for
the detection of antibodies (Toplak et al, 2002, Toplak, 2004). Young bulls are tested for BVD
antibodies and BVDV genome just before entering the breeding centres and later once per year in
centres. Before 2014 only few reports of successful elimination of BVDV was published for
Slovenia (Toplak et al., 2001; Toplak, 2004; Starič et al., 2010).
Molecular methods are used for molecular epidemiology studies to confirm the transmission of the
BVDV between infected herds. Comparison of the nucleotide sequences of BVDV strains obtained
from different infected herds in the 5'-non-coding or Npro region of the viral genome reviled not
only identical strains in different herds, but also introduction of new strains from abroad (Toplak et
al., 2002, Toplak, 2004). The results of genetic typing of 52 BVDV strains collected between 2004
and 2013 from infected Slovenian herds have shown that the identified strains of BVD virus were
classified into the same subgroup of genotype 1 (1b, 1d, 1f, 1g) as have been detected in years 1998
to 2004 confirming that the same strains have been circulating more than a decade in infected herds
(Toplak, 2004, Toplak et al., 2014). BVDV strains of genotype 2 were not identified in Slovenia.
Although it is difficult to estimate the disease cost because of variable infection many cost-benefit
analysis proved the positive impact of BVDV elimination at herd and eradication at national level
(Houe, 2003, Häsler et al., 2012). With the beginning of 2014, new national rules determining the
conditions for recognition, acquisition and maintenance of a herd status, free of BVDV (Uradni list
Republike Slovenije no. 107/2013) were introduced. This program is modification of the BVDV
eradication programs successfully established at national level in Sweden, Norway, Finland,
Denmark and Austria (Lindberg and Alenius, 1999; Obritzhauser et al., 2005). After one year of
experiences some breeders already achieved significant progress in health status, implement
preventive measures and maintenance of BVDV free status in the herds. In this paper, we would
like to present some experiences with new rules and some preliminary results.
Material and Methods
1. Basic principles of voluntary program for control and eradication of BVDV infections in
Slovenia
The program is running on the voluntary basis and breeders can officially acquire the status of a
herd free of BVD. This new regulation helps Slovenian farmers to take a decision how they want to
regulate the health status of their herds. Recognition, acquisition and maintenance of status are
based on the results of laboratory tests, as well as the farmer who is obliged to implement all
measures to prevent re-introduction of BVDV into herd. Vaccination is not practiced as an option in
the rules by the program running in Slovenia.
37
1.1 Recognition of a herd - free of BVDV
If the farmer decides to apply for recognition of status, it must comply with the following: during
the past 12 months no BVD infection has been confirmed in the herd, no animal shows clinical
signs of disease, animals shall be separated by a physical or natural barriers from herds with a lower
status, only negative animals may be introduced into herd through quarantine, for insemination only
semen obtained from bulls free of BVD is used. In addition to these conditions, the herd owner
should provide two consecutive BVD antibody ELISA testing at intervals of at least six months in
all animals in the age group 7 to 13 months (Scheme 1). Into the second sampling, other groups of
animals, which at the time of samples collection are aged 7 to 13 month, are included. If the
laboratory results of these tests are negative in all animals, the herd owner may submit an
application for recognition of a herd free of BVD. Rules allow the recognition of the status also for
herds that do not have young animals, as in this case, samples are collected in the next age group of
animals, e.g. the age group 14 to 20 months. The holder of the animals submits an application for
recognition to the Regional Office of Administration for Food Safety, Veterinary and Plant
Protection. List of herds free of BVDV is published on the website and it is free available to
farmers.
1.2 Acquisition of a herd - free of BVDV
For BVDV infected herds recognition of BVDV free status is possible on voluntary basis after
removing BVDV positive animals from the herd. The most important measure is to eliminate all
persistently infected animals (PI) from the herd. In the first step, blood samples (serum) should be
collected and tested from all animals in a herd to determine the presence of BVD virus
(identification of PI). PI animals must be immediately slaughtered. In addition, samples of all
newborn calves (blood sample - serum) in the first week of age should be sampled in a herd for a
period of one year and tested for BVD genome. All BVDV positive calves must be removed from
the herd. One year after the elimination of the last PI from the herd all animals in the age group of 7
to 13 months are tested for the presence of BVD antibodies by ELISA. If the results of all animals
are negative, the same herd is tested after six months again to prove the stability of the herd. If all
animals in the age group of 7 to 13 months are negative and farmer implemented preventive
measures, the herd owner may apply for recognition of the status free of BVD (Scheme 1).
1.3 Maintenance of a herd - free of BVDV
Recognition of a herd free of BVD is granted for a period of one year. The farmers successfully
maintain the status by the implementation of preventive measures and status need to be renewed
again after one - year period. To keep the status farmer should ensure the laboratory testing for
BVD antibodies in all animals in the herd, which are at that time aged 7 to 13 months. Sampling in
the herd is repeating at intervals of 12 months, thus confirming the absence of BVDV infection.
1.4 Withdrawal the status - free of BVDV
If is determined that the herd has no longer qualifications for the status, the veterinary
administration made a decision which status is withdrawn. Farm, which has been deprived of the
status, is deleted from the list on the website. If a farmer wants to renew the status, must fulfill the
conditions laid down for the recognition of BVDV free status.
Results
During the period between January 2014 and April 2015 in total 1.138 samples were collected from
99 individual cattle herds and tested for antibody detection. Sera samples were tested by ELISA
38
(Svanovir BVDV® Ab, Svanova) for detection of specific antibodies against BVDV and samples
were interpreted according to producer instructions. First herd with BVDV free status was officially
confirmed in July 15, 2014. Until April 2015, official recognition of BVDV free status was
confirmed already for 13 individual herds.
Between January 2014 and April 2015 in total 2.186 individual serum samples from newborn calf
and cattle were collected from 86 different herds and tested for BVDV genome by RT-PCR as
previously described (Toplak et al., 2015). Out of 2.186 tested animals 64 (2.92 %) were detected
BVDV genome positive animals by RT-PCR method.
Scheme 1: The algorithm for recognition, acquisition and maintenance of BVD free status - the basic
principles for voluntary program, which was started in Slovenia in January 2014.
Discussion
The first voluntary program providing officially recognized BVDV free status for individual herds
in Slovenia started in January 2014. Veterinary faculty from Ljubljana together with Administration
for Food Safety, Veterinary and Plant Protection and private practitioners prepared the conditions
for farmers, which want to apply for BVDV free status. The basic principle of first voluntary
program is laboratory testing of young stock together with preventive actions of farmer to prevent
reintroduction of BVDV. Although the BVDV infection is endemic in Slovenia with about 30 % of
infected cattle herds, still more than half of herds are free of BVDV. For this reason, more than
hundred cattle farmers already started with first step for recognition of BVDV free status.
According to herd size, number of animals which need to be tested, is usually between 5 and 10.
This is important information for farmers who covers all costs, including laboratory testing. After
39
first negative results in a herd, the same herd needs to be tested after six month to prove the stability
of herd. First 13 herds which have already been officially recognized with BVDV free status are
promising, that new rules were accepted from farmers and recognition of BVDV free status have
important value to improvement the production on a farm. The initial testing of herds for antibodies
gives also opportunity for farmers to start with the control BVDV in infected herd. The BVDV
positive herds can take the actions through the identification and elimination of persistently infected
animals from a herd. These animals secreted the BVD virus into the environment, especially with
nasal discharge, saliva, feces and urine. In BVDV infected herd newborn calf are constantly
infected within a few months after the birth, expressing clinical symptoms and get sick because of
acute phase of the disease. After two to three weeks, specific antibodies can be detected by ELISA
and those antibodies are present in the blood throughout life. If the mother is positive for BVD
antibodies, calves receive colostral antibodies, by sucking colostrum, which remain in the blood up
to 3 months, in rare cases up to 8 months of age. During the laboratory testing it was recognized
that individual animals from the sampled age group of 7 to 13 months were low antibody positive in
ELISA test, usually with percent positive values between 10 and 20, so we suggest retesting them
after one month. The important preventive measure is also that animals must be separated by
physical or natural barriers from herds with a lower status and only negative animals may be
introduced into herd through quarantine. Farmer needs to be careful about this, not to lose BVDV
negative status. In the infected herd, BVDV is quickly transmitted between animals of all ages and
this feature of BVD virus can be successfully used in laboratory tests. Screening of the age group of
young animals in the herd (aged 7 to 13 months) assures us that based on the results specific
antibodies the current situation regarding BVDV infection in the whole herd is recognized.
BVDV positive animal may not be sold, because those animals are the main source for spreading
BVDV infection into new herd. The identification of PI in infected herds was successful and 64
new PI animals were detected last year. Our system is still without record of slaughtering those
BVDV positive animals and these needs to be corrected as soon as possible. At the moment the
action and responsibility is on farmer. If such BVDV positive animal is send to other herd this will
act like a boomerang, because it will extended the circulation of virus in population and maintain
the BVDV infection in the herds.
Conclusion
New rules which were adopted for the first time in Slovenia define the conditions for recognition,
acquisition, maintenance and withdrawal of BVDV free status. Veterinary Faculty is offering to a
breeder special package of discounts for laboratory testing of samples and reduced the time from
sample reception to the results on one week. For the recognition and maintenance of BVDV free
status, only a small proportion of the entire herd need to be tested, which allows lower costs and the
recognition of the BVDV free status for many breeders. Program is on voluntary basis and gives no
compulsion to breeders which can at any stage of the control decide to deviate from the target. The
common aim for farmers and veterinarian is healthy herd and healthy animals. Breeders who are
selling and buying animals will benefits based on BVDV free status. They can decide what kind of
animals want buy for a fixed price. The new rules based on voluntary program have already been
implemented and widely accepted and we believe that it provides a good solution for our farmers.
Our neighbouring countries such as Austria, Italy and Switzerland were already implemented or
finished similar BVD national program. Thus, the next step will be moving from voluntary to
national program.
Acknowledgments
Special thanks to Breda Hrovatin, Jedrt Maurer Wernig and group from The Administration of the
Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection, private veterinary
40
practitioners and farmers for collaboration in program. Thanks to Polona Berčon for technical
assistance during sample testing. This research was financially partly supported by the Slovenian
Research Agency, program group P4-0092 (Animal Health, Environment and Food Safety).
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41
Invited lecture
COMPARISON OF MACROSCOPIC AND MICROSCOPIC LESSION IN MESENTERIAL
LYMPH NODES IN PIGS NATURALLY INFECTED WITH MYCOBATERIUM AVIM
SUBSP HOMINISSUIS
Vladimir Polaček1*, Jasna Prodanov-Radulović1, Dejan Vidanović2 Sanja Kovačević-Aleksić3
1
2
Veterinary Specialized Institute „Kraljevo“, Kraljevo, Serbia
3
Faculty of Veterinary Medicine, Belgrade, Serbia
* Corresponding author: vlade@niv.ns.ac.rs
Abstract
Mycobacterium avium, belonging to the Mycobacterium avium complex (MAC), is divided into the
subspecies avium, paratuberculosis and silvaticum. More recently, M. avium subsp avium has been
further divided into M. avium subsp avium and M. avium subsp. hominissuis. M. avium subsp
hominissuis (MAH) is a bacterium which causes mycobacteriosis both in pigs and humans and most
common diseases of children and immune-compromised humans. The most important
morphological characteristic of infections caused by MAH and other mycobacteria is granuloma
formation. Granuloma is a unique structure and within it the growth of mycobacteria is in
accordance with anti-bacterial effector mechanisms of the host.
In our study we compared macroscopic and microscopic lessons in formalin fixed paraffin
embedded (FFPE) samples of mesenterial lymph nodes from tuberculin positive pigs. FFPE lymph
nodes were also examined by real-time PCR for IS1245. Post mortem examination was performed
on 100 pigs. Granulomatous lesions compatible with mycobacterial infection were found in
mesenterial lymph nodes in 88 pigs. Macroscopic lesions were detected in 43 pigs, while 45 pigs
manifested only microscopic lesions. In 12 pigs, no lesions were detected. The granulomas were
composed of focal accumulation of inflammatory cells in which macrophages, epithelioid cells,
multinucleated giant cells and lymphocytes predominated. Caseous necrotic areas were present in
some granuloma. Numerous eosinophils and myofibroblasts were present within the granuloma.
Our results show that routine meat inspections at slaughterhouses may fail to detect microscopic
lessons caused by M. avium subsp hominissuis and in certain cases could pose a threat for consumer
health.
Keywords: Mycobaterium avium subsp hominissuis, mycobacteriosis, granuloma, meat inspections
Introduction
Mycobacterium avium, belonging to the Mycobacterium avium complex (MAC), is divided into the
subspecies avium, paratuberculosis and silvaticum (Inderlied et al., 1993). More recently, M. avium
subsp avium has been further divided into M. avium subsp. avium and M. avium subsp. hominissuis
(Mijs et al, 2002; Turenne et al, 2007). M. avium subsp. hominissuis (MAH) is a ubiquitous
microorganism which lives both in water and soil and causes mycobacteriosis of pigs and human
population, mostly in children and immunocompromised humans (Agdenstein et al., 2014; Alvarez
et al., 2011; Domingos et al., 2009; Falkinham et al 1996; 2001; Johansen et al., 2014; Matlova et
al., 2004, 2004a, 2005; Biet et al., 2005; Hilbor et al., 2008; Yajko, 1995). One of major hallmark of
MAH infections in pigs are localized granulomatous lesions on lymph nodes of digestive tract
or/and on lymph nodes in the head (Thorel et al, 2001; Hybia et al, 2008; Polaček, 2010).
Generalized disease is less common and can affect internal organs such as liver, spleen or kidneys
though not always, are found at the meat inspection in slaughterhouses, without a previous detection
42
of clinical symptoms of the disease (Agdestein et al., 2012; Thoen, 2006). An annual study of a
slaughterhouse in the Netherlands shows that caseous changes have been detected in 0.5% of
examined lymph nodes of slaughtered pigs out of which 50% were infected with Mac (Komijn et
al., 2007). The available data suggests that certain herds of pigs exhibited granulomatous changes
in 30-50% of slaughtered pigs where M. avium was detected (van Ingen et al., 2010). As a routine
tuberculin skin test is not performed on pigs in most countries, the main source of information about
the occurrence of tuberculosis is the data gathered at meat control in slaughterhouses (Cvetnić et al.,
2007; Agdestein et al., 2012). On the other hand, a large number of infected animals may fail to
detect by routine meat inspection at slaughterhouses since the changes at some stages of Mac
infections aren’t macroscopically visible (Komijn et al., 1999; Tirkkonen et al., 2007; Polaček,
2010). This paper will present the results of macroscopic and microscopic changes on mesenteric
lymph nodes of the pigs naturally infected with M. avium subsp. homminissuis.
Examinations have been performed on the samples of mesenteric lymph nodes of 100 pigs
(Norwegian Landrace breed), 5-10 months old. The pigs originating from Lithuania were kept in
quarantine. During the quarantine period, tuberculin skin tests with bovine and avian tuberculin
were carried out. These tests were positive. According to the ruling issued by Veterinary Directorate
of the Ministry of Agriculture, Forestry and Water Management, swine euthanasia was authorized.
Immediately after the euthanasia autopsy and sampling of mesenteric lymph nodes were performed.
Euthanasia was performed by preparation T-61 (Intervet, Holand) as described in previous study
(Agdestain et al., 2011). The samples of mesenteric lymph nodes for pathohistological and
molecular examinations were fixed for 24-48 hours in 10% buffered formalin. After the standard
processing in automatic tissue processor they were embedded into paraffin blocks. Then the 4-5
µmn thick sections were stained according to hematoxylin-eosin method (HE). Mesenteric lymph
nodes of 10 healthy pigs were used as a control.
Molecular examination
Real time PCR from FFPE for IS 1245 and lymph nodes has been performed by the Norwegian
Veterinary Institute described in previous study (Agdestain et al., 2011, Polacek, 2010).
Macroscopic examination
The analysis of macroscopic lesions has been performed by indentation of the nodes in several
parallel sections, followed by classification into two groups:
A no macroscopic lesions detected
B macroscopic lesions detected
Microscopic examination
Granulomas were classified into three following histological groups by microscoping. These
included the granulomas with:
1. No necrosis
2. Initial phase of necrosis
3. Distinctive necrosis and calcification.
Results
Macroscopic examination showed that mesenteric lymph nodes were enlarged with or without
bordered areas of caseous necrosis. I.e. calcification. The diameter of the limited areas of caseous
43
necrosis, i.e. calcification was most commonly 1-2 mm, although in some cases it was larger than
10 mm (Image 1.).
The presence of M. avium subsp. hominissuis has been confirmed in a previous study by real time
PCR detecting IS1245 (Agdestein et al. 2011, Polaček 2010).
Picture 1. Macroscopic changes on mesentric lyph nodes infected with M. avium complex A limited area of
caseous necrosis on the cross section of a mesenteric lymph node (A-B). Disseminated areas of caseous
necrosis and dystrophic calcification on the cross section of a mesenteric lymph node (C-F).
The results of pathohysiological examinations
The microscopic examination of lymph node samples stained by the routine hematoxylin-eosin
(HE) method showed the presence of granulomatous lymphadenitis. According to morphological
charactersitics, granulomas were classified into three histological groups.
44
The first group included granulomas dominated by macrophages (epithelioid cells) and
lymphocytes. Caseous necrosis in granuloma centre was not detected in this group. Eosinophil
granulocytes were located between macrophages and lymphocytes, diffusely dispersed in a
granuloma. Epithelioid cells have light cytoplasm and vesicular nucleus with scarce chromatin. In
order to expand phagocytic capacity, several epiteloid cells are merged into polynuclear giant cells.
At this stage, granulomas contain 1-2 giant cells on average. Out of the total of 100 examined
samples of swine mesenteric lymph nodes, 19 belonged to the first histological group- 15 of them
were from macroscopic group A (no visible changes on lymph nodes) and 4 were from the group B
(with visible changes on lymph nodes).
The second group contained granulomas with morphological characteristics of circumscript lesions
with outer layer formed of myofibroblasts. These cells are commonly elongated or oval, located on
the periphery of granulomas, which separates it from the surrounding tissue. Individual
myofibroblasts with different phenotype are located diffusely in a granuloma. The central part of
the granulomas from the second group is modified by the areas of caseous necrosis. In some cases
dystrophic calcification in the form of dark blue deposits of irregular shape and uneven size is
detected. The thickness of connective tissue capsule is more conspicuous in big granulomas in the
areas of central necrosis. The number of polynuclear cells is significantly smaller than in the
granulomas from the first group. Morphological characteristics of granulomas from the second
group were found in 8 samples- 5 of them belonged to the macroscopic group A (with no visible
changes on lymph nodes), while 3 samples were classified into macroscopic group B (with visible
changes on lymph nodes).
Granulomas with distinctive and ever-present caseous necrosis and/or calcification in the centre
were in the third group. Necrotic area in the centre and clearly conspicuous connective tissue layer
on the periphery narrow the zone of epithelioid cells and lymphocytes and form a layer where
eosinophil granulocytes are found. Their distribution is distinctive on the periphery which is why
these cells from the 3rd group are peripherally located, between myofibroblasts, with irregular shape
in the connective tissue outer layer. They can even be found outside the belt. Giant polynuclear cells
were not found in these granulomas. A great majority of mesenteric lymph node samples (61)
belonged to the third histological group. 25 of them were in the macroscopic group A (with no
visible changes on lymph nodes), while 36 were in group B (with visible changes on lymph nodes).
(Table 1)
Morphological characteristics of the above mentioned granuloma types, in different stadiums of the
same tissue samples overlap in some cases. The criterion for classification in such situations was the
presence of dominating changes on a sample. Out of the total number of examined samples there
were 12 pigs that showed neither macroscopic nor microscopic changes typical of tuberculosis.
These lymph nodes were classified into macroscopic group A (Table 1).
Table 1. Overall results of both macrocopic and microscopic examinations of pig mesenteric lymph
nodes
microSCOPIC
changes
No changes
(Group A)
scopic
MACRO
No
changes
With changes
(Group B)
Group 1.
changes
Group 2.
Group 3.
12
15
5
25
57
(45)
-
4
3
36
43
12
19
8
61
45
Picture 2. Microscopic changes on mesenteric lymph nodes of the pigs infected with Mycobaterium avium
complex : A-microscopic group 1, B,D-microscopic group 2, C, E- multinucleated giant cells and Fmicroscopic group 3.
Discussion
This paper deals with morphological changes on mesenteric lymph nodes in pigs that were
previously diagnosed with positive tuberculin skin reaction. Molecular method determined the
presence of M. avium subsp hominissuis genome.
Granuloma is a unique structure and within it the growth of mycobacteria is in accordance with
anti-bacterial effector mechanisms of the host (Co et al., 2004). Granulomatous tip of inflammatory
46
reaction is a basic morphological characteristic of immune response of an organism to the infection
caused by mycobacteria. Formation and sustainability of granulomas is a complex process
undergoing several phases of immune response of the host (Hybia et al 2008). Immune reaction
against persistent bacteria is a sensitive and delayed reaction, mediated by T lymphocytes (Th1),
which respond with antigen presenting cells (APC), followed by a transformation of macrophage
proliferation. Mycobacteria found in a granuloma are affected by a low oxygen concentration and
very restricted nutrition. Some researchers suggest that in such conditions mycobacteria activate the
genes that initiate alternative metabolic pathways, one of them being the alternative citric acid cycle
for using lipids found in necrotic centre of a granuloma in great quantities. It is also assumed that
M. tuberculosis which is in a ‘hibernation’ state changes the structure of cell wall in a granuloma
since it loses the staining feature as an acid resistant bacterium (Co et al., 2004),
Neither macroscopic nor microscopic examinations of the samples detected changes in mesenteric
lymph nodes in 12 pigs (12%) that were infected with tuberculosis. All 12 pigs were positive to
tuberculin, 8 out of which showed a positive reaction to tuberculin B as well. Mesentric lymph
nodes of 7 pigs selected randomly from this group of animals were additionally tested using real
time PCR method and one turned out to be positive to IS1245 (M. avium). This implicates a low
specificity of tuberculin skin test.
Out of 57 (57%) pigs that had a positive reaction to avian turbeculin and which did not have any
changes on mesenteric lymph nodes, 45 (45%) had specific granulomatous lymphadentitis,
according to histological examinations. In a large number of cases (25/45) the specific
granulomatous lymphadenitis had the characteristics in the later stage of chronic granulomatous
process with massive necroses and very conspicuous dystrophic calcification. These results
unequivocally support the fact that tuberculin tests are highly sensitive. The results of examination
of 43 pigs with positive tuberculin reaction and visible macroscopic changes on mesenteric lymph
nodes speak in favour of test sensitivity. Microscopic examination of these animals also detected
the changes typical of tuberculosis.
The analysis of the tuberculin skin test results and morphological examinations raises a question of
the extent to which the test is an indicator of pathological changes in an organism as it certainly
detects the existence of immune response to the presented mycobacteria. The data about occurrence
and extent of tuberculosis are most commonly obtained from the meat control in slaughterhouses or
from a quarantine of imported animals a since tuberculin skin test is often one of the compulsory
diagnostic measures during the quarantine period of most categories of imported pigs in the
Republic of Serbia. Although imperfect, tuberculin test is a significant measure in the control of
micobacterioses in pig production (Cvetnic et al., 2007; Polacek, 2010). The results we have
obtained suggest that the use of tuberculin skin test method is more reliable compared to a routine
examination at the meat inspection in abattoir, particularly in cases when tuberculous changes are
not macroscopically visible and detectable by routine meat control in slaughterhouses, which results
in the failure to spot infected pigs during the control process (Polacek, 2010; Agdestein et al.,
2012). In our opinion, the control on the presence of tuberculosis cause needs to be tightened;
having in mind that the number of positive tuberculin skin tests performed on imported animals in
our country during quarantines is on the increase. The introduction of more reliable measures for
determination of positive reactors and their restriction from food chain will significantly decrease
the occurrence of disease in human population.
Acknowledgements:
This study was supported by Project TR 31084 of Ministry of Education, Science and
Technological Development, Republic of Serbia. Also we would like to express our grateful to
47
Tone Bjordal Johansen and her colleagues from Norwegian Veterinary Institute (Oslo) for help with
molecular examinations and Danka Vukašinović for help with manuscript and translation.
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infection in mammals. Rev Sci Tech. 2001; 20:204–218.
24. van Ingen J, Wisselink HJ, van Solt-Smits CB, Boeree MJ, Van Soolingen D. Isolation of mycobacteria
other than Mycobacterium avium from porcine lymph nodes. Vet Microbiol. 2010; 144:250–253.
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26. Turenne CY, Wallace R Jr, Behr MA. Mycobacterium avium in the postgenomic era. Clin Microbiol
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27. Yajko D. M., Chin D. P., Gonzalez P. C., Nassos P. S., Hopewell P. C., Reingold A. L., Horsburgh C.
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Deficiency Syndromes and Human Retrovirology. 1995; 9(2):176–182.
49
IMMUNE RESPONSE AND PROTECTIVE EFFECT OF VACCINE AGAINST
LISTERIOSIS IN SHEEP IN SERBIA
Dragan Bacić1*, Blagoje Dimitrijević1, Mila Savić1
1. University of Belgrade, Faculty of veterinary medicine, Serbia
* Corresponding author: bacicd@vet.bg.ac.rs
Abstract
In this study the protective effects of two bivalent inactivated vaccines were evaluated. Vaccines
were prepared from Listeria monocytogenes, serotypes 1/2a and 4b, as the most frequent in our and
surrounding epidemiological areas. Vaccine A consists of whole L. monocytogenes bacteria cells,
inactivated with 0.4% formaldehyde and aluminium hydroxide as a carrier. Vaccine B contains
0.1% saponin in addition to ingredients of vaccine A. Evaluations of these vaccines were performed
in 60 sheep, divided into four groups (n=10) with a corresponding negative control group (n=5).
After 14 days, boosterisation of all animals was performed. In order to evaluate the immune
response, blood samples were obtained every 14 days during the next 6 months. Antibody titres
were determined by microaglutitation (MAT) and complement fixation tests (CFT).
Comparative analyses of antibody titres, induced by vaccines A and B, show that the later (with
saponine) significantly increased the level of antibody titres (p<0.01). The levels of immune
response were also significantly impacted by the total number of bacteria and vaccine dosages
(p<0.01).
The bivalent vaccine containing 0.1% saponin (vaccine B) in 5.0 ml x 106 cfu/ml (colony-forming
units per milliliter) dosage shows a protective effect after challenge with L. monocytogenes. The
protective levels of antibody were 1/80 and 1/16, determined by MAT and CFT, respectively.
Antibody titres were significantly higher after boosterisation (p<0.01) and protective levels could be
detected in sera of vaccinated animals during the next 6 months.
Therefore, it is strongly recommended to perform boosterisation two weeks after initial vaccination.
Keywords: Listeria monocytogenes, vaccine, immune response, protective effect, saponin, sheep
Introduction
Listeria monocytogenes, a gram-positive, facultative intracytosolic bacteria, is the causative agent
of listeriosis, a severe disease associated with a high mortality rate. Nearly all domestic animals are
susceptible to L. monocytogenes infection, but animal listeriosis most commonly occurs in
ruminants. Listeriosis is also an important food-borne zoonosis (Farber and Peterkin, 1991; Regan
et al., 2005). In ruminants, among which sheep are the more commonly affected, listeriosis is
usually represented by three distinct clinical syndromes; septicemia, encephalitis
(meningoencephalitis), and abortion. Ovine listeriosis has also been associated with mastitis, some
ophthalmic lesions, such as keratoconjunctivitis and gastroenteritis (Wagner et al., 2000; Clark et
al., 2004; Otter et al., 2004; Gasanov et al., 2005).
Listeria is widely distributed in the natural environment and has been isolated from soil surface,
rotten vegetables and pasture herbage. Domesticated ruminants play a key role in the maintenance
of Listeria spp. in the rural environment through a continuous faecal–oral enrichment cycle (Picoux,
2008). The main source of infection in ruminants is poorly fermented silage with a pH above 5.0–
5.5 and within pockets of aerobic deterioration (microaerophilic microorganisms). In general, the
disease is more frequent in winter and early spring. This risk during the winter season is associated
50
with the fact that Listeria can grow at temperatures where growth of other pathogens is inhibited
due to excessive cold (psychrophilic microorganisms) (Radostits et al., 2007).
L. monocytogenes can be distinguished in 16 serovars on the basis of somatic and flagellar antigens
and there is genetic diversity between serovars (Beumer and Hazeleger, 2002). Two virulent
serovars (l/2a and 4b) are most commonly isolated in farm animal diseases in Serbia (unpublished
data). These strains can multiply in macrophages and monocytes and produce a haemolysin,
listeriolysin O (LLO), which is the virulence determinant of the organism (Bennett and Weaver,
2001; Amagliani et al., 2006).
The recovery rate is higher if treatment is administered early in the course of the disease.
Antibiotics (ampicilline and gentamicin) must be administered for a prolonged period, because
recovery can take as long as one month (Low and Donachie, 1997). Treatment of severely ill or
recumbent animals with listeriosis is rarely successful (George, 2002). Immunoprophylaxis (i.e
vaccination) has proved itself over the past 120 years to be by far the most efficient and costeffective method of controlling infectious diseases. Nevertheless, vaccination is not always an
innocuos procedure, and its use must always be accompanied by a careful assessment of the risks
and benefits of the procedure (Tizard, 2006).
Taking into account all of the above mentioned, the objective of this study was to evaluate the
protective effects of two inactivated, bivalent vaccines prepared from two of the most frequent
serotypes of L. monocytogenes in our and surrounding epidemiological areas.
Material and methods
Isolation and serotypization of isolated L. monocytogenes strains
L. monocytogenes was isolated from samples of aborted fetuses or brains of dead sheep on tryptone
agar (Torlak, Belgrade-Serbia) according to the standard procedure. Serotypization of the isolated
strains was determined by the method of fast agglutination on the microscope slide coated with antiO and anti-H immunoserum of L. monocytogenes (Bioveta, Czech Republic).
Preparation of vaccines
Vaccines were prepared from whole L. monocytogenes bacterial cells, serotypes 1/2a and 4b,
inactivated with 0.4% formalin with the addition of aluminium hydroxide as a carrier (vaccine A).
Vaccine B, beside above mentioned constituents also, contained 0.1% saponin.
Examining the sterility and toxicity of vaccines
The examination of the sterility of prepared vaccines was performed by sowing on nutritive agar,
5% blood agar and tryptone agar. During the five day incubation period (37 oC), the substrates were
inspected on a daily basis, no growth of aerobic and anaerobic microorganisms having been
detected.
The toxicity of vaccines was also examined on laboratory mice. Briefly, after the subcutaneous
application of the examined vaccine, the dose being 0.3 ml, the mice were monitored daily for any
possible changes in the general condition during a period of four weeks. After this period, the mice
were sacrificed and a suspension of parenhimatic organs was sown on tryptone and blood agar.
During this period of monitoring, no deaths of mice were recorded.
Serological reactions
Titre determination of the antibodies after immunization was performed by the method of slow
agglutination (MAT), according to the instructions of the manufacturer (Bioveta, Czech Republic).
51
For performing serological reactions, personally prepared and standardized antigenes of L.
monocytogenes, serotypes 1/2a and 4b were used.
Examination of the prepared vaccines on sheep
The examination of vaccines (A and B) was performed on 60 sheep of the Würtenberg breed aged
between three and four months, with a average body mass of 30-35 kg. The sheep were divided into
four groups (n=10), each group having its own negative control group (n=5). Before applying the
analysed vaccines, subcutaneously into the knee fold, blood samples from the sheep were obtained
by puncture of v. jugularis with the aim of detecting the possible presence of antibodies of the
studied L. monocytogenes serotypes. The first group of animals was vaccinated with 2.5 ml × 106
cfu/ml, the second group with 5.0 ml × 106 cfu/ml of vaccine A. The third group was vaccinated
with 2.5 ml × 106 cfu/ml, while the fourth group received 5.0 ml × 106 cfu/ml of vaccine B,
respectively.
Sheep from the negative control groups received subcutaneous injections of physiological solution
(Hemofarm, Vršac).
Two weeks after immunization, the sheep were revaccinated according to the same protocol.
Determination of the antibody titre in the blood serum after immunization was performed at
fortnightly intervals, commencing from the second week and ending on the 24th week.
All experimental procedures were performed according to our institutional guidelines for animal
research and principles of the European Convention for the Protection of Vertebrate Animals Used
for Experimental and Other (Official Daily N. L 358/1–358/6, 18, December 1986).
Statistical analysis
Statistical significance of differences of all examined parameters was determined by means of the
ANOVA, followed by the Tukey test. Data were expressed as means ± standard error. The level of
significance was set at p < 0.05. Statistical analysis was performed using the Graph Pad Prism 5.0
Software, CA, USA.
Results
Examining the blood serum on day zero, (before commencing with the vaccination), employing the
method of slow agglutination, it was determined that the sheep included in the experiment were
seronegative for 1/2a and 4b L. monocytogenes serotypes.
Results of sheep antibody titres obtained by the method of microagglutination
Titres of agglutinized antibodies in sheep vaccinated with vaccine A ranged within the interval from
1/20 to 1/80, while in sheep which received vaccine B, the titres ranged from 1/40 to 1/320 (results
not shown).
It has been determined by statistical analysis that the average antibody titre was highest in the
animal group IV which received the vaccine B, the dose being 5.0 ml (17.69 ± 1.11), while the
lowest titre was in the animal group I which received the vaccine A, the dose being 2.5 ml (12.96 ±
1.25) (Fig. 1). Comparing the average values of antibody titres, a high statistical difference was
determined between all the studied groups (p<0.01). Comparing antibody titre values between the
groups that received different doses of vaccine B, no statistical differences were detected (p>0.05).
Analyzing the trend of agglutinized antibody titre movement, after applying vaccine A in a dose
range from 2.5 to 5.0 ml during 24 weeks, it has been detected that the titre gradually increases,
reaching maximum values on the sixth and fourth week respectively, followed by a fall (Figure 2).
52
A very similar tendency of movement has the antibody titre after the application of vaccine B with a
dose of 2.5 ml. The titre gradually rises and reaches its peak on the eigth week after vaccination,
followed by a downfall (Figure 2).
Analyzing the tendency of agglutinizing antibody titre movement after application of vaccine B,
with doses of 2.5 and 5.0 ml, during 24 weeks, it has been determined that the titre gradually
increases, reaching its maximum value on the sixth week followed by a gradual fall (Figure 2). A
very similar tendency of movement is shown by the antibody titre after applying the vaccine B with
a dose of 2.5 ml. The titre gradually rises, reaching its highest level on the eight week after
vaccination, followed by a gradual fall (Figure 3).
Figure 1. Average titres of antibody following the application of vaccines A and B in different
dosages, determined by MAT. Group I versus group II, p<0.01; Group II versus group III, p<0.01; Gruop
III versus group IV, p>0.05
Figure 2. The trend of agglutinized antibody titres movement during 24 weeks period following the
application of vaccine A in 2.5 and 5.0 x 106 cfu/ml, determined by MAT. CI – Confidential Interval
53
Results of artificial infection
Artificial infection was performed in five sheep from group IV with the antibody titre ranging
within average values (1/80-MAT and 1/16 –CFT). Sheep were infected with 5.0 ml x 106 cfu/ml of
fresh bujon, 24 h culture containing 1/2a and 4b strains, applied subcutaneously in the knee fold.
The animals were monitored daily the next 30 days for possible changes in their general health
conditions. In the studied sheep, neither body temperature increase nor changes in the general health
have been recorded. Bacteriological analyses of fecal samples, chemoculture of blood, nasal and
buccal smears were negative for the presence of the studied L. monocytogenes strains.
Figure 3. The trend of agglutinized antibody titres movement during 24 weeks period following the
application of vaccine B in 2.5 and 5.0 x 106 cfu/ml, determined by MAT. CI – Confidential Interval
Discussion
Vaccination is a very useful tool in preventing infectious diseases, if there is an elicitation of
effective, protective antibodies. Two major criteria must first be satisfied in determining whether
vaccination should be used to control a specific disease. First, it must be established that the
immune system can protect against the disease in question. Second, before using a vaccine, we must
be sure that the risks of vaccination do not exceed those associated with the chance of contracting
the disease itself (Tizard, 2006; Radostits et al., 2007).
Since the therapy of listeriosis is currently under discussion and does not always offer good results,
listeriosis being a serious zoonosis (Braun et al., 2002), the aim of this study is to evaluate the
efficiency of our experimental inactivated vaccine in contributing to the control and suppression of
listeriosis. On the basis of isolation and serotypization, it has been determined that serotypes 1/2a
and 4b L. monocytogenes dominate on our epizootological region, as well as in the surrounding
countries (unpublished data). This is the reason why in our study we selected the above mentioned
L. monocytogenes strains.
The key factors for the vaccine quality and adequate immune response are: the number of
microorganisms in the vaccine, degree of attenuation or means of inactivation, combination of
bacterial antigens in the vaccine, as well as the type of added adjuvant, time of vaccination and
revaccination (Gudding, 1989).
It has been established that the total number of microorganisms in the vaccine, as well as the vaccine
dose, have an effect on the immune response (Linde et al., 1995). For this reason, the animals in our
54
experiment were vaccinated with various doses of vaccine, 2.5 and 5.0 ml × 106 cfu/ml. On the basis
of the fact that we used pathogen L. monocytogenes serotypes, their inactivation was achieved with
formalin with the addition of aluminium hydroxide as the carrier (Szemeredi and Padanyi, 1989).
We determined that the antibody titre is significantly higher if the studied vaccine is applied as a dose of
5.0 ml × 106 cfu/ml, in relation to the dose of 2.5 ml × 106 cfu/ml (Figs 1 and 3). Comparing antibody
titres in animals immunized with vaccines A and B, it has been determined that the addition of saponin
in vaccine B, at a dose of 0.1%, significantly supports the immune response (Lhopital et al., 1993;
Linde et al., 1995). These results are expected due to the fact that saponin is a nonspecific activator of
the immune system (Tizard, 2006).
Antibody titres obtained by vaccines with saponin differ significantly when compared to titres obtained
by applying vaccines without saponin. Titre levels of agglutinated antibodies applying the vaccine
containing saponin ranged from 1/40 to 1/320, while with vaccines lacking saponin, the values ranged
from 1/20 to 1/80.
The aim of revaccination is the boosterization, i.e. obtaining higher antibody titres (Tizard, 2006). In the
present study, 14 days after vaccination, a revaccination has been performed. It is evident that the
agglutinated antibodies persisted in the serum of animals six months after vaccination, specifically with a
higher titre after the application of vaccine B with a dose of 5.0 ml 106 cfu/ml (Figs 4, 5 and 6). The
vaccinal titre obtained in this way was in the range of 1/80 to 1/320, determined by MAT. Our results
are in agreement with the results of other authors (Ivanov, 1985; Gudding, 1989; Linde, 1995), who
suggested revaccination for a more efficient protection from L. monocytogenes.
With the intention of checking the quality and efficiency of the prepared vaccine, an artificial
infection was initiated (Mencikova et al., 1989). Taking into account that by applying vaccine B,
with a dose of 5.0 ml 106 cfu/ml, the highest antibody titre was obtained. The examination was
performed on five sheep with average titre levels for this group of animals. Monitoring the health
conditions of the studied sheep during the period of 30 days, we determined that the bivalent vaccine
prepared from inactivated listeria serotypes with saponin and dose of 5.0 ml × 106 cfu/ml, protected the
studied animals after artificial infection, the protective titre having a value of 1/80 for MAT, i.e. 1/16
applying CFT. These results are in agreement with those previously reported by Nowacki et al. (1987)
and Tzora et al. (1998).
Since the studied vaccine has not shown toxicity and was efficient in the protection of sheep after
artificial infection, such a vaccine with additional research and evaluation on a larger number of
animals could find its application in the control of listeriosis in flocks where listeriosis appears
more frequently resulting in greater economic losses (Ivanov et al., 1977; Mencikova et al., 1989;
Regan et al., 2005).
In conclusion, in this study, we demonstrated that antibody titres were significantly higher after
boosterisation and that protective levels could be detected in sera of vaccinated animals during the
next 6 months. Therefore, it is strongly recommended to perform boosterisation two weeks after
initial vaccination.
Acknowledgements
Financial support for this study was provided by the Ministry of Education Science and
Technological Development, Republic of Serbia (Grant No 31085).
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investigations into Listeria encephalitis in sheep. Small Rumin Res, 71, 293–297, 2007
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sheep with listeriosis. J Clin Microbiol, 31, 1537–1540, 1993
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Mencikova E., Snirc J., Gubran D., Smola J., Mara M.: Experimental listeriosis in immunized sheep.
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Nowacki J., Lewandowska S., Konopa M., Przymus J.: Studies on the safety and efficacy of a live
vaccine against Listeria infection in lambs. Medycyna Weterynaryjna, 46, 259–261, 1991
Otter A., Houlihan M.G., Daniel R.G., Kirby F.D., Shock A., Higgins R.J.: Ovine gastrointestinal
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Radostits O.M., Gay C.C., Hinchcliff K.W., Constable P.D.: Veterinary Medicine. A Textbook of the
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Szemeredi G., Padanyi M.: A ten years experience with inactivated vaccine against listeriosis of sheep.
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Tizard I.:, In: Veterinary Immunology, WBC Saunders 7th Ed, 2006, 365-392.
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56
ORAL FLUID AS A POTENTIAL SAMPLE FOR VIRAL DISEASES
DETECTION IN PIGS
Vesna Milicevic1*, Branislav Kureljusic1, Jelena Maksimovic-Zoric1, Ljubisa Veljovic1, Vladimir
Radosavljevic1, Nemanja Jezdimirovic1, Bozidar Savic1
1. Institute of Veterinary Medicine of Serbia, Belgrade, Serbia
* Corresponding author: virusologija@nivs.rs
Abstract
Diagnosing infectious diseases in pigs is laborious work, as it involves taking blood or swab
samples which is very stressful for pig; it is time consuming, independently whether piglets or
adults are meant to be sampled. Since the most of viruses are excreted orally, and taking into
consideration experiences in human medicine, oral fluid started to be investigated as a potential
sample for diseases detection.
There are many advantages of this kind of sampling. By testing oral fluid more animals can be
screened, than by individual testing of blood or swabs. This method is welfare – friendly means of
monitoring disease, working towards better productivity. The results represent the status of the herd
not of the individual animals. Having in mind that most of viruses can be excreted before clinical
signs; this is a very useful tool for early detection of the disease. Oral fluid is collected by giving the
cotton ropes to the animals to chew it. The ropes are afterwards processed in the laboratory in order
to extract oral fluid which then could be tested for the presence of different markers, such as
viruses, bacteria, antibodies etc. On the selected pig farms, we installed the ropes in the pens, to be
easily accessed and attractive for pigs. Collected oral fluid samples have been tested on PRRS,
PCV2 and SIV. Initial results suggest that oral fluid sampling could provide a low-cost, noninvasive method to assess the health status of pigs. Additional work on technique validation is
needed.
Keywords: non invasive sampling, oral fluid, PRRS, PCV2, SIV
Introduction
Accurate and reliable laboratory diagnosis in modern swine production is the request. It is of same
importance for early disease detection, surveillance and monitoring purposes. Although pigs are
intelligent animals which remember rewards, sampling for them is very stressful and affects their
welfare. Traditional sampling methods require skilled personnel and is time consuming since is
individually based. Serum samples are, the most often, used for infectious diseases diagnosis. But
there are many limitations in serology results interpretation that are linked with estimation of time
when infection occurred, presence of maternal antibodies, but also with early stages when
antibodies are not detectable. In particular, the need to handle and bleed animals presents a
significant risk for further spread of diseases (Mur et al., 2013).
Gold standard in infective disease diagnosis is agent detection. Permanent infectious disease
monitoring, molecular epidemiology and tracking disease back, which are of high priority in high
density farms, are possible only by capturing the virus or viral genome. Many causative agents of
infectious diseases are excreted even before onset of clinical symptoms, making it important to
detect them as early as possible.
57
For these purposes, live animals are needed to be sampled; swabbing is the most often used
technique. Taking nasal swabs is not difficult, but needs individual sampling. Oral fluid is an
alternative specimen that could potentially replace nasal swabs.
In human medicine, oral fluid specimens have been used for infective agents detection but also for
hormones, toxins and drugs (Prickett and Zimmerman, 2010). Nowadays, for HIV (Connolly et al.,
2004) and measles (Carr et al., 2009) surveillances, oral fluid samples are preferred because of it
safety, efficiency, cost effectiveness and large number of samples availability.
Porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2),
swine influenza virus (SIV), foot and mouth disease virus (FMDV), classical swine fever virus
(CSFV) and African swine fever virus (ASFV) are shed in oral fluid which has been proved to be
accurate diagnostic specimen (Ramirez et al., 2012). It has been shown that oral fluid can be used
also for antibody detection. There are many advantages of oral fluid collection: requires no catching
or killing of animals and is feasible to get it from farmed pigs (Hoffman et al., 2008) and from wild
boar (Chichkin et al., 2012).
Rope sampling is a non-invasive method of oral fluid collection which allows detection of various
infectious agents and assists with disease surveillance. It is less laborious to perform and causes less
stress to animals. Basically, oral fluid samples are collected from individual pigs or pens of pigs by
allowing them to chew on cotton rope placed in the pen for 20–30 min. Oral fluid is extracted
manually from the rope and further processed in the laboratory (Prickett et al., 2008). Once
collected, oral fluid must be cooled immediately and transported to the laboratory as soon as
possible because of very active saliva enzymes which tend to inactivate viruses, in particular if virus
isolation test is going to be performed.
Rope sampling and oral fluid testing is, at some circumstances, even more sensitive than swab
testing. It has been shown that rope sampling was able to detect FMDV 6 days prior to visual
clinical observations in unvaccinated and infected pigs. However, saliva swabs were positive 3 days
before the rope samples (Vosloo et al, 2012).
However, this method has its limitations. The amount of oral fluid sample collected is dependent on
animal behavior. Sick animals show less interest in the ropes. When sampling was performed in
pens, it has been seen that sick animals come to chew on ropes only after healthy pigs got bored
(Vosloo et al, 2013).
How much important control of infective disease, in particular transboundary ones, in big farms is
well known. But, majority of swine population in the world is kept in small size, backyard holdings,
with low biosecurity that, despite almost perfect tools for disease control in developed countries,
heavily can affect the risk of disease spreading (Depner et al., 2013). Wild life plays the same role.
Rope sampling of free ranging pigs and wild boar can possible overcome these limitations for
disease control in this epizootic niche.
New techniques always arise suspicion on their improvement comparing to traditional ones.
Usually, people are distrust when ever new technique requires no contact with animals. Such
reactions were seen when surveyed owners were asked about oral vaccination (Mogner et al., 2015).
Infective disease surveillance in swine sector in Serbia is at very low level. This is possibly due to
the demanding sampling but also to the cost of individual testing. Swine production is a kind of
production where herd level of diseases status should be assessed, so therefore there is no need for
individual testing. In this case, group sampling sensitivity is the most important factor.
In this experiment, we wanted to show that disease detection is possible with alternative sampling
methods which do not require animal catching, in local, field conditions. For this purposes, we
selected 2 farms where respiratory diseases were previously confirmed. The objective of this study
was to conduct a preliminary assessment of the feasibility of detecting PRRSV, PCV2 and SIV in
58
oral-fluid samples. Fine tuning of the method, depending on the disease stage and number of
animals per pen, is still needed.
To test potential use of oral fluid for virus detection in the field conditions, we selected 2 pig farms
with recently reported respiratory disorders. On the both farms, pigs were kept in pens capacity 510 animals. All animals were clinically examined before oral fluid collection. Two pens with pigs
showing different respiratory symptoms from each farm were chosen for sampling. The ropes that
we used were made of pure cotton, 50 cm length, not treated with any chemicals or dyes, and with
high absorbance capacity. Cotton ropes have been fixed in pens at places easily accessible for pigs
and left for 30 minutes that pigs could chew on it. Pigs were observed during the all collection time
and their reaction on ropes and how attractive ropes were for them were recorded. After exposure
time, ropes were stored in plastic bags, cooled at +4° C and transported to the laboratory.
Oral fluids were extracted manually, squeezing cotton ropes. The fluid was centrifuged for 10
minutes at 1500 g and used in subsequent analyses. Molecular methods, RT-PCR and PCR, were
used for viral genome detection. Total RNA was extracted from 150 µl of oral fluid using
commercial kit (Isolate RNA Kit, Bioline, USA) according to the manufacturer’s instructions. DNA
was isolated using QIAamp® DNA Mini (Qiagen, USA).
For PRRSV genome detection one-step RT-PCR (Verso 1 Step RT-PCR Reddymixx Kit, Thermo
Scientific) was performed using primers targeting ORF 7 (Donadeu et al., 1999). The reaction was
carried out in a total volume of 25 µl. The RT-PCR program included a reverse transcription stage
at 50 °C for 30 min, followed by an initial PCR activation step at 95 °C for 15 min. and 40 cycles of
94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min and a final extension step at 72 °C for 10 min.
The RT-PCR products were visualized in 2% agarose gel with 0.5 µg/ml ethidium bromide.
A PCR technique for PCV2 detection was performed. The primers and conditions used for PCV2
have been previously described (Sandvik et al., 2001). The amplified products were run in a 2%
agarose gel and visualised by staining with 0.5 µg/ml of ethidium bromide.
Partial matrix gene of Influenza A virus was amplified using published primers (WHO, 2009) by
real time RT-PCR (Verso I Step qRT-PCR ROX Kit, Thermo Scientific). Thermal cycling was done
under the following conditions: 15 min at 50 °C; 15 min at 95 °C; 50 cycles of 15 s at 95 °C; and
60 s at 60 °C. The specimen was considered positive if Ct value was ≤ 38.0.
Results
From each farm, 2 samples were tested. One sample was taken from animals old 12-16 weeks, and
another one from pigs older than16 weeks.
Oral fluid collection on both farms, in the field conditions, using ropes was very easy. Pigs were
very interested in a new object in their pens and chew on ropes continuously for 30 minutes,
replacing one another. All ropes stayed fixed in pens, not damaged. Each rope was quite well
sodden with saliva which volume was enough for performing the laboratory tests. There was no
difference in behaviour and interest in ropes between different age groups.
Clinically, farm I reported respiratory disorders in all age categories 6 weeks ago. Pigs were treated
with antibiotics and on sampling day all pigs had no fever, occasionally coughing was heard and
pigs were in recovery stage.
On second pig farm, respiratory disease was in acute phase. Pigs had fever, anorexia, nasal
discharge, sneezing and labored breathing.
59
Results of molecular tests showed that samples from the farm in recovery stage were negative on
PRRV, PCV2 and SIV. In the farm with acute respiratory disease, PRRS and SIV infections were
confirmed.
In one oral fluid sample, on real time RT-PCR genome of Influenza Virus A was detected, Ct value
28,7. In two samples, genome of PRRSV was confirmed. All samples, from both farms were
negative on PCV2 (Table 1).
Table 1: Results of oral fluid samples tested on SIV, PRRSV and PRV2 presence
Virus
Result (%)
PRRSV
PCV2
SIV
2/4 (50)
0/4 (0)
1/4 (25)
Farm I
Pen 1 12-16 w
negative
negative
negative
Pen 2 >16w
negative
negative
negative
Farm II
Pen 1 12-16 w
positive
negative
negative
Pen 2 >16w
positive
negative
positive
Discussion
Swine Influenza Virus was detectable in porcine oral fluids by real-time RT-PCR. SIV is
transmitted via nasopharyngeal route, in direct contact between animals by droplets and aerosols of
nasal secretions. Virus excretion begins within 24 hours from infection and lasts 7-10 days (OIE,
2009) although Blaskovic et al. (1970) reported a case of a pig which had been shedding SIV over 4
months. Furthermore, swine influenza H1N1 virus has been recovered from pigs with no signs of
disease (Hinshaw et al., 1978). Carrier animals are rarely described but also considered as
responsible for Influenza virus introduction into uninfected herds (Merck Manual, 2008).
Pigs from the pen in which we confirmed SIV showed typical clinical signs of SIV. The infection
was in acute phase when amount of excreted virus is at highest level. Influenza A is present in pig
farms year round, but the outbreaks are most common in cold weather time, mainly in finishing pigs
(OIE, 2009).
Influenza in pigs can be diagnosed confirming presence of virus itself or immune response of the
host. Diagnostic tests that detect virus, viral proteins (antigens), or viral nucleic acid are Fluorescent
antibody (FA) test, AgELISA, RT-PCR and Virus isolation. Detection of circulating antibodies to
SIV can be performed by Hemagglutination inhibition test or Ab ELISA. Preferred samples are
nasal swabs, lung tissue and serum samples (Janke, 2000).
The results of the recent studies indicate that pen-based oral fluids provide an easy, effective, and
safe collection method for the detection of SIV with rapid testing methods such as real-time RTPCR (Detmer et al., 2011).
Oral fluid represents group sample and therefore its sensitivity should be considered. Romagosa et
al (2011) showed that pen-based collection of oral fluids is a sensitive method to detect influenza
even when within pen prevalence is low and when pigs have been vaccinated and highlight the
potential use of oral fluids for influenza surveillance (Romagosa et al., 2011).
Influenza A situation in Serbia is unknown. The virus presence has been rarely reported although
respiratory disorders are common in pig farms. Cost of individual tests and labour to get samples
for virology tests are possible reasons for this.
Another respiratory disease which heavily can affect health status of pigs is PRRS. However, in
some herds, infection can be asymptomatic. The virus is shed via saliva, milk, colostrum, urine,
faeces. Since proven that viral RNA could be detected in oropharingeal region of growing pigs up
to 251 days post infection nasal swabs are the sample of choice (OIE, 2008). Though, detection of
PRRS virus is the most reliable during the early stages of PRRS infection.
60
Viral antigen can be identified in infected fresh tissue, preferably lung, by direct fluorescent
antibody test. PCR or in situ hybridization is used to demonstrate viral RNA. Serology may be
helpful in confirming the presence (seropositivity) and stage (high levels of antibody in recent
infections) of PRRS infection in the herd.
In experimental conditions, PRRSV can be detected in oral fluid for approximately 4 weeks after
exposure ( Opriessnig et al., 2006). Field data showed that this period can be prolonged up to 8
weeks (Prickett et al., 2008a). Since the diagnostic window for PRRS is much larger than for SIV,
we succeeded to detect PRRV in oral fluid samples in both oral fluid samples from one farm. PRRS
surveillance if individually based is very costly. Using oral fluid for surveillance purposes, more
animals can be tested whereas number of samples and tests is less. Making surveillance and disease
control plans, sempling method sensitivity should be considered in order to be replaced with
appropriate number of animals to be samples in order to achieve the aim of the plan.
Porcine circovirus 2 is ubiquitous in domestic pigs. At some circumstances, PCV2 cause
postweaning multisystemic wasting syndrome (PMWS), reproductive failure, enteritis, respiratory
disease, porcine dermatitis and nephropathy syndrome (PDNS) or a combination of these
(Opriessnig et al.,2007). PMWS most commonly affects 2-4 month-old pigs, although the disease
has been also described in younger and older animals (Segalés et al., 2005). PCV2 is transmitted
horizontally excreting virus in nasal secretions, saliva, faeces and serum (Shibata et al. 2003) and
vertically (Pensaert et al. 2004).
Limited data on the amount of PCV2 in nasal fluid and saliva following experimental infection is
available. The internationally accepted criteria to diagnose PMWS include the presence of
compatible clinical signs, lesions and moderate to high amount of the virus. Detecting the virus
itself doesn’t` mean that it caused the disease but only evidence of virus infection (Caprioli et al.,
2006)
The positive and significant correlation found between amounts of PCV2 detected in lymphoid
tissues, sera and swabs suggests that nasal and rectal swabs are suitable indicators of the level of
PCV2 excretion (Lorenc et al., 2009)
Oral fluid sampling for PCV2 detection has been studied recently; showing that with these samples
PCV2 is detectable longer than 8 weeks. However, we didn`t detect PCV2 in oral fluid samples
probably because of low level of excreted virus in the stage where no other disorders have been
seen which we could linked to PCV2.
Diseases surveillance is fundamental to the control, elimination, or eradication of an infectious
agent and often requires large number of samples. Large number of samples requires labour and
time associated efforts. Due to these restrictions, in many farms, surveillance is abandoned or
inefficient. Having available and accurate alternative sampling methods that are cost and time
effective, simply to be performed, we believe that surveillance and disease control can be improved.
Detecting PRRSV and SIV in oral fluid, we showed that depending on stage, early disease detection
in farm condition is feasible. For precise characterization of method sensitivity, more experiments
are needed to be performed.
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63
MYCOBACTERIA IN ANIMALS IN SLOVENIA: FROM CATTLE TO AQUARIUM FISH
Mateja Pate1*, Darja Kušar1, Urška Zajc1, Vlasta Jenčič1, Diana Žele1, Gorazd Vengušt1, Jože
Starič1, Jožica Ježek1, Katarina Logar1, Tina Pirš1, Petra Bandelj1, Brane Krt1, Matjaž Ocepek1
1. University of Ljubljana, Veterinary Faculty, Gerbičeva 60, SI-1115 Ljubljana, Slovenia
* Corresponding author: mateja.pate@vf.uni-lj.si
Abstract
Slovenia was officially declared free of bovine tuberculosis (bTB) in 2009 although the proportion
of tuberculin test positive herds and animals in the past 40 years did not exceed 0.08% and 0.07%,
respectively. In the last two decades, a single case of bTB in cattle of Slovene origin was confirmed
by the culture method. However, bTB was detected recently in cattle imported from an EU member
state with a relatively high prevalence of bTB.
Among nontuberculous mycobacteria (NTM) in farm animals, the majority of isolates identified in
the past 15 years belonged to Mycobacterium (M.) avium which was mostly isolated from pigs.
After 2003, when compulsory slaughterhouse inspection of pigs was abolished, sporadic M. avium
isolates were obtained from cattle, poultry, pet birds, reptiles and zoo animals.
Aquarium fish are also an important reservoir of NTM. Two studies on NTM in ornamental fish in
Slovenia (2001-2004 and 2009-2011), which included 142 fish from several pet shops, revealed that
the majority (~80%) of aquarium fish harbour NTM. The predominant species was M. fortuitum.
The proportion of M. marinum, the most important zoonotic pathogen among NTM in fish, was
relatively high (~10%).
Between 2010 and 2013, the first survey on mycobacterial infections in wildlife in Slovenia was
conducted, including 306 apparently healthy free-range wild animals belonging to 13 species.
Members of M. tuberculosis complex were not detected, but a number of NTM were identified in
11.8% animals, with M. peregrinum being the most prevalent, followed by M. avium and M.
intracellulare. Some of the NTM were most probably reported for the first time in certain wildlife
hosts.
Paratuberculosis has become a common disease of ruminants in Slovenia. After a few
seroprevalence studies showing relatively low prevalence of disease in cattle herds in the past, it
recently became clear that presumably a lot more dairy cattle herds are affected and that up to 89%
of the animals within a herd may be infected with M. avium subsp. paratuberculosis. These facts
call for immediate implementation of measures necessary to control the disease.
Keywords: Mycobacterium, farm animals, wildlife, fish
Introduction
Bacteria of the genus Mycobacterium commonly cause pathologies or mortality in humans and
animals worldwide. Their infections are notoriously difficult to treat due to the unique cell wall of
mycobacteria and their natural resistance against many chemical or physical agents. Three main
groups of mycobacteria can be identified, namely the obligate pathogens from the Mycobacterium
tuberculosis complex (MTBC; e.g. M. tuberculosis, M. bovis, M. caprae) causing tuberculosis, the
obligate pathogens M. leprae and M. lepromatosis causing leprosy, and the nontuberculous
mycobacteria (NTM) causing skin/soft tissue disease, pulmonary disease, lymphadenitis or
disseminated disease. NTM are also called mycobacteria other than tuberculosis (MOTT), atypical,
opportunistic or environmental mycobacteria as being ubiquitous in soil and water (Dawson, 2000).
64
They usually do not cause disease in the immunocompetent humans despite the continuous
exposure to low levels, since the majority of human-mycobacteria interactions are self-curing
colonizations (Primm et al., 2004). Not to be overlooked, however, the release of potent
immunomodulators during the immune system activation may result in downstream effects like
allergies and irritations of the bowel (Primm et al., 2004). Some species of mycobacteria are very
difficult to grow in vitro as they are fastidious and take long periods to develop in culture; species
can be divided into slow and fast growers. They are widespread and typically found in water and
food sources, with the exception of obligate pathogens not found as free-living mycobacteria.
Cultivation and identification of mycobacteria
Cultivation of mycobacteria, performed in our laboratory, follows the sample homogenization,
concentration and decontamination protocol described by Kent and Kubica (1985). The processed
samples are inoculated onto five selective growth media: Löwenstein-Jensen (one slant
supplemented with pyruvate and one slant supplemented with glycerin), Stonebrink, Middlebrook
7H10 and BBL Mycobacteria Growth Indicator Tube (MGIT, Becton Dickinson). The tubes are
incubated for eight weeks at 37°C. For the detection of mycobacteria from fish, the media are
incubated also at 30°C and at room temperature. In case of suspected paratuberculosis, the samples
are processed as described by Logar et al. (2012). The inoculated Herrold’s egg yolk agar slants,
supplemented with mycobactin J, amphotericin B, nalidixic acid, and vancomycin (HEYA, Becton
Dickinson) are then incubated at 37°C for over 16 weeks.
Identification of the isolates is based on the colony morphology, PCR-hybridization assays
(GenoType Culture Identification Kits MTBC, CM and AS, Hain Lifescience), PCR assays and 16S
rRNA gene sequencing. PCR tests targeted at mycobacteria-specific insertion sequences (IS), short
DNA fragments which are capable of transposition and are abundant in the mycobacterial genome,
are used for identification of M. avium subsp. avium (IS901; Kunze et al., 1992), M. avium subsp.
paratuberculosis (IS900, Logar et al., 2012) and members of M. tuberculosis complex (IS6110,
Thacker et al., 2011). A real-time PCR assay targeting the erp gene and IS2404 is employed for
differentiation of M. marinum from other members of the M. marinum group (Slany, 2013). To
determine the phylogenetic affiliation of isolates, PCR amplification using the mycobacteriaspecific primer pair 285/264, targeting a 1037-bp segment of the 16S rRNA gene, is performed
(Kirschner et al., 1993). When 285/264 amplicons cannot be obtained or the sequencing generates
poor or ambiguous results, isolates are subjected to PCR amplification of ribosomal genes using the
commonly employed eubacterial primer pair 27F/1492R (Lane, 1991). Ribosomal PCR amplicons
are sequenced in both directions. The retrieved forward and reverse sequence fragments are edited
and assembled employing the SeqMan II v.5.05 program (DNASTAR Inc.) and submitted to
megablast classification accessed through NCBI, EzTaxon and RIDOM websites.
Bovine tuberculosis: Slovenia is officially free of the disease
The first records of bovine tuberculosis (bTB) in Slovenia date back in 1851. Several decades later,
between 1891 and 1900, the disease was reported to be spread among cattle population and the first
tuberculin skin testing (ST), which was performed on some large farms in 1922, revealed 70% of
animals as positive reactors. Nevertheless, the proportion of positive animals in abattoirs did not
exceed 1% in the period between the world wars. After the 2nd world war a planned ST on the
community farms started and a federal eradication program was established in 1954. Systematic
monitoring came into force in 1962 and has been carried out ever since. In 1968, the OIE-generated
animal health code was adopted, providing basic principles of bTB eradication. In addition to
regular ST and examination of carcasses at slaughter, bTB control included ST in quarantine for all
imported cattle. As a result, between 1974 and 1976 the proportion of positive herds fell under
65
0.1%. A decade later it dropped to 0.01%. Slovenia was declared officially free of bTB only in 2009
due to administrative reasons even though a single case of bTB in cattle of Slovene origin has been
culture-confirmed in the last 25 years (Ocepek et al., 2014). However, due to abolished quarantines
in the European Union, the situation can change at any time. Indeed, a case of bTB has already been
detected, caused by M. caprae in a herd consisting of cattle imported from an EU member state
with a relatively high prevalence of bTB (Pate et al., 2012).
Even though M. tuberculosis primarily causes TB in humans, several cases of infections have been
described also in domestic animals, mostly in those living in close contact with diseased humans.
Therefore, humans with active TB are regarded as the main source of infection for animals, which
may sometimes develop classical form of TB. With regard to domestic animals, M. tuberculosis
infection was most frequently reported in cattle. We described such a case ten years ago; our case
was the first human-to-cattle transmission of M. tuberculosis to be proven by a modern genotyping
method (Ocepek et al., 2005).
Mycobacteria in wildlife: evidence of a variety of nontuberculous mycobacteria
One of the greatest threats to any control programme in domestic animals is infection in wildife
maintenance hosts that cannot be controlled and can re-introduce infection in livestock. Until
recently, the only known significant mycobacterial infection in wildlife in Slovenia was an
M. caprae outbreak in a zoo in 2004, affecting bisons and dromedary camels (Pate et al., 2006).
However, as the countries bordering Slovenia have all recorded cases of bTB in free-range wild
animals, particularly in deer and wild boar, a survey on mycobacteria in Slovenian wildlife was
undertaken. The study was conducted on hunting grounds across the entire territory of Slovenia,
using the most important game animals as target species with a particular focus on wild boar and
red deer as potential reservoirs. The survey included a total of 306 apparently healthy free-range
wild animals of 13 species: red deer, roe deer, wild boar, wolf, badger, chamois, red fox, fallow
deer, mouflon, polecat, ibex, stone marten and jackal. Mycobacteria were isolated from 11.8%
animals, namely from red deer, roe deer, wild boar, fallow deer and jackal. The presence of the
causative agents of bTB was not demonstrated in any wildlife species. In contrast, a variety of
potentially pathogenic environmental mycobacteria (NTM) were identified: M. peregrinum,
followed by M. avium subsp. hominissuis, M. intracellulare, M. confluentis, M. fortuitum,
M. terrae, M. avium subsp. avium, M. celatum, M. engbaekii, M. neoaurum, M. nonchromogenicum
and M. vaccae. Despite their omnipresence in the environment, it seems that some of the NTM
found in this study have not been described before in the respective hosts. Nevertheless, the
significance of their presence in wildlife hosts in the absence of evident clinical signs and gross
pathology has yet to be assessed.
Mycobacteria in fish: the presence of potentially pathogenic species for humans
Mycobacterial infections are one of the most common infections of aquarium fish. Since their initial
discovery at the end of 19th century, fish mycobacterial diseases have been reported to occur
worldwide in more than 150 species. The mycobacteria generally accepted as fish pathogens are
M. marinum, M. fortuitum and M. chelonae. Several other species are listed in the literature but
most isolates belong to one of the mentioned species. Fish mycobacteriosis is a typically chronic
progressive disease that may take years to develop into clinically noticeable illness, recognized by
listlessness, lethargy, pigment alternation, sluggish movements, open lesions and ulcerations, skin
inflammation, exophthalmia, and signs of emaciation. Gross or microscopic greyish-white tubercles
may be found scattered or grouped in any parenchymatous tissue, but are usually seen on the liver,
kidney and spleen. Fish mycobacteriosis is also an important zoonosis and poses a significant risk to
all human beings working with the affected fish or the aquaria. Fish mycobacteria are capable of
66
causing both localized and disseminated infections in man. M. fortuitum and M. chelonae usually
cause superficial lesions via skin wounds, but pulmonary disease and cervical lymph node infection
may also occur. Most M. marinum infections are cutaneous, with the hands being commonly
affected (Dolenc-Voljč and Žolnir-Dovč, 2010).
The first study on mycobacteria in fish in Slovenia was conducted between 2001 and 2004, when 35
aquarium fish were investigated and 29 were positive for mycobacteria. M. fortuitum, M. marinum
and M. gordonae were most frequently isolated, followed by M. chelonae and M. peregrinum.
Approximately one fifth of fish harboured M. marinum (Pate et al., 2005). The latest study
encompassed 107 apparently healthy ornamental fish belonging to 20 species that were investigated
in the period from 2009 to 2011. Almost 80% of the investigated fish were positive for
mycobacteria, among which the common fish pathogens M. fortuitum, M. marinum and
M. chelonae were discovered, in addition to M. peregrinum/septicum, M. gordonae, M. arupense,
M. kansasii, M. ulcerans and M. setense (Pate et al., 2014).
The results of our studies demonstrate that the vast majority of ornamental fish available in pet
shops in Slovenia harbour mycobacteria, among which the potentially pathogenic species for
humans are predominant. A particularly striking fact is a relatively high percentage of M. marinum,
which is the main causative agent of cutaneous mycobacteriosis in humans. Therefore, it is of great
importance to increase the awareness of workers in fish industry, aquarium hobbyists and medical
staff. The most important step towards decreasing the possibility to acquire mycobacterial
infections from fish is to wear waterproof gloves whenever cleaning the aquarium and handling the
fish, even when preparing them for a meal. With this simple precaution, many could avoid a longlasting therapy with antimicrobials.
Nontuberculous mycobacteria in farm animals: the predominance of M. avium
In the past, M. avium was the most frequently encountered NTM in samples from farm animals
(especially pigs) sent to our laboratory. M. avium is the causative agent of avian tuberculosis, a
chronic contagious disease of poultry and birds with a fatal outcome. Pigs are susceptible to
infection caused by members of the M. tuberculosis and M. avium complex, and by several
opportunistic mycobacterial species. However, infections caused by M. avium complex are the most
common in pigs and have been described worldwide. M. avium-induced disease in pigs usually
manifests in the form of lymphadenitis. Environment (water, soil, sawdust, feed, bedding, birds etc.)
is a risk factor for human and animal infections caused by the M. avium complex, comprising
M. avium and M. intracellulare. During 2000-2011, M. avium was by far most frequently isolated
from pig samples sent to our laboratory; however, most isolates date before 2003 when the
compulsory slaughterhouse inspection of swine lymph nodes was abolished. In other farm and
companion animals (horses, cattle, cats and dogs), a disease due to M. avium is considered to be
very uncommon. However, we described M. avium-related lung tuberculosis in a cow showing
clinical signs and lesions closely resembling those due to M. bovis (Ocepek et al., 2003). Apart from
this case, M. avium has been isolated from cattle in several other cases during 2000-2011, as well as
from poultry and pet birds. In the last few years, sporadic infections in various domestic and wild
animals (goose, snake, elephant, deer, and jackal) were recorded.
Paratuberculosis: a threat to dairy cattle farming
Paratuberculosis (paraTB), a chronic granulomatous enteritis caused by M. avium subsp.
paratuberculosis (Map) which affects many animal species, is a common disease of ruminants in
Slovenia. The first case was detected in 1961 in imported Jersey cows. No other cases were reported
until 1993, when paraTB was found in a sheep flock; since then, several outbreaks of the disease in
cattle, goats and sheep have been documented. Systematic screening of paraTB was carried out
67
between 1995 and 2001, using various serological tests and including different categories and
numbers of cattle (Ocepek et al., 1999, 2002). For the period between 2002 and 2007, no data on
paraTB prevalence are available. In 2008, a seroprevalence study was conducted in which animals
older than two years were screened for Map in randomly selected cattle herds. The study showed
that the true prevalence at the herd level was almost the same as in 1999 and that it was fairly low
(18.49%) compared to many European countries (Kušar et al., 2011).
The results from our previous studies, concerning the comparisons of different diagnostic tests for
paraTB, suggested that the specificity of the ELISA kits used is questionable and showed a need for
highly sensitive and specific method to detect subclinically infected animals which are the main
cause of maintained herd infection and of spread of infections between herds and premises. Lack of
sufficiently sensitive, specific, reliable and fast laboratory tests can lead to underestimation of the
proportions of Map shedders in herds.
Therefore, we then introduced a high-yield DNA extraction method coupled with quantitative realtime PCR (qPCR) to detect Map in cattle faeces. The study, which included faecal culture, milk
ELISA, milk qPCR and faecal qPCR, was performed on the samples of 141 subclinically infected
dairy cattle of all age categories from a farm with a history of paraTB. The results suggested that
the proportion of low-level Map shedders in cattle populations tested in the past was most likely
underestimated. In addition to detecting a surprisingly high proportion of Map shedders within a
herd (89%), we demonstrated a negative influence of Map infection to milk production already in
the first lactation heifers (Logar et al., 2012). Furthermore, later studies indicated that presumably a
lot more dairy cattle herds are infected with Map. In the last study, eight randomly selected big
dairy herds were monitored for the presence of Map-antibodies. These were detected in all herds
while in half of them culture-positive animals were found (Starič et al., 2011).
Vaccination can be used as a measure to control the disease; however, as it hampers diagnostics of
paraTB and interferes with tuberculin skin testing, the opinions on its use vary. Vaccination
prevents the occurrence of clinical signs and reduces the shedding of Map in faeces and milk. The
latter was observed during our vaccination trial performed in two heavily infected dairy herds; in
addition, the vaccination contributed to increased annual milk yield (Ocepek et al., 2014). However,
hygienic measures are of crucial importance for controlling the disease.
As Slovenia has not yet implemented any measures for the prevention and control of the disease, a
significant increase in the prevalence of paraTB in the majority of big dairy herds is expected in the
forthcoming years. Certain EU member states have already implemented strict preventive measures
which request paraTB negative status of the herds providing milk for public consumption.
Economic losses faced by the Slovenian breeders of Black and White cattle due to paraTB are
estimated at 1 to 5 million euro per year. Therefore, it would be reasonable to introduce the control
and eradication program which would encompass systematic screening of the disease and
certification of infection-free herds. This would enable the farmers to buy non-infected animals and
increase the animal value in trading. However, this goal cannot be successfully pursued without
participation of the government, veterinarians, cattle breeding associations and the cattle breeders
themselves.
Acknowledgements
Mycobacteria research was funded by the Slovenian Research Agency (Grant P4-0092). Milojka
Šetina, Alenka Usenik and Nataša Peterka are thanked for their technical assistance over the years.
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70
SANITARY AND QUALITY CONDITIONS OF IMPORTED BULLS SEMEN IN SERBIA
ANALYSED AT "NIV-NS" (RETROSPECTIVE FROM 2010-2014)
Aleksandar Milovanović 1*, Tomislav Barna 1, Milovan Jovičin1, Jelena Apić1, Sava Lazić1, Igor
Stojanov1, Miroslav Urošević2
1
2
Scientific Veterinary Institute "Novi Sad", Novi Sad, Serbia
Scientific institute of reproduction and artificial insemination for domestic animals "Temerin", Temerin,
Serbia
*Corresponding author: aca@niv.ns.ac.rs
Abstract
During the period from 2010 to 2014 Scientific veterinary institute "Novi Sad" (NIV-NS) was
responsible for quality control of 44 quarantines with 344,485 imported doses in 710 batches from
248 bulls, originating from 10 different countries (EU countries, USA and Canada) and 18 different
breeding centers.
Samples were analyzed for bacterial count (expressed as colony forming units - CFU/mL) and
bacterial types, virus gene presence of IBR/IPV, BVD and Schmallenberg virus. Semen quality was
tested by CASA (Computer Assisted Sperm Analyzes), flow citometry and cytology sperm quality
parameters. Complete analyses were performed on 550 semen samples of 248 bulls.
Dominantly, semen traits were in accordance to relevant national and international standards
(Official Journal of R. Serbia № 38/2014 and OIE). However, 43 batches (32 bulls from 15
imports), with total of 21.826 straws (6.33% of all imported straws) were rejected/destroyed
because of different and prominent discrepancy, related to improper transportation (liquid nitrogen
leaking), presence of the genome of Bovine viral diarrhea virus, opportunistic pathogen bacteria
(Citrobacter freundii), elevated bacterial count (CFU from 40-780103), but mostly were rejected
because of low semen quality (10 of 15 cases). E.g., recorded sperm total motility ranged between
9.30-56.70% (average 35.709.60%), progressive motility 1.7-28.8% (16.775,20%), total motile
cells per straw 8.11106 (1,70-15,10106), progressive motile cells 0.36-7.0106 (3.761.40106,
CASA) and live sperm cells with intact acrosome (LIA) 4-55% (33.679.75%) and pathologic
forms (5-65%) from cyto-morphological studies. LIA cells by flow cytometry were 15.22 to
64.26% with average of 39.2412.02%.
EU semen trading policy is devoted only to sanitary conditions and semen quality is a matter of
standard operative procedure (SOP) of breeding center. Semen of lower quality (up to dead-non
motile semen) could be expected from imported semen.
Keywords: bull, semen, quarantine, quality and sanitary condition
Introduction
Republic of Serbia has a long tradition of cows farming, bulls semen production and semen import.
First semen production and insemination started in 1951 with fresh semen (Perković, 2011) and first
import of frozen semen in 1976 (frozen semen of Simmental bulls for insemination of sires’
mothers). Commercial semen imports started from 1989-1990 with Montbéliarde frozen semen
from France (Perković, unpublished data), and then continued with Simmental breed from West
Germany and Austria, together with Holstein breed from USA.
From that period, semen import, storage, breeding plans and distribution were a privilege and
obligation of breeding centers. After 2011 first distributive centers were registered and semen
import became more intensive and versatile, including many worldwide breeding centers and local
71
companies involved in semen trading and several cattle breeds become available to our market.
Nowadays semen import in Serbia is carried out trough 3 registered breeding centers and 10 semen
distributive companies. Most of significant producers of semen from the globe are presented on our
market now, selling semen of the best genetic value and those with commercial value.
General directives for intra-Community semen trading are regulated by 88/407/EEC directive.
Intercommunity trading between non EU trading is regulated by Commission for implementing
decision 2011/630/EU (bovine species from third countries). Regulations of semen storage centers
(semen distributive centers) are regulated with Directive 88/407/EEC.
None of this directive is regulating minimal hygiene conditions of produced semen (level of
bacterial contamination and bacterial type) or minimal technical guidelines of produced semen (e.g.
minimal number of live motile cells, maximal percentage of pathology forms).
OIE Terrestrial Animal Health Code from 2003 had guidelines related to count of saprophytic
bacteria in semen doses. Thawed semen should not contain more than 5,000 CFU per semen
sample, including saprophytic bacteria, fungi, moulds and yeasts. Regulations in some countries are
even stricter (2), and allows up to 500 CFU/ml of saprophytic microorganisms. Presence of
opportunistic pathogen bacteria is not recommended. Explanation was that presence of
opportunistic pathogen bacteria in the sterile uterus of potentially immunocompromised individuals
or herd cattle may present a risk for the occurrence of reproductive and other health problems in
animals or humans (3, 4, 5). Current OIE Terrestrial Animal Health Code from 2010 is missing
these paragraphs. Reason for this is not stated and it is not known to this the first author of this
article.
Some countries had established their own health criteria for bulls' semen clearing semen quality and
hygiene. Generally, Republic of Serbia is following these positive EU recommendations, but
current status of non-EU country allow to have national regulations that more precisely regulate
semen import, stressing on semen quality traits (minimal producing requirements regarding hygiene
and semen quality; Official Journal of R. Serbia № 38/2014).
Before semen import, importers have to acquire positive opinion from Scientific-expert Council and
to fulfil veterinary requirements regarding Veterinary Health certificate of animal that were used to
produce bovine semen as well as for frozen semen. After import semen is held in quarantine during
maximal period of 21 day which is provided for semen sanitary and quality control. Control is
under supervision of Republic veterinary inspectors and samples are submitted to reference
veterinary institutions (Faculty of Veterinary medicine in Belgrade or to regional Veterinary
Institutions). Most batches imported in the province of Vojvodina (north Serbia) were directed by
decision of Veterinary Directorate to be tested in Laboratory of Reproduction at Scientific
Veterinary institute in Novi Sad. Method of sampling (number of batches to be tested and number
of doses per batch) was determined to be in accordance of the criteria of the representing Institutes.
In our case, all bulls from the list and almost all batches were subjected to analyses (exception was
if the bulls had import of several batches and some batches were with small number of straws-e.g.,
less than 50 doses). In most cases 3 straws per analyze were sampled for viral, bacterial and quality
investigations.
Bulls’ semen quality control consisted of:
1. CASA (Computer Assisted Sperm Analysis, ISAS, Proiser, Spain) for assessing
concentration, total and progressive motility and spermatozoa speed values;
2. flow cytometry analyses (Guava Millipore-IMV, USA) for sperm chromatin structure assay
- SCSA test (acridine orange, Invitrogen), and test of membrane and acrosome integrity
72
(PNA-FITC/PI, Invitrogen, USA) for synthetic extenders, or vitality test-membrane integrity
(Sybr14-PI, Invitrogen, USA) for milk or egg-yolk extenders;
3. Detailed cyto-morphological examination of stained sperm sample with eosine-nigrosinetripan blue staining (Alfapanon, Novi Sad) under oil immersion with phase contrast
objective, 1000x magnification (Olympus BX-40, Japan). The spermatozoa morphology was
assessed according to Barth and Oko (1989) and semen was classified according to quality
criteria by Jovičin et al. (1992) in four class (I, II, III and „out of class“).
Qualitative determination of bacteria was performed by cultivation onto the Columbia agar plates
supplemented with 5% sheep blood (Oxoid, Basingstoke, UK), MacConkey agar (Oxoid,
Basingstoke, UK) and Sabouraud dextrose agar (Torlak, Serbia). For anaerobic and microaerophilic
bacterial determination the thioglycollate broth was used. Tests for total aerobic microorganisms
were carried out in accordance with the international standard (ISO 8607: 2003). Briefly, series of
dilution (101-105) were prepared in buffered peptone water (CM1049, Oxoid, Basingstoke, UK),
and 0.5 mL were inoculated on Petri dish and Tryptone Soya Agar (CM131, Oxoid) supplemented
with 5% of sheep erythrocytes was poured over. The plates were incubated for 48h at 37oC under
aerobic conditions.
Imported semen was also subjected to virological testing for the presence of IBR/IPV (BHV-1),
BVD and Schmallenberg virus. Identification of the virus was carried through susceptible cell
culture (MDBK cell line; OIE Terrestrial Manual, Chapter 2.4.8, 2008) or molecular biological
study PCR technique. For identification of BVDV and Scmallenberg agent in the bulls' semen realtime ART-PCR was used, and PCR molecular method for IBR (Applied Biosystem 7500, Life
Technology, MA, USA).
Semen must be free of virus genome presence, or pathogen bacteria. Before national directives for
semen quality (declared in 2014) bacterial loads was tolerated up to 10.000 of total CFU/ml at NIVNS. Doses also were discarded (scored as “out of class”) in the following situation: CASA
parameters-total motility spermatozoa 30%, CASA progressive motile under 20%, total motile
cells in a dose less than 4×106 sperm cells per doses, progressive motile cells less than 3×106. Also,
semen should have at least 40% of live normal sperm cells with intact acrosome and less than 40%
of abnormal forms (cito-morphology after supravital staining).
From April 2014, official guidelines for semen quality were established (Official Journal of
Republic of Serbia" no. 38 from April 4, 2014). Main quality parameters of bull semen extracted
from this regulative are now more demanding and are as follows:
1) progressive sperm motility: at least 50% (for our sperm motility test that was performed
by CASA, we considered progressive motility at least of 30% and total motility at least of
50%);
2) the percentage of morphologically altered sperm up to 30%;
3) the number of (progressively) motile and morphologically normal spermatozoa in a dose
after thawing: at least 10 million;
4) the total number of the bacteria up to 500 CFU/ml (if semen had good quality traits we
tolerate up to 10,000 of saprophytic bacteria).
In cases with very good semen quality and less sperm cells in doses semen could pass analyses
because some elite bulls breeding centers were producing doses not more than with 6-9 ×106 sperm
cells per doses. There were rare cases of compensation of semen quality with higher number of
spermatozoa. Sexed semen is not covered by this quality regulation but all other regulations
regarding bacterial and viral presence were done as for conventional semen.
In the case that semen is not in accordance to relevant quality or sanitary requirement semen must
be safely destroyed or turned back to producer. Importers and producers accepted semen destroying
in such cases. Semen destroying was carried out by autoclaving at 128C for 1h and10 minutes.
73
Results
During the period from 2010 to 2014 Scientific Veterinary Institute "Novi Sad" (NIV-NS) was
responsible for quality control of 44 quarantines with 344,485 imported doses in 710 batches from
248 bulls. Out of 710 imported batches, 562 were analyzed (79.15%). Complete analyses were
performed on 550 conventional and 22 sexed semen samples of 248 bulls. Sexed semen represented
only 3.85% of all analyzed semen batches.
Semen originated from 10 different countries (Austria, Germany, Denmark, Croatia, Czech
Republic, Slovakia, Netherlands, USA and Canada) and from 18 different breeding centers. Most
often imports were from Croatia (15 times), Netherlands (7), Switzerland and Canada (5).
Table 1. Overview of the basic details of the number of quarantines, imported of conventional semen
batches, total bulls’ semen doses, performed analyses and rejected semen doses by exporting countries
subjected to analyses at NIV-NS during period 2011-2014.
Imported
Country
Austria
Canada
Croatia
Czech Rep.
Denmark
Germany
Netherlands
Slovakia
Switzerland
USA
TOTAL 10
Analyzed:
№ of imports
Samples
Batches bulls № of doses bulls
(quarantines)
(batches)
3
57
4
28,009
4
24
5
86
54
53,950
54
78
15
225
73
115,464
73
207
1
10
2
4,790
2
10
1
24
5
6,300
5
8
2
82
20
40,100
20
52
7
55
27
22,157
27
52
1
7
1
4,455
1
7
5
114
37
54,350
37
67
4
50
25
14,910
25
45
44
710
248 344,485 248
550
№ of rejected:
semen
bulls batches
doses
14
23
13,376
1
1
502
8
10
4,070
4
4
1,967
1
1
690
1
1
500
2
3
721
21,826
31
43
Table 2. Imported semen samples/analyses according to bulls breed and imported countries for conventional
semen (N=550).
Breed
Country
Austria
Canada
Croatia
Czech Rep.
Denmark
Germany
Netherlands
Slovakia
Switzerland
USA
TOTAL
Angus Belgian blue Brown Swiss Holstein Limousine Simmental
1
2
3
2
2
6
4
10
74
87
20
10
7
28
34
31
34
251
4
4
24
180
37
7
32
280
Table 3. Imported semen samples/analyses according to bulls breed and imported countries (sexed semen),
N=22.
Breed
Holstein
Simmental
Country
Canada
Denmark
Germany
Netherlands
USA
TOTAL
2
1
3
15
21
1
1
Table 4. Basic details of the rejected bulls semen doses (main reasons for rejection, number of
quarantines/imports, imported batches, bulls and number of doses) that was subjected to analyses at NIV-NS
during the period from 2011 to 2014.
Imported
№ of imports
doses % of all
batches
bulls
№ of doses
(quarantines)
rejections
Viral presence (BVD)
1
1
1
502
2.30%
Bacterial unsoundness
3
10
5
2,916
13.36%
Semen quality criteria
10
29
23
16,741
76.70%
Improper transport (N2 leaking)
2
3
3
1,667
7.64%
TOTAL
100%
16*
43
32
21,826
*15 imports were rejected, in one a combination of high bacterial load and low semen quality was noted
Reason for rejection
Reasons for semen rejection were classified as viral presence, bacterial unsoundness, low semen
quality or improper transport (liquid nitrogen leaking).
In 15 cases from 44 imports (quarantines) one of the mentioned reason for rejection were noted,
presenting incidence of 34.09%. One quarantine had combination of low semen quality in 2 batches
and high bacterial presence in 6 batches.
Viral genome presence of BVD by RT-PCR technique was noted in one batch of one imported
bulls' semen.
Bacterial unsoundness represented in 3 imports, on 5 bulls and 10 batches, representing 23.25%
(10/43) of rejected batches and 13.36% of all rejected doses (2,916/21,826; Table 5). From all
semen analyzes this is represented 1.81% of all batches (10/550) and only 0.85% of all imported
semen.
Bacterial contamination with Citrobacter Freundii (an opportunistic microbe that is accountable for
quite a few significant opportunistic infections) isolated in monoculture, in concentration of 1.000
CFU/ml was noted in one case and rejected as a potential risk for uro-genital infections (Milanov
and all., 2012; Joaquin and all., 1992; Badger et all, 1999). Other 9 rejections were because of high
bacterial load of saprophytic and/or opportunistic microorganism (40,000 to 780,000 CFU/ml,
averaging 237,778232,313 CFU/ml). This was several times over technical normative for semen
quality.
Main reason for semen rejecting was improper semen quality (29/43 cases; 67.77%), with total
rejections of 16,741 doses from 21,826 rejected doses (76.70%; Table 4). Low semen quality was
registered in 10 imports, in 29 batches and in 23 bulls. This represented 5.27% of all imported
semen batches (29/550) and 4.86% of all imported semen doses (16,741/344,485).
75
Table 5. Details of rejected bulls’ semen doses regarding bacterial contamination (number of imports, bulls,
batches, doses, bacterial load and isolated strains) during the period from 2011 to 2014.
Import
Bull
Batches
Import A
Bull A
Batch 1
Batch 2
Batch 3
№ of
doses
300
375
150
Batch 4
410
Batch 5
325
Batch 6
375
Batch 7
260
Batch 8
Batch 9
Batch 10
10
185
300
236
2.916
Bull B
Import B
Bull C
Bull D
Import C
TOTAL: 3
Bull F
5
Bacterial load
Isolated bacterial strain
(CFU/ml)
1.000 Citrobacter Freundii
350.000 Enterobacter aerogenes and Bacillus sp.
200.000 Corynebacterium sp. and Bacillus sp.
Corynebacterium sp., Bacillus sp., and
160.000
Staphylococcus sp.
Bacillus sp., Staphylococcus sp. and
130.000
Micrococcus sp.
Enterobacter aerogenes, Bacillus sp. and
780.000
Corynebacterium sp.
Bacillus sp., Staphylococcus sp. and
340.000
Micrococcus sp.
80.000 Bacillus sp. and Staphylococcus sp.
60.000 Enterobacter aerogenes
40.000 Enterobacter aerogenes
-
Regarding CASA parameters, average total motility of 29 rejected semen was 35.479.6%
(minimal value 9.30%, maximal 56.70%), progressive motility 16.775.50% (from 1.70% to
28.80%) and fast moving spermatozoa 19.10% (from 2.00-35.70%). Average total motile sperm
cells were 8.11106 (1.70-15.10106).
Table 6. Descriptive statistic of the CASA parameters of rejected deep frozen bulls' semen (29 semen
samples)
Descriptive
statistic
values
Mean
Minimum
Maximum
Std.Dev.
Coef.Var.
St. Error
1. Number of
spermatozoa
million million in
in ml
dose
90.90
22.72
29.10
7.30
133.90
33.50
22.26
5.56
24.48
24.47
3.87
0.97
2. Total motility
after
thawing
35.47%
9.30%
56.70%
9.61%
27.08
0.02
million
in dose
8.11
1.70
15.10
2.95
36.35
0.51
3. Progressive
motility
after
million
thawing
in dose
16.77%
3.76
1.70%
0.36
28.80%
7.00
5.50%
1.40
32.81
37.32
0.01
0.24
4. Percent of
fast moving
spermatozoa
19.10%
2.00%
35.70%
6.83%
35.77
0.01
On cyto-morphological analyses of deep frozen bull semen (29 semen samples) average percentage
of normal live sperm cells was 33.6712.01 (4.00-55.00%) and total abnormalities 42.7612.01%
(5.00-65.00% ).
Sperm chromatin structure assay (SCSA) of rejected deep frozen bulls' semen indicated average
sperm chromatin defects in 9.509.00% of spermatozoa, ranging from 0.40-32.10% (29 semen
samples) detected by flow cytometry. On membrane permeability test (sperm vitality test, livedead), intact sperm membrane was detected in 30.48±9.67% sperm cells, ranging from 1.60-43.80%
(23 semen samples). On sperm membrane and acrosome integrity assay (24 semen samples) live
spermatozoa with intact acrosome was detected in 39.24±12.02% sperm cells, ranging from 1,6043,80% (23 semen samples); booth detected by flow cytometry. Total damaged acrosomes (DA)
in sperm cells were present in 31.51±14.30% (ranging from 2.52-65.38%).
76
Table 6. Descriptive statistic of cyto-morphological analyses of deep frozen bull semen (29 semen samples).
Subpopulation of sperm cells (%)
Descriptive statistic
values

Mean
Minimum
Maximum
Std.Dev.
Coef.Var.
St. Error
Normal sperm morphology
Total
Live spermatozoa
LIА
LDA
L
 DА
Abnormal sperm morphology
Total, live and death
I АBN
II АBN
 PPD
ABN
1.
2.
3.
4.
5.
6.
7.
8.
42.76
5.00
65.00
12.01
28.10
2.09
33.67
4.00
55.00
9.75
28.97
1.70
2.39
0.00
26.00
4.51
188.54
0.79
32.67
13.00
64.00
10.60
32.45
1.85
2.00
0.00
8.00
2.26
113.19
0.39
11.88
2.00
30.00
7.02
59.11
1.22
6.88
0.00
35.00
7.81
113.53
1.36
42.76
5.00
65.00
12.01
28.10
2.09
( L=Total live/unstained spz.; LIA=Live spz. with intact acrosome; LDA=Live spz. with damaged acrosome;  DA=Damaged acrosome - total; 
PPD=Protoplasmatic droplet-total; I АBN=Primary abnormalities; II АBN= Secondary abnormalities; ( ABN= Total abnormalities).
Table 7. Descriptive statistic of sperm chromatin structure assay (SCSA) of rejected deep frozen bulls' semen
(29 semen samples) by flow cytometry
1. DNA status
values
(DNA – %)
undamaged
damaged
Mean
90.51
9.50
Minimum
67.93
0.40
Maximum
99.60
32.10
Std.Dev.
8.97
9.00
Coef.Var.
9.91
94.68
St. Error
1.61
1.62
Table 8. Descriptive statistic of membrane permeability test (sperm vitality test, live-dead) for rejected deep
frozen bull semen (24 semen samples) by flow cytometry
1. Membrane status (%)
values
Undamaged
Damaged
(live)
(dead)
Mean
30.48
69.52
Minimum
1.60
56.20
Maximum
43.80
98.40
Std.Dev.
9.67
9.67
Coef.Var.
31.72
13.91
St. Error
1.97
1.97
Table 9. Descriptive statistic of sperm membrane and acrosome integrity assay for rejected deep frozen bull
semen (23 semen samples) by flow cytometry
Detection of specific subpopulations of spermatozoa (%)
values
LIA
DIА
LDA
DDS
L
 DA
1.
2.
3.
4.
5.
6.
Mean
43.33
39.24
16.56
16.88
27.34
31.51
Minimum
22.94
15.22
0.60
0.34
2.02
2.52
Maximum
66.42
64.26
47.42
51.00
56.18
65.38
Std.Dev.
13.06
12.02
15.03
16.05
12.73
14.30
Coef.Var.
30.15
30.64
90.71
95.10
46.56
45.39
St. Error
2.72
2.51
3.13
3.35
2.65
2.98
( L=Total live spermatozoa; LIA=Live spermatozoa with intact acrosome; DIA=dead spermatozoa with intact acrosome; LDA=Live spermatozoa
with damaged acrosome; DDS= dead spermatozoa with damaged acrosome;  DA=Damaged acrosome - total).
77
Discussion
Council directive 88/407/EEC is laying down the animal health requirements applicable to intraCommunity trade in and imports of semen of domestic animals of the bovine species. Bulls semen
produced for artificial insemination should fulfil sanitary requirements before the quarantine period
(requirements of the farm, AI station or quarantine), at herd and individual level (diagnostic tests on
the bull).
Intercommunity trading and EU to non EU trading is regulated by Commission for implementing
decision 2011/630/EU on imports into the Union of semen of domestic animals of the bovine
species from third countries. In both cases no strict or any technical guidelines for intra community
trade is mentioned that could serve as a general guideline regarding semen quality requirements or
for bacterial count in extended semen.
Quality of imported semen analyzed at NIV-NS dominantly was in accordance to relevant national
and international standards (Official Journal of R. Serbia № 38/2014, Council directive 88/407/EEC
and OIE). However, 43 batches (32 bulls from 15 imports), with total of 21,826 straws (6.33% of all
imported straws) were rejected/destroyed because of different and prominent discrepancy, related to
improper transportation (liquid nitrogen leaking – 1,667 straws), presence of the genome of Bovine
Viral Diarrhea Virus, opportunistic pathogen bacteria (Citrobacter freundii - 300 doses), elevated
bacterial count (CFU from 40-780103; 2,616 doses), but mostly were rejected because of low
semen quality (10 of 15 cases 16,741 doses).
France has established automatically generated system of on field data collecting trough coding of
cows and semen straws. Information of cows, used semen, insemination or rebreeding is
automatically sent to main computers by internet on the moment of insemination which allows that
fertility problems are quickly recognized. Serbia without this system is relaying on obligation of
veterinary service to inform breeding or distributing center on pregnancy rate of purchased bulls'
semen. Practically, this system is not working in most regions (lack of data collecting and data
summering). Complains often are coming directly from farmers instead of veterinarian
practitioners. Other way to have better insight of semen quality is to test semen with up to date
laboratory system taking in account that biology traits are not always in accordance with laboratory
results (Milovanović and Barna, 2011), what was strong argument of importers when semen of low
quality was established, before declaration of national direction for semen quality (Official Journal
of R. Serbia № 38/2014. With modern, up-to-date system of semen quality control and strong
argumentation (semen quality tested by several methods, photo and movie documentation, ISO
standardized bacteriology and virology laboratories/tests), semen rejection was accepted as normal
issue, and most domestic importers and foreign breeding centers as sellers are easier accepting
reasons for semen destroying.
Conclusion
EU semen trading policy is devoted only to sanitary conditions and semen quality is a matter of
standard operative procedure (SOP) of breeding center. Semen of lower quality (up to dead-non
motile semen) could be expected from imported semen.
Routine semen testing at the import and national recommendations of semen quality traits can be
comprehended as a good method for reduction of fertility problem generated by male infertility on
cattle farms in Serbia.
Acknowledgements
This work was supported by a grant from scientific project TR 031071 of Ministry of Education and
Science of Republic of Serbia
78
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Badger LJ, Stins F. Monique, Kim KS; Citrobacter freundii Invades and Replicates in Human Brain
Microvascular Endothelial Cells, Infection and Immunity, 1999, Vol. 67, 8, 4208-15.
Barth AD, Oko RJ, 1989. Abnormal Morphology of Bovine Spermatozoa. Iowa State University Press,
Ames, IA, US.
Collection and processing of Bovine, small ruminant and porcine semen, OIE - Terrestrial Animal
Health Code,Chapter 4. 6, 2010.
Dr Stevan Perković, 60 godina rada stočarsko-veterinarskog centra „Krnjača“, 1951-2011, Мonografija,
publisher: Stočarsko veterinarski Centar Krnjača, print, Naučna KMD, Beograd, 2011.
ISO 8607:2003(E): Artificial insemination of animals -- Frozen semen of breeding bulls -- Enumeration
of living aerobic microorganisms, www.iso.org
Joaquin A, Khan S., Russell N., Al Fayez N: Neonatal Meningitis and Bilateral Cerebellar Abscesses
due to Citrobacter freundii, Pediatr Neurosurg 1991/1992; 17:23-4.
Jovičin M, Nemeš Ž, Boroš I, Jakovljević G, Kašić M, Salma J, Glavonić L, 1997. Steonost krava u
zavisnosti od citološkog i mikrobiološkog kvaliteta zamrznutog semena bikova. Zbornik naučnih radova
PKB Agroekonomik, 329-39.
Milanov D., Stojanović D., Barna T., Milovanović A.: Izolacija Citrobacter freundii iz duboko
zamrznutog semena bika - prikaz slučaja. Naučni simpozijum Reprodukcija domaćih životinja i bolesti
novorođenčadi, Divčibare, 4-7. oktobar 2012, urednik Miodrag Lazarević, Zbornik predavanja, Str. 125129, Beograd, Naučna KMD, 2012 (srp)
Milovanović A., Barna T.: The modified thermoresistance test is not siutable for fertility prediction of
frozen-thawed bull semen. The 15th Annual Conference of the European Society for Domestic Animal
Reproduction (ESDAR), Antalya, Turkey 15-17 September 2011, Guest Editor Detlef Rath,
Reproduction in Domestic Animals, Str. 130-130, Berlin, Wiley-Blackwell, 2011.
Minimalni
pogoji
za
oploditveno
sposobnost
semena:
http://www.uradnilist.si/1/objava.jsp?urlid=200751&stevilka=2725
OIE Terrestrial Manual 2008, Chapter 2.4.8. Bovine Viral Disease
"ОIE
Terrestrial
Animal
Health
Code
2003",
http://www.oie.int/eng/normes/mcode/code2003/A_00127.htm
Pravilnik o uslovima u pogledu objekata, opreme, sredstava za rad i stručnog kadra koje mora da
ispunjava centar za skladištenje i distribuciju semena za veštačko osemenjavanje (Objavlјeno u
„Službenom glasniku RS”, broj 6/11 od 4. februara 2011. godine
79
THE HEALTH STATUS OF BREEDING STALLIONS FOR NATURAL BREEDING AND
ARTIFICIAL INSEMINATION: REGULATORY COMPLIANCE IN EUROPEAN UNION
AND WEST BALKAN
Miroslav I. Urosević1*, Luis Losinno2, Aleksandar Milovanović3, Dragiša Trailović4, Slobodanka
Vakanjac4, Jelena Petrović3, Natasa Filipović5
1. Scientific institute of reproduction and artificial insemination for domestic animals "Temerin" Temerin, Serbia
2. Fac. Agronomia y Veterinaria, Universidad Nacional de Rio Cuarto, Rio Cuarto, Argentina
3. Scientific Veterinary Institute “Novi Sad”, Novi Sad, Serbia
4. Faculty of veterinary medicine, University of Belgrade, Belgrade, Serbia
5. Faculty of agriculture, Veterinary medicine Dep., University of Novi Sad, Novi Sad, Serbia
*Corresponding author: urosevic.miro2012@gmail.com
Abstract
Diagnostic and health condition control procedures of breeding stallions in Serbia are in accordance
with Regulation on the establishment of animal health care measures for 2014. Diagnostic tests for
breeding stallions are performed on each breeding animal one time in a year, and those include only
tests for Equine infectious anaemia. Nevertheless, the majority of horse owners and keepers tend to
follow their previous routines and preventive measures. When it comes to stallions, tests which are
conducted on horse-farm prior to breeding season include serum neutralization test for Equine
Herpes Virus -1 (EHV-1) and Equine viral arteritis.
What is the looming problem nowadays is that there is no existing system control whether would
that involve certain institutions or associations (sport or breeding animals). Examination of equine
sperm for arteritis is not a common procedure - however, in justified cases of doubt solely it is. On
the other hand, as an example of regulations in South America, we shall mention compulsory
measures in Argentina about the obligation of examination of Equine Viral Arteritis on stallions.
These are following: Every breeding association must submit a certification of negativity (serum
neutralization test) for Equine Viral Arteritis of all active stallions before the annual breeding
registration or AI. Frozen semen samples imported from foreign countries must be checked by state
laboratories for detection of viral presence by PCR before commercialization and insemination. On
balance, it can be emphasized, that in relation to further development of veterinary profession and
horse industry in Serbia as well, it is not recommended to “a priori” implement every EU model
currently, and completely, due to the difference in horse breeding between our system and similar in
EU countries. Nevertheless, in relation to mentioned legal framework and de facto situation in horse
breeding in Serbia, we would recommend thoroughly planned control and primarily to make
evidence of stallions used for breeding of mares in sport and rural areas.
Keywords: equine, semen, legislative, Serbia, EU
Introduction
The state of horse breeding and reproduction in Serbia
Horse breeding as an animal husbandry branch has a long and significant tradition in Serbia through
the use of horses in work, sport and nowadays increasingly more as a hobby. According to the
records from the Bureau for statistics of Serbia from 2014 here are 16.000 horses, which include
8.000 of mares and fillies. However, when it comes to the number of stallions there are no official
records. Although, those are the official records we consider them as not viable enough and they are
80
taken with caution owing to the fact that those do not reflect the actual state especially regarding the
breeding animals.
Moreover, the additional records available to us are containing the information about the population
size on the Province Vojvodina territory (Trivunovic, 2014). This report, from main breeding
organisation in the animal husbandry at the Faculty of Agriculture of Novi Sad from 2014, includes
only stallions that are registered, and are under the regular productivity control. The active
population counts 184 animals. Thus, there is an assumption that for breeding is used a larger
number of the breeding animals, however the breeders are not completely adjusted to breeding
programmes from the unknown reason whether they are not keeping records or they do not hand in
breeding reports to basic breeding organisations. In addition to those mentioned facts there have not
been a proper number of scientific publications when it comes to fertility and problems in horse
reproduction due to infectious disease in Serbia. In the continuation of this work we are going to
describe what are the laws that should be obeyed regarding the health conditions of stallions, if they
are compared with the legislation in Serbia (West Balkan) and EU.
Legislation about breeding stallions in Serbia
Diagnostic and health condition control procedures of breeding stallions in Serbia are in accordance
with Regulation on the establishment of animal health care measures for 2014 (Anonymous,
2014A). Diagnostic tests for breeding stallions are performed on each breeding animal one time in a
year, and those include only tests for Equine infectious anaemia (EIA). Nevertheless, the majority
of horse owners and keepers tend to follow their previous routines and preventive measures. When
it comes to stallions, tests which are conducted on horse-farm prior to breeding season include
serum neutralization test in blood sample for Equine Herpes Virus-1 (EHV-1) and Equine viral
arteritis (EVA).
Nevertheless, the present Program of measures states that in order to do establishment, monitoring
and prevention of Leptospirosis specific diagnostic examination (microscopic agglutination testMAT), is biannually conducted in stallions for natural mating and for semen production for
artificial insemination. Furthermore, there are no precise data about the number of breeding mares
and stallions that are under regular serological control for Leptospirosis in the herds in Serbia.
Owing to that, we cannot accurately determine the nature of epizootic situation for this contagious
disease.
What is the looming problem nowadays is that there is no existing system control whether would
that involve certain institutions or associations (sport or breeding animals). Examination of equine
sperm for EVA is not a common procedure - however, in justified cases of doubt solely it is.
In any case, in our country no systematic control of breeding animals for CEM (Contagious equine
metritis) is done which is hardening the insight on the field. However, there is existing risk of CEM
transmission during the import of breeding stallions and mares from the neighbour countries and
EU, as well as the stallions provided and borrowed for breeding.
Compulsory blood examination for EIA with the Coggins test has to be conducted in the following
cases: once in a year in the breeding horse-farms, horse-farms owned by Serbian Military Force,
sports horse-farms, stud sections, forests and other places of work, as well as in every herd
containing more than 10 horses; 90 days prior their arrival to hippodromes, fairies, exhibitions,
reviews, sporting competitions etc. If the owner of an animal sells the animal or alienates it in any
other way, the certificate about the examination of EIA must not be older than 30 days. Diagnostic
examination can also be done with ELISA test, with confirmation Coggins test in addition.
Equine viral arteritis
All newly purchased horses should be kept in quarantine and subjected to serological testing
81
(serum neutralisation test), necessarily paired blood samples.
In relation to the regulations on the health status of breeding stallions in Serbia rules about semen
quality ought to be mentioned (Anonymous, 2014B).
It is necessary to obtain certificate of the official veterinary organization claiming that the samples
are taken from studs with high in quality with well-familiar health status and that the standard
measures are conducted which are in accordance with program of measures of animal health care;
Certificate of the official veterinary organization claiming that standard clinical examination is
conducted and that no symptoms are established responsible for causing diseases which can be
transmitted by the semen of the donor animal.
Such records are kept for each production run of conserved semen, and in a manner when it is
possible at any time to determine the origin of semen and it is kept for at least five years from the
date of placing on the market of the last batch produced.
The semen meets the requirements in terms of quality if: it is produced in a centre for reproduction
and artificial insemination; it meets the health and hygiene conditions in accordance with
recognized international standards; it is taken from an animal that has been clinically examined and
at which no symptoms of diseases which can be transmitted by semen of an animal which the
semen is taken from; it is taken from the high in quality breeding animals with known health status
over which are implemented the measures prescribed by a special regulation governing the program
of measures of animal health care;
European Union legislative
An interesting example of legislation is in Croatia, where the Regulations on traffic equine semen
are in fact the provisions of Directive 92/65/ EEC (Anonymous, 1992) on health requirements for
trade and imports into the European Union (EU) of animals, semen, ova and embryos. Regardless of
its issuing in 2009, the mentioned Regulations came into force on the day of Croatia entering the
EU in 2013.
As in the whole EU veterinary certificate for trade of semen of the equine animal is issued or
authorized by the official veterinarian and above all he specifies the type, race and identity of donor
semen, as well as the time of collection of semen. The official veterinarian confirms that the Centre
for semen collection in which that semen was collected, processed and stored for transport, meets
the following requirements: that is situated on the territory or in the region of the Member State on
the day of collection of semen until the date of dispatch of semen or until the expiry of a period of
30 days mandatory storage of frozen semen that is free of African horse sickness (AHS) in
accordance with special regulations; that during the period commencing 30 days prior to the date of
semen collection until the date of dispatch of fresh / chilled semen or until the expiry of a period of
30 days mandatory storage of frozen semen fulfil the requirements of the Ordinance on the
veterinary conditions for the transfer of the equine animal and imports from third countries. This
Ordinance applies to most contagious animal diseases, in accordance with Council Directive EC. In
the first place, the definition of "third country" means States that are not members of the EU. But in
the narrow sense of "third country" in relation to the status of AHS, means a Member State of the
EU free from AHS, or most precisely where no clinical, serological (in unvaccinated equidae) or
epidemiological evidence of AHS have not been established in the past two years and in which in
the last 12 months has not implemented a vaccination against this disease. The term "official
veterinarian" means that the veterinarian is appointed by the Minister in accordance with the
provisions of the Veterinary Act.
The official veterinarian must during the inspection and on the basis of declarations by the owner or
breeder to establish the absence of any reason to suspect that the equidae have been in contact with
animals suffering from infectious or contagious diseases during the 15 days immediately prior to the
82
examination. This applies to a negative status of the animal from a disease which should be
reported: Dourine, Glanders, Equine encephalomyelitis (all types including Venezuelan
encephalomyelitis), EIA, rabies, anthrax, AHS, Vesicular stomatitis.
The certificate of health in the case of a registered equine must be issued within 48 hours before
loading or no later than the last working day prior to loading, in the language of the country of
export of semen and at least one of the official languages of the EU. The health certificate is valid
for 10 days and consists of a single sheet. Interestingly, the experts of the European Commission, in
cooperation with the Ministry of Agriculture (as the competent body of the exporting country), may
do the examination on the spot, to the extent that is necessary to ensure the implementation of this
Regulation. The competent body shall provide all necessary assistance to the experts referred to in
paragraph in carrying out their work. If it is about the import of equidae from the third countries to
Croatia it is only allowed if those countries are officially enlisted by the European Commission.
This approval relates to the whole territory of a third country or to only a part of its area. We should
not neglect the legislation of the third country which applies in relation to animal health and
welfare.
The conditions to be met by the Centre for artificial insemination in EU are: That during the period
commencing 30 days prior to the date of semen collection until the date of dispatch of fresh / chilled
semen or until the expiry of a period of 30 days of mandatory storage of frozen semen possessed
only equidae which were free of clinical signs of CEM and EVA. That the above described semen
comes from donor stallions, which: on the day of collection of semen have not shown clinical signs
of an infectious or contagious disease; during at least 30 days prior to collection of semen have not
been used for natural mating; during at least 30 days prior to collection of semen have been kept on
farms where no equine animal showed clinical signs of EVA; during at least 60 days prior to
collection of semen have been kept on farms where no equine animal showed clinical signs of
CEM; according to the information / check the official veterinarian of the 15 days immediately
prior to collection of semen have not been in contact with equidae suffering from an infectious or
contagious disease; that they are subjected to the following tests in a laboratory approved by the
competent authority in accordance with a test program: Coggins test for EIA with negative result,
serum neutralisation test with serum dilution of 1 in 4, or with virus isolation test on an aliquot of
the entire semen of the donor stallion, with the negative result; a test for CEM carried out on two
occassions with an interval of seven days by isolation of Taylorella equigenitalis from preejaculatory fluid or a semen sample and from genital swabs taken at least from the penile sheath,
urethra and urethral fossa with negative result in each case.
That the stallions donors undergo one of the following testing programs: that at least 30 days prior
to and during the collection of semen continuously resident in the collection centre of semen and in
that period no equine animal in the centre has not come into direct contact with equidae of lower
health status than the one in the centre. Certainly, the exact date is stated when they carried out
indicated tests on samples of semen or blood.
During the completion of this certificate, parts that are not related to the shipment must be crossed
out. In order to prevent forgery of documents, the colour of the seal and signature of veterinarian
should differ from the colour of the printed text.
Discussion
Unfortunately, for now, in Serbia some kind of diagnostics includes only sport horses or other
categories of horses that are exposed to diagnosis because of bringing them to the event, fairs or
export.
83
The impression is that the most widely implemented serological diagnosis is the one of EIA.
Although the Ordinance on eradication EIA specifies the compulsory serological diagnostic
measures in horses once in five years it is highly dubious that this is fully carried out. This is
indicated by the occasional discovery of seropositive animals in the past 5 years. In contrast, most
sports horses are regularly controlled, sometimes several times a year, and it is partly regulated by
law, and partly by sporting regulations.
Regardless of whether it is possible titre of antibodies against influenza, herpes virus infection, or
EVA result of vaccination or infection it raises the question of what this finding means when we are
aware that a large number of horses are positive to all these diseases, without evidence of
vaccination.
There are some interesting experiences and research on the occurrence of EVA in Croatia in the
period from 2009 to 2012 (Kolaković, 2013). According to the legislation in Croatia, EVA is
mandatory controlled with serological examinations of all stallions before the breeding season.
Serologically positive animals then are subjected to an overview of at least two semen samples for
the presence of the virus, with the PCR method. Stallions with a positive test for EVA in the sperm
must be excluded from breeding. The author concludes that this reduction in the number of stallions
serologically examined presents a significant risk of uncontrolled spread of EVA in the horse
population in Croatia. It underlines the need to careful approach when it comes to the
implementation of vaccination against this disease, because the mentioned measure cannot
eliminate the cause of the permanently infected stallions. Therefore, it is recommended to provide
vaccination of only seronegative animals under strictly controlled conditions with the required stay
in quarantine after the vaccination. In addition, vaccination, particularly in countries with unreliable
labelling and record-keeping of horses (i.e. Croatia and Serbia), completely loses the ability to
control the disease with serological examination, primarily because of the problems in
differentiating infectious and vaccination titre. Moreover, epizootic situation in Serbia, together
with the manner of keeping, manipulating and breeding animals, is not suitable to disease control
implementing vaccination against EVA.
As an example of regulations in South America (Argentina), we would mention compulsory
measures (National Animal Sanitary Service-SENASA- 434/01) in Argentina about the obligation
of examination of EVA on stallions. These are following: Every breeding association must submit a
certification of negativity (serum neutralization test) for EVA of all active stallions before the
annual breeding registration or artificial insemination. Frozen semen samples imported from foreign
countries must be checked by state laboratories for detection of viral presence by PCR before
commercialization and insemination.
In relation to the occurrence of Dourine, interesting is the situation in Italy (Calistri et al. , 2013),
where the disease was diagnosed in the regions Sicilia and Campagna in 2011. In Italy, the program
of measures requires examination of about 4000 breeding stallions annually to this parasitic
infection. Having established five outbreaks of Dourine in spring 2011, competent institutions have
made a plan to eradicate and above all to provide the determination of the prevalence of this disease
in Italy. Consequently were found two, but later seven outbreaks. It has been discovered that the
infection is created directly from infected individual coitus. Although Italy is considered the country
with developed horse breeding, this prevention of Dourine has been used to detect a number of
deficiencies in recording and registration of horses in the Central Database in relation to the
situation on the field. All this made it difficult to implement measures on eradication Dourine and
identify the source of infection. This refers to an uncontrolled mating mares (without proper
records), non-implementation of the prescribed veterinary measures and on the impossibility of
monitoring the movement of animals. This example confirms that effective control of infectious
diseases that occur occasionally involves primarily the establishment of effective communication
between veterinarians, authorized diagnostic institutes and laboratories, veterinary inspection,
84
government and international institutions which are responsible for recording the occurrence of
certain diseases and regular reporting when that is the case.
Conclusion
It can be concluded that the aforementioned regulations differ significantly only in the control of
CEM and EVA, which are the most actual epizootiological problem in Serbian horse population,
because we have no real information about their presence in horse studs in Serbia.
Actually it is understood the free status of centres for artificial insemination and horse studs of
especially dangerous infectious diseases, such as AHS. We believe that more important would be
consistent implementation of regulations related to the diagnosis of infectious diseases in stallions.
On balance, it can be emphasized, that in relation to further development of veterinary profession
and horse industry in Serbia as well, it is not recommended to “a priori” implement every EU model
currently, and completely, due to the difference in horse breeding between our system and similar in
EU countries. Nevertheless, in relation to mentioned legal framework and de facto situation in horse
breeding in Serbia, we would recommend thoroughly planned control and primarily to make
evidence of stallions used for breeding of mares in sport and rural areas.
Thus, based on these data, we propose appropriate measures. First of all, audits (as amended)
regulations and their alignment with the current EU directives, bearing in mind the epizootiological
epizootiological situation in the country and the region. As a prerequisite, it should be the training
of certified laboratories for fast and effective diagnosis of infectious diseases of horses, those that
are regularly or occasionally occurs in our country, as well as diseases that can potentially introduce
horses or otherwise from other countries. This mainly refers to identifying EVA in the sperm
(PCR), as well as with evaluation of genital swabs for CEM. However, as far as it is possible we
would not recommend vaccination against EVA. The reason for this is that the disease situation in
Serbia, the way of keeping animals, the use and breeding horses, including the records and marking
of animals, are not appropriate and in accordance with control of the disease by introducing just
these preventive measures.
To ensure effective diagnosis, it is necessary to review and offer laboratory analysis of all
veterinary institutes related to infectious diseases of horses. Constant communication and exchange
of information and experience would ensure effective coordination of all involved in the health care
of horses, and certainly with international organizations such as OIE and the relevant EU bodies.
Because of the introduction of legal solutions for immunoprophylaxis should enable simpler
procedure of import of vaccines for infectious diseases of horses. And last but not least, a crucial
measure for combating infectious diseases, breeding a horse and raising awareness of the
importance of this issue, both for the owner or holder of the animals themselves and veterinarians
who care about their health condition.
Acknowledgements
The presented work is part of the research done in the scientific projects TR-31084 and III-46005
granted by the Serbian Ministry of Education and Science.
Reference:
1. Anonymous: Council Directive 92/65/EEC, of 13 July 1992 laying down animal health requirements
governing trade in and imports into the Community of animals, semen, ova and embryos not subject to
85
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animal health requirements laid down in specific Community rules referred to in Annex A (I) to
Directive 90/425/EEC. (OJ L 268, 14.9.1992, p.54), 1992.
Anonymous: Veterinary Law ("Off. Gazette of RS" No 91/2005, 30/2010), 2010.
Anonymous: Ordinance on animal health requirements when moving and imports equidae from third
countries ("Official Gazette of Croatia" No. 78/12), 2012.
Anonymous: Regulation on the establishment of animal health care measures for 2014. year ("Off.
Gazette of RS" No. 24/14), 2014A.
Anonymous, Regulation of marking semen, making evidence about producing semen, as well as
requirements when it comes to fulfilment of semen quality (Offizial Gazette of Serbia 38/14 from april
2014), 2014B.
Aurich Ch, Reproduktionsmedizin beim Pferd. Gynäkologie – Andrologie – Geburtshilfe. Stuttgart,
Parey Verlag, 2010.
Bazanow AB, Jackulak, AN, Frącka BA, Staroniewicz MZ: Abortogenic viruses in horses, Equine Vet
Educ, 26, 48-55, 2014.
Calistri P, Narcisi V, Atzeni M: Dourine reemergence in Italy. J Equine Vet Sci, 33, 2, 83–89, 2013.
Đuričić B, Petrović T, Urošević M, Dimić-Jakić D, , Appearance and spread of Equine Viral Arteritis
(EVA) Republic in Serbia. Proceedings of the 10th International Congress of World Equine Veterinary
Association, Moscow, Russia, February 2008, 624.
Kolaković J: "Serosurveillance of equine viral arteritis in Croatia and evaluation of control measures
during period 2009. – 2012.", Graduate thesis. University of Zagreb, Faculty of veterinary medicine,
Croatia, 2013.
Trivunovic S.: The Main breeding organisation in the animal husbandry of the Faculty of Agriculture,
University of Novi Sad, 2014. https://sites.google.com/a/stocarstvo.edu.rs/glavna-odgajivackaorganizacija/publikacije
Urošević I.M., Trailović R.D., Salajpal K., Jajić I., Ilić S.: Infectious diseases of equine reproductive
organs: preventive measures and erradication. Proceeding, The Horseville, Science and Profession, third
Regional Symposium „Breeding, reproduction and healt care horses’’. Novi Sad, Serbia, 1-3 October
2012, 711-19
86
SPERM PATHOLOGICAL FORMS AND ACROSOMAL MEMBRANE INTEGRITY IN
BOAR AI DOSE ON PIG FARMS IN AP VOJVODINA (SERBIA)
Jelena Apić1*, Aleksandar Milovanović1, Tomislav Barna1, Milovan Jovičin1
1. Scientific Veterinary Institute "Novi Sad", Novi Sad, Serbia.
*Corresponding author: jelena.a@niv.ns.ac.rs
Abstract
The success of sows artificial insemination (AI) significantly depends on the quality of sperm used.
The presence of morphologically abnormal sperm significantly reduces the boar semen fertilization
potential. Additionally, the presence of such sperms indicates on various diseases and disorders, or
adverse effects of unfavourable environmental factors. Therefore, the aim of this study is to analyze
the presence of sperm morphological abnormalities, detected in the boars used for AI on farms in
AP Vojvodina (Serbia). Extended semen samples, from 111 AI boars, was tested on sperm and
acrosomal membrane integrity by flow cytometry. Supravital staining (eosin-nigrosin) according to
Bloom, was used to determinate the percentage of live, dead and pathological forms of sperm, as
well as sperm with intact or damaged acrosome. In average, live sperm was average 71%, of which
59% was with intact acrosome. Damaged acrosome was found in 13% live and in 17% dead sperm.
Pathological forms (PF) were found in 32.5% of sperm (20.7% primary and 11.5% secondary PF).
It is important to point out that only 19% of AI doses were categorized as a "first class", while 49%
of the AI doses were "outside of class" (not for use in AI). These results indicate a very poor quality
of examined AI doses. In conclusion, factors that can affect AI dose fertilizing capacity, in the on
farm artificial insemination technology, must be determined in detail.
Keywords: sperm, morphological abnormalities, AI, boars.
Introduction
In modern intensive pig production, more than 90% of sows are artificially inseminated (AI), by
using liquid diluted semen (Stančić and Dragin, 2011; López Rodríguez, 2012; Kalifa et al., 2014).
Fertilizing capacity of AI dose significantly depends on the quality of used semen. Sperm
concentration, progressive motility, percentage of viable cells and acrosome morphology are the
basic parameters of semen quality evaluation (Knox, 2004; Mircu et al., 2008; Maes et al., 2011).
To selection of the boars with a good reproductive performance, the prediction of sperm fertilizing
ability has a great economic importance, when expensive genetically superior boars is used in sowʼs
artificial insemination (Gadea et al., 2004). For maximal reproductive exploitation of genetic
quality boar, large number of AI doses per one ejaculate, are often produced (Stančić, et al., 2012).
This results in excessive semen extension (Johnson, 2000; Stančić et al., 2012). However,
overextension of semen is the most common cause of reduced progressive motility and increased
number of abnormal sperm in AI doses (Maxwell et al., 2007; Caballero et al., 2008; Stančić et al.,
2012), particularly sperms with acrosome damaged or disintegrated acrosomal membrane (Perez
Marcos et al., 1991; Kommisurd et al., 2002). Therefore, to achieve high fertility rate in artificially
inseminated sows, it is necessary to accurately control the quality of each ejaculate and extended
semen in AI dose.
The aim of this study was to analyze the presence of morphologically abnormal sperm in the
extended boar semen, used for AI on intensive pig production farms in Vojvodina Province
(Republic of Serbia).
87
Quality testing of diluted boar semen was performed in the Laboratory of reproduction in domestic
animals, Scientific Vterinary Institute, Novi Sad (Republic of Serbia). Extended semen samples
were transported from the farm to the Institute in thermo-box, at +17oC. After arrival to the
laboratory, the semen samples were prepared according to the usual protocol for testing by CASA
(computer assisted semen analysis) and for flow cytometry methods. Total of 111 samples of
diluted boar sperm (AI doses) during the year 2014 and 2015 was tested.
Flow cytometry (Guava-IMV Millipore, USA) was used to test the sperm cell membrane integrity
and akrosome membrane integrity (a combination of fluorometric colors PNA-FITC and propidium
iodide). Percentage ratio of live and dead sperm, sperm with intact acrosome, protoplasmic droplet,
as well as pathological forms of spermatozoa was determined by cytomorphological examination of
supravital stained sperm preparation (eosin-nigrosin staining method according to Bloom). Based
on cytomorphological findings (according to Jovičin et al., 1997), as well as on internal
classification according to the number of progressively motile sperm cells (determined by CASA
method), AI doses were classified as class I, II, III or "out of class" (Milovanović et al., 2013).
Table 1. Reference values for classification of diluted boar semen*
Sperm subpopulation in examined semen samples
1. Total live (L)
2. Live with intact acrosome (LIA)
Normal sperm
3. Live with damaged acrosome (LDA)
forms
4. 4. Total with damaged acrosome (DA)
5. Total with protoplasmic droplet (PPD)
6. Primary abnormal (I ABN)
Pathological
7. Secondary abnormal (II ABN)
sperm forms
8. Pathological forms total (PFT)
Class of sperm qality1
I class
II class
III class
51-69%
40-50%
 70%
46-59%
40-45%
 60%
6-9%
10-15%
 5%
11-20%
21-30%
 10%
11-20%
21-30%
 10%
6-10%
11-15%
 5%
6-10%
11-15%
 5%
16-30%
31-7.0%
 15%
*Laboratory for Reproduction in Domestic Animal, Scientific Veterinary Institute, Novi Sad.
1
Semen samples with parameters above or below of these values are classified as "out of class"
Results
The obtained parameters of tested diluted boar semen samples (AI doses), are shown in Table 2.
From the total of 71% live (unstained) sperm, 59% of them was with intact acrosome. Damaged
acrosome was found in 13% of live and in 17% of dead sperm cells. Various pathological forms
were found in 32.2% of sperm cells (abnormalities of head and/or principal piece of the sperm cell,
primary abnormalities - I ABN, in 20.7% and abnormalities of middle or rear part of the sperm tail,
secondary abnormalities - II ABN, in 11.5%).
From the total number of AI doses (n = 111), 49% of them was categorized as "out of class" (not for
use in AI), while only 19% of AI dose was a "first class", 16% of AI doses was "second class" and
16% of AI doses was "third class" (Fig. 1).
88
Table 2. The parameter values of tested diluted semen samples (AI doses)
Parameter value
Average
Variation
71.4±13,98
35.6 - 91.9
58.7±16,00
17.3 - 87.7
11.2±13,15
1.8 - 46.3
12.7±13,22
0.2 - 53.7
17.3±13,40
0.0 - 49.7
30.0±16,43
0.2 - 68.2
20.7±17,49
1 - 64
11.5±16,33
0 - 68
32.2±19,87
2 - 85
Parameter of sperm subpopulation in
examined semen samples
1. Total live, unstained (L)
2. Live with intact acrosome (LIA)
3. Dead with intact acrosome (DIA)
4. Live with damaged acrosome (LDA)
5. Dead with damaged acrosome (DDA)
6. Total with damaged acrosome (DA)
7. abnormalities (I ABN)
Primar
8.
Secondar
abnormalities (II ABN)
9.
Pathological
forms total (PFT)
Total semen samples / boars tested (n)
111
I ABN - Pathological changes of head and/or uper part of the sperm tail.
II ABN - Pathological changes of midle and/or rear part of the sperm tail.
Semen samples, %
60
49
50
N = 111
40
30
19
20
16
16
Second
Third
10
0
Out of class
First
Semen class
Fig. 1. Classes’ distribution of tested diluted semen samples
Fig. 1. Classes distribution of tested diluted semen samples
Discussion and Conclusion
The results of our research show a very poor quality of tested AI doses. Based on the presence of
pathological sperm forms (32.2%) and the number of sperm with damaged acrosomal membrane
(30%), up to 49% of AI doses were categorized as "out of class" (not for use in artificial
insemination).
There are many factors that can influence AI dose fertilizing capacity: factors associated with the
boar and factors associated with AI dose (volume, sperm numbers and progressive motility, the
percent of abnormal sperm, contaminants within the dose, and even the amount of sperm cell
agglutinatio) (Knox, 2004; Yeske, 2007). Progressive motility and acrosomal membrane integrity
are the main parameters of AI dose fertilizing capacity, and can be significantly decrease by
overdilution of ejaculate or by impact of inadequate storage and/or transport condition (darkness
and temperature +17oC) (Johnson et al., 2000; Stančić et al., 2012). The results which were
obtained by Kommisurd et al. (2002), indicate that the acrosome is more susceptible to damage
during storage than the organelles being the structural basis of motility. Using over diluted AI doses
is frequently demonstrated as a reason for reduced fertility in the artificially inseminated, compared
89
to naturally inseminated sows (Zvekić, 2003; Gadea, 2005; Alm et al., 2006). On the other hand, it
has been shown that there is considerable variation among boars concerning the fertilizing capacity
of semen during storage (Waberski et al. 1994; Stančić et al., 2003b).
In conclusion: to detected the reasons for large number of poor AI doses, following should be done:
(1) evaluate boar ejaculate quality, (2) check the boars healt status, (3) determine dilution rate of
each AI dose, and (4) check the storage and/or transport conditions of AI doses.
Acknowledgments
This work was supported by a grant from scientific project TR 031071 of Ministry of Education and
Science of Republic of Serbia.
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Mircu C., Cernescu H., Knop I.Violeta, Frunză Ilinca, Ardelean V., Bonca G.H., Otava G., Zarcula
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91
ECHINOCOCCUS GRANULOSUS OF DOMESTIC PIGS: A CASE CONTROL STUDY
Doroteja Marčić*1, Jasna Prodanov-Radulović1, Igor Stojanov1, Siniša Grubač1,
Ivan Pušić1, Vladimir Polaček1
1 Scientific Veterinary Institute “Novi Sad”, Novi Sad, Serbia
* Corresponding author: dora@niv.ns.ac.rs
Abstract
Internal parasites of swine are very common in swine worldwide, sometimes compromising
production and occasionally the cause of clinical disease. One of the most important zoonotic
parasites that can be found in swine is a larval stage of Echinococcus granulosus. Adult
Echinococcus spp. cestodes occur in carnivores, and their larval cysts (hydatids) occur in various
herbivores and omnivores, including humans and swine. In carnivores, egg-laden proglottids pass
with the faeces, contaminating the environment. When ingested by a pig, the larva (oncosphere)
hatches in the small intestine, penetrates the intestinal wall, and enters the circulation. The liver
stops about 70% of the oncospheres, where they nidate and develop into hydatids. Hydatic disease
in pigs is not common finding on large swine farms, because biosecurity measuers are taken to
prevent the contact of swine with carnivores. However, this is not a case with the backyards and
free-range pigs, and they present the most frequent way of pig production in villages. The aim of
the paper is to present a finding of intensive parasitic infection (hydatic disease) in backyards pigs.
The material for this research included pigs from one backyard, where certain disorders and health
problems in weaned pigs were detected. The applied research methods included: anamnestical and
clinical evaluation, pathomorphological examination, parasitological laboratory testing for detection
and determination the presence of parasites in the organs and tissue samples (liver, lung) derived
from died pigs. Since in the evaluated backayard a sudden death in 10 weaners occurred, with
unspecific clinical signs, the gross pathology examination of 2 dead weaners was performed. The
nacropsy findings revealed the presence of enormous number of hydatid cysts on and in the liver
tissue. Also, a large number of free parasitic cysts were found in the abdominal cavity. By
parasitological examination of the liver and abdominal content, the presence of small parasitic cyst
of Echinococcus granulosus (2-5 mm) was detected. Based on the achieved results, it can be
concluded that Echinococcus granulosus infection is present in the backyards and free-roaming
pigs, where husbandry methods allow direct contact with domestic and wild carnivores.
Keywords: backyards, domestic pigs, Echinococcus granulosus
Introduction
Hydatidosis in pigs is caused by larval form of Echinococcus granulosus.
Echinococcosis/hydatidosis is a zoontic parasitic disease that poses a major health as well as
economic threat all around the world. In the etiology of the disease, two stages can be discerned: 1)
hydatid stage (cystic echinococcosis), which occurs in intermediate hosts (domestic and wild
omnivores, herbivores and humans); and 2) echinococcosis stage, which occurs in the “dead-end”
(definitive) hosts (animals from the families Canidae and Felidae), caused by the adult
Echinococcus spp. (Brunetti and Filice, 2008).
In Serbia, the highest percentage of infected pigs can be found in backyard and free-range pig
production systems (up to 75%). The percentage of infected pigs found on large swine farms (with
biosecurity measures) is also not negligible and adds up to 10% (Pavlović et al., 1997).
92
A certain number of wild boars in our country are reared in controlled and enclosed hunting
grounds, while a number of free-ranging populations are mainly unknown. One of the
characteristics of outdoor swine production in some regions is raising free-roaming domestic pigs,
where they share forest habitat with wild boars. Contacts between wild boars and domestic pigs
kept in outdoor farms may occur occasionally. Since both animals have the same susceptibility to
various infections including hydatidosis, there is a major concern to monitor the epidemiological
situation of wild boars especially when control measures in domestic pigs are implemented
(Prodanov-Radulović et al., 2014).
Uncharacteristically for tapeworms, the adult tapeworm is very small, only 5 mm in length. The
eggs produced by the parasite are very light and resilient (Laaksonen and Paulsen, 2015). The
infection of intermediate hosts occurs exclusively through the use of the contaminated water and
food. The source of the Echinococcus eggs contamination is to be found in the faeces of “dead-end”
(definitive) hosts, which is continuously being released into the external environment and ultimately
consumed by the pigs (Pavlović and Ivanović, 2006).
The development process of the hydatic cysts is the same in all intermediate hosts, including
humans. Once the digestive tract is reached, oncospheres (hexacanths) are released from the eggs,
penetrating through the intestinal mucosa and reaching the capillaries, from where, through vena
porta, the primary implantation site is reached - the liver (up to 70% share). Certain number of
oncospheres that mash the liver, arrive at the right heart via vena cava, proceeding towards the
lungs, the second most frequent implantation organ for hydatic cysts. The remaining hexacanths are
transferred, through the left heart, towards the other parts of the organism – implanting kidneys,
eyes and seldom brain and bones. When oncospheres (hexacanths) are fixed to the mucous
membrane, the development of the cysts can begin. The cysts are continuously growing throughout
the whole life of the intermediate host (Pavlović et al., 2011). Their size varies, depending on the
duration of the infection, from barely noticeable to cysts of 150 mm in diameter. The cysts are often
pearly grey colour and contain clear liquid with thousands of small, infectious larval forms of the
parasite (Laaksonen and Paulsen, 2015). They are under considerable pressure and in the case of
their rupture, the outcome is fatal.
In most of the cases the disease in pigs is asymptomatic and the losses primarily occur due to organ
failures caused by the implanted cysts. There are three main factors contributing to the spreading of
this infection in pigs: 1) the lack of pig breeders’ and dog owners’ knowledge concerning the life
cycle of the parasite and concerning its epidemic potential; 2) the percentage (share) of the infected
dogs as “dead-end” (definitive) hosts; and 3) hygienic circumstances, especially in pig breeding
practices.
The aim of this paper is to widen the awareness of the possibility of Echinococcus granulosus
epidemic mostly among the pigs bred in backyard and free-range pig production systems.
The material for the investigation included one small family owned backyards swine farm (capacity
of 5 sows), where the free-range was also practiced.
The Veterinary Inspection had informed the Scientific Veterinary Institute “Novi Sad” about the
suspicion of the possible outbreak of classical swine fever in the family owned swine farm engaged
in extensive swine breeding in the Srem region. Since in the evaluated backayard a sudden death in
10 weaners occurred, with unspecific clinical signs, the gross pathology examination of 2 dead
weaners was performed. The samples from dead weaners were tested by RT-PCR method, in order
to exclude the possibility of infection with classical swine fever virus.
93
Since gross pathology findings had indicated the presence of significant pathomorphological
changes, an additional parasitological testing was performed.
Results
During the external gross pathological pathomorphological examination of dead pigs, carried out in
the backyard of the pig owner, a dirty slimy discharge of the nostrils was discovered at both pigs, as
well as a strong cyanosis of the skin in the area of proboscis and earlobe base with visible yellow
discoloration of the skin and mucous membranes. Mild palpebral oedema was also detected at one
of the dead pigs. The internal gross pathological pathomorphological examination, on the other
hand, revealed an amber coloured, free liquid, with fibrin strands in the chest cavity (Picture 1).
Picture 1. Chest and abdominal cavity: free liquid with fibrin strands
The examined tissues were also remarkable for mucosal and serosal yellow discolouration.
Individual bleeding on the side parts of the diaphragmatic lobes were present in the lungs, as well as
the hepatisation of apical and cardiac lobes. A few examples of Metastrongylus spp. were found in
the lumens of bronchi and bronchioles. The mediastinal lymph nodes were unchanged. Tonsils,
epiglottis, pharynx and larynx were also unchanged. The mandibular lymph nodes were unchanged.
Extremely yellow discolouration of endocardium was discovered. In the abdominal cavity, the free
liquid mixed with large amount of fibrin strands and free whitish, almost translucent, cystic
formations, like “drops”, the size of grain of wheat or rice, also was found (Picture 2a and Picture
2b).
Picture 2a. Abdominal cavity: free liquid with fibrin
strands and cystic formations
Picture 2b. Abdominal cavity: free liquid with fibrin
strands and cystic formations
94
The liver was significantly enlarged with rounded edges and with a small but very numerous cystic
formations protruding and dropping out of the liver tissue after cutting (Picture 3a). Hepatic
hemorrhage with considerable structural defects was present (Picture 3b).
Picture 3a. Liver: hemorrhage and cystic formations
dropping out of the liver tissue after cutting
Picture 3b. Liver: hemorrhage and cystic formations
The stomach was nearly empty, with expressed folds of mucous membranes and without changes in
the mucosa. The small and large intestines were almost empty, with only a small amount of content
and without changes in the mucous membrane. There was yellow discolouration of the kidneys with
no changes either under the capsule or on the transverse section. The bladder was full of dense,
distinctly yellow liquid content and without changes in the mucosa (Picture 4).
Picture 4. Slit bladder: dense, distinctly yellow liquid content
Discussion and conclusions
In Serbia, there are no official datasets of infection rates available for all the farms applying the
backyard and free-range pig production systems, i. e. the slaughter is often carried out nonsupervised by inspection service and without being reported. According to some recent
investigations, infection rates vary widely across country between 4.6 and 57.6% (Ivanović and
Pavlović, 1999). It has been established that around 75% of the infected pigs come from mini farms,
applying the backyard and free-range pig production systems, mostly not maintaining strict
biosecurity measures. Socio-economic factors as well as the lack of breeders’ and dog owners’
knowledge concerning the epidemiological features of the infection and the lack of adequate
cemeteries for disposal of the deceased animals all contribute to the spread of this zoontic disease.
95
Considering the fact that echinococcosis/hydatidosis poses a major global threat, infection
monitoring must be carried out with care and perseverance all over the world. For example, on the
occasion of a pig slaughter in Lithuania, the examination of 684 pig livers revealed that
significantly more infected animals came from mini farms (applying the backyard and free-range
pig production systems and thus not maintaining strict biosecurity measures) then from large-scale
farms (Bruzinskaite et al., 2009).
In order to make progress in the fight against echinococcosis/hydatidosis, it is necessary to cut off
every rout of transmission of the infection. To this end, it should be made mandatory, that livestock
slaughter process is always carried out in slaughterhouses, and under constant supervision of
veterinary inspection services. It is also of paramount importance to report every case of the
infection to the veterinary inspection service from where animal originate and to document it. The
hygiene of the pig production practices and methods also affects the spread of the infection, which
makes it extremely important to maintain strict biosecurity measures, which also means control on
livestock movement, so that, in this particular case, the contact between the pigs and the
intermediate and definitive hosts (dogs, i. e. their faeces) would be made impossible. The
importance of widening of the global awareness of the problem as well as widening the access to
the knowledge of infection should also be emphasised. Through education, which should start from
school and also encompass instructing livestock breeders, butchers and dog owners on how to
prevent the infection, everyone should be familiar with the basic facts concerning this infection.
This education also means making wider public familiar with measures that should be undertaken,
in order to combat this zoonosis more successfully. The measures also include the dog
dehelmintisation, the control of dog movement and secure disposal of the organs contaminated with
hydatic cysts (by boiling, burning or handling in animal shelters).
Acknowledgements
This paper is a result of the research within the project TR 31084, financed by the Ministry of
Education, Science and Technological Development, Republic of Serbia.
References
1. Brunetti E., Filice C.: Echinococcus Hydatid Cyste Medicine Specialities „Infectious Diseases“ Parasitic
Infections, http://emedicine.medscape.com/article/216432 - overview, 2008
2. Bruzinskaite R., Sarkunas M., Torgerson P. R., Mathis A., Deplazes P.: Echinococcosis in pigs and
intestinal infection with Echinococcus spp. in dogs in southwestern Lithuania. Vet Parasitol., 160, 3-4,
237-241, 2009
3. Ivanović S., Pavlović I.: Raširenost ehinokokoze kod svinja u ekstenzivnom držanju na području srednjebanatskog okruga. Tehnologija mesa, 40 (6), 302-303, 1999. Beograd, Srbija
4. Laaksonen S. and Paulsen P.: Hunting hygiene, Wageningen Academic Publishers, 2015
5. Pavlović I., Ivetić V., Valter D., Lončarević A., Kulišić Z., Dimitrić A.: Ehinokokoza svinjaepizootiološki, epidemiološki i ekonomski značaj. Zbornik radova 4. savetovanja veterinara Republike
Srpske sa međunarodnim učešćem, 116, 1997. Teslić, Republika Srpska
6. Pavlović I., Ivanović S.: Ehinokokoza/hidatidoza, bolest životinja i ljudi, izd. Naučni institut za
veterinarstvo Srbije i Ministarstvo poljoprivrede, šumarstva i vodoprivrede, 1-32, 2006. Beograd, Srbija
7. Pavlović I.,Hadžić I., Žugić G., Anđelić-Buzadžić G., Vaić D., Jovčevski S.: Hidatidoza aktuelan problem
stočarske proizvodnje. Zbornik naučnih radova sa XXV savetovanja agronoma, veterinara i tehnologa,
Institut PKB Agroekonomik, Vol.17, 3-4, 133-139, 2011. Beograd, Srbija
8. Prodanov-Radulović J., Došen R., Petrović T., Polaček V., Lupulović D., Stojanov I., Grubač S.:
Antibodies to selected viral disease agents in hunted wild boars in Vovodina region, 3rd International
symposium on hunting, Modern aspects of sustainable management of game population, 149-153, 2014.
Zemun-Beograd, Srbija
96
THE PREVALENCE OF ASPERGILLOSIS IN POULTRY AND CONTROL MEASURES OUR EXPERIENCE
Miloš Kapetanov¹*, Dragana Ljubojević¹, Igor Stojanov¹, Milica Živkov-Baloš1, Miloš Pelić¹,
Marko Pajić¹
* Corresponding author: milos@niv.ns.ac.rs
Abstract
The fungi of the genus Aspegillus are ubiquitous saprophytic microorganisms which are, in certain
circumstances, responsible for clinical infections of respiratory tract in all poultry, particularly in
young birds. Aspergillus fumigatus, A. niger and A. glaucus are the most common isolated fungi in
the cases of pulmonary aspergillosis in poultry. Poultry is constantly exposed to these fungi in its
environment, and predisposing factors, such as long exposition, highly contaminated environment
and litter, high humidity in poultry houses, poor ventilation, as well as malnutrition and stress
contribute to the development of clinical manifestations of aspergillosis. The aim of the present
paper was to examine the effects of some factors on the incidence and prevalence of the clinical
form of aspergillosis in poultry. The possibility to control the disease by introduction of
prophylactic measures was also considered. The presence of Aspergillus sp. in poultry was analyzed
based on the results of clinical and laboratory tests carried out during 2000, 2010 and 2014. The
prevalence of aspergillosis was noted in poultry of different age, such as hatched eggs, young and
adult birds. Aspergillus sp. was isolated in the range of 3.85% to 36.14% from unhatched eggs, litter
and environmental and hatchery swabs. An increased trend in number of infected poultry flocks was
observed, especially in young birds. During the years 2000, 2010 and 2014, acute aspergillosis was
detected in 12, 16 and 21 commercial flocks of chickens and turkeys, respectively. Application of
proper sanitary-hygiene measures on poultry farms and hatcheries, as well as microbiological
control of feed are considered essential for an efficient control of spreading of infection.
Keywords: aspergillosis in poultry, humidity, temperature, feed
Introduction
Aspergillosis is a fungal disease of all poultry species, particularly of young birds. Most frequently
it occurs in turkey poults, chicks, ducklings and goslings (Kunkle, 2003; Beytut et al., 2004).
Aspergillus fumigatus is considered to be the most pathogenic and is the most frequent isolate from
pathologic lesions, while others like A. niger, A. flavus, A. terreus and A. glaucus can induce the
disease, too. The fungal spores are ubiquitous in nature. Exposure of poultry to fungi or spores
occurs after the introduction of contaminated litter and feed. Early infection is possible in hatcheries
if fungal contamination occurs.
Certain infectious diseases may contribute to aspergillosis, e.g. infectious bronchitis, coryza,
chronic respiratory disease, laryngotracheitis, Newcastle disease and fowlpox. It has been
speculated that extremely dry air and dust can cause the infection with Aspergillus because they dry
out the respiratory mucosa and protective effect of mucus is absent (Kristensen and Wathes, 2000).
Some geographic and seasonal regularity are observed in relation to the distribution of aspergillosis
outbreaks. Incidence of the disease decreases on poultry farms with stringent hygiene and good
nutrition management.
97
Influence of certain factors on the outbreak and spreading, as well as clinical features of
aspergillosis, were investigated in this paper. The possibility to control the disease by introduction
of prophylactic and intervention measures was discussed.
The presence of Aspergillus spp. on poultry farms was analyzed from data collected after the
clinical and laboratory investigations performed during the three selected years, 2000, 2010 and
2014.
Poultry flocks were clinically observed and postmortem examination of chickens and turkeys of
different age was carried out. If aspergillosis was detected in the findings, the attacked tissues were
taken for microbiological investigations. Having been processed first, tissue samples were cultured
on solid media using standard methods. Mycological investigations were done in laboratory at the
Veterinary Institute Novi Sad.
Litter taken directly from different surfaces in poultry houses and swabs was cultured on solid
media. Unhatched eggs, hatchery waste and environmental swabs taken from different surfaces in
hatcheries were sampled in order to determine the presence of fungi and its frequency, as well as for
the comparison of mycological contamination with the environmental conditions.
The influence of different factors on the occurrence, spreading and source of aspergillosis was
investigated using data from the official report of the Republic Hydro meteorological Service of
Serbia, regarding the outer temperature and rainfalls in 2000, 2010 and 2014, for the purpose of
comparison with the control period from 1961 to 1990.
Results and Discussion
The spread out of aspergillosis was noted in poultry of different age, from very young birds to adult
ones. It is well known that the disease can occur in other young and adult poultry species, including
poults, goslings, ducklings, swans, wild and pet birds, particularly if kept in intensive manner.
While resistant and healthy poultry can overcome the infection with high number of Aspergillus,
week and young birds generally become ill easily. It is experimentally demonstrated that chickens
up to three days of age are the most susceptible to the infection, while older are more resistant.
Islam et al. (2009) found significant differences in morbidity (up to 70%) and mortality rate
depending on age and category; aspergillosis was most frequently detected in cockerels (9.03%),
then in broiler chickens (5.48%) and in laying hens (1.92%). The disease was clinically present in
broilers at the earliest age (13 days) and in layers and cockerels at the oldest age (76 weeks). In
epizootiological investigation Sajid et al. (2006) found the majority of sick flocks at the age of 14
days. Aspergillosis appears in two clinical forms: 1) acute form with high morbidity and mortality
rates, the disease persists from only few days to two weeks, and 2) subacute and chronic form in
adult poultry, disease being persistent several weeks or rarely several months.
During the years 2000, 2010 and 2014, acute aspergillosis was detected in 12, 16 and 21
commercial flocks of chickens and turkeys, respectively. In 2010 and 2014 the disease was acute
with high morbidity and mortality within only few days. Numerous flocks suffered detrimental
losses in a short period of time and were destroyed because their further raise was economically
unjustified. Clinical symptoms during the acute source of infection are described in literature,
including depression, inapetence, thirst and hyperventilation with dyspnoe (Akan et al., 2001; Islam
et al., 2009; Stoute et al., 2009). Chickens have ruffled feathers; they drowse, get weaker and, in
complicated cases of dyspnoe, pathologic wheese is observed, which differs from other respiratory
diseases. Besides the symptoms previously mentioned, the investigation of mass pneumomycoses
98
discovered one distinctive finding which was grouping of the chickens toward the source of fresh
air ("hunger for oxygen"; Figures 1 and 2).
Besides respiratory form, infection of eyes can also occur (Akan et al., 2001). In that case
protruding eye lids are observed because of formation of yellowish-cheesy small pellets around the
membrane nicticans with central ulceration (Figures 3 and 4). In our investigations, ocular infection
with Aspergillus was determined in two broiler flocks, at the age from 7 to 10 days. In subacute and
chronic infection, only one or several individuals in flock became ill. Clinical signs of depression,
inapetence, ruffled feathers and emaciation are common, sometimes chronic bronchopneumonia,
too (Martin et al., 2007; Islam et al., 2009) during the year 2010 and 2014 subacute and chronic
aspergillosis was not detected.
In the investigated period, acute aspergillosis was clinically noted. Postmortem findings included
nodules - aspergillus granuloma, oval or round shaped, single or in conglomerate, size of a pin head
to pea, located on air sacs, lungs and on visceral serosae of abdominal cavity, liver and intestines
(Figures 5 and 6). Granulomas, oval or round, single or in group of conglomerate, are typical
section finding in Aspergillus infected birds. They are located in thoracic and abdominal cavity and
on the liver surface. Their appearance resembles the one found in avian tubercullosis, but with
radial hyphae nets surrounded by reactive zone like granulation tissue. Generally, nodules are
present in almost all tissues, even in eyes and brain (Throne Steinlage et al., 2002; Mukaratirwa,
2006; Martin et al., 2007; Cacciuttolo et al., 2009; Islam et al., 2009; Stoute et al., 2009).
Figures 1 and 2. The infected chickens from aspergillosis – prominent dyspnea.
Figures 3 and 4. Ocular form of aspergillosis - yellowish cheesy pellets around the membrane nicticans.
99
Clinical signs and pathologic findings are not sufficient for diagnose, so laboratory confirmation by
isolation of Aspergillus sp. is necessary. The spores germinate well in laboratory conditions, on
standard media and at room temperature, producing green to green-blue colour colonies that become darker, even black over time (Figures 7 and 8). In order for the disease to be diagnosed other
respiratory agents need to be excluded, such as infectious bronchitis, coryza, chronic respiratory
disease and Newcastle disease, infectious laryngitis, tuberculosis and fowlpox.
In intensive poultry keeping, there are numerous sources of this important fungal infection. Feed,
particularly mashed, can be contaminated with high number of fungi and moulds (Akanetal., 2001;
Nešić et al., 2005; Škrinjar et al., 2009; Kapetanov et al., 2012; Kapetanov et al., 2013).
Figures 5 and 6. The infected turkeys from aspergillosis; post-mortem finding: disseminated nodulesaspergillus granulomas in the lungs and serosal surfaces of organs in the abdominal cavity.
Figures 7 i 8. The growth of Aspergillus flavus colonies on Sabouraud agar.
The infection in poultry may occur if litter, environment or hatcheries are contaminated. Fertile
eggs and embryos can be contaminated before or during the incubation, and Aspergillus sp. is found
in tissues of unhatched eggs and hatchery waste (Jacobsen et al., 2010). Warm and humid air in
hatchery provides ideal conditions for the survival of Aspergillus. Infected embryos die between
15th and 18th incubation day, which may cause a decrease in hatch-ability of up to 30%. Dust
containing more than 800 colonies per gram leads to prospective embryo infection (Kozić, 1967).
100
Eggshell can be contaminated in case of inadequate collection and storage of eggs. The spores are
most frequently isolated from alanto-chorion liquid, producing green colonies up to 2 cm in size.
During the years 2000, 2010 and 2014, Aspergillus sp. colonies were often detected in unhatched
eggs and swabs taken from the hatchery (Table 1). In order to prevent the contamination of embryos
and chickens at hatch, different sanitation programs are applied (Ivanov, 2008). The sanitation
programs are designed in such manner that the number of fungi and other potential pathogens is
maintained at an acceptable level. However, their total elimination is not realistic. Lately, some
reports have brought the attention to possible infection of reproductive tissues of hens and
concomitant disease (Egg Borne Aspergillosis or congenital aspergillosis). It is demonstrated that
intravenous and inhalation infection is possible (Femenia et al., 2007). Still, under natural
conditions, infection is predominantly induced by air and orally.
Using inadequate litter increases the possibility of infection at early age. Higher number of fungi,
longer exposition, particularly in combination with stressing procedures, like vaccination, beek
trimming etc., all contributes to aspergillosis. Different types of litter may be contaminated
unequally. Sajid et al. (2006) found significantly higher rate of infected eggs in flocks kept on
sawdust (67.74%) in comparison to rice hulls (32.26%). On the other hand, Islam et al. (2009) could
not clearly relate the incidence of aspergillosis to the type of litter used. In our investigations,
mycological control of litter and swabs taken from surfaces in poultry houses points to oversights
during sanitation and disinfection (Table 1). With respect to year 2000, the total number of samples
and the number of mycologically inadequate samples was nominally higher in 2010 and 2014.
Raising poultry under higher fungal contamination increases the risk of aspergillosis, especially in
immune compromised or sick birds. The influence of particular outer and environmental factors to
fungal survival are numerous (De Bey et al., 1995; GigIi et al., 2005; Karwowska, 2005; Nichita
and Tirziu , 2008). The spores of Aspergillus germinate better when the air is dry. Aspergillus sp. is
often isolated from dust, and since the largest proportion of dust particles is inhalable, dust is
considered to be the predisposing factor for aspergillosis outbreak (De Bey et al., 1995). The type of
ventilation and air humidity are also relevant (De Bey et al.. 1995; Gigli et al., 2005; Sajid et al.,
2006) as they can impact the concentration of Aspergillus in the poultry house (Nichita and Tirziu,
2008).
By using available data on meteorological conditions in the region, this paper aims at establishing
the connection between clinical aspergillosis and overall laboratory findings, as well as the climatic
factors. The official report of Hydrometeorological Service of the Republic Serbia, for the year
2010, shows extremely warm percentile distribution. During the summer of 2010 maximal daily
temperatures were often above 30 C, and the total number of tropical days was higher than the usual
in most parts of Serbia, except in mountain regions. However for 2014 besides the very hot days,
abundant precipitations were also characterized, leading to flooding of the rivers from their banks
and further to (some sort of) natural disasters.
Table 1 - Contamination of unhatched eggs, litter, swabs taken from the poultry houses and
hatcheries by Aspergillus sp.
Sample type
Year 2000
Number and
Total number
%of positive
of samples
samples
Year 2010
Number and
Total number
% of positive
of samples
samples
Year 2014
Number and
Total number
% of positive
of samples
samples
Unhatched eggs
481
33 - 6.86
617
62 - 10.05
746
74 – 9.91
Litter
39
9 - 23.07
42
15-35.71
112
32-28.57
Swabs from houses
94
34 - 36 17
111
39-36.14
171
31-18.13
Hatchery swabs
182
7-3.85
268
15 - 5.60
311
101
11 - 3.53
The Hydro-meteorological reports also show rainfalls of totally 110 to 150% of the average values
(taken from the period between 1961 and 1990) in most parts of Serbia, and the percentile
distribution was rainy, very rainy and extremely rainy. The rainfalls were extremely rainy in all
regions. During the selected years (2000, 2010 and 2014) average outer temperatures were high
throughout the whole year. The rainfalls, watery alluvium and relative humidity of air, were
significantly higher than usual. In order to survive and reproduce, fungi need certain humidity and
temperature level which is around and higher than 37°C (De Bey etal., 1995; Karwowska, 2005;
Gigli et al., 2005; Sajid et al., 2006; Chate and Bhivgade, 2010).
The distribution of the aspergillosis outbreaks is influenced by some geographic and seasonal
differences, so it is more frequent in areas with high outer temperature and rainfalls (De Bey et al.,
1995; Karwowska, 2005; Madscn , 2006; Sajid et al., 2006). On the other hand, during winter, due
to inadequate environmental conditions and the fact that sometimes it is impossible to provide
optimal ambient for poultry, aspergillosis incidence increases. Some daily aberrance in the number
of Aspergillus in poultry houses was determined by Nayak et al. (1998). In our country, aspegillosis
outbreaks are more frequent in areas northern from the rivers Sava and Danube.
Microflora in poultry houses and farms in general, in our case the "population" of Aspergillus sp.,
contributes to the emission of biosol into atmosphere, especially during the warmer months
(Karwowska, 2005). The concentration of unwanted and potentially harmful gasses, dust, bacteria
and fungi in poultry house are changes depending on the age of poultry, particularly in broiler
chickens (Vučemilo et al., 2007). The air pollution impacts the health and productivity of poultry
(Nešić et al., 2005). Also, certain species of fungi have toxinogenic and allergenic features so they
can disturb the health of people, either directly, in case of farmers, or in case of the rest of the population, indirectly, after emission into atmosphere (Škrinjar et al., 2009; Chate and Bhivgade , 2010).
It seems to be necessary to create and implement standards for adequate hygiene, epizootiological
and epidemiological conditions on farms and their environment.
Conclusions
The global warming and high relative humidity induced increase of incidence of some diseases,
including aspergillosis.
Poultry is constantly exposed to Aspergillus sp. in its environment, so high contamination and long
exposal contributes to clinical aspergillosis.
The success of therapy basically depends on the source of disease and degree of dissemination of
process. In acute cases, particularly in very young chickens, it would be wrong to rely on antifungal
therapy solely. Implementation of sanitary-hygiene measures in poultry houses and hatcheries, as
well as microbiological control of feed, is essential for prevention of significant losses.
Acknowledgement
Education, Science and Technological Development, Republic of Serbia
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104
________________________________________________________________________
Session № 2
FOOD AND FEED SAFETY AND QUALITY
Full papers
_______________________________________________________________________
105
Invited lecture
GAME MEAT SAFETY – WILD BOARS
Jelena Petrović*, Milica Živkov Baloš, Živoslav Grgić
1
Scientific Veterinary Institute „Novi Sad“, Rumenacki put 20, Novi Sad, Serbia
*Corresponding author: jelena@niv.ns.ac.rs
Abstract
Wild animal meat harvesting and processing is significantly different from classical livestock meat
production and represents a challenge by itself. Implementation of the concept "from forest to fork"
encompasses epidemiological difference between wild animals and livestock, influence of hunting
ground ecology, type of hunting, field evisceration of hunted game, meat inspection after shooting
or transport etc. The objective of this paper was to identify biological and chemical hazards
important for wild boar meat safety. Several hazards were analysed: Mycobacterium spp.,
Salmonella spp., Trichinella spp.,Toxoplasma spp., Alaria alata, microelements, and heavy metals.
Two criteria were established for hazards identification: evidence of shared pathogens presence in
wild boar population in specific geografical region and evidence of hazardeous pathogens spread
during handling, processing and consumption of wild boar´s meat. The research on the presence of
food borne pathogens in wild boar´s meat is still scarce, while the Trichinella spp. life cycle is
distinctly described and there are relevant data about epidemiology and natural reservoirs of the
parasite in this part of the Europe, however little is known about other food borne pathogens in wild
boar population. The presence of Salmonella spp., Toxoplasma gondii, Alaria alata is identified in
wild boar population in our region. The analysis of the results of the microelements and heavy
metals in wild boar meat has shown that no samples exceeded legally set limits, but our results
suggest that pollution of the biosphere with chemical contaminants should be systematically
monitored to identify potential increasing contamination tendencies. Programme for monitoring and
control of game meat safety should include control measures for live animals, control measures
during hunting and after shooting, guidelines for official meat inspection, control measures for
carcass processing and surveillance of chemical residues.
Keywords: meat, wild boar, food borne pathogens, microelements, heavy metals
Introduction
Wild boar (Sus scrofa) distribution covers the entire continental Europe; it is missing in British
Isles, Scandinavia and northern part of the European Russia. Wild boars in Serbia are native and
very abundant big game species. Excessive and irresponsible hunting resulted in demographic
decline in early 20th century. However, thanks to substantial changes of agricultural practice,
reduced number of natural enemies as well as game feeding policies in last four decades the game
population has recovered and is still increasing, not only in Serbia but also around Europe (Rippa et
al., 2012).
Game meat production chain is substantially different from the conventional production of meat
from domestic animals, thus presenting an unique challenge. The concept “from forest to fork”
encompasses the effects of hunting ground ecosystems, type of hunting/shooting, evisceration in the
field, meat inspection after slaughtering, transportation etc. The risk assessment regarding
alimentary pathogens in wild boars implies elucidation of some basic issues, i.e., identifying the
epidemiological difference between domestic and wild animals and determining the criteria for
evaluating the safety of wild game meat.
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When speaking of wild animals, the disease control measures encompass the control of the diseases
that are transmissible from wild to domestic animals or directly to humans. Potential negative
environmental impact of measures and actions taken to the purpose of disease control is a specific
issue within the program of wildlife disease control.The opinion that complete eradication of
infectious and zoonotic diseases among wild animal population is virtually impossible is nowadays
widely accepted.
Domestic animals are raised for food and food production in controlled conditions, thus undergoing
the range of measures for health status control and ante and post mortem examinations at slaughter.
Contrary to that, game undergoes only a post mortem examination. In that respect, information on
certain diseases or mortality rates is lacking as well as the veterinary treatment, specific laboratory
testing, etc., thus restricting the proper evaluation of animals’ health status and prevention of their
entrance into the food chain.
The food safety criteria for assessing the safety of wild game meat are still lacking. They represent a
complex food-safety issue, since the majority of criteria relies on the average daily food intake,
which is highly specific in wild animals. The majority of the population consumes minor amounts
of game meat, but small population consumes pretty large quantities of game meat. The average
consumption of game meat among hunters’ families is estimated to be some 4kg meat per person,
i.e., family member (Ramanzin i sar., 2010).
The objective of this paper was to identify biological and chemical hazards of importance for wild
boar meat safety. Moreover, control measures for particular hazards are described, too.
Hazard identification and control options
Our identification of hazards of importance for wild boar meat relied on two main criteria. Is there
evidence for hazard transmission during handling, preparing and consuming of wild boar meat and
is there evidence of the presence of the pathogen among the game population in our region? The
following hazards were categorized:
 High-priority hazards: Mycobacterium spp., Salmonella spp., Trichinella spp. Toxoplasma
gondii
 Low-priority hazards: chemical hazards, Alaria alata
 Hazards of undefined priority, i.e. hazards that still lack sufficient data Campylobacter spp.
Y. enterocolitica, pathogenic verotoxic E. coli
The guidelines of Codex Alimentarius Commission (CAC, 2005) put the emphasis on hygiene and
inspection surveillance of hunted game at the primary stage of meat production chain (including
transport) as the critical points for game meat control. The recommendations are implemented
through Directive Regulations (EC) 853/2004 and 854/2004.
The measures are basically distributed into two groups:
 identification of the diseases and all major changes by visual inspection ( in case of apparent
pathological changes, severe contamination from the environment or suspected specific
biohazard, additional laboratory testing is recommended)
 application of practical skills and knowledge aimed at preventing the spread or increase of
biohazards (such as Salmonella spp.) on/in edible tissues
Mycobacterium bovis
M. tuberculosis is considered the most important biological hazard in large game. Among the
wildlife population of Europe, three animals are considered major hosts of tuberculosis: badger,
wild boar and deer from the subfamily Cervinae. Wild boar can act as a direct source of human
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tuberculosis infection, as well as the reservoir of infection in domestic animals (Gortazari i sar,
2008). Transmission of tuberculosis to humans occurs mainly by inhaling infectious aerosols,
consuming raw milk and, though somewhat less frequently, by consuming meat products
originating from infected animals. Some literature reports described potential infection routes from
wild boars to humans, including contact via the raw meat (Ashford et al., 2001).
Some 90% of known-infected wild boars with tuberculosis manifest calcified granulomatous lesions
in the mandibular lymph nodes. Moreover, 60% of these animals have generalized tuberculosis, i.e.,
lesions in multiple organs, predominantly in the head, thorax and abdomen. However, even
generalised tuberculosis in wild boar rarely causes visible loss of body condition (Martín-Hernando
et al., 2007). Hence, tuberculosis in wild boars is not manifested by specific clinical picture and
only the pathoanatomical examination can reveal suspect disease.
Wild boar tuberculosis has been reported in the last decades in at least 10 European countries:
Bulgaria, Croatia, France, Germany, Hungary, Poland, Portugal, Slovakia, Spain and the UK
(Gortázar et al., 2012). Wild boar experience much higher levels of exposure than deer (Vicente et
al., 2006). M. bovis prevalence in wild boar ranged from 46 to 52 % in three different surveys in
the Iberian Peninsula (Gortázar et al., 2008), where this host is considered the main carrier of
wildlife tuberculosis and a key factor in cattle tuberculosis eradication (Naranjo et al., 2008). In the
region of South Bačka, three endemic foci of tuberculosis were recorded located in municipalities
of Žabalj, Novi Sad and Titel. The percentage of infected animals in individual herds ranged
between 11.10% and 59.18% (Pušić et al., 2007; Pušić et al., 2009; Pušić et al., 2013). So far,
relevant data on the presence of tuberculosis in wild boars in this region are not available and the
research is ongoing. The occurrence of tuberculosis in cattle was recorded in the village of Kovilj
(the territory of Novi Sad municipality), which suggests potential contacts between wild and
domestic animals and thus presence of tuberculosis in wild game.
Eradication programmes in domestic animals rely on annual diagnostic examination of cattle using
the method of intradermal tuberculinization reaction, computed records and animal tracking, testing
positive herds and those who were in close contact with them at short intervals, and mandatory
slaughter of positive animals (Pušić et al., 2008; Pušić et al., 2009a,b,c). When speaking of wild
animals, the surveillance is practically impossible, so meat inspection is the only potential current
source of information and measure for control of tuberculosis.
Trichinella spp
Trichinellosis is endemic in almost all European countries, affecting also the regions of Srem and
river valeys of Danube, Drina and Kolubara in Serbia (Petrović et al.,2012a). The analysis of
recorded trichinellosis epidemics in Vojvodina in the period 2001-2011 identified domestic pig as
the main reservoire of Trichinella. The infection is commonly acquired by consuming raw or
undercooked meat infected with living Trichinella larvae. Documented cases of human infections
after consuming wild boar meat were reported in Serbia (Urošević et al., 2013). In Europe, wild
animals are considered the main reservoir of Trichinella, which makes the eradication of the
infection impossible in spite of its pretty low prevalence in wild game (Rafter et al., 2005).
According to Petrović et al. (2012 a, b, c), high incidence of trichinellosis was established in the
territory of Vojvodina among several animal species such as jackals (7.89%), foxes (4.76%) and
wild boars (0.53%). In countries in which trichinellosis of domestic animals has been fully
eradicated, such as Danemark, the prevalence of sylvatic trichinellosis is extremely low (0.001%)
(Enemark et al., 2000). Moreover, an average infection rate in carnivores in Vojvodina (3
larvae/10g) is significantly higher than that recorded in Danemark (1 larva/10g). In some cases,
extremely high infection rates has been established in wild boars in Vojvodina region, ranging even
up to 1100 larvae/g (Petrovic et al., 2013a, b). High incidence of sylvatic trichinellosis in some
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geographic regions poses substantial risk of infection spreading to domestic pigs grazed in sylvatic
habitats. The potential transmission routes of Trichinella spp. in pigs include canibalism, ingestion
of synanthropic and sylvatic animals as well as of the faeces of pigs that have been infected some 12 days earlier (Petrović et al., 2014).
According to Petrovic et al. (2014), life cycle of T. spiralis in Vojvodina region includes circulation
from domestic pigs to wild boars and vice versa, which is assotiated with specific behaviour of this
animal species. Wild boars are very tolerant to the presence of humans, often commingling with
domestic pigs on common pastures and have access to laystall and food waste. Improper disposal of
pig carcasses and offals in the field, is the greatest risk factor for trichinellosis maintenance and
spread within pig population.
The diaphragms of all killed wild boars must be examined by artificial digestion method, for
estimating the presence of Trihinela spp larvae. This is the most effective measure for the control of
meat safety when speaking of this pathogen. However, it should be emphasized that appropriate
disposal of meat originating from infected animals represents an important step in preventing
further spreading of trichinellosis.
Salmonella spp.
Salmonella has long been recognised as an important zoonotic pathogen of economic significance
in animals and humans. Human salmonellosis is usually characterised by the acute onset of fever,
abdominal pain, nausea and sometimes vomiting. Symptoms are often mild and most infections are
self-limiting, lasting a few days. The common reservoir of Salmonella is the intestinal tract of a
wide range of domestic and wild animals, which may result in a variety of foodstuffs of both animal
and plant origin becoming contaminated with faecal organisms either directly or indirectly (EFSA,
2013).
Finding of Salmonella spp in wild boars has traditionally been associated with S. Typhimurium;
however, in the past few decades, the spectrum of serotypes isolated from carcasses, tonsils, faeces
and lymph nodes is much more diverse. Great differences in the prevalence of Salmonella spp. in
game were reported between individual species (e.g., the rates are higher in wild boars than in
ruminants) as well as between particular regions (e.g., higher prevalence rates were recorded in
southern countries of the EU) (Table 1). According to data reported by EU MSs in the framework
of the Zoonoses Directive (2003/99/EC) in 2004–2011, 1.1 % of deer, 11.1 % of reindeer, 18.3 % of
wild boar, 1.8 % of ostrich and 2 % of rabbit faecal samples were positive for this organism.
Salmonella was therefore shortlisted for risk ranking (EFSA, 2013)
Investigation of wild game pathogens in our country were mainly aimed at wild birds and other
enteropathogens (Stojanov et al., 2012; Velhner et al., 2012). The prevalence of salmonellas on wild
boar carcasses is relatively low, i.e. below 10% (unpublished data). Wild boars are more frequently
the carriers of Salmonella spp. than wild ruminants. Thus, an inadequate evisceration and/or bad
shot (e.g. shooting wound in abdominal region) increases the risk of meat contamination with
Salmonella spp. (Wisniewski, 2001).
Table 1. Prevalence of Salmonella spp. in wild boars in some European countries (Paulsen et al., 2012)
Animal species and sample
Wild boars, faeces
Country
Number of samples
Number of positive
samples
Italy
2365
441 (18.7%)
Portugal
Switzerland
77
73
17 (22.1%)
4 (5.5%)
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There are few practical options for eradication of Salmonella and other zoonotic diseases in
wildlife, with the possible exception of vaccination or culling in geographically isolated areas.
Regulating animal density (to avoid crowding and overabundance), can contribute to disease
control; also, when game is eviscerated on the spot, the offal should be removed from wildlife
(Gortazar et al., 2006). However, there are no records indicating that such measures would have
successfully eradicated Salmonella in the wild.
In actual scientiffic literature there are no published recommendations (at least to our knowledge)
on Salmonella spp., control measures in live wild boars. Therefore it is important to control hygiene
during hunting, evisceration, bleeding and cleaning of carcasses, cooling and transportation,
coupled with sampling of processed carcasses for Salmonella spp. presence.
Toxoplasma gondii
T. gondii infection is common in animals and humans. T. gondii is an obligate intracellular
protozoan parasite. Nearly all warm-blooded animals can act as intermediate hosts, and almost all
animals may be carriers of tissue cysts of this parasite. However, the parasite matures only in
domestic and wild cats, which are the definitive hosts (EFSA, 2013). In pregnant women, the
parasite can cause congenital infections resulting in abortion, stillbirth, mortality and hydrocephalus
in newborns. The parasite can also cause severe disease in immune compromised individuals such
as organ graft recipients and individuals with AIDS or cancer (EFSA, 2013). In immune-competent
individuals, 80–90 % of cases of T. gondii infection are asymptomatic and the majority of the
remainder result in only mild, self-limiting symptoms.
Common infection routes for wild boars include ingestion of oocysts from the environment,
ingestion of infected rodents and birds or cannibalism (Tenter et al., 2000). An interesting
observation was reported by Dubey et al (1992) suggesting significantly higher seroprevalence of
Toxoplasma among pigs raised outdoor than in those reared in conventional settings. The natural
production system is conducive for exposure of the pigs to various known risk factors for
Toxoplasma infestations such as cats (the definitive host) and other species that can be harbouring
cysts in their musculature as compared to the indoor conventional production system. In addition,
the outdoor environment with open access to soil, vegetation, and moisture allows viable
environments for Toxoplasma oocysts. The similar trend of higher seroprevalence of Toxoplasma
and Trichinella in outdoor-reared swine was reported in the Netherlands (van der Giessen et al.,
2007). The frequency distribution of Toxoplasma seroprevalence reported in Dubey et al (1992)
study ranged from 0.38% in intensive production to 5.62% in outdoor-reared system.
T. gondii is common in hunted wild boars in EU, where the seroprevalence has been reported to
vary between 8% and 38% (Lutz, 1997; Gauss et al., 2005; Antolova et al., 2007). The
seroprevalence in farmed wild boar has been reported to be 33 % (EFSA, 2007). Given the high
incidence in wild boar, this organism is significant hazard for wild boar meat safety.
Toxoplasmosis in animal commonly takes an asymptomatic course, which makes the diagnostics in
living animals impossible. To detect T. gondii infection in livestock, serologic assays can be
applied. With the exception of cattle, the presence of antibodies and tissue cysts is assumed to
correlate well. Meat or tissue samples can be tested by bioassay or PCR. T. gondii oocysts cannot be
differentiated from Hammondia or Neospora oocysts morphologically, thus molecular techniques
need to be applied. If infected meat is consumed without prior freezing or proper heating (core
temperature over 67 °C) T. gondii can be transmitted. Salting, fermenting, drying, and smoking also
reduce tissue cyst viability, but the exact conditions needed to inactivate T. gondii are less wellestablished (Opsteegh et al., 2013).
110
Alaria alata
Alaria alata is a trematode parasite, and the transmission of this parasite occurs when humans eat
undercooked game or frog meat infected with the mesocercarial stage of this parasite. The
epidemiology of Alaria infection is not well-understood (Moehl et al., 2009a). The reported cases of
human larval alariosis are most likely due to mesocercariae from Alaria species other than A.
americana, but primates can be infested by A. americana (Moehl et al., 2009b). A study in
Germany (Riehn et al., 2012) found a high prevalence of A. alata in wild boar (11.5%). Although
specific methods for targeted detection of A. alata are available, its diagnosis is feasible during the
official Trichinella inspection in the competent veterinary inspection offices. Recent studies
conducted in the eastern parts of Austria indicated an overall prevalence of A. alata mesocercariae
in wild boar of 2%, when lean muscle (M. masseter) tissue was tested (Sailer et al., 2012) or 6.7%,
when a muscle – fat tissue mixed sample was tested (Paulsen et al., 2012).
Jakšić et al. (2002) and Grosse and Wüste (2006) pointed out that the parasite represents a potential
source of infection for both humans and animals, and that consumption of wild boar meat can be an
important factor in the epidemiology of this zoonosis (Moehl et al., 2009b). However, to date, there
has been no report on human alariosis cases due to consumption of wild boar meat and thus Aaria
alata was ranked as hazard with low priority.
Chemical hazards
EFSA (2013) ranked chemical residues and contaminants on the basis of bioaccumulation,
toxicological profile and likelihood of occurrence, and they took into account the findings from the
NRCPs for the period 2005–2010. The ranking results were as following:
- No substances were classified in the high potential concern category for game.
- Within the category of medium potential concern for farmed game is cadmium.
- All other substances listed in Council Directive 96/23/EC was ranked as being of low or
negligible potential concern
- Potentially higher exposure of consumers to these substances from game meat takes place
only incidentally, as a result of mistakes or non-compliance with known and regulated
procedures.
The analysis of the results of the microelements and heavy metals in wild boar meat in Serbia (non
published data), has shown that no samples exceeded legal limits, but our results suggest that
pollution of the biosphere with chemical contaminants should be systematically monitored to
identify potential increasing contamination tendencies.
Conclusion
The absence of characteristic clinical picture is common to all aforementioned hazards, thus a
thorough post mortem examination for the presence of tuberculosis and artificial digestion to
confirm the presence of Trichinella but also Alarai alata is of outmost importance. However, some
hazards are not detectable by these procedures (Salmonella, Toxoplasma, chemical hazards) and
designing and implementation of appropriate hazard control programs is necessary.
The presence of alimentary pathogens and contamination of boar meat with chemical hazards in
Serbia has not yet been fully elucidated. While relevant data on Trichinella spp are available and
the mechanisms of the maintenance of its life cycle in our region are well known, the data on other
hazards are still unclear. A number of ongoing research-scientific projects in our country address
the prevalence of selected hazards among wildlife population with an aim of obtaining
111
scientifically-based risk analysis as a corner-stone for implementing appropriate programmes and
measures for the control of alimentary hazards. The programme for control and monitoring of wild
boar meat should encompass control measures for live animals, control measures during and after
hunting, guidelines for veterinary inspection of meat, control measures for carcass processing and
monitoring over chemical residues.
Acknowledgement
This work was supported by the Ministry of Science and Technological Development of the
Republic of Serbia, grants TR 31084.
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Invited lecture
THE DETERMINATION OF THE QUALITY IN CEREAL SILAGES SUPPLEMENTED
WITH PUMPKIN AND CARROT
Milica Zivkov-Balos1, Sandra Jakšić1, Milovan Jovicin1
1
Scientific Vetereinary Institute „Novi Sad“, Novi Sad Republic of Serbia
* Corresponding author: milica@niv.ns.ac.rs
Abstract
The aim of this work was to evaluate the nutritional composition of silages based on the cereals
supplemented with carrot as well as pumpkin. For the ensilage, the following combinations of raw
materials were used: whole corn plant and carrot, oat grits and carrot and corn grits and pumpkin.
Determination of moisture, fat, crude fiber, ash, calcium and phosphorus in the samples was
performed by standard methods, while the protein was analyzed by measuring total nitrogen by total
combustion (according to Dumas). Content of mineral matters was measured applying the atomic
absorption spectrometry. The results obtained from this research reveal that cereals silages
supplemented with pumpkin and carrot can be considered a good source of nutrients.
Key words: silages, cereals, pumpkin, carrot, dairy cows
Introduction
Plant species that are suitable for silage have a higher dry matter yield in the field and higher
digestibility, low buffer capacity and a higher amount of water-soluble carbohydrates. Whole plant
maize silage is used worldwide in the rations for cattle. However, β-carotene content in maize
silage, which is a popular main feed for dairy cows, is very low. The majority of feeds for dairy
cows contain low levels of β-carotene (Nozière et al. 2006). The contents of β-carotene in grass
vary depending on the plant species and growth stage. The highest levels of β-carotene in pasture
grass, maize, alfalfa, etc. are found in young plants and their content decreases with plant ageing.
Plant carotenoids are transferred to the products of animal origin with the different absorption rates.
Thus, egg yolk demonstrates high absorption rate, whereas carotenoids transfer is somewhat less
pronounced in ruminants and affects the colour of milk and dairy products and body fat as well.
Consumers mostly assess the initial quality of animal products according to its colour, though their
preferences regarding the colour and thus the acceptability of milk and dairy products differ
between the countries and even between the regions in one country. Yellow colour of milk and
dairy products is often associated with grass-feeding systems, which are considered „natural“ in
most countries. Thus, the carotenoids content in milk can be considered an indicator of pasturebased farming (Nozière et al., 2006).
The only „natural“ source of vitamin A for ruminants is that occurring by β-carotene cleavage and
its absorption by enterocytes and, to some lesser extent, as the product of liver metabolism. To that
end, adequate β-carotene content in the colostrum is a prerequisite for providing adequate levels of
vitamin A (β-carotene). Furthermore, its absorption by the enterocytes is of vital importance for calf
development (Kaelawmun et al., 2011). Peripartal period in dairy cows is highly stressful period
associated with intensive physiological changes and metabolic adaptation during pregnancy period
and lactation. The energy requirements increase because of accelerated foetal growth and milk
production. This period is characterized by increased immunosuppression and thus higher
susceptibility to disease. Metabolic changes are associated with rapid foetal growth, parturition and
115
inducing of lactation, which results in increased generation of free radicals. When the production of
free radicals exceeds the antioxidant defence mechanisms of the body, the cow is in oxidative stress
(Kaelawmun et al., 2011). Oxidative stress is an important factor contributing to disease
susceptibility. β-carotene as an antioxidant can affect the functionality of host’s immune system
through its positive effects on membrane fluidity (Chew and Park, (2004); Tjoelker et al., (1988,
1990)). Chawla and Kaur (2004) reported that β-carotene supplementation to cows in drying off
period is required to improve the antioxidative plasma status and overall health status post partum,
to improve the milk production and quality, and to reduce the incidence of mammary infections.
Kawashima et al. (2010) reported that β-carotene supply improved embryo production and quality
in super ovulated cows. Among the role of retinol in ruminants, as in other mammals, its influence
on reproduction has been extensively studied. Retinol deficiency may reduce reproductive
efficiency in dairy cows, especially through impaired ovarian function and increased incidence of
abortion (Hurley and Done, 1989). Retinol is also involved in various functions, such as vision,
growth and male fertility. In addition, the conversion rate of β-carotene to vitamin A in granulose
cells has been enhanced by follicular growth, and intrafollicular concentrations of vitamin A
correlated positively with those of estradiol and follicle diameter (Schweigert et al., 1987).
Some crops, which are used as feed alternative for domestic animals, are rich sources of this
vitamin precursor. In our region, carrot and pumpkin can be considered alternative cultures and
good sources of vitamin A and β-carotene for cattle. The use of agricultural residues and surplus
products as well as alternative crops is often a useful way of overcoming the shortage of animal
feedstuffs and has been intensively addressed by some recent researches and projects worldwide.
Agricultural surplus, low purchase price of agricultural products and inability of selling the products
on the market result in the disposal and loss of substantial amounts of agricultural products.
Carrot is good source of carotene, but it conteins also xanthophyll, though in some lesser amounts.
At the food market, the carrots of poor quality (some 22% of total production) are returned to the
producers mainly because of undersize, deformity, breakage or root diseases/infection. The rejected
crops that are returned to the supplier are considered goods of poor economic value and are used
rather as animal feed than the source of carotenoids. Potential way of using the health-nutritional
components of „rejected“ crops and thus increasing their economic value is their application in
organic egg production (Hammershøj et al., 2010). Carrots have become a common animal feed in
egg production in Danemark. In the U.S., carrot root has been used in cattle breeding as the winter
feed for dairy cows, in the production of yellow-colour butter and cream (Watson, 1994). Some
novel researches indicated that carrot supplementation to the diet of dairy cows increases the
content of vitamin A and lactic acids in milk (Nalecz-Tarwacka et al., 2003).
Beside for human consumption, pumpkin (especially some particular varieties) is used as animal
feed. Flashy and cellulose portions of pumpkin are one of the most important edible agricultural
residues (Razzaghzadeh et al., 2007). In the U.S., pumpkin varieties are grown as decorative plants
as well as for human nutrition. Pumpkins with spots or blemish as well as those broken or damaged
during harvesting are discarded as inacceptable for the market. Moreover, after October 31
(Halloween), the demand for decorative pumpkins decreases, thus substantial amount of the crops is
left in the fields. Some farmers use pumpkin fields for grazing (in combination with maize-stalk),
but in general, very little information is available on the nutritive value of pumpkin in cattle
nutrition. Some researches pointed out that pumpkin is good source of energy with adequate protein
content for the cattle (Jenkins, 2010).
It is to be emphasized that including of these alternative crops into the diet for dairy cows could
contribute to the increase of organic milk production in line with the basic principles of organic
production. Organic milk production is based on organic principles and objectives including
naturalness and recycling of nutrients. According to current regulations, the feed used in organic
milk production must be 100% organic-grown (EC, 1999). However, the integrity of organic
116
production could be further enhanced if frequent use of vitamin supplementation with artificial fatsoluble vitamins is substituted by vitamins from natural sources (Mogensen et al., 2012).
Preservation of alternative crops
Carrot and pumpkin are highly moist materials. Although these materials have nutritive value, they
are difficult to incorporate into a commercial feeding program because of preservation problems.
This means that these need to be treated for storage if they are to be made available as a feed
resource during lean season. Ensiling can be considered as a method for making use of wet raw
materials more effectively. Prior to silage making, drying and chopping are also required, which
makes the method somewhat expensive and cumbersome for farmers.
Silage making involves harvesting crops by mechanical harvester, usually with considerable
reduction of the size of particles, which accelerates the release of plant cell contents. The harvested
materials are placed in a silo, which is sealed to produce anaerobic conditions. The silo is opened
after several weeks or months and the silage is removed for use as feed. Preservation of ensiled
materials is achieved by the conversion of plant water-soluble carbohydrates – glucose, fructose,
fructans and sucrose to organic acids, principally lactic acid. There is also a variable degree of
degradation of proteins to amino acids and other nitrogenous compounds (Wilkinson, 1999).
The advantages of ensiling alternative crops and root crops include (Moran, 2005):
•
•
•
•
•
•
•
•
feeding is possible when such by-products are not being produced
increasing feed resources and an insurance for high nutrient demands, such as milking cows
reducing demands on home-grown forages
if low cost, reducing total feed costs
can improve their palatability
can reduce toxicity to safe levels (in vegetables)
can destroy harmful bacteria (in food by-products)
can constitute a major proportion of diets
Another aspect of using carrot and pumpkin in cattle nutrition that is to be emphasized is the fact
that using agricultural surplus products for animal feed is a specific recycling process, since such
surplus, when accumulated, can cause environment pollution and increased costs of waste disposal.
The basic aim of these researches was finding new options for balanced livestock diet avoiding the
competition with human nutrition, and thus changing the perception of economic value of food.
Using alternative crops as animal feed can provide competitive alternatives to traditional feed
sources and potentially reduce the expenses and impact on the environment. At the same time,
agricultural surplus products should be used with an aim of increasing the self-sufficiency and
stability of food supply. Such raw materials can be used not only as the source of nutrients for
ruminants but also as the substitute for commercial and imported feeds, which leads to further
savings in energy and transportation. Moreover, potential harmful environmental effects of waste
incineration, i.e., landfills for plant material surplus are greatly reduced.
The aforementioned strongly suggests the soundness and necessity of our research that was aimed at
alternative crops and agricultural surplus products from our region, which could be used as feed for
dairy cows and an alternative to synthetic vitamin A and β-carotene in organic milk production.
Materials and Methods
Material:
For ensilaging, the following combinations of raw materials were used:
117
1. Vitaminized silage of whole maize plant and carrot
Silage maize mass was 72 000 kg and carrot mass (whole root) was 3500 kg. The ensilaging was
performed in a trench silo using an inoculant (Start supplement for silage, Natura point, Novi Sad).
2. Vitaminized silage of carrot and oat grits
Carrots (14 kg) were chopped in small particles (2 cm), and then 12.5 kg of oat-grits were added
along with 1.5 l water. The ensiling was performed in a PVC barrel.
3. Vitaminized silage of maize grits and pumpkin, variety Ishicu Kuru (used for seed production)
was prepared in a trench silo. Pumpkin’s pulp (720 kg) was pulverized using a tractor chips and
mixed with 480 kg of maize grits. Silage was carried out in six plastic barrels of 200 L
Sampling procedure:
Silage samples were taken from the top, centre and bottom parts of the trench silo, i.e. the barrel.
The samples were collected into sterilized bags, kept in iceboxes and transported to the laboratory
for analysis.
Animal experiment:
Four cows in late pregnancy and four recently calved cows originating from the same dairy farm
were fed vitaminized silage of whole maize plant and carrot. Blood samples were collected before
and after silage supplementation to the diet.
Methods of chemical analysis:
Determination of moisture, crude fat, crude fibre, crude ash, calcium and phosphorus in the
samples was performed by standard methods (ISO 6496; 6492; 6865; 5984; 6490-2; 6491), while
crude protein was determined by measuring total nitrogen using total combustion according to
Dumas (EN ISO 16634-1) and applying Elementar Rapid N Analyzer. Content of β-carotene was
determined by spectrophometric method. Content of mineral matters (manganese, zinc, copper,
iron, magnesium, sodium) were determined using atomic absorption spectrophotometry applying
Varian SpectrAA-10. The serum content of β–carotene was determined using colorimetry.
Data on average chemical composition of the examined silage samples are displayed in Table 1.
According to the research of Horrocks and Vallentine (1999), the desirable content of dry matter
(DM) of maize plant for ensiling is 35%. This percentage provides an optimal proportion of starch,
which is the „carrier“ of energy value and soluble sugars essential in production of adequate
amounts of lactic acid. Moisture content in the examined samples ranged from 45.33% to 76.92%.
Reduced fermentation in carrot/oat grit silage is most probably due to the high content of dry
matter, which inhibits the microbial activity (Živkov-Baloš et al., 2013, 2014).
Table 1. Chemical composition of vitaminized silages (on dry matter basis, %) (Živkov-Baloš et al.,
2014)
Sample
Dry matter
Organic matter
Crude Protein
Crude fat
Crude fiber
Crude ash
(DM)
(OM)
(CP)
(EE)
(CF)
(CA)
1
27.58 ± 4.50
95.29 ± 4.44
9.32 ± 0.36
1.20 ± 0.23 23.86 ± 1.73 4.71 ± 0.06
2
53.02 ± 5.59
96.72 ± 5.12
13.45 ± 0.59
0.79 ± 0.30 10.49 ± 1.02 3.28 ± 0.65
3
55.13 ± 1.79
89.70 ± 3.60
8.11 ± 0.28
0.54 ± 0.02 8.98 ± 0.13 10.30 ± 1.82
Sample No: 1. whole corn plant and carrot; 2. oat grits and carrot; 3. corn grits and pumpkin; Results are
presented as means±SD.
118
The CP content was 2.29-2.84% on fresh matter basis (average 9.32% in DM) in silage of whole
maize plant and carrot, 7.13-7.52% (average 13.45% in DM) in silage of carrot and oat grits and
4.19-4.75% (average 8.11% in DM) in silage of maize grits and pumpkin (Table 1). Our results of
silage CP were in between the results of other authors. Demirel et al. (2011) noted that silages of 12
maize hybrids have CP content from 4.07-6.91% in DM. Kamalak et al. (2002) findings revealed
the content of CP from 8.5 to 9.6% for control and transgenic maize varieties. The effect of stage of
maturity on the ensiling properties of whole crop maize was studied under laboratory conditions.
The CP content decreased from 80 to 58 g/kg DM (Filya, 2004). Vranic et al. (2008) reported that
the CP content in samples of maize silage for dairy cows nutrition, was 61.6 g CP / kg DM. Similar
findings (68.72 g/kg DM) were reported by Đorđević et al. (2009). Carrots have 85-90% moisture
content and about 10% crude protein in dry matter (Rust and Buskirk, 2008). Carrot silage has 27.1
% of DM and 13.8% of CP (Laflamme, 1992). Content of proteins in pumpkins (carving and pie
pumpkins) is 14.3-14.4% in DM (Jenkins, 2010) and 16% (Davis et al., 2012).
The CF content in silages changed from 4.82 to 8.45% on fresh matter basis (8.98-23.86% in DM).
The highest content of CF has been established in vitaminized silage of whole maize plant and
carrot, and the lowest in vitaminized silage of maize grits and pumpkin. Our results are in
compliance with the results of other authors. Demirel et al. (2011) established CF content in maize
silages ranging between 17.96 and 27.28% DM. Đorđević et al. (2009) reported that the content of
CF in DM of whole plant maize silage is 17.65%, whereas Galila et al. (2012) reported the content
of CF in maize stalks, after the process of fermentation (that lasted 28 day), ranging between 24.54
and 27.20%. Carrot root contains 8.1-12.1% crude fibre in DM (Nonaka et al., 1994). The carrot
roots and carrot heads contain 9.1% and 18.1% of CF, respectively (Davis et al., 2012). Fresh
pumpkin (Cucurbita sp.) contains averagely 13.2% CF in DM (Enishi et al., 2004).
Ether extract (EE) ranged between 0.54% and 1.20% DM. Our silage results were lower than the
results of other authors. The maize silages EE content changed from 2.68% to 4.40% in DM
(Demirel et al., 2011). Djordjević et al. (2009) reported that EE content in dry matter of maize
silages is 8.76%. EE content in carrot is on average 1.0% (0.2-1.9%) in DM (Nonaka et al., 1994).
Fresh pumpkin contains about 2.8% of crude fat in DM (Enishi et al., 2004).
The CA content in silages changed from 3.28 to 10.30 % in DM. Highest ash content was measured
in the silage of whole maize plant and pumpkin as a consequence of higher proportion of maize
stalks in the silage. Fresh pumpkin contains about 2.8% of crude fat in DM (Enishi et al., 2004). CA
in whole plant maize silages changed from 4.64 to 7.94% in DM (Demirel et al., 2011). According
to the report of Djordjević et al. (2009), CA content in dry matter of maize silages is 5.04%. Filya
(2004) reported CA contents in maize silages in different stages of fermentation ranging 4.0213.57% in DM. The average CA content in carrot is 7.4% (3.1-10.4%) in DM (Nonaka et al., 1994).
Enishi et al. (2004) reported that fresh pumpkin contains about 7.9% CA in DM.
Calcium (Ca) and phosphorus (P) content in investigated silages is displayed in Table 2. The
chemical analysis revealed abundant amounts of these essential macro elements for animal nutrition
on DM basis ranging between 900-4300 mg/kg (Ca) and 1600-2200 mg/kg (P). The highest content
of Ca was measured in vitaminized silage made of whole maize plant and carrot. Calcium content
in whole maize plant is 800 mg Ca/kg DM (Galila et al., 2012). The average calcium and
phosphorus contents in fresh carrot roots are 3800 mg/kg and 2900 mg/kg DM, respectively
(Nonaka et al., 1994). Idi et al. (2005) reported Ca and P contents in dry matter of carrot of 0.36%
and 0.26%, respectively. The pumpkin Ca and P contents are averagely 3900 mg/kg and 2600
mg/kg in DM, respectively (Enishi et al., 2004). Hashemi and Razzaghzadeh (2007) reported Ca
and P contents in dry matter of silages from Cucurbit residues of 0.94% and 0.09%, respectively.
The DM mineral content of our silages was within the range of average values reported by other
authors. The variability in the contents of mineral elements in plants results from a number of
119
factors, such as plant species, soil properties and application of agro technical measures (ŽivkovBaloš et al., 1999). Macro- and microelements analyses of soils, feed (alfalfa and maize) and food
(wheat) indicated a high variability in the contents of these elements between some locations in
Vojvodina Province (Živkov-Baloš et al., 2000, 2011).
Table2. Mineral content of vitaminized silages (on dry matter basis)
Mineral,
unit of measure
1
Type of vitaminized silage
2
3
Calcium, %
0.36-0.43
0.09-0.13
0.13-0.16
Phosphorus, %
0.18-0.22
0.21-0.26
0.14-0.18
Manganese, mg/kg
47.1-61.3
2.1-10.3
7.6-8.9
Zinc, mg/kg
22.8-24.3
18.7-20.4
15.4-19.8
Coper, mg/kg
30.8-31.2
7.2-7.9
4.5-21.6
Iron, mg/kg
126.9-147.6
70.5-95.1
99.8-111.2
Magnesium, mg/kg
2175-3912
754.-802
805-898
Natrium, mg/kg
798-1131
660-753
3283-17187
Type of vitaminized silage: 1. whole corn plant and carrot; 2. oat grits and carrot; 3. corn grits and pumpkin;
Results are presented as interval of variation
The highest manganese (Mn) levels were measured in the samples of maize silage (whole plant) and
carrot, being averagely 54.2 mg/kg of DM. This is significantly higher value as compared to that
reported by other authors. Thus, Demirel et al. (2011) reported Mn contents in samples of diverse
maize hybrids silage ranging from 4.09 to 6.16 mg/kg of DM. Blackwood (2007) stated that silage
made of whole plant maize contained 34.0 mg/kg Mn in DM. The content of Mn in carrot ranges
between 34.4 mg/kg DM in the root and 198 mg/kg in leaves (Intawongse and Dean, 2006). The
results of their investigation showed that uptake of some microelements by plants corresponded to
the increasing level of soil contamination, and that soil-to-plant transfer factor values decreased
from Mn>>Zn>Cd>Cu>Pb. Higher manganese contents in our samples are most probably due to
the high soil Mn levels. The highest zinc (Zn) levels were determined also in silages based on whole
maize plant and carrot. Our results on zinc levels correspond with the results of other authors
(Demirel et al., 2011; Intawongse and Dean, 2006). Iron contents in the examined samples of
vitaminized silages were within the range of available literature data. Vitaminized silages,
particularly those based on pumpkin pulp and maize grout, are very rich in sodium and magnesium.
The most important attribute of these silages is their richness in vitamins and β-carotene. The
average content of this provitamin in maize and carrot based silages was about 386.60 mg/kg.
According to the data from the literature, the level of β-carotene in maize ranges from 24 to 35
mg/kg, whereas average β-carotene contents in green grass, dry forages, silages and hay are 196,
159, 81 and 36 mg/kg, respectively (Noziere et al., 2006).
Total 8 sera of cows from two production groups, i.e., cows in late pregnancy and recently calved,
were tested before and during feeding vitaminized silage made of maize (whole plant) and carrot
(Table 3.).
120
Elevated contents of β-carotene were measured in the blood of all cows fed vitaminized silage
based on maize and carrot. Such high levels of β-carotene in vitaminized silage, that is, blood sera
of the cows, contributed to improved production results and significantly reduced administration of
veterinary drugs.
Table 3. The content of β-carotene in blood serum before and during feeding vitaminized silage
Characteristic
Reference value
Sample No.
1.
2.
3.
4.
n=4
x
 SD=
x
 SD=
Sample No.
1.
2.
3.
4.
n=4
Content of β-carotene
winter 2.79-9.30; summer 9.30-27.90
The values in samples before feeding
vitaminized silage
7.22
8.23
7.59
7.98
7.76  0.44
The values in samples during feeding
vitaminized silage
36.83↑
28.64↑
42.59↑
36.07↑
36.03  7.01
Conclusions
Vitaminized silages based on grains, carrot and pumpkin that were used in the present experiment
were assessed to be of good quality. With regard to their nutritional composition, the investigated
vitaminized silages are potential resource to be used in bovine diets. Vitaminized silages based on
grains, carrot and pumpkin ensure good results in cattle reproduction and better milk yield. The
entire technological process is designed to provide highest level of preserving the quality and
nutritive properties of feed. The selected alternative crops are widely grown in the territory of
Serbia and do not require any substantial economic investments, which guarantees a sustainable
production of safe organic food of animal origin and satisfactory production outcomes without
significantly affecting the farming technology.
Acknowledgments
The research was financially supported by the Ministry of Education, Science and Technological
Development of the Republic of Serbia (Project No TR31071)
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Invited lecture
MYCOTOXICOLOGICAL ASSESSMENT OF FEED IN SERBIA IN 2014 IN THE LIGHT
OF NEW LEGISLATION
Ksenija Nešić1*, Sandra Jakšić2, Milica Živkov-Baloš2, Bojana Prunić2
1
2
Institute of Veterinary Medicine of Serbia, Belgrade, Serbia
Scientific Veterinary Institute “Novi Sad”, Novi Sad, Serbia
* Corresponding author: ksenija_n@yahoo.com
Abstract
Under favorable environmental conditions, when temperature and moisture are suitable, fungi
proliferate and may produce secondary metabolites well known as mycotoxins. They commonly
enter the food chain through contaminated food and feed crops, mainly cereals, which get infested
prior to and during harvest, or during (improper) storage. But neither all molds are toxigenic, nor all
secondary metabolites are toxic. Therefore, although more than 300 mycotoxins have been isolated
and chemically characterized, worldwide research has focused on those forms causing significant
injuries to humans and animals. There is a small number of toxins that are of practical relevance, as
well as small number that has been officially regulated. European Union and Serbian regulations
treat only several mycotoxins by giving maximal permitted limits in feed and food. Regarding feed,
recently revised directive was published in Serbia in March 2014.
The aim of this paper was to present the mycotoxicological assessment of feed in Serbia in 2014 in
the light of this newly established regulation. Here shown results indicate that mycotoxins are
present more than they used to be, so more attention has to be paid to this problem, especially
keeping on mind their global impact on food safety, with possible serious effects on animal and
human health. So there is strong need for preventive measures, as well as regular and
comprehensive monitoring of feed as important step for struggle and control of these natural
contaminants.
Keywords: feed safety, monitoring, mycotoxins, regulations
Introduction
Mycotoxins have globally significant human and animal health, economic and international trade
implications (Bryden, 2012). The supply of meat, milk and eggs, the human food of animal origin,
can be adversely affected by mycotoxins. This is followed by the impact on animal health and
production (Shier et al, 2005).
Fungi are ubiquitous and all feedstuffs can be contaminated with mycotoxins. Formation of
mycotoxins is not restricted to any component of the animal feed supply chain, but the level of
contamination varies with location and reflects different agronomic practices and climatic
conditions, which dictate the fungi that are present in a farming system (Bryden, 2009).
The fungal species most often encountered with intoxications belong primarily to genera
Aspergillus, Fusarium and Penicillium. These moulds produce many different toxic compounds but
not all isolates of the same species produce toxins (Cole et al, 2003; Brase et al, 2009). The major
toxins produced by these three genera include: aflatoxins, ochratoxins, trichothecenes, fumonisins
and zearalenone.
Fungi are a normal part of the microflora of standing crops and stored feeds, but the production of
mycotoxins depends upon the fungi present, agronomic practices, the composition of the
commodity and the conditions of harvesting, handling and storage (Bryden, 2009). The amount of
124
toxin produced will depend on physical factors (moisture, relative humidity, temperature and
mechanical damage), chemical factors (carbon dioxide, oxygen, composition of substrate, pesticide
and fungicides), and biological factors (plant variety, stress, insects, spore load).
The accumulation of mycotoxins, both before and after harvest, largely reflects climatic conditions.
Fusarium toxins are produced in cereal grains during high moisture conditions around harvest
(Nesic et al, 2014), whereas pre-harvest aflatoxin contamination of crops is associated with high
temperatures, insect damage and prolonged drought conditions. Moreover, because Aspergillus can
tolerate lower water activity than Fusarium, it is more likely to contaminate commodities both preand post-harvest, whereas Fusarium is more likely to be found as a contaminant pre-harvest
(Bryden, 2009). These examples demonstrate that although it may be convenient to describe fungi
as either pre- and post-harvest organisms, the actual colonisation and proliferation of fungi is not
clear cut but depends on the environmental and ecological circumstances and the resulting toxins
will differ accordingly.
Stored grain is not static as well. It is in a dynamic state and may become infested with fungi and
insects. These interrelationships are affected by climatic factors such as temperature and humidity,
by geographical location, by the type of storage container and by grain handling and transport.
Moisture depends mostly on water content at harvest, the amount of drying, aerating, and turning of
the grain before or during storage as well as the respiration of insects and microorganisms in the
stored grain. If provided grain is dry when placed in storage, moisture content can only rise from
leaks or condensation. Grain may go into storage at a uniform temperature but over a period the
grain mass will cool at a different rate in the centre than at the periphery. As a result of temperature
differentials moisture migrates through the storage bin, resulting in condensation and the provision
of ideal conditions for mould growth or the development of ‘hot spots’ in localised areas. Microbial
and insect growth in stored grain also results in moisture condensation and the potential
development of ‘hot spots’. The minimum critical levels for the growth of fungi are 70–150 g/kg
moisture (depending on commodity) and 80–85% relative humidity. Temperatures at which toxin
production can take place vary from 0◦ C to 35◦ C, depending on fungal species. Most mycotoxins
are very stable chemically and once formed in a feedstuff will continue to contaminate that
commodity and feeds manufactured from it (Bryden, 2012).
However, the major problem associated with mycotoxin contamination of the animal feed supply
chain is not acute disease episodes but reduced animal productivity. Low level toxin ingestion may
cause an array of metabolic disturbances which may or may not be accompanied by pathological
change (Haschek et al, 2002). One of the first indications of a chronic mycotoxicosis is growth
depression, which may result from reduced feed intake, impaired nutrient utilisation, changes in
feed quality or toxicity per se. Many studies have demonstrated that mycotoxin ingestion reduced
feed conversion efficiency and may reflect impaired nutrient utilisation. The perturbations of
metabolism that occur following mycotoxin ingestion may affect reproductive efficiency of both
males and females (Nesic et al, 2008) and in the pregnant animal, mycotoxins may disturb
embryonic and foetal development. Mycotoxins are immunomodulators, mostly
immunosuppressive and it has been shown they increase the susceptibility of animals to infectious
disease (CAST, 2003).
It is important to know the fate of mycotoxins after ingestion, as contamination of animal products
with mycotoxins or their metabolic products has significant public health implications. In this
regard, transfer of aflatoxin into milk and ochratoxin A into meat have been the issues of most
concern. Available evidence suggests that tissue accumulation of aflatoxin or its metabolites is very
low and that residues are excreted in a few days. Animals are effective toxin eliminators with milk,
the animal product most likely to contain aflatoxin residues. The hydroxylated metabolite of
aflatoxins B1, aflatoxins M1 is excreted into milk from 1 to 6% of dietary intake (Fink-Gremmels,
2008).
125
Ochratoxin A has been detected in blood, kidneys, liver and muscle tissue from slaughtered pigs in
several European countries. There are significant differences among pig and poultry tissue
deposition studies and this is presumably due to differences in absorption and metabolism of the
toxin. It has been shown that the half-life of ochratoxin A in pigs and chickens is 180–140 h and
approximately 4 h, respectively (Petterrson, 2004).
Residues of other mycotoxins including zearalenone, trichothecenes and fumonisins are not
considered to be of public health importance as only very low levels of the toxins have been found
in the tissues of animals that had been fed very high levels of the toxins in experimental situations
(Petterrson, 2004). However, caution should be exercised when extrapolating or predicting tissue
residues as there is presently insufficient data on which to anticipate the outcome of any field
toxicosis. There are some reports of synergistic effects of different mycotoxins which suggest that
contamination of e.g. dairy rations with other mycotoxins, in particular ochratoxin, may decrease
the excretion of aflatoxin B1 by dairy cattle (Bryden, 2012).
Keeping on mind worldwide impact of these natural contaminants on feed and food safety, with
possible serious effects on animal and human health, the aim of this paper was to present the
mycotoxicological assessment of feed in Serbia in 2014 in the light of actual legislation and to
highlight the need for greater attention focused on the prevention.
Meterial and Methods
Monitoring of the presence of mycotoxins was done performing 1315 analysis of feedstuffs and
complete feed for different animal species produced in Serbia during 2014. The contents of
aflatoxin B1, ochratoxin A, zearalenone, deoxynivalenol and T2 toxin in feed samples were
determined in the Institute of Veterinary Medicine of Serbia in Belgrade and in the Scientific
Veterinary Institute Novi Sad. Both institutions perform ISO17025 accredited methods, using
commercial ELISA tests produced by Neogen®, USA (Veratox HS for Aflatoxin B1, product
8031B; Veratox for Ochratoxin, product 8610; Veratox for Zearalenone, product 8110; Veratox for
DON, product 8331NE; Veratox for T-2/HT-2 toxin, product 8230, with detection limits: 1.0 µg/kg,
2 µg/kg, 25 µg/kg, 250 µg/kg and 25 µg/kg, respectively) and R-Biopharm®, Germany (Ridascreen
AflatoxinB1 30/15, Art. No. R1211; Ridascreen FAST Ochatoxin A, Art.No. R5402; Ridascreen
FAST Zearalenon, Art. No. R5502; Ridascreen FAST DON SC Art. No. 5905 and Ridascreen
FAST T-2 toxin, Art.No. R5302, with detection limits: 1.0 µg/kg, 5 µg/kg, 60 µg/kg, 74 µg/kg and
33 µg/kg, respectively). Results were compared and interpreted according to new Serbian regulation
(Sl.glasnik RS, 27/2014).
Results
Results of mycotoxicological examination of feed samples during the year 2014 showed low
presence of ochratoxin A (Table 1), as well as T-2/HT-2 toxin (Table 2).
While 25.4 % of 138 analysed samples were slightly positive, detected concentrations of ochratoxin
A were far below maximum permitted levels given in Serbian directive (Sl.glasnik RS, 27/2014),
which is more strict compared with the previous one (Sl.glasnik RS, 4/2010). Most of the values
were below the detection limits of the used ELISA protocols. The highest concentration of
ochratoxin was detected in the mixture for poultry and it was 0.0275 mg/kg.
Regarding T-2/HT-2 detection no comment could be given in respect of new regulation (Sl.glasnik
RS, 27/2014), as no limits are proposed here. But directive from 2010 (Sl.glasnik RS, 4/2010), that
is not valid since March 2014, prescribed levels of 1.0 mg/kg for adult pigs and poultry and 0.5
126
mg/kg for young animals. Both were high above determined concentrations. Maximal content of
0.143 mg/kg among 169 samples was discovered in feed for pigs.
With regard to aflatoxin crisis in 2013, special attention was paid to the presence of this group of
toxins. Therefore, the largest number of samples were examined, a total of 668. Overall results
(Table 3) indicated that aflatoxin was still there in 59.1 % of performed analysis with 5.1 % of
samples that exceeded limits permitted by now actual legislation (Sl.glasnik RS, 27/2014). The
largest content was quantified in the corn sample (0.0322 mg/kg) which was above regulated limit
of 0.03 mg/kg, but which would be tolerable according to previous permitted limit of 0.05 mg/kg
(Sl.glasnik RS, 4/2010). Generally, by the adoption of new Directive more stringent criteria for
allowable levels of aflatoxin were introduced, thereby specifying limits for B1 from group of
aflatoxins. During the whole year, decreasing trend of recorded concentrations of aflatoxin B1 was
noted, with very few positives at the end of the year.
Table 1. Ochratoxin A in feed samples
Type of feed
Number of
samples
Positives
[%]
Poultry
Pigs
Cattle
Corn
Silage
Sunflower meal
Wheat
OVERALL
27
62
4
39
3
1
2
138
29.6
30.7
0
20.5
0
0
0
25.4
Range of
concentrations
[mg/kg]
0.0037 – 0.0275
0.003 – 0.0182
< 0.002
0.0052 – 0.0153
< 0.002
< 0.002
< 0.002
0.003 – 0.0275
Mean value
[mg/kg]
0.0084
0.0096
< 0.002
0.0082
< 0.002
< 0.002
< 0.002
0.0087
Above
permitted
limits [%]
0
0
0
0
0
0
0
0
Maximum
permitted
level [mg/kg]
0.2
0.1
0.25
0.25
0.25
0.25
-
Table 2. T-2/HT-2 toxin in feed samples
T 2 toksin
Number of
samples
Positives
[%]
Poultry
Pigs
Cattle
Corn
Wheat
OVERALL
38
77
5
48
1
169
39.5
26.0
20.0
18.8
0
26.6
Range of
concentrations
[mg/kg]
0.029 – 0.095
0.026 – 0.143
0.060
0.039 – 0.098
< 0.025
0.026 – 0.143
Mean value
[mg/kg]
0.0514
0.0546
0.060
0.0681
< 0.025
0.0585
Above
permitted
limits [%]
-
Maximum
permitted
level [mg/kg]
-
But, examining feed throughout 2014, while the situation with aflatoxin B1 turned down, the results
related to Fusarium toxins, zearalenone and DON, were changing and becoming more complex.
Over time more positive samples were found.
Results of zearalenone analysis are presented in Table 4. Among 165 samples in 51.5 % of them
certain concentrations were detected. The highest level of 2.389 mg/kg was found in corn, but there
were 4.8 % of samples with contamination above permitted level and all of them were for feeding
pigs.
Deoxynivalenol (Table 5.) was found in the highest percentage of analysed samples (64 %) and
11.4 % of them were highly contaminated, more than it was allowed by regulation (Sl. glasnik RS,
27/2014). As the most sensitive species, pigs were strongly affected and the largest mean value was
determined in feed for those animals (1.159 mg/kg), as well as the biggest number of inadequate
samples (27.3 %). Maximal detected concentration was found in the corn sample (12.242 mg/kg)
and 76 % of them were found positive, while all samples of wheat were also contaminated.
127
Table 3. Aflatoxin B1 in feed samples
Type of feed
Number of
samples
Positives
[%]
Range of
concentrations
[mg/kg]
Mean value
[mg/kg]
Above
permitted
limits [%]
Poultry
189
73.0
0.0011 – 0.030
0.0037
7.9
Pigs
190
58.4
0.0011 – 0.024
0.0060
6.8
Cattle
103
59.2
0.0010 – 0.0082 0.0034
1.9
Lambs
23
30.4
0.0011 – 0.0072 0.0046
8.7
Premix and
39
71.8
0.0011 – 0.013
0.0038
0
additional mix
Corn
100
37.0
0.0010 – 0.0322 0.0065
2
Silage
14
71.4
0.0011 – 0.0013 0.0040
0
Sunflower
8
15.0
0.0020 – 0.0016 0.0063
0
meal
Wheat
2
50.0
0.0050
0.0050
0
OVERALL
668
59.1
0.0010 – 0.0322 0.0048
5.1
*Double permitted level: for young animals (lower level) and adult animals (higher level)
Maximum
permitted
level
[mg/kg]
0.005-0.02*
0.005-0.02*
0.005-0.02*
0.005
0.01
0.03
0.03
0.03
0.03
-
Table 4. Zearalenone in feed samples
Type of feed
Number of
samples
Positives
[%]
Range of
concentrations
[mg/kg]
Mean value
[mg/kg]
Above
permitted
limits [%]
Poultry
14
28.6
0.046 – 0.249
0.145
Pigs
77
48.1
0.031 – 0.574
0.168
10.4
Cattle
9
77.8
0.067 – 0.593
0.258
0
Corn
52
53.9
0.063 – 2.389
0.309
0
Silage
8
100.0
0.062 – 1.542
0.494
0
Sunflower
2
0
< 0.025
< 0.025
0
meal
Wheat
3
33.3
0.083
0.083
0
OVERALL
165
51.5
0.031 – 2.389
0.243
4.8
Maximum
permitted
level
[mg/kg]
0.2-0.5*
1
6
6
4
4
-
Table 5. Deoxynivalenol (DON) in feed samples
Type of feed
Number of
samples
Positives
[%]
Range of
concentrations
[mg/kg]
Mean value
[mg/kg]
Above
permitted
limits [%]
Poultry
32
53.1
0.079 – 1.770
0.931
0
Pigs
66
63.6
0.079 – 5.852
1.159
27.3
Cattle
5
40.0
0.290 – 1.47
0.88
0
Corn
50
76.0
0.091 – 12.242
1.792
2
Silage
12
66.7
0.170 – 10.377
3.980
8.3
Sunflower
5
0
< 0.074
< 0.074
0
meal
Wheat
5
100.0
0.370 – 3.612
1.9
0
OVERALL
175
64.0
0.079 – 12.242
1.774
11.4
128
Maximum
permitted
level
[mg/kg]
5
0.9
2-5*
12
12
8
8
-
Discussion
Taking into consideration all these collected data, one fact cannot be neglected: mycotoxins are
more present than they used to be. In Serbia during previous years rare or strictly experimental
reports were given regarding this topic, which stayed captured in professional circles until the
attention of broad public was drawn to the case of aflatoxin in 2013, solely because of its presence
in milk (Kos et al, 2014). Also, in terms of aflatoxin presence in feed, situation was quite different
and very unlike years ago (Jakic-Dimic et al, 2010; Jakic-Dimic and Nesic, 2011). Most of the
analyses presented by Nesic and Pavlovic in early 2013, showed in 75 % of them significant
contamination, while as many as 35 % exceeded the maximum permitted levels, so did not
correspond to the Serbian regulation (Sl.glasnik RS, 4/2010). The highest percentage of
contaminated samples (100%) and also the maximal concentration (0.263 mg/kg) was detected in
feed for piglets, while 70 % of dairy mixtures were positive and 47.5 % over regulated level.
Extremely hot and dry conditions followed by drought were noted during maize growing season
2012 (Republic Hydrometeorological Service of Serbia, 2012), so aflatoxins occurrence in 68.5% of
samples harvested in 2012 could be contributed to weather conditions favourable for mould growth
and mycotoxins production (Kos et al, 2013). Comparing the results shown in Table 3 it can be
noted that the percentage of positive samples decreased in 2014, as well as detected aflatoxin
concentrations (the largest content was 0.0322 mg/kg quantified in the corn sample). Although
greater number of examinations done, only 5.1 % were above limits permitted by new legislation
(Sl.glasnik RS, 27/2014), which prescribed more strict criteria for aflatoxin. Approaching the end of
the year downward trend for this toxin had been increasingly apparent.
As aflatoxin crisis began to subside, the public interest in the problem of mycotoxins declined, but
not the problem itself. It has only modified in accordance with the new conditions of climate and
environment with lot of rain and moisture (Republic Hydrometeorological Service of Serbia, 2014).
Therefore, testing of maize and wheat during 2014, showed a large percentage of positives on
presence of new group, Fusarium mycotoxins: zearalenone and deoxynivalenol. Although these
moulds and their secondary metabolites are typical for our region, however, the frequency of their
presence and level of contamination exceeded previous data (Nesic and Pavlovic, 2013). Residues
of those mycotoxins are not considered to be of public health importance as only very low levels of
the toxins have been found in the tissues of animals that had been fed very high levels of the toxins
in experimental situations (Petterrson, 2004). But, from animal health and production point of view
their presence should not be neglected. The economic consequences can be very serious, especially
in pigs as the most sensitive species. Common clinical signs of zearalenonetoxicoses are vaginal
and vulvar swelling, enlargement of mammary glands and testicular atrophy, as well as other
reproductive effects, such as decreased fertility, increased number of resorptions and reduced litter
size (EFSA 2011; Nesic et al, 2008). Recent studies have demonstrated the potential for ZEA to
stimulate growth of human breast cancer cells (Yu et al, 2005). Intoxication with deoxynivalenol
(vomitoxin) is manifested by a decrease in food intake or its refusal, vomiting and digestive
disorders with subsequent losses of weight gain (Nesic et al, 2014). The gastrointestinal system is
the target organ of this toxin. In practice, the co-occurrence of DON and ZEA, or even additional
mycotoxins in contaminated cereals exacerbates the management of affected animals (Döll and
Dänicke, 2011).
Due to synergistic interactions co-contaminated samples might elicit adverse effects even the
concentrations of the individual mycotoxins do not surpass legal guidance values. However, in their
present state regulations do not address co-contamination and associated risks. For example, there is
data that a 5% growth depression is to be expected in pigs fed naturally deoxynivalenol (DON)contaminated diets containing 0.6 mg kg−1 DON, whereas 1.8 mg kg−1 DON is required to obtain
a similar effect if pure toxin is used. Serbian Directive as well as the European Union’s guidance for
DON in complete feed for pigs is 0.9 mg/kg (Streit et al, 2013).
129
Based on the discussion above it is also important to emphasize the process of detection of
mycotoxins. Mycotoxins are a major analytical challenge because of the range of chemical
compounds they represent, and the array of feed matrices in which they are found. Analysis is
essential for determining the extent of mycotoxin contamination, for risk analysis, for confirming
the diagnosis of a mycotoxicosis and for monitoring mycotoxin mitigation strategies. Quantifying
these compounds requires sophisticated laboratory equipment and trained people (Krska et al, 2008;
Rahmani et al, 2009). The development of immunological methods for mycotoxin detection,
especially enzyme-linked immunosorbent assays (ELISA), although screening test, was a major
step toward developing rapid, repeatable and sensitive assays. But there are other influences that
can disturb the validity of the results. Sampling is the greatest source of error when quantifying
mycotoxin contamination. It is difficult to obtain feed samples representative of what may have
caused a mycotoxicosis incident or to represent large grain consignments. These difficulties arise
because of the uneven distribution of toxin within a commodity, in which mycotoxins occur (CAST,
2003; Whitaker, 2003, 2006). Also, it has recently become apparent that there is a connection
between ‘masked’, ‘hidden’, ‘bound’ and/or conjugated mycotoxins in feedstuffs and the potential
for animals to perform poorly. Mycotoxin conjugates may be formed as a result of plant metabolism
(Berthiller et al, 2007), but are not detected by using conventional analytical procedures. These
conjugates will be hydrolyzed following ingestion, thereby increasing exposure to the precursor
toxin.
As the latest laboratory results showed permanent presence of some mycotoxins and the cooccurance of few of them, it is important to establish better prevention (Nesic et al, 2013), as well
as the predictive models in regard to meteorological forecasts. However, special attention has to be
paid to the control system and regular monitoring of cereals, feed and food has to be organized.
Since managing impact of these contaminants is still great practical challenge, only timely and
adequate reaction is the right solution. There are a number of approaches that can be taken to
minimize mycotoxin contamination in the food chain: prevention of fungal growth and therefore
mycotoxin formation, strategies to reduce or eliminate mycotoxins from contaminated feedstuffs or
diverting contaminated products to low risk uses. A “field to table” combating program against
mycotoxins should involve Good Agricultural Practice, Good Manufacturing Practice, Good
Storage Practice and applying the seven HACCP (Hazard Analysis Critical Control Points)
principles within the framework of the quality systems, as well as Good Laboratory Practice and the
methods for mycotoxin detection which must meet the highest standards.
Acknowledgments
This paper is published as part of the projects of the Ministry of Education, Science and
Technological Development of Republic of Serbia No. III46009 and 031071.
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132
EFFECTS OF FED DIETS WITH A DIFFERENT N-6/N-3 PUFAS RATIO ON OXIDATIVE
STABILITY, AND PHYSICO-CHEMICAL PROPERTIES OF CHICKENS MEAT
Dragan Milićević1*, Dejana Trbović1, Zoran Petrović1, Breda Jakovac-Strajn2, Ivan Nastasijević1,
Nenad Parunović1, Mirjana Lukić1
1
2
Institute of Meat Hygiene and Technology, Belgrade, Republic of Serbia
Faculty of Veterinary Medicine University of Ljubljana, Ljubljana, Republic of Slovenia
*
* Corresponding author: dragan@inmesbgd.com
Abstract
The objective of this study was to determine the fatty acid (FA) profile, oxidative stability of lipids,
measured by the thiobarbituric acid (TBA) and physicochemical properties of raw and heatprocessed meat of chickens fed with a different n-6/n-3 PUFAs ratio (14.38, 12.91, 13.40 and
12.85, respectively), resulting from the different dietary ingredient (particularly soybean meal and
sunflower oil). Samples of breast and thigh muscles of broilers equal to 48 from two farms (Farm
I and Farm II) were taken during 2012. The chickens were fed ad libitum on commercial diets for
growing broilers which contained different feed ingredients. Generally, all parameters measured
were influenced by the interaction of growth performances, the quality characteristics of the
chicken meat, and treatment group (farms and diets) by at most 5%. The farm treatments modify
significantly growth performance and feed intake of the broilers between farms. The
physicochemical properties, including cooking loss, color, pH, lipids and thiobarbituric acid (TBA)
in raw and heat-processed breast and drumstick meat samples, differed significantly. Content of
total iron (Fe) did not differ significantly between meat samples. Comparing FAs composition in
diet with drumstick and breast muscle, the diet was comprised mainly of polyunsaturated FA
(PUFA) then monounsaturated FA (MUFA) and saturated FA (SFA) (100:47:27%) versus
drumstick and breast muscle, where the predominant FAs was MUFA, in comparison to SFA and
PUFA (100:78:55%), although the proportion of FAs with four or more double bonds was
metabolized specific. It may be concluded that, higher n-6/n-3 PUFAs ratio in the diets improved
the FAs ratio in the meat samples, however, the higher n-6/n-3 PUFAs ratio in meat adversely
affected the oxidative stability as manifested by the significantly higher TBA concentrations in
heat-processed breast and drumstick meat (4.69  0.93 to 5.52  1.44 and 5.87  1.27 to 8.63 
0.88 MAL/kg, respectively), compared with those of the group where are low n-6/n-3 PUFAs ratio
in meat samples (2.49  1.08 to 5.46  0.19 and 3.06  0.99 to 4.53  0.10 MAL/kg, respectively).
Therefore, our results suggest that poultry meat quality is a complex and multivariate property,
which is affected by multiple interacting factors.
Keywords: chicken meat, physicochemical properties and oxidative stability.
Introduction
Effect of diet on human health is a major problem not only in developing countries but also in
highly industrialized countries, where consumers are becoming more demanding in relation to food
products. Chicken meat is the fastest growing component of global meat demand. In Serbia the
consumption of chicken meat is higher compared to other meats, as it is preferred over other meat
for its health appeal because of its nutritional value, but also its taste and appearance
(Bogosavljevic-Boskovic et al., 2010). Compared to other meats, chicken lipids are characterised
by relatively high levels of unsaturated fatty acids, especially polyunsaturated fatty acids (PUFA),
which are considered as a positive and healthy aspect by consumers (Bonoli et al., 2007).
However, commercial chicken meat is known to be deficient in n-3 FA and rich in n-6 FA. From
133
the human health point of view, it is important to have low saturated fat and adequate amount of
PUFA preferably less n-6 FA, balancing n-3 FA. The modern human diet is deficient in n-3 FAs,
which has been linked to the increase in several degenerative diseases such as cardiovascular
disease, diabetes, arthritis, cancer and mental illness (Nichols et al., 2010). Therefore it is important
to enrich chicken meat with n-3 FA and lower the saturated and n-6 FA content. Decreasing the n6/n-3 FA ratio in chickens is important in order to make chicken meat a functional food (Rondelli et
al., 2004). Of all the meat constituents, the lipid fraction has the highest susceptibility to
modification, in contrast to protein with aminoacidic composition determined by the genetic code.
As a result, feeding strategies have been adopted to alter lipid composition of broilers meat (Kouba
and Mourot, 2011). However, the increasing amount of PUFA content in poultry diets could
increase the degree of broilers fat unsaturation and probably the rate of tissue lipid peroxidation,
which reflects the so-called thiobarbituric acid reactive substance (TBA) value of broilers tissue
(Cherian et al., 1996). Lipid oxidation is responsible for the production of undesirable compounds
(from both a toxic and sensory point of view) which is of significant importance in consumer
purchase decisions for fresh meat (Risvik, 1994), and in general causes shelf-life quality
deterioration in meat (Bou et al., 2009). Therefore, the present study was conducted to determine
the fatty acid (FA) profile, oxidative stability of lipids, measured by the thiobarbituric acid (TBA)
and physicochemical properties of raw and heat-processed meat of chickens fed with a different n6/n-3 PUFAs ratio and a possible interaction between these factors.
Animals and diets
A total number of 48 samples of breast and thigh meat were taken from chilled broiler carcasses
collected during 2012. Two homogeneous groups of male and female (50%:50%) Ross 508 and
Hubbard broilers were reared under commercial conditions on two farms in Vojvodina in the
northern part of Serbia, and fed ad libitum commercial diets for growing broilers with different
ingredients (Table 2). All diets were formulated to meet the minimum requirements for broilers
and were proved as mash feed.
Sample preparation
At the age of 39 day, 12 birds selected on the basis of live weight within as wide range as
possible, were slaughtered at a commercial abattoir. Feed gain and body weight (b.w.) had been
recorded at weekly intervals. Feed conversion ratio corrected for mortality was then calculated
(Table 1). After slaughtering and dressing, the hot carcasses were chilled for 2 h at 4°C . The
carcasses were weighed and refrigerated for 24 h.The breast with skin (Mm. pectoralis major) and
thigh meat with skin (Mm. of regio tibio-femoralis) were cut, separated and weighed (Table 1).
Analyses
Meat color was evaluated immediately after deboning using colorimeter (Minolta Chroma Meter
RC-400, Japan). The CIE system color profile of lightness (L*), redness (a*) and yellowness (b*)
was measured by reflectance colorimeter using illuminant source D. The colorimeter was calibrated
throughout the experiment using a standard white ceramic tile. Color was measured on skin side
surface of each left breast in area free of obvious color defect, bruises and blood spots, while pH
value was measured 24 h postmortem, by inserting a portable pH meter (Testo 205, AG, Germany)
along with a temperature probe into the cranial part of each left breast sample. For determine
cooking loss whole samples of both muscles (about 20 g) were placed in open aluminium pans
and roasted in an electric oven pre-heated to 220°C for 30 min. Then the samples were
equilibrated to room temperature and reweighed. Cooking loss determined as percentage of weight
134
lost during cooking. Total lipid was determined in raw and roasted breast and thigh meat with
skin. Total lipid content was determined by extraction of fat by petroleum ether and Soxhlet
extractor after acid hydrolysis of the samples (SRPS ISO, 1992). Total lipids were extracted from
the diet, thigh and breast muscle samples and further converted to fatty acid methyl esters (FAMEs)
(0.25 M trimethylsulfonium hydroxide - TMSH in methanol as the methylating agent). FAMEs
were determined by capillary gas chromatography on GC Shimadzu 2010 (Kyoto, Japan) equipped
with flame ionization detector and capillary HP-88 column (100 m × 0.25 mm × 0.20μm, J&W
Scientific, USA). Thiobarbituric acid (TBA) was determined according to the method proposed by
Tarladgis et al. (1964) and Holland (1971). TBA content was expressed as mg of malondialdehyde
per kg of meat (mg MAL/kg). Determinations of Fe in meat samples were performed by flame
atomic absorption spectrophotometry (Analyst 300, Perkin Elmer, USA) following the methods
described by Jorhem (2000).
Data from the experiment was analysed by descriptive statistics (mean, standard deviation, and
range). ANOVA with Tukey's test was performed, in order to determine statistical differences
among examined parameters between farms (P < 0.05).
Results
Growth performance of the broilers
The Growth performances of broilers fed various dietary and composition of the commercial
broiler diets which were used for feeding chicks, are shown in Tables 1 and 2. Average live
weight, daily gain, carcass, breast, and drumstick weights were different among the groups of
broilers. At the same age, the highest average daily body gains (57 g), average daily feed intake
(103.9 g) and carcass (1764.87  227.83 g), breast (647.95  78.70 g), and drumstick weights
(536.40  88.41 g) were in chickens from Farm I in the autumn. Despite the different diet
compositions, nutritive values were similar during the investigation (Table 2). In regard to fatty
acids content, n-6/n-3 PUFAs ratio were different (14.38, 12.91, 13.40 and 12.85, respectively),
resulting from the different dietary ingredient, particularly soybean meal and sunflower oil.
Physicochemical properties, FAs rations and TBA values in broiler meat
Physicochemical properties, FAs rations and oxidative status of raw broiler meat are shown in
Table 3 and Figs 1 and 2. The physicochemical properties, including cooking loss, color, pH, lipids
and thiobarbituric acid (TBA) in raw and heat-processed breast and drumstick meat samples,
differed significantly between treatment group. Content of total Fe did not differ significantly
between samples. Comparing FAs composition in diet with drumstick and breast muscle, the diet
was comprised mainly of PUFA then MUFA or SFA (100:47:27 %) versus drumstick and breast
muscle, where the predominant FAs was MUFA, in comparison to SFA and PUFA (100:66-77:5670 %), except in the treatment group were is sunflower oil added in diets in 4.45 % (100:71-96:3751 %). Although the proportion of FAs with four or more double bonds was metabolized specific,
the n-6/n-3 PUFAs ratio in breast and drumstick muscle samples was higher in broilers fed with
higher level of the sunflower oil and the soybean meal (29.01:1 and 20.84:1 respectively). These
results indicated that n-6/n-3 PUFAs ratio in broiler meat large in magnitude, was significantly
different (P<0.001). The average TBA values in drumstick and breast muscle (0.04 and 0.02 mg
MAL/kg, respectively) in treatment group Ib was significantly (P<0.001) lower than those from the
other treatment group. The average TBA values in drumstick and breast muscle in other group
varied between 0.067 to 0.09 MAL/kg, both in breast and drumstick muscle.
135
Table 1. Growth performances of broilers fed various dietary ( X ± Sd, Range)
Farm
Parameter
(range)
a
Age at slaughter (d)
Average daily body gain (g)
Average daily feed intake (g)
Feed gain (g:g)
Carcass weight (g)
Breast weight (g)
Thigh weight (g)
I (n = 12)
38
57
103.9
1.82
1279.45±174.02
(1021.7- 1681.2)
452.5 ± 80.17
(339 - 628)
389.89 ± 51.19
(316.8 - 494)
a
II (n = 12)
38
39
84.33
2.16
1223.3 ± 116.38
(1055 - 1486.4)
427.70 ± 53.44
(337.1 - 547.2)
392.9 ± 39.20
(354 - 472.7)
Ib (n = 12)
39
57
100.51
1.76
1764.87 ± 227.83
(1498.8 - 2226.7)
647.95 ± 78.70
(510 - 775.1)
536.40 ± 88.41
(443.2 - 703)
IIb (n = 12)
38
52
106.1
2.04
1400.70 ± 130.01
(1186 - 1712.7)
470.62 ± 61.15
(393.4 - 614.1)
476.78 ± 48.20
(398.6 - 572.4)
I, II - farms, n - number of samples, a - summer, b - autumn/winter
Table 2. Composition and main characteristics of the commercial broiler diet
Ingredients/
Amount (%)
Corn
Starter (0 - 15 d)
Ia
55.2
IIa
55.12
Ib
50.06
IIb
49.45
Grover (16 - 32 d)
Farm
a
a
I
II
Ib
IIb
56.03
56.03 39.08
49.6
Finisher (33 - 38 d)
Ia
61.1
IIa
55.8
Ib
55.8
IIb
52.75
SbM
34.8
34.8
18
30.9
30.9
25.4
29.8
(46% CP)
SbM
15
33.60
31.60
29.4
23.5
24.5
(44% CP)
Wg
3.0
3.0
6.0
6.0
6.0
6.0
20.0
10.0
6.0
6.0
10
10
SfM
3.0
(33% CP)
Yeasts
1.5
CG
2.0
2.0
2.0
SfO
1.65
1.65
2.5
2.62
2.4
2.4
4.42
3.45
3.05
3.45 3.45
4.45
L-Lysine
0.27
0.27
0.29
0.36
0.02
0.02
0.08
0.11
0.16
0.05 0.24
0.11
DL-Meth.
0.28
0.28
0.30
0.28
0.15
0.15
0.17
0.15
0.15
0.11 0.14
0.15
Treonine
0.12
0.12
0.13
0.14
0.03
0.03
0.05
0.07
0.06
Ch. 60%
0.13
0.13
0.13
0.14
0.10
0.10
0.10
0.10
0.10
0.10 0.10
0.10
VMP 0.5%
0.60
0.60
0.60
0.60
0.60
0.60
0.50
0.50
0.50
0.50 0.50
0.50
Chemical composition (%)
Crude protein 22.32 22.82 22.46 22.25
19.24
20.39 20.1
20.62 18.71 18.83 19.12 19.82
Water
11.07 10.23 11.50 13.19
10.69
10.73 11.40 12.79 10.30 11.46 11.11 11.57
Crude fibre
1.15 1.68
1.57
1.88
1.73
1.50
1.66
1.75
1.34
1.55
1.63
2.68
Fat
4.75 4.48
4.56
4.60
4.69
4.30
4.92
4.90
6.32
5.95
6.10
6.20
Ash
5.48 5.80
5.63
5.61
5.32
6.04
5.52
5.21
4.62
4.88
4.98
5.44
Ca
0.82 0.95
0.86
0.88
0.74
1.03
0.86
0.81
0.81
0.74
0.78
0.81
Available P
0.71 0.70
0.68
0.65
1.73
0.69
1.05
0.73
0.58
0.70
0.64
0.63
Sums of fatty acids
SFA
15.3 16.05 16.0 16.62
15.7
16.83 16.83 16.95
15.6
16.06
15.8
15.8
MUFA
27.6 26.83 28.09 29.85
27.9
27.79 28.40 29.53
27.2
25.95
27.0 27.88
PUFA
57.1 57.12 55.92 53.53
56.4
55.4 55.77 55.53
57.2
58.01
57.2 56.38
n-3
3.49 3.65
3.58
3.61
3.53
4.14
3.67
3.36
3.72
4.17
3.98
4.07
n-6
53.6 53.47 52.33 49.92
52.87
51.25 51.45 50.17 53.48
53.84
53.21 52.31
n-6/n-3
15.3 14.65 14.61 13.83
14.98
12.38 14.10 14.93 14.38
12.91
13.40 12.85
PUFA/SFA
3.73 3.56
3.49
3.22
3.60
3.29
3.38
3.27
3.66
3.61
3.62
3.58
MUFA/
1.65 1.67
1.75
1.79
1.77
1.65
1.72
1.72
1.74
1.61
1.71
1.77
SFA
ME (MJ/kg)
12.65 12.65 12.65 12.65
13.00 13.00 13.00 13.00 13.45 13.45
13.45 13.45
I, II - farms, a - summer, b - autumn/winter, SbM - soybean meal, Wg - wheat grain, SfM - sunflower meal, CG - corn
gluten, SfO - sunflower oil, DL Meth–DL - methionine, Ch - choline, VMP - vitamin + mineral premix, SFA - saturated
FA, MUFA - monounsaturated FA , PUFA - polyunsaturated FA
136
Table 3. Chemical composition and physicochemical properties of raw and heat-processed breast
and drumstick meat
Treatment group
Parametar
Breast
Ia
Ib
IIa
IIb
36.11  1.70 ***
(33.58-39.71)
58.08  3.71 *
(52.30-66.08)
1.40  0.67,***
(0.14-2.54)
4.31  1.77 ***
(1.20-6.99)
5.40  0.20 ***
(5.11-5.71)
4.06  1.37
(2.26-7.74)
5.53  0.61 ***
(5.01-6.78)
6,08  0.34 ***
(5.50-6.55)
0.08  0.036 ***
(0.04-0.16)
29.06  2.80 ***
(24.04-32.86)
53.96  3.70 **
(48.70-60.78)
-0.172  0.76 ***
(-1.41-1.47)
3.40  1.51 **
(0.59-5.55)
5.90  0.18 ***
(5.57-6.15)
3.94  0.42 *
(3.44-4.76)
4.19  1.15 ***
(2.30-5.62)
6.25  0.78***
(5.00-7.00)
0.018  0.012 ***
(0.000-0.03)
35.67  3.18 ***
(30.32-40.12)
56.72  2.90 **
(51.94-63.64)
0.47  0.37 **
(0.01-1.11)
2.00  0.70 ***
(0.76-2.86)
5.89  0.11 ***
(5.70-6.10)
4.34  0.55
(3.62-5.14)
3.05  1.11 ***
(1.80-5.79)
3.57  0.155***
(3.42-4.00)
0.086  0.05 ***
(0.03-0.17)
31.70  2.73 ***
(25.60-34.83)
58.26  3.20 **
(53.94-62.82)
0.46  0.92 *
(-0.64-2.36)
4.93  1.47 ***
(2.78-8.91)
5.84  0.14 ***
(5.62-6.15)
4.30  0.31 *
(3.90-4.90)
2.61  0.77 ***
(1.32-3.92)
3.72  0.61***
(3.04-4.90)
0.067  0.012 ***
(0.05-0.09)
32.60  2.10 **
(29.96-36.10)
57.20  1.87 ***
L*
(54.66-60.05)
2.83  1.67 *
a*
(1.10-7.48)
4.71  1.95 **
b*
(2.44-7.63)
5.71  0.29 ***
pH 24
(5.21-6.10)
7.88  1.41
Fe total (mg/kg)
(6.03-10.34)
9.63  0.64 ***
raw
Lipids
(8.18-10.81)
(%)
heat14.73  0.55 ***
processed
(14.00-15.50)
TBA (mg MAL/kg)
0.07  0.02 **
raw
(0.05-0.12)
* P < 0.05; ** P < 0.01; *** p  0.001
29.69  3.48 *
(23.73-36.25)
52.93  2.58 ***
(49.01-56.36)
2.02  0.57
(1.18-3.39)
5.91  1.22 ***
(4.36-8.20)
6.45  0.25 ***
(6.02-6.77)
8.42  1.46
(6.08-11.18)
9.85  1.98 ***
(8.12-15.29)
13.37  0.95 ***
(12.00-15.00)
0.04  0.014 ***
(0.004-0.06)
33.34  4.58 **
(24.78-41.34)
58.82  2.21 ***
(54.91-62.33)
1.26  0.79 ***
(0.25-3.11)
2.86  1.10 ***
(1.10-4.60)
6.44  0.17 ***
(6.15-6.74)
7.45  0.98
(6.07-9.24)
5.20  0.45 ***
(4.33-5.70)
10.54  0.22***
(10.00-11.00)
0.087  0.064 *
(0.03-0.24)
29.60  2.37
(25.06-33.36)
58.10  3.22 ***
(49.54-61.84)
3.10  1.07 ***
(1.30-4.65)
7.67  2.77 ***
(1.47-11.55)
6.51  0.09 ***
(6.38-6.68)
8.12  0.69
(7.00-9.10)
8.16  2.26 ***
(5.27-11.26)
11.07  2.04 ***
(8.11-14.11)
0.09  0.01 ***
(0.07-0.12)
Cooking loss (%)
L*
a*
b*
pH 24
Fe total (mg/kg)
Lipids
(%)
raw
heatprocessed
TBA (mg MAL/kg)
raw
Drumstick
Cooking loss (%)
The concentrations of total lipid and TBA values in heat-processed broiler meat are shown in
Table 3 and Figure 3. Heat-processed broiler meat (Table 3) belonging to chickens from Farm II
contained lower quantities of total lipids in breast (3.57 to 3.72%) and drumstick (10.54 to 11.07
%) than samples from Farm I in breast (6.08 to 6.25 %) and drumstick (13.37 to 14.73%). These
differences were significant (P < 0.001). TBA values as a measure of lipid oxidation in heatprocessed breast and drumstick chicken meat are shown in Figure 3. A higher (P  0.001) TBA
values was found in the muscle from the Farm II. The average TBA values in samples of heatprocessed drumstick muscle of chickens from Farm II ranged from 5.87 ± 1.28 (mg MAL/kg) in
summer to 8.63 ± 0.88 (mg MAL/kg) in autumn, compared to those from the Farm Ib, where the
average TBA values in samples of heat-processed breast muscle was 2.49 ± 1.08 (mg MAL/kg)
and 3.06 ± 0.99 (mg MAL/kg) in drumstick muscle. These differences (p < 0.001) could be
137
attributed to initial higher level of TBA values in samples from farm II, as well as high n-6/n-3
PUFAs ratio in breast and drumstick muscle.
100%
PUFA
90%
MUFA
80%
SFA
70%
60%
50%
40%
30%
20%
10%
0%
B (Ia)
D (Ia)
B (Ib)
D (Ib)
B (IIa)
D (IIa)
B (IIb)
D (IIb)
Fig. 1. The fatty acid (FAs) rations (% of total fatty acids) in raw breast (B) and raw drumstick (D) meat
from farms I and II during summer (a) and autumn (b) fed dissimilar diets
40
12
A
35
(mg MAL/kg)
n-6/n-3
30
25
20
12
B
10
10
8
8
6
6
4
4
2
2
15
10
B (I a)
D (I a)
B (I b)
D (I b)
B (II a)
D (II a)
B (II b)
D (II b)
B (Ia)
D (Ia)
B (Ib)
D (Ib)
B (IIa)
D (IIa)
B (IIb)
D (IIb)
Fig. 2.(A) n-6/n-3 PUFA ratio in raw breast (B) and raw drumstick (D) meat from farms I and II during
summer (a) and autumn (b) fed dissimilar diets (B) Malondialdehyde (mg MAL/kg) content in heatprocessed breast (B) and drumstick (D) meat with skin from farms I and II during summer (a) and autumn
(b)
Discussion
In the present study, we analyzed the effect of diets with a different n-6/n-3 PUFAs ratio resulting
from the different dietary ingredient (particularly soybean meal and sunflower oil), on the fatty acid
(FA) profile, physicochemical properties and oxidative status of raw and heat-processed breast and
drumstick meat. The results of the study indicate that all parameters measured were influenced by
the interaction of growth performances, the quality characteristics of the chicken meat, and
treatment group (farms and diets). Because of inclusion of grain, corn, plant seeds or oils, which
138
are rich in C18:2, poultry meat of different kinds generally has as high n-6/n-3 ratio as pork and
higher than beef and lamb, whereas the P/S ratio is closer to 1.0 (Li et al., 1998). These results are
in contrast to those observed in our study where P/S ratio was below of 1.0 (0.39 to 0.97 in breast
and 0.76 to 1.04 in drumstick). However, P/S ratio is better in samples originating from farm I then
from farm II. A lower dietary n-6/n-3 PUFA ratio provides health benefits to consumers. In typical
Western diets, the n-6/n-3 PUFA ratio is high, ranging between 10:1 and 30:1 (Hibbeln et al.,
2006), while it should remain in the 1:1–1:4 range (Lands, 2005). The ideal dietary n-6/n-3 PUFA
ratio, recommended by an international panel of lipids experts, is approximately 2:1 (Simopoulos et
al., 1999). In the present experiment, the ideal n-6/n-3 PUFA ratio was not achieved and was far
from recommended, ranged from 14.1:1 to 29.01:1 in breast meat, versus 18.08:1 to 20.84:1 in
drumstick meat. High n-6/n-3 PUFA ratio corresponded to low P/S ratio in samples originating
grom farm II.
Concerning results in the present studies, this result suggests that dietary lipid source or n-6/n-3
PUFA ratio did not influence its color characterized in scale L*,a*, and b* (data not presented). For
example, Bianchi et al., (2009) reported that dietary use of vegetable oils produced darker and a
higher yellowness of breast meat than animal fats. In regarding to diferences between FAs
composition in diet with thigh and breast muscle, the results in this study supported the hypothesis
that chicks can better assimilate FAs from fat sources that are rich in UFA than from fats that are
rich in SFA (Smits et al., 2000). Friedman and Nylund (1980), reported that the absorption of long
chain SFA is limited by their incorporation rate into micelles. Saturated FA are less rapidly
incorporated into micelles than polyunsaturated FA because of their non- polarity, which makes
them rely on an adequate presence of bile salts for efficient emulsification (Polin et al. 1980,
Dänicke, 2001).
The TBA test is a sensitive and useful method for monitoring lipid oxidation in many systems and
under a wide variety of conditions. The increase in the TBA content in heat-processed breast and
drumstick meat noted in the present study, was accompanied by a higher proportion n-6/n -3 PUFA
ratio in the total FA pool. Several studies have reported, an increase in n-6/n -3 PUFA ratio in meat
was followed by an increase in the concentrations of lipid oxidation products (measured as TBA
content) or cholesterol oxidation products, both in raw meat (Zanini et al., 2006: Cortinas et al.,
2004) and heat-processed meat (Eder et. al., 2005). Also, the results (Fig. 2B) revealed that the
initial MAL levels in meat, which strongly depend on fat content, composition, and fatty acid
composition, determine the absolute MAL levels after cooking. Similar results have been reported
by Vasavada et al. (2006).
It is important to point out the role that haemoglobin can perform in the beginning of
lipoperoxidative process. In the case of meat, it has been found that the haeme group (contains iron,
Fe) present in some proteins would have an important catalytic effect in the oxidative
decomposition of PUFA. In addition, Kranen et al. (1999) suggested that poultry leg and breast
contain significant concentrations of haemoglobin, 0.67 mg/g and 0.24 mg/g, respectively and in
consequence, when the animals are slaughtered, the biochemical processes that turn the muscle into
meat allow haemoproteins to control the lipoperoxidative processes that definitively accelerate the
deterioration of the meat (Alayash et al., 2001). Concerning the differences in the MAL levels in
meat between farms and season it is important to emphasise that this differences can be attributed to
the differences in the type of ingredient added to feeds, particularly corn, soybean meal and
sunflower oil, the main source of dietary fat.
In fact, it was noted that production parameters for broilers from the farms were very different. The
performance of the birds was not within the expected range of the breed standard (Aviagen, 2007),
and so the study did not reflect commercial performance. These data show much worse production
parameters for broilers at Farm II compared with Farm I, probably due to the problems induced by
microbiological factors (high mortality rate – 28.37%, data not presented), which consequently
139
might alter the chemical composition, and physicochemical properties of the meat. In addition, to
explain some discrepancy in obtained results with other authors, linked to possible microbiological
factor which could induce worst production results, cells involved in the inflammatory response are
typically rich in the n-6 fatty acid. PUFA are important constituents of the phospholipids of the
membranes of inflammatory cells. Typically these contain a relatively high proportion of the n-6
PUFA (Calder, 2012).
Conclusion
This study contributes an update to the literature data about fatty acid (FA) profile, oxidative
stability of lipids, measured by the thiobarbituric acid (TBA) in the meat cuts obtained from
chickens from the conventional commercial rearing system. From the results of the present study, it
can be concluded that, higher n-6/n-3 PUFAs ratio in the diets improved the FAs ratio in the meat
samples. However, the higher n-6/n-3 PUFAs ratio in meat adversely affected the oxidative stability
as manifested by the significantly higher TBA concentrations in heat-processed breast and
drumstick meat. Results from the current study can be used as a feeding strategy in order to improve
poultry production. Therefore, further studies should be encouraged to evaluate the nutritive value
of chicken meat produced through new and alternative rearing systems, the utilisation of animal
feed fortified in some nutrients, and better slaughtering practices, which currently also contribute to
the variability of the nutrient composition of meat.
Acknowledgements
This study was supported by the projects no. TR 31008, funded by the Ministry of Education,
Science and Technological Development of the Republic of Serbia.
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141
ECOLOGICALLY SAFE PRODUCTION OF SMOKED COMMON CARP MEAT
Jelena Babić1*, Brankica Kartalović1, Jelena Petrović1, Okanović Đorđe2, Biljana Božić1, Miloš
Pelić1, Miroslav Ćirković1
1
2
Institute of Food Technology in Novi Sad, Serbia
*Corresponding author: jelenababic@niv.ns.ac.rs
Abstract
Recently, the production of smoked common carp meat has suffered some changes in the production
process that include changing production conditions, as well as adapting recipes and quality to
industrial production conditions. The product obtained in this way has not all those sensory
characteristics of the products which consumers are given by craft production. The aim of this work
was to prove quality, safety and the lower PAHs content of a product obtained in ecological safe
production of smoked common carp meat.
The sensory, chemical and microbiological analyses of the ecologically produced smoked common
carp meat which were done at the Scientific veterinary institute "Novi Sad" confirm quality and
safety of the product and prove that the application of gravel filter gets smoked product without the
presence of carcinogenic polycyclic aromatic hydrocarbons (PAH).
Keywords: ecological, smoking, common carp, gravel filter, safety
Introduction
Production of smoked meat is common in Serbia and the region, which makes smoked products
very popular and consumed quite often. In this region, smoked fish is mostly eaten during the
Christmas fastening period. The consumption of smoked fish in our country is lower compared to
other countries in the EU, but it shows the tendency for the significant growth. The taste of smoked
carp meat resembles the taste of other traditional smoked products and therefore affects the increase
of the fish consumption in our market.
The main technological problem when it comes to the fish meat is that it goes bad very quickly
(FAO, 1995; Adelaja et al, 2013; Rzepka et al, 2013; Cirkovic et al, 2015). This characteristic is
mainly due to the lack of activity of the milk acid, that is, the pH value being close to the neutral
value and high activity of water (aw) being higher than 0,95 (Milijasevic et al, 2010).
In order to make the fish meat more durable (better preserved) as the product, we have to take
knowing and professional care of the fish from the moment they are caught until the moment they
are sold to the consumer. Processing of the fish meat creates adverse conditions for survival of
microorganisms, the main cause of fish meat going bad, and they act in a bacteriostatic,
bactericidical and the combined way (Cirkovic et al, 2002). The fish processing enables selling it on
a much bigger market, that is, it makes it more available for the consumers. The fish processing
industry is not well developed in our country because for many years the amount of fish was just
not enough and also because of the consumers' habits. This led to selling all the fish raw (Cirkovic
et al, 2002; Cirković et al, 2015). The main purpose of the smoking process is to preserve the
product. This is partially done by drying it out and partially through the transfer of antimicrobiological compositions, such are the phenols in smoke. Smoked fish is considered to be a
ready –to- eat meal (Ready-To-Eat, RTE), and lately, its consumption has increased significantly in
many European countries (Gallart-Jornet et al, 2007). Approximately 15% of the fish for human
consumption is available in either cold or hot variant (Stolyhwo and Sikorski, 2005). In contrast to
142
the times when the smoked fish meat was considered to be an item of luxury, today this is a
common product, available to the great number of people. Within this type of the fish products, the
smoked carp, silver carp and trout meat is available in our country (Pavlicevic et al, 2013).
Recently, the production of smoked common carp meat has suffered some changes in the
production process that include changing production conditions, as well as adapting recipes and
quality to industrial production conditions. The product obtained in this way has not all those
sensory characteristics of the products which consumers are given by craft production. The standard
quality of the hot smoked fish meat is not easily sustainable. It depends on the number of factors
related to the fish farming, fishing, transport, raw material, stunning, slaughtering, cutting off the
head and gills, descaling, exentration, washing, halving, brining, washing under pressure and
draining, drying, hot smoking, cooling, packaging and storing.
The acceptance of smoked fish in developed countries is based primarily on the sensory
characteristics (Yanar et al, 2006), while group of authors Akintola et al. (2013) confirmed the
nutritional qualities and adequacies.
It is known that the smoke, consisting of hundreds of components, also contains at least 100
polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons, PAH) and their alkylated
derivatives. Some of them are proven to have carcinogenic characteristics (Stolyhwo and Sikorski,
2005). According to the latest classification of the carcinogenic PAH compounds, benzo[a]pyrene
(BaP) is a definite carcinogenic (group 1), dibenz[a,h]anthracene is probably carcinogenic (group
2A), but naphthalene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene,
benzo[j]fluoranthene and indenol[1,2,3-c,d]pyrene are classified as possible human carcinogens
(group 2B) (International Agency for Research on Cancer, 2012). Wood smoke has also been
classified as definite carcinogenic (group 1) (International Agency for Research on Cancer, 2012;
Essumang et al, 2014).
PAH compounds reach their maximum levels during burning of materials, wood or cigarettes at
500-700 °C (Wenzl et al, 2006). This carcinogenic effect needs to be minimized by technological
procedures in the production of the smoked carp meat. These procedures imply construction of craft
smokehouses and chambers that serve the same purpose, choosing the materials with limited fat
content, choosing the most adequate type of wood, the height of the kiln in relation to the fish,
temperature of pyrolysis, the duration of drying and smoking, the temperature in the central part of
the fish and finally, using the different types of filters (Yurchenko and Mölder, 2005; Anggraini and
Yuniningsih, 2013).
The carp fish was farmed on the fish farm „Ečka“ in Lukino Selo. The feed with 30% protein was
used for farming. The fish meat contained 6% fat. The fishing was done in autumn period, and the
fish was stored at the temperature of 4 °C before it was processed.
Brining was done with a dry method with 2 % NaCl for 12 hours at room temperature. After that we
did washing under pressure and draining, then drying in the smoke generator with the gravel filter
for 1 h at 55 ° C, followed by hot smoking using beech sawdust until the temperature in the centre
of the product, which was continuously monitored, reached 65 ° C. This value was kept for 1h.
After that we continued with cooling down to the temperature of 10 ° C or less within 3 hours after
smoking, and then down to the temperature of 3 ° C or less within 12 hours. The analyses of the
smoked carp meat were done at the Scientific Veterinary Institute "Novi Sad". 20 samples of the
smoked carp meat were tested in total. The quality of the product was tested with the sensory,
chemical and microbiological methods.
Chemical analyses included determining of the PAH levels. The identification of PAHs was done
with an analytical method gas chromatography–mass spectrometry (GC-MS) (Agilent
143
7890B/5977A MSD), based on comparison of the retention times of the peaks and target ions with
those obtained from standard mixture of PAHs (standards supplied by instrument manufacturer).
Quantification was based on external calibration curves prepared from the standard solution of each
of the PAHs. The coefficients of determination (r2) for the PAH standard calibration plots were
pyrene (0.99975), benzo[b]fluoranthene (0.99945), benzo[k]fluoranthene (0.99950), acenaphthylene
(0.99982), indenol[1,2,3-c,d]pyrene (0.99999), benzo[a]anthracene (0.99926), acenaphthylene
(0.99928) and flourene (0.99963).
Microbiological analyses were done according to the current Regulation (Regulation on General
and Special Conditions of Hygiene of Food at any Stage of Production, Processing and Transport,
“the Official Gazette SRJ” 72/2010) by determining/detecting Listeria monocytogenes (SRPS EN
ISO 11290-1/2010) in the final product.
Sensory characteristics of the final product, the smoked carp meat, were determined in a
quantitative-descriptive test (SRPS EN ISO 6658/2002), at the interval scale ranging from 1 to 5.
Five sensory evaluators, who had passed the sensory evaluation training in detecting and
recognizing smells (SRPS ISO 5496/2002) and whose senses had previously been tested using the
test for determining the sense of taste (SRPS ISO 3972/2002), were giving marks in sensory
characteristics.
Results
The total PAHs concentration in ecological smoked common carp meat was 279.60 μg/kg. Maximal
concentrations of PAHs detected in the final product are: fluoranthen (63.93μg/kg), naphtalene
(61.18 μg/kg), acenaphthylene (60.97 μg/kg), fluorene (50.58 μg/kg), anthracene (21.56 μg/kg),
acenaphtene (8.13 μg/kg), fluoranthene (7.66μg/kg ), pyrene (3.33 μg/kg). The most important
thing is that PAHs from the groups 1, 2A i 2B (International Agency for Research on Cancer, 2012)
were not detected in the samples.
The reference method for determining the presence of Listeria monocytogenes (SRPS EN ISO
11290-1/2010) did not reveal any of its presence in all the samples. The results of the sensory
characteristics of the smoked carp meat are shown in Figure 1. The obtained results of chemical,
microbiological and sensory tests confirm the quality and safety of the smoked carp meat processed
in this ecological way.
Figure 1. The results of the sensory marks of the ecologically smoked carp meat
Sensory
characteristics
Appearance
Colour
Smell
Colour at the
intersection
Texture
Taste
Sd
CV%
4,51
4,67
4,83
4,67
0,60
0,52
0,49
0,66
13,30
11,13
10,14
14,13
4,83
4,76
0,86
0,42
17,81
8,82
X
Many studies done on animals have shown that dietary intake of BaP causes increased levels of
tumors, especially in the upper gastrointestinal tract (Kazerouni et al, 2001).
144
In order to determine PAH compounds in food, various methods are used. They are:
fluorometricand spectrofotometric, high performance liquid chromatography, gas chromatography
mass spectrometry (GC-MS) or gas chromatography with flame ionization detector (Yurchenko and
Mölder, 2005). Gas chromatography is widely used to determine PAH compounds in food (Simko,
2002).
Non-processed fish can have small or undetectable amounts of different PAHs absorbed from the
environment, most commonly from the contaminated water because fish rapidly metabolizes PAHs
(Stolyhwo and Sikorski, 2005; Yurchenko and Mölder, 2005).
The levels of PAH in smoked fishery products from modern smoking kilns with external smoke
generation are higher than the levels in smoked fishery products from traditional smoking kilns
(Karl and Leinemann, 1996).
Temperature of smoke plays an important part in the amount of carciogenic PAHs that are created
during pyrolysis. The amount increases linearly with the smoking temperature within the interval of
400- 1000°C (Simko, 2002). Direct exposure to smoke leads to a product with higher
concentrations of PAHs, whereas non-direct exposure to smoke leads to a product with lower
concentrations of PAHs (Simko, 2002; Duedahl-Olesen et al, 2010). Many studies have been
investigating the influence of different combustion woods and smoking duration on smoked food
PAH content and confirmed that soft resinous woods and longer smoking durations resulted in food
with higher PAH content (Stumpe-Viksna et al, 2008; Duedahl-Olesen et al, 2010; Forsberg et al,
2012)
The sum of PAH in smoked fish products in Denmark ranged from 22 μg/kg in smoked mackerel
prepared in an electric oven to 1387 μg/kg in herring smoked by direct smoking (Duedahl-Olesen et
al, 2006).
Traditional sauna smoked products are marketed as special delicacies, but during an official control
programme of BaP levels in Swedish smoked meat and fish, six samples of smoked fish which were
produced by traditional ‘‘sauna’’ smoking, had high BaP levels ranging from 8,4 to 14,4 μg/kg.
Samples of meat and fish smoked by indirect technique all had BaP levels below limit of
quantification. Regional initiatives of Sweden support further development of the traditional sauna
smoking process and improvement of the quality of the smoked products (Wretling et al, 2010).
Forsberg et al. (2012) reported that traditional smoking of fish is still practiced, and is still a
significant component of the CTUIR’s cultural and spiritual identity in the USA, but smoking
processes can introduce potentially harmful combustion by-products into the smoked fillets. That
was confirmed by their study in which the carcinogenic and non-carcinogenic PAHs in traditionally
smoked salmon were 40–430 times higher than those measured in commercial products.
Investigation of potential carcinogenic PAH in two different smoked fish in Turkey showed a
significant correlation between the fish fat and the total PAHs level. Also, the content of PAH in
smoked fish depends on the concentration of these compounds in the wood smoke, smoking
temperature and smoking time. The concentration of total PAHs varied between 23.83- 79.74 μg/kg
(Basak et al, 2010).
The results of Essumang and the group of authors (2014) have shown the statistically significant
difference in PAH levels between smoking with no charcoal as a filter and smoking with charcoal
as a filter.
Yusuf et al. (2015) investigated the effect of fish smoking methods on dietary exposure to PAHs
and potential risks to human health. They proved that traditional method of smoking had 7
genotoxic PAHs.
Data reported in the literature on PAHs in smoked fish are highly variable and that can be attributed
to the differences in the procedures used for smoking. Some of variables are: the type and
145
composition of wood, type of generator, oxygen accessibility, temperature of smoke generation,
smoking time, fat content, preparation of fish etc (Basak et al, 2010; Yusuf et al, 2015).
We proved the quality, safety and the lower PAHs content of product obtained in ecologically safe
production of smoked common carp meat. By producing the smoked carp meat in this way, we
obtain a product that has all the sensory characteristics provided by the traditional craft industry,
while at the same time, we obtain a safer product because it contains lower levels of PAHs which
minimizes the carcinogenic effect if consumed. This method of smoking can be applied to other
smoked products in craft industry.
Food safety is one of the most important policies of the EU, especially when it comes to the policy
of protecting the consumers. Beside this, the EU pays a lot of attention to the production of
traditional-craft products because it enables the development of villages, rural tourism and higher
profit for the producers. Considering that the traditional-craft production of the smoked meat is
typical for our country, it is necessary to continue our work on improving and securing the quality
and safety of smoked products and also, protecting the geographical origin.
Acknowledgments
This paper is a result of the research within the project TR 31011 “The influence of the quality of
the food components for cyprinid fish species on the quality of meat, losses and the profitability of
production”, financed by the Ministry of Science and Technological Development, Republic of
Serbia.
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147
EFFECT OF THE DIET ON IMPROVEMENT OF THE FATTY ACID COMPOSITION OF
PIG MEAT
Radmila Marković1*, Milica Todorović1, Srđan Pantić3, Milan Baltić1, Jelena Ivanović1, Dobrila
Jakić-Dimić2, Dragan Šefer1, Branko Petrujkić1, Stamen Radulović1
1
Faculty of Veterinary Medicine, University of Belgrade, Serbia
2
Veterinary Scientific Institute, Belgrade, Serbia
3
DOO Nutritio, Bijeljina, BiH
* Corresponding author: radmilam@vet.bg.ac.rs
Abstract
Numerous studies have confirmed the correlation between the prevalence of chronic disease and
human nutrition. In these studies, special emphasis is placed on the use of fats in human nutrition,
especially n-3 and n-6 fatty acids and their relation. The importance of conjugated linoleic acid
(CLA) for human health is also highlighted. Furthermore, the content and a ratio of fatty acids in
pig meat may be affected by choice of feeds for pigs. The aim of our research was to investigate the
impact of commercial preparations linseed, or of commercial preparations of CLA supplementation
in diets for pigs on fatty acid composition of pig meat. Fatty acid composition of feed for pigs and
pig meat was determined by gas chromatography.The use of linseed or CLA in the diet of pigs
significantly influenced the content of saturated, monounsaturated and polyunsaturated fatty acids
in pig meat. The meat of pigs fed with these supplements had significantly better ratio of n-6/n-3
fatty acids, compared to the meat of pigs fed without the addition of linseed preparation, or CLA.
The meat of pigs fed with the addition of CLA preparations revealed the presence of both isomers
of this preparation. The presence of these isomers has not been proven in the meat of pigs fed
without the addition of CLA.Adding linseed preparation, or preparation of CLA in food for pigs
improves the nutritional value of pigs meat.
Keywords: pigs nutrition, linseed, fatty acids, meat quality
Introduction
Numerous medical findings show that a significant role in the development of cardiovascular and
other chronic diseases in humans has a relationship between the two groups of polyunsaturated fatty
acids in the diet: n-6 acids, whose main representative is linoleic acid (C18: 2 n-6) and n-3 fatty
acids, whose main representative is alpha linolenic acid (C18:3 n-3).
Unsaturated fatty acids have mutually differences in chain length, number and position of double
bonds in molecules. The abbreviation "n-6" (or n-6) indicates that the first double bond in the
molecule of fatty acid is at the sixth carbon atom, as for "ω-3 '(or n-3) indicates that the first double
bond in the molecule of fatty acid is at the third carbon atom, counting from the methyl group. In
the natural resources most often reported unsaturated fatty acids belong to the n-6, n-3 and n-9
series.
The human body cannot synthesize essential fatty acids, but retains the ability to translate basic
essential fatty acid ingested with food (linoleic and α-linolenic) in the so-called long-chain
polyunsaturated fatty acids with specific roles in the body. In these biotransformation are involved
enzymes desaturase and elongase and throughout their activities, as well as the amount of substrate,
depends the intensity and efficiency of these reactions (Šobajić, 2002).
148
Numerous studies confirm that increased intake of n-3 fatty acids can affect on decreasing the risk
of heart disease and vascular disorders, but also alleviating symptoms and improving the clinical
picture in some autoimmune and inflammatory disorders.
Interest in the role of polyunsaturated fatty acids has been launched by investigations in 70 th of the
last century, which have revealed a very low occurrence of vascular disease in the population of
Greenland Eskimos, despite their diet rich in fat and based on marine mammals and fish whose food
chain is based on algae and plankton rich in n-3 polyunsaturated fatty acids. Similar observations
were later confirmed by epidemiological studies in other populations with a similar diet, for
example, in coastal areas of Japan (Karolyi, 2007).
The ability of producing n-3 enriched meat products of pigs is very interesting for many producers
and consumers, as noted above. In monogastric animals such as pigs, fatty acids are absorbed from
the gastrointestinal tract with small changes. In fact, the fatty acid profile of tissue directly reflects
the profile of fatty acids in animal nutrition. In ruminants, however, the fatty acids from food in the
digestive tract are changed, under the influence of the process of microbial fermentation and
biohydrogenation, prior to absorption from the gastrointestinal tract (Baltic et al., 2011b; Markovic
et al., 2011b).
In modern human nutrition the ratio of n-6/n-3 polyunsaturated fatty acids is relatively wide (1015:1), so that today in human diet efforts is made in order to enrich food with n-3 fatty acids, so that
this relationship could be approximate to optimal (4:1).
In the pig nutrition soybean, sunflower and other oilseeds that contain fatty acids of n-3 series and
fatty acids of n-6 series are used (Baltic et al., 2011; Markovic et al., 2011).
Linseed contains about 35 to 45% oil compared to the mass of dry material (Karleskind, 1996).
More than 70% of this oil contains polyunsaturated fatty acids, primarily alpha-linolenic acid
(ALA), an essential ω-3 fatty acids and linoleic acid (LA), essential n-6 fatty acids.
It should be noted that linseed meal contains the fatty acid profile that is similar, if not identical, as
linseed, considering that the total oil content of the meal is variable depending on the method used
for extraction of oil, which affects the energy value of this nutrient.
The CLAs are a mixture of positional and geometric isomers of linoleic acid (9c,12c C18:2), which
were first identified in rumen fluid as an intermediate in the biohydrogenation process (Bartlett and
Chapman, 1961). In synthetic CLA preparations the 9c,11t and 10t,12c isomers are predominant
(often in a 1:1 ratio). It appears that the 9c,11t isomer has positive effects on some types of cancer
by inhibiting tumorogenesis, while 10t,12c isomer could be responsible for changes in whole-body
fat deposition (Pariza et al., 2001).
In addition, dietary CLA seems to be highly deposited in body tissues of monogastric animals and
as a result, in pork and meat products (Corino et al., 2005; Jiang et al., 2010).
The aim of this study was that in two experiments on pigs’ choice of ingredients, meal examination
to influence the fatty acid composition and meat quality of pigs.
Materials and methods
The aim of the research in the Experiment 1 was to investigate the effect of different sources of fat
in the diet on fatty acid composition and meat quality of fattening pigs.
The trial was conducted on 30 crossbreeds pigs (♂Yorkshire × ♀Landrace), with an initial body
weight of 60 kg. The pigs were divided into three groups of ten animals and the experiment lasted
46 days until a mean weight of about 100 kg. They were fed with standard mixture for pigs in the
final stage of fattening, provided that the groups differed only in the fact that the first experimental
149
group (E-I) had a grain sunflower, second experimental group (E-II) preparation of linseed
(Vitalan®, Vitalac, France) in the recommended inclusion rate of 2.5% in the mixture, and the third
experimental group (E-III) full-fat soybean meal included in the diet (Table 1). The fatty acid
composition of feed and meat content of n-3 and n-6 fatty acids, as well as their relationship with
feed and meat were investigated.
In the Experiment 2 the aim of the research was to investigate if the addition of CLA in the diet of
fattening pigs, affects the fatty acid composition of meat. Sixty crossbreed pigs (♂Yorkshire ×
♀Landrace), with an initial body weight of 60 kg were used in this study. The pigs were divided
into two groups (Control-C and Experimental-E) of 30 pigs in each and fed with a standard mixture
(NRC, 1998), from 60 to 110 kg (fattening period of 60 days). The diet between groups differed
only in the fact that the experimental group had commercially prepared conjugated linoleic acid
60% CLA (Lutalin®, BASF, Germany), added in the feed, at the recommended rate of 2.0% of the
mixture. The mixtures were balanced and completely satisfied the needs of the animals in this
production stage. The nutrient composition of the diets is detailed in Table 1.
Table 1. Raw material of the final feed for pigs fattening (%)
Feed
Corn, grain
Wheat, grain
Barley, grain
Full-fat soybean meal
Soybean meal
Sunflower, grain
Wheat bran
Vitalan®
Lutalin®
Dicalcium phosphate
Chalk
Salt
Vitamin mineral premix
∑
E-I
Experiment 1
E-II
E-III
46.7
15.0
11.5
7.5
16.2
-
50.80
14.0
13.0
16.50
2.5
51.0
14.0
14.60
1.0
16.20
-
0.50
1.20
0.40
1
100
0.60
1.20
0.40
1
100
0.60
1.20
0.40
1
100
Experiment 2
C
E
48
28
16
5
3
100
46
28
16
5
2
3
100
The total lipids, for fatty acid determination, were extracted from pig muscle tissue with
hexane/isopropanol mixture by accelerated solvent extraction (ASE 200, Dionex, Germany). After
the evaporation of solvent to dryness under the stream of nitrogen, total lipids were converted to
fatty acid methyl esters (FAME) by trimethylsulfonium hydroxide. FAMEs were determined by
Shimadzu 2010 gas chromatograph equipped with flame ionization detector (FID) and cianopropyl
HP-88 capillary column (100m × 0.25 mm × 0.20μm) (Trbović et al., 2011).
Meat samples (M. longissimus dorsi) from each pig in all groups for analysis of fatty acid
composition analysis were taken at the end of the both experiments, after slaughtering, processing
and chilling of the carcasses.
Results
In these studies, we have investigated the quantities and proportions of fatty acids in pork meat
when fed diets with variable fatty acid content. In the examined total fatty acid content of pig feed,
(Table 2) it was noticed that there was a statistically significant (p<0.01) difference between groups
(Experiment 1). The complete feed mixture of E-II group, which contained the preparation of
150
extruded linseed, had a significantly lower content of SFA and significantly higher PUFA content
(p<0.01), compared to the other two groups.
Since the fatty acid content was significantly different, that resulted in different ratios of n-6/n-3. In
the Experiment 1, the most beneficial ratio of n-6/n-3 was observed in E-II group (5.57) (a group
that was fed linseed preparation), compared to E-I group (52.65) and E-III group (11.00) (p <0.01)
(Table 2).
Table 2. The fatty acid composition of feed (%), and the ratio n-6/n-3
FA
SFA
MUFA
PUFA
n-6
n-3
n-6/n-3
c9t11CLA
t10c12CLA
c9t11CLA+
t10c12CLA
E-I ( X SD)
20.60B ±0.46
35.29A,B±0.56
43.54AB±0.76
42.73AC±0.76
0.81AC ±0.1
52.65AC±1.10
-
Experiment 1
E-II ( X SD)
18.38AB±0.60
25.46B±0.26
55.99A±0.70
47.46AB±0.60
8.53AB±0.22
5.57AB±0.14
-
E-III ( X SD)
20.17A±0.32
25.51A±0.32
54.33B±0.48
49.79BC±0.41
4.54BC±0.22
11.00BC±0.54
-
-
-
-
Experiment 2
C ( X SD)
E ( X SD)
22.21A±0.40 17.93A±0.27
38.31A±0.20 25.71A±0.24
39.48A±0.60 56.36A±0.32
37.98A±0.55 53.94A±0.18
1.47A±0.07
2.38A±0.22
25.78a±1.11 22.80a±2.21
ND
2.57±0.02
ND
2.55±0.01
ND
5.12±0.03
Legend: A, B, C the same letter indicates significant difference of p<0.01; a the same letter indicates significant difference
of p<0.05
In the Experiment 2, significant differences between the contents of fatty acid in the feed of control
and experimental groups were observed. SFA and MUFA in the feed of the control group were
significantly higher (p<0.01) than in feed of the experimental group. The content of n-3 and n-6
fatty acids, was significantly higher (p<0.01) in the feed of the experimental group. Similarly the
most beneficial ratio of n-6/n-3 was observed in the feed of the experimental group (p<0.05) (Table
2). CLA isomers c9t11CLA and t10c12CLA were detected in the feed supplemented with CLA
(experimental group) and were below the detection limit in the control group (Table 2).
The average content of SFA and MUFA in the meat (Experiment 1) was higher in the group of pigs
fed with linseed preparation (E-II), but on the other hand the average content of PUFA in this group
was lower as well as the n-6/ n-3 ratio compared to the other two groups of pigs (p<0.01) (Table 3).
In the Experiment 1, meat from the group which was fed with linseed preparation, E-II group, had
the highest percentage of SFA (37.91±0.75%), which was significantly higher (p<0.01) compared to
the E-I group, while between E-II and E-III groups no significance was observed (Table 3).
Table 3. Fatty acid composition in meat (%), and ratio
Experiment 1
FA
SFA
MUFA
PUFA
n-6
n-3
n-6/n-3
c9t11CLA
t10c12CLA
c9t11CLA+
t10c12CLA
Legend: A the same
p<0.05
Experiment 2
E-I ( X SD)
36.98A±0.25
39.59AC±0.12
23.17AC±0.28
22.53AC±0.28
0.69AC±0.01
32.40AC±0.51
-
E-II ( X SD)
37.91A±0.75
43.71AB±0.28
17.99AB±0.44
16.98AB±0.56
1.23AB±0.03
13.67AB±0.08
-
E-III ( X SD)
37.54±0.05
41.42BC±0.05
20.87BC±0.08
19.76BC±0.07
1.12BC±0.02
17.84BC±0.55
-
C ( X SD)
43.33A±1.38
46.72A±1.88
9.95a±0.60
9.51±0.57
0.32±0.04
29.72a±3.60
ND
ND
E ( X SD)
53.48A±1.07
37.53A±0.28
8.99a±0.92
8.56±0.90
0.36±0.03
24.58a±2.97
2.37±0.01
1.19±0.01
-
-
-
ND
3.56±0.71
letter indicates significant difference of p<0.01; a the same letter indicates significant difference of
151
Meat from E-II group (Experiment 1) of pigs contained 43.71±0.28% of MUFA, which was
significantly higher (p<0.01) and 17.99±0.44% of PUFA, which was significantly lower (p<0.01)
compared to E-I and E-III group. The meat of pigs that were fed with soy (E-III group) was
41.42±0.05% of MUFA and 20.87±0.08% of PUFA, which was significantly higher (p<0.01)
compared to the E-I group that was fed with sunflower grain, as a source of fatty acids (Table 3).
It was found that the average content of saturated fatty acids in the meat of the experimental group
of pigs (53.48±1.07%) was significantly higher (p<0.01) than the average content of saturated fatty
acids in the meat of pigs of the control group (43.33± 1.38%). The average content of
monounsaturated or polyunsaturated fatty acids (37.53±0.28%, 8.99±0.92%, respectively) in the
experimental group pig meat was significantly lower (p<0.01, p<0.05, respectively) than the
average content of monounsaturated or polyunsaturated fatty acids in the meat of pigs of the control
group (46.72±1.88%, 9.95 ± 0.60, respectively). There was no statistically significant difference
between the average content of n-3 and n-6 fatty acids in meat sample (0.36±0.03%, 8.56±0.90%,
respectively) and a control group of pigs (0.32±0.04%, 9.51±0.57%, respectively).
The average ratio of n-6/n-3 fatty acids in meat pigs experimental group (24.58±2.97) were
significantly lower (p<0.05) than the same ratio in the meat of the control group pigs (29.72 ± 3.60)
as shown in Table 3.
The average content of conjugated linoleic acid c9t11, t10c12 and total CLA content in the meat of
pigs of the experimental group was 2.37±0.01%, 1.19±0.01% and 3.56±0.71% (respectively). In the
meat of the control group of pigs the CLA was not detected (Table 3).
Discussion
A negative attitude towards the consumption of meat has many different causes. One of the most
common is the health concern. Today, virtually everyone knows that there is a connection between
diet and health. Scientific discoveries and better informed consumers have contributed to the
understanding of these connections. Among the risk factors of numerous chronic noncommunicable diseases (cardiovascular and cerebral diseases, hypertension, malignant neoplasms,
diabetes, obesity, nephrolithiasis, bile duct diseases, osteoporosis, dental caries), which are
numerous (more than 200), nutrition is of the great importance.
The fatty acid profile of the meat directly reflects the fatty acid profile in feed (Eastwood, 2008).
Since linseed has a desirable fatty acid composition, many producers are interested to include
linseed in swine finisher in order to improve the fatty acid composition of meat.
In this study (Experiment 1) results indicate that by changing sources of fat and fatty acid
composition of feeds, the fatty acid composition of meat can be affected. The average content of
saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids, as well as the
average contents of n-6 and n-3 fatty acids in the feed, between groups, were statistically
significantly different.
It has been found that the average content of saturated fatty acids in pork meat of pigs fed with the
addition of linseed was significantly higher compared to the SFA content of the meat from pigs fed
with the addition of sunflower. The average content of polyunsaturated fatty acids in pork from pigs
fed with the addition of linseed was significantly lower compared to average content of these acids
in pork from pigs in the other two groups (p <0.01) (Todorovic, 2014).
The average content of n-6 fatty acids in meat was significantly lower, and content of n-3 fatty acids
significantly higher in pork meat of pigs fed with the addition of linseed (p<0.01). Pork from pigs
fed with additions of linseed had a statistically significantly better ratio of n-6/n-3 fatty acids.
152
Apart from the optimal quantity of essential fatty acids in nutrition, their ration is also very
important. The ratio of n-3/n-6 fatty acids is optimal if it is in a range 1:4 to 1:5. It is recommended
that polyunsaturated fatty acids present 10 to 20% of total daily intake of lipids. Fatty acids n-3
have a protective effect on the cardiovascular system and the decrease of frequency of deaths
caused by cardiovascular diseases. These acids decrease triglycerides in blood, and blood pressure,
regulate the activity of protein kinase C that have a role in angiogenesis and slow down the growth
of tumor metastases. An important source of n-3 fatty acids is the fish meat of northern sea fish
(mackerel, herring, sardines, salmon) (Losso, 2002; Pariza, 1997).
One of the first studies about the effects of linseed on meat lipid profile was performed by Cunnane
et al. (1990). In this experiment, from the age of two till the tenth week of age feed was
supplemented with 5% of linseed. Piglets had significantly higher levels of ALA in the liver,
kidney, heart, skin, subcutaneous adipose and muscle tissue.
Later, other researches performed trials in order to determine the optimal level and the length of
supplementation of linseed in feed, in order to ensure that the enrichment of n-3 fatty acids does not
negatively affects meat quality. Romans et al. (1995a) studied the influence of different
concentrations of linseed in feed (5, 10 and 15%) 25 days before the slaughter. These authors
concluded that linseed should not be used in the feed of finisher pigs at the level higher than 15% in
order to maintain the meat sensory properties. In the other study, Romans et al. (1995b) studied the
different length of linseed use in feed (15% linseed) 7, 14, 21 and 28 days before the slaughter. The
ALA content of back fat adipose tissue significantly linearly increased with the increase in the
length of feed supplementation with linseed. A large number of experiments were performed in
final pig fattening stage (Raj et al., 2010; Matthews et al., 2000; Václavkova and Bečkova, 2007;
Enser et al., 2000 and Thacker et al. 2004).
Okanović et al. (2010) investigated the impact of feed enriched with linseed on the content of n-3
fatty acids in pork, from pigs with an average weight of 110 kg. Feed containing linseed resulted in
higher concentrations of n-3 fatty acids (> 7 mg/100 g), which decreased ratio of n-6 and n-3 fatty
acids in meat (<3) making it better for human health.
The CLA concentration in muscle tissue may be further enhanced when CLA supplementation is
combined with additional dietary fat (Gatlin et al., 2002). Adding CLA in the mixture for pigs
(Experiment 2) has significantly influenced the fatty acid composition of the mixture. It was found
that the experimental group of pigs had significantly increased the content of polyunsaturated fatty
acids (PUFA), while the amount of saturated and monounsaturated fatty acids was reduced in the
same group. Adding a CLA mixture to the feed of experimental groups of pigs increased content of
n-6 fatty acids, as well as the void content of n-3 fatty acids. The swine feed by adding CLA to
change the attitude of n-6/n-3 fatty acids, which is caused by the content of these acids in mixtures.
Results of n-6/ n-3 fatty acid ratio in the diets of the control and experimental groups of pigs are
shown in Table 2. The average content of isomers c9t11CLA and t10c12CLA in feed for the pigs of
the experimental group were nearly identical, which is understandable, given the fact that the
Lutalin® preparation, according to the producer statement contains 50% and 50% c9t11CLA
t10c12CLA (Table 2).
As a part of these investigations have been two isomers of CLA, c9t11CLA and t10c12CLA, as a
supplement mixture for pigs. Both of these acids were found in the meat of pigs, provided that the
content c9t11CLA was almost twice the content t10c12CLA (Table 3).
It is notable that it is not the same degree of adoption and incorporation of both isomers of
conjugated linoleic acid were observed in the feed. Isomer c9t11 is more suitable for installation in
intramuscular fat depots (Pantić, 2014).
A number of the current research is directed at manipulating the fatty acid content of meat animals
in order to increase n-3 and CLA content (Enser et al., 1998). The cereal-based diet commonly
153
offered to poultry and pig supplies mainly n-6 PUFA and a small amount of n-3 PUFA. This is
reflected in the fatty acid composition of the animal product. Dietary modification of poultry meat,
eggs or pork meat in order to increase the content of n-3 PUFA requires a supply of n-3 PUFA from
the diet (Nieto and Ros, 2012). Many studies in order to manipulate the fatty acid composition of
the meat using whole oilseeds have been conducted.
Several reports indicate that CLA supplementation increases the amount of saturated fatty acids
(C14:0, C16:0, and C18:0) and decreases the amount of MUFA fraction (mainly C18:1) in pig the
tissues by down-regulating the D9-desaturase activity (Eggert et al., 2001; Gatlin et al., 2002; Joo et
al., 2002; Lauridsen et al., 2005; Smith et al., 2002; Thiel-Cooper et al., 2001).
Conclusion
A diet enriched with extruded linseed had a beneficial impact on the content of n-3, n-6 acids and
the ratio of n-6/n-3, which is important for the health of consumers. Chemical analysis of the fatty
acid composition of complete mixtures found that the feed which was included preparation of
linseed had significantly lower SFA content, and content is significantly higher PUFA mixture of
full fat soybean meal. Also, the ratio of n-6/n-3 fatty acids in feeds linseed group was lower (5.567)
in comparison to a composition for a full fat soybean meal group (11.00) (p<0.01). By adding a
mixture of linseed to the mixture for finishing pigs the meat with favorable fatty acid composition
and favorable ratio of n-6/n-3 fatty acids compared to pigs which had no linseed in the diet was
obtained.
The inclusion of high dose of dietary CLA in pigs of conventional genotype (♂Yorkshire ×
♀Landrace) increased the CLA content in pig meat. Also, the addition of CLA in the diet of pigs
increases the content of the SFA and reduces the content of MUFA and PUFA in the pig meat.
The results from this study show that the pig products can be modified to in order to provide a
significant increase the functional lipids, which can have a positive influence on the human health.
Acknowledgment
The study was conducted within the project "The selected biological threat to the safety/quality of
food of animal origin and control measures from the farm to the consumer", TR 031034 financed by
the Ministry of Education, Science and technological development of the Republic of Serbia.
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156
EFFECTS OF SOME DIEATARY SUPPLEMENTATION WITH PHYTONUTRIENTS ON
SELECTED BIOCHEMICAL PARAMETERS AND GROWTH PERFORMANCE IN
BROILER CHIKENS
Milanka Jezdimirović1*, Blagoje Dimitrijević1, Saša Ivanović1, Nemanja Jezdimirović2, Mila
Savić1, Dragan Bacić1, Slavoljub Jović1
1
University of Belgrade, Faculty of veterinary medicine, Serbia
2
* Corresponding author: milanka@vet.bg.ac.rs
Abstract
Maintaining bird health, regarding diseases or agents acting on the gastrointestinal tract, is crucial in
broiler production, since this is the entry route of nutrients for bird development. Also, it is well
known that broiler production is impossible without antibiotics, which are used as growth promoters
and in order to suppress overgrowth of pathogenic microflora in the gastrointestinal tract. Taking
into account this, we investigated the growth promoting and beneficial effects of three
phytoadditives (Ocimum basilicum, Thymus vulgaris and Pimpinella anisum) on growth
performances (body weight, total feed intake, feed conversion ratio and carcass yield),
concentrations of glucose, total proteins, albumin, total bilirubin, triglycerides and activity of
creatine kinase enzyme, in broiler chicks at 56 days of age. On the day of hatching 150 broiler
chicks, were divided into five equally groups (n=30). The control group (1st group) of broilers
received a basal diet (BD) without any feed additive. The second, third and fourth groups of chicks
were fed BD enriched with 1% of Ocimum basilicum pulverised herba, 0.5% of Ocimum basilicum
and 0.5% of Thymus vulgaris pulverised herba and 0.5% of Thymus vulgaris herba and 2% of
Pimpinella anisum pulverised fructus, respectively. The fifth group was fed with BD mixed with
Salinomycin (60 ppm). The results indicated that feeding the diets enriched with selected herbal
supplement failed to affect the growth performance of chickens at 56 days of age. In addition, this
supplementation had no influence on glucose metabolism, but we observed beneficial effects on
some parameters of protein metabolism. The concentration of total proteins was significantly higher
in chicks fed with phytoadditives; 41.71±1.08 vs 35.88±1.04 g/L; p<0.01. Albumin concentration
was also significantly higher in experimental groups (28.29±1.32 vs 19.73±0.51 g/L; p<0.001).
Creatine kinase activity and total bilirubin concentration were significantly reduced in broilers fed
with phytoadditives; p<0.05. Ocimim basilicum in combination with Thymus vulgaris decreased the
concentration of tryglicerides (0.89±0.02 vs 1.23±0.05 mmol/L; p<0.001), but this was not the case
with other phytoadditives.
It could be concluded that supplementation of the diet with phytoadditives has a potential to
improve health status in broilers. Further examinations are needed in order to elucidate the exact
mechanism of action.
Key words: phytonutrients, growth performance, blood chemistry, broiler chickens
Introduction
Expansion of the poultry industry holds the greatest promise for bridging the animal protein gap in
the world. This is mainly due to the short generation interval of poultry, particularly of broilers.
However, the rising cost of poultry feed, problems of drugs residues, microbial resistance and
diseases have become major problems militating against the industry (Puvača et al., 2013).
Maintaining bird health, regarding diseases or agents acting on the gastrointestinal tract, is crucial in
157
broiler production, since this is the entry route of nutrients for bird development. Also, it is well
known that broiler production is impossible without antibiotics and coccidiostats, which are used as
growth promoters and in order to suppress overgrowth of pathogenic organisms in the
gastrointestinal tract.
The prophylactic use of antibiotics in poultry nutrition in order to cause improvements in growth,
feed consumption, feed utilization and decreased mortality from clinical diseases is well
documented. But, the growing concern over the transmission and the proliferation of resistant
bacteria via the food chain has led to a ban of the feed use of antibiotic growth promoters in
livestock within the European Union since 2006 (Brenes and Roura, 2010). As a result, new
commercial additives derived from plants including aromatic plant extracts and their purified
constituents have been examined as a part of alternative feed strategies for the future. Such products
have several advantages over commonly used commercial antibiotics since they are residue free and
they are also, generally recognized as safe and commonly used items in the food industry (Lv et al.,
2011). These botanicals have received increased attention as possible growth performance
enhancers for animals in the last decade. Also, „natural“ methods are likely to play an increasing
role in the control of the disease since they are well accepted by consumers. Plant products are
residue-free, and function by mechanisms other than those of chemotherapeutics, involving new
therapeutic molecules to which resistance has not yet developed (Orengo et al., 2011).
Plants and their extracts have been used for many centuries as treatments for ailment of many
pathological conditions, yet only in the past 20-30 years have scientists seriously begun to
determine whether plant-derived traditional remedies are effective, and, if so, their mode of action.
Less than 10% of approximately 250000 of the world’s flowering plant species have been
investigated scientifically for their pharmacological properties but almost 25% of active medical
compounds currently prescribed in the USA and UK were isolated from higher plants. Plants are an
important source for drug discovery – particularly for parasites because of the long association
between the coexistence of parasites, humans and herbal remedies (Anthony et al., 2005). Recently,
a number of plants and their extracts have been used in poultry nutrition. Their functional
substances (such as flavonoids, polyphenols and terpenoids) are mainly secondary metabolites
synthesized by plants to deter herbivorous predators, repel competitors and attract pollinators
(Petrović et al., 2011).
Taking into account this, we investigated the growth promoting and beneficial effects of three
phytoadditives (Ocimum basilicum, Thymus vulgaris and Pimpinella anisum) grown and harvested
in Serbia, on growth performances, concentration of glucose, total proteins, albumin, total bilirubin,
triglycerides and activity of creatine kinase enzyme, in broiler chicks at 56 days of age.
Plant materials
The herba of basil (Ocimum basilicum) and thyme (Thymus vulgaris) and fructus of Pimpinella
anisum were collected in Vojvodina (North Province of Serbia), dried at room temperature away
from sunlight. The dried leaves and fructus were pulverised and kept at 8 oC before mixing with
broiler diets.
Experimental animals and diets
On the day of hatching 150 broiler chicks, were randomly divided into five equally groups (n=30).
The broilers were kept in large pens on wood shavings. On the day of hatching, the room
temperature was kept at 32 oC and then was gradually decreased by 3 oC per week to a final
temperature of 23 oC on the day 21, which was then maintained constantly. Continuous lighting
158
regimen (24 h of light per day) was kept throughout the fattening period. Relative humidity in the
room was maintained at 70%.
All birds were fed ad libitum with the commercial standard diets for broilers (Veterinarski ZavodZemun, Serbia): „Starter“ from day 1 to 14; „Grower“ from day 15 to 35 and „Finisher“ from day
36 to 56. The control group (1st group) of broilers received a basal diet (BD) without any feed
additive. The second, third and fourth groups of chicks were fed BD enriched with 1% of Ocimum
basilicum pulverised herba, 0.5% of Ocimum basilicum and 0.5% of Thymus vulgaris pulverised
herba and 0.5% of Thymus vulgaris herba and 2% of Pimpinella anisum pulverised fructus,
respectively. The fifth group was fed with BD mixed with Salinomycin (60 ppm). The broilers had
free access to feed and water.
All experiments were performed according to our institutional guidelines for animal research and
principles of the European Convention for the Protection of Vertebrate Animals Used for
Experimental and Other (Official Daily N. L 358/1–358/6, 18, December 1986).
Sample collection
None of broiler chickens in any group died during the trial. The body weight of broilers, feed
conversion ratio and carcass yield were among the growth parameters studied and recorded on the
day 56 of life. On day 56 of age all broilers were slaughtered by decapitation. Blood was taken
without anticoagulants’ presence, enabling the separation of blood serum. Sera were obtained after
a spontaneous blood coagulation attained by centrifugation lasting 10 min at 3000 rpm. Thus
obtained blood sera were frozen at -20 oC until further analysis.
Biochemical assaying
The kinetic method was used to determine creatine kinase (CK) enzyme activity, while triglycerides
concentration was determined after enzymatic hydrolysis with lipases and quinoneimine as
indicator. The biuret method was used to determine total protein concentration, while bromocresol
green was used to determine albumin concentration. Total bilirubin (the sum of conjugated and
unconjugated bilirubin) was determined in the reaction with diazonium ion of sulphanilic acid. All
of the above mentioned biochemical parameters were determined using commercial kits (Bayer
Diagnostics, Germany). Spectrophotometric measurements were performed with Cecil CE
2021UV/VIS spectrophotometer. Blood glucose concentration was determined by using PrecisionXtra plus test strips.
Statistical significance of differences of all examined parameters were determined by means of the
ANOVA, followed by the Tukey test. Data were expressed as means ± standard error. Significance
level was set at p < 0.05. Statistical analysis was performed using the Graph Pad Prism 5.0
Software, CA, USA.
Results
The effect of diet supplementation with 1% of Ocimum basilicum, 0.5% of Ocimum basilicum and
0.5% of Thymus vulgaris and 0.5% of Thymus vulgaris herba and 2% of Pimpinella anisum, in
experimental groups, as well as in control groups (C1 – fed with BD; and C2 – fed BD plus
Salinomycin) on the body weight, total feed intake, feed conversion ratio and carcass yield are
shown in Table 1.
159
Our results showed no statistically significant differences (p > 0.05) in the growth performance
indices (such as body weight, total feed intake, feed conversion ratio and carcass yield), between
groups of chicken.
Table 1. The effects of diet supplementation with 1% of Ocimum basilicum (I group), 0.5% of Ocimum
basilicum and 0.5% of Thymus vulgaris (II group) and 0.5% of Thymus vulgaris herba and 2% of Pimpinella
anisum (III group); Control groups (C1 – fed with BD and C2 – BD plus Salinomycin) on growth
performance of broilers at the age of 56 days
C1
I group
II group
III group
C2
2602±64.99
2654±70.48
2573±75.04
2375±55.25
2694±91.49
Total feed intake (g)
8405
7214
6985
7365
8210
Feed conversion ratio
2.15
1.99
1.87
1.89
2.05
Carcass yield
1605
1794
1768
1601
1715
Body weight
Concentrations of total proteins (Fig. 1) were statistically significant higher in groups of chickens
supplemented with phytoadditives; the higher concentrations were recorded in I (41.02±0.76 g/L; p
< 0.01) and III groups (41.71±1.08 g/L; p < 0.001) compared to C2 group (35.26±1.06 g/L).
The albumin concentrations (Fig. 2) were also statistically significant higher in broilers fed with
phytoadditives. Also, in this case the higher albumin concentrations were determined in I
(28.29±1.32 g/L; p < 0.001) and III groups (26.50±0.89 g/L; p < 0.001) compared to C1
(19.73±0.51 g/L) and C2 (19.31±0.53 g/L) groups. But it is also worth to mention that we
determined higher values of albumin in I group (fed BD plus 0.5% basil) compared to II group
(24.39±0.85 g/L; p < 0.05).
Total proteins g/L
50
**
+
***
++
*
40
30
20
10
2
C
gr
ou
p
III
ro
up
II
g
1
C
Ig
ro
up
0
Fig. 1. The effects of diet supplementation with 1% of Ocimum basilicum (I group), 0.5% of Ocimum basilicum and
0.5% of Thymus vulgaris (II group) and 0.5% of Thymus vulgaris herba and 2% of Pimpinella anisum (III group); C1 –
control group fed with BD; C2 – control group fed with BD plus Salinomycin) on concentration of total proteins in
broilers at the age of 56 days; + p<0.05; ++ p<0.01 vs. C1 control group; ** p<0.01; *** p<0.001 vs. C2 control group
Creatine kinase activity (Fig. 3) is another biomarker which can be used for evaluation, not only of
functional status of hepatocytes, but also for skeletal muscle integrity. In this experimental design
we determined the lowest activity in group of broilers fed BD with 0.5% of Ocimum basilicum and
0.5% of Thymus vulgaris (403.4±30.55 U/L; p < 0.01) and broilers fed BD with 0.5% of Thymus
vulgaris herba and 2% of Pimpinella anisum (470.1±41.06 U/L; p < 0.05), compared to C2 group
(695.8±74.5 U/L). There were no differences between C1 group (BD only) and I group of broilers
(BD plus basil), p > 0.05.
160
40
#
***
+++
Albumin g/L
30
***
+++
**
++
20
10
2
C
III
gr
ou
p
ro
up
II
g
Ig
ro
up
C
1
0
control group fed with BD; C2 – control group fed with BD plus Salinomycin) on albumin concentration in broilers at
the age of 56 days; ++ p<0.01; +++ p<0.001 vs. C1 control group; ** p<0.01; *** p<0.001 vs. C2 control group; # p<0.05 I
group vs. II group
Creatine kinase U/L
1000
800
600
*
**
400
200
2
C
III
gr
ou
p
ro
up
II
g
Ig
ro
up
C
1
0
control group fed with BD; C2 – control group fed with BD plus Salinomycin) on creatine kinase activity in broilers at
the age of 56 days; * p<0.05; ** p<0.01 vs. C2 control group
Total bilirubin mol/L
15
10
**
+
*
5
2
C
gr
ou
p
III
ro
up
II
g
Ig
ro
up
C
1
0
control group fed with BD; C2 – control group fed with BD plus Salinomycin) on concentration of total bilirubin in
broilers at the age of 56 days; * p<0.05; ** p<0.01 vs. C2 control group
161
Concentrations of total bilirubin (Fig. 4) were the lowest in III group of broilers (8.11±0.16 µmol/L;
p < 0.01) and in II group (8.27±0.31 µmol/L; p < 0.05) compared to C2 group. It is of interest to
note that we did not find statistically significant difference between C1 (BD only) and C2 groups
(BD plus Salinomycin); p > 0.05.
Triglycerides concentration (Fig. 5) was the lowest in II group of broilers fed with addition of 0.5%
of Ocimum basilicum and 0.5% of Thymus vulgaris (0.89±0.02 mmol/L; p < 0.001), compared to C1
(1.23±0.05 mmol/L) and C2 (1.14±0.03 mmol/L). The same statistical differences (p < 0.001) were
noticed between I (1.20±0.02 mmol/L) and II group (1.25±0.06 mmol/L) of broilers.
We did not find any significant differences (p > 0.05) between broiler groups regarding
concentration of glucose (Fig. 6)
Triglycerides mmol/L
1.5
###
***
+++
1.0
0.5
2
C
III
gr
ou
p
ro
up
II
g
1
C
Ig
ro
up
0.0
control group fed with BD; C2 – control group fed with BD plus Salinomycin) on concentration of triglycerides in
broilers at the age of 56 days; +++ p<0.001 vs. C1 control group; *** p<0.01 vs. C2 control group; ### p<0.001 I group vs.
II group
Glucose mmol/L
15
10
5
2
C
gr
ou
p
III
up
ro
II
g
Ig
ro
up
C
1
0
control group fed with BD; C2 – control group fed with BD plus Salinomycin) on concentration of glucose in broilers at
the age of 56 days
Discussion & Conclusion
The practice of feeding livestock with subtherapeutic levels of antibiotics has been in use for over
fifty years. But, usage of antibiotics has negative effects on animal’s health and production
162
(Marković et al., 2009). Furthermore, now days consumers request poultry products that are free
from residual chemotherapeutics. The use of natural products as an alternative to drugs may be the
best solution to this consumer demand (Harper and Makatouni, 2002).
The herbal products are complex mixtures of ingredients, where the relative concentrations of each
phytomolecule may vary considerably (Orengo et al., 2012). Instead of testing each and every one,
our study focuses on three plants used alone or in combination. The aim of this research was to find
what plant and effective doses of herbal additives could have a beneficial impact on the growth
performance and health status of broilers.
Plants and their extracts (called essential oils) possess antibacterial, antifungal and antiviral
properties and have been screened worldwide as potential sources of novel antimicrobial
compounds, alternatives to treat infectious diseases and agents promoting food preservation
(Solorzano-Santos and Miranda-Novales, 2011). Weight gain is one of the most sensitive and
informative measure of efficacy of certain additives (Conway et al., 1999). From productive point
of view, it is of interest to mention that our results showed that phytoadditives can improve growth
performance in broilers, although there were no statistically significant differences between control
and experimental groups (Table 1). But, it is obvious from results showed in Table 1, that broilers
received BD with 0.5% of Ocimum basilicum and 0.5% of Thymus vulgaris had a better feed
conversion ratio and carcass yield compared to other groups of broilers. Our trial was performed
under highly hygienic conditions, which could have alleviated the animals’ response.
Environmental conditions, such as density and stress status of the animals, are important for
detecting performance responses to plants used as feed additives (Catala-Gregori et al., 2008).
According to Ertas et al. (2005), the combination of some plants extracts could present better effect
on the growth performance in poultry in comparison with their individual supplementation.
Synergism among some herbal constituents was highlighted in the in vitro studies performed by
Montes-Belmont and Carvajal (1998). Moreover, Burt (2004) reported that an antagonistic effect
has been expected as well. We presumed that potential synergistic effects between basil and thyme
could result in beneficial effect on both the growth performance of broilers and their
hepatoprotective effects.
Although total protein concentrations were also affected by phytoadditives in BD (Fig. 1), the most
remarkable are results in albumin concentrations (Fig. 2). Our results showed that 1% of basil had
the best stimulatory effect on hepatocytes to synthesized albumin, since the liver is the only site for
its synthesis. It is possible that eugenol and carvacrol, two major ingredients of basil (Lv et al.,
2011), are responsible for such liver response. Total proteins, also were much higher in herbal
supplemented broilers; the most higher values were recorded in group supplemented with mixture
of 0.5% basil and 0.5% of thyme (Fig. 1). If one takes into account that globulins are the second
protein fraction, very important as a part of defensive systems of an organism (and that they are
elevated in this case), the contribution of this herbal combination should not be neglected in
stimulation of broilers immune system.
Dietary supplementation of our plants combination to chickens for 8 weeks did not cause a
significantly lower blood plasma glucose concentration, and in this respect our results are
contradictory. Namely, a number of spices and herbs have a long history of traditional use in
treating elevated blood sugar levels. For example Jarvill-Taylor et al. (2001) reported that cinnamon
stimulated glucose uptake, glycogen synthesis, and activated glycogen synthase in 3T3-L1
adipocytes. Later studies of Bakirel et al. (2008) showed that ethanolic extracts of rosemary leaves
lowered blood glucose in normoglycemic and glucose-hyperglycemic rabbits.
In the present experiment, there was a significant decrease in total bilirubin concentration in birds
fed diets supplemented with 0.5% basil and 0.5% thyme (II group) and 0.5% basil and 2% anis (III
group). A decrease in total bilirubin level in the blood sera in the current study could be explained
by stimulation of uridine diphosphate glucuronyltransferase enzyme (UDP-glucuronyltransferase),
163
or by protective effects of essential oils presents in our combination of plants on wall of liver cells
(Figs. 3 and 4). There are several reports concerning an inhibition or activation of hepatic UDP
glucuronyltransferase by certain plant constituents. Siraki et al. (2005) found out that borneol
inhibited glucuronidation of non steroidal antiinflammatory drugs in isolated rat hepatocytes. This
could be also applied to activity of creatine kinase activity. Namely, we found significantly decrease
of its activity in the blood sera of broilers fed with combinations of basil and thyme (II group) and
basil and anis (III group). Undoubtedly, these findings coupled with total bilirubin concentration,
suggest that wall of liver cells was strengthened in some way (Bakkali et al., 2008).
Little is known about the effects of herbs on lipid metabolism of broilers. The effect of some
functional herb substances (cyclic terpenes) on serum cholesterol and total lipids in poultry were
reported by Faxova et al. (2009). On the other hand, no changes in serum cholesterol and plasma
lipids because of dietary supplementation of herb extracts (some monoterpens and essential oils) to
broilers were observed. Najafi and Torki (2010) found that total cholesterol, triglycerides and highdensity lipoproteins in the blood of broilers did not respond to the dietary supplementation of clove
extract. Our results are in line with these findings, except for the combination of 0.5% basil and
0.5% thyme (Fig. 5), where this combination of phytoadditives significantly decreased the
concentration of triglycerides in the blood of broilers.
It could be concluded that supplementation of the diet with phytoadditives has a potential to
improve health status in broilers. Further examinations are needed in order to elucidate the exact
mechanism of action.
Acknowledgment
This work was granted by Ministry of education, science and technological development, Republic
of Serbia; Grant No III 46009.
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Current Opinion in Biotechnology, 23, 1-6, 2011
165
THE INFLUENCE OF CLIMATIC FACTORS IN SERBIA ON MYCOTOXIN
PRODUCTION
Sandra Jakšić1*, Milica Živkov-Baloš1, Nadežda Prica1, Zoran Mašić1, Ksenija Nešić2, Igor Jajić3,
Biljana Abramović4
1
3
Faculty of Agriculture, University of Novi Sad, Serbia
4
Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia
* Corresponding author: sandra@niv.ns.ac.rs
2
Abstract
Marked climate changes led to frequent droughts and high temperatures that favor the growth of
molds, occurrence of plant diseases and pests and consequently, the toxin production. Having in
mind characteristic climatic conditions in Serbia in 2011/12 and 2013/14 production years, our
research was aimed at investigating their influence on contamination of animal feed and potential
consequent poisoning of milk by specific mycotoxins. The available results on crops contamination
for each year were correlated with the climatic conditions characteristic for the relevant year.
Extremely warm and dry 2011/12 production year (according to the report of Republic
Hydrometeorological Service of Serbia) was characterized also by high aflatoxin levels in maize.
Out of 67 examined maize samples, 61.2% were contaminated with aflatoxins, whereas 24% of
examined 75 milk samples contained aflatoxin M1 at concentrations above 0.5 µg/kg. Contrary to
that, extremely humid climate conditions in 2013/14 production year resulted in particularly high
concentration of Fusarium toxins. Thus, 100% of 21 examined maize samples were contaminated
with deoxynivalenol and fumonisins, and 90% with zearalenone. Consequently, high percentage of
16 examined samples of complete feed mixtures for pigs were declared unsafe, that is, contaminated
with deoxynivalenol (75%) and zearalenone (44%). Comparison of the data on climatic conditions
with the levels and incidence of samples contaminated with aflatoxins and Fusarium toxins
revealed negative effects of climatic deviations (precipitation rate and temperature) in the territory
of Serbia on mycotoxicological safety of maize, and consequently animal feed and milk.
Keywords: mycotoxins, climate, feed, milk
Introduction
Mycotoxins are toxic compounds produced by filamentous fungi, which often contaminate cereal
food and feeds. Mycotoxins may possess carcinogenic, cytotoxic, immunosuppressive, neurotoxic,
estrogenic or teratogenic activity (Nešić et al., 2014). The mycotoxins of greatest concern in Serbia
usually were Fusarium toxins such deoxynivalenol (DON, Jajić et al., 2008) and zearalenone (Jajić
et al., 2013), but also ochratoxin A as Aspergillus toxin (Jakšić et al., 2011). In warm and humid
subtropical and tropical conditions, maize ears are ideal conditions for colonization and dominance
of Aspergillus flavus/parasiticus species, resulting in the formation of aflatoxins (Sanchis and
Magan, 2004). Until now, aflatoxins were not identified as common contaminant of cereals in the
territory of Serbia (Jakšić et al., 2011; Kos et al., 2013). Moreover, high concentrations of
fumonisins were not recorded either (Jakšić et al., 2012), thus, they were not addressed in legal
regulations pertaining to animal feed (Official Gazette RS, 2014). Mycotoxins cause individual
toxic effects to human and animal health; however, cumulative effect of low mycotoxin
166
concentration and potential synergistic effects of low levels of multiple mycotoxins are issues that
are receiving growing attention (Stojanov et al., 2013).
Production of mycotoxins on crops depends on climatic factors such as temperature and relative
humidity, thus, changing climate has a direct impact on mycotoxin production (Paterson and Lima,
2011). Accurate information is therefore needed on the impact of and relationship between key
factors related to climate conditions (water availability and temperature), and which are marginal
and which are optimum for germination, growth and toxin production (Sanchis and Magan, 2004).
Table 1 shows the optimal temperatures and water activity (aw) for mycotoxins production and
growth in vitro for some important plant pathogenic fungi.
Table 1. Optimal temperature (ºC) and water activity (aw) for mycotoxin production and growth on
different substrates (Sanchis and Magan, 2004).
Fungus species
Aspergillus flavus
Aflatoxins
33
(0.99 aw)
Mycotoxins production
Ochratoxin
Zearalenone DON
15−30
(0.93 aw)
Fusarium
verticillioides,
F. proliferatum
25
(>0.98 aw)
F. graminearum
A. ochraceus
Penicillium
verrucosum
Fumonisins
25−30
(0.98 aw)
25
(0.90−0.95 aw)
30
(0.99 aw)
Growth
35
(0.95 aw)
30
(0.90 aw)
20−22
(0.98-0.995 aw)
30
(0.96−0.98 aw)
25
(0.95 aw)
Conditions adverse to the plant (drought stress, temperature stress, stress induced by pest attack,
poor nutrient status, etc.) encourages the fungal partner to develop more than under conditions
favourable to the plant with the expectation of greater production of mycotoxins. On the other side,
factors influenced by climate such as insect and other pest attack, soil condition and nutrient status
and agro-industrial methodology are potential and indirect triggers of fungal colonization and
mycotoxins production (Tirado et al., 2010). Temperature and rainfall are the climatic factors that
are most likely to be affected widely by future global change, and alterations in these are expected
to have a wide range of impacts on plants and on mycotoxins concentrations in plants (Miraglia et
al., 2009).
Maize can support different mycotoxin-producing molds, such as F. graminearum, F.
verticillioides, and A. flavus, and the dominant species is determined by meteorological conditions.
F. verticillioides, a producer of the fumonisin toxins, is the most common species on maize in
Southern Europe. Fumonisins have been associated with dry weather during grain fill and late
season rains. Therefore, the production of the toxins will be favored by the foreseen climate change.
While drought years may take most of crop value in the case of aflatoxin problems, mild
temperature and rain during maize plant growth is conducive to plant infection by F. graminearum
and DON levels (Miraglia at al., 2009). Therefore, changes in global temperature would directly
affect their growth and mycotoxins production capacity.
According to Paterson and Lima (2010) the biggest risk with respect to mycotoxins from climate
change will be found in developed countries with temperate climates (e.g. parts of Europe and the
United States of America, etc.) and the anticipated climate changes will present numerous
challenges for those involved in mycotoxins research and crop production in the near future.
167
A high priority over the next decade is the collation of accurate contamination and weather data,
together with the development of models to forecast the effects of climate change on mycotoxins,
with a view to providing the necessary foresight for strategic adaptation to climate change (Miraglia
et al., 2009; Paterson and Lima, 2010). Modelling studies provide increasingly realistic scenarios
for the influence of changes in the magnitude and variability of precipitation, temperature, etc. on
mycotoxins contamination.
Having in mind characteristic climatic changes that resulted in specific extreme conditions in Serbia
in 2011/12 and 2013/14 production years, our research was aimed at investigating their influence on
contamination of maize, feed and potential consequent poisoning of milk by specific mycotoxins.
The results of determining the contents of total aflatoxins in 67 maize samples, harvest 2012 are
presented in this article. The level of aflatoxin M1 was examined in 75 milk samples. The contents
of Fusarium toxins: deoxynivalenol, zearalenone and total fumonisins were examined in 21 maize
samples (harvest 2014) as well as 16 samples of complete pig mixtures and 7 complete poultry
mixtures. The samples originated from different localities of Serbia. The investigated samples were
a part of animal feeds that were submitted to our laboratory for regular control, yet suspect because
of evident health problems on related pig and poultry farms. Milk samples were collected from milk
collecting points or dairy plants, directly on the production line.
The presence of total aflatoxins, aflatoxin M1, deoxynivalenol, fumonisins (FB) and zearalenone in
corn, feed, and milk were analyzed by enzyme-linked immunosorbent assay methods, using
Ridascreen®FAST Aflatoxin (Art. No. R5202), Ridascreen®Aflatoxin M1 (Art. No. R1121),
Ridascreen®FAST DON (Art. No. R5901), Ridascreen®Fumonisin (Art. No. R3401) and
Ridascreen®FAST Zearalenon (Art. No. R5502) test kits (R-Biopharm, Germany). The color
intensity is measured photometrically at 450 nm (Multiskan FC, Thermo Scientific, China) and is
inversely proportional to the mycotoxin concentration in the sample. According to the
manufacturer´s description, the detection limits (DL) were 1.75 µg/kg (ppb) for aflatoxins, 5 ng/kg
(ppt) for aflatoxin M1, 0.2 mg/kg (ppm) for DON, and 25 µg/kg for fumonisins. Laboratory
detection limit for zearalenone was 60 µg/kg. The analytical quality of the ELISA method was
assured by the use of certified reference materials: TR-A100, lot #A-S-267, TR-F100, lot #F-C-439,
TR-D100, lot #DW-174 and ТR-Z100, lot #Z-C-320 (Trilogy Analytical Laboratory, Washington,
USA), as well as by participation in proficiency testing scheme (milk powder sample FAPAS
04224). Recovery was 101% for aflatoxins, 105% for aflatoxin M1, 103% for DON, 99% for
fumonisins, and 90% for zearalenone. Special software, the Rida®Soft Win (Art. No. Z9999, RBiopharm, Germany), was used for the evaluation of enzyme immunoassays.
Results and discussion
Contamination of maize and milk by aflatoxins in 2012
Having in mind characteristic climatic conditions in Serbia during 2012, which particularly affected
maize production, aflatoxin concentration has been examined in selected maize samples (Table 2).
Table 2. Aflatoxin content in maize samples in Serbia, harvest 2012.
DL
Min
Mean value
Positive/
Positive samples
Toxin
Max (µg/kg)
(µg/kg)
(µg/kg)
(µg/kg)
total no. of samples
(%)
Aflatoxins
1.75
2.11
156
37.36
168
41/67
61.2
As obvious from Table 2, 61.2% of samples revealed aflatoxin levels above DL. Out of 67
examined samples, 14 (21%) were unacceptable for animal feed, i.e., the level of total aflatoxins
exceeded 50 µg/kg, which was maximum permitted level in animal feed according to the relevant
Regulation in Serbia of that time (Official Gazette RS, 2010). Namely, pursuant to new Regulation
(Official Gazette RS, 2014), maximum permitted content of aflatoxin B1 in animal feed is 30 µg/kg,
thus in that case 22.4% of examined samples would have been declared unacceptable. Researches
on aflatoxin in maize (harvest 2012) in Serbia of other authors revealed similar results: 56%
positive samples out of which 5.4% with concentration above 50 µg/kg and with a maximum
concentration reaching even 145.8 µg/kg (Škrinjar et al., 2013); 68.5% positive samples with 29.5%
revealing toxin content >50 µg/kg (Kos et al., 2013).
Maize contamination with high levels of aflatoxins has lead to consequent milk contamination with
M1, a metabolic product of aflatoxin B1. The results of the examination of milk samples are
displayed in Table 3. Eighteen (24%) examined samples contained more than 0.5 µg/kg aflatoxin
M1, thus not complying with the legislative regulations in Serbia of that time (Official Gazette RS,
2013). When analyzing the obtained results in relation to EU regulations (EC, 2010), 73.3% of the
samples would have been declared unacceptable for human consumption because of aflatoxin
concentration above 0.05 µg/kg
Table 3. Contents of aflatoxin M1 in milk samples in Serbia in 2013.
No. of samples
Toxin
Aflatoxin M1
DL*
(µg/kg)
Positive/
total no. of
samples
Positive
samples
(%)
0.05−0.50
(µg/kg)
0.50−0.80
(µg/kg)
> 0.80
(µg/kg)
0.05
55/75
73.3
37
8
10
* Because of high toxin concentration and max permitted level of 0.5 µg/kg, the samples were diluted, thus
determination range encompassed concentrations from 0.05 to 0.80 µg/kg
According to the report of the Republic Hydrometeorological Service of Serbia, 2012-production
year was characterized by pronounced climatic changes, i.e., hydro-meteorological extremes
ranging from heat and cold waves to severe and prolonged drought. Three heat waves were
recorded in Serbia in the period from June to August 2012. Positive deviation of maximum daily
temperature as compared to the annual range of average daily temperatures was even up to 13°C.
Prolonged periods of extremely high air temperatures during June, July and August 2012 as well as
precipitation deficit resulted in severe and extreme draughts in many regions of Serbia.
Consequently, dramatically accelerated ripening of the majority of crops has occurred. Poorer
qualitative and quantitative crop yield can be attributed to both unfavourable combination of
temperature and humidity conditions in the periods of the year coinciding with the most critical
stages of plant development and inadequate implementation of appropriate agro-technical measures.
Such conditions favored the occurrence of Fusarium infection and increase in pest population.
Weather conditions negatively affected the tasseling, silk production and fertilization of corn. Such
conditions were, however, ideal for the development and activity of plant pests. During this period,
the worst situation was recorded in Vojvodina region, with an average precipitation rate of only
25%. Maize was the most endangered crop of all, and was ready for harvesting as early as end
August (RHMZS, 2012).
According to the aforementioned report of the RHMZS, one can conclude that summer 2012 was
the hottest one in Serbia since record began, and with very little precipitation. The hottest and driest
period (30 June-25 July) in the major part of the territory coincided with the most important
generation phases of spring crops, thus causing substantial damage and losses in agricultural crops
production manifested by high concentration of aflatoxins in maize and consequently in milk.
169
Contamination of maize and animal feed with Fusarium toxins in 2014
The results on the contents of some Fusarium toxins in maize (harvest 2014) and animal feed are
presented in Table 4.
Table 4. Concentration of Fusarium toxins in samples of animal feed and maize (harvest 2014), in Serbia
Corn
DON
Zearalenone
Fumonisins
(mg/kg)
(µg/kg)
(mg/kg)
21
21
21
Total number of samples
21
19
21
Number of positive samples
Number of inappropriate
0
0
0
samples
7.6
2389
10.2
max content
0.207
83
0.449
min content
Average
samples
max content
min content
Average
samples
max content
min content
Average
2.12
433.4
3.28
Complete swine mixtures
DON
Zearalenone
Fumonisins
(mg/kg)
(µg/kg)
(mg/kg)
16
16
16
16
16
16
12
7
0
5.82
0.47
574
144
2.15
0.901
2.82
337.6
1.42
Complete poultry mixtures
DON
Zearalenone
Fumonisins
(mg/kg)
(µg/kg)
(mg/kg)
7
Not
7
legislated
6
7
0
0
3.17
0.311
4.24
0.837
1.82
1.83
According to the results presented in Table 4, high incidence of samples positive to Fusarium toxins
was recorded in maize samples. DON and fumonisin contamination was confirmed in 100% of
examined maize samples, whereas 90.5% of samples were contaminated with zearalenone.
Although the concentrations of the examined mycotoxins in maize did not exceed the maximum
permitted levels (8 mg/kg for DON, 4000 µg/kg for zearalenone and 60 mg/kg for fumonisins
(Official Gazette RS, 2014; EC, 2006)), maize contamination with these mycotoxins consequently
resulted in large number of contaminated samples of complete pig mixtures (100% samples
contaminated with all three toxins) and complete poultry mixtures (DON and fumonisin
contamination of 85.7% and 100% samples, respectively). As complete feed mixtures contain large
proportion of maize, high percentage of pig mixtures were declared inappropriate according to the
Regulation because of increased contents of DON (75.0%) and zearalenone (43.7%). Fumonisin
concentration in examined samples did not exceed the maximum permitted values according to EU
170
regulations (EC, 2006); however, their presence should not be neglected because of their potential
cumulative and synergistic toxic effects with DON and zearalenone.
According to the report of RHMZS, weather conditions in the territory of Serbia in 2014 revealed
several extreme deviations as compared to the annual range of average values for this climatic
region. By mid May, the territory of Serbia was characterized by abundant rainfall, which has never
been recorded so far. The summer 2014, (June-August) was characterized by moderately warm and
extremely rainy and humid season. During the vegetative period (April-September) 2014, the
average rainfall recorded in Serbia was 700 mm, which is the highest rate of rainfall during
vegetation period recorded in the last 45 years. In the major part of the country, the precipitation
rate was 2-3 times higher as compared to the multi-year annual average. Such extremely humid and
rainy weather brought about a number of plant diseases such as blight and decaying fungi as well as
intensive activity of the European corn borer (Ostrinia nubilalis). Frequent rains and increased air
humidity contributed to significantly slower ripening of maize, thus resulting in delayed harvesting.
Even though this rainy year contributed to record maize yields, increased grain moisture content as
well as grain damage caused by mouldy corn agents such as Fusarium and Penicillium was
recorded (RHMZS, 2014).
The presented results indicated that humid climatic conditions in 2014 significantly contributed to
the development of Fusarium moulds and consequent mycotoxin production in maize.
Conclusion
Based on the analysis of the content of examined mycotoxins and agrometeorological factors we
can conclude that temperate climate is the determining factor for mycotoxicological safety of
cereals in the region of Serbia. The deviation from annual average precipitation and temperature
values, that is, precipitation surplus or droughts as well as prolonged periods with extremely high
temperatures, impose the necessity of applying all available agro technical measures to prevent
development of fungi, maize diseases and insects and thus reduce the risk of mycotoxin
contamination. The obtained data on the level of mycotoxins and correlation of their contents are
useful instrument for developing a model to improve prediction of risks for mycotoxin
contamination in Serbia.
Acknowledgments
The work was financially supported by the Ministry of Education, Science and Technological
Development of the Republic of Serbia (Project No 172042).
References
1.
2.
3.
4.
EC (European Commission): Commission Recommendation of 17 August 2006 on the presence of
deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 and fumonisins in products intended for animal
feeding. 2006/576/EC, O. J., L 229: 79, 2006
EC (European Commission): Commission Regulation 165/2010 of 26 February 2010 amending
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172
RADIOACTIVITY OF THE SOIL IN VOJVODINA
(NORTHERN PROVINCE OF SERBIA)
Željko Mihaljev1*, Dragana Ljubojević1, Miroslav Ćirković1, Milica Živkov-Baloš1, Sandra
Jakšić1, Brankica Kartalović1, Nadežda Prica1
* Corresponding author: zeljko@niv.ns.ac.rs
Abstract
Peaceful uses of nuclear energy (nuclear weapons testing, nuclear reactor accidents, industrial and
medical use of radioactive compounds) and application of phosphate mineral fertilizers in
agricultural production lead to substantial environmental contamination. Land contaminated with
radionuclides represents the first link in the food chain and hence the radioactive contamination of
crop and livestock production. To determine the soil levels of natural radionuclides the samples
were collected from 11 localities in the territory of Vojvodina. The measurements were performed
applying inductively coupled plasma with mass spectrometry. The values for concentration of
radionuclide activity measured in the examined samples do not significantly diverge from standard
values for agricultural soils.
Key words: natural radionuclides, soil, ICP-MS
Introduction
Natural radioactivity implicates presence of radioactive elements that have been present in nature
since the formation of Earth and the very beginning of formation of its entire living world
(biosphere). Abundant researches revealed substantial differences in the levels of natural
radionuclides between particular localities at Earth’s surface, and their amounts vary according to
the locality (Mitrović et al., 1996). The largest source of radiation activity of the soil is a natural
radionuclide potassium-40 (Dželalija, 2006). Some other natural radionuclides, which have always
been present on Earth, include also uranium-235, uranium-238, thorium-232, radium-226 and
radon-222 (Levant, 1996). The exposure to low-level radiation originating from these natural
elements has been always affecting all living beings on Earth, and it is considered background
radiation or natural phon.
The basic component of the biosphere is lithosphere (Earth’s crust), which represents the first link
of the ecology chain: soil-vegetation-animals-man. The soil plays a crucial role in the process of
radionuclide distribution and transfer, thus, knowing of radioactive contamination of the soil is of
great importance for radiation safety issues in biotechnology (Petrović et al., 1994). Radioactive
contamination of the soil is either natural (formed without man’s activity) or human produced
(nuclear testing, reactor accidents, radioactive waste) (Dobrić et al., 2006).
The “technologically enhanced naturally occurring radioactivity” is attributed to uranium, which is
brought to the environment through diverse technological procedures and agro technical measures
(Rajković, 2001). Technological development resulted in substantial increase of natural soil
radioactivity, predominantly through intensive application of agro-technical measures based on the
use of artificial phosphate fertilizers that contain substantial amounts of natural uranium. Moreover,
mining of ore containing heavy metals (zinc, copper, led) from deep layers of lithosphere and
processing thereof, as well as the thermal power stations producing huge amounts of solid waste
(ash, cinder) that contain natural radionuclides such as uranium, thorium and their prodigy (Kisić et
al., 2013) increase the levels of soil radioactivity. Uncontrolled application of phosphate fertilizers
173
implicates substantial potential for undermining of ecological balance as they represent the most
powerful source of 238U and 232Th content in the soil, and hence in plants and other links within the
food chain. In a superphosphate, uranium is deposited as highly water-soluble uranyl sulphate UO2(SO4) and urano-sulphate - U(SO4) (Mitrović et al., 2011). Contrary to other radioisotopes,
radioactive decay of 238U and 232Th results in formation of the series of unstable nuclei, 226Ra and
222
Rn being the most dangerous members of uranium chain. 226Ra is a long half-life -emitter,
manifesting affinity for accumulating in bones, while gaseous 222Rn is responsible for internal
irradiation of lung tissues. Emissions of those radionuclides into the environment represent potential
significant risk factors for the exposure of local inhabitants to ionizing radiation, as well as for
increased levels of natural radiation in particular regions.
Involvement of particular radionuclides into biological cycle is associated with plants ability to
absorb radioactive elements from the soil via their root systems. The amount of radioactive material
from the soil absorbed by plants is directly proportional to the pollution emission density at
particular territory (Simić M., 2001). The transfer of radionuclides from the soil into the plants is
substantially dependant on the soil type, that is, its physico-chemical properties that significantly
affect the resorption rate of radioactive material. The most important physico-chemical properties
include chemical composition (concentration of minerals and content of organic matter), structure
(mechanical composition), pH, moisture content and crop density.
Soil samples were collected from 11 localities in the territory of Vojvodina (Table 1) during 2014.
All samplings (at each of 11 localities) were performed by collecting soil samples from 10-15
different points at the total surface of 100m2. The samples were collected only from flattened soil
surface at the depth of 10-20 cm. The majority of soil samples were of “chernozem”-type (Živković
et al., 1972). The samples were dried at 1050C until reaching the constant mass (IAEA, 1989).
Subsequently, removal of mechanical impurities (mainly stones and plant particles) has been
performed. Dried soil samples were mechanically crushed (ground) to obtain fine powder. The
amount of 1g of homogenized sample was weighed and decomposed in HNO3 and H2O2 mixture
using the wet digestion method and the system Ethos, Microwave Labstation, Milestone. Uranium
content was determined using Agilent 7700x Series ICP-MS, and data analysis was performed by
MassHunter Workstation software.
Figure 1. Geographical view of sampling locations
174
The activity levels of uranium-235 and uranium-238 in soil samples were determined according to
total uranium concentration using mass activity values 0.570 Bq/mg U for 235U and 11.10 Bq/mg U
for 238U (Eisenbud, 1973). Potassium content was determined using the method of emission
spectrophotometry on Spectr AA–10, manufactured by Varian, at wavelength 766.5 nm and using
cesium as the ionization-suppressor. The soil levels of potassium-40 activity were calculated from
total potassium, using the mass activity value for potassium being 31.561 Bq/g K (Eisenbud, 1973).
Results
The soil in Vojvodina region undergoes radioactive contamination from diverse sources. Above all,
emissions from nuclear plant reactors in neighboring regions cause contamination of the air and
water in a wide area. Intensive application of phosphate fertilizers with high uranium content
potentially causes gradual increase in the activity level of uranium chain in the soil. Moreover,
potential contamination of the soil in Vojvodina with depleted uranium caused by NATO bombing
in 1999 is widely accepted public opinion. All aforementioned factors strongly suggest the
importance of comprehensive assessment of the soil radioactivity status in Vojvodina (Bikit et al.,
2010). Table 1 displays the results on potassium and uranium concentrations, as well as the activity
levels of natural radionuclides 40K, 235U and 238U.
Table 1. Contents of potassium and uranium and concentration of potassium-40, uranium-235 and
uranium-238 activity in soil samples
No.
Locality
1.
Bač
2.
Zmajevo
3.
Despotovo
4.
Temerin
5.
Čenej
6.
Lovćenac
7.
Čurug
8.
Kisač
9.
Bački Brestovac
10.
Bački Jarak
11.
Rumenka
Average value ± SD
K content
[g/kg]
16.37 ± 0.54
15.08 ± 0.24
10.91 ± 0.06
15.92 ± 0.30
11.40 ± 0.29
12.84 ± 0.17
17.72 ± 0.58
21.88 ± 0.35
17.57 ± 0.33
22.03 ± 0.42
23.49 ± 0.45
16.84 ± 4.28
40
K activity
[Bq/kg]
516.6 ± 17.0
476.1 ± 17.6
344.3 ± 12.1
502.4 ± 19.6
359.8 ± 19.4
405.4 ± 15.3
559.3 ± 18.6
690.6 ± 11.0
554.5 ± 10.5
695.3 ± 13.2
741.4 ± 14.1
531.4 ± 134.9
U content
[mg/kg]
4.76 ± 0.24
3.55 ± 0.18
3.36 ± 0.17
2.65 ± 0.13
3.12 ± 0.16
3.89 ± 0.19
3.06 ± 0.15
2.87 ± 0.14
2.80 ± 0.14
2.76 ± 0.14
3.24 ± 0.16
3.28 ± 0.62
235
U activity
[Bq/kg]
2.71 ± 0.14
2.02 ± 0.10
1.91 ± 0.10
1.51 ± 0.08
1.78 ± 0.09
2.22 ± 0.11
1.74 ± 0.09
1.63 ± 0.08
1.60 ± 0.08
1.57 ± 0.08
1.85 ± 0.09
1.87 ± 0.35
238
U activity
[Bq/kg]
52.84 ± 2.64
39.40 ± 1.97
37.30 ± 1.86
29.42 ± 1.47
34.63 ± 1.73
43.18 ± 2.16
33.97 ± 1.70
31.86 ± 1.59
31.08 ± 1.55
30.64 ± 1.53
35.96 ± 1.80
36.39 ± 6.83
The results displayed in Table 1 revealed potassium contents in the examined soil samples ranging
from 10.91 to 23.49 g/kg with an average value for all localities being 16.84±4.28 g/kg. These
values correspond with the potassium-40 activity concentration in a range 344.3 – 741.4 Bq/kg,
with an average activity level for all localities being 531.4 ± 134.9 Bq/kg. The obtained levels of Kconcentration and 40K activity are considered common values for the soil and are in full accordance
with the results of other authors (Bikit et al., 1990). One can also conclude that potassium-40 is
predominant natural radionuclide in the soil as compared to other radionuclides. Obvious variations
in potassium content in soil lead to the conclusion that incorporation of radionuclides from the soil
into the plants largely depend on pedological properties, soil exploitation, agrotechnical treatments
as well as on the type of plants, what is in accordance with the results of our previous researches
(Ćupić et al., 2005; Mihaljev et al., 2011).
175
Uranium contents in soil samples were uniform, ranging within an interval 2.65-4.76 mg/kg with an
average value for all localities being 3.28 mg/kg (Table 1). Based on these values, the following
activity concentrations for uranium were calculated: 235U = 1.51-2.71 Bq/kg, average value 1.87 ±
0.35 Bq/kg and 238U = 29.42-52.84 Bq/kg, average value 36.39 ± 6.83 Bq/kg. Similar results on
uranium concentration in the soil and the activity concentration for radioactive uranium-238 were
reported by other authors (Skipperud et al., 2011).
The importance of natural radionuclides as potential pollutants in agricultural and livestock
production is addressed in the latest addendum to the Ordinance on the limits of radionuclides in
drinking water, food, animal feed, drugs, general use items, building materials and other goods that
are placed on the market (Official Gazette of RS, No. 97/13). This addendum introduced the new
article, 11a, establishing limits for the contents of natural radionuclides (238U, 226Ra, 40K) in mineral
and phosphate fertilizers as potential sources of radioactive contamination of soil.
We also may conclude that ICP-MS (inductively coupled plasma mass spectrometry) proved very
sensitive method for quantitative determination of uranium concentration in soil samples (Sahoo et
al., 2011). It is therefore an attractive alternative for monitoring uranium because can detect the
normal environmentally caused concentrations (Schramel et al., 1997).
Systematic monitoring of radioactivity level of agricultural soil is of paramount importance in
Vojvodina, as the region with extremely high potential for production of safe food. Soil
contamination with wet or dry atmospheric precipitation and materials characterized by
technologically increased level of natural radioactivity can represent a permanent reservoir of
radionuclides that significantly contribute to the overall radiation exposure and total population
radiation dose.
Acknowledgments
The work was financially supported by the Ministry of Education, Science and Technological
Development of the Republic of Serbia, project No TR 31084.
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177
PHYSICOCHEMICAL ANALYSIS AS AN INDICATOR OF THE QUALITY OF HONEY
ORIGINATING FROM VOJVODINA REGION
Nadežda Prica1*, Milica Živkov-Baloš1, Sandra Jakšić1, Željko Mihaljev1, Dragana Ljubojević1,
Branka Vidić 1, Sara Savić1
1
Scientific Veterinary Institute ’’Novi Sad’’, Novi Sad, Serbia
* Corresponding author: nadja@niv.ns.ac.rs
Abstract
Physicochemical analysis of honey plays an important role in determining the overall characteristic
of honey and final assessment of its quality. This study was aimed at investigating the
physicochemical properties of local honeys collected from different flora from Vojvodina. The
physicochemical parameters such as moisture content, titratable acidity, HMF determination and
ash content were analyzed. The physicochemical characteristics of 35 out of 40 honey samples
(87.5%) analyzed in this study completely correspond with the national Regulation No. 45/2003 and
Codex Alimentarius, indicating adequate processing, good maturity and freshness. Five samples
(12.5%) did not meet standards established in the Regulation No. 45/2003 and Codex standards. In
40 samples analyzed, the HMF content was elevated in one sample (2.5%) exceeding the limit of 40
mg/kg, and 29 samples (72.5%) revealed values lower than 10 mg/kg, which is typical for fresh
unheated honeys, according to the current quality criteria. The moisture content exceeded the
maximum level permitted by the Regulation in only one of 40 analyzed honey samples (2.5%).
Moreover, in only three samples (7.5%), ash content exceeded the maximum level permitted by the
Regulation. The titratable acidity of all samples was lower than the limit of 40 mmol of acid per
1000 g of sample. Research findings pointed out that the physicochemical properties of local honey
from Vojvodina were in accordance with the Codex standard and the products meet significant
quality criteria for a high-quality honey.
Key words: honey, quality, moisture content, titratable acidity, HMF content, ash content
Introduction
Honey is a sweet liquid produced by honeybees using nectar from flowers through a process of
regurgitation and evaporation. The possible health benefits of consuming honey have been
documented in early Greek, Roman, Vedic, and Islamic texts and the healing qualities of honey
were referred to by philosophers and scientists all the way back to ancient times, such as Aristotle
(384 - 322 BC) and Aristoxenus (320 BC). Modern science is finding that many of the historical
claims that honey can be used in medicine may indeed be true. In the Bible (Old Testament), King
Solomon said, "My son, eat thou honey, for it is good", and there are a number of reasons why it
may be good ( Nordqvist, 2014).
Honey also possesses antiseptic and antibacterial properties. In modern science, we have managed
to find useful applications of honey in chronic wound management. However, it should be noted
that many of honey's health claims still require further rigorous scientific studies to confirm them
(Nordqvist, 2014).
According to the Regulation ("Official Gazette of SCG", No. 45/2003), honey is defined as “sweet,
dense, crystallized, viscous product produced by honeybees from the nectar of honey plant flowers
or from secretions of living parts (conifer or hardwood species), which the bees collect, transform
by combining with specific substances of their own, and deposit in honeycombs to mature”. In
178
Codex standard (2001), honey is defined as “natural sweet substance produced by honey bees from
the nectar of plants or from secretions of living parts of plants or excretions of plant sucking insects
on the living parts of plants, which the bees collect, transform by combining with specific
substances of their own, deposit, dehydrate, store, and leave in the honey comb to ripen and
mature”. Chemical composition of honey implicates highly complex mixture of more than 200
different substances (Ferreira et al., 2009). Some of these substances are produced by honeybees,
and some originate from honey plants, whereas some are produced during the maturation process in
the honeycomb (Krell, 1996).
Serbia has a very long tradition of beekeeping. Its favourable climate, good geographical condition
and a variety of botanical species provide great potential for the development of apiculture
(Mačukanović-Jocić, 2008).
Serbian honey could potentially be very interesting for the EU market, so it is very important to
verify its compliance with the quality specifications of the European Union (European Economic
Community, 2002).
To the purpose of determining the moisture content, ash content, hydroxyl methyl furfural (HMF)
content and total acidity, 40 samples of different honeys originating from Vojvodina region were
collected. All samples were in their original packages and were transferred to the laboratory and
stored in a cold and dark place. The investigated samples included 12 samples of meadow honey, 14
samples of acacia honey, 14 samples of linden honey, 4 samples of multiflower honey, 5 samples of
sunflower honey and 1 sample of forest honey.
Moisture content was determined by the refractometric method (Off.Gazette of SFRJ, 1985), using
an Abbe refractometer (Model RMT, Optech, Italy). All measurements were performed at 200C
after equilibrium, and obtaining the corresponding % of moisture from the refractive index of the
honey sample was calculated by consulting a standard table for this purpose.
The acidity of honey was determined by volumetric method (Off. Gazette of SFRJ, 1985). Ten
grams of honey were dissolved in 75 ml of distilled water and alcoholic solution of phenolphthalein
was added. The solution was titrated with 0.1 mol/dm3 NaOH. Acidity (milimol of formic acid per
kg of honey) was determined as 10 times the volume of NaOH used in titration.
For determination of ash content, the method Off. Gazette of SFRJ, 1985, was followed. According
to the method, 5 g of each sample was weighed in a ceramic plate. The plate was heated in a muffle
furnace for about 3 to 5 h at 600°C. It was cooled in desiccators and weighed. The weight of ash
gave the ash content and was calculated by the following formula:
Ash (%) = Weight of sample after ashing × 100 /Weight of fresh sample taken
For the HMF determination use was made of an HPLC Dionex UltiMate 3000 Series system with a
diode array detector (DAD-3000, Thermo Scientific, Germany), consisting of an autosampler WPS3000, degasser, quaternary pump, and Hypersil GOLD column (150×3 mm, particle size 3 μm). The
system was controlled by Chromeleon® 7 software (Thermo Scientific). The mobile phase was
MeOH−water (10+90, v/v) filtered through 0.22-μm membrane filter, at a flow rate of 1.0 ml/min.
Results and discussion
The obtained results on moisture content, total acidity, HMF content and ash content in the
examined honey samples are displayed in Table 1.
179
Moisture content is one of the most important compositions to be considered as a quality parameter
of honey. The maximum moisture content was found to be 34.00 mmol of acid/1000 g in linden
samples, whereas 2.2 mmol of acid/1000 g moisture content was observed in multiflower variety.
Table 1. Results of determining moisture content, HMF, ash content and total acidity in diverse
honey samples
TYPE OF
HONEY
No. of
samples
Meadow
Moisture content
(%)
Acidity
(mmol of acid/1000 g)
Ash content
(%)
HMF content
(mg/kg)
Range
Average
±SD
Range
Average
±SD
Range
Average
±SD
Range
Average
±SD
12
14.6 – 18.6
17.33 ± 1.13
13.75 – 26
20.34 ± 3.79
0.07 – 2.96
0.48 ± 0.81
3.84 – 34.75
10.81 ± 8.20
Acacia
10
14.2 – 18.4
16.2 ± 1.42
9.5 – 18.75
13.17 ± 3.07
0.0 – 0.41
0.09 ± 0.13
6.78 – 43.78
15.87 ± 12.64
Linden
8
15 – 18.6
16.67 ± 1.36
10.5 – 34.0
15.9 ± 7.44
0.04 – 1.27
0.38 ± 0.40
4.69 – 16.13
8.40 ± 3.54
Multi
flower
7
15.8–21.4
17.66±1.93
2.2 –27.7
18.98 ±9.10
0.07 –-0.36
0.22 ± 0.10
5.69 –10.7
7.48 ±1.75
Sunflower
3
16 – 16.8
16.33 ± 0.42
10 – 15.00
12.42 ± 2.50
0.02 –0.275
0.11 ± 0.15
5.23 – 7.45
6.47 ± 1.13
Average values obtained in our research (Table 1.) are in agreement with the findings of Cantarelli
et al. (2008), who reported that the moisture content in honey was in the range of 14 to 18%;
however, it depends upon the season and geographic condition. Furthermore, these results are also
in agreement with those of Nuru (2002) and Downey et al. (2005), who reported that the range of
moisture content of pure honey is 16.10 to 23.36%. Fredes and Montenegro (2006) reported that
honey with lower moisture content would have a longer shelf life. The maximum ash content
(2.96%) was found in one sample of meadow honey, another sample of meadow honey revealed ash
contents of 0.86%, which exceeds the maximum permitted level of 0.50%. Total ash contents
measured in other honey samples were in accordance with the limits prescribed by relevant
Regulation. , These findings are in agreement with those of Ihtisham-ul-haq (1997), who analyzed
different varieties of honey and determined the ash content range of 0.008 to 0.49% in honey
samples. These results are also in agreement with those of White (1975a), who worked on different
varieties of honey and obtained ash content in the range of 0.020 to 1.028%. The variation may be
due to many factors such as soil conditions, atmospheric conditions and physiology of each plant.
The composition of organic acids in honey has not yet been adequately investigated; however, some
evidence (Rogulja et al., 2009) suggest that acacia, chestnut and meadow honeys are characterized
by particularly low contents of organic acids, whilst darker honeys in general appear to be higher in
acidity.
The research of Prica et al. (2014.) also demonstrated low acidity of acacia honey as compared with
other examined honey types. The results obtained for meadow honey do not correspond with the
aforementioned evidence, yet the acidity was within the proper range. Our results correspond with
these reports. HMF represents the freshness of honey and depends on adequate beehives and harvest
practice. The majority of honey samples had HMF contents in line with the maximum permitted
levels prescribed by the Regulation ("Official Gazette of SCG", No. 45/2003); maximum HMF
content in honey put in the market is fixed to 40 mg/kg. According to the obtained results, HMF in
honey content exceeded maximum permitted value in only one sample of acacia honey.
180
Conclusion
The physicochemical characteristics of 35 out of 40 honey samples (87.5%) analyzed in this study
completely correspond with the national Regulation No. 45/2003 and Codex Alimentarius,
indicating adequate processing, good maturity and freshness. Five samples (12.5%) did not meet
characteristics established in the Regulation No. 45/2003 and Codex standards. In 40 samples
analyzed, the HMF content was elevated in one sample (2.5%) exceeding the limit of 40 mg/kg, and
29 samples (72.5%) revealed values lower than 10 mg/kg, which is typical of fresh unheated
honeys, according to the current quality criteria.
The moisture content exceeded the maximum level permitted by the Regulation in only one of 40
analyzed honey samples (2.5%). Moreover, in only three samples (7.5%), ash content exceeded the
maximum level permitted by the Regulation. The titratable acidity of all samples was lower than the
limit of 40 mmol of acid per 1000 g of sample.
Research findings pointed out that the physicochemical properties of local honey from Vojvodina
were in accordance with the Codex standard and it meets significant quality criteria for a highquality honey.
Acknowledgments
This work is supported by a grant from the Ministry of Education, Science and Technological
Development, Republic of Serbia, Project number TR 31084
References
1. Cantarelli M.A., Pellerano R.G., Marchevsky E.J., Camiña J.M.: Quality of honey from Argentina: Studi
of chemical composition and trace elements. The Journal of the Argentine Chemical Society, 96, 1/2, 3341, 2008
2. Codex Alimentarius Commission: Revised Codex Standard for Honey, Codex STAN 12-1981, 2001.
3. Downey G., Hussey K., Jelly J.D., Walshe T.F., Martín P.G.: Preliminary contribution to the
characterization of artisanal honey produced on the island of Ireland by palynological and
physicochemical data. Food Chemistry, 91: 347-354. 2005
4. European Economic Community: EEC Council Directive of 20 December 2001 relating to honey.
Official Journal of the European Communities, 110, 47-50, 2002
5. Ferreira I.C.F.R., Aires E., Barreira J.C.M., Estevinho L.M.: Antioxidant activity of Portuguese honey
samples: Different contributions of the entire honey and phenolic extract. Food Chemistry, 114, 14381443, 2009
6. Fredes C., Montenegro G.: Heavy metal and other trace elements contents in honey bee in Chile. Cien.
Inv. Agric., 33, 50-58, 2006
7. Ihtisham-ul-haq: Effect of floral type, geographical locations and honey bee species on the water activity
and other physico-chemical parameters of honey. M.Sc. Thesis. Deptt. Food Sci. Technol., Faculty of
Nutrition Sciences, NWFP Agricultural University, Peshawer, Pakistan, 1997
8. Krell R.: Value-added products from beekeeping, FAO Agricultural Services Bulletin No. 124, Food and
Agriculture
Organization
of
the
United
Nations
Rome
1996,
Dostupno
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www.fao.org/docrep/w0076e/w0076e00.htm. Pristupljeno 12.11.2014.
9. Mačukanović-Jocić M.: The biology of melliferous plants with an atlas of Serbian apiflora. Faculty of
Agriculture (on Serbian), Beograd, Serbia, 2008
10. Nordqvist J., Reviewed by Megan Ware, RDN, LD, registered dietitian and nutritionist, What are the
health benefits of honey?; 26.09.2014. Dostupno na:
http://www.medicalnewstoday.com/articles/264667.php
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11. Nuru A.: Geographical races of the Honeybees (Apis mellifera L.) of the Northern Regions of Ethiopia.
Ph.D. dissertation. Rhodes University, South Africa. 2002
12. Prica N., Živkov-Baloš M., Jakšić S., Mihaljev Ž., Kartalović B., Babić J., Savić S.: Water content and
acidity as an indicator of the quality of honey originating from Vojvodina region, Archives of Veterinary
Medicine, 2, vol 7, 99-110, 2014
13. Rogulja D., Vahčić N., Matković D.: Kemijske, fizikalne i senzorske značajke meda, 2009, Dostupno na
www.pcelinjak.hr/OLD/index.php/Prehrana-i-biotehnologija/kemijske-fizikalne-i-senzorske-znaajkemed.html. Pristupljeno 12.11.2014.
14. Sl. list SCG, br. 45/2003, Pravilnik o kvalitetu i drugim zahtevima za med, druge pčelinje proizvode,
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London, pp. 157-206,1975a
182
EFFECTS OF DIETARY HOT RED PEPPER ADDITION ON PRODUCTIVE
PERFORMANCE AND BLOOD LIPID PROFILE OF BROILER CHICKENS
Nikola Puvača1*, Ljiljana Kostadinović2, Dragana Ljubojević3, Dragomir Lukač1, Sanja Popović2,
Jovanka Lević2, Olivera Đuragić2, Rade Jovanović4
1
University of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia
University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia
3
4
Institute for Science Application in Agriculture, Belgrade, Serbia
* Corresponding author: nikola.puvaca@gmail.com
2
Abstract
Experiment was conducted to investigate the effect of hot red pepper in broiler nutrition on
productive performances and blood lipid profile. For biological research three treatments with the
total of 450 broilers were formed, within four replicates. Control treatment (CON) of chickens were
fed with mixture based on corn flour and soybean meal of standard composition and quality, while
the experimental treatments were fed with the same mixture only with addition of two levels of hot
red pepper 0.5 (HRP-0.5) and 1.0 g/100g (HRP-1.0). Addition of hot red pepper in the amount of
0.5 g/100g has led to the highest final body weight of chickens (2460.6 g), followed by the addition
of 1.0 g/100g (2442.4 g) with significant differences (p<0.05) compared to a control treatment
(2075.8 g). The lowest amounts of triglycerides, total cholesterol, low density lipoprotein (LDL)
and non-high density lipoprotein (non HDL) was recorded in broilers in treatments with hot red
pepper with statistically significant (p<0.05) differences compared to a control treatment. The
highest share of high density lipoprotein (HDL) with statistical significance (p<0.05) was
determined also in hot red pepper treatments. Based on the obtained results, it can be concluded that
the addition of hot red pepper in broiler chicken nutrition has positive effects on production
performances and in improvement of chicken blood lipid profile.
Key words: Hot red pepper, cholesterol, nutrition, chickens, food
Introduction
Beside of an important role of hot red pepper in daily human nutrition for enhancement of taste,
aroma and colour of food, this spice have also been efficiently used in animal nutrition for
improvement of animal health and production of healthier meat and eggs. With the ban of
antibiotics use in animal nutrition due to the emergence of microbe resistance, alternative growth
promoters must be found (Steiner, 2009). Removal of antibiotics as growth promoters has led to
animal performance problems, increase of feed conversion ratio, and a rise in the incidence of
certain animal diseases (Wierup, 2001). The alternatives to antibiotics as growth stimulators are
numerous (Steiner, 2009; Puvača et al., 2013). Hot red pepper (Capsicum annuum L.) plays an
important role in decreasing the deposition of cholesterol and fat in the body, contributes to
decreased levels of triglycerides and supports the vascular system in the body. Efficient hot red
pepper compounds consist of capsaicin, capsicin and capsanthin. Hencken, (1991) explained that
hot red pepper is rich in vitamin C, which have a considerable impact in improving production
through contributes the reduction of heat stress (Yoshioka et al., 2001). A recent studies involved in
chicken performance have shown that blends of active compounds for hot red pepper have
chemopreventive and chemotherapeutic effects. In research of Al-Kassie et al. (2012) addition of
hot red pepper had significant effect on the heterophil/lymphocytes (H/L) ratio, which reflects the
183
role of hot red pepper, especially its active compound capisicine, which is involved in stress
hormones, and which supports the immune system of birds and enhances its resistance against
disease through decreasing (H/L) ratio.
The aim of this study was to investigate the effect of hot red pepper in broiler nutrition on blood
lipid profile and productive performances.
Determination of bioactive components in hot red pepper
Content of capsanthin or colored matter in a sample of hot red pepper powder is determined by the
reference method SRPS EN ISO 7540 (2012). The method is based on extraction of colored
substances from a sample of hot red pepper with benzene and then spectrophotometric measurement
of maximum absorbance at a wavelength of 477nm. Content of capsanthin in samples of ground
pepper is expressed in g/kg of dry matter of the sample. The content of capsaicin in a sample of
pepper is determined according to the method described in the manual for quality control of fresh
and processed fruits, vegetables and mushrooms and non-alcoholic beverages (Vračar, 2001). The
method is based on extraction of capsaicin from a sample of hot red pepper, separation of colouring
matters and the development of colour characteristic of capsaicin, followed by spectrophotometric
measurement of maximum absorbance at a wavelength of 433nm. The colour intensity of the
solution is proportional to the concentration of capsaicin. The content of capsaicin in samples of hot
red pepper powder is expressed in g/100g dry matter of the sample. Concentration of capsanthin and
capsaicin is given in Table 1.
Table 1. Concentration of capsanthin and capsaicin in experimental hot red pepper
Hot red pepper powder samples
(n=3)
LSM
SELSM
Bioactive compounds
Capsanthin, g/kg
Capsaicin, g/kg
a
3.31
0.96b
0.58
0.04
Treatments with different letter indexes in the same row are statistically significantly different (p<0.05)
Animal trials
Biological tests were carried out under production conditions at the experimental farm "Pustara" in
property of the Faculty of Agriculture from Novi Sad. At the beginning of the experiment, a total of
450 one-day old Hubbard broilers were distributed into three dietary treatments with four replicates
each. Every dietary treatment included 150 chickens, which were divided in four pens with 37-38
chicken per each pen. Chickens were reared on floor holding system with the chopped straw as litter
material. Chickens were provided with the light regime of 23h of day per entire experimental period
of 42 days with incandescent light source. For nutrition of chicks three mixtures were used, starter,
grower and finisher through pan feeders. For the first 14 days, during the preparatory period, chicks
were fed with starter mixtures. Following the preparation period, chicks were fed with grower
mixtures for the next 21 days, and then for the last 7 days of fattening period with finisher mixtures
according the experimental design given in Table 2 and dietary chemical composition of used
starter, grower and finisher mixtures which is given in Table 3.
During the experiment chicks were fed and watered ad libitum. Chickens were watered throught the
nipple water system. Microclimate conditions were regularly monitored. Body weight was
monitored at an individual level during the entire experimental period every seven days, while the
feed consumption and feed conversion ratio were monitored at the pen level also every seven days.
184
Table 2. Experimental design with chickens
Experimental
treatmens
CON
HRP-0.5
HRP-1.0
Concetration of additives in chicken diets
In starter,
In grower,
In finisher, g/100g
g/100g
g/100g
Additive
1 – 14 days
15 – 35 days
36 – 42 days
Control treatment
0.0
0.0
0.0
Hot red pepper
0.0
0.5
0.5
Hot red pepper
0.0
1.0
1.0
Table 3. Chemical composition of dietary mixtures, g/100g
Nutrients
Dry matter
Moisture
Crude protein
Crude fat
Crude fibre
Crude ash
Ca
P
Metabolic Energy, MJ/kg
*Hot red pepper is added on top on the basic diet
Starter
89.4
10.5
21.1
3.9
3.5
5.0
0.8
0.6
12.5
Diet mixtures
Grower
89.3
10.7
20.7
3.9
3.5
4.8
0.9
0.6
12.8
Finisher
89.4
10.5
17.3
4.7
3.6
5.6
1.1
0.5
13.3
Blood lipids
At the end of 6th week, twelve birds were randomly chosen from each treatment and bled via wing
vein puncture to obtain blood samples. Serum samples from blood were separated by centrifugation
(4000 rpm for 5 min at 20°C). Commercially available kits (Randox Laboratories Limited - United
KIngdom) were used to analyse the serum for triglycerides, total cholesterol, HDL and LDL on an
biochemical autoanalyzer Cobas Mira Plus (Roche Diagnostics). Values were expressed as mg/dl.
Statistical analyses
Statistical analyses were conducted within statistical software program Statistica 12 for Windows,
to determine if variables differed between treatments. Significant effects were further explored
using analysis of variance (ANOVA) with repeated measurements, least square means (LSM) and
standard errors of least square means (SELSM), as well as Fisher's LSD post-hoc multiple range test
with Bonferroni corrections to ascertain differences among treatment means. A significance level of
p<0.05 was used.
Capsanthin and capsaicin content
From the results given in Table 1 it can be seen the concentration of capsanthin (3.31 g/kg) and
capsaicin (0.96 g/kg) as the main bioactive components in hot red pepper. According the Serbian
regulation (Off. Gazette of SFRY, No. 1/79), hot red pepper on 1 kg of dry matter should comprise
at least 2 g of capsanthin and capsaicin between 0.5 to 0.7 g. As it can be seen from the results
shown in Table 1, samples of hot red pepper correspond to quality parameters requirements of
Serbian regulations, except for the content of capsaicin, which in the tested samples was higher for
185
0.26 g of dry mater. Taking into account that the capsaicin is alkaloid responsible for the hot taste of
pepper, this result was expected because the hot red pepper is recognizable its pungent quality.
Similar result was obtained by Dang et al. (2014) in their study of three-liquid-phase extraction and
separation of capsanthin and capsaicin from Capsicum annum L. The highest content of capsaicin
was found in the placenta, as well as dihydrocapsaicin, 10.48 and 6.43 g/kg, respectively, while the
highest ratio of 3.71 estimated from the quantity of capsaicin and dihydrocapsaicin was calculated
in the pericarp. The determined pungency level in placenta of 272 211 SHU was almost five times
and two times higher than the pungency level in the seed and pericarp, respectively (Simonovska et
al., 2014).
Productive performance of broilers
Based on the obtained results it can be concluded that the addition of hot red pepper in the diet of
broiler chickens led to a statistically significant (p<0.05) differences in body weight (Table 4).
Chickens have finished the preparatory period with uniform body weight with no statistical
significant differences (p>0.05). At the end of the second fattening period, addition of hot red
pepper exerted the stimulating effect and led to statistically significant differences (p<0.05) in body
weight in relation to the control treatment. After the completion of the experimental period, the
highest achieved body weight of chicken was in treatment HRP-0.5 (2460.6 g) which was followed
by treatment HRP-1.0 (2442.4 g) with statistically significant differences (p<0.05) compared to
control treatment.
Table 4. Body weight of chickens in experiment, g
Experimental
treatments
CON
LSM
HRP-0.5
LSM
HRP-1.0
LSM
Pooled SELSM
1 day
42.8a
42.5a
42a
0.47
7 days
162.7a
162.5a
161.6a
1.6
Age of chickens
14 days
21 days
28 days
388.6a
785.6a
1162.4a
a
a
385.3
770.5
1193.6a
a
a
385.1
762.4
1183.6a
3.87
8.38
11.84
35 days
1643.8b
1815.1a
1812.1a
12.2
42 days
2075.8b
2460.6a
2442.4a
24.33
Treatments with different letter indexes in the same column are statistically significantly different (p<0.05)
Our study has shown that the addition of hot red pepper has positive effect on production results of
chickens, which is also in agreement with previous findings of Alaa (2010), Al-Kassie et al. (2012)
and Puvača et al. (2014c) with the use of hot red pepper in broiler chicken nutrition. Research of AlKassie et al. (2011) revealed that the inclusion of hot red pepper at levels of 0.5%, 0.75% and 1% in
the diets of broiler chicken of hybrid line Ross 308 improved body weight gain and feed conversion
ratio. Investigation of Thiamhirunsopit et al. (2014) with the different forms of hot red peppers
showed better growth performance results of chickens on experimental hot red pepper treatments in
comparison to control treatments.
Lipid profile in broiler chickens
Addition of hot red pepper as feed additives to broiler chicken nutrition in this experiment led to
high improvement of lipid profile of chickens. From the results given in Table 5 it can be noticed
that the highest amounts of triglycerides (65.9 mg/dl), total cholesterol (97.2 mg/dl) and LDL (36.7
mg/dl) were in control treatment with statistically significant (p<0.05) differences in comparison to
treatments with the dietary addition of hot red pepper.
This effect can be explained by the possible inhibition of the Acetyl CoA syntheses enzyme that is
necessary for the biosynthesis of fatty acids. Afzal et al. (1985) reported that polyunsaturated fatty
acids prevent atherosclerosis through the formation of cholesterol esters. Both levels of hot red
pepper in our study decreased LDL levels compared to the levels in chickens of the control
186
treatment. This effect can be explained by the possible mechanism of antioxidant and antiperoxide
lowering action on LDL or the decrease in hepatic production of very low density lipoprotein
(VLDL) which serves as the precursor of LDL in the blood circulation (Kim et al., 2009).
Table 5. Biochemical blood parameters and lipid profile, mg/dl
Experimental
treatments
CON
LSM
HRP-0.5
LSM
HRP-1.0
LSM
Pooled SELSM
Triglycerides
a
65.9
16.7b
17.7b
0.8
Total
cholesterol
97.2a
52.4b
54.3b
0.9
HDL
LDL
b
a
19.2
35.5a
35.7a
1.16
36.7
9.4b
10.3b
1.01
non HDL
a
78.0
16.9b
18.6b
1.03
HDL/LDL
0.5b
3.8a
3.6a
2.33
Treatments with different letter indexes in the same column are statistically significantly different (p<0.05)
Addition of hot red pepper to the broiler diet in different amounts from 0.25 to 1% had influence on
decreased concentration of blood cholesterol, and other blood biochemical parameters (Alaa, 2010;
Al-Kassie et al., 2012). Furthermore, addition of spice herbs and medicinal plants can facilitate
activity of enzymes which are involved in the conversion of cholesterol to bilious acids and
subsequently will result in lower cholesterol concentration in the carcass. Similar results with the
lowering effects of total cholesterol in red and white meat and skin of chickens fed with dietary
garlic powder was obtained by Stanaćev et al. (2012). Spice herbs in human nutrition had a very
large influence in health promotion and lowering concentration of blood cholesterol and lipid
oxidation (Ahuja and Ball, 2006). Beside the hot red pepper, garlic (Puvača et al., 2014a) and black
pepper (Puvača et al., 2014b) had a high impact on alteration of blood lipid profile of chickens.
Capsinoids present in red peppers causes pungent, hot tasting sensations when consumed as a part
of the diet in addition to sensory properties of chicken meat that may be affects human health,
because capsinoids includes antimicrobial activities against disease caused by bacteria. Meat
obtained by chickens fed with hot red pepper poses better lipid profile and can be successfully used
in daily human nutrition as a dietetic food.
Conclusions
Based on the obtained results, it can be concluded that the addition of hot red pepper in broiler
chicken nutrition has positive effect on production performances. Addition of hot red pepper in the
amount of 0.5 g/100g has led to the highest final body weights of chickens. Also it can be
concluded that significant lowering of plasma cholesterol, triglycerides, LDL and increase of HDL
by this spice herb supplementation in broiler diet could indicates effective in regulation of lipid
metabolism in a favourable manner for prevention of atherosclerosis or coronary heart diseases in
humans who use this kind of chicken products in their daily nutrition.
Acknowledgments
This paper is a part of the project III 46012 which is financed by Ministry for Science and
Technological development of the Republic of Serbia. Also the realization of one part of this
experiment was supported by the Perutnina Ptuj – Topiko a.d., Petefi brigade 2, 24300 Bačka
Topola, Serbia.
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________________________________________________________________________
Session № 3
VECTOR BORNE INFECTIONS
Full papers
_______________________________________________________________________
189
Plenary lecture:
MOSQUITO-BORNE FLAVIVIRUSES IN EUROPE: CURRENT PERSPECTIVES
Juan Carlos Saiz
Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria (INIA). Carretera de la Coruña Km 7.5, 28040, Madrid (Spain)
Corresponding author: jcsaiz@inia.es
Abstract
Flaviviruses (Flaviviridae family) constitute a genus of viruses that contains more than 50 different
species, many of which are important human and animal pathogens. Flavivirus genome is
constituted by a single-stranded RNA molecule of positive polarity (≈ 11,000 nt) that encodes a
polyprotein in a single open reading frame. Flaviviruses are maintained in nature in an infectious
cycle between wild animals, which act as their natural host, and insects (mosquitoes and ticks) that
act as their vectors. These vectors can transmit the virus to other animals or to humans, causing
zoonoses. Flaviviruses can infect the host nervous system, thus inducing severe neurological
diseases that can lead to death. Global climate warming together with changes in land use are
helping to change the distribution patterns of flaviviruses. Nowadays, 3 mosquito-borne flaviviruses
have been detected in Europe: West Nile virus (WNV), Usutu virus (USUV) and Bagaza virus
(BAGV), being WNV the most clinically relevant. WNV lineage 1 has been circulating in Europe
since 1960´s, but from the 1990´s the number, frequency and severity of the outbreaks increased
greatly. Earlier in this century, lineage 2 strains entered the continent, being already responsible for
hundreds of human cases and dozens of deaths. Lineage 2 strains are currently circulating in central
and south-eastern Europe, including Serbia. Usutu virus (USUV), was reported for the first time in
Austria in 2001 and quickly spread across Europe, causing a considerable number of bird deaths
and neurological disorders in a few immunocompromised patients, and it is now considered a
European resident pathogen. BAGV was reported for the first time in Europe when caused a deadly
outbreak among partridges and pheasants in Spain in 2010. Here, different aspects of mosquitoborne flaviviruses pathogenesis and clinical consequences, distribution, epidemiology, diagnosis,
and prophylaxis (vaccine and antivirals) will be discussed.
Keywords: Flavivirus, mosquitoes, Europe, West Nile virus, Usutu virus, Bagaza virus
Introduction
Flaviviruses (Flaviviridae family) constitute a genus of viruses that contains more than 50 different
species, many of which are important human and animal pathogens, such as Dengue virus (DENV),
West Nile virus (WNV), Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Usutu virus
(USUV), or Tick-borne encephalitis virus (TBEV), among others (Weaver and Reisen, 2010). Most
flaviviruses are maintained in nature in an infectious cycle that includes transmission between wild
animals, which act as their natural host, and insects (mosquitoes and ticks) that act as their vectors,
being thus arboviruses (arthropod-borne viruses). These vectors can transmit the virus to other
animals or to humans, causing zoonoses. A characteristic shared by a wide variety of flaviviruses is
that they can infect cells from the host nervous system, thus inducing severe neurological diseases,
which often include encephalitis (Sips et al., 2012). Global climate warming together with changes
in land use are helping to change the distribution patterns of some flaviviruses, so that, they are
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currently being detected in new geographical zones, where they cause infectious outbreaks that
affect both human and animals (Gould and Higgs, 2009).
Genomic organization and structure
Flavivirus genome is constituted by a single-stranded RNA molecule of positive polarity of about
11,000 nucleotides in length that encodes a polyprotein in a single open reading frame flanked by
two non-coding regions (NCRs) located at the 5’ and 3’ ends of the genome (Martín-Acebes and
Saiz, 2012). The RNA presents a cap structure at 5’ end, but lack a 3’ poly (A) tract, and a single
open reading frame that encodes for a polyprotein that is proteolytically processed by viral and
cellular proteases rendering ten major viral proteins: three structural (C, prM and E) and seven nonstructural, NS (NS1, 2A, 2B, 3, 4A, 4B y 5) involved in different aspect of the viral cycle (MartínAcebes and Saiz, 2012).
The genomic RNA is enclosed within a nucleocapsid formed by the capsid (C) protein that
constitutes the core of the virion and is enveloped by a lipid bilayer derived from the host cell
(Martín-Acebes and Saiz, 2012). Mature virions display a smooth outer surface with no projections
or spikes constituted by the membrane (M) protein and the E glycoprotein, resulting in a particle of
icosahedral symmetry. The E glycoprotein is exposed on the surface of the virion and is the main
responsible for the induction of neutralizing antibodies.
Ecology
Flaviviruses are grouped according to their vectors, mosquitoes, ticks, or those for which the vector
is still unknown. Those transmitted by mosquitoes are subdivided into 2 clades depending on the
vector genus (Culex or Aedes) (Beck et al., 2013). Culex-clade viruses usually have birds as natural
reservoirs, are neurotropic, and may cause encephalitis. Aedes-clade viruses have primates as main
reservoir and are not used to be neurotropic, but frequently cause hemorrhagic diseases. Tick-borne
flaviviruses are also grouped into 2 clades, those that circulate among seabirds and those that
produce encephalitis and which main natural reservoir are rodents, being the most clinically
relevant the TBEV (Beck et al., 2013).
Within a mosquito genus, different species show diverse competence for transmission of the
different flaviviruses (Martín-Acebes and Saiz, 2012). For instances, more than 11 species have
been described as WNV competent vectors in U.S., being the most efficient ones those of the Culex
species, although other species (such as Aedes or Ochlerotatus) may also play a role on viral
transmission as bridging vectors that can transmit the virus to mammals. In Europe, the virus has
been isolated from more than 40 different species, being again those of the Culex species the main
vectors. Several other species have been also described as competent vectors in other geographical
areas.
Climate (temperature, humidity, rainfall rate, etc.) and landscape conditions (altitude, latitude, water
sources, forestation, etc.) directly affect arthropod populations, density and stability, thus playing a
pivotal role in arboviral dissemination and evolution (Gould and Higgs, 2009; Beck et al., 2013;
Weaver and Barret, 2004). Climate change affects the geographical and seasonal distribution of
arthropod vectors, a fact that has many consequences. Expansion of mosquito species to more
extreme latitudes and elevations allows them to reach new host populations to feed on, and to share
new ecological niches with other vector species. Heavy rainfall and warm temperatures benefit the
increase of mosquitoes populations, and has been positively correlated with arbovirus transmission
(Martín-Acebes and Saiz, 2012; Weaver and Barret, 2004). Likewise, overabundance of birds that
share urban and rural areas with ubiquitous fastidious mosquitoes that feed on birds and mammals
plays an important role in flavivirus transmission. In fact, in two of the major urban WNV
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outbreaks reported in Europe, which took place in Bucharest (Romania), and Volgograd and
Volzhskiy (Russia), the regions were heavily infested by potential mosquito vectors. Thus, arboviral
epidemics are probably more related to the concomitant profusion of hosts and vector populations in
the same area, generally wastelands and mashes where migratory birds are abundant, than to other
factors (Brault, 2009).
An important factor in arboviral evolution is socio-economic development since it has led to
increased and faster travelling, urbanization, deforestation, irrigation, and migratory activities
leading to overpopulation. For instance, and because JEV outbreaks were mainly restricted to Japan
before the second world war, the conflict seems to have favoured virus spread (Gould and Higgs,
2009). Hence, new human and animal behaviours and climate changes are facilitating contact
between vectors and hosts that, together with viral genetic mechanisms, may lead to the emergence
or re-emergence of new arbovirus with expanded tropism and host-range and, thus, represents an
important threat for human and animal health.
Flavivirus circulation in Europe
In Europe, only a few flaviviruses are considered endemic (WNV, USUV and TBE), although
imported infections by other members of the family (DENV, YFV, etc.) are occasionally reported
(Beck et al., 2013). Among tick-borne flaviviruses, TBVE is the most clinically relevant in Europe
(Amicizia et al., 2013). The virus is transmitted by Ixodes ricinus and I. persulcatus and infects
more than 10.000 humans every year around the world. In Europe, hundreds of cases are reported
every year, even though it is estimated that between 70%-95% of them are asymptomatic. Most
cases occur in Finland, central Europe, and the European regions of Russia. Besides TBVE,
Looping ill virus (LIV), which infects mainly sheep and it is transmitted by I. ricinus ticks, has been
reported in several countries (Spain, Norway, Ireland, Turkey and Bulgaria), but where it is more
common is in the British Islands (Amicizia et al., 2013).
Regarding mosquito borne flavivirus circulating in Europe, West Nile virus is the more clinically
important, being now considered endemic in the continent (Martín-Acebes and Saiz, 2012).
Recently USUV seems to have also colonized the continent, and the Bagaza virus (BAGV) was
recently detected in Spain (Beck et al., 2013). Presence of WNV in Europe is known since the
1960´s, but it was in the 1990´s when lineage 1 strains colonized south Europe and the
Mediterranean basin, originating a very significant increase in the number, frequency and severity
of the outbreaks (Martín-Acebes and Saiz, 2012). At the beginning of this century, lineage 2 strains
entered the continent and, since then, they have been isolated in birds, mosquitoes, sentinel
chickens, and humans in several countries, being already responsible for hundreds of human cases
and dozens of deaths (Lani et al., 2014). Lineage 2 strains have already been detected in central and
south-eastern Europe. Nowadays, viruses from the two lineages are currently circulating in Italy. In
Serbia, the first outbreak of WNV clinical infection in humans was reported in 2012 with a total of
70 West Nile fever cases, 41 clinically and laboratory confirmed of which 9 resulted fatal (Dauphin
and Zientara, 2007). One year later over 300 new human cases were reported in the country, in what
was the largest human WNV outbreak in Europe during that year (http://ecdc.europa.eu/). Before
that, anti-WNV antibodies had been detected in humans, horses and birds, and WNV-RNA was
amplified from mosquito and bird tissue pools, from which the first WNV strain was isolated in the
country (Lani et al., 2014).
As mentioned early, only other two mosquito borne flaviviruses have been detected in Europe
(Beck et al., 2013). BAGV was reported to cause a deadly outbreak among partridges and pheasants
in Spain in 2010, but it has not been detected again since then. In contrast, USUV, which was
reported for the first time in Austria in 2001, has quickly spread across the continent, causing a
considerable number of bird deaths. The virus has been detected in mosquitoes, birds, bats, and
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equids, and it has been recently associated with a few cases of neurological disorders in
immunocompromised patients in Italy and Croatia (Beck et al., 2013).
Clinical manifestations and pathogenesis
Most of WNV infections in humans are asymptomatic, but an estimated 20% of the infected people
develop clinical symptoms, although severe neurological diseases are observed in less than 1% of
them (Sips et al., 2012; (Martín-Acebes and Saiz, 2012). West Nile fever (WNF) is characterized by
several non-specific flu-like symptoms, which usually develop between 2 to 15 days after infection
and last for 2 to 5 days. Severe West Nile disease (WND) is associated with neurological disorders,
with an estimated 10% of the neuroinvasive cases resulting fatal. A fully functional immune (innate
and adaptive) response (humoral and cellular) is essential to fight WNV infection, although, overall,
humoral immune response is capable of control viral load and dissemination; Nevertheless, WNV is
capable to directly infect neurons, brain stem and spinal cord. The pathogenesis of WNV infection
is similar to that of other Flaviviruses. After primary inoculation, WNV is believed to replicate in
resident skin Langerhans dendritic cells before it traffics to the lymph nodes and blood stream from
where it reaches the spleen and kidneys and, finally penetrates the CNS resulting in inflammation of
the medulla, brain stem and spinal cord (Martín-Acebes and Saiz, 2012).
As in humans, the majority of WNV infections in horses are usually not accompanied by
presentation of clinical signs, which are only observed in around 10% of the animals and are
characterized by fever and, sometimes, by ataxia and muscular weakness, which usually last for 3
weeks (Martín-Acebes and Saiz, 2012). Even though outbreaks resulted in 25 to 45% of mortality
rates among affected horse, implementation of vaccination campaigns has strongly reduced these
mortality rates. However, contrary to other Flavivirus infections, clinical cases of WND in horses
do not precede that in humans, discarding their use as sentinels.
Typical clinical signs in birds are neurological, such as ataxia and paralysis, as well as nonneurological, such as depression, lethargy, ruffled feathers, weight loss and myocarditis (MartínAcebes and Saiz, 2012). When birds die from WND, they use to do it in the first 24 h after the onset
of clinical signs. WNV infection causes damage in multiple bird organs. Monitoring of crow
mortality has been successfully adopted as an epidemiological indicator for tracking WNV activity
in the USA, predicting an increase of the risk for human infections (Martín-Acebes and Saiz, 2012).
Diagnosis
Flavivirus laboratory diagnosis relays on isolation of virus, detection of viral antigens or RNA in
blood or tissues, or detection of virus-specific IgM antibody that should be further confirmed by
detection of IgG antibody in the same or a subsequent sample.
Virus isolation in susceptible cell culture is the gold standard for virus detection, but it is usually
hampered by the typical short duration and low levels of viremia and by the need of BSL-3
facilities. In this regard, several methods for detection of viral RNA have been applied for
Flavivirus surveillance and diagnosis, mainly reverse transcription (RT) polymerase chain reaction
(PCR) assays and quantitative real-time RT-PCR (Martín-Acebes and Saiz, 2012; Dauphin and
Zientara, 2007).
Cross-reactivity between Flavivirus antigens is the greatest drawback for proper serological
diagnosis and epidemiological studies and, thus, sera have to be tested against different related
viruses and results have to be subsequently confirmed by different assays, namely hemagglutination
inhibition (HI), immunoflourescence (IF) or plaque reduction neutralization test (PRNT),
considered as the gold-standard (Martín-Acebes and Saiz, 2012; Dauphin and Zientara, 2007). A 4193
fold increase in PRNT titers between 2 sequential serum samples collected 2-3 weeks apart usually
confirms an acute Flavivirus infection, and viral neutralizing titers 4-fold higher than titers to other
related-flavivirus is usually taken as a probe of the specificity of the infection. Different ELISA
formats, both commercial and in-house, are currently applied for serological testing using
inactivated whole virus as antigen; however, its production implies risks for laboratory personnel
and needs highly sophisticated biosafety level 3 (BSL-3) containment facilities to grow the virus,
and then, several ELISAs have been developed using recombinant viral proteins expressed in a
variety of systems (bacteria, mammalian cells, insect cells, and larvae) (Martín-Acebes and Saiz,
2012; Dauphin and Zientara, 2007).
Prophylaxis
Until now, no WNV or USUV vaccine or specific therapy has been approved for humans, and
clinical treatment is only supportive; however, promising approaches are being developed (MartínAcebes and Saiz, 2012; Dauphin and Zientara, 2007). In this regard, and even though there are
effective, licensed WNV vaccines for horses, no approved vaccines exist for human use, although
some have undergone clinical trial. These candidates are based on the use of either live attenuated
or chemically inactivated virus, or recombinant viral proteins (or fragments of them) synthesized in
diverse systems (from bacteria to insect cells and larvae) (Dauphin and Zientara, 2007).
The search for antiviral compounds active against Flavivirus infection includes the identification of
those targeting distinct aspects of the viral replication cycle (Martín-Acebes and Saiz, 2012;
Dauphin and Zientara, 2007). The candidate may be targeted against the viral particle itself,
impairing its entry and/or infection, or it may block multiplication of the virus within infected cells.
This search has leaded to the identification of multiple promising compounds interfering with these
processes, including the repositioning of drugs approved for other purposes that have also shown
antiviral activities against Flavivirus (Martín-Acebes and Saiz, 2012; Dauphin and Zientara, 2007).
Recently a new approach in antiviral search has focused on cellular factors (proteins and lipids)
implicated in the viral life cycle, which may constitute potential targets for antiviral activity. In any
case, progress in Flavivirus therapies should pass through a combination of drug strategies that
targets viral replication, boosts protective immune responses, minimizes neuronal injury, and limits
the development of resistant variants. However, long way remains to be completed before these
novel compounds could to be administered to humans or animals, as they should be first tested for
antiviral activity in animal models and adverse effects should be ruled out.
Conclusions
Although our knowledge about Flavivirus infections has greatly increased during recent years, some
aspects of WNV activity still need to be further addressed: the ways WNV colonizes new ecological
niches and the role that climate and anthropogenic factors play; the differences in WNV disease
manifestations between different regions of the world; the better understanding of WNV immunity,
pathogenicity, and the molecular basis of virulence; the development of national and international
surveillance programs to monitor WNV spread and to take appropriate measures to control it; the
search for more efficient, rapid, and specific diagnostic assays; and the search for cost-effective
human vaccines and antiviral targets for therapeutic usage.
Acknowledgments
Supported by grants RTA-2011-0036 and E_RTA2013-00013-C04-2014 (INIA), and PLATESAP2013/ABI-2906 (Comunidad Autónoma de Madrid).
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Invited lecture
AN OVERVIEW OF THE RECENT STUDIES ON TICK BORNE PATHOGENS IN
SERBIA
Sara Savić1, Branka Vidić1, Aleksandar Potkonjak2, Željko Čonka3
1 Scientific Veterinary Institute „Novi Sad“
2 Department of veterinary medicine, Faculty of agriculture, University of Novi Sad,
2 Zito Dunav Agroveterina DOO, Voganj
* Corresponding author: sara@niv.ns.ac.rs
Abstract
Tick borne diseases are one of the key issues within a „One Health“ concept. A triangle between
humans, animals and ticks has been a challenge for researchers for some time now. In Serbia,
research on ticks as vectors, tick borne pathogens and tick borne diseases has started approximately
a decade ago. The first studies were on the presence of Borrelia burgdorferi sensu lato starins in
ticks, followed by research on Babesia spp. During the last few years a lot of work has been done,
not only in Serbia, but in the whole region – on the topic of the presence of Anaplasma
phagocytophilum, Coxiella burnetii and Francisella tularensis in ticks.
Tick species found in different regions of Serbia so far are the following: Ixodes ricinus,
Dermacentor marginatus, Dermacentor reticulatus, Rhipicephalus sanguineus, Hemaphysalis
punctata, Haemaphysalis concinna
For B.burgdorferi s.l. more genospecies were found in ticks during the last few years: B. afzelii,
B,burgdorferi s.s, B. lusitaniae, B. garinii and B. valaisiana. Monitoring of the prevalenece of
B.burgdorferi s.l. in ticks is constant, but it differs among different regions in Serbia (Vojvodina,
Belgrade region and middle Serbia). The prevalence for B.burgdorferi in ticks varies from 23% to
42.5%.
Babesia spp is found to be present in ticks with two different methods, in different tick species with
different prevalence varies from 10.61% to 46.40% in different regions. In another study,
Anaplasma phagocitophylum has recently been found in 13.9% ticks in Serbia. Coxiella burnetii
was found in less than 7% in ticks in different regions of Serbia. The prevalence for Francisella
tularensis microorganisms has also been found as 3.8% in ticks.
All of the monitored pathogens are causative agents for more or less dangerous zoonozes, which
can be found in the region of Serbia and almost all of them have already been reported in humans.
Key words: tick borne pathogens, zoonozes, borreliosis, anaplasmosis, ricketiosis, babesiosis
Introduction
Arthropods as vectors of pathogens are causing vector-borne diseases, an important threat to public
health. The pathogen carried by the arthropod vector is transferred during blood feeding, the closest
interaction between the arthropod vector and the vertebrate host. Whether or not the transferred
pathogen is actually causing a disease, depends on a complex interaction of many factors, most
important the pathogen, the host and the host’s immune system (Mencke, 2013).
A research on vector borne infections and pathogens has started two decades ago. Vector borne
pathogens are often known as emerging pathogens. Emerging pathogens include infectious agents
that were previously un recognized and have been identified and associated with a new disease or
an illness with a previously unknown aetiology; organisms that have been described in other regions
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and imported into areas where they were previously unknown, or agents that were constantly
present in the affected area at a low level or in a different host and due to some change have become
more widely spread in the population under concern (Harrus and Baneth, 2005). Important tickborne zoonoses such as Lyme disease, human granulocytic anaplasmosis (HGA) caused by A.
phagocytophilum, human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis, and
human babesiosis caused by B. microti were all described in the US during the second half of the
20th century and later in other countries and continents. Candidates Neoehrlichia mikurensis is an
intracellular bacterium member of the Anaplasmataceae family which causes severe disease with
fever and septicaemia in humans. It is an example of an “emerging” tick borne infection that has
been known only for a decade and appears to be dispersed in multiple regions. It has been identified
throughout Europe, in patients in Sweden, Switzerland, Germany, Czech Republic, China (Foldvari,
2014). Similarly to other tick borne infections agents, it is likely to have been circulating among
wildlife animals and ticks long before it emerged as a recognised clinical cause of human disease.
Tick borne diseases are one of the key issues within a „One Health“ concept. A triangle between
humans, animals and ticks within the environmental conditions, has been a challenge for
researchers for some time now.
Figure 1 – Overlaping of tick borne infections among different species (Baneth, 2014)
Ticks as vectors
Approximately 900 species of ticks have been described to date, of which more than 700 belong to
the Ixodidae (hard ticks); approximately 200 belong to the Argasidae (soft ticks). The lifestyle of
ticks which includes uptake of blood from hosts, secretion of saliva into the host tissues, movement
between different hosts and production of eggs from which a new generation of ticks develops,
inevitably makes them suitable to host other organisms. A wide variety of pathogens is transmitted
from ticks to vertebrates including viruses, bacteria such as rickettsiae and spirochetes, fungi,
protozoa and helminths, of which most have a life cycle which requires passage through the
vertebrate host (Dantas-Torres et al., 2012).
Several factors contribute to the change in geographic ranges of tick borne infections. Ticks cannot
fly or move long distances by themselves and, therefore, their transfer from one location to another
depends mostly on movement of hosts including birds which can fly long distances (Mathers et al.,
2011 and Hasle, 2013), migrating wild animal species such as jackals, animals introduced into new
areas by humans such as farm animals imported into areas where they were not present before,
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rodents travelling accidently with ships or trucks, or imported pet animals. Climate change may also
facilitate migration of vertebrate tick hosts, allowing dispersal of ticks and pathogens into new
territories (Estrada-Peña et al., 2012). Transmission of tick borne infections can also occur by
contaminated blood transfusions and needles. Babesia of different species have been shown to be
transmitted by blood transfusion in both humans and dogs (Stegeman et al., 2003). Such artificial
transmission may result in spread of tick borne infections to new areas, if competent tick vectors
and reservoir hosts are found in those areas. Although the majority of tick borne infections are
transmitted via the tick saliva during the course of the blood meal, there are some pathogens of
veterinary importance which are transmitted naturally by other mechanisms. For example,
Hepatozoon spp. is protozoa that belongs to the Apicomplexa and infect vertebrates by oral
ingestion of an arthropod host containing infective sporozoites. Hepatozoon spp. infect dogs that
orally uptake and ingest ticks harbouring mature sporozoites.
Ixodes ticks are abundant throughout Europe, but I.ricinus nortward shift has been noted, all the
way up to Norway and Sweden (Jaenson and Lindgren, 2011). I. ricinus has also been recorded at
1200-1300m above sea level in Check Republic and Austria (Daniel et al., 2003; Stuenzner et al.,
2006).
Factors that influence the distribution of I. ricinus in Europe have been identified as the following:
(1) factors directly related to climatic change (affecting the tick, host or habitat), (2) factors related
to changes in the distribution of tick hosts (which may be directly or indirectly a consequence of
human interventions) and (3) other ecological changes. Considering the large size of the global
human population, the high density of humans in some areas, and the surface size of the adult
human body, humans would be expected to be one of the most common blood sources for
ticks. Certainly most tick borne infections circulate between wildlife animals and ticks, and may
affect humans or domestic animals, but do not rely on infecting people for their persistence. For
example, Lyme disease circulates mostly among rodents, and humans or domestic dogs are just
incidental hosts that could suffer from clinical disease but do not play an important role in the
enzootic transmission and epidemiology of this infection (Radolf et al., 2012). Despite the global
abundance of humans and their presence in a variety of climates and ecological conditions, they are
not major reservoirs for tick borne infections. Tick borne infections of humans, farm animals and
companion animals such as dogs and cats, may overlap, and some agents such as B.
burgdorferi and Anaplasma phagocytophilum are able to infect hosts belonging to more than one of
these categories, however all of these zoonotic agents are associated with wildlife reservoirs
Tick borreliosis
One of the most prevalent vector borne disease in humans in Europe is borreliosis. In Europe
several Borrelia species have been described, which are considered to cause human borreliosis with
clinical symptoms. These species are referred to collectively as Borrelia burgdorferi sensu lato, and
B.afzelii, B.garinii and B.valesiana. It is proposed to use term borreliosis or tick borreliosis for
Borrelia infection in Europe with B.burgdorferi s.l. instead of Lyme borreliosis which is used in
North America.
The risk of tick bite during outdoor activities for humans and dogs was studied in hunters and
compared to humans without association to hunting. The study indicated a higher prevalence of
borreliosis in owners of hunting dogs (15%) compared to non hunters (9%), but in more than 90%
of hunters, there was no evidence of clinical symptoms. There is an association between
occupations such as forestry workers, military personnel, rangers and borreliosis (Nohlmans et al.,
1991).
Information on the role of dogs in tick borreliosis, as infected patient or as a sentinel for tick
borreliosis in Europe is rather limited. The data for seroprevalence of borreliosis in dogs in Europe
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vary from only several percentages in Czech Republic, Poland and Romania, to 50% in Slovakia
(Pelchalova et al., 2006; Scotarczak and Wodecka, 2003; Kiss et al., 2011; Stefancikova et al.,
1998)
Tick-borne rickettsiales
Rickettsiales transmitted by ticks in Europe and causing clinical signs in dogs are Anaplasma
phagocytophilum and Anaplasma platys. There is also Erlichia canis, belonging to the
Anaplasmataceae family. Anaplasma phagocytophilum has been found all over Europe in the
following countries: Austria, Belgium, Czech Republic, Finland, France, Germany, Hungary, Italy,
Luxembourg, Netherlands, Norway, Poland, Russia, Slovakia, Slovenia, Spain, Sweden,
Switzerland and United Kingdom (Jahfari et al., 2014)
Rickettsia conorii is the most important tick borne rickettiosis with public health impact in Europe.
It is the pathogen of „Mediterranean spotted fever“. The main vector for Rikettsia conorii is
Rhipicephalus sanguineus, but also Ixodes ricinus and Dermacentor spp have been described
(Benianti et al., 2002). Dogs have been identified as reservoirs of Rickettsia conorii (Levin et al.,
2012)
Tick borne babesiosis
In temperate areas of Europe, Dermacentor reticulatus is a quite common tick species affecting
dogs and is the primary vector of canine babesiosis due to Babesia canis
Canine babesiosis is a tick-borne disease caused by intraerythrocytic protozoa of the genus
Babesia which can cause severe clinical illness. In recent years, the geographic distribution of
babesiosis has expanded from western and central Europe toward northern Europe, probably due
both to changes in the climate which has increased tick survival and due to an increase in
companion animal travels.
Human babesiosis is caused by the intraerythrocytic parasite of the genus Babesia (phylum
Apicomplexa). Humans are commonly infected by the bite of Ixodid ticks. Rarely, transmission does
occur perinatal or via contaminated blood transfusion. Human babesiosis is a zoonotic disease with
a worldwide increasing importance according to the increasing number of immunocompromised
patients. Clinical symptoms have a wide range from asymptomatic to severe and letal cases. So far,
the detection of the parasites in ticks and seroepidemiological data in Europe identified 3
humanpathogenic species: B. microti, B. divergens und B. venatorum (EU1-3). The relative small
number of approximately 50 documented human cases is probably due to the lack of knowledge of
the disease and the availability of diagnostic tools. Comprehensive systematic investigations of the
prevalence in ticks, seroepidemiological data and improved diagnostic tests are urgently needed to
evaluate the importance of the parasite.
Review of current situation in Serbia
The development of molecular diagnostic tools such as conventional PCR, real-time PCR, DNA
sequencing and other methods have not only enhanced the capacity of diagnostic laboratories to
detect the presence of infection, but have also expanded the capability of detecting new, previously
unknown, pathogens and distinguishing between species and strains of microorganisms, which was
difficult and sometimes impossible prior to the advent of molecular biological techniques.
Furthermore, these molecular capabilities have become accessible and affordable to diagnostic
laboratories in the last decade, and not merely restricted to research facilities.
199
In Serbia, research on ticks as vectors, tick borne pathogens and tick borne diseases has started
approximately a decade ago. The first studies were on the presence of Borrelia burgdorferi sensu
lato strains in ticks, followed by research on Babesia spp. During the last few years a lot of work
has been done, not only in Serbia, but in the whole region – on the topic of the presence of
Anaplsma phagocytophilum, Coxiella burnetii and Francisella tularensis in ticks.
Tick species found in different regions of Serbia so far are the following: Ixodes ricinus,
Dermacentor marginatus, Dermacentor reticulatus, Rhipicephalus sanguineus, Hemaphysalis
punctata, Haemaphysalis concinna (Tomanović, 2013).
For B.burgdorferi s.l. more genospecies were found in ticks during the last few years: B. afzelii,
B,burgdorferi s.s, B. lusitaniae, B. garinii and B. valaisiana (Tomanović et al., 2014; Potkonjak et
al., 2014. Monitoring of the prevalenece of B.burgdorferi s.l. in ticks is constant, but it differs
among different regions in Serbia (Vojvodina, Belgrade region and middle Serbia). The prevalence
for B.burgdorferi in ticks varies from 23% to 42.5% in different regions (Milutinović et al., 2008;
Savić et al., 2010). Babesia spp is found to be present in ticks with two different methods, in
different tick species. It was found in Dermacentor reticulatus (21.57%) and Haemophysalis
concinna (8.57%) with one group of authors, by molecular detection in ticks collected from the field
(Mihaljica et al., 2012). The other group of authors searched for Babesia by microscopic analysis of
the midgut content of ticks in R. sanguineus, D. marginatus and D. reticulatus ticks, collected from
dogs with clinical sighs of babesiosis. The prevalence in ticks in the second study was quite higher
46.40% (Pavlović et al., 2002). Both studies were done in the same region. In another study,
Anaplasma phagocitophylum has recently been found in 13.9% ticks in Serbia, including strains
with proven pathogenicity for humans (Tomanović et al., 2010; Potkonjak et al., 2013). Coxiella
burnetii was found in less than 7% in ticks in different regions of Serbia (Tomanović et al., 2013).
The prevalence for Francisella tularensis microorganisms has also been found as 3.8% in ticks
(Milutinovićet al., 2008).
All of the monitored pathogens are causative agents for more or less dangerous zoonozes, which
can be found in the region of Serbia and some of them have already been reported in humans (Savić
et al., 2012).
Table 1 – Zoonotic tick borne diseases and hosts found in Serbia
Disease
Borreliosis
Bartonellosis
Ehrlichiosis
Rickettsiosis
Anaplasmosis
Tularaemia
Coxiellosis
Tick-borne encephalitis
Louping ill
*unpublished data, research still going on
Vector
Tick
Flea, Tick
Tick
Tick, Flea
Tick
Tick
Tick
Tick
Tick
Detection in Serbia
Ticks, dogs, humans
Cats
*
Ticks
Ticks, dogs
Ticks
Ticks, sheep, humans
*
No
Conclusion
Vector-borne diseases are important infectious disease of human and veterinary medicine across
Europe. From all the published data recently, it is concluded that further research is needed and
field studies on vector borne diseases in Serbia. The epidemiology of ticks, the dynamics of ticks
spreading into new habitats due to climate changes and land use is not enough known.
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Veterinary medicine in general, needs to be included in any action of European bodies regarding
tick borreliosis and also this disease is an important canine vector borne disease. Infections with
Rickettsiales, like Anaplasma phagocytophilum or Rickettsia conorii are emerging and re-emerging
vector borne diseases in Europe.
Vector borne diseases shared between humans and dogs, point clearly towards the necessity for a
joint effort under the ‘One health’ concept in all aspects, from epidemiology, clinic, diagnostic,
therapy and prevention. From a veterinary medicinal perspective the importance of a ‘One health’
concept was recently addressed by the World Small Animal Veterinary Association (WSAVA) by
initiating the ‘One Health Committee’ ( Day, 2010). It is the aim of this committee to position small
animal veterinary medicine as an integral part of the ‘One health’ concept in terms of zoonotic and
vector-borne diseases (Day, 2011).
The question at the moment is weather is the global prevalence of tick borne infections increasing
or are improvements in the ability to detect infection using sensitive and specific new techniques,
and an increased awareness, responsible for more detection of disease? It is most likely that both an increase in the spread of tick borne infections and an improved capability of detection are the
answer for the global rise in reporting of tick borne infections. Human tick borne infections cannot
be described without studying and understanding their relationship to animal hosts and reservoirs.
Unlike some of the major human vector-borne diseases associated with flying insect vectors, where
infection could be independent of association with animals, tick borne infections are zoonoses and
control efforts must consider this when programs are developed to limit or eradicate them.
Integration of veterinary and human reporting systems, surveillance in wildlife and tick populations,
and combined teams of experts from several scientific disciplines such as entomology,
epidemiology, medicine, public health and veterinary medicine are needed for the formulation of
regulations and guidelines for the prevention of tick borne infections (Baneth, 2014).
Acknowledgments
This work was supported by the grant TR31084 of the Ministry of Education, Science and
Technological Development of the Republic of Serbia.
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Invited lecture
WNV IN SERBIA: UPDATE OF CURRENT KNOWLEDGE
Tamaš Petrović1, Dušan Petrić2, Diana Lupulović1, Ivana Hrnjakovic Cvetkovic3, Vesna
Milosevic3, Sava Lazić1, Juan Carlos Saiz 4
1. Scientific Veterinary Institute “Novi Sad”, Novi Sad, Serbia
2. Laboratory for medical and veterinary entomology, Faculty of Agriculture, University of Novi Sad, Novi Sad,
Serbia
3. Institute of Public Health of Vojvodina, Medical Faculty, University of Novi Sad, Novi Sad, Serbia
4. Department of Biotechnology, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA),
Madrid, Spain
Corresponding author: tomy@niv.ns.ac.rs
Abstract
West Nile virus (WNV) is a neurovirulent mosquito-borne Flavivirus with zoonotic potential.
Recently, the number, frequency and severity of outbreaks of infections caused by WNV, with
neurological consequences for birds, humans and horses have increased dramatically throughout
central and south Europe, including Serbia, constituting a serious veterinary and public health
problem.
The emergency of WNV infections in Serbia is described through the current epidemiology
situation based on recent data on the presence, prevalence and incidence of WNV infection among
virus natural hosts and vectors; sentinel (horses) and other animal species, and in human population.
The short overview of the WNV serology studies conducted on horse blood samples collected in
different occasions during the last six years, and the results of the serology studies conducted
among other animal species like pigs, wild boars, and roe deer in Serbia are presented and
discussed. The results of the first studies on WNV presence in mosquito vectors, and in wild birds
as virus natural hosts in Serbia are presented and analyzed. Also, the data on the WNV serology
studies conducted in human population in Serbia in the last few years, and the existing data of
WNV outbreaks in 2012, 2013 and 2014 are included.
In addition, the national program for WNV monitoring launched by the veterinary service in Serbia
in April 2014 is presented. The program was funded by the Veterinary Directorate, and it was
implemented on the field by veterinary service in collaboration with entomologists and
ornithologists. The main objective of the monitoring program was early detection of WNV presence
in nature, and consequently timely alerting of human health services and local governments in order
to control the mosquito population and to inform the local communities. The monitoring program
was based on the direct and indirect monitoring of WNV presence in nature. Indirect monitoring of
virus presence was performed by serological testing of WNV seronegative - sentinel horses and
backyard chickens hatched during 2014, and direct monitoring was done by molecular testing of
WNV presence in pooled mosquito’s samples and in wild birds. Number of tested samples is
defined at the level of each district of the Republic of Serbia in relation to the risks of WNV
infection.
Keywords: West Nile virus, antibody and virus detection, domestic and wild animals, human
population, mosquitoes, surveillance program, Serbia
Introduction
West Nile virus (WNV) is a neurovirulent mosquito-borne Flavivirus with zoonotic potential,
which is maintained in nature in an enzootic transmission cycle between avian hosts and
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ornithophilic mosquito vectors. The virus occasionally infects other vertebrates, including humans
and horses, in which it may cause sporadic disease outbreaks that may result fatal. West Nile virus
(WNV) was first isolated from a febrile woman in the West Nile district of Uganda in 1937
(Smithburn et al., 1940) and today is considered as the most widespread flavivirus in the world,
endemic in Africa, Asia, Europe, Middle East, Australia and Americas (Trevejo and Eidson, 2008;
Calistri et al., 2010; Weissenböck et al., 2010; Papa et al., 2011).
WNV infections have been described in a wide variety of vertebrates (Komar et al., 2003). The
virus is maintained in an enzootic cycle between ornithophilic mosquitoes, mainly of the Culex
genus (Hayes et al., 2005; Ziegler et al., 2012), but also Aedes and Ochlerotatus genus, and certain
wild bird species (Savini et al., 2012; Ziegler et al., 2012). WNV was found in more than 150
species of wild and domestic birds (van der Meulen et al., 2005). Wild birds are important to public
health because birds migrating across national and intercontinental borders and becoming a longrange virus vectors (Linke et al., 2007). Following infection, many bird species produce levels of
viraemia that are sufficient for transmitting the virus to mosquitoes (Komar et al., 2003). Human
and mammals, especially horses, are occasional, dead end hosts and play limited roles in the natural
cycle because viraemia is generally too low to infect mosquitoes (Dauphin et al., 2004; Valiakos et
al., 2011), however severe neuroinvasive disease and occasionally with fatal outcomes can occur.
In Europe, until the 1990´s WNV had caused sporadic outbreaks with rare reports of encephalitis
but its epidemiological behaviour changed when it re-emerged in Romania, Russia and the
Mediterranean basin causing dozens of humans and horses deaths (Castillo-Olivares and Wood,
2004; Blitvich, 2008; Calistri et al., 2010). Also, only recently the strains of WNV lineage 2 were
identified in Europe: in 2004 and 2005 in goshawks and birds of prey in Hungary, in 2007 in
Volgograd, Russia, and in 2008 and 2009 in goshawks and a falcon in Austria (Bakonyi et al., 2006;
Erdélyi et al., 2007; Platonov et al., 2008; Wodak et al., 2011). Since 2008, WNV has been heavily
spreading throughout central and southeastern Europe, constituting a serious veterinary and public
health problem for Europe (Barbić et al., 2012; Ziegler et al., 2012).
The history of West Nile fever in Serbia is largely unknown. Due to absence of routine diagnose
praxis and limited resources in hospitals of Serbia the human cases of meningoencephalitis of
“unknown” origin, that should be submitted to laboratory testing for WNV presence, had been
neglected up until 2012. In addition, with the exception for research purpose, the regular, program
based WNV surveillance in sentinel chickens, horses or mosquitoes did not exist before 2013. Only
scarce historical data exists about the presence of WNV in human population and indicate
seroprevalence of WNV in republics of former Yugoslavia of 1-3% in Croatia, 1% in Bosnia and
Herzegovina and Kosovo, 1% in Montenegro and 1-8% in Serbia (Vesenjak-Hirjan et al., 1991).
WNV epidemiology in human population in Serbia
First serological investigation was conducted in 1972 and antibodies against West Nile virus
(WNV) were found in 2.6% - 4.7% of human sera (Bordjoški et al., 1072). After almost a 30 years
gap, ELISA IgG testing revealed the seroprevalence of WNV in 6.67% of human sera of the 45
patients who were hospitalized for encephalitis or meningoencephalitis in the period 2001-2005 and
3.69% of 406 samples taken from healthy persons. Average seroprevalence of WNV in samples
taken from 2001-2010 was 3.99% (18 out of 451). A total of 337 persons tested in 2010 were
exposed to at least one mosquito exposure related risk factor. Among them, 5.04% were
seropositive to WNV. Most of the probably infected people did not screen windows and doors in
their houses, while in the group using window screens only 0.88% were seropositive to WNV
(Petrić et al., 2012). The obtained seroprevalence in this study still didn’t suggest more intensive
circulation of WNV in Serbia. Except this data, as to our knowledge, no clinical manifestation of
disease was ever reported in Serbia until 2012.
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In August 2012, an outbreak of WNV infection in humans, was reported for the first time ever in
Serbia (EpiSouth Weekly Epi Bulletin - N°232, - N°240; ECDC, 2012; Obrenović et al., 2013;
Popović et al., 2013), being the first time that WNV infections in the country have been associated
with clinical symptoms. As of November 30, 2012, a total of 71 West Nile fever cases were
reported, among which 42 were clinically and laboratory confirmed, and in 9 cases resulted fatal
(lethality of 12.7%). All the cases were detected in central and northern part of the country, 72% of
them in the Beograd district (ECDC, 2012; Obrenovic et al., 2013; Popovic et al., 2013). This
epidemic continued, and became even more severe during 2013. As of November 2nd, 2013, a total
of 303 West Nile fever cases were reported, among which 202 were clinically and laboratory
confirmed, and 103 were classified as probable cases. Infection in 35 cases resulted fatal (lethality
of 11.6%). Almost all of the cases were also detected in central and northern part of the country
(Institute of Public Health of Serbia, 2014).
The epidemic also continued during 2014. The outbreak characteristic was similar as those from
2012. In total 76 clinical cases were reported and 9 cases resulted fatal. Almost all the cases were
detected in central and northern part of the country, and 65 out of 76 (86%) of them in four counties
(Belgrade, South Banat, South Backa and Srem county) (ECDC, 2014). In Europe during 2014 in
total 163 human cases were reported, so 47% of cases were from Serbia. It can be concluded that
WNV infection in Serbia become endemic and can be assumed that it will be public health problem
in the coming years also.
WNV epidemiology situation in horses
Serological analysis by ELISA based on WNV recombinant envelope E (rE) protein and PRNT
showed for the first time in Serbia that 12% of 349 tested horses from northern part of country,
sampled during 2009-2010, presented specific neutralizing WNV antibodies (average PRNT90 =
120, range: 42–650), which in one case also cross-neutralized Usutu virus. This was the first time
that anti-USUV high neutralizing antibody titers have been reported in horses. Positive horses were
found in 14 of the 28 municipalities studied, which are up to 200km distant (Lupulović et al., 2011).
In our another previous study (Medić et al., 2014), the presence of WNV specific antibodies was
examined in 252 horse sera samples collected from 7 different stables and locations in Vojvodina
province and Belgrade area, during 2007-2011. WNV antibodies were found in 72 (28.6%) sera
samples. The higher level of 28.6% anti-WNV antibody positive horses obtained in that study
comparing to 12% reported in our first investigation could be explained by the fact that in the first
study the horse sera were collected randomly from the whole territory of Vojvodina province, often
individually reared, and in the second study the blood sera were taken from horses situated in the
stables, with high number of horses in the same location. Also, most of the examined horse sera in
this study were sampled during 2010 and 2011 that could imply on possible more intensive WNV
circulation during year 2011. WNV seroprevalence ranged per stable from 13.3% up to 40%
seropositive animals. The highest prevalence of anti-WNV antibody positive animals was found in
stable near Romanian border (40%) and near Belgrade (35.5%). The high WNV prevalence was
assumed to be the results of intensive WNV circulation that was confirmed in Romania during 2008
- 2010 and the close proximity of river Danube with a high circulation of migratory wild birds
(Medić et al., 2014).
In addition, to asses WNV presence in the environment immediately after the human WNV
outbreak in 2012, during November and December of 2012, presence of anti-WNV IgG antibodies
were examined by commercial ELISA test in blood sera samples of 130 horses from 6 stables and 1
settlement in Vojvodina province, northern Serbia (Petrović et al., 2014). Positive results were
obtained in 49.23% (64/130) samples. Per stable, percent of seropositive animals was from 35% to
64%. This prevalence (49.23%) obtained in horses during 2012 was much higher than that found in
horses during 2009 and 2010 (12%), including the confirmed seroconversion in at least 8 horses that
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tested negative in 2010, thus confirming an intensive WNV circulation in 2012 on the territory of
Serbia. Very recent WNV infection theory is also supported by findings that among young, up to 3
years old animals, almost 57% tested positive on anti-WNV antibodies (Petrović et al., 2014).
Similarly, 96 horses from 5 tested stables during 2012 were tested again during 2013 with the same
methodology. High prevalence of 46.88% (ranged between stables from 23.53-75.0%) with new
cases of seroconversion were detected also indicating an intensive WNV circulation in 2013
(unpublished data). The very recent data suggests that more than 50% of horses from Northern and
central part of Serbia, the areas around the rivers Danube, Sava and Tisza, are seropositive
(unpublished data).
Presence of WNV in natural host species and vectors in Serbia
Presence of WNV was also studied in susceptible wild bird’s species and mosquitoes as virus
natural hosts and vectors in the last few years in Serbia. WNV circulation was examined by ELISA
and PRNT in 92 blood sera and 81 pooled tissues from 133 wild resident and migratory birds from
45 species within 27 families collected from January until September 2012 in Vojvodina Province northern part of Serbia (Petrović et al., 2013). WNV antibodies were detected in 7 (7.6%) blood
sera of: 4 Mute swans (Cygnus olor), 2 White-tailed eagles (Haliaeetus albicilla), and 1 Common
pheasant (Phasianus colchicus). Three of the seropositive birds found here were resident birds (two
White-tailed Eagles and one Common Pheasant), while the other four (Mute Swans) are considered
both migratory and resident birds in Serbia. Viral RNA was detected, for the first time in Serbia, by
RT-qPCR in 9 birds: 3 Northern goshawks (Accipiter gentilis), 2 White-tailed eagles, 1 Legged gull
(Larus michahellis), 1 Hooded crow (Corvus cornix), 1 Bearded parrot-bill (Panurus biarmicus),
and 1 Common pheasant. Seven of these birds died during the summer of 2012 while two (a
pheasant and one goshawk) died during winter-early spring. Eight of the nine WNV RNA positive
birds were strictly resident, suggesting that they became infected in the country. Moreover, isolation
of WNV-RNA from dead predators (5 of the 9 WNV positive birds) provides more evidence that
birds of prey play a key role in virus transmission (Petrović et al., 2013).
All the isolates were classified by phylogenetic analysis of partial E region sequences as lineage 2
WNV strains and they were closely related to those responsible of recent outbreaks in Greece, Italy,
and Hungary. Further on, West Nile virus from one Northern goshawk (SRB-Novi Sad/12) was
isolated on Vero cell culture and its full genome sequenced. Phylogenetic analysis of this complete
genomic sequence showed a lineage 2 strain that clusters with the viruses responsible for the most
recent human and animal outbreaks reported in neighbouring countries, however, SRB-Novi Sad/12
isolate was unique, as it showed a total of 29 distinctive nucleotides when compared to those
circulating in Europe. Comparison of partial sequences of the E region from five additional WNV
sequences recovered from respective birds in this study shows that at least two different groups of
lineage 2 strains, which simultaneously circulated during summer of 2012, can be distinguished
(Petrović et al., 2013). These results suggest that WNV has reached the country in, at least, two
different events and suggest that the virus not only has become endemic in Serbia and surrounding
countries, but that it is also evolving while circulating in the area. According to these findings, it
seems logically to think that since its original detection in Hungary, WNV lineage 2 has expanded
southwards and reached Serbia recently, but it cannot be ruled out that there had been prior sporadic
human and animal cases that have gone unnoticed.
Mosquitoes were sampled on the spots where possible circulation, based on serological testing of
humans and horses, was detected in order to optimally utilize available number of test kits and
minimize the number of pools for mosquito surveillance. Data obtained from human serological
surveillance in 2009 had indicated seven “hot spots” of possible WNV transmission in the
municipality of Novi Sad (capital of Vojvodina Province). In 2010 mosquito trapping was focused
207
on these “hot spots” revealing WNV RNA in three out of 50 pools of Cx. pipiens pipiens, the first
finding of WNV in mosquitoes in Serbia (Petrić et al., 2012). During this experiment, a total of
56757 mosquitoes (841 pools of 50 individual insects) originating from 66 localities in 29
settlements in Vojvodina were examined. The presence of WNV genome was established in only
three pooled-samples of mosquitoes collected during 2010 in the territory of Detelinara (part of the
city of Novi Sad). The isolate was typed as lineage 2 WNV (Petrić et al., 2012). During the year
2012 WNV outbreak in Serbia, mosquitoes were collected at 62 sites in 31 municipalities in Serbia.
West Nile virus RNA was detected in 9.55% of 314 mosquito pools (11113 specimens) from 9
municipalities in Cx. pipiens pipiens, Aedimorphus vexans and Culiseta annulata (Petrić et al., in
press). In addition, presence of WNV genome was confirmed in 28 (9.2%) out of 306 mosquito
pools collected and tested from 20 localities in Vojvodina Province of Serbia during 2013
(unpublished data). The very recent testing of WNV presence in mosquito vectors (Cx. pipiens
pipiens) was done during the first WNV surveillance program during 2014 in Serbia. The Cx.
pipiens pipiens mosquitoes were collected from the whole territory of Serbia and out of 995 tested
mousquito pools WNV were found present in 22 (2.21%) samples (unpublished data). These data
point on slightly less intensive WNV circulation during 2014 in Serbia, but still the virus circulation
in vectors is endemically present for the least four last years.
WNV seroprevalence in different domestic and wild animal species
Presence of anti-WNV antibodies in blood sera of different animal species detected recently also
represents the evidence of intensive circulation of WNV in the last few years in Serbia. Out of
tested blood sera of 66 donkeys, 1076 dogs, 318 poultry, 102 sheep, 6 goats, 30 cattle, and 5 deer,
collected between 2008 and 2012, presence of anti-WNV antibodies was found in 0.93% dogs and
0.31% poultry (Đuričić et al., 2013). To assess WNV circulation among mammals in the country,
688 samples obtained from 279 farm pigs, 318 wild boars, and 91 roe deer were investigated for the
presence of antibodies to WNV by ELISA and viral neutralization test. ELISA-reactive sera were
identified in 43 (15.4%) pigs, 56 (17.6%) wild boars, and 17 (18.7%) roe deer. Of these, 6 (14%),
33 (59%), and 4 (23.5%) respectively, neutralized WNV. One out of the 45 ELISA negative sera
tested, from a roe deer, neutralized WNV (Escribano-Romero et al., 2015).
Methodology of WNV surveillance program in Serbia
Veterinary Directorate of the Ministry of Agriculture and Environmental Protection infront the
Veterinary Service launched and funded the national WNV surveillance program starting from
April 2014. The surveillance program encompassed sentinel species (poultry and horses),
mosquitoes (particularly species Culex pipiens, which were confirmed as most prevalent WNV
vectors in our region) and wild bird species, which are natural virus reservoirs and populate the
natural habitats in Serbia, either temporarily or permanently.
The surveillance program was conducted throughout the year according to the provided guidelines.
Active surveillance was performed by serological examination (by ELISA) of sentinel poultry and
horses and by testing of virus presence in samples of mosquito vectors (sampled by dry-ice baited
traps in the period of most prominent vector activity using special traps), as well as in the samples
of all collected dead wild birds belonging to the species susceptible to WNV (tested throughout the
year). The detection of virus presence in birds and mosquitoes was done by molecular diagnostic
(RT-PCR or real-time RT-PCR). Passive surveillance encompassed serological (testing of paired
serum samples) and virological examination of clinically ill horses manifesting signs of CNS
dysfunction.
208
The active and passive surveillance encompassed all municipalities in the Republic of Serbia. The
selection and distribution of sampling localities in each county-region is defined by epizootiological
services of Scientific and Specialized Veterinary Institutes according to the assessment of the risk of
exposure to WNV (according to the human cases in the last 2 years, and previous studies performed
in horses). The whole Program was described in details by Petrović et al. (2014a). The basic
methodology of WNV surveillance program in Serbia in 2014 is described in the Table 1.
Table 1. WNV surveillance program (sampling /testing) in Serbia in 2014 (Petrović et al., 2014a)
High-risk regions/Counties
Lower-risk regions/Counties
1. Testing of sentinel animals (domestic poultry and horses) aimed at early detection of WNV
circulation
Surveillance of
sentinel poultry on
rural households –
poultry hatched in
current year
(backyard poultry)
Surveillance of
sentinel horses
Serological testing at the authorized
institute in the period May-September
from 10 settlements / County; 5 samples /
settlement from et least one household
according to described schedule.
6 samplings (1 in May; 1 in June; 2 in
July; 1 in August – by middle; 1 in
September (until 15 Sept)
Serological testing of 50 sentinel horses in
the authorized institute, sampling from
minimum 3 localities per County.
Sampling and blood testing of same
horses to be performed three times (in
three occasions) (June-July-August)
Serological testing at the authorized
institute in the period June-September
from 6 settlements / County; 5 samples /
settlement from et least one household
according to described schedule.
4 samplings (1 in June; 1 in July; 1 in
August – by middle; 1 in September
(until 15 Sept)
Serological testing of 30 sentinel horses
in the authorized institute, sampling
from min 3 localities per County.
Sampling and blood testing of same
horses to be performed three times (in
three occasions) (June-July-August)
2. Testing aimed at early detection of WNV in natural reservoirs and vectors
Virus surveillance
in wild birds
Virus surveillance
in vectors mosquitoes
(Culex pipiens)
Application of RT-PCR or real time RTPCR methodology for testing samples of
dead susceptible bird species throughout
the year, or up to 100 samples of
purposely hunted birds or live captured
susceptible bird species per County during
the period May - October
Collecting mosquitoes at 2-week intervals
in the period May-September at 10
localities within the County and testing
the virus presence by RT-PCR or real time
RT-PCR methodology (7 samplings in the
period from end May to the first half of
September)
RT-PCR or real time RT-PCR
methodology for samples of up to 50
dead birds (WNV-susceptible species)
per County during the period May October
Collecting mosquitoes at monthly
intervals in the period May-September at
5 localities per County and testing the
virus presence by RT-PCR or real time
RT-PCR methodology (5 samplings
once a month in the period from second
half May to the first half of September)
Future steps and expectations
The aforementioned serological and virological examinations confirmed active circulation and
endemic presence of WNV in the territory of the Republic of Serbia. Based on the obtained results
and anticipated intense circulation of WNV that poses substantial risks for both public and animal
209
health in Serbia, there is a need for further studies and continuous monitoring and surveillance of
WNV infection and virus epidemiology in Serbia in the coming years.
Acknowledgments
This work is conducted within the project TR31084 funded by the Serbian Ministry of Education,
Science and Technological development
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PATHOMORPHOLOGICAL FINDINGS IN ORGANS OF SHEEPS DIED OF
BLUETONGUE DISEASE
Ivan Dobrosavljevic1*, Milena Zivojinovic1, Slavonka Stokic-Nikolic1, Milica Lazic1, Dragan
Rogozarski1
1
Vetrinary specialistic institute “Pozarevac”, Pozarevac, Serbia
* Corresponding author: i.dobrosavljevic@vsipozarevac.com
Abstract
Bluetongue is an infectious arthropod-borne viral disease primarily of domestic and wild ruminants.
Infection with bluetongue virus (BTV) is common in a broad band across the world. Since the
1990s, BTV has extended considerably north of the 40th and even the 50th parallel in some parts of
the world (eg, Europe).
The etiological agent of the disease is Orbivirus, family Reoviridae. There are at least 24 serotypes
worldwide. Although not all serotypes exist in any one geographic area, virus has high capacity to
quickly spread across the area.
During the late summer and early fall 2014, emerging of the disease was observed in the eastern and
southeastern part of Serbia. It was confirmed as BTV Serotype 4. The first cases in sheep in
Branicevo district were recorded in September. The sheeps have had fever, lameness and
depression. Edema of lips, nose, face, submandibular area and eyelids were seen in most of the
cases. Edema of the head was followed by plentiful salivation. On the tongue and gums there could
be seen erosions and in one sheep it was found edematous tongue with mild cyanosis (blue tongue).
The first autopsy findings of dead sheeps did not clearly indicate blue thongue disease: several
erosions on the tongue and gums with plenty of saliva, slightly swollen subcutaneous tissue in the
chest region. The most pronounced lesions were in the thoracic cavity: hyperemia and pulmonary
edema, massive subpleural hemorrhages. The lesions of the proximal part of the pulmonary artery
were slightly pronounced (hardly visible changes).
Subsequent autopsy cases were revealed pathognomonic lesions on the proximal part of the
pulmonary artery (ring bleeding under the adventitia of the pulmonary artery), edema and
hemorrhages of the papillary muscle of the left heart ventricle. These gross lesions are nearly
certain sign of the disease. The severity of lesions depends on the virulence of the virus, the period
of time of it presents on some area and the immune status of the host. Laboratory confirmation is
essential for final diagnose.
Key words: Orbivirus, sheep, gross lesions.
Introduction
Bluetongue (BT) or ovine catarrhal fever is an arthropod-borne viral disease of domestic and wild
ruminants, particularly sheep. It is characterised by inflammation, haemorrhage, excoriations,
erosion and cyanosis of the mucous membranes of the oronasal cavity, coronitis, laminitis, oedema
of the head and neck, and torticollis. The name of the disease derives from cyanosis of the tongue.
Bluetongue is most prevalent in tropical and subtropical climate area. It was first described in South
Africa (in the late eighteenth century) in Merino sheep (Verwoerd and Erasmus, 2004). During the
twentieth century, the disease have been reported in Cyprus, Israel, USA, Portugal, Spain, Pakistan,
India, Middle East, China, Malaysia, Indonesia, Australia and in now days in Southeast Europe.
The etiological agent of the disease is a virus which is composed of a segmented, double-stranded
RNA enclosed in a double-layered capsid. It was classified in genus Orbivirus, family Reoviridae.
There are at least 24 serotypes worldwide. In blood and tissue specimens it is very stable at 20 °C, 4
°C and -70 °C, but not at -20 °C. The virus is unstable below pH 6,5 (Owen, 1964).
213
Bluetongue virus (BTV) first multiplies in the regional lymph nodes after virus inoculation by the
bite of a midge before spreading to the rest of the body. Viral replication occurs primarily in
endothelial cells and pericytes of capillaries and small blood vessels. Pathological changes in these
cells leads to hyperplasia of the endothelium and consequently vascular occlusion, stasis and
exudation, which eventually gives rise to hypoxia, oedema and haemorrhage as well as to secondary
lesions in the overlying epithelium (Verwoerd and Erasmus, 2004). The most sever lesions occur in
areas where the temperature is lower than that of circulating blood (Verwoerd and Erasmus, 2004).
There is also evidence for a highly selective involvement of endothelial cells in certain blood
vessels, such as the pulmonary artery and those where the most severe lesions are found. The
mechanism of these lesions is not understood.
The aim of this paper was not to elucidate the new facts about BT, but to remind and emphasizes
the importance of gross lesions in the diagnosis of this disease.
Photos used in this paper were taken during necropsies of sheep, in Veterinary institute
“Požarevac”, in September 2014. In all cases BTV was confirmed in full blood samples by ReverseTranscription Polymerase Chain Reaction (RT-PCR).
Results
During the late summer and early fall 2014, emerging of BT was observed in the eastern and southeastern part of Serbia. It was confirmed BTV, serotype 4. The first cases in sheep in Branicevo
district were recorded in September. The sheep have had fever, lameness and depression. Oedema
of lips, nose, face, submandibular area and eyelids were seen in most of the cases. Oedema of the
head was followed by plentiful salivation. On the tongue and gums there could be seen erosions and
in one sheep it was found oedematous tongue with mild cyanosis (blue tongue).
The first autopsy findings of dead sheep did not clearly indicate Blue tongue disease: several
erosions on the tongue and gums with plenty of saliva (Fig. 1, 2 and 3), slightly swollen
subcutaneous tissue and few ecchymoses of the intermuscular connective tissues in the chest region
(Fig. 4 and 5). The most pronounced lesions were in the thoracic cavity: hyperaemia and pulmonary
oedema, massive subpleural haemorrhages. The lesions of the proximal part of the pulmonary artery
were slightly pronounced (hardly visible haemorrhages) (Fig. 6, 7, 8 and 9).
Figure 1. Erosion and haemorrhage
on the mucosa of lower lip.
Figure 2. Erosive glositis and hyperemia of corner of the
lips.
214
Figure 3. Erosions and haemorrhages on
dental pad.
Figure 4. Slightly swollen subcutaneous tissue in
the chest region.
Figure 6. Hyperemia and pulmonary edema with
lot of foamy exudate in bronchus
Figure 5. Ecchymoses of the intermuscular
connective tissues in the chest region.
Figure 8. Hardly visible subepicardial
petechiae and petechiae at the proximal part
of pulmonary artery
Figure 7. Massive subpleural hemorrhages
215
Figure 9. Hardly visible petechiae in tunica
media of proximal part of pulmonary artery
(inner side).
Figure 10. Erosions on dental pad covered with
diphtheritic deposits.
Figure 14. Disseminated subepicardial ecchymoses
216
Figure 12. Gelatinous oedema of connective
tissue in the surroundings of the esophagus
Figure 11. Oedema of subcutaneous tissue
in the submandibular area.
Figure 13. Hydrothorax
Figure 15. Ring bleeding in the tunica
media of proximal part of pulmonary
artery.
Figure 16. Edema and hemorrhages of the papillary muscle
of the left heart ventricle. Disseminated subendocardial
ecchymoses.
Figure 17. Subcapsular haemorrhages of the spleen
Subsequent autopsy cases were revealed more pronounced and characteristic lesions: erosions on
dental pad covered with diphtheritic deposits – Fig.10; oedema of subcutaneous tissue in the
submandibular area – Fig. 11; gelatinous oedema of connective tissue in the surroundings of of the
esophagus – Fig. 12; hydrothorax - Fig. 13; sprayed subepicardial bleeding which cover a large area
of the heart - Fig. 14; pronounced ring bleeding in the tunica media of proximal part of pulmonary
artery - Fig. 15; edema and hemorrhages of the papillary muscle of the left heart ventricle - Fig. 16;
subcapsular petechial hemorrhages of the spleen – Fig. 17.
Discussion
Previously described gross lesions correspond largely with the clinical signs (Moulton, 1961).
Ecchymoses and larger haemorrhages in the tunica media of the pulmonary artery near its base are
characteristic of BT (Fig. 8, 9 and 15). These lesions are sometimes described as being
pathognomonic for BT, but they have also been seen on rare occasions in other infections such as
217
Rift Valley fever, heart-water and pulpy kidney disease in sheep. These haemorrhages may be
difficult do see and it may be necessary to stretch the pulmonary artery and to hold it against the
light (Fig. 9). Sometimes epicardial and endocardial haemorrhages as well as focal necrosis of the
papillary muscle can be seen in the left ventricle (Fig. 16). Bleedings in the wall of the base of the
pulmonary artery and lesions of the papillary muscle of the left ventricle are highly characteristic
for BT and they are usually obvious in severe clinical infections but as shown in Figures 8 and 9,
they may be barely visible in mild or convalescent cases (Lear and Callan, 2014).
Lymph nodes are commonly swollen and oedematous and at the spleen can be seen subcapsular
haemorrhages (Fig. 17).
Oral lesions which have been seen in the initial stage of the disease consist of slightly expressed
hyperemia, erosions and haemorrhages of the mucous membrane (Fig. 1, 2 and 3). The mucous
membrane was covered with a large amount of saliva. Destruction of epithelial cells lead to
excoriation and erosions (especially in areas of most friction: inside of the lips, dental pad, cheeks
and tongue, particularly adjacent to molars). Secondary bacterial infecton is often responsible for
the diphteric necrosis of the erosions (Martens et al., 1989), which have been observed in latter
clinical cases and necropsies (Fig. 10).
Hyperaemia, petechiation and erosions of the mucosa of the forestomachs, particularly of the
papillae, rumenal pillars, reticular folds and oesophageal groove also can be seen in gastrointestinal
tract (Erasmus, 1990).
The most severe pathology changes occur in the lungs. Severe hyperaemia and oedema of the lungs
accompanied by copious amounts of froth in bronchi and trachea, as well as hydrothorax, occur
especially in acute fatal cases (Fig. 6). Aspiration pneumonia may occasionally be found (Luedke et
al., 1964).
The muscle lesions comprise ecchymoses (Fig. 5) and gelatinous oedema of the intermuscular
connective tissue, particularly in the neck and the dorsal thoracic region.
Each of described lesions for himself are not sufficient for diagnosis of BT, but all together in
correlation with clinical signs and epizootic anamnesis are certain milestone in the right direction.
First cases of the disease should be differentiated from early signs of hepatogenous photosensitivity
caused by a variety of plant and mycotoxin poisonings (Kellerman et al., 1988), vesicular diseases
such as foot-and-mouth disease and contagious ecthyma. In sheep that die after typical oral lesions
of BT have regressed, it may be difficult to differentiate BT from disease such as heartwater and
pulpy kidney disease (Verwoerd and Erasmus, 2004).
A presumptive diagnosis of BT can be made from previously described lesions in affected sheep,
especially in those areas where the disease is endemic. In nonendemic areas and in other ruminant
species laboratory conformation by either virus isolation, conformation of the virus genome or
serology is necessary. Laboratory conformation is also necessary to identify the serotype involved
in order to establish specific control measures.
References:
1. Erasmus B.J.: Bluetongue. In: Virus infections of Ruminants, Vol. 3, ed. Dinter Z and Morein B.,
Amsterdam: Elsevier Science Publishers, 1990.
2. Kellerman T.S., Coetzer J.A.W. and Naudé T.W.: Plant Poisonings and Mycotoxicoses of Livestocks in
Soutern Africa. Cape Town, Oxford University Press Soutern Africa, 1988.
3. Lear A.S. and Callan R.J.: Overview of Bluetongue. In: The Merck Veterinary Manual, ed. Aiello S.E.
and Moses M.A., Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Whitehouse Station,
N.J., U.S.A., 2014,
http://www.merckmanuals.com/vet/generalized_conditions/bluetongue/overview_of_bluetongue.html
218
4. Luedke A.J, Bowne J.G., Jochim M.M. and Doyle C.: Clinical and pathologic features of bluetongue in
sheep. American Journal of Veterinary Research, 25, 963-970, 1964.
5. Martens P.P.C., Pedley S., Cowley J., Burroughs J.N., Corteyn A.H., Jeggo M.H., Jennings D.M. and
Gorman B.M.: Analysis of the roles of bluetongue virus outer capsid protein VP2 and VP5 in
determination of virus serotype. Virology, 170, 561-565, 1989.
6. Moulton J.E.: Pathology of bluetongue of sheepin California. Journal of the american Veterinary Medical
Association, 138, 493-498, 1961.
7. Owen N.C.: Investrigation into the pH stability of bluetongue virus and its survival in mutton and beef.
Onderstepoort Journal of Veterinary Research, 31, 109-118, 1964.
8. Verwoerd D.W. and Erasmus B.J.: Bluetongue. In: Infectious diseases of livestock, 2 nd edition, ed.
Coetzer J.A.W. and Tustin R.C., Cape Town, Oxford University Press Soutern Africa, 2004, 1201-1220.
219
SPECIES DIVERSITY AND LIFE STAGES DOMINANCE OF HARD TICKS
(Acari: Ixodidae) AT VOJVODINA HUNTING RESORTS
Aleksandra Petrović1*, Aleksandar Jurišić1, Ivana Ivanović1, Aleksandar Potkonjak2, Vuk Vračar2,
Dragana Rajković1
1 Department of Environmental and Plant Protection, Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia
2 Department of Veterinary Medicine, Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia
* Corresponding author: petra@polj.uns.ac.rs
Abstract
All hard ticks species are obligatory parasites, often host nonspecific, and therefore capable of
parasitizing a numerous species of vertebrates. Their relatively slow feeding process on the hosts,
the absence of digestive enzymes and relatively adaptable life cycles enable acquisition,
maintenance and transmission of a variety of the pathogens and thus cause severe diseases in
humans and animals. The aim of the study is to determine the hard tick species diversity and the
dominance of the certain life stages on the hosts and on the vegetation cover at the hunting resorts
of Vojvodina. Two methods were used for tick sampling: from the vegetation and from the body of
the hunted roe deer (Capreolus capreolus Linnaeus, 1758) and wild boars (Sus scrofa Linnaeus,
1758). The study was conducted from 2011 to 2014 at 20 hunting resorts in Vojvodina, all
agroecosystems with wide belts of deciduous forests and sporadic shrub vegetation. Eight tick
species from four genera were identified on the hosts: Ixodes ricinus Linnaeus 1758,
Haemaphysalis concinna Koch 1844, H. punctata Canestrini & Fanzago 1878, H. sulcata
Canestrini & Fanzago 1878, Dermacentor marginatus Sulzer 1776, D. reticulatus Fabricius 1794,
Rhipicephalus bursa Canestrini & Fanzago 1878 and R. sanguineus Latreille 1806, but only five
species from three genera on the vegetation: I. ricinus, H. concinna, H. punctata, D. marginatus and
D. reticulatus. Depending on the season, locality and the sampling methods, the dominance of the
certain life stages was significantly different. The adult stages of all tick species were found on the
hosts, nymphae of I. ricinus, H. concinna and H. punctata and larvae only of I. ricinus and H.
punctata. However, on the vegetation cover, adults were found for five tick species, but nymphal
and larval stages only of I. ricinus and H. concinna. Due to its wide home ranges, seasonal
migrations and population fluctuations most of the vertebrate species found at hunting resorts
represent an "epidemiological bridge" among hosts, parasites and pathogens, responsible for the
maintenance of the high tick density populations in certain habitats and therefore seasonal, spatial,
transstadial and transovarial transmission of important pathogens for human and animal health.
Key words: ticks, Ixodidae, diversity, life stages, hunting resorts, Vojvodina
Introduction
All hard ticks species are obligatory parasites, often host nonspecific, and therefore capable of
parasitizing a numerous species of vertebrates. Their relatively slow feeding process on the hosts,
the absence of digestive enzymes and relatively adaptable life cycles enable acquisition,
maintenance and transmission of a variety of the pathogens and thus cause severe diseases in
humans and animals (Sonenshine, 1991). High tick infestation could cause the death of the host due
to blood loss (Balashov, 1968; loc. cit. Evans, 1992), but more important is the role of ticks as
vectors of numerous pathogens, viruses, bacteria and protozoa, transmitted to the host during the
feeding phase. During the last two decades, cases of zoonoses transmitted by ticks, such as Lyme
borreliosis, Tick-borne encephalitis, anaplasmosis, babesiosis and others, are on the rise, and
220
represent the leading health problem in many parts of Europe and North America (Paulauskas,
2009). Additionally, the further increase of tick-borne disease incidences could be expected as a
result of global climate change, as well as the expansion of vectors and pathogens in the new areas.
Due to their vector role, ticks are the subjects of many studies. To understand the complex
interactions among ticks, their hosts and pathogens, and to determine the potential risk of the
contact between humans and animals with infected ticks, a good knowledge of all biotic and abiotic
factors that affect the population of these arthropods is needed. Furthermore, continuous and
systematic monitoring of species abundance, seasonal and spatial dynamics of ticks and their hosts
is crucial.
Acarological studies in our country are mainly focused on urban and suburban habitats, although a
few numbers of studies were conducted at the natural habitats of ticks, such as hunting and natural
resorts (Jurišić et al., 2011, Jurišić et al., 2012). The rural habitats have low anthropogenic
influence, so floristic and faunistic communities are rich and diverse and provide favourable
conditions for maintaining the tick populations. The main host for the adult stages of Ixodes ricinus
in Central Europe is roe deer (Capreolus capreolus Linnaeus, 1758) (Pintur et al., 2012), followed
by the wild boar (Sus scrofa Linnaeus, 1758). Several studies have confirmed the high infestation of
roe deer and wild boars in Europe (Fuente et al., 2004; Ruiz-Fons et al., 2006; Kiffner et al., 2010;
Vor et al., 2010; Pintur et al., 2012; Carpi et al., 2008), stating that its abundance can affect the
population dynamics of I. ricinus ticks. Although the role of roe deer and wild boars in maintaining
the agents of some diseases in natural habitats (such as Lyme borreliosis and Tick-borne
encephalitis) is still unclear, there are numerous evidences of a positive correlation between the
number of roe deer and ticks (Carpi et al., 2008), which confirms the importance of understanding
the complex mechanisms of transmission of these potentially serious diseases. The role of wild boar
as a reservoir of Anaplasma phagocytophilum is confirmed by Michalik et al. (2009). Due to its
wide home ranges, seasonal migrations and population fluctuations most of the vertebrate species
found at hunting resorts represent an "epidemiological bridge" among hosts, parasites and
pathogens, responsible for the maintenance of the high tick density populations in certain habitats
and therefore seasonal, spatial, transstadial and transovarial transmission of important pathogens for
human and animal health.
In order to obtain the most accurate data on tick species diversity and density in practice, it is
necessary to use different methods of tick sampling. Due to different host seeking strategies, the
estimated relative abundance of tick populations is in most cases the direct result of the applied
sampling methods and as such do not represent a real tick species diversity at a certain locality. To
avoid the bias and to obtain the actual state of tick species diversity and their spatial dispersion, two
different methods of tick sampling were applied in this study. The aim of the study is to determine
the hard tick species diversity and the dominance of the certain life stages found on the hosts and on
the vegetation cover at the hunting resorts of Vojvodina.
Tick sampling and identification
Ticks were collected during the four year study (2011-2014) using two methods: from the nature
and from the hunted carcasses of roe deer (Capreolus capreolus Linnaeus, 1758) and wild boars
(Sus scrofa Linnaeus 1758). Frome nature, ticks were sampled according to „Flag-hour“ method
(Maupin et al., 1991), i.e. by dragging the white flannel cloths (1 x 1.6 m) through the vegetation
and the soil surface in total length of 100 m for an hour (Sonenshine, 1993). Both sides of the cloth
were carefully examined every 20 m. The sampling was conducted at five chosen transects of each
hunting resort. Ticks were sampled monthly, from 10 am till 6 pm, if weather conditions were
suitable. This method was not used if the average daily temperature values were under 5°C. The
221
collection of ticks from the carcasses of roe deer and wild boars was performed immediately after
the hunt. Each carcass was systematically and thoroughly inspected using palpatory technique by
three observers using latex gloves. All tick specimens were collected using acarological tweezers
and placed into plastic tubes (5 ml) with a small cotton ball soaked in water to maintain the
humidity and closed with perforated plastic stopper for sufficient ventilation. The collected
specimens were properly labeled and transported to the laboratory and maintained in induced
diapause at 5°C till examination and identification. Tick species were identified up to species level
according to identification keys: Nosek & Sixl (1972), Estrada-Peña et al. (2004) and Walker et al.
(2007).
Localities
The study was performed at 20 localities on the territory of Autonomous Province of Vojvodina, 15
in Bačka: Ada (13.277 ha, N45°46`522`` E20°09`230``), Apatin (28.812 ha, N45°41`593``
E18°55`340``), Bač (30.149 ha, N45°20`447`` E19°13`108``), Bačka Palanka (52.204 ha,
N45°14`198`` E19°20`239``), Bačko Gradište (6.548 ha, N45°33`336` E20°05`512``), Bačko
Petrovo Selo (11.304 ha, N45°41`144`` E20°05`283``), Bečej (30.147 ha, N45°36`536``
E20°04`419``), Futog (14.413 ha, N45°14`005`` E19°41`093``), Kanjiža (39.856 ha, N46°03`271``
E20°04`422``), Mol (9.429 ha, N 45°44`103`` E20°09`462``), Nadalj (3.646 ha, N45°32`005``
E19°54`542``), Senta (25.500 ha, N45°58`399`` E20°05`316``), Srbobran (19.441 ha, N45°33`331`
E19°46`052``), Titel (26.070 ha, N45°12`338`` E20°19`075``), Turija (5.343 ha, N45°32`509``
E19°51`239``); four in Banat: Čoka (23.139 ha, N45°57`301`` E20°05`469``), Novi Bečej (60.745
ha, N45°35`509`` E20°07`219``), Pančevo (69.565 ha, N44°51`530`` E20°37`453``), Novi
Kneževac (30.539 ha, N46°01`456`` E20°04`598``); and one in Srem: Ruma (50.560 ha,
N45°01`570`` E19°49`189`` (area according to Antonić & Beuković, 2007). All studied localities
were described as agroecosystems with wide belts of mixed deciduous forests and sporadic shrub
and bush vegetation.
Statistical analyses
The obtained values were tested using software Statistica 12 (StatSoft, licensed to University of
Novi Sad, Faculty of Agriculture). In order to statistically analyze the data, distribution fitting tests
(χ2 and Kolmogorov-Smirnov tests), one-way ANOVA (tested by Fisher’s LSD test for significance
level of p<0.05 as statistically significant and p<0.01 as highly statistically significant) were
applied.
Results
The qualitative and quantitative studies of tick species and life stages collected from vegetation and
from the carcasses of the hosts have shown the differences in relative abundance of identified tick
genera and species (Tab 1.).
Table 1. Differences in abundance of identified tick species, life stages and localities depending on sampling
method
Ticks collected
from the
vegetation
Ticks collected
from the hosts
Total
number
Number
of tick
species
The mean of
collected
ticks
Minimum
collected
ticks
Maximum
collected
ticks
Variance
Standard
deviation
Standard
error
20035
5
50.0875
0
569
8641.057
92.957
4.648
7267
8
11.3547
0
256
1084.236
32.928
1.302
222
Ticks collected from the vegetation
The totals of 20035 ticks were sampled from the vegetation (Tab 2.). Five tick species from three
genera were identified: Ixodes ricinus Linnaeus 1758 (82.98%), Dermacentor marginatus Sulzer
1776 (9.99%) and D. reticulatus Fabricius 1794 (1.20%), Haemaphysalis punctata Canestrini and
Fanzago 1878 (5.43%) and H. concinna Koch 1844 (0.39%). The most abundant were the larvae of
I. ricinus (32.5%). The larval stages of other four species as well as the nymphal stages of D.
marginatus and D. reticulatus have not been found using this method. The highest number of
collected tick specimens was obtained at locality Pančevo (6.42%) and the lowest at hunting resort
Bačko Gradište (4.07%).
Table 2. The number of ticks collected from the vegetation
Locality
l
273
245
321
I. ricinus
n
♀
♂
152 217
73
145 154
89
169 269
85
Σ
715
633
844
l
D. marginatus
n
♀
♂
0
0
48 27
0
0
55 23
0
0
59 25
Σ
75
78
84
l
D. reticulatus
n
♀ ♂ Σ
0
0 11
8 19
0
0
0
0
0
0
0
0
0
0
l
H. punctata
n
♀ ♂
11 32 10
13 35 11
15 21
8
Σ
53
59
44
l
H. concinna
Total
n
♀ ♂ Σ
0
0
2
1
3
865
0
0
1
0
1
771
0
0
2
0
2
974
%
Ada
0
4.32
Apatin
0
3.85
Bač
0
4.86
Bačka
256 189 198
96
739
0
0
72 26
98
0
0
0
0
0
0 12 34
9
55
0
1
0
0
1
893
4.46
Palanka
Bačko
259 175 178
59
671
0
0
56 35
91
0
0
0
0
0
0 12 29
8
49
0
0
3
1
4
815
4.07
Gradište
Bačko
Petrovo
358 211 159
63
791
0
0
65 24
89
0
0 12
9 21
0 11 26
7
44
0
0
1
0
1
946
4.72
selo
Bečej
211 148 265
84
708
0
0
69 29
98
0
0 18
7 25
0 12 32
9
53
0
2
1
0
3
887
4.43
Futog
345 201 245 102
893
0
0
78 38 116
0
0 12
5 17
0 11 31
8
50
0
0
5
1
6 1082
5.40
Kanjiža
326
36 240
68
670
0
0
71 21
92
0
0 21 11 32
0 18 30 12
60
0
0
0
0
0
854
4.26
Mol
211 184 211
75
681
0
0
84 33 117
0
0 13
5 18
0 13 25
2
40
0
1
2
1
4
860
4.29
Nadalj
198 196 256
79
729
0
0
92 22 114
0
0 10
6 16
0 19 27 24
70
0
0
1
0
1
930
4.64
Senta
371 258 242
96
967
0
0
69 28
97
0
0
0
0
0
0 19 24 21
64
0
0
0
0
0 1128
5.63
Srbobran
386 234 214
96
930
0
0
67 31
98
0
0 14
5 19
0 18 29
8
55
0
2
3
2
7 1109
5.54
Titel
345 265 198
93
901
0
0
99 34 133
0
0
5
3
8
0 21 34
9
64
0
0
4
2
6 1112
5.55
Turija
245 233 206
85
769
0
0
48 39
87
0
0
6
1
7
0 19 36 15
70
0
0
5
2
7
940
4.69
Ruma
425 245 254
86 1010
0
0
87 29 116
0
0
2
0
2
0 15 29 16
60
0
1
1
0
2 1190
5.94
Čoka
398 257 214
82
951
0
0
84 26 110
0
0
8
1
9
0 21 24 28
73
0
2
5
2
9 1152
5.75
Novi Bečej 387 311 236
76 1010
0
0
76 34 110
0
0 14
8 22
0 20 21 22
63
0
1
7
4 12 1217
6.07
Pančevo
569 254 197
58 1078
0
0
88 26 114
0
0 19
7 26
0 11 30 21
62
0
0
5
2
7 1287
6.42
Novi
387 224 226
99
936
0
0
59 25
84
0
0
0
0
0
0
0
0
0
0
0
2
1
0
3 1023
5.11
Kneževac
Total
6516 4087 4379 1644 16626
0
0 1426 575 2001
0
0 165 76 241
0 291 549 248 1088
0 12 49 18 79 20035 100.00
%
32.52 20.40 21.86 8.21 82.98 0.00 0.00 7.12 2.87 9.99 0.00 0.00 0.82 0.38 1.20 0.00 1.45 2.74 1.24 5.43 0.00 0.06 0.24 0.09 0.39 100.00
Obtained data had normal distribution, and ANOVA did not emphasize any statistical significances
regarding the different localities as independent variables on the number of collected ticks as a
dependent variable (pl=0.99999 for p˂0.05). However, ANOVA proved high statistical significance
between the total number of collected ticks and their species and life stages (psp=0.00000 and
pst=0.00475 for p<0.01) (Graph 1. and 2.). The Fisher's LSD test demonstrated high statistical
differences (for p<0.01) between the number of collected I. ricinus and all other species and D.
marginatus comparing to D. reticulatus and H. concinna. Furthermore, the number of sampled
males had high statistical differences (for p<0.01) comparing to collected larvae and females.
Ticks collected from the hosts
The total number 7267 specimens of ticks were sampled from the roe deer (64 carcasses) and wild
boars (29 carcasses) (Table 3.). Eight tick species from four genera were identified: I. ricinus
(69.00%), D. marginatus (13.76%), D. reticulates (4.06%), H. punctata (2.11%), H. concinna
(1.22%), as well as Haemaphysalis sulcata Canestrini & Fanzago 1878 (2.53%), Rhipicephalus
sanguineus Latreille 1806 (6.47%) and R. bursa Canestrini & Fanzago 1878 (0.85%). The most
abundant were the females of I. ricinus (49.33%). The larval stages of all identified species, as well
223
as the nymphs of D. marginatus, D. reticulatus, H. punctata, H. sulcata and R. bursa have not been
found on the hosts. The highest number of sampled ticks was at hunting resort Novi Kneževac
(6.12%) and the lowest at locality Mol (3.59%).
Table 3. The number of ticks collected from the hosts
Locality
I. ricinus
l
n
♀
D. marginatus
♂
Σ
l
♀
n
♂
D. reticulatus
Σ
l
♀
n
H. punctata
♂
Σ
l
♀
n
H. concinna
♂
Σ
l
♀
n
H. sulcata
♂
Σ
l
♀
n
R. sanguineus
♂
Σ
l
♀
n
R. bursa
♂
Σ
l
♀
n
♂
Total
Σ
%
Ada
0
22
125
58
205
0
0
24
18
42
0
0
2
0
2
0
0
5
1
6
0
0
8
3
11
0
0
0
0
0
0
2
6
3
11
0
0
0
0
0
277
3.81
Apatin
0
24
129
54
207
0
0
28
16
44
0
0
6
1
7
0
0
0
0
0
0
0
0
0
0
0
0
5
1
6
0
5
10
5
20
0
0
2
1
3
287
3.95
Bač
Bačka
Palanka
Bačko
Gradište
Bačko
Petrovo
selo
0
19
256
62
337
0
0
35
24
59
0
0
9
6
15
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9
11
3
23
0
0
0
0
0
434
5.97
0
25
247
51
323
0
0
36
21
57
0
0
11
5
16
0
0
0
0
0
0
2
1
0
3
0
0
0
0
0
0
8
8
5
21
0
0
1
0
1
421
5.79
0
23
211
25
259
0
0
29
18
47
0
0
19
5
24
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
11
5
3
19
0
0
0
0
0
349
4.80
0
35
241
32
308
0
0
41
31
72
0
0
9
1
10
0
0
9
4
13
0
0
0
0
0
0
0
0
0
0
0
16
6
4
26
0
0
0
0
0
429
5.90
Bečej
0
26
158
57
241
0
0
27
15
42
0
0
10
8
18
0
0
0
0
0
0
0
0
0
0
0
0
8
2
10
0
12
11
5
28
0
0
5
1
6
345
4.75
Futog
0
58
132
48
238
0
0
39
15
54
0
0
19
4
23
0
0
0
0
0
0
0
5
2
7
0
0
0
0
0
0
11
9
5
25
0
0
0
0
0
347
4.78
Kanjiža
0
24
154
34
212
0
0
34
14
48
0
0
5
1
6
0
0
24
18
42
0
0
7
0
7
0
0
0
0
0
0
10
5
0
15
0
0
2
0
2
332
4.57
Mol
0
26
126
25
177
0
0
29
13
42
0
0
8
5
13
0
0
8
1
9
0
0
2
1
3
0
0
0
0
0
0
9
5
1
15
0
0
1
1
2
261
3.59
Nadalj
0
35
191
11
237
0
0
31
5
36
0
0
14
8
22
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
3
6
2
11
0
0
0
0
0
307
4.22
Senta
0
45
174
24
243
0
0
40
18
58
0
0
15
5
20
0
0
9
4
13
0
2
0
0
2
0
0
0
0
0
0
8
8
1
17
0
0
0
0
0
353
4.86
Srbobran
0
47
152
37
236
0
0
25
13
38
0
0
9
6
15
0
0
0
0
0
0
0
4
3
7
0
0
9
2
11
0
17
11
5
33
0
0
2
1
3
343
4.72
Titel
0
39
147
36
222
0
0
29
5
34
0
0
10
8
18
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
15
10
7
32
0
0
2
0
2
309
4.25
Turija
0
50
205
41
296
0
0
36
17
53
0
0
11
4
15
0
0
0
0
0
0
2
0
0
2
0
0
11
5
16
0
9
8
5
22
0
0
0
1
1
405
5.57
Ruma
0
25
185
21
231
0
0
41
16
57
0
0
8
8
16
0
0
14
8
22
0
0
8
4
12
0
0
16
6
22
0
14
15
9
38
0
0
5
3
8
406
5.59
Čoka
0
26
164
26
216
0
0
48
6
54
0
0
17
9
26
0
0
0
0
0
0
0
0
0
0
0
0
24
9
33
0
15
8
6
29
0
0
1
0
1
359
4.94
Novi Bečej
0
38
184
34
256
0
0
50
25
75
0
0
12
4
16
0
0
0
0
0
0
0
7
2
9
0
0
21
11
32
0
10
11
8
29
0
0
5
5
10
427
5.88
Pančevo
Novi
Kneževac
0
34
182
45
261
0
0
23
11
34
0
0
5
8
13
0
0
17
9
26
0
4
9
2
15
0
0
19
18
37
0
9
18
8
35
0
0
6
4
10
431
5.93
0
31
222
56
309
0
0
36
18
54
0
0
0
0
0
0
0
14
8
22
0
0
6
3
9
0
0
11
6
17
0
9
8
4
21
0
0
7
6
13
445
6.12
777 5014
0
0 681 319 1000
0
0 199
96 295
0
0 100
53 153
0
10
57
22
89
0
0 124
89 470
0
0
39
23
62
Total
%
0 652 3585
60 184
0 202 179
7267 100.00
0.00 8.97 49.33 10.69 69.00 0.00 0.00 9.37 4.39 13.76 0.00 0.00 2.74 1.32 4.06 0.00 0.00 1.38 0.73 2.11 0.00 0.14 0.78 0.30 1.22 0.00 0.00 1.71 0.83 2.53 0.00 2.78 2.46 1.22 6.47 0.00 0.00 0.54 0.32 0.85 100.00
Tested values had normal distribution, and there were no significant differences among collected
ticks concerning different localities (pl=1.00000 for p<0.05). Nevertheless, ANOVA emphasized
high statistical associations between the number of collected ticks and species and life stages
(psp=0.00000 and pst=0.00000 for p<0.01) (Graph 3. and 4.). The Fisher's LSD test proved high
statistical differences (for p<0.01) between the number of collected I. ricinus ticks and all other
species, as well as D. marginatus with H. concinna and R. bursa. The statistical significances (for
p<0.05) were obtained for comparing the numbers of D. marginatus with D. reticulatus, H.
punctata and H. sulcata. No statistical significances were found in the number of D. marginatus and
R. sanguineus. Moreover, the high statistical differences (for p<0.01) were found in the number of
sampled larvae comparing to females and males, and females compared to nymphs and males.
224
90
45
80
40
70
35
60
30
50
Number of ticks
Number of ticks
40
30
20
25
20
15
10
10
5
0
0
-10
I. ricinus
D. reticulatus
H. concinna
R. sanguineus
D. marginatus
H. punctata
H. sulcata
R. bursa
Mean
Mean±0.95 Conf. Interval
-5
Larvae
Species
Mean
Graph 3. The number of different ticks species
collected from the hosts
Mean±0.95 Conf. Interval
Nymphs
Females
Males
Stage
Graph 4. The number of different ticks stages
collected from the hosts
Discussion & Conclusion
The obtained differences in relative abundance of identified tick species collected from the
vegetation and from the hosts were the direct results of applied sampling methods, as the various
tick species demonstrate a different host seeking strategies. According to Crooks & Randolph
(2006) ixodid ticks could achieve contact with a host by active hunting (Hyalomma, Amblyomma),
by waiting in nests or lairs (I. hexagonus), or by ambushing a passing animal from a vantage point
on the vegetation cover (I. ricinus). Furthermore, they emphasize that active host questing
behaviour increase desiccation, so these ticks have to return periodically to the moist litter layer in
order to reabsorb needed water to maintain a certain level of humidity. Three species were collected
only from the hosts: H. sulcata (females and males), R. sanguineus (nymphs, females and males)
and R. bursa (females and males). R. sanguineus demonstrates an active host seeking behavior,
although it could also find an appropriate host by ambush strategy – questing behaviour (DantasTorres, 2010).
The most abundant species collected by both methods at each prospected locality was I. ricinus.
The same results were published by Barandika et al. (2008) and by Mihalca et al. (2012). According
to these authors, I. ricinus is the predominant tick species in Europe found by both methods,
flagging the vegetation or on the hosts. The determining factors for its abundance are microclimate
characteristics and host densities. Barandika et al. (2008) proved that although the flagging method
is suitable for sampling several tick species like I. ricinus and other exophilic ticks, the efficiency of
this technique varies substantially with different tick species and life stages. Higher adult tick
species diversity found on the studied hosts could be explained by the fact that the immature tick
stages are capable of feeding on almost any vertebrate, but adults require the blood meals of more
than 1 ml, especially from the animals that could provide it better, such as ungulates (Donzé et al.,
2004).
The adults of D. marginatus and D. reticulates were collected from the vegetation and from the
hosts. D. marginatus follows the distribution pattern of I. ricinus in Vojvodina as the second most
abundant tick species. Its presence is registered at all prospected localities, as well as in deciduous
forests, pastures, meadows, all kinds of shrub vegetation along the river banks, even in the urban
areas (Jurišić et al., 2010). D. reticulatus, on the other hand, is sporadically found on the vegetation,
but more often on the hosts. This species has been registered on the vegetation cover at 14
localities. The results of this study demonstrate that the habitats of this species are restricted to river
basins, wet/flooded forests, grassland and meadows, rich in bush and shrub vegetation, which is
confirmed by similar conclusions of Mihalca et al. (2012).
225
The nymphs and adults of H. punctata were obtained from the vegetation, but only adults were
found on the host. This species is confirmed at 19 localities as a host-seeking tick, and only at 7 as
parasiting on the hosts. More frequent, but less abundant was H. concinna, which nymphal stages
and adults were found both on the vegetation and the hosts. H. concinna was identified at all
prospected localities when sampled the vegetation and at 14 localities when sampled from the hosts.
These two species have preferences to open, warm and mild humid areas, with overlapping niches
with I. ricinus, and according to Nosek (1971, loc. cit. Mihalca et al. 2012), these species prefer
pastures, forest margins and forest-steppes. H. sulcata was the most abundant species of
Haemaphysalis genus, found as adults only on the hosts. Its presence is confirmed at 9 localities.
Similarly to findings of Mihalca et al. (2012), this species prefers warm and humid areas in
Vojvodina.
The species of genus Rhipicephalus were found only on the hosts. The nymphs and adults of R.
sanguineus were obtained from all studied localities. However, only adults of R. bursa were
collected at 13 localities. Although R. sanguineus is an endophilic, monotropic and three-host tick
species, it is also able to survive and maintain its population in the outdoor environments (DantasTorres, 2010) and therefore parasite on cattle or wild ungulates (Estrada-Peña et al., 2004). R.
bursa, on the other hand is a typical representative of tick fauna commonly found on ungulates,
with preferences to areas well covered with bush and steppe vegetation (Fuente et al., 2004).
Statistical differences obtained from studying the appearance of species on the vegetation and on
the hosts are mainly caused by ticks preferences to suitable habitats and hosts, their adaptability and
different host seeking strategies. Furthermore, the absence of D. marginatus and D. reticulatus
larval stages on the vegetation cover could be explained by their induced endophilic behaviour, as
they are found in the vicinity or in the lairs of rodent and insectivore species. In addition, the larval
stages of all collected tick species from the hosts are absent too, as they parasite only on rodents and
other small mammals, reptiles and birds, although Vor et al. (2010) have reported their presence on
the heads of the roe deer in Germany.
The most abundant tick stages obtained from the vegetation were larvae and females. Statistical
significance calculated for the male could be explained by their low abundance and specific sex
ratio characteristics typical for the ambush host-seeking behaviour confirmed by Hornok (2009).
However, statistical differences obtained for females comparing to nymphs and males from the
hosts proves the fact that females are obligated to take the blood meal in order to oviposit.
The presence and abundance of certain tick species at studied localities have not emphasized any
statistical significance in both types of sampling. The total number of collected ticks was evenly
distributed. The number of ticks collected from the vegetation varied from 4.07% at Bačko Gradište
to 6.42% at Pančevo and from the hosts from 3.59% at Mol to 6.12% at Novi Kneževac.
In order to obtain the most accurate data on tick species diversity and density in practice, to avoid
the bias and to obtain the actual tick population densities and their spatial dispersion it is necessary
to use different methods of tick sampling and to include a wider range of tick hosts such as other
ungulate species, carnivores, rodents, reptiles, birds, bats. Due to consistent population densities,
wide home ranges and seasonal migrations, most of the vertebrate species found at hunting resorts
are responsible for the maintenance of the high tick density populations in certain habitats and
therefore seasonal, spatial, transstadial and transovarial transmission of pathogens important for
human and animal health.
Acknowledgments
The presented work is part of the research done in the project TR31084 granted by the Serbian
Ministry of Education and Science and project No: 114-451-1293/2014-03 granted by the
Provincial secretary for science and technological development of AP Vojvodina
226
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228
CROSS-REACTIONS IN SEROLOGICAL DIAGNOSIS OF FLAVIVIRUS INFECTIONS
Ivana Hrnjakovic Cvjetkovic1a.1b*, Dusan Petric2, Tamas Petrovic3, Gordana Kovacevic1a, Jelena
Radovanov1a, Aleksandra Jovanovic Galovic1a, Dejan Cvjetkovic4, Sandra Stefan Mikic4,
Aleksandra Patic1a.1b, Natasa Nikolic1a.1b, Vesna Milosevic1a.1b
1a Institute of Public Health of Vojvodina, Novi Sad, Serbia
1b Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia
2. Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia
3. Scientific Veterinary Institute ’’Novi Sad’’, Novi Sad, Serbia
4. Clinic for Infectious Diseases, Clinical Centre of Vojvodina, Faculty of Medicine, University of Novi Sad, Novi Sad,
Serbia
*Corresponding author: ivana.hrnjakovic@izjzv.org.rs
Abstract
The most important flaviviruses are mosquito-borne viruses responsible for severe encephalitis in
humans: Japan encephalitis virus (JEV), Saint Louis encephalitis virus, West Nile virus (WNV) and
Dengue viruses (DV-1 through 4). The most important mammalian tick-borne flavivirus is tickborne encephalitis virus (TBEV). Nowadays, in early illness phase, diagnosis is based on detection
of viral RNA by PCR/real time PCR. In the late phase of infection serologic diagnosis is a method
of choice to establish the diagnosis of flavivirus infection. Significant problems in serological
diagnosis are cross-reactions between members of Flavivirus genus due to antigenic similarity. The
aim of the study was to demonstrate the extent to which the use of multiple serological testing is
able to contribute solving the cross-reaction issues between some members of genus Flavivirus.
Eighteen ELISA IgG-positive sera on WNV were tested on Dengue virus by ELISA as well as on 8
flaviviruses (TBEV, WNV, JEV, YFV, DV 1-4) by indirect immunofluorescent test (IIFT)
Flavivirus Profile 2 (Euroimmun, Germany).
All of 18 ELISA IgG- positive sera on WNV were simultaneously ELISA IgG- positive on Dengue
virus. Considering the fact that WNV circulates in Serbia among mosquitoes, birds, horses and
humans unlike Denga virus, it was assumed that there were cross-reactions and sera from 18
patients were tested on eight flaviviruses by IIFT. In 12 sera specific IgG antibodies to WNV were
confirmed by determination of antibody titres. Among those 12 sera, 5 sera were only positive to
WNV by IIFT (but not to other flaviviruses), whereas the testing of the other 7 sera showed the
presence of both the antibodies against WNV and antibodies against Dengue viruses 1-4 (although
the latter in lower titers). Six (33.3%) patients need to be additionally tested for setting the final
diagnose. Cross-reaction issues between WNV and Dengue virus can be solved in some cases by
IIFT.
Keywords: flaviviruses, cross-reaction, serological diagnosis
Introduction
Flavivirus genus consists of more than 70 viruses. More than thirty of them are pathogens
significant for human medicine. Flaviviruses are 40 - 60 nm in diameter and have a single-stranded,
positive-sense RNA of 11 kb. There is glycoprotein E inserted in the viral lipid envelope (Jawetz,
Melnick & Adelberg's, 2013). The antigenic determinants of glicoprotein E are responsible for
production of neutralisation antibodies in the host. On the basis of antigenic characteristics
flaviviruses can be divided into eight antigenic groups. The most important antigenic group is the
complex of Japan encephalitis (JE) whose members are mosquito - borne viruses that can cause
229
severe cases of encephalitis in humans: Japan encephalitis virus (JEV), Saint Louis encephalitis
virus (SLEV), West Nile virus (WNV), Murray Valley virus and the complex of Denga viruses
whose members are DV-1 through 4.
Four denga viruses are widely distributed in the tropics (between 35 north and 35 south latitude)
where principal vector Aedes aegypti is present. WNV is the only mosquito borne flavivirus which
activity in humans and animals is detected in Serbia (Petrović et al., 2014; Petric et al ., 2012). The
vast majority of WNV infections in humans are asymptomatic. Approximately 20% of infected
humans suffer from WNV fever and less than 1% develops neuroinvasive disease resulting in
encephalitis and meningitis. Myocarditis, pancreatitis and fulminant hepatitis have been described
in some symptomatic persons infected with WNV (Petersen and Marfin, 2002). Human clinical
cases associated with WNV infections have been reported from many countries in Africa, southern
Europe, south-western and south-central Asia and Australia (Hubálek and Holouzka, 1999). Cases
of yellow fever virus (YFV) infection have been recorded in subtropical areas of Africa and South
America (Jerant Patić, 2007). Since 2000, circulation of YFV has been increased significantly in
Africa. Epidemics of YFW infection occurred in unimmunized population of Africa in 20th century;
morbidity rate was very high, ranging from 51 - 89% (WER, 2005). The most remarkable clinical
feature is haemorrhagic syndrome. Neurological disorders, meningitis, kidney and liver failure may
also occur.
Among mammalian tick-borne flaviviruses, the most important is tick-borne encephalitis virus
(TBEV). TBEV infections are endemic in large parts of Europe (Southern Germany, Austria,
Switzerland, Czech Republic, Slovakia, Hungary, Slovenia, Baltic countries, Poland, parts of
Scandinavia, European Russia (Heinz et al., 2013). Neighbouring countries like Bosnia, Croatia,
Bulgaria, and Romania are at high risk due to high prevalence of the virus in ticks (WHO, 2011.).
Japanese encephalitis is zoonosis widely distributed in Asia caused by Japanese encephalitis virus
(JEV). Most JEV infections are asymptomatic, but in some cases the virus can cause systemic
febrile illness with the central nervous system involvement and possible fatal outcome.
Surprisingly, Ravanini reported detection of JEV RNA in mosquitoes collected in northern Italy.
Further investigations are needed for confirmation of spreading JEV in Europe (Ravanini et al.,
2012).
Nowadays, in early illness phase, diagnosis is based on detection of viral RNA by PCR or real time
PCR or serologic tests. However, serologic tests appear to be the method of choice for establishing
the diagnosis in the late phase of virus infection. Significant problems in serological diagnosis are
cross reactions between members of Flavivirus genus because of antigenic similarity. Viral isolation
from blood or cerebrospinal fluid is usually unsuccessful even in the early stage of infection
because of low viral loud in humans.
The aim of the study was to examine the value of multiple serological testing in solving the crossreaction issues between flaviviruses.
Eighteen sera ELISA IgG-positive on WNV were tested on Dengue virus by ELISA. The same sera
were tested on 8 flaviviruses (TBEV, WNV, JEV, YFV, DV, 1-4) by indirect immunofluorescent
test (IIFT) Flavivirus Profile 2 (Euroimmun, Germany).
For purpose of WNV IgG antibodies detection, commercially available ELISA was used (produced
by Euroimmun, Lübeck, Germany). Testing, calculation and interpretation of results were
performed strictly on the automatic device Euroimmun Analyzer I-2P following manufacturer
instructions. Results were evaluated semiquantitatively by calculating a ratio of the extinction value
of patient sample over the extinction value of the calibrator 2 which was included into the test.
230
Results were considered as positive if ratio was equal to or greater than 1.1, intermediate if ratio
was between 0.8 and 1.1 and negative if ratio was less than 0.8. All samples were tested in the
Institute of Public Health of Vojvodina and all were positive on WNV IgG antibodies.
Eighteen ELISA WNV IgG-positive sera were tested by commercially available ELISA IgG for
denga (Euroimmun, Germany) and IIFT Flavivirus Mosaic 1 (Euroimmun, Germany) against
TBEV, WNV, JEV, YFV, DV 1-4. For IIFT, serum samples were diluted 1:10 and applied on the
slides coated with TBEV, WNV, JEV, YFV, DV 1-4 antigens. The results were read under a
fluorescent microscope (Olympus BH2), objective 40X.
Results
All of 18 ELISA WNV IgG-positive sera were simultaneously ELISA IgG- positive on Dengue
virus (table 1). Considering the fact that WNV circulates in Serbia among mosquitoes, birds, horses
and humans unlike Dengue virus, it was assumed that there were cross-reactions.
Table 1.Results of ELISA IgG on Denga virus in IgG WNV positive serum samples
Serum
samples
116
159
8
14
91
31
11132
5291
11582
11553
11542
5409
11796
688
626
667
11528
11561
Origin of
samples
ND
ND
MBD
MBD
MBD
MBD
SBD
SBD
SBD
SBD
SBD
SBD
SBD
SBD
SBD
SBD
SBD
SBD
History of
WNV
infection
recent
past
past
past
past
past
past
past
recent
past
past
past
past
past
past
past
recent
recent
ELISA
WNV
IgG
4,46 IgM +
5,56
2,98
5,73
IgG+
1,96
3,25
2,60
5,13 IgM+
5,97
5,04
3,79
4,60
1,28
5,83
5,85
I1,53 IgM+
4,69 IgM+
ELISA
Denga
IgG
1.28
3.28
1,16
3,43
3,17
2,89
0,88
1.70
2,29
4,52
3,23
4,62
3,18
1,94
2,97
2,16
3,15
3,35
Legend: ND - Nisava district, MBD - Middle Banat District, SBD - South Backa District
Between WNV and Denga virus by ELISA IgG test independent whether it is recent or past WNV
infection. Serum sample No 116, 11582, 11528 and 11561 were positive on WNV IgM antibodies
that indicated recent infections. In the other serum samples past WNV infection were detected.
Cross reactivity of the WNV positive sera observed in ELISA against DV was 100%.
All 18 sera positive against WNV were tested on eight flaviviruses by IIFT. In 6 (33.3%) sera
specific WNV IgG antibodies were confirmed and there were no cross-reactivity with other
flaviviruses. In 12 serum samples cross reactivity was observed (table 2). The highest crossreactivity was observed with DV, in 11/18 (61.11%)
231
Table 2.Cross-reactivity between flaviviruses
lavivirus
Percentage (number of cross reactive
samples/number of total tested)
DV
11/18 (61,11)
YFV
4/18 (22,22)
TBEV
3/18 (16,67)
JEV
1/18 (5,55)
Legend: DV- Denga virus, YFV - Yellow fever virus,
TBEV - tick-borne encephalitis virus, JEV - Japanese encephalitis virus
Cross-reactivity was observed for all denga virus serotypes (table 3). The highest cross-reactivity
(33.33%) was observed with DV4. Among those 12 sera, where cross-reactivity was observed in,
11 sera showed the presence of both the antibodies against WNV and antibodies against Dengue
viruses 1-4 (although the latter in lower titers).
Table 3. Cross-reactivity between denga virus (1-4) by IgG IIFT in serum samples positive on
WNV by ELISA IgG
Flavivirus
Percentage (number of cross reactive
samples/number of total samples tested)
DV1
DV2
DV3
DV4
5/18 (27,77)
5/18 (27,77)
3/18 (16,67)
6/18 (33,33)
Legend: DV1-4 Denga virus, serotype 1-4
Discussion and conclusion
Flaviviruses are important human pathogens distributed worldwide. Laboratory diagnosis of
flavivirus infection can be made by isolation from CSF or blood on cell culture in BSL 4 in
reference laboratories. Neutralisation test, IFT, ELISA as well as PCR test can be applied for
identification of isolates. Molecular techniques are the tests of choice to detect viremia in all
flavivirus infections. Molecular tests are important because of their sensitivity and specificity but in
the late phase of infection they are negative. For this reason, detection of specific antibodies to
flaviviruses in late phases of infection by serologic tests is widely used for routine diagnosis of
flavivirus infection. The serum and/or CSF (in case of neuroinvasive infections) can be tested by
ELISA, IIFT, complement fixation test or hemagglutination inhibition test. ELISA and IIFT are
commercially available. The cross-reactivity within the flavivirus group must be considered in
setting up the diagnosis (Jawetz, Melnick & Adelberg's, 2013). In areas where many ARBO viruses
are present simultaneously, cross-reactivity is important problem in establishing accurate diagnosis.
In this study, cross-reactivity among the Flaviviridae family members was observed using ELISA
and IIFT. The study results reported from Makino indicated that the cross-reactivity among
flaviviruses has been observed quite often (Makino et al., 1994).
In our study, IIFT showed a better discrimination between specific IgG antibodies to DV and
specific IgG antibodies to WNV than did ELISA IgG specific for these viruses. Cross-reactivity
among DV and WNV was 100% by ELISA and 66.6% by IIFT. Such results are in agreement with
the study results provided by Koraka in which DV antigen was responsible for lower rate of crossreactions by IIFT than by ELISA (Koraka et al., 2002).
232
Applying IIFT in our study, WNV, JEV, YFV and TBEV antigens gave lower rate of crossreactions than DV antigens by the same test. DV4 antigen gave the highest rate of cross-reactivity.
Acknowledgments
The presented work is part of the research done in the project TR31084 and TR43007 granted by
the Serbian Ministry of Science and Technological Development and 114-451-2142/2011-01 (20112014) by Provincial Secretariat for Science and Technological Development.
References
1.
Heinz F., Stiasny K., Holzmann H., Grgic-Vitek M., Kriz B., Essl A., and Kundi M.: Vaccination and
tick-borne encephalitis, Central Europe. Emerg Infect Dis,19,1,69-76,2013.
2. Hubálek Z., Holouzka J.: West Nile fever – a re-emerging mosquito-borne viral disease in Europe.
Emerg Infect Dis , 5, 5, 643-650, 1999
3. Jawetz, Melnick & Adelberg's.: Medical Microbiology.26th Edition. New York, McGraw Hill Lange,
2013
4. Jerant Patić V. Medicinska virusologija..Novi Sad, Ortomedics, 2007.
5. Koraka P., Zeller H., Niedrig M., Osterhaus A., Groen J. Reactivity of serum samples from patients with
a flavivirus infection measured by immunofluorescence assay and ELISA.Microbes and Infection,
4,1209-1215,2002
6. Makino Y., Tadano M., Saito M., Maneekarn N. , Sittisombut N.,Sirisanthana V., et al. Studies on
serological cross-reaction in sequential flavivirus ections, Microbiol. Immunol, 38, 951–955, 1994.
7. Petersen LR., Marfin AA. West Nile virus: A primer for clinician. Ann Inter Med, 137, 3, 173 179,
2002
8. Petric D., Hrnjakovic Cvjetkovic I., Radovanov J., Cvjetkovic D., Jerant Patic V., Milosevic V., et
all.West Nile virus surveillance in humans and mosquitoes and detection of cell fusing agent virus in
Vojvodina province (Serbia). HealthMed 6, 2, 462-468, 2012;
9. Petrović T., Lupulović D., Petrić D., Vasić A., Hrnjaković Cvjetković I., Milošević V. i ost. Groznica
zapadnog Nila - značajna vektorska virusna infekcija u Srbiji:aktuelna situacija. Veterinarski glasnik,
69,1-2, 2015 (prihvaćeno za štampu)
10. Ravanini P., Huhtamo E., Ilaria V., Crobu MG., Nicosia AM., Servino L., et all. Japanese encephalitis
virus RNA detected in Culex pipiens mosquitoes in Italy. Euro Surveill,17,28,2012,:pii=20221
Available on line: http://www.eurosurveillance.org/ViewArticle.
11. WHO.Vaccines against tick-borne encephalitis:WHO position paper.Wkly WHOEpidemiol Rec, 86, 24,
241-56, 2011.
12. WER...The yellow fever situation in Africa and South America in 2004. Weekly epidemiological
record. 80, 29, 249-256, 2005 Available on line:http://www.who.int/wer
233
VECTOR BORNE INFECTION (EHRLICHIA CANIS) – CLINICAL CASE
Zlatko Dimeski1*, Biljana Petrovska1, Zivko Gacovski1, Goce Cilev1,
Elena Buntevska2, Bojan Stamadziovski2
1
Univesity St. Kliment Ohridski - Faculty of Veterinary Medicine, 7000 Bitola, R. Macedonia
2
Pet Land – Veterinary Hospital, 7500 Prilep, R. Macedonia
*Corresponding author/ e-mail: zlatko_raja@yahoo.com
Abstract
Vector borne infection with Ehrlichia canis is a systemic disorder that manifests with fever,
bleeding tendencies, with thrombocytopenia and platelet dysfunction and non-regenerative anaemia.
The disease is transmitted through the saliva of brown tick, Rhipicephalus sanguineus, which is
widespread in the world. The etiologic agent of canine monocytic ehrlichiosis (CME) is rickettsia
Ehrlichia canis (E. canis). It is a small gram negative coccoide bacteria that parasitizes in
circulating monocytes as a group of organisms called morulae. The clinical manifestation of the
disease is known as ehrlichiosis in dogs (CME). This disease is known in the world, also under the
following names dog’s rickettsioses, canine hemorrhagic fever, canine typhoid, tropical
pancytopenia.
Diagnostic and treatment of canine ehrlichiosis is described in a male dog, of Labrador breed, about
2.5 years old. Clinical symptoms in this dog were depression, reduced limb function after long
inactivity, disbalance in the movement of the rear limbs, nervous symptoms, loss of weight.
After performing the complete blood count, blood examination showed reduced number of
granulocytes (41.7 - %), increased number of lymphocytes (49.6 + %) and monocytosis (8.7 + %).
Diagnosis was not based on blood tests, rapid tests were also done for diagnostics of giardiasis,
distemper, parvovirosis, leishmaniasis and ehrlichiosis, after which it was concluded that the dog
had a positive finding only for ehrlichiosis.
Keywords: Ehrlichia canis, Labrador, Vector borne infection, Ehrlichiosis
Introduction
In our case, it is the treatment of Ehrlichiosis, male breed dog Labrador, about 2.5 years old, with
signs of depression, reduced limb function in long inactivity, imbalance in the movement of the rear
limbs, nervous signs, reduced body weight.
Ehrlichiosis in dogs is usually caused by Ehrlichia canis, E. chaffeensis, E. ewingii, and possibly E.
ruminantium. There are several types of Ehrlichia, which affect various species of animals. Some
affect humans. Some organisms that were formerly classified as Ehrlichia now reclassified as
Anaplasma. Ehrlichia organisms are called rickettsia that the evolutionary scale between bacteria
and viruses (Tuna and Ulutas, 2009). Canine ehrlichiosis is a disease of dogs and wild canids (e.g.,
wolves) and located around the world. Canine ehrlichiosis is also known by other names such as
"tracker dog disease", "tropical canine pancytopenia, ''canine hemorrhagic fever, 'and' dog typhoid."
Ehrlichia are transmitted by ticks including Brown dog tick, Rhipicephalus sanguineus. When the
mature form of a tick feeds on another animal, Ehrlichia are transmitted to the animal.
Ehrlichiosis can have three stages. Signs of the acute phase of the disease usually develop 1-3
weeks after being bitten by triggers. The acute phase of the illness usually lasts 2-4 weeks. Platelets
small cell fragments that help blood clotting, often destroyed. As a result of infection, lymph nodes,
liver and spleen are often enlarged. Anemia, fever, depression, lethargy, loss of appetite, shortness
234
of breath, joint pain and stiffness and bruising are common symptoms. Many dogs will be able to
fight off the infection. If not, enter the subclinical stage.
In subclinical stage of the animal may appear normal or mild anemia. At this stage of Ehrlichia is
inside the spleen. This stage can last for months or years. The dog will either eliminate Ehrlichia
body or infection can progress to chronic phase.
Chronic phase may have moderate or severe symptoms. Weight loss, anemia, neurological signs,
hemorrhage, eye inflammation, edema (fluid accumulation) of the hind limbs, and can’t be seen,
and fever. Blood tests show that one or all types of blood cells are reduced. One type cells,
lymphocytes can be increased and abnormal. This can sometimes interfere with some types of
leukemia. If the dog became chronically infected, the disease can be restored, especially during
periods of stress. In some cases, arthritis or kidney disease called "glomerluonephritis" which can
develop (Yğci et al., 2010).
Reducing the number of platelets (platelets that assist in blood clotting) is the most common
laboratory finding in all stages of disease. Changes in the level of proteins in blood are different.
The most common protein albumin is reduced, and globulin increased (Bulla et al., 2004).
Since one tick can infect more than one disease (e.g., haemobartonellosis or babesiosis), dogs
infected with more than one of these diseases at a time, which generally causes serious symptoms
(Mylonakis et al., 2010).
The diagnosis is based on results of special blood tests. One is known as ELISA test, and the other
is a quick test, which we did when the dog was brought to the veterinary clinic, he was positive on
E. canis. The antibodies can be detected at an early stage of the disease, because it takes a while for
the body to make. A positive result indicates that the dog is affected by Ehrlichia. In the acute phase
of disease, antibody levels increased significantly. A dog with an active infection showed a
significant increase in antibodies present.
For treatment we used tetracycline or doxycycline, for 3-4 weeks, even though the dog's symptoms
generally improve after several days of therapy. We gave supportive therapy, including intravenous
infusion and vitamins (B – complex and C vitamin).
The rapid test that we did was positive (Figure 1) and in addition we show the results of blood
(Table 1)
Table 1. Blood results-clinical case-patient dog ID 3197 (date of analysis 18.02.2015)
Parameter
Leucocites
Eritrocites
Hemoglobine
Hematokrit
MCV
MCH
MCHC
PLT
Result
9
11.3x10 /L
5.30x109/L
11.9
36.7
69.2
22.5
32.4
220
235
Reference
4-9x10^9/L
4.2-5.5x10^12/L
14-18g/dl
37.0-54.0%
76.0-96.0um^3
27.0-32.0pg
30.0-35.0x10^6/L
140-340x10^6/L
Blood results were small increase in leucocytes, while the remaining parameters were within the
normal reference values.
Figure1. Rapid test
Conclusion
Ehrlichia canis is the cause of classical Ehrlichiosis in dogs. This Ehrlichia targets moncytes and is
transmitted by Rhipicephalus sanguineus, the "brown dog tick." The therapy that was given to the
dog, his general condition improved, neurological symptoms and also improved weight gain.
Diagnosis is usually made on the basis of a combination of clinical signs, hematologic
abnormalities and serologic findings.
References
1. Tuna E. G. and Ulutas B. (2009). Prevalence of Ehrlichia canis infection in trombocytopenic dogs.
Lucrări Stinifice Medicină Veterinară Vol. XLII, 2009, Timisoara.
2. Yğci B. Ba. et al. (2010) The spread of canine monocytic ehrlichiosis in Turkey to Central Anadolia.
Israel Journal of Veterinary Medicine Volume 65 (1).
3. Bulla C. et al. (2004). The relationship between the degree of thrombocytopenia and infection with
Ehrlichia canis in an endemic area. Vet. Res. 35 (2004).
4. Mylonakis M., Siarkou V. and Koutinas A. (2010) Myelosupressive caninemonogytic ehrlichiosis
(Ehrlichia canis): An update on the pathogenesis, diagnosis and management. Israel Journal of the
veterinary medicine; Volume 65 (4) 2010.
236
LESHMANIASIS IN 12 YEARS OLD MALE PEKINGESE-CLINICAL CASE
Zlatko Dimeski1*, Biljana Petrovska1, Zivko Gacovski1, Goce Cilev1,
Blagica Trajanoska2, Martin Kamceski2, Bojan Stamadziovski2, Elena Buntevska2
1
Univesity St. Kliment Ohridski - Faculty of Veterinary Medicine, 7000 Bitola, R. Macedonia
2
Pet Land – Veterinary Hospital, 7500 Prilep, R. Macedonia
* Corresponding author: zlatko_raja@yahoo.com
Abstract
Leishmaniasis is a protozoan disease transmitted among domestic and wild animals, as well as
humans. It is widespread in tropical, subtropical regions, but is prevalent in temperate regions.
Leishmaniasis is a transmissible disease caused by protozoa of Leishmania genus, and it is primarly
spread with a bite of female sand fly - Phlebotomine (Phlebotomus). The disease occurs in many
forms of which the most common are two following forms: visceral (caused with Leishmania
donovani) and cutaneus form (caused by Leischmania tropica).
In this report we will present the diagnosis of leishmaniosis and treatment in a dog of Pekingese
breed, male, about twelve years old. The only distinctive symptom in the dog, observed by the
owners and according to the anamnesis - was sharply aging, during a week and was mostly visible
on the head of the dog. There were no characteristic signs as alopecia or skin changes, lethargy and
hyperthermia. Due to the wide spreading of leishmaniasis in our region, a rapid immunological test
for leishmaniosis was conducted and the result was positive finding. Also, laboratory tests were
made such as haematological analysis (WBC 31.27x109/L, Lymph 21.04x109/L, Gran.8.60x109/L,
Lymph 67.3% Mon 5,5%, Gran. 74,9%, RBC 5,04x1012/L, HGB 100g/L, HCT 30,04%, MCV
65,5fL, MCHC 333 g/L, RDW 15,7%, PLT 154x109/L, MPV 8,9 fL, PDW 39,6, PCT 0.079%) and
blood biochemistry test (creatinine 168,6 µmol/L, urea 10.8 mmol/L, АSТ 124 U/L, ALT 62 U/L,
ALP 23,55 U/L) in the blood. Despite these trials a fast test for leishmaniasis was further made.
According to the test, the dog was positive to leishmaniasis. We chose therapy with allopurinol
(20mg/kg, P/O in period of 9-12 months), Rubenal 300, Ipaktin phosphate reducer and Milteforan
(2mg/kg body weight, for 28 days).
Keywords: Leishmaniasis, Pekingese dog, haematological and biochemical profile, fast
immunological test, Allopurinol, Milteforan
Introduction
Canine leishmaniasis (CanL), cause Leishmania infantum is the largest global zoonosis potentially
fatal for humans and dogs, which is the main reservoir of infection in humans. CanL is endemic in
more than 70 countries worldwide (Shaw et al., 2009). CanL manifest a wide range of clinical signs
and degrees of severity, and although there is an insufficient number of research contracts for the
management of this disease. CanL is systemic disease that may potentially relate to any organ or
tissue, presents with nonspecific clinical signs.
Some dogs show clinical signs, while others remain asymptomatic carriers and are infectious for
sand fly. Infected animals are usually affected with lymphadenopathy, anemia, desquamated and
ulcerative lesions of the skin and glomerulonephritis with proteinuria (Zatelli et al., 2003). Ocular
lesions as blepharitis, uveitis, corneal edema or chorioretinitis, can also be present. Unspecific
symptoms may occur frequently, making it difficult to diagnose the disease. Renal lesions are most
commonly affected organs present in dogs with leishmaniasis (Baneth et al., 2008). Proliferation of
glomeruli appears, because circulating immune complexes deposited, which leads to proteinuria.
237
These lesions may progress to chronic renal failure or nephritic syndrome. Renal failure is one of
the classic general symptoms that appear in leishmaniasis, but rarely only manifestation of the
disease.
Complete blood count was performed in blood samples in EDTA tubes, using automated blood
analyzer which is calibrated specifically for dog blood. Although we did biochemical profile and a
rapid test for leishmaniasis (rapid test kit), the test results were positive. The test for leishmaniasis
was made for high prevalence of this disease in our region.
Treatment was initiated with Allopurinol. Allopurinol belongs to a class of drugs called xanthine
oxidase inhibitors which prevent the accumulation of uric acid. If uric acid accumulates in the body,
crystals form in the urine which may congregate to produce kidney or bladder stones. It is used in
dogs alone or in combination with pentavalentantimonials, for its limited toxicity, efficiency and
low cost. The most common dosage is 5-20 mg per day, divided into two doses every 12 hours, over
a long period of time. Side effects are very rare. Allopurinol therapy for Pekingese is (25 mg, 8-11
months, ¼ of the tablet 100mg) was selected, with a recommendation for controls every 3 months
(http://www.1800petmeds.com/Allopurinol-prod10042.html). Also the patient was given a dietary
supplement to support renal function - Rubenal 300. Rubenal 300 is a new aid to maintaining
normal kidney structure which is important in kidney function and is an issue for dogs and cats with
kidney problems. Rubenal 300 helps by maintaining the normal fibrous architecture of the kidneys
(Dosage: Dog 3 - 6kg 1 x Rubenal tablet twice daily). Epakitine phosphate reducer that helps
kidney function in support of renal function in case of chronic renal failure in dogs 5 g twice daily,
mixed with food for up to 6 months from the beginning. Epakitin should be recommended at the
first signs of hyper-phosphatemia in cases of CanL. For best results, it is suggested to use Epakitin
in combination with a low phosphorous diet. However, Epakitin’s highly palatable formulation can
be used with their regular food in animals that refuse to change their diet.
Table 1.Acknowledged forms of currently available treatment
The dog was treated with Milteforan analkylphosphochorine, it is currently the standard treatment
of leishmaniasis Dog. The drug was administered orally for 28 days. The drug is in the form of
liquid, administered orally. The most common contraindication is vomiting, which is transient.
238
Also, the drug showed no adverse effects on the liver and kidneys, and it can be attributed to dogs
with renal failure and avoiding danger, if the dogs do not have renal insufficiency treatment. This
drug is an orally bio-available chemical that was originally studied as an antitumor agent.
Subsequent to the serendipitous laboratory finding that Milteforan was active against Leishmania, it
was tested in dogs in vivo and found to be an effective agent at eliminating parasitemia. The
advantage of this drug is that its direct anti-parasitic activity is not dependent on a functional
immune system.
The haematological and biochemical results after the first clinical examination are given in the
Table 2 and 3.
Table 2. Hematological finding in the 12 year old Pekingese, on the day of receiving in the clinic
Parameter
WBC
Lymph.
MCH
Gran.
Lymph.%
Mon. %
Gran. %
WBC
RBC
HGB
HCT
MCV
MCH
MCHC
PLT
MPV
PCT
Result
9
Ref.values
4.1 x10 /L
0.7 x109/L
0.2 x109/L
3.2 x109/L
16.40%
3.80%
69.90%
3.08x109/L
4.1 x109/L
71g/l
15.7%
89.5fL
29.3
358g/L
103x109/L
8.3 fL
0.079%
6.0-17.0
0.8-5.1
0.0-1.8
4.0-12.6
12.0-30.0
2.0-9.0
60.0-93.0
5.50-8.50
6.0-17.0
110-190
39.0-56.0
62.0-72.0
20.0-25.0
300-380
117-460
7.0-12.0
Parameter
Result
Ref.values
Creatinin
ALP
Urea
AST
ALT
315.9µmol/L
72.64 U/L
45.0mmol/L
54.1 U/L
35.7 U/L
44-138
<136
3.1-9.2
9-49
8-57
Table 3. Biochemical analysis
From the tables we conclude that the dog was severally affected. The dog had overall leukopenia
with low number of leucocytes, and also got severally anaemia because of eritropenia and
hypohaemoglobulinemia, wich probably results from reduced synthesis of erythropoietin in
damaged kidneys and reduced intake of food. The kidneys are severally affected and they can’t
normally clear the creatinine and urea from the body. AST (asparat aminotransferase) showed
increased acctivity, probably because of kidney tissue inflammation. In the specific treatment, we
have included also some maintenance therapy for kidneys (Rubenal 300 and Epakitin).
239
After 28 days of therapy has been shown to improve the general condition of the dog and improved
renal function and liver function. The leukocytes count has normalized, the finding of anemia has
also improved, with normal number of erythrocytes, hemoglobin concentration and hematocrit
value. Platelet count is also near normal value. AST level is normal. Creatinine is in normal values,
wich means that kidneys are working well in respect of clearence. Urea in the serum show normal
levels.
Table 4: Blood results 28 days of therapy with Milteforan and other supportive therapy results
after blood analysis and biochemistry
Parameter
WBC
Lymph.
MCH
Gran.
Lymph.%
Mon. %
Gran. %
RBC
HGB
HCT
MCV
MCH
MCH
PLT
MPV
PCT
Result
9
Reference
8.5 x10 /L
1.1 x109/L
0.9 x109/L
6.3 x109/L
21.10%
4.30%
77.40%
6.06x1012/L
137g/L
41.6%
68.3fL
27.3
315g/L
239x109/L
8.9fL
0.071%
6.0-17.0
0.8-5.1
0.0-1.8
4.0-12.6
12.0-30.0
2.0-9.0
60.0-93.0
5.50-8.50
110-190
39.0-56.0
62.0-72.0
20.0-25.0
117-460
117-460
7.0-12.0
Result
57µmol/L
64.52 U/L
8.2mmol/L
30.1 U/L
32.4 U/L
Ref.values
44-138
<136
3.1-9.2
9-49
8-57
Table 5: Biochemical analysis
Parameter
Creatinin
ALP
Urea
AST
ALT
Conclusion
In this case report, we have concluded that: Allopurinol together with Milteforan is acceptable
specific therapy for canine Leishmaniasis. Supportive therapy for kidney is also important,
especially in cases in wich there is kidney damage. Maybe we should also include some other
reasonable drugs (namely to support kidney function) like diuretics (furosemide and
spironolactone), but also synthetic erythropoietin and antimonials.
References
1. Shaw SE, Langton DA, Hillman TJ (2009) Canine leishmaniosis in the United Kingdom: a zoonotic
disease waiting for a vector? Vet Parasitol 2009.
2. Zatelli A, Borgarelli M, Santilli R, Bonfanti U, Nigrisoli E, Zanatta R, Tarducci A, Guarraci A (2003)
Glomerular lesions in dogs infected with Leishmania organisms. Am J Vet Res 2003.
240
3. Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L (2008) Canine leishmaniosis-new
concepts and insights on an expanding zoonosis: part I. Trends Parasitol 2008.
4. http://www.1800petmeds.com/Allopurinol-prod10042.html
241
________________________________________________________________________
Session № 4
WILDLIFE DISEASES AND PATHOGENS IN ENVIRONMENT
Oral presentations
_______________________________________________________________________
242
Plenary lecture:
INFLUENZA IN BIRDS AND OTHER ANIMALS
Vladimir Savić
Croatian Veterinary Institute, Poultry Centre, Zagreb, Croatia
* Corresponding author: v_savic@veinst.hr
Abstract
There are three types of influenza viruses: A, B, and C; and the latter two are primarily of a human
health importance. In contrast, influenza A viruses have been isolated from a variety of birds and
mammals, nevertheless the natural hosts of the virus are wild waterfowls, gulls and related birds.
Other species that are infected with influenza A viruses, particularly chickens, turkeys, swine,
horses, and humans, are considered aberrant hosts. Majority of influenza A viruses are fully adapted
to the natural hosts in which they multiply mainly in intestines and the infection causes no
symptoms. Influenza A viruses in their natural hosts are in evolutionary stasis. On the other hand,
the infection in aberrant hosts usually results in rapid evolution due to selection pressure driven by
the virus adaptation to a new host. Such rapid evolution can result in high virulence for the new
host, and sometimes even for other species. Emergence of highly virulent influenza A viruses is of a
particular concern for the poultry industry because such viruses cause up to 100% mortality in
chickens and turkeys. Few influenza viruses are well adapted and established in mammalian hosts,
causing primarily respiratory disease like swine influenza, equine influenza and human influenza.
Swine, as an aberrant host, plays an important role in ecology and epidemiology of influenza A
viruses because this species is prone to infection with viruses originating from wild birds, domestic
poultry and mammalian hosts. Such a universal host can serve as a vessel for mixing of the genetic
material of different viruses which can result in new influenza A viruses with unpredictable
features.
Keywords: Influenza A, virus, birds, mammals
Introduction
Influenza viruses are classified into types A, B and C, according to the antigenic properties of
matrix proteins or nucleoproteins. The majority of human influenza virus epidemics, including all
pandemics, are caused by influenza virus type A, which is also the type associated with influenza of
avian, equine and swine species (Webster et al. 1978.). Influenza B and C viruses are isolated
mainly from humans and are less pathogenic than influenza A viruses (McCauley and Mahy, 1983).
Influenza A viruses can be further divided into sub-types on the basis of the antigenic reactivity of
the surface glycoproteins, the haemagglutinin and neuraminidase molecules. So far there are 16
haemagglutinin (H) and nine neuraminidase (N) subtypes (Fouchier et al., 2005) and each virus
possesses one H and one N subtype, apparently in any of 144 possible combinations from H1N1 to
H16N9. Recently, two influenza-like virus genomes designated H17N10 and H18N11 were
identified in South American bats, but attempts to propagate this virus in cell cultures and chicken
embryos were unsuccessful (Wu et al. 2014).
According to numerous authors, influenza A virus was first isolated from chickens at the beginning
of the 20th century (A/Brescia/1902 [H7N7]). Although there is some dispute on a later year of the
isolation of the virus, another isolate of the same subtype (A/FPV/Dutch/27 [H7N7]) dates back in
1920s (Alexander, 2009). This was followed by isolation of influenza virus from pigs (Shope, 1931)
and humans (Smith et al., 1933) in 1930s and two decades later from horses (Sovinova et al., 1958).
243
Influenza virus isolates from chicken were initially called fowl plague virus because until 1955 it
was not known that they were actually influenza viruses (Schäfer, 1955). Subsequent extensive
research revealed more or less barriers to frequent interspecies transmissions of influenza A viruses.
They are influenced by the virus composition and the host specific receptors (Ito eta al., 2000;
Suarez, 2000; Matrosovich et al., 2009). Despite these barriers, influenza A viruses occasionally
transmit from its natural host to other species in which usually cause short-term spread and soon
vanish. In extremely rare cases these viruses adapt to the new host, and such adaptation usually has
far-reaching consequences.
Natural and aberrant hosts of influenza A viruses
Influenza A viruses infect numerous avian and mammalian species, but the natural hosts of the virus
are aquatic birds from orders Anseriformes (ducks, geese and swans) and Charadriiformes (gulls,
shorebirds and terns), particularly ducks. Other species that are infected with influenza A viruses
are considered as aberrant hosts. The most common aberrant hosts are domestic poultry (chicken
and turkey), domestic mammals (pig and horse) and man (Figure 1). The distinction between the
natural and aberrant host is essential for understanding the ecology of these viruses. The vast
majority of influenza A virus is fully adapted to aquatic birds in which infection does not cause
virtually any disease and such viruses in these hosts are in the evolutionary stasis. In contrast, very
intense evolution in aberrant hosts is attributed to selection pressures for adaptation of these viruses
the new host. This adaptation may result in very high virulence for the new host, and sometimes for
other species. It is important to emphasize that influenza viruses multiply in natural hosts, i.e.
aquatic birds primarily in the intestines being excreted via feces. In contrast, influenza viruses in
aberrant hosts usually multiply in the respiratory system (Suarez, 2000; Capua and Alexander,
2007).
Figure 1. The most important hosts and transmission of influenza A viruses. Solid line shows usual
transmission. Dotted line shows transmission which occurs rarely or only presumed transmission.
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Wild birds as natural hosts of influenza A viruses
While only few H and N subtypes are found in aberrant hosts, all 16 H and 9 N subtypes in the
majority of possible combinations are isolated from waterfowls. Wild ducks are the primary natural
reservoir for influenza A viruses in which most of the H subtypes are found (Capua and Alexander,
2007) while H13 and H16 subtypes are isolated almost exclusively from gulls and related birds
(Kawaoka et al., 1988). Influenza A virus excretion from infected duck lasts for just two to four
weeks, but the virus is excreted in high concentration, even in hundreds of millions of infectious
particles per gram of faeces (Webster et al., 1978). Avian influenza viruses have been isolated from
freshly deposited faecal material and from unconcentrated lake water, thus waterfowl, as gregarious
species, have a very efficient way to transmit viruses; i.e., via fecal material in the water supply
(Webster et al., 1992). The successful spread of the influenza A virus among waterfowl is
contributed by their seasonal migrations when they can transmit viruses over long distances (Gilbert
et al., 2006). Migratory bird flyways can be roughly divided into two groups as a result of
geographic ecozones: flyways of the Eastern Hemisphere (Eurasia, Africa, and Australasia and the
flyways of the Western Hemisphere (Americas). In most cases, there is no or very limited overlap
between these two groups of flyways. Consequently there is no or very limited virus exchange
between waterfowl populations the Eastern and Western Hemisphere. This resulted in separate
influenza A virus evolutions in waterfowl in these two hemispheres. With addition of unique
influenza A viruses from gulls (H13 and H16 subtypes), nucleotide sequence analysis of viruses
from the natural host reservoir shows three distinct categories of avian viruses: North American,
Eurasian and gull lineages (Suarez, 2000). Separate evolution and ecology of avian influenza
viruses in the Eastern and Western Hemisphere is further emphasizes by complete absence of H14
and H15 subtypes in America (Krauss et al., 2007). However, infrequent incursions occur and
mainly incursions of avian influenza viruses of Eurasian lineage into North America are
documented (Pearce et al., 2010) including recent incursions of Asian H5 viruses of high
pathogenicity into Canada and USA. Beside genetic differences (Figure 2) there are also significant
antigenic differences between same H subtypes from different hemispheres.
Influenza A viruses in poultry
Domestic poultry species such as chicken and turkey are susceptible to only a limited range of
circulating influenza virus subtypes (Brown et al., 2006). These two avian species are aberrant hosts
and influenza A viruses in chickens and turkeys are under continuous selection pressure (Suarez,
2000). According to pathogenicity in these two hosts, influenza A viruses can be divided into two
clearly distinct groups. Highly virulent viruses replicates in virtually all organ systems and cause
mortality to 100%. This form of the disease is termed highly pathogenic avian influenza (HPAI).
These viruses are of either H5 or H7 subtype, but not all viruses of these groups cause HPAI.
Another group is comprised of viruses causing mild, primarily respiratory disease, termed low
pathogenic avian influenza (LPAI). It may not be neglected that LPAI viruses, if exacerbated by
other infections or environmental conditions, may cause serious disease. More important is that
LPAI viruses of H5 and H7 subtype are potential precursors for HPAI viruses whereas some of
them need only a slight mutation to turn them into very virulent viruses. These mutations occur
after a number of passages in the chicken or turkey (Capua and Alexander, 2007). HPAI virus of
H5N1 subtype that emerged in mid 1990s in the Far East has undergone additional passages trough
domestic poultry and became pathogenic for ducks which were considered until 2002 to be
unsusceptible to HPAI (Sturm-Ramirez et al., 2004). It became pathogenic even to carnivore
mammals, particularly cats, tigers and leopards, which normally do not contract the disease
(Alexander and Capua, 2008). The virus also caused serious fatally in infected humans (Li et al.,
2008). The situation with the H5N1 HPAI was even more worsening by virus spill-over from
245
domestic poultry into the ecosystem which resulted in migratory waterfowl infection. Seasonal
migration during 2005 and 2006 resulted in spread of the virus from China to the rest of Asia,
Europe and Africa (Salzberg et al., 2007). This virus still circulates among migrating wild birds in
Asia, Europe and Africa. Similar scenario happened with a HPAI virus of H5N8 subtype, which is a
reassortant of H5N1 and therefore also poultry origin influenza virus. The novel reassortant virus
occurred in 2014 among poultry and wild birds in South Korea (Lee et al., 2014) and afterwards in
Japan and China. This H5N8 virus probably spread by wild birds further into Europe and North
America and consequently infected the local poultry. So far there is no evidence of infection of
mammals including humans with this virus.
Figure 2. Phylogenetic tree from the NS gene of different subtypes of influenza A viruses. Thirty-three
isolates from the most important host of influenza A virus with different geographic origin in the period from
1956 to 2011 were compared. Groups of viruses with respect to the host, geographical origin and
evolutionary features are marked on the right side of the image. Viruses from different host within the
respective groups are underlined. The tree was made using Maximum Composite Likelihood and Neighbour
Joining algorithm with 1000 repetitions, and the tree is midpoint rooted.
Swine influenza
Commonly circulating subtypes in pigs are H1N1, H1N2, and H3N2 influenza A viruses which are
adapted to this species. Nevertheless, pigs have receptors for avian and human influenza viruses.
Therefore, if the waterfowl is considered a natural host of influenza A viruses, the pig can be
246
considered as a universal host of this virus (Figure 1). Hence the same subtype of swine influenza
virus can be of a different origin, genetic makeup and antigenic reactivity. Swine influenza virus of
H1N1 subtype which is swine entity and evolutionarily has a common origin with the Spanish flu
virus prevails in the North American pig population. In contrast, avian origin swine adapted H1N1
virus prevails in the pig populations in Europe and Asia which is also referred as “avian-like” H1N1
(Figure 2). The North American and Eurasian swine H1N1 viruses, although of the same subtype,
can be easily distinguished by serology. Beside these two H1N1 viruses, human H1N1 viruses (both
seasonal and pandemic) can be found in pigs which is usually result of a direct transmission from
humans. Similarly to H1N1 subtype, viruses of H3N2 subtype create a very complex
epizootiological situation in pigs (Figure 2) including “human-like” and “avian-like” H3N2
variants. Swine H1N2 viruses are various reassortants from mentioned H1N1 and H3N2 viruses.
Occasionally, true avian and even equine influenza viruses could be found in pigs, but they usually
did not establish in this species and soon disappear from the swine populations (Brown et al., 2000).
Other subtypes, like H2N1, H2N3 and H3N1 were also found in pigs.
Swine influenza is generally mild and short-lasting illness manifested similarly to a mild clinical
picture of human influenza. More important is the role of pig as an intermediate host for avian and
human influenza viruses (Figure 1). Simultaneous infection of pigs with two or more different
influenza viruses may result in reassortment of the gene segments between viruses of different
origin. This can result in a new progeny virus with genetic makeup of both (or all) parental viruses.
The new progeny virus may have possibility to cross host species barrier and even to cause
pandemic (Ito et al., 2000; Matrosovich et al., 2009).
Influenza in horses and other equids
Equine influenza is a common acute respiratory disease of horses, donkeys and mules with
symptoms resembling human influenza. In fully susceptible animals, clinical signs include pyrexia
and a harsh dry cough followed by a mucopurulent nasal discharge (Gerber, 1970). There is also a
report on equine influenza with neurological signs due to viral-type non-suppurative encephalitis
(Daly et al., 2006). As influenza in other species, equine influenza spreads rapidly in a susceptible
population. Vaccination provides protection against clinical disease given that the appropriate
vaccinal strain is used (Daly et al., 2011) yet vaccinated infected horses can still shed the virus.
Before the virologic era, equine epizootics highly suggestive of influenza were documented
repeatedly, beginning in 13th century (Morens and Taubenberger, 2010), but the virus was first
isolated in 1956 (Sovinova et al., 1958) during the epizootic equine influenza of H7N7 subtype,
formerly known as “equi-1” or “Prague type”. This subtype in equine population is now likely
extinct. Another subtype formerly known as “equi-2” or “Miami type” emerged in early 1960s in
equine population in North America (Waddell et al., 1963). This H3N8 subtype still circulates in
equine populations worldwide and it is currently present in Croatia (unpublished). Both subtypes
are at the antigenic and genetic level specific for horses (or equids in general), and differ
significantly from H3N8 and H7N7 viruses isolated from other hosts i.e. birds. In addition to this,
great difference between these two equine influenza subtypes (Figure 2) implicates two long-term
separate evolutions with no reassortment events between these two subtypes as well as with other
influenza A viruses. Therefore, horses were considered as a dead-end host, but infection of dogs in
2004 with influenza A virus of H3N8 subtype genetically closely related to contemporary strains of
equine influenza virus (Crawford et al., 2005) related racing greyhounds with infected horses at
hippodromes in Florida. The virus was later found in nongreyhound dogs indicating horizontal dogto-dog transmission (Payungporn et al., 2008). Similarly to interspecies transmission of equine
influenza virus in Florida, cohabitation of horses and camels in Mongolia resulted in isolation of
equine influenza virus (H3N8) from one healthy Bactrian camel out of 460 tested (Yondon et al.,
2014).
247
Influenza A in other animals
Besides already mentioned mammals, influenza A viruses have been sporadically isolated from
other species such as whales, seals and minks i.e. water related mammals. Isolation of H13 subtype
from stranded whales can be associated with direct transmission of the virus from seagulls, but it is
not known whether the infection stranding was related (Hinshaw et al., 1986). Influenza A viruses
of H7N7 and H4N5 subtypes have been isolated from seals (Geraci et al., 1982) and the virus of
H10N4 from minks (Klingebron et al., 1985) caused severe and fatal disease in the these species.
These viruses were phylogenetically closely related to avian influenza A viruses. Such findings
indicate a relatively frequent transmission of influenza A virus among different species, mostly
from birds to mammals, but such infections are restricted and the virus is not maintained in the new
species for a longer period (Webster et al., 1992). A range of carnivorous mammals that were
infected with the Asian H5N1 HPAI virus was fed by infected poultry or wild birds. Transmission
of the H5N1 virus from mammal to mammal was not yet indisputably documented.
Primates are sensitive to human influenza virus. Like other human respiratory viruses, human
influenza virus has repeatedly caused outbreaks of flu-like disease with high morbidity and even
deaths amongst chimpanzees and gorillas (Ryan et al., 2011). Non human primates that have
contact with humans can be naturally infected with seasonal endemic human influenza viruses and
with emerging pandemic-risk avian influenza viruses (Karlsson et al., 2012). Like humans,
macaques infected with influenza virus exhibit fever, malaise, nasal discharge, and nonproductive
cough (Berendt, 1974). It remains unknown whether non human primates are infected with
influenza viruses in nature (Karlsson et al., 2012).
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Invited lecture
VIRUS INFECTIONS OF THE HONEYBEE (APIS MELLIFERA L.) IN CENTRAL
EUROPE
Tamás Bakonyi1,2*, Petra Forgách1, Aleš Gregorc3, Ivana Tlak Gajger4, László Békési5, Miklós
Rusvai1, Norbert Nowotny2,6
1
Faculty of Veterinary Science, Szent István University, Budapest, Hungary
2
University of Veterinary Medicine, Vienna, Austria
3
Agricultural Institute of Slovenia, Ljubljana, Slovenia
4
Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia
5
Institute of Apiculture and Bee Biology, Research Centre for Farm Animal Gene Conservation, Gödöllő, Hungary
6
College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman
* Corresponding author: Bakonyi.Tamas@aotk.szie.hu
Abstract
Viral infections of the honeybee (Apis mellifera L.) became into the scope of the veterinary interest
in the last two decades due to their pathogenic role and the increasing importance of the host
species. Since 2004 extensive losses of honeybee colonies have been observed in North America
and Europe, but its aetiology remained unclear. The phenomenon was named to Colony Collapse
Disorder, and viruses were also considered as causative agents. Moreover, winter losses of
honeybees seemed to be increasing everywhere, and resulted in decline of managed honeybee
population. The presence of bee viruses has been reported from several countries worldwide. Many
of them can be detected in apparently health colonies. Subclinical infection, however, might be
activated by predisposing- and weakening factors, which lead to depopulation or sudden collapse of
the colonies. The most important predisposing factor is the parasitic mite Varroa destructor, which
is a vector of several honeybee viruses, and also weakens the pupae and adult bees by its feeding.
The presence of other pathogens, parasites (i.e. Nosema apis), intoxications and environmental
pollution may also act as an activator of viral infections. The conventional diagnostic methods have
limited value in the case of bee virus infections and diseases; therefore, molecular methods,
predominantly reverse-transcription polymerase chain reaction (RT-PCR) assays are used to detect
viral RNA in honeybee samples.
Our research groups are investigating honeybee virus infections in central-European countries since
1997. We have been detecting the presence of Acute bee paralysis virus, Black queen cell virus,
Chronic bee paralysis virus, Deformed wing virus, and Sacbrood virus in Hungary, Austria,
Slovenia and Croatia. The frequencies of the viruses in different countries varied significantly.
Phylogenetic comparisons revealed closer genetic relationships between bee viruses collected in the
Carpathian basin. Although the prevalence of bee viruses in central-European countries was lower
than those reported in Western Europe, a steady increase has been observed in Hungary between
2004 and 2007. Besides increasing bee colony density and problems with varroa-control, the
domestic and international trade of bees and bee products may also contribute to the aggravation of
virus-associated problems in beekeeping in the region.
Key words: honeybee, virus infections, Varroa destructor, RT-PCR
Introduction
Approximately 25,000 species of bees have been identified worldwide. Nevertheless, only ten
species can be regarded as honeybees. Since bees are highly adaptable insects, they are able to
251
adjust to a wide variety of climates and geographic regions. The habitat of the western honeybee or
European honeybee (Apis mellifera L.) ranges from the tip of southern Africa to southern
Scandinavia, and from Americas, through Europe to western Asia (Whitfield et al., 2009).
Beekeeping is deeply rooted in human society. The apiculture provides full or additional family
income with a considerable market for bee products used as food, pharmaceutics and medical
products. Bee products (i.e. honey, wax, propolis, royal jelly and bee venom) have been used since
the prehistoric times for consumption and for therapeutic purposes. In 2012 the total world honey
production exceeded 1,500,000 tonnes (FAO, 2013). The two leading producers of honey are China
and the European Union (Word Trade Daily, 2012). The direct value of the produced honey is about
140 million EUR, but the total added crops due to pollination services has estimated 14,2 billion
EUR in 2005 in the EU (Gallai et al., 2008).
Honeybees are key pollinators native to Europe and have great impact for many agricultural crops
and conservation of natural plant diversity. In light of the decline of wild insect pollinators the
importance of managed beekeeping is greater today than ever. In the last decade extensive losses of
honeybee colonies have been observed in North America and Europe (Communication from the
Commission on honeybee health, 2010), but the aetiology of the losses remain unclear (Le Conte et
al., 2010; Neumann and Carreck, 2010; Moritz et al., 2010). The phenomenon was named to
Colony Collapse Disorder (CCD) with defined signs and was characterized by „disappeared”
colonies, which were not typically found later in many parts of the world. However, winter losses of
honeybees seemed to be increasing everywhere, and resulted in decline of managed honeybee
population. Honeybee experts in the USA and Europe formed networks to collect more exact data to
identify factors that seem to be not only multifactorial, but interact with individual situations by
countries. The European concerted action was designed in 2008 as a COST action FAO803 by the
name of „Prevention of honeybee COlony LOSses” (COLOSS).
The Working Group 1 (WG1) of the COLOSS epidemiological unit developed a detailed selfadministrated questionnaire to collect exact data on losses. The first results were published recently
(van der Zee et al., 2012) with the analysed information from 12 countries in 2009 and 24 countries
in 2010. According to the survey the mean losses varied between 7-22 % in 2009 and 7-30 % in
2010 winter. An important finding was that for all countries which participated in 2009, winter
losses were found to be substantially higher in 2010. Beekeepers in the majority of the countries
who reported disappeared colonies experienced higher winter losses compared with beekeepers that
experienced winter losses but not reported disappeared colonies. The same was noticed in the USA
where survey responders had lost an average 38.4 % of their colonies in 2011 (van Engelsdorp et
al., 2012). Studies focusing on infectious agents in the background of CDD identified viruses,
microsporidia and parasites as most important factors.
Although more than 20 different viruses were detected from bees, only a few of them are
considered to be able to cause severe diseases in honeybees, and hence are relevant in beekeeping.
The most important beeviruses in Europe are Acute bee paralysis virus (ABPV), Black queen cell
virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus (DWV), Israeli acute
paralysis virus (IAPV), Kashmir bee virus (KBV), and Sacbrood virus (SBV).
ABPV is commonly present in apparently healthy bees; however, this virus may play a role in cases
of sudden collapse of honeybee colonies infested with the parasitic mite Varroa destructor. Due to
the world-wide spread of the varroa mite in the last decades, also ABPV gained more and more
importance (Ball, 1997). On one hand, the mite is a possible vector for the virus; on the other hand
it weakens the bees and activates viral infections (Nordstom et al., 1999).
BQCV causes frequent subclinical infections in adult bees; however it affects clinically prepupae or
pupae of the queen especially in spring and early summer (Laidlaw, 1979). Infected queen pupae
252
die and darken, and the cell walls get black. Nosema apis infestation is a suspected predisposing
factor for disease manifestation (Allen and Ball, 1996).
Two manifestations of CBPV infection have been noted: I. the bees are unable to fly, they are
trembling and crawling and the wings are asymmetric outspread. They often suffer from dysentery
and die within a few days; II. the bees look black because of hair loss. The guard bees do not
recognize them and dismiss them because of their altered look. Although in some cases up to 30%
of worker bees are affected, CBPV infection remains sometimes undetected, and the colonies
usually recover spontaneously from the disease (Bailey, 1967).
DWV is mostly detected in varroa-infected bees (Bowen-Walker et al., 1999). The virus propagates
slowly, and pupae infected at the white-eyed stage of development may have malformed wings and
shortened life span. Adult bees carry the virus without apparent signs.
IAPV was discovered in Israel, but its presence was also demonstrated in other countries. Because
IAPV infections were diagnosed more frequently in CCD-affected honeybee colonies, compared to
healthy colonies in the USA, the role of this virus was hypothesized in the background of CCD
(Cox-Foster et al., 2007; Chen et al., 2014). IAPV is genetically closely related to ABPV and KBV.
KBV is closely related to ABPV and IAPV, however it has been rarely reported in Europe so far
(Allen and Ball, 1996; Siede and Buchler, 2004.). It affects brood and adult bees. Adult bees usually
die within a few days after infection, but larvae may survive following ingestion of the virus, and
some of them become inapparently infected adults.
SBV affects primarily the brood of the honeybee and results in larval death (Ritter, 1996). Infected
larvae fail to pupate and the ecdysial fluid aggregates around the integument, forming the "sac" for
which the disease is named. Larvae change in colour from pearly white to pale yellow and after
death they dry out and change to a dark brown ship-shaped scab. Infection of adult bees is possible,
the viruses are able to propagate in them, but the bees remain apparently healthy. Sacbrood appears
mainly in spring, when the brood-season begins and large numbers of infected young adults are
present (Ritter, 1996).
The virological diagnostic methods have limited value in the case of bee viruses. Because bees are
colonial animals, investigations of individuals are usually less informative. Possibilities for
observation of clinical signs are often complicated, and signs of different virus infections may be
similar. Due to the size of bees, gross pathology is rarely informative. Histopathology is
complicated by the presence of chitin, causing poor penetration of tissue fixation substances, and
causing problems with sectioning of embedded tissues. Identification of beeviruses on
morphological basis, using electron microscopy is on one hand expensive and time-consuming, on
the other hand poorly selective, because of similarities in size and virion morphology. Virus
isolation based on serial passages of viruses in cell cultures is not applicable due to the lack of beespecific and beevirus-susceptible cell cultures. The only possibility for beevirus isolation is
experimental infection of larvae, pupae or newly emerged bees. This technique is, however,
laborious and time consuming. Additionally, the availability of brood is seasonal, and because
viruses frequently cause inapparent infections in honeybee colonies, finding virus-free brood for
experimental virus isolation is rarely possible. Serological methods using virus-specific antibodies,
raised in experimental mammals have been developed in specific laboratories. Viral antigens were
detected and characterized with these methods using agar-gel immunodiffusion (AGID) or enzymelinked immunosorbent assay (ELISA) platforms (Bailey, 1967; Békési et al., 1999). However, the
availability of such sera is limited, and their specificity – due to virus mixtures used for
immunization – might be impaired. Because the immune system of invertebrates is mainly based on
non-specific, innate immunity; classical serological methods are not applicable for indirect virusdiagnostic approaches, due to the lack of adaptive immunity, specific anti-viral antibodies and
immune memory in bees (Azzami et al., 2012). The advances of developments of molecular
diagnostic techniques had a revolutionary effect on honeybee virology diagnostics. As more and
253
more beeviruses were genetically characterized, specific, sensitive and rapid diagnostic assays were
developed on the basis of the polymerase chain reaction (PCR) technology. These techniques are
widely used in the last decade for the diagnosis, surveillance and molecular epizootiology of virus
infections in honeybees. In this paper, our studies on certain honeybee viruses in Europe are
summarized.
Sample collection
The collection methodology was depending on the aim of the investigations. In the case of diseased
colonies (i.e. winter losses, colony collapses), usually dead bees were collected in the hives or from
their surroundings. Sometimes bees with neurological signs (altered behaviour, crawling, inability
of flying), deformed wings, changed colour, or altered brood were collected. For the surveillance
studies usually healthy bees were collected from the hives, and – if it was available – 10 × 10 cm
covered brood was also collected. Bee samples usually contained at least 50 individuals from each
colony. Samples were stored and transported at 4ºC, however, sometimes -20ºC storage was applied
for longer periods (few weeks) at the beekeepers.
Sample processing
Bees were observed at the laboratory, and if they were infested with V. destructor, mites were
collected from the samples. Approximately 25 to 50 bees were pooled from the same colonies and
were crashed in ceramic mortars using sterile quartz sand, and were homogenised in 10 × volume
phosphate buffered saline (PBS). After centrifugation (~1,500 ×g, 10 minutes) viral RNA were
extracted from the supernatants using the QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany)
according to the manufacturer's instructions. Viral RNA was reverse-transcribed and amplified in
continuous reverse transcription PCR (RT-PCR) method by using the One Step RT-PCR kit
(Qiagen, Hilden, Germany) following the manufacturer's recommendations. Virus-specific primers
are listed in Table 1.
The reverse transcription at 50°C for 30 min was followed by a denaturation and polymerase
activation step at 95°C for 15 min and by 40 cycles of PCR with 30 s at 94°C, 50 s at 55°C, and 1
min at 72°C. Reactions were completed by a final elongation step for 7 min at 72°C. The PCR
products were electrophoresed in a 1.2% TAE-agarose gel, stained with ethidium bromide, and
were photographed under UV light. Fragment sizes were determined with reference to molecular
size markers.
Genetic comparisons
Selected amplification products were subjected to fluorescence-based direct sequencing (Sanger’s
method) in both directions. Nucleotide sequences were identified by the Basic Local Alignment
Search Tool (BLAST, 2), and aligned using the Align Plus program (Scientific and Educational
Software, version 4.1).
Multiple alignments for phylogenetic analyses were created with the help of the ClustalX program
(Thompson et al., 1997). Phylogenetic analyses were conducted by the PAUP*4.0 beta 10 version
(Swofford, 2002), PHYLIP package 3.6 beta version (Felsenstein, 2004), and Mega 5 (Tamura et
al., 2011) software using maximum parsimony, distance (neighbor-joining) and maximum
likelihood criteria. Bootstrap analyses were performed on 1000 replicates. Trees were drawn with
the help of the TreeView 1.6.6. software.
254
Table 1: Oligonucleotide primer pairs used in RT-PCR assays.
Primer name
ABPV 23fa
ABPV 24ra
BQCV 3fb
BQCV 4rb
CBPV 111fc
CBPV 426rc
DWV 2345fd
DWV 2779rd
IAPV 3042fe
IAPV 3799re
KBV 5406ff
KBV 5800rf
SBV 1fg
SBV 2rg
Primer sequence (5´to 3´)
Primer position
on the genome
GTGCTATCTTGGAATACTAC
AAGGYTTAGGTTCTACTACT
AGTAGTTGCGATGTACTTCC
CTTAGTCTTACTCGCCACTT
TGTCGAACTGAGGATCTTAC
GACCTGATTAACGACGTTAG
ATTGTGCCAGATTGGACTAC
AGATGCAATGGAGGATACAG
ATTGAGAGTTGCCAAGGAGT
GTCTGTGCTTCGATCACAAT
GATGAACGTCGACCTATTGA
TGTGGGTTGGCTATGAGTCA
ACCAACCGATTCCTCAGTAG
CCTTGGAACTCTGCTGTGTA
7928-7947
8527-8546
252-277
710-729
111-130
407-426
2345-2364
2760-2997
3042-3061
3780-3799
5406-5425
5781-5800
221-240
689-708
Length of the
amplified
product (bp)
618
472
315
434
758
395
487
Nucleotide positions refer to the published sequences of aABPV (GenBank accession number AF150629), bBQCV
(accession number AF125252), cCBPV (accession number AF375659), dDWV (accession number AJ489744), eIAPV
(accession number EF219380), fKBV (accession number AY275710) and gSBV (accession number AF092924). f:
forward primer; r: reverse primer
Results
Occurrence of honeybee viruses in Central European countries
Between 1999 and 2010 we have been investigating the occurrence of the most important honeybee
viruses in four central European countries: Austria, Hungary, Slovenia and Croatia (Berényi et al.,
2006, Forgách et al., 2008, Gregorc and Bakonyi, 2012, Tlak Gajer et al, 2014). Four honeybee
viruses, ABPV, BQCV, DWV and SBV were detected in all four countries. The occurrence of
CBPV was tested and detected in Austria, Hungary and Croatia. The presence of KBV and IAPV
were tested in Austria, Hungary and Croatia, but were not found in any of the investigated samples.
The detection frequencies are summarised in Table 2.
Table 2: Occurrence of honeybee viruses in four central European countries
Country
n*
year
ABPV
2003Austria
131
68°%
2004
199952
37%
Hungary
2004
72
2007
71%
81
2006
12%
Slovenia**
72
2008
10%
Croatia
82
2010
11%
*n= number of samples; NT= not tested
**Newly mated queens were investigated
BQCV
CBPV
DWV
IAPV
KBV
SBV
30%
9%
91%
0%
0%
49%
54%
0%
72%
NT
0%
2%
40%
0%
24%
29%
6%
NT
NT
10%
49%
7%
58%
95%
0%
NT
NT
0%
0%
NT
NT
0%
61%
1%
11%
40%
255
Genetic relationships between central European honeybee viruses
Our phylogenetic studies with ABPVs from Austria, Hungary and Poland revealed that the central
European viruses are genetically distinct from ABPVs detected in the USA, in the United Kingdom
and in South Africa. Hungarian ABPVs formed a monophyletic group, closely related to Polish
ABPVs, while Austrian and further Polish viruses clustered together (Bakonyi et al., 2002). In the
case of BQCV, the South African reference sequence was genetically distant from the central
European viruses. Hungarian Austrian and Polish viruses formed common groups, while some
BQCVs from Poland formed a separate group. Some Polish BQCVs, however clustered differently
depending on the investigated genome region. This finding indicates genetic recombination between
the viruses (Tapaszti et al., 2009). Phylogenetic analysis of CBPVs revealed that Austrian,
Hungarian and Polish viruses formed a common genetic lineage with German, Swiss, and French
viruses, but were distinct from other three genetic lineages containing CBPVs from France, Spain,
Belgium and Uruguay (Blanchard et al., 2009). The genetic relationships between DWVs collected
in Austria, Canada, Germany, Hungary, Japan (“Kakugo virus”), Nepal, New Zealand, Poland,
Slovenia, Sri Lanka, and the United Arab Emirates were investigated. Despite the great diversity of
collection origin, the sequences has shown high level of genetic conservation, therefore
phylogenetic analysis could not reveal statistically supported clustering of the sequences. The
DWVs, however were genetically distinct from Varroa destructor virus 1 (Berényi et al., 2007). The
phylogenetic relationship of one Austrian SBV was investigated. It was found closely related to
SBVs from Germany, but more distantly related to SBVs from the United Kingdom, India, Nepal
and South Africa (Grabensteiner et al., 2001).
Discussion
Our studies described the presence of five different honeybee viruses (Acute bee paralysis virus –
ABPV, Black queen cell virus – BQCV, Chronic bee paralysis virus – CBPV, Deformed wing virus
– DWV, and Sacbrood virus – SBV) in four central European countries (Berényi et al., 2006,
Forgách et al., 2008, Gregorc and Bakonyi, 2012, Tlak Gajer et al, 2014). Simultaneous infections
with different virus species in colony level have been frequently diagnosed. The investigations
revealed that the infection rate of the Hungarian honeybee colonies have been increasing within the
last years (Tapaszti et al., 2010). Usually, honeybee virus infections are not apparent as they remain
in a latent state within individuals and spread among bee populations at a low level. This means that
clinical signs appear only when virus replication is initiated and infection becomes systemic. When
cells of honeybees are infected with virus particles, they cannot work efficiently; this inevitably
causes vital organs to dysfunction (Békési et al., 1999). This can lead to outbreaks of disease and
induce significant losses. Infestation with the parasitic mite Varroa destructor is a major
predisposing factor which has a weakening effect on the honeybee (Yang and Cox Foster, 2005)
and viruses may directly spread through the lesions induced by the mite (Ball, 1989). Also, it has
been experimentally proven that these mites carry ABPV (Békési et al., 1999), DWV (Tentcheva et
al., 2004) as well as KBV (Chen et al., 2004). Other predisposing factors include, for example
nosema infestation, intoxications, environmental pollution, cold weather, dysentery, overcrowding
and further infectious agents such as bacteria, fungi, and parasites. With the spread of Varroa
destructor almost worldwide, attention has been focused on viruses of bees because of the role of
the mite in transmission of a number of these infections and their connection with colony mortality
(Ball and Allen, 1988). This has led to use of the term “bee parasitic mite syndrome” (Hung et al.,
1996). Therefore, the control of honeybee virus infections must be based on optimal keeping and
breeding conditions (avoiding the above-mentioned predisposing factors) with a particular attention
to varroa-control. Additionally, the possibility for specific treatment or prevention for certain
honeybee virus infections (IAPV and DWV) were reported by using RNA silencing technologies
256
(Maori et al., 2009; Desai et al., 2012; Chen et al., 2014). Because the dominance of innate
immunity and lack of immune memory in bees, active immunisations (vaccinations) are not
applicable for the prevention of bee colonies from virus infection or from the development of virusassociated disease manifestations. The results of our studies indicate that movement and
importation of non-apparent infected stocks of bees may introduce viruses into areas where they are
not endemic. It is therefore important to identify bee virus infections and take this aspect into
consideration in domestic and international transport and trade of honeybees and bee products.
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Invited lecture
THE MOST COMMON HEALTH DISTURBANCES DETECTED IN WILD BOARS IN
ENCLOSED HUNTING GROUNDS IN VOJVODINA PROVINCE
Jasna Prodanov-Radulović1*, Radoslav Došen1, Igor Stojanov1, Tamaš Petrović1
1 Scientific veterinary Institute “Novi Sad”, Novi Sad, Serbia
* Corresponding author: jasna@niv.ns.ac.rs
Abstract
The control of health status of wild boar population is quite demanding and it is not easy to achieve.
Also, often is not possible entirely to perform a complete diagnostic examination in wild boars in
each evaluated case. The aim of this research was to evaluate the most common health disturbances
detected in wild boars in enclosed hunting grounds in Vojvodina Province. The material for this
research included enclosed hunting grounds, where clinical signs of health disorders and dead of
different categories of wild boars were recorded. The following research methods were applied:
epidemiological investigation, clinical examination of live and gross pathological examination of
dead and/or shot diseased wild boars. The clinical examination was performed from the safe
distance at the feeding place. In the laboratory, the applied research methods included:
bacteriological testing, virusological testing (ELISA test, HI test and RT-PCR) and parasitological
examination. On the basis of the achieved results it may be concluded that wild boars could be
source of different endoparasites species. By gross pathological examination it was discovered that
in the largest number of animals the health problems were mainly connected to the parasitic
infestations and bacterial infections of digestive and respiratory organs.
Keywords: wild boars, enclosed hunting grounds, Vojvodina Province
Introduction
Wild boar (Sus scrofa scrofa) numbers have dramatically increased over the past 60 years and the
species also shows a more widespread distribution (Artois et al., 2001; Sedlak et al., 2008; Wu et
al., 2011), which not only means a larger number of hosts available for the transmission of disease,
but also a higher contact rate between hosts (Ruiz-Fons et al., 2008). The population of wild boar in
15 member states of European Union (EU) has been roughly estimated between 800,000 and 1
milion heads, but its density varies from country to country (Laddomada, 2000). Increasing food
availability and climatic change provide optimal conditions for a rapid wild boar multiplication and
expansion (Wu et al., 2011). According to available data of the Veterinary directorate of the
Ministry of Agriculture and Environmental protection, in the Republic of Serbia, the population of
wild boars was aproximatelly estimated on 30.000,00 heads and the density of the wild boar
population ranges from a minimum of 0.2/km2 to over 20/km2. In Serbia, there are about 300
hunting grounds with wild boars. Hunting grounds are managed by two public enterprises, also two
hunting association, four National parks in wich hunting is allowed and 5 hunting grounds are
managed by Ministry of Defence. In Vojvodina Province, there is one public enterprise
„Vojvodinašume“ with 17 hunting grounds, one National park Fruška gora, one private hunting
ground and 86 hunting associations.
Wild boar pathogens are highly relevant not only for the livestock industry but also for wildlife
conservation and for the hunting industry (Došen et al., 2013; Laaksonene and Paulsen, 2015;
Prodanov-Radulović et al., 2014a). Knowledge of diseases circulating in wildlife populations can be
260
important not only for conservation and livestock production but also for public health (Boadella et
al., 2012; Meng et al., 2009). Because several million wild boars are harvested and consumed yearly
in Europe, wild boar meat and derivates are a likely source of human infections (Boadella et al.,
2012).
Wildlife is a reservoir for several economically important diseases and epizootiologic surveys are
needed, especially in large wild boar populations (Garcia-González et al., 2013). Many diseases
may cause external symptoms and abnormal behaviour in game animals. They can also diminish the
value of the end products obtained from game. In the most of the cases, the assessment of game
animal health is based on medical history of the game and, after shoting, observations made of the
carcass and viscera (Laaaksonen and Paulsen, 2015; Prodanov et al., 2009a; Prodanov-Radulović et
al., 2013a). Migrating and roaming animals can carry pathogens over long distances (Laaaksonen
and Paulsen, 2015). The parallel increase of outdoor piggeries has led to a higher risk of contacts,
and thus of disease transmission, between wild boars and domestic pigs. Because pigs and wild
boars belong to the same species, they share the same pathogens (Prodanov et al., 2009a; Prodanov
et al., 2009b; Wu et al., 2011). The overabundance of wildlife, recognized as a relevant risk factor
for disease transmission between wildlife and domestic animals, compromises the health
surveillance programs carried out both populations (Frölich et al., 2002; Ruiz-Fons et al., 2008).
Hunting and consumption of wild boar meat enables direct contact of humans and wild pigs,
providing ideal conditions for pathogen transmission from wild to domestic pigs and humans. It
should be taken into consideration that domestic animals and humans usually have never been
exposed to pathogens common for wild pigs, and thus are highly susceptible to infection (Ruiz-Fons
et al., 2008). Hunters have a greater than average risk of encountering various pathogens
communicable from animals to people. The ways of handling the carcass, meat, offal and hides
have an effect on this risk (Laaaksonen and Paulsen, 2015).
Animal health surveillance is routinely applied to domestic animals, but limited data exist on the
prevalence and distribution of infectious agents of wild boars in Vojvodina Province. Also, often is
not possible entirely to perform a complete diagnostic examination in wild boars in each evaluated
case (Petrović et al., 2012, Prodanov-Radulović et al., 2013a; Prodanov-Radulović et al., 2013b). In
Vojvodina Province a certain number of wild boars is controlled and reared in an enclosed hunting
ground while the number of free-ranging population is mainly unknown (Prodanov et al., 2009b).
One of the characteristics of outdoor swine production in some regions of Vojvodina is raising freeroaming domestic pigs, where they share forest habitat with wild boar population. It can be assumed
that direct contacts between wild boars and domestic pigs kept in outdoor farms occur occasionally
(Došen et al., 2013; Prodanov-Radulović et al., 2011; Prodanov-Radulović et al., 2014a).
In some regions, especially near the river banks, domestic pigs are kept outdoors on the pasture,
which provides favourable conditions for infection transmission. Domestic pigs move freely in the
woods, thus getting in contact with wild boars. However, after summer pasture, domestic pigs are
returning into the pens. It is especially important that owners of the free-roaming animals in the
same time have backyard pigs (Prodanov et al., 2009b). In areas in which traditionally raising freeroaming domestic pigs has been introduced in the woods, hybridization with the wild boars has led
to crossbreeding, production which is often referred to as a feral pig or feral hog (Ruiz-Fons et al.,
2008). In our research during the epizootiological and clinical examination, we discovered the
existence of this type of animal hybrids in the backyards of the owners who practice extensive
grazing (Prodanov et al., 2009b).
The objective of our study was to evaluate the most common health disturbances detected in wild
boars in enclosed hunting grounds in Vojvodina Province, focusing on detected diseases that are
epidemiologically and economically important to the health of both wild boar and domestic swine
populations.
261
The material for this research comprises two different sources with the aim to perform health
control wild boars population in enclosed hunting grounds in Vojvodina Province. As a part of
health control program of wild boars, regularly shot by hunters, the examination of the trunci and
internal organs deriving from wild boars was carried out. The detailed gross pathological
examination, according to the specially adopted protocol, with an aim to not interfere with regular
hunting procedure, and taking the samples for laboratory examination, was done. In order to check
the wild boars in which the hunters noticed some signs of abnormal behaviour and/or signs of
diseases, the gross pathological examination of carcasses of dead or diseased animals shot by
hunters and laboratory testing of their organs and tissue samples was performed. The following
research methods were applied: epidemiological evaluation, clinical examination of live and gross
pathological examination of dead wild boars, standard laboratory testing for detection the presence
of aerobic and anaerobic bacteria in tissue samples (mediastinal and mesenterial lympho nodes,
lungs, heart, liver, spleen, kidney) deriving from dead wild boars, patohistological examination
(lungs) and serology testing (sera samples).
Isolation of bacteria from tissue samples deriving from dead pigs was performed by standard
aerobic and microaerophilic cultivation. Microscopic examination determined whether the isolated
bacteria were Gram positive or not and whether it is a coccoid or rod-like organisms. The
determination was carried out by determining the biochemical characteristics of the isolated
bacteria. Beside this, each animal carcass was thoroughly analysed by gross pathology examination
for the presence of helminths. Lung, digestive tract, liver, gall-bladder and renal pelvis were
examined with routine techniques for the detection of helminth parasites. A parasitological
examination was carried out with fecal material extracted from the rectum of each animal after
necropsy (zinc sulphate flotation, sedimentation and McMaster’s method). In cooperation with the
hunting societies and local veterinary service gathering of sera samples of hunted wild boars was
organized. The serology testing included classical swine fever (indirect immunoenzyme test kit:
HerdChek CSFVAb, IDEXX Laboratories, USA) and porcine parvovirus (haemagglutination
inhibition test).
Wild boars diseases caused by bacteria
On the several examined hunting grounds, the case history data revealed the problem in piglets
category with clinical signs of growth retardation. The piglets were reluctant to move, fatigued,
easy to catch and a large number of dead piglets were discovered. As a part of health control
program, clinically diseased wild boars (animals showing staggering gait, with long bristling hair
and arched back) were shot by hunters. The gross pathological examination revealed changes
dominantly in the respiratory tract: severe necrotizing pleuropneumonia and the presence of
multiple abscesses in the lung tissue (Pneumonia apostemosa disseminata). In few animals, purple
to gray areas of consolidation of lung tissue were detected (Pneumonia fibrinosa in statu
hepatisatiois rubrae et griseae). Macroscopically, the lung lobes were very similar to the hepato or
pancreatic tissues. In addition, the trachea and bronchi were filled with a foamy exsudate mixed
with small number of lung worms in the respiratory pathways. By standard bacteriological testing
on tissue samples (lungs, mediastinal lymph nodes) Pasteurella haemolytica, Streptoccocus alfa
haemolyticus, Streptococcus beta haemolyticus and Actinobacillus pleuropneumoniae were
detected.
262
In various game animal species, the bacteria from the genus Pasteurella can cause epidemics with
high mortality rate (Laaaksonen and Paulsen, 2015). The bacteria from the genus Pasteurella is
probably the most frequent and damaging invader in the lung i.e. tipically secondary invader and
was never isolated in the bronchial tree of healthy pigs. Even the most pathogenic strains are not
capable of infecting a healthy lung, unlike A. pleuropneumoniae (Le Potier et al., 2006). It is
consider that the outbreak of the disease in wild boars requires stressful and predisposing factors,
such as poor state of nutrition, overexertion, overdensity of population, unfavourable weather
conditions and great number of parasites. Common clinical symptoms in wild boars include hanging
of head and severe functional disorder of respiratory organs (Laaaksonen and Paulsen, 2015;
Prodanov-Radulović et al., 2013b).
Actinobacillus pleuropneumoniae is considered an obligate parasite of the porcine respiratory tract
and there are no other natural hosts. Transmission occurs via aerosols or direct contact between
pigs. The clinical and pathologic outcome of infection depends on serovar and virulence factors, but
also on age, immunity, hygiene, infectious pressure, breed, and stress factors (Reiner et al., 2010).
Pleuropneumonia caused by A. pleuropneumoniae is one of the important bacterial diseases of the
respiratory tract of the pig and occurs in most pig-keeping countries (Boadella et al., 2012).
Subclinically and chronically infected pigs may enter a permanent carrier state, harbouring A.
pleuropneumoniae in sequestra or well-encapsulated abscesses in the lung and in tonsillar crypts
(Le Potier et al., 2006). Survivors of acute infections become carriers, and the infectious agent is
located mainly in nerotic lung lesions and/or in the tonsils, less frequently in the nasal cavity
(Boadella et al., 2012). Reiner et al. (2010) discovered that the overall prevalence of wild boars that
were PCR-positive for A. pleuropneumoniae DNA was 35.8%. Actinobacillus pleuropneumoniae
DNA was amplified from tonsils of 34.8% and from lungs of 6.4% of wild boars. The discrepancy
between A. pleuropneumoniae loads in lungs and tonsils likely demonstrates the higher colonization
of tonsil tissue with A. pleuropneumoniae in wild boars, although it cannot be excluded that lungs
were categorized as false negatives. Prevalence of A. pleuropneumoniae increased with age and
body weight. Higher prevalences in tonsils without gross pathologic signs of pleuropneumoniae
suggest colonization of most of these animals by non-pathogenic or low-pathogenicity serotypes
(Reiner et al., 2010).
In Europe, most attention has been devoted to diseases that are under official surveillance and
control in either pigs, wild boar or both, while other infections have received comparatively less
effort. Monitoring wildlife diseases faces a number of wildlife-specific constaints, including
sampling difficulties regarding proper sample and site stratification, consistent sampling of the
same sites, and limitations of the diagnostic test available for wildlife (Boadella et al., 2012). It
should be stressed that overdense regional game populations are a predisposing factor for the
disease (Laaaksonen and Paulsen, 2015).
In only few examined enclosed hunting grounds, by clinical examination of wild boars during the
feeding, the clinical signs (greyish to brownish diarrhea) of the enteric infection were observed.
Applying bacteriological testing on tissue samples (kidney, liver, spleen, mesenterial lympho nodes)
derived from diseased shot and dead wild boars the presence of Escherichia coli and Escherichia
coli haemolytica was detected. Most common gross pathology findings included moderate signs of
gastric and intestinal infection and enlarged mesenterial lymph nodes.
Escherichia coli bacteria cause a group of enteric diseases mostly found in young wild boars
(Laaaksonen and Paulsen, 2015). Disease outbreak usually requires predisposing factors, depending
on the virulence of bacterium. Starvation and poor condition of mothers/dams set the scene for an
outbreak of the disease. Infection pressure is also increased when the number of diseased animals is
high and the population is dense (Laaaksonen and Paulsen, 2015; Prodanov-Radulović et al.,
2013a). In the treatment, usually the same antibiotics are used in the treatment of bacterial diseases
of domestic pigs and wild boars. Bacteria have a genetic ability to mutate and develop strains that
263
are resistant to antibiotics. This, together with poorly planned treatment or mass uncontrolled use of
antibiotics, may cause great problems in the health care of humans and animals (Došen et al., 2014).
In the control, important precautions include game animal feeding ground hygiene and prevention
of contacts between game and production animals (Laaaksonen and Paulsen, 2015).
Wild boar diseases caused by parasites
By gross pathology examination, the adult forms of several gastrointestinal parasites were
discovered: Ascaris suum, Macracanthorhinhus hirudinaceus, Trichuris suis. However, no gross
pathology lesions were detected in the gastrointestinal tract of examined wild boars. Applying
parasitological control of the intestinal content and/or faecal material extracted from the rectum, the
presence of eggs from several parasites was discovered: Trichuris suis, Oesophagostomum sp. and
Hyostrongylus sp. Also, in most of the examined cases, the presence of Protozoa of genus Eimeria
(Coccidia) was detected. Parasites, and changes caused by them, are the most common findings that
a hunter confronts in game handling. It is important to distinguish between a parasitic infection,
which practically all animals have, and a disease caused by parasites. The manifestation of disease
is often directly comparable to the severity of parasitic infection (Laaaksonen and Paulsen, 2015;
Prodanov-Radulović et al., 2011).
The parasite from genus Coccidia reproduces in the intestines and damages the intestinal lining,
causing diarrhea and intestinal dysfunction of host animals and produce egg-like oocysts to their
feces. The infection is transmitted to another animal via feces-contaminated food or water. In dense
populations, the transmission of the parasite increases and infection pressure grows. Consequently,
coccidian may become a significant cause of mortality in young animals. In the prevention, correct
location and hygiene of game feeding grounds is important (Laaaksonen and Paulsen, 2015).
Roundworms (Trichostrongylidae, Trichuroidea, Strongylidae and Ascaroidea) that parasitize in
the gastrointestinal tract are frequently found in wild boars (Prodanov-Radulović et al., 2013a). An
adult parasite inhabiting the stomach or intestines of a host animal produces eggs that are passed to
the environment in feces. In general, parasites of the gastrointestinal tract do not cause problems
under normal conditions, and their spread is self-limiting. However, these parasites are significant
in dense animal populations, as well as when starvation occurs. In that case, diseases may be caused
especially by large roundworms (Ascaroidea), they can cause weakening of the animal and
significant mortalities. In addition, animals with a parasitic infection can present a weight loss,
wasting, swollen abdomen, dry and dull hair and diarrhea (Laaaksonen and Paulsen, 2015).
In most of the shot healthy, diseased or dead found wild boars originated from different enclosed
hunting areas the presence of lung worms was established. Clinically, the case history data revealed
the problem in piglets category (signs of growth retardation). The piglets were described as
reluctant to move, fatigued, easy to catch. In several examined hunting grounds, by clinical
examination clinical signs of respiratory infection in young wild boars were detected: dispnoea and
intensive coughing („thumping“). The wild boars were emaciated and slow gait, with shrunken eyes
and tough dry hair, and lagging behind the pack were noticed. As part of health control program of
clinically diseased wild boars shot by hunters, gross pathological examination of trunci and internal
organs deriving from shot wild boars was performed. In all examined cases, the gross pathology
examination revealed changes dominantly in the respiratory tract: purulent nasal discharge,
effervescent content in bronchi and bronchioles, mixed with a large number of lung worms, which
were like mucoid plugs filling the respiratory pathways (Metastrongylosis pulmonum summ). All
lobes of the lungs were diffusely swollen, edematous and reddened with marginal emphysema and
consolidation. A large amount of clear, foamy fluid and numerous slender, white nematodes 4-7 cm
long were visible in the trachea and bronchial trees. In several cases, pathomorphologically the
presence of numerous abscesses (Pneumonia apostematosa disseminata) in lung tissue were
detected. In the cases when macroscopically changes on the lung tissue were noticed, the
pathohystological examination were performed and in two examined shot wild boars Pneumonia
264
interstitiais was diagnosed. By parasitological examination the presence of lung worms
(Metastrongylus spp.) in the trachea, bronchi and in posteroventral parts of the diaphragmatic lung
lobes were detected (Pneumonia verminosa).
Lungworms are often encountered as highly prevalent helminthes in wild boars (ProdanovRadulović et al., 2011; Senlik et al., 2011). In Europe, these parasites have a high prevalence,
affecting more than 80% of pigs created in extensive system and considered one of the main causes
of respiratory changes of these animals (Da Silva et al., 2013). This result might be explained by the
wide geographical distribution of different earthworm species, which form part of the diet of wild
boars and act as intermediate hosts for these parasites (Senlik et al., 2011). An adult parasite dwells
in the bronchial tubes of wild boars, where it produces eggs. The eggs are coughed up to the throat
and travel via the pharynx to the intestines. In the intestines, the eggs develop into larvae and are
passed in feces to the ground. New animals are infected after ingestion of food that contain larvae.
Inside the new host, the larvae penetrate the intestinal wall and migrate via lymphatic vessels to the
lungs, thus completing the parasitic life cycle (Laaaksonen and Paulsen, 2015; Prodanov-Radulović
et al., 2011). Mild infections are often asymptomatic. Severe infections cause caught and
inflammation of the lungs, the symptoms of which may include deterioration of general condition,
slow growth, weight loss and mortalities. By gross pathological examination, adult parasites can be
seen in the bronchial tubes. The scars left by them are greenish areas of 0.5 to 2 cm in size in the
lungs (Laaaksonen and Paulsen, 2015). The verminous processes are mainly located dorsocaudally
in the lung (Prodanov-Radulović et al., 2013b).
Lung parasites of the genus Metastrongylus are considered one of the most important selective
factors acting on wild boar population, increasing the mortality of weaker young and adult animals
because they may cause dyspnea, bronchopneumonia, and permanent weight loss in addition to
inflicting tissue damages which allow opportunistic infections of viruses and bacteria (Da Silva and
Muller, 2013). Young wild boars are thought to ingest a higher number of earthworms than adults
and therefore may have a higher level of parasitism (Garcia-González et al., 2013; Järvis et al.,
2007). In Europe, these parasites have high prevalence, affecting more than 80% of pigs created in
extensive system and considered one of the main causes of respiratory changes of these animals (Da
Silva and Muller, 2013; Yoon et al., 2010). Despite the limited number of wild boars examined, our
study suggests these species are common and enzootic in wild boars in Vojvodina region
(Prodanov-Radulović et al., 2013a; Prodanov-Radulović et al., 2013b). The reason for the high
prevalence of lungworms may be the density of game population, which often is the natural
explanation of the spread of a parasite transmitted from animal to animal (Laaaksonen and Paulsen,
2015; Järvis et al., 2007). In control, infection pressure can be diminished by preventing the
development of dense game populations and maintaining good game feeding ground hygiene
(Laaaksonen and Paulsen, 2015). The uncontrolled use of anthihelmintics can lead to an excessive
selection of resistant parasites, which may cause an increase in the infection intensity (Da Silva and
Muller, 2013).
Sporadically, by gross patology examination of shot wild boars, the presence of parasitic cysts in
the abdominal cavity were detected. Tapeworms (Taenia hydatigena) is a ubiquitous parasite, the
definitive hosts of the adult parasite are carnivores such as wolf, dog, lynx and fox. The parasites
intermediate hosts are herbivores and wild boars, which get the parasites eggs to their intestines
through food. The larvae burrow through the intestinal wall and form cysts in the body of
intermediate host (Cysticercus tenuicollis). Each cysts contains one infectious larva. When the cystbearing animal is eaten by a carnivore, the parasites life cycle is completed. The pearly gray, liquidfilled cysts of the T. hydatigena tapeworm, 1.5 to 10 cm in diameter, are most commonly found in
the omentum, liver or peritoneum of wild boars. Single, smaller cysts go easily unnoticed, since the
parasite cysts, as far as is known, do not cause symptoms to the intermediate host. In order to break
a cycle, an infected animal’s raw meat or organs should not be given to dogs or wild animals
(Laaaksonen and Paulsen, 2015)
265
The other detected parasitic agent in examined wild boars is cysts of a tapeworm, Echinococcus
granulosus. The definitive hosts of E. granulosus are canines, often wolves, whose intestines are
inhabited by adult parasites. The eggs produced by the parasite are spread to the environment
through the host animals feces, and further via food to the intermediate host. Inside the intermediate
host, the parasite forms cysts that contain a large number of infectious larvae. When the animal and
cysts are ingested by carnivores, the parasites life cycle is completed (Rojo-Vazquez et al., 2011).
Typical cysts (hydatid cyst) are commonly found in the liver of the wild boars. Their size varies,
depending on the duration of the infection. The cysts are often pearly grey colour and contain clear
liquid with thousands of small, infectious larval forms of the parasite. The number of cysts may
vary from a single one to over a hundred cysts. An animal suffering from severe infection and large
cysts in abdominal cavity or lungs is more easily killed by carnivores. This assists in the completion
of the parasites cycle. E.granulosus infection can be contracted by humans, who ten act as
intermediate hosts and the infection causes a so called hydatid disease (hydatidosis) (Laaaksonen
and Paulsen, 2015).
Mycotoxicoses in wild boars
In two examined enclosed hunting areas, the problem of diarrhoea, slow growth and mortality in
piglets was discovered. The clinical examination was performed from the safe distance at the
feeding place. The signs of weakness and growth retardation, staggering gait, trace of fecal content
on the hind body parts and on the ground were found. In the feed control a significant quantity of
moldy corn was discovered. Having in mind all the facts, the potential problem of mycotoxicosis
was suggested.
Different fungal species (Fusarium, Aspergillus, Penicillium genera) produce mycotoxins in food
and cause intoxication to those animals that eat the contaminated food. On entering the body, the
toxins possibly produced by fungi may cause a group of different symptoms that are often difficult
to recognize (Prodanov-Radulović et al., 2014b). Mycotoxins can get to wild animal food from
grass of fodder made from grass, from silage and grain feed (Laaaksonen and Paulsen, 2015).
Symptoms caused by mycotoxins in animals depend on the toxin and the ingested dosage. Clinical
symptoms vary from organ-destroying acute intoxications to chronic states of intoxication that may
include reduced appetite, diarhoea, weight loss and reproductive disturbances. Diagnosis is difficult
to make as the mycotoxin content is very small and several toxins can be found in the same sample
(Prodanov-Radulović et al., 2014b). Fungal intoxication has rarely been reported in game animals,
perhaps due to difficulty of reaching a diagnosis. In nature, the occurrence of mycotoxins cannot be
controlled. However, the entrance of mycotoxins into the nourishment of game animals can be
avoided with careful hygiene of game animal feeding grounds, using only feed that would be
acceptable for production animals, do not offer feed from the ground but from a feeding device that
prevents the feed from getting wet (Laaaksonen and Paulsen, 2015).
Wild boar diseases caused by viruses
In cooperation with the hunting societies and local veterinary service gathering of sera samples of
hunted wild boars was organized. Serological examination in year 2009 on the presence of specific
antibodies against CSFV (ELISA test) comprised only 259 blood samples obtained from wild boars
in the hunting area of Vojvodina region and it revealed negative result. In year 2010 the serological
control of CSFV antibodies in wild boars population included significantly larger number of
animals i.e. in total 471 tissue samples and 455 blood samples were examined. From examined
blood samples, in 36 positive results i.e. the presence of specific antibodies against CSFV was
detected. However, applying reverse transcription-polymerase chain reaction (RT-PCR) analysis the
presence of viral genome was not established in tissue samples deriving from shot wild boars. The
serological control of the CSFV presence in wild boars population in Vojvodina region was even
more intensified during years 2011-2012. In total 2038 samples were examined: 996 sera samples
266
and 1042 tissue samples (spleen, lymph node, and kidney). Once again, in 33 sera samples the
presence of CSFV specific antibodies was detected. Clinically, no abnormal mortality has been
reported in the analyzed districts of Vojvodina region (Prodanov-Radulović et al., 2013a; ProdanovRadulović et al., 2014a). By additional epidemiological evaluation, it was discovered that some of
the examined sera samples from certain hunting grounds that tested positive were a consequence of
previous vaccination against CSFV in the past, with modified live (China strain) vaccine. Although
prohibited from 1983 in Serbia, CSFV vaccination of wild boars may have been applied for a while
after vaccine ban (Prodanov et al., 2009b). Therefore, we cannot exclude the possibility that
vaccinated wild boars may have been sampled and detected as positive in the survey. This could
explain the presence of antibodies against CSFV in some of examined sera samples. In research
conducted in 2013, applying RT-PCR analysis the presence of CSFV genome was not established in
tissues samples deriving from shot wild boars in Vojvodina (Prodanov-Radulović et al., 2014a).
However, the results of the epizootiological questionnaire indicated that CSFV may be present in
hunting grounds in the region of Danube River, implying that the wild boars population represents
also a source of infection with CSFV (Prodanov et al., 2009b).
Classical swine fever (CSF) is a viral disease caused by a member of Flaviviridae family, genus
Pestivirus and of great economic concern for the pig farming industry (Artois et al., 2002; Rossi et
al., 2005). The disease in the wild boar population was diagnosed and/or serologically confirmed in
several Central and Eastern European countries (Artois et al., 2002; Montagnaro et al., 2010;
Prodanov et al., 2009b; Roic et al., 2012; Rossi et al., 2005; Vengust et al., 2006). In some
European countries, CSFV has been reintroduced periodically into domestic pigs via contact with
infected wild boars (Le Potier et al. 2006; Rossi et al., 2005). Moreover, epidemiological links
between CSFV infections in wild boars and domestic pigs have been repeatedly reported, mainly in
Germany (Ruiz-Fons et al., 2008). At present, CSF monitoring program in Serbia is primarily
focused on the serological investigations of blood samples and control of tissue samples by RTPCR from hunted wild boar. Sampling is performed randomly based on the density of the wild boar
population in different regions (Prodanov –Radulović et al., 2014a). The inveterate tradition to
keep domestic pigs at free range and the consequent contacts with the wild boars are considered the
major cause of outbreaks of CSF, which seems to facilitate disease persistence (Laddomada, 2000;
Rossi et al., 2005).
In one enclosed hunting ground, according to the history data and clinical signs detected in wild
boars (a small number of newborn piglets, stillborn and mummified piglets in the litters), the
problem of PPV infection was suggested. However, the clinical examination was feasible only in a
certain number of wild boars located in specially separated, fenced area. During the visit of hunting
area it was noticed that the fence was not entirely surrounding the area and there were parts without
fence. Having in mind migratory characteristics of population, this could facilitate contact with
domestic pigs located in forest habitat. Applying serological examination (HI test) of blood
samples, antibodies against PPV were widely distributed among the wild boar population in
Vojvodina province: 148 (49.33%) of the 300 examined samples tested PPV positive. The highest
prevalence of seropositive animals was associated with the hunting areas in Bačka and Srem
districts. We believe that this is connected with the tradition of keeping domestic pigs in woods
(especially in Srem district), thus increasing possible contact and transmission of diseases between
wild boars and domestic swine (Prodanov et al., 2009b; Prodanov-Radulović et al., 2014a). The
high prevalence of PPV antibody suggests this virus is endemic in our wild boar populations. In
Europe, PPV is highly prevalent in wild boars, with an incidence ranging from 14 to 57 % (RuizFons et al. 2008; Vengust et al., 2006), except Italy, where low prevalence of antibody to PPV was
reported (Montagnaro et al., 2010).
267
Conclusion
On the basis of the achieved results we can conclude that wild boars could be source of different
endoparasites species. The gross pathological examination discovered that in the largest number of
animals the health problems were mainly connected to the parasitic infestations and bacterial
infections of digestive and respiratory organs. Discovered parasitic infestations in the evaluated
wild boars are economically significant because of retardation in the growth and weigh gain. The
obtained serological results suggest that wild boars have direct or indirect contact with domestic
pigs, which facilitate transmission of pathogens. Avoiding close contact between wild boars and
domestic animals is of logical importance in disease control and eradication programmes. Having in
mind this fact, the special attention should be given to active surveillance of wild boars population
in the areas where close contact with domestic swine is possible. The measures should include the
serological monitoring of wild boars and free-roaming domestic swine, even the prohibition of
extensive grazing, pathological examination of the trunci deriving from shot wild boars. The study
underlines the importance of improving surveillance strategies for pathogens shared between
wildlife and domestic animals and the need to increase disease awareness of hunters, farmers and
veterinary practitioners. In the future, a better connection between veterinary service and experts
from hunting area is needed in order to solve to problems comprehensive way.
Acknowledgements
Education, Science and Technological Development, Republic of Serbia
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270
CONTAMINATION OF PUBLIC PLACES AT CENTRAL BELGRADE
MUNICIPALITIES WITH DOGS PARASITES IN PERIOD 2013-2014
Ivan Pavlović 1, Dubravka Jovičić2, Vladimir Terzin3, Dragana Petković4, Dragana Terzin3, Miloš
Pavlović5, Zoran Tambur6, Snežana Radivojević7, Borivoje Savić8, Slobodan Stanojević1
1. Scientific Veterinary Institute of Serbia, Belgrade, Serbia
2. Faculty Futura, University Singidunum, Belgrade, Serbia
3. Veterinary Ambulance Pet&Vet, Belgrade, Serbia
4. Veterinary Ambulance Petweellnes Eva, New Belgrade, Serbia
5. Faculty of Veterinary Medicine, University in Belgrade, Serbia
6. Military Medicine Academy, Belgrade, Serbia
7. City Institute of Public Health, Belgrade, Serbia
8. Clinical Center of Serbia, Belgrade, Serbia
* Corresponding author: dripavlovic58@gmail.com
Abstract
A steady increase in the number of dogs is a serious sanitary-epidemiological problem of urban
areas. Those animals permanent contaminated those areas with faeces which present a significant
health problem from human. From these reason at Belgrade we had parasitological research of that
places continuously since 1993. Here we presented results of our examination performed at central
Belgrade municipalities in period 2013-2014. Parasites contamination were detected at 45.91%
(45/98) soil samples and 72.5% (58/80) dog faeces. Toxocara canis were found at 31.63% soil
samples and 72.5% dog faeces, Dipyllidium caninum at 19.9% and 25.6%, Ancylostomidae spp. at
18,36% and 22,5%, Trichuris vulpis at 16.32% and 21.25%, Toxacaris leonina at 14.28% and
18.75%, and Taenia-type helminths at 5.1% and 6.6%, Giardia intestinalis at 16.32% and 22.5%,
Amoebae spp at 6.6% and 7.5%, Eimeria canis 7.14% and 12.5% and and Isospora canis 10.28%
and 15.0%.
Keywords: dogs, parasites, zoonoses, environmental contamination
Introduction
The presence of a large number of dogs, pets and stray, whose droppings dirty sidewalks and green
areas, is the current epidemiological problem in urban areas. In close cohabitation stray animals and
pets oriented on the same surface movement in the immediate environment of people and
contamination of public areas eggs zoonotic parasite species leads to constantly attend on the
possibilities of human infection (Mangaval and Pavlović, 2005; Pavlović, 2006; Lalošević and
Laošević, 2008; Elaine et al., 2011). Commenting on the results of parasitological examination of
green areas in in cities around the world, we will see that those polution in Madrid is 9%, London
15-17 % , Michigan 19 % , Kansas 20.6% , Utrecht 23% , Belgrade, Paris and Prague 28 %, Dublin,
32 %, Tokoshima 63 % and etc (Gothe and Reichler, 1990; Jansen et al., 1993; Cielecka et al.,
2005; Demirci et al., 2010; Tylkowska et al., 2010). If it is known that more than 5% of the
contaminated urban areas pose a serious threat to people's health is our opinion that this is not to
comment (Woodroff, 1976).
In order to monitor the contamination in the Belgrade area since 1993 continuously monitors the
contamination of public areas for the presence of parasites originating from dogs (Pavlović et al.,
1997, 2009; Pavlović and Surla, 2003). In our paper we presented results of our examination
performed at central Belgrade municipalities in period 2013-2014.
271
Samples of grass and soil with green areas in our climate condition were collected from March to
October. Material for examination was taken on the basis of indicators of bioclimatic conditions
prevailing in the same area leading to the method of bioclimatograme by Uvarov. Samples of dog’s
faeces are raised with the same location in the same intervals as samples of soil, grass and sand.
The examination is carried using native preparation, with sedimentation method, the flotation
method, and the sedimentation - flotation method (Euzeby, 1981; Pavlović and Stevanović, 2005;
Pavlović et al., 2007). Assessment of the infection will be performed by the methodology described
by McMaster and Stoll and determination of eggs and oocysts based on morphological
characteristics (Beugnet et al., 2008).
Results
Parasites contaminations were detected at 45.91% (45/98) soil samples and 72.5% (58/80) dog
faeces. Toxocara canis were found at 31.63% soil samples and 72.5% dog faeces, Dipyllidium
caninum at 19.9% and 25.6%, Ancylostomidae spp. at 18,36% and 22,5%, Trichuris vulpis at
16.32% and 21.25%, Toxacaris leonina at 14.28% and 18.75%, and Taenia -type helminths at 5.1%
and 6.6%, Giardia intestinalis at 16.32% and 22.5%, Amoebae spp at 6.6% and 7.5%, Eimeria
canis 7.14% and 12.5% and and Isospora canis 10.28% and 15.0%.
Discusion
Epidemiological study of parasites contamination in the area of Belgrade is the research carried out
continuously since 1993. Throughout this period, this study was performed for scientific purposes
and were unprofitable (which is sometimes caused misunderstanding in the institute), exception of
three occasions when the city government paid for this study (in 2002, 2011 and 2012).
On the basis of the control of parasitic contamination of land from parks and other green areas
between 1993 and 2002 (Pavlović et al., 1997, 2000), the presence of the locations parasite eggs
was found 65.90% of the examined samples. This was followed by the first reactive in terms of
cleaning the playgrounds so that in the period 2003-2007. The contamination has been established
at 45.90% of the area (Pavlović et al., 2007).
The following progress has been made by introducing during 2008-2009 in central city districts has
taken root system of baskets with plastic bags from dog feces (Dogi-pot system). During 2011 in
some parks are form of eco zones or parks for dogs (Pavlović and Terzin, 2012)
A special segment of solving this problem in Belgrade has been the adoption of problem-solving
strategies non-owner of dogs and cats in the city of Belgrade, which was adopted at the Belgrade
City Assembly held 21.9.2011. The city of Belgrade has become one of the few cities that have a
strategy to solve the problem of non-proprietary dogs and it is a document that defines the
principles, objectives and measures to solve the problem in terms of non-owner dogs carry out
administration of local government. In developing this strategy for the City of Belgrade and its
implementation was guided by the principle of humanity, combining the method of euthanasia
without (no kill strategy) and CNR method "catch - treat - let " ( CNR - catch - neuter -release),
with special emphasis on the protection of human and animal health. At the same time more shelters
for dogs has been built and a regular parasitological examination of parks and green areas in the city
was introduced (Pavlović and Terzin, 2012; Terzin et al., 2012).
On the basis of parasitic control of soil contamination from the parks during the 2012 presence of
parasite eggs was found in over 40% less than in the period 2008-2009.
272
At last two years (2013-2014), activities related to the parasitological control of green areas will
terminate and implementation of Strategies for addressing non-owner of dogs in the City of
Belgrade will partially apply. Those result with increase of number of non-owner dogs, dogs fecal
and parasites contamination of public places (parks, green areas and etc.) for more than 30%
compared to the previous period and high possibility to human infection. To solve that problem we
must to back to permanent parasitological control of public places.
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274
RISK FACTORS IN DOMESTIC AND WILD CYCLES OF TRICHINELLA SPECIES
Milena Zivojinović1*, Ivan Dobrosavljevic1, Ljiljana Sofronic Milosavljević2, Budimir
Plavsic3 Zoran Kulisic4, Sonja Radojicic4
1
2
Veterinary Specialistic Institute ‘‘Pozarevac’’, Dunavska 89, 12000 Pozarevac, Serbia
Institute for the Application of Nuclear Energy – INEP, University of Belgrade, Belgrade, Serbia
3
Veterinary Directorate, Ministry of Agriculture and Environmental protection, Serbia
4
Faculty of Veterinary medicine, University of Belgrade, Belgrade, Serbia
* Corresponding author: povetinst_milenaz@hotmail.com
Abstract
In the Balkan region of Europe, Trichinella spp. infections are endemic. These zoonotic parasites
are a serious problem for the human health and animal husbandry in Serbia. The aims of the present
study were to define risk factors in transmission of Trichinella spp. in domestic animals and
synanthropic and wild animals.
Through the surveillance and monitoring of Trichinella spp. in wild boars (Sus scrofa), red foxes
(Vulpes vulpes), golden jackals (Canis aureus), wolves (Canis lupus), stray dogs (Canis familiaris)
and domestic pigs (Sus scrofa domestica) we investigated presents and possible pathways for
transmission of Trichinella species. After examination of muscle samples from wild and domestic
animals for presents of Trichinella larvae, genotyping was performed by multiplex PCR. GIS
(Geografical Information System) was used for mapping the spatial distribution of Trichinella spp.
infected animals and all defined point of interest (pig farms, hunting section, slaughterhouses, and
dumps).
Trichinella spp. infection was detected in domestic and wild animals. Trichinella spiralis and
Trichinella britovi were the only two species identified in the isolates as single or mixed infections.
The identification of Trichinella spp. positive animals allowed to identify the foci of transmission
and to inform the veterinary services, the owners of pig farms and slaughterhouses and hunter’s
associations about the risk of transmission of these zoonotic agents.
The results point out the circulating of Trichinella species by a domestic or a sylvatic cycle, the
transmission between these two cycles and the role of some host species as reservoirs of T. spiralis
or T. britovi or of both species in Serbia.
Key words: Trichinella spp., zoonotic, domestic, wild, transmission
Introduction
In the Balkan region of Europe, Trichinella spp. infections are endemic. These zoonotic parasites
are a serious problem for the human health and animal husbandry in Serbia (Cuperlovic et al.,
2005). Human health problem is mainly the consequence of high prevalence of infection in
domestic animals, especially swine. According to the most important risk factors for trichinellosis
in the domestic and sylvatic cycles (Pozio, 2007), many are present in the endemic district of
Branicevo and Podunavlje, and include intentional feeding of food waste containing pork scraps,
scavenging on garbage dumps by pigs and dogs, and disposing of animal carcasses in the field
which allows sylvatic animals to scavenge on carcasses of infected domestic swine and increases
the probability of transmission to new hosts. The aims of the present study were to define risk
factors in transmission of Trichinella spp. in domestic pigs and synanthropic and wild animals
through surveillance and monitoring of Trichinella spp. among wild and domestic animals in this
region.
275
Through the surveillance and monitoring of Trichinella spp. we investigated presents and possible
pathways for transmission of Trichinella species in domestic and wild animals. During the period
2010, muscle samples were collected from 192.707 domestic pigs, 30 stray dogs and from 338 wild
animals including 174 wild boars (Sus scrofa), 118 red foxes (Vulpes vulpes), 42 golden jackals
(Canis aureus), and 4 wolves (Canis lupus). Muscle samples from wild animals provided by hunters
were analyzed by artificial digestion at the VSI of Pozarevac, according to Regulation (EC) No.
2075/2005 (European Community) and the Manual of Standards for diagnostic tests and vaccines
for terrestrial animals of the World Health Organization for Animal Health (OIE, 2008). Pig meat
samples were examined by trichinoscopy and/or artificial digestion at veterinary stations, veterinary
ambulances and slaughterhouses. When the sample size or types of muscle were inadequate, the pig
owner supplied a new sample. In the case of a positive finding, information was submitted to
veterinary inspectors. The worm burdens were expressed as the number of larvae per gram of
muscle tissue (LPG).
Trichinella larvae recovered after artificial digestion were washed three to five times in cold water,
counted in triplicate and fixed in absolute ethyl alcohol for molecular typing. Larvae were identified
at the species level by multiplex PCR at the International Trichinella Reference Center (ISS, Rome,
Italy) and at the INEP, according to the protocol accredited at the European Union Reference
Laboratory for Parasites (http://www.iss.it/crlp).
The epizootiological surveillance of swine trichinellosis was performed using data from the Animal
Notification System, veterinary services, laboratory results, geographical and epizootiological
surveillance data.
GIS (Geographical Information System) was used for mapping the spatial distribution of Trichinella
spp. infected animals and all defined point of interest (pig farms, hunting section, slaughterhouses,
and dumps).
Trichinellosis is the one of most important zoonosis in the regions Branicevo and Podunavlje. The
re-emergence of trichinellosis was officially recognized in 1999 and in 2003, government declared
parishes Pozarevac, V.Gradiste, Golubac, Kucevo, M. Crnice and Zabari as endemic Trichinella
districts and established measures for control (Zivojinovic et al., 2009).
Figure 1 – Trichinella prevalence in the region of Branicevo and Podunavlje in period 1995-2012.
276
Results from meat inspection at Branicevo and Podunavlje regions over an 18-year period (1995–
2012) indicated that the level of Trichinella prevalence in slaughter domestic swine was relatively
steady from 1995 to 1998 (Figure 1).
A significant increase (0.839%) was observed from 1999 to 2004 followed by a significant decrease
in 2005 (0.27%) (Zivojinovic et al., 2009). Thereafter, the incidence declined from 2006 to 2010
with low increase in 2011 (0.15%) (Fig. 1). The decrease in prevalence can be explained by the
continued application of the control program organized at the VSI which is based on: the
identification of all the pig farms; systematic rodent control and improvement of biosafety measures
at pig production facilities; antihelminthic treatment of pigs with mebendazole; and the traceability
of infected animals (Zivojinovic et al., 2010). The education of farmers, hunters and consumers was
also included in the control program.
During the period 2010, from 192.707 muscle samples Trichinella infections were detected in 136
(0.07%) pigs. At slaughterhouses from 116.966 samples, 13 samples (0.01%) were positive, while
from 75.741 households, Trichinella infection has been detected in 123 (0.16%) pigs. Swine that
have been examined at slaughterhouses are mainly from big farms with high level of biosecurity
measures which is in accordance with determined t value (t=10,118) and statistically significant
difference (p<0.01) in percent of positive findings at slaughterhouses and slaughtering at
households (Table 1).
Table 1 – Results of examinations domestic pigs in slaughterhouses and households in districts of Branicevo
and Podunavlje in 2010
Location of examination
Slaughterhouses
Households
In total
No of examinated pigs
116.966
75.741
192.707
No of Trichinella infected
13
123
136
%
0,01
0,16
0,07
We isolated 211 samples of domestic pigs which originated from households where Trichinella
infection was found in 2010 and 150 samples of pigs from big industrial farms. Trichinella infection
was detected in 25 (11.85%) samples, while 186 (88.15%) samples were negative. In fact, from the
total number of pigs from households, 40.98% were with Trichinella infection, while at industrial
farms that percent was 0%. The larvae isolated from 25 pigs were identified as T. spiralis. The
worm burdens were expressed as the number of larvae per gram of muscle tissue (LPG) and it was
from 0.03 to 20.
In the last 100 years, the increase in forests and fallow land, concomitant with a decrease in farms,
in Europe over the past 100 years has facilitated a great expansion of wild boar (Sus scrofa)
populations in some regions and increased transmission of Trichinella to animals and humans. The
common habit of hunters is leaving animal carcasses in the field after skinning or removing and
discarding the entrails, which increases the probability of transmission to new hosts (Pozio and
Murrell, 2006).
In total of 30 dogs from Branicevo and Podunavlje districts Trichinella infection were found in 7
(23.33%). All of them were stray dogs from municipality Pozarevac. T. spiralis was the only specie
found in dog population. The LPG was from 0.04 to 3.2.
During the same period, Trichinella infections were detected in 27 wild animals (7.99%), which
included 7 (4.02%) wild boars, 8 (6.78%) red foxes, 8 (19.05%) golden jackals and 4 wolves (Table
2).
T. spiralis was detected in 5 red foxes and 3 wild boars; T. britovi was detected only in population
of wild animals: 1 wild boar, 1 golden jackal, 1 red fox and 4 wolves; mix infection with T. spiralis
277
and T. britovi was detected in 2 red foxes and 1 golden jackal. T. spiralis and T. britovi are the two
main species circulating in Europe (Pozio et al., 2009) and in particular in the Romanian counties
bordering Serbia (Blaga et al., 2009).
Table 2. Results of examinations wild animals by artificial digestion in districts of Branicevo and Podunavlje
in 2010.
Animal species
No of examinated samples
No of Trichinella infected animals (%)
Wild boars (Sus scrofa)
Red foxes (Vulpes vulpes)
Golden jackals (Canis aureus)
Wolves (Canis lupus)
In total:
174
118
42
4
338
7 (5,93%)
8 (6,78%)
8 (19,05%)
4 (100%)
27 (7,99%)
The larval burdens related to T. spiralis ranged from 0.12 to 8.12, T. britovi from 0.11 to 19.45 and
mixed infections with both T. spiralis and T. britovi from 0.97 to 7.2. The lowest and the highest
worm burdens were detected in a wild boar and in a golden jackal, respectively.
According to Pozio and Murrell, 2006, the most important risk factors for domestic cycle are:
1) the intentional feeding of food waste containing pork scraps (Gamble et al., 2000) or intentional
or unintentional exposure to carcasses of dead swine or wildlife; these risks are usually
encountered in free-range pasturing
2) pigs allowed to scavenge on garbage dumps
3) feeding of wild game carcasses or scraps from hunting
4) horses fed with pork scraps or with carcasses of fur animals
5) sled dogs fed with carcasses of other dogs or of game in the arctic
6) the use of carcasses of slaughtered fur animals as food for other fur animals present at the farm
7) the use of meat of slaughtered crocodiles to feed other farmed crocodiles as observed in
Zimbabwe
8) the use of pork scraps to feed young crocodiles as demonstrated in Papua New Guinea.
In our work, epizootiological data were obtained by a questionnaire which included data on the type
of farm, number and species of all animals on farm, swine age category, type of production
(fattening or reproduction), breeding conditions, presence of stagnant water, water currents in the
backyard and near the backyard, contact with swine from other husbandries or with wild animals,
whether breeding of swine was the only occupation of the owner, feeding practices (whether swine
are fed with scraps or whether they have access to free ranging), former parasitological
examinations of slaughtered swine from the same farm, existence of Trichinella infected swine in
the yard, anti-helminthic treatment of swine. Introduced to the Animal Notification System (ANS)
and approached only by licensed users, data collected contributed to a better knowledge on endemic
foci and traceability. Since 2006, the spatial and temporal patterns of Trichinella infections in
domestic pigs have been recorded for Branicevo and Podunavlje area (Zivojinovic et al., 2010). We
identified risk factors such as the behavior to raise pigs near rivers (e.g., Danube, Morava, Mlava,
Pek, Jasenica, Kubursnica, Veliki Lug), small waterways and ponds where they can be in touch
with wildlife, which explained the finding of T. spiralis in domestic pigs as well as wild animals
hunted near human settlements. Also, the husbandry conditions on 90% of these backyard farms are
very poor due to the intentional feeding of food waste containing pork scraps, scavenging of pigs in
garbage dumps and the improper disposing of pig carcasses in the field. Furthermore for 10
settlements of this region, the Danube River forms a natural border between Serbia and Romania
where T. spiralis and T. britovi circulate among wild and domestic animals (Blaga et al., 2009). Our
results, as well as those from other authors (Cvetkovic et al., 2011), support the role of wolves as
278
the main reservoir of T. britovi in Serbia. In Serbia, it is mandatory to report Trichinella findings in
pigs to the veterinary services (Plavsic et al., 2009) because of their zoonotic potential.
Application of tools for epizootiological investigations in veterinary medicine, such as
Geographical Information Systems (GIS), offered a new approach and possibilities for the
eradication or control of infectious diseases. GIS is particularly useful for research conducted in
small areas strongly impacted by man. The first report of GIS application in Serbia was for
surveillance and monitoring of Trichinella spp. (Zivojinovic et al., 2010) at district Branicevo. We
used geographical data included location (latitude, longitude) of farm/ premises where Trichinella
infection has been detected, object relationship (proximity of piggeries to stagnant water, water
currents, garbage dumps), points of interest – proximity of main roads, hunting sections, forests
with wild animals, industrial farms and slaughterhouses (Figure 1, and 2) all collected by
application of GARMIN Map Source (Garmin, Taiwan) that contains maps, waypoints, routes and
track.
Figure 1 – Geographical location of big industrial swine farms (n = 5) in Branicevo and Podunavlje region
Figure 2 – Geographical location of hunting sections (26) in Branicevo and Podunavlje region
279
Spatial and temporal patterns of Trichinella infection were made for the Branicevo and Podunavlje,
all settlements were recognized for the presence of Trichinella infection. According to geographical
distribution at Branicevo district, we identified presence of T.spiralis and T.britovi, but at
Podunavlje district only T. spiralis was present. From 11 investigated municipalities, at territory of
nine (Pozarevac, Kucevo, Veliko Gradiste, Malo Crnice, Golubac, Zabari, Smederevo, Velika Plana
and Smederevska Palanka) which present 81.82% of investigated territory we confirmed presents of
Trichinella species (Figure 3).
Figure 3 – Geographical location of Trichinella infected domestic pigs in Branicevo and Podunavlje region
Figure 4 – Geographical location of Trichinella species in Branicevo and Podunavlje region (
T. britovi,
Mix infection with T. spiralis and T.britovi)
280
T.spiralis,
Trichinella infection hasn’t been confirmed in two municipalities Petrovac and Zagubica. Five big
industrial farms were recognized as Trichinella free (Figure 1). We also marked all 26 hunting
sections at this territory in aim to investigate Trichinella infection in wild animals, necessary for
monitoring parasite flow between the domestic and sylvatic cycles (Figure 2). In population of wild
boars, red foxes, golden jackals and wolves Trichinella infection has been confirmed in seven
municipalities (Pozarevac, Veliko Gradiste, Kucevo, Malo Crnice, Golubac, Petrovac and
Zagubica). Trichinella infection only in population of wild animals at territory of those two
municipalities Petrovac and Zagubica indicated high risk for domestic pigs, especially for free range
pigs which are common in this area.
Trichinella spiralis and Trichinella britovi were the only two species identified in the isolates as
single or mixed infections (Figure 4).
The identification of Trichinella spp. positive animals allowed to identify the foci of transmission
and to inform the veterinary services, the owners of pig farms and slaughterhouses and hunter’s
associations about the risk of transmission of these zoonotic agents.
Conclusion
According to our results, we concluded that risk factors for circulating Trichinella species between
domestic and wild species are numerously at Branicevo and Podunavlje district. The finding of T.
spiralis in domestic pigs as well as wild animals hunted near human settlements can be explained by
the behaviour to raise pigs near rivers (e.g., Danube, Morava, Mlava, Pek, Jasenica, Kubursnica,
Veliki Lug) small waterways and ponds where they can be in touch with wildlife (Zivojinovic et al.,
2013). Pigs and dogs allowed scavenging on garbage dumps (Zivojinovic et al., 2010). The
husbandry conditions on 90% of backyard farms with Trichinella infection are very poor. There is
intentional feeding of food waste containing pork scraps, scavenging of pigs in garbage dumps and
the improper disposing of pig carcasses in the field (Zivojinovic et al., 2013). Possibility of sylvatic
animals fed with carcasses of infected swine is present. There is a influence of human action such as
the habit of living animal caracasses in the field, which increase the probability of transmission to
new hosts. For 10 out of 256 settlements of this region, the Danube River forms a natural border
between Serbia and Romania where T. spiralis and T. britovi circulate among wild and domestic
animals (Blaga et al., 2009).
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S.: Veterinary information management system (VIMS) in the process of notification and management
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G.: Hosts and habitats of Trichinella spiralis and 196 Trichinella britovi in Europe. Int. J. Parasitol. 39,
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282
INSIGHT INTO THE PRESENT PESTICIDE CONTAMINATION AND COPEPODS
STATUS (Crustacea: Copepoda) OF SURFACE WATER IN IRRIGATION CANALS IN
VOJVODINA
Vojislava Bursić1*, Gorica Vuković2, Aleksandra Petrović1, Maja Meseldžija1, Tijana Zeremski3,
Aleksandar Jurišić1, Dragana Rajković1
1
Department of Environmental and Plant Protection, Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia
2
Institute of Public Health of Belgrade, Serbia
3
Institute of Field and Vegetable Crops, Novi Sad, Serbia
* Corresponding author: bursicv@polj.uns.ac.rs
Abstract
The concern about environmental and health issue related to pesticides makes it important to
understand the possible hazards associated with their use and how to minimize risks. The use of
agrichemicals has been steadily increasing in the country during the last three decades and has
continued to grow in recent years. The fate of pesticides in the environment depends, above all, on
their persistence, but also on the characteristics of soil and water. In order to avoid harmful effects
on health and the environment due to the exposure to pesticides and to obtain more data which
would be the basis for the list-legislation setting the maximum residue levels of pesticides in surface
waters, the work on it needs to be ongoing and thorough. In order to gain the insight into the present
pesticide contamination of surface water, two main irrigation canals in Vojvodina (Serbia) Čelarevo
and Vrbas were examined for the pesticide residue content and presence of copepods as
bioindicators by sampling water monthly from December 2013 to November 2014. The aim of this
work was to determine the trace levels of twenty-one pesticides and the products of their
transformation in irrigation canals, from triazine and urea herbicides and their influence on
copepods species diversity and abundance in canal water. A simple multiresidue method was used
for the determination of herbicides in surface water using LC-MS/MS with ESI. The water analyses
pointed at the detection of the pesticides whose detected values are above the maximum available
concentrations – MACs in the period from May to October. Species identification and zooplankton
abundance calculation were conducted by the filtration of the water samples and examination of the
residues using a stereo camera BTC TCA-3.0C with the maximum magnification of 160x. The
water samples obtained from the locality Čelarevo had high abundance of copepods from two
genera (Diaptomus and Cyclops) in summer. However, in the water samples from the locality Vrbas
no copepod species were found due to the pesticide residues presence.
Keywords: pesticide residues, copepods, fresh water, canals
Introduction
Among all anthropogenic chemicals, the pesticides may cause the most serious problems in the
environment as they are chemicals specifically designed to kill organisms (both the pest target and
other non-target organisms) and they are released into the natural environment intentionally
(Hanazato, 2001). The pesticides used in agriculture, forestry and water management demonstrate
multiple effects on molecules, cells, tissues, organs, individuals, populations and communities.
The presence of zooplankton is crucial in aquatic ecosystems because of their leading role in
maintenance of freshwater food chains as a necessary link between the primary producers, primary
and secondary consumers and decomposers. They provide food for the predator species, especially
283
amphibians and fish, and due to their fast and strong metabolic activity they also recycle certain
nutrients, and therefore feed on living material or detritus dissolving it. In aquaculture, copepods
have a multiple role: food for small fish, micropredators of fish and other organisms, fish parasites,
intermediate hosts of fish parasites and as hosts and vectors of human and animal diseases (Piasecki
et al., 2004).
According to Hanazato (2001), zooplanktons are one of the most sensitive animal groups to the
toxic effects of the chemicals. However, Braginskiiet al. (1979) have concluded that depending on
concentration, the pesticides may either suppress or stimulate the plankton organisms, although,
regarding the zooplankton, pesticides almost always lead to their elimination in freshwater
biocenoses. The abundance, species diversity and seasonal population dynamics of zooplankton
represent very sensitive and accurate bioindicators of freshwater ecosystems conditions, such as
water quality, eutrophication, pollution levels and presence of contaminants. Zooplanktons as well
as other aquatic organisms prefer habitats which have stabile and constant physical, chemical and
biological features. Variations in one or more of these abiotic and biotic factors could lead to stress,
migrations or even death of zooplankton individuals. Balakrishna et al. (2013) emphasize that
zooplankton has been widely used in assessment of aquatic pollution, because of their sensitivity to
small changes in the environment, short generation time and possible parthenogenesis. According to
Day (1990), zooplankton are known to accumulate persistent lipophilic chemicals, particularly the
organochlorine pesticides to concentrations greater than those found in their environment, and
therefore contribute the pesticide residues increase and maintenance in the higher trophic levels.
A lot of studies worldwide have confirmed the negative effect of pesticide residues on the
zooplankton in freshwater bodies worldwide (DeLorenzo et al., 2002; Mukherjee et al., 2010;
Stampfli et al., 2011; Dhembare, 2011), as well as on the freshwater copepods (Day, 1990;
Hanazato, 2001; Piasecki et al., 2004; Yiğit, 2006).
The intensive use of pesticides during recent decades has led to the accumulation of their residues in
the environment which especially endangers water quality in the canals in Serbia. Since the water of
rivers and canals are used for drinking and irrigation purposes not only in Serbia but in the whole
world as well, it has become imperative to study the extent and magnitude of pesticides in these
water bodies (Bursić et al., 2013). Data on pesticide residue effects on zooplankton in smaller water
bodies in Serbia are rare, so this study was aimed in obtaining preliminary insight into the present
pesticide contamination of two irrigation canals in Vojvodina and its effect on copepod species and
populations.
In order to gain the insight into the present pesticide contamination of surface water and copepod
status, two main irrigation canals in Vojvodina, Čelarevo and Vrbas were examined by sampling
water monthly from December 2012 to October 2013, away from the zone of the direct influence of
waste water and tributaries influx. The water samples for analytical analysis (500 ml) were taken
according to the guidelines for taking samples of surface water from rivers and streams SRPS ISO
567-6. This water samples were stored in amber bottles in the dark, at 4 °C, during transport to the
laboratory until further analyses. The trace levels of twenty-one pesticides and the products of their
transformation in irrigation canals, from triazine and urea herbicides were determined. The water
samples (1000 ml) for qualitative and quantitative analysis of copepod status were taken using
Standard Ruttner Water Samplers (1000 ml volume). In order to increase positive sampling results
plankton nets were used for qualitative determination of species diversity. All samples were stored
in plastic bottles, properly labelled and transported to the laboratory for the further analysis.
284
Analytical method
The sample preparation was performed by OASIS HLB cartridges (200 mg). They were conditioned
with 2 ml of methanol and 2 ml of HPLC - grade water. After conditioning step, 250 ml volume of
water was enriched on OASIS HLB cartridge with the flow rate settled between 3 and 10 ml/min.
The cartridge was flushed with 10 ml of HPLC - grade water. Pesticides were eluted from the
sorbent with 5 ml dichloromethane and collected in the 12 ml amber glass vial. The solvent was
evaporated in the stream of nitrogen in the Techne - Dry block and the residue was dissolved in the
0.25 ml of initial mobile phase. An amount of 10 µl was injected into LC-MS-MS system.
Stock standard solutions for each of the analyses were prepared in acetonitrile at 100 µg/ml and
stored in the dark at 4 °C. Standard solutions of the mixtures of all compounds at concentrations
ranging between 10 ng/ml and 200 ng/ml were prepared by appropriate dilution of the stock
solutions in acetonitrile. High purity standards (mixture of pesticides) were purchased from LGC
Standards.
The analytical conditions for the determination of pesticides are shown in Table 1. In LC-MS-MS,
data acquisition was performed in multiple reaction monitoring (MRM) modes.
Table 1. LC-MS/MS l conditions
HPLC
Column
Column temperature
Mobile phase
Flow rate
Injection
MS
Ionisation
Nebulizer gas
Dry gas
Vaporiser
Charging Voltage
Capillary
Agilent 1200
XBridge C18, 150 x 3.0 mm, 3.5 µm, Waters
40 °C
A = 0.1 % HCOOH in methanol
B = 0.1 % HCOOH in water
A: B = 70 : 30
0.5 ml/min
10 μl
Agilent 6410
ESI
50 psi
5 L/min at 350 °C
250 °C
2000 V
3500 V
Copepode sampling
The sampling of copepod species was conducted by the filtration of the water samples (1000 ml)
and examination of the residues, using a stereo camera BTC TCA-3.0C with the maximum
magnification of 160x. The copepod species were identified up to genera level according to Dussart
(1969) identification keys.
Results
Analytical method
MRM conditions for QQQ and coefficients of correlation with validation parameters are presented
in Table 2.The obtained limits of quantification - LOQs for all twenty-one investigated pesticides
were 0.020 µg/l (Fig. 1).
285
In the samples obtained from the canal Vrbas twelve pesticides were detected during the whole
research period. The highest concentrations have had terbuthylazine-desethyl in May and prometryn
in July 2013 (Graph. 1.).
Fig. 1. TIC MRM chromatogram from NE7500 0.1 μg/mL
Table 2. MRM conditions for QQQ and coefficients of correlation with validation parameters
Pesticide
MRM transition
(m/z)
MW
Atrazine
215
Carbetamide
237
Chloridazon
221
Chlorotoluron
212
Cyanazine
240
Desethylatrazine
187
Desisopropyl-atrazine
173
Dimefuron
338
Diuron
232
Ethidimuron
464
Isoproturon
206
Linuron
248
Metabromuron
258
Metamitron
202
Metazachlor
277
Methabenzthiazuron
221
Metolachlor
283
Propazine
229
Simazine
201
Terbuthylazine
229
Terbuthylazine-desethyl
201
216→174
216→ 96
238 → 72
238 →192
222 →104
222 → 92
213 → 46
213 → 72
241 →214
241 →104
188 →146
188 →104
174 →104
174 → 68
339 →256
339 →140
233 → 72
233 →160
265 →208
265 →162
207 → 72
207 →165
249 →160
249 →182
259 →170
259 →148
203 →104
203 →175
278 →134
278 →210
222 →165
222 →150
284 →252
284 →176
230 →146
230 →188
202 →132
202 → 96
230 →174
230 →104
202 →146
202 →110
286
tR (min)
R2
Recovery±RSD
(%)
16.724
1.000
82.93±5.77
10.464
0.9998
91.10±5.17
4.959
0.9998
91.20±5.10
16.209
0.9998
85.83±4.90
10.912
0.9998
82.30±6.17
6.165
0.9997
88.63±5.93
3.036
0.9999
76.43±6.73
22.103
0.9981
76.27±6.97
18.474
0.9999
76.57±3.97
3.628
0.9987
107.77±6.93
18.056
1.000
68.87±8.93
22.163
0.9998
74.37±6.57
16.469
0.9997
73.33±6.00
4.401
0.9998
72.00±4.63
18.067
1.000
81.63±5.50
16.830
1.000
83.83±4.97
24.409
0.9996
74.47±5.80
22.054
0.9993
73.53±6.77
11.658
0.9999
83.93±5.23
22.499
0.9996
70.70±7.60
13.948
0.9997
85.63±8.62
The water samples from the canal Čelarevo have had eight detected pesticides, only from May till
October 2013, when the concentration of prometryn was the highest (10.57μg/l). High
concentrations were obtained also for terbuthylazine-desethyl in May (Graph. 2.)
Graph. 1. Detected pesticides in the samples from canal Vrbas (detected concentration – μg/l)
Graph. 2. Detected pesticides in the samples from canal Čelarevo (detected
concentration – μg/L)
Copepode samplings
During one year research period, no copepod species were found in the water samples obtained
from the canal Vrbas. Only two genera, Cyclops and Diaptomus were detected in the canal
Čelarevo, with very low population densities (Tab. 3.).The highest abundance for Cyclops sp. was
detected during September and for Diaptomus sp. during August 2013.
287
Čelarevo
Vrbas
Tab. 3. The copepods population densities (ind/l)
dec. 12
jan. 13
feb. 13
mar. 13
apr. 13
maj. 13
jun. 13
jul. 13
avg. 13
sep. 13
okt. 13
nov. 13
Cyclops sp.
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
Diaptomus sp.
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
Cyclops sp.
1.33333
0.66667
1.33333
2.00000
3.33333
3.33333
6.66667 14.00000 16.66667 17.33333 12.00000 10.00000
Diaptomus sp.
0.00000
0.66667
0.66667
1.33333
2.66667
4.00000
5.33333
9.33333 13.33333 12.66667
9.33333
6.66667
Based on the results of the pesticide residues analyses in the canal water and in accordance with
Directive 2008/105/EC, which by Annex X defines the list of priority substances in water
management policy comprising 33 pollutants out of which 14 are pesticides. Our research work
pointed at the detection of the pesticides which are not on that list but whose detected values are
above the maximum available concentrations – MACs.
Twelve herbicides were registered in the water samples from the canal Vrbas and eight from the
canal Čelarevo. Although water samples obtained from the canal Čelarevo have had lower number
of detected pesticides, their concentrations were higher (over 10 μg/L) comparing to results
acquired from the canal Vrbas.
The small values for population densities of copepod species in spring could be explained by the
fact that neonates are most sensitive to pesticides (Hanazato, 2001). Therefore, populations
composed of a large proportion of neonates are more sensitive to pesticides, which are frequently
happened in a growing phase, when the production of the neonates is intensive. Low population
densities and species diversity of copepods could be also conditioned by their annual fluctuations.
Normally, zooplankton abundance is higher in spring and autumn and lower in summer and winter,
as their abundance is limited by nutrient availability (Yigit, 2006).
The pesticides that have had the highest concentration at both localities were terbuthylazinedesethyl and prometryn. According to Kegley et al. (2014) there are no valid data for aquatic
ecotoxicity ground water contamination of terbuthylazine-desethyl, although terbuthylazine itself is
highly toxic for zooplankton and moderately toxic for fish. The same authors emphasize that this
pesticide demonstrates multiple effects on zooplankton, such as accumulation, intoxication and
mortality. Prometryn is slightly too moderately toxic for zooplankton, causing intoxication and
mortality and potential ground water contaminant (Kegley et al., 2014).
However, all pesticides have had low concentrations at both localities, so apparently they are not
the main reason for none (Vrbas) or small copepod population densities (Čelarevo). According to
Day (1990), organophosphate insecticides, the synthetic pyrethroids, many herbicides and their
residues may not be detectable in the organisms (not bioaccumulated to any extent) and have no
observable biological effects, but the organism may experience a toxic effect. Furthermore, such
chemicals present a problem in the assessment of environmental damage from pesticide
contamination and techniques to detect the effects of very low concentrations of these nonlipophilic pesticides in natural waters on biota need further development (Day, 1990). Braginskii et
al. (1979) state that low concentrations of pesticides could have a stimulating effect on the
functional activity of bacterial plankton and changes coupled with this in biogeochemical cycles of
nitrogen and phosphorus, as well as the elimination of the water fleas from the zooplankton
promotes the development of the “secondary” eutrophication and increase the biomass of the
phytoplankton.
288
The main environmental hazard resulting from accumulated residues of pesticides in zooplankton is
their transfer to higher trophic levels (Day, 1990). In conclusion, pesticides along with the abiotic
and biotic factors play a crucial role in copepods species diversity and population dynamics.
Consequently, analysis of pesticides and their residues in the water, as well as their interaction with
zooplankton are important issues in ecotoxicologial and environmental studies.
Acknowledgement
The authors acknowledge the financial support of the Ministry of Education and Science, Republic
of Serbia, Project Ref. III43005 and TR31072.
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289
A SEROLOGICAL SURVEY ON AUJESZKY'S DISEASE IN WILD BOARS IN THE
REGION OF VOJVODINA IN SERBIA
Sava Lazić1*, Vesna Milićević2, Gospava Lazić1, Ljubiša Veljović2, Diana Lupulović1, Jasna
Prodanov-Radulović1, Jelena Maksimović-Zorić2, Siniša Grubač1, Radoslav Došen1,
Tamaš Petrović1
1
2
* Corresponding author: lazic@niv.ns.ac.rs
Abstract
The article presents the results on the presence of specific antibodies against Aujeszky's disease
(AD) virus in blood samples of wild boars hunted in the territory of the Autonomous Province of
Vojvodina in R. Serbia during hunting season 2013-2014. Blood samples were collected during the
evisceration of internal organs in hunted wild boars, from the abdominal vein or heart chamber.
Examination for the presence of specific antibodies against AD was carried out in two virology
laboratories: Scientific Veterinary Institute of Serbia (Belgrade) and Scientific Veterinary Institute
"Novi Sad" (Novi Sad), using commercial ELISA kits for the detection of antibodies against AD.
The study included 434 blood samples of wild boars, originating from 6 locations, that is, epizootic
units in Vojvodina. According to the evaluation of the hunting organizations for the aforementioned
hunting season, the total population of wild boars in the 6 epizootic units included 7,065 animals.
Thus, this examination encompassed 6.14% of the total number of wild boars in the area of
Vojvodina Province.
Antibodies against AD were detected in 179 samples, making 41.24% of the total number of blood
samples. The greatest number of seropositive wild boars was established in epizootic unit of Srem,
where specific IgG anti-AD antibodies were detected in 68 (51.52%) of 132 tested samples. Also,
high percentage of seropositive samples was determined in the epizootic unit of West Bačka. In
total, 118 samples were analyzed, and 49.15% thereof reacted positive. The lowest rate of
seropositive samples was detected in the unit of Severna Bačka. In this epizootic unit, 24 blood
samples were tested, and antibodies against AD were found in 6 samples, which are still considered
high seropositivity percentage with 25% of positive animals.
The results of this examination indicated that Aujeszky's disease virus infection is widespread in the
population of wild boars in the territory of Vojvodina.
Keywords: Wild boar, Seroprevalence, Aujeszky’s disease, ELISA
Introduction
Wild boar (Sus scrofa) populations are found in many regions worldwide. The role of wild boar as a
reservoir of some viruses, and thus a possible source of infection for domestic swine and other
animals is still unclear (Müller et al., 2011; Ruiz-Fons et al., 2008). Aujeszky’s disease (AD,
pseudorabies) is a notifiable disease caused by Suid herpesvirus 1 (SuHV), syn. Aujeszky’s disease
virus (ADV), which belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus
Varicellovirus (Mettenleiter, 2000). The Aujeszky’s disease virus (ADV) is an important pathogen
of pigs and infects almost all mammalian species except man (Keros et al., 2014; Pannwitz et al.,
2012). However, pigs are the only animal species that can survive the infection with ADV, which
accounts for its ability to be subclinically (latently) infected (Martinez-López et al., 2009) while
infected individuals of all other animal species succumb to the disease without shedding the virus
(Komáromi and Szabó, 2005). Because of the substantial economic losses AD causes to the pig
industry, it represents one of the most dangerous diseases in domestic pigs (Pannwitz et al., 2012).
290
In Europe, ADV has been eliminated from domestic pig populations in many countries (Müller et
al., 2011). However, despite the tremendous progress made to control and eliminate the disease in
domestic pigs, ADV infections appear to be widespread in populations of non-domestic swine,
including feral pigs, wild boar and hybrids, across the world (Pannwitz et al., 2012). First evidence
for the occurrence of ADV in wild boars was reported from the USA, Italy, and the Netherlands in
the mid-1980s (Müller et al., 2000). In recent years, ADV infections in wild boar populations have
also been reported from many European countries, including the Czech Republic, France, Slovenia,
Croatia, Poland, Russia, Switzerland and Spain (Albina et al., 2000; Sedlak et al., 2008; Župancic et
al., 2002; Vengust et al., 2006). Direct impact of AD in wild boar population dynamics is
considered to be low, but AD outbreaks with associated wild boar mortality have been reported and
restrictions to wild boar movements may also have an impact on wild boar production for hunting
(Ruiz-Fons et al., 2008).
There are no recently published data concerning detection the ADV in wild boars population in
Vojvodina province. However, Müller et al. (2011) have reported an ADV infection in border
regions between Hungary and Serbia, and Keros et al. (2014) suggested the possibility of virus
spread, especially among wild boars, between the borders of Croatia and Serbia. It should be
stressed that domestic pig population in Vojvodina is enzootically infected with ADV (Pušić et al.,
2011). One of the characteristics of outdoor swine production in some regions in Vojvodina
Province is raising free-roaming domestic pigs, where they share forest habitat with wild boars
(extensive grazing) (Prodanov-Radulović et al., 2011). In such conditions, the contacts between
wild boars and domestic pigs kept in woods (free range) may occur occasionally (Albina et al.,
2000). Since wild boars and domestic pigs have the same susceptibility to various infections,
including Morbus Aujeszky virus, there is a major concern to monitor the epidemiological situation
of wild boars especially when control measures in domestic pigs are implemented. The aim of the
current study was to investigate the presence of ADV antibodies in hunted wild boars of Vojvodina
region.
Blood samples and sampling procedure
Blood sampling was performed within the framework of monitoring of classical swine fever in wild
boar population pursuant to the “Instruction for monitoring of classical swine fever in wild boars in
2013 and 2014” (Veterinary directorate, No. 323-02-8407/2013-05, dated 28/10/2013). During
evisceration of internal organs of hunted wild boars, determination of animal’s age and blood
sampling was performed. The age was determined according to the number of molar teeth in the
lower jaw. All hunted animals were distributed into 4 age categories: 0-6 months, 6-18 months, 1.52.5 years and older than 2.5 years. Blood sampling was performed by puncture of abdominal vein
and/or heart chamber using disposable sterile plastic syringes and injection needles. The samples
were transferred into sterile 5 mL tubes and transported to the laboratories. Each sample was
accompanied by a Form containing relevant data on the hunting ground, shooting date,
identification number of hunted boar, material sample and animal’s age. Separation of blood serum
was performed by the method of spontaneous coagulation and/or centrifugation. Separated blood
serum was poured into the 2mL „eppendorf“ cuvettes and frozen at -20°C until testing. In this
manner, 434 wild boar blood serum samples were collected for the purpose of this research.
Localities for blood sampling
The territory of Vojvodina is divided into 6 epizootiological units that correspond with the
administrative regions, except for Banat epizootiological unit composed of two administrative
regions (North Banat and Middle Banat). The number of collected samples according to
291
epizootiological units was as following: Srem -132; West Bačka – 118; South Bačka – 117; North
Bačka – 24; Banat - 22 and South Banat – 21.
Blood sample examination
Detection of antibodies against Aujeszky’s disease virus was performed in the laboratories of the
Institute of Veterinary Medicine of Serbia, Belgrade and Scientific Veterinary Institute “Novi Sad”
in Novi Sad using commercial ELISA kits Ingezim ADV TOTAL and IDEXX PRV/ADV gB Ab
test.
Results
The obtained results were displayed in Tables. Data on the presence of ADV-specific antibodies in
the examined samples in relation to the number of examine samples and number of wild boars in
epizootiological units in Vojvodina, are presented in Table 1.
Table 1. Number of wild boars, number of examined blood sera and findings according to epizootiological
units in Vojvodina
No.
Epizootiological
unit
Srem
1
West Bačka
2
South Bačka
3
North Bačka
4
Banat
5
South Banat
6
Total
No. of wild
boars
No. of examined
blood sera
2,080
1,445
1,670
130
540
1,200
7,065
132
118
117
24
22
21
434
% of examined
samples compared
with the total number
of wild boars
6.35
8.17
7.00
18.46
4.07
1.75
6.14%
Finding: Number (%)
Positive
Negative
68 (51.52)
58 (49.15)
34 (29.06)
6 (25.00)
6 (27.27)
7 (33.33)
179(41.24)
64 (48.48)
60 (50.85)
83 (70.94)
18 (75.00)
16 (72.73)
14 (66.66)
288(58.76)
According to the data in Table 1, the greatest number of examined wild boar blood samples
originated from the epizootiological unit of Srem, which is characterized by the largest wild boar
population in the territory of Vojvodina. The number of samples examined in this epizootiological
unit was 132, and ADV-specific antibodies were detected in 68 samples indicating a seroprevalence
rate of 51.52%. High ADV seroprevalence of 49.15% was established in wild boars from West
Bačka epizootiological unit. However, in other epizootiological units, the ADV seroprevalence
ranged between 25% and 33%, indicating that more than a quarter of wild boar population in
Vojvodina is infected with Aujeszky’s disease.
The finding of antibodies against Aujeszky’s disease was presented according to the age of
examined wild boars. The data on the total number of examined blood samples and antibody finding
according to epizootiological units and animals’ age are displayed in Table 2.
Table 2: The finding of antibodies against Aujeszky’s disease according to the age of examined wild boars
from epizootiological units of Vojvodina
Epizootiological
unit
Srem
West Bačka
South Bačka
North Bačka
Banat
South Banat
Total
0-6 months
Exam Pos. (%)
14
6 (42.9)
4
0
8
2 (25)
4
0
3
1 (33)
0
33
9 (27.3)
Neg.
8
4
6
4
2
24
6-18 months
Exam. Pos.(%)
40
16 (40)
40
20 (50)
53
11(20.8)
14
2 (14.3)
9
1 (11)
10
2 (20)
166
52(31.3)
Neg.
24
20
42
12
8
8
114
292
1.5-2.5 years
Exam Pos.(%)
32
17 (53.1)
26
10 (38.5)
22
8 (36.4)
1
1
3
0
7
3 (42.6)
91
39(42.9)
Neg.
15
16
14
0
3
4
52
> 2.5 years
Exam Pos.(%)
46
29 (63)
48
28(58.3)
34
13(38.2)
5
3 (60)
7
4 (57)
4
2 (50)
144 79(54.9)
Neg.
17
20
21
2
3
2
65
As obvious from Table 2, antibodies against Aujeszky’s disease were detected in all age categories
of wild boars in the epizootiological units of Srem and South Bačka. In Srem epizootiological unit,
the highest percentage of seropositive wild boars, 63%, was recorded in the age category above 2.5
years. However, the percentage of seropositive animals of all age categories was quite high in this
region, ranging from 40% to 53%. In South Bačka, the highest prevalence of seropositive animals
was established in the age category > 2.5 years, being 38.2%, whereas seropositivity rate among
other age categories ranged between 20% and 36%. In the unit of West Bačka, high percentage of
seropositive animals was detected in age categories > 2.5 years and 6-18 months, being 58.3% and
50%, respectively. As regards the age category, the majority of examined wild boars were from the
age category 6-18 months (166) and > 2.5 years (144), whereas only 33 examined animals were
piglets from age category below 6 months. The percentage of seropositive wild boars gradually
increases with increase of animals’ age. The lowest and highest seroprevalence rates were
established in piglets below 6 months and boars > 2.5 years old being 27.3% and 54.9%,
respectively.
Discussion
The presented results indicate the presence of ADV infection in the population of wild boars in the
territory of Vojvodina, Republic of Serbia. The occurrence of the disease in wild boars was
confirmed and reported by several authors from neighbouring countries such as Croatia and
Hungary (Keros et al. 2014; Župancic et al., 2002; Komáromi et al., 2005) as well as from
numerous countries in Europe and America (Müller et al., 2011). The infection has commonly been
confirmed by serological examination; however, virus identification in hunted wild boars using
molecular methods has been increasingly reported during the past few years. The seroprevalence of
Aujeszky’s disease in hunted wild boars in several countries and regions in Europe is variable, yet
generally high. Thus, the following seroprevalence rates were reported in some countries: 51% in
central Italy (Lari et al., 2006); 54.54% in Maslovačka Gora in Croatia (Župančić et al., 2002); 26%
in Slovenia (Vengust et al., 2006), and 30% in Slovakia (Sedlak et al., 2008). The analysis of the
results on seroprevalence of Aujeszky’s disease in Germany, which was monitored throughout 24
years in 66 regions, ranges between 0.4 and 15.9%. However, in some regions, the seroprevalence
rate reached even more than 30% during 2006 and 2007 (Pannwitz et al., 2012). The results on
seroprevalence of Aujeszky’s disease among the wild boar population in Vojvodina region,
Republic of Serbia, presented in this article are similar to the results reported in the majority of
European countries.
Analysis of presented data on seroprevalence of Aujeszky’s disease according to age category of
examined wild boars indicates close correlation of our results with those reported in the literature.
The results of several authors suggested the highest seroprevalence rates in oldest age categories of
wild boars (Müller et al., 2012; Lari et al., 2006; Vengust et al., 2006). According to our results, the
seroprevalence of Aujeszky’s disease in hunted wild boars older than 2.5 was 54.9%, whereas
lowest rates were recorded in piglets below 6 months, being 27.3%
Further research of Aujeszky’s disease in wild boars is necessary, especially in the aspect of the
spread and persistence of the virus among the wild boar population. Wild boars are potential
infection reservoir for domestic pigs but also for other animals such as hunt dogs, wild carnivores,
etc. Molecular characterization of isolates of ADV in wild and domestic pigs as well as of virus
isolates in other animal species could contribute to better understanding and elucidation of
numerous epidemiological aspects of persistence and spread of Aujeszky’s disease in the
environment.
293
Acknowledgments
The research was carried out in the framework of the Project No TR 31084, which was financially
supported by the Ministry of Education, Science and Technological Development of the Republic of
Serbia.
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M., dedek J., Hoffmann L., Kramer M., Selhorst T., Conraths F.J., Mettenleiter T. and Müller T.: A
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characteristics of Aujeszky's disease in Vojvodina region and biosecurity concerns. Biotechnol. Anim.
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population dynamics and reservoirs role. Vetrinary Journal, 176, 158–169, 2008
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Czech Republic. Journal of wildlife diseases, 44, 777-780, 2008
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294
VIRUSES IN ENVIRONMENT: SITUATION IN VOJVODINA PROVINCE OF SERBIA
Gospava Lazić1*, Siniša Grubač1, Dejan Bugarski 1, Diana Lupulović1, Sava Lazić1, Petar
Knežević2, Tamaš Petrović1
1
2
University of Novi Sad, Faculty of sciences, Department of Biology and Ecology, Novi Sad, Serbia
* Corresponding author: gospaval@gmail.com
Abstract
The environment receives, maintains and transports etiological agents to susceptible hosts. The
presence of animal and human, especially zoonotic viruses in environment is intensively studied
and monitored in a lot of countries worldwide. Viruses are shed in extremely high numbers in the
faeces of infected individuals. Surface water is subject to faecal contamination from a variety of
sources. Water could be contaminated through direct inflow of untreated sewage. There is also more
direct faecal contamination of the environment from humans and animals, for example by bathers or
by defecation of farm animals and free-range or wild animals onto surface waters or soil. After the
enteric viruses are discharged into water, they can survive for prolonged periods in the aquatic
environment. Some of the human, animal and zoonotic viruses were chosen to estimate the possible
existence of route of human and animal faecal contamination, as well as indicate whether the
examined surface waters could present a possible hazard for animal and public health.
In Vojvodina Province in Serbia, in total 60 surface water, and 6 untreated sewage samples were
collected at 33 locations during two sampling periods: summer (July-October), and autumn
(November-December). The presence of seven ((human (HAdV, NoV GI, NoV GII and HAV),
animal (PAdV and BPyV) and zoonotic (HEV)) viruses in surface water and sewage samples was
tested by real-time PCR (qPCR) and reverse transcription real-time PCR (RT-qPCR) assays. The
results show that the potential risk for public and animal health exists if the examined surface
waters are used in agricultural and recreational purposes and present the need for assessing water
sources for viral contamination to help protect public health.
Keywords: environment; human, animal and zoonotic viruses; Vojvodina; Serbia
Introduction
The environment receives, maintains and transports etiological agents to susceptible hosts. Viruses
can be transmitted through various environments (water, sewage, soil, air, or surfaces) and persist
enough in these vehicles to represent a health threat. Pathogenic viruses are routinely introduced
into the environment through the discharge of treated and untreated wastes, as current treatment
practices are unable to provide virus-free wastewater effluents (Bosh et al., 2006). The animal,
human and especially zoonotic viruses in environment is intensively studied and monitored in a lot
of countries worldwide. They are shed in extremely high numbers from infected individuals and
they are stable in the environment for extended periods of time. Human exposure to even low levels
of some pathogenic viruses in the environment, such as norovirus, adenovirus, hepatitis E and A
viruses, can cause infection and disease. Some of these viruses have been suggested as a parameter
for evaluating viral pollution of environmental waters (La Rosa et al., 2012). The viruses
transmitted by the respiratory and faecal-oral routes, transport and persistence in environment is
directly related to the potential for and risk of transmission, host exposure, infection and disease
(Sobsey and Meschke, 2003). Enteritic viruses can survive the water disinfection processes that
eliminate bacteria (Carratala et al., 2013). Several waterborne outbreaks of viral gastrointestinal
295
illness have been documented. Besides gastrointestinal illnesses, enteric viruses have been linked to
more acute conditions, including meningitis and paralysis (Spinner and di Giovani, 2001). Many of
the viruses are important pathogens of their human and animal hosts, although some of them do not
always cause severe illness or high mortality rates (Sobsey and Meschke, 2003).
Faecal Contamination of the Environment
The presence of viruses in water is evidence of faecal contamination because they are excreted by
infected individuals and do not belong to natural microbial population (Faccin-Galhardi et al.,
2013). Especially important are a variety of non-enveloped enteric and respiratory viruses. These
include adenoviruses, astroviruses, caliciviruses, papovaviruses, parvoviruses, picornaviruses
(enteroviruses and hepatitis A virus), and other nonenveloped viruses that can be shed in faeces
(and in some cases urine) from infected individuals and can be present in faecal wastes and faecally
contaminated environmental samples (Sobsey and Meschke, 2003). Sewage, especially from
slaughterhouses, may contain animal adenoviruses, sapoviruses, and HEV, which may also be
zoonotic (Wyn-Jones et al., 2011; Petrović, 2013). The prevalence and distribution of animalspecific viruses in environmental waters must be determined in order to validate the use of these
viruses for source tracking purposes (Fong and Lipp, 2005).
The treatment of waste water and sewage, especially from small towns and villages, as well as from
some large cities in Serbia, is seldom implemented, or works only with partial function. Rivers,
lakes, streams, and coastal waters are regularly contaminated by septic tanks, storm water runoff,
agricultural run-off or run-off of the animal manure used in agriculture and effluents from
inefficiently operated sewage treatment plants. Additionally, water could be also contaminated from
overflows of treatment plants impacted by flooding events, or through direct inflow of untreated
sewage (Lazić et al., 2015).
Viral pathogens transmitted through water
Water is an important vehicle for the transmission of enteric viruses. These agents are adapted to
the hostile environment of the gut and in most cases, can persist for a very long time in water.
Adenovirus
Adenoviruses are members of the family Adenoviridae and the genus Mastadenovirus (Sinclair et
al., 2009). Human adenoviruses (HAdV) have been proposed as indicators of the presence of human
viral pathogens in the environment, because they are prevalent in environmental waters and
successfully detected in different environmental samples, such as waters in lakes and rivers, sewage
systems, treated waters and swimming pools (Bofill-Mas et al., 2010; Silva, 2011). HAdV has also
shown to be very stable in the environment and resistant to water treatments (Rodr guez-Lazaro et
al., 2011). In spite of the existence of reported cases of pneumoenteritis or encephalitis, porcine
adenoviruses (PAdV) do not normally produce clinically severe pathologies (Maluquer de Motes et
al., 2004). PAdV have been often isolated from apparently healthy pigs (Fong and Lipp 2005).
PAdV have been proposed as porcine faecal contamination indicators (Maluquer de Motes et al.,
2004; Girones and Bofill-Mas, 2013).
Norovirus
Noroviruses belongs to the family Caliciviridae and has its own genus Norovirus. Since less than
10 virus particles can lead to infection and disease, noroviruses (NoV) are very common cause of
both endemic and epidemic gastroenteritis (Teunis et al., 2008; Atmar, 2010). These viruses are
extremely contagious. The burden of calicivirus (including NoV) has been clearly documented in
296
numerous geographical areas worldwide (Rodr ´guez-La´zaro et al., 2012) In US, noroviruses
causes an estimated 23 million cases per year (Sinclair et al., 2009). In Europe NoV epidemics
have been reported to increase in both incidence and severity, probably as a result of an increased
pathogenicity and/or transmissibility of new strains (La Rosa et al., 2012).
Hepatitis E virus
Hepatitis E viruses (HEV) excreted in faeces and urine constitutes a significant proportion of
pathogens present in sewage (Sinclair et al., 2009). The four major genotypes (GI to GIV)
belonging to a single serotype. While GI and GII are restricted to humans, GIII and GIV are
zoonotic and may infect animals (La Rosa et al., 2012). HEV has been detected in different water
environments, including urban sewages, in Spain, Italy, France and the United States (La Rosa et
al., 2012). Moreover, infectious HEV particles have been reported to occur in sewage, indicating
the existence of a potential public health risk from the contamination of surface water with HEV
(La Rosa et al., 2012).
Hepatitis A virus
Hepatitis A virus belongs to the family Picornaviridae and has its own genus Hepatovirus (Sinclair
et al., 2009). HAV infections result in numerous symptoms, including fever, malaise, anorexia,
nausea and abdominal discomfort, followed by jaundice; the infection can also cause liver damage.
HAVs have been detected in different water environments: wastewaters, treated effluents, surface
waters and drinking waters (La Rosa et al., 2012). Water is considered to be the most important
source of infectious virus because it can survive for long periods in this environment. For example,
the virus can survive over 6 weeks in river water (Rodr ´guez-La´zaro et al., 2012).
Polyomaviruse
Bovine polyomaviruses (BPyVs) are members of the family Poliomaviridae and the genus
Poliomavirus. BPyV do not lead to clinically severe diseases in cattle (Girones and Bofill-Mas,
2013). These viruses have been suggested as potential bovine markers (Hundesa et al., 2006;
Girones et al., 2010; Girones and Bofill-Mas, 2013).
Situation in Vojvodina Province comparing to the situation in Europe
A number of studies have been carried out with the aim of estimating the risks from viral
contamination related to the release of wastewater into surface waters. In Serbia, untreated urban
sewage and wastewater inflow directly into the different surface waters. The sewage treatment
plants usually do not exist, or there are only mechanical and partial- treatment sewage systems on
place. The surface water samples were collected at 30 locations during two sampling periods:
summer (July-October), and autumn (November-December). The sampling locations for surface
waters were chosen near all larger towns and as close as possible to a few intensive animal
production farms. Among these water samples, 10 samples were collected from 5 urban beaches
and 5 locations in protected natural areas. In total, 60 samples of untreated surface water and 6
samples of untreated urban sewage samples were tested by qPCR and RT-qPCR. The most
prevalent virus found was HAdV which was detected in 43.33% (26/60) samples in Danube, Sava,
Begej and Krivaja Rivers, and DTD, Great Backa, KCIII canals and Palic Lake. HAdV has been
proposed as an indicator of the presence of human viral pathogens in the environment (Bofill Mas et
al., 2006; Silva et al., 2011). Results of our study confirm this observation, and are in line with the
results of many other studies conducted around the world (Rusiñol et al., 2014; Kern et al., 2013;
Silva et al., 2011; Pina et al., 1998). In Poland, 60 water samples were collected in 2007 from four
sampling sites situated along the river Wieprz. Human pathogenic viruses were detected in 35% of
297
samples. HAdVs were detected in 28.3% of samples, and were present throughout the whole year
(Kozyra et al., 2011). In Spain, 23 river water samples from two sites with different levels of faecal
pollution were tested, and the human virus most often detected was HAdV, being present in 15 of
the 23 samples (65%) (Pina et al., 1998).
NoV GII was found in 40% (24/60) samples in Danube, Sava, Begej and Krivaja Rivers, and DTD,
Great Backa, KCIII canals, Palic Lake, and Rakovac stream. NoV GI was found in 10% (6/60)
samples in Danube and Sava Rivers, and Great Backa Canal. Our results are in accordance with the
results of the studies conducted in Hungary, where noroviruses were detected in 30% examined
surface water samples (Kern et al. 2013) and in the region of North-Eastern Greece where NoV GII
strains were found in 34% samples (Parasidis et al., 2013b).
PAdV, BPyV and HEV were detected in 5 (8.33%), 4 (6.67%) and 2 (3.33%) samples in Krivaja,
Sava, Danube and Begej Rivers, DTD and KCIII canals. In our study we found PAdV and BPyV
presence in tested surface waters in Serbia in much lower extent than it is published in some other
studies in Europe. One of the possible reasons for these results is because the animal production in
Serbia has been quite low in recent years due to the economic crisis (Lazić et al., 2015). As HEV is
highly prevalent in pig population in Serbia (Lupulović et al., 2010; Petrović et al., 2014); the
source of the HEV detected in the samples could be infected humans and / or pigs.
HAV was not found in any of analyzed surface waters or urban sewage samples. Viruses were not
detected in 25% (3/12) of the examined surface waters (Tisa and Jegricka Rivers, and the Special
Nature Reserve Obedska bara - wetland).
Among 6 analyzed sewage samples, 5 (83.33%) were found positive for the target viruses. On both
sampling occasions in two urban sewage systems HAdV and NoV GII and in one occasion NoV GI
were detected, and in the third urban sewage system only NoV GII was found in one of two
sampling occasions. Viruses such as norovirus and adenovirus may be highly prevalent in sewagepolluted recreational waters (Wyn-Jones et al., 2011). Autochthonous strains of HEV have been
reported in urban sewage in several highly developed countries, as well as related cases of sporadic
acute hepatitis caused by these non-imported strains (Pina et al., 2000).
Conclusion and recommendations
The surface water samples tested positive for human, animal and zoonotic viruses indicating that
the contamination must have originated from a variety of sources. The results show that the
potential risk for public and animal health exist if the examined surface waters are used in
agricultural and recreational purposes, and suggest the necessity for further and more extensive
studies. Methods related to virus purification and detection of viral particles should be improved
such that survival of human pathogenic viruses in the environment can be followed reliably.
Legislation on handling and treatment of water and sewage should be adapted as needed to reduce
the risk of environmental virus contamination.
Acknowledgements
This work is conducted within the project TR31084 funded by the Serbian Ministry of Education,
Science and Technological development.
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DEVELOPMENT OF A MULTI-RESIDUE METHOD FOR THE DETERMINATION OF
INSECTICIDES IN ANIMAL FAT BY LC-MS/MS
Vojislava Bursić1, Gorica Vuković2, Tijana Zeremski3, Dejan Beuković1, Miloš Beuković1,
Aleksandra Petrović1, Magdalena Cara4
1
Faculty of Agriculture, University of Novi Sad, Serbia
2
Institute of Public Health, Belgrade, Serbia
3
Institute of Field and Vegetable Crops, Novi Sad, Serbia
4
Faculty of Agriculture and Environment, Agricultural University of Tirana, Albania
* Corresponding author: bursicv@polj.uns.ac.rs
Abstract
Agricultural intensification has resulted in the increased mechanization and use of chemical
compounds, and in dramatic changes of landscape characteristics such as the decrease in permanent
vegetation cover, the increase in field size and the reduction of habitat diversity. The occurrence of
pesticide poisoning in wildlife is a matter of species susceptibility (hazard of a compound) and
likelihood of exposure. The most common pesticides in animal origin food are compounds that tend
to concentrate and remain in fatty food. The aim of this work was to develop a liquid
chromatography tandem mass spectrometry method (LC-MS/MS) for the simultaneous
identification and quantification of 18 insecticides in adipose tissue of brown hare, in a single run.
Insecticides were detected by triple quadrupole mass spectrometer (Agilent 6410B Triple Quad
Mass Spectrometer, USA) in positive electrospray ionization using multiple reactions monitoring
mode (MRM). The method was validated for accuracy, precision, linearity, LODs and LOQs. The
calibration was performed as matrix calibration, by means of spiking the calibration samples before
extractions and preparing them in the same way as the test samples. The calibration range was from
0.01 to 0.5 µg/ml. The obtained R2 was higher than 0.99 for all the studied pesticides. The LODs
were below 0.005 mg/kg and the LOQs were set on 0.01 mg/kg. For the recovery, the samples were
spiked with the analytes at three concentration levels (0.05, 0.1 and 0.2 mg/kg). The average
recoveries for all analytes were in the range from 67.0 to 101.3% (RSDs 6.73-10.98%). The
obtained mean values of the responds were with RSD<20%. An efficient, sensitive and reliable LCMS/MS method has been developed which can be applied in the analysis of real samples to 20
insecticide residues in brown hare fatty tissue.
Keywords: insecticide residues, LC-MS/MS, validation, brown hare, fat
Introduction
A brown hare (Lepus europaeus Pallas 1778) is one of the most wide-spread and hunted game in
Europe (Beuković et al., 2011). The most numerous brown hare populations are in agricultural
regions on small agricultural plots with various crops situated among woods meadows and
shrubberies (Šelmić & Gačić, 2011). By drastic increase in areas under crops where the pesticides
are intensively used along with the decrease in areas under fodder crops, the possibility for proper
brown hare nutrition is reduced while its typical habitats have been changed (Beuković & Popović,
2014).
It is considered that the excessive use of pesticides is responsible for 18-68% cases of animal
poisoning (Berny, 2007). Berny (2007) also stated that highly toxic and „rapid-acting“ substances
such as organophosphoric insecticides and carbomates cause the kill-off.
The paper will validate the QuEChERS multi-residue method for the determination of insecticide
residues in fatty tissue by liquid chromatography tandem mass spectrometry (LC-MS/MS)
according to SANCO/12571/2013.
301
Within the validation the recoveries of extraction, detection limits (LOD), quantification limits
(LOQ) and linearity with carbofuran-D3 addition as an internal standard (IS) will be determined.
Since 2014 in the European Union the validation of analytical methods is performed according to
directives of SANCO/12571/2013 document. Based on the document for the analysis of pesticide
residues in various matrices and for the improvement of selectivity and sensibility it is necessary to
apply gas (GC) or liquid (LC) chromatography with mass spectrometry (MS) or tandem mass
spectrometry (MS/MS). QuEChERS (Quick Easy Cheap Effective Rugged and Safe) is the
abbreviation of: quick, easy, cheap, effective, rugged and safe method of extraction which has been
developed aimed at the determination of pesticide residues in agricultural products. This method has
become the official AOAC method No. 2007.01 and BSEN 15662:2008 for the determination of
pesticide residua in food (Bursić et al., 2012).
Chemicals
The analitical insecticide standards were manufactured by Dr. Ehrenstorfer GmbH, Germany. As an
internal standard, carbofuran-D3 (99.7%) was purchased from Pestanal, Fluka (Germany), and was
used in the concentration of 1 mg/ml of the basic standard in acetonitrile with the dilution up to 1.0
g/mL. The stock standard solutions were prepared by dissolving an analytical standard in
acetonitrile while the working solution i.e. the mixture of the studied pesticides was obtained by
mixing and diluting the stock standards with acetonitrile resulting in the final mass concentration of
10 g/mL.
Instruments
For LC analysis, an Agilent 1200 (Agilent Technologies, USA) HPLC system with a binary pump
was used. This was equipped with a reversed-phase C8 analytical column of 150×4.6 mm and 5 m
particle size (Agilent Zorbax Eclipse XDB). The mobile phase was methanol and Milli-Q water
with 0.1% formic acid in gradient mode, with the flow rate 0.6 ml/min. For the mass spectrometric
analysis, an Agilent 6410 Triple-Quad LC/MS system was applied. Agilent Mass Hunter Data
Acquisition, Qualitative Analysis and Quantitative Analysis software were used for method
development and data acquisition.
10 g sample + 10ml MeCN + 100l ISTD (10 µg/ml Carbofuran-D3)
 Shake vigorously for 1 min
Add 4 g MgSO4, 1 g NaCl, 1 g Na3Citrate dihydrate, 0.5 g Na2HCitrat sesquihydrate
Shake tube immediately for 1 min
 Centrifuge for 5 min at 3000 g
Transfer 5 ml of the extract into a PP tube contained MgSO4, PSA, C18
Shake for 30 s
 Centrifuge for 5 min at 3000 g
Transfer 200 l into a vial, evaporate to dryness
Reconstitute in 200 l of mobile phase
LC-MS/MS
Graph. 1. QuEChERS extraction
Validation
The limit of detection - LOD was determined as the lowest concentration giving a response of three
times the average baseline. The ratio signal/noise in the obtained chromatograms for the LOD was
302
calculated by MassHunter Qualitative Software. The linearity was checked using matrix matched
standards (MMS) at the concentrations of 5.0, 10.0, 25.0, 50.0 and 100.0 ng/ml. The recovery was
checked by enriching 10 g of a blank sample with the mixture of pesticide standard of 10 g/ml in
the amount of 100 and 50 μl (final mass concentration 0.10 and 0.05 mg/kg) and with the mixture of
pesticide standard of 1 g/ml in the amount of 100 μl (final mass concentration 0.01 mg/kg) with
the addition of the internal standard carbofuran-D3 (Graph. 1.).
Results
Before the calibration and quantification of pesticides it was necessary to set an acquisition method.
The determination of the acquisition method comprises: setting chromatographic conditions,
determining the precursor and product ion so called monitoring mode of ion transfer (MRM or
SRM), determining the fragmentation energy (Frag.) and the energy of collision cell (CE). For
setting the SRM MassHunter Optimizer Softvere Version B03.01 (Agilent Technologies 2010) and
Agilent G1733AA MassHunter Pesticide Dynamic MRM Database were used (Tab. 1.).
Table 1. Retention times, MRM and collision cell energy of studied pesticides
Pesticide
Acetamiprid
Carbofuran
Clothianidin
Fenpropathrin
Chlorpyrifos
Tefluthrin
Thiodicarb
Carbosulfan
Cyromazine
Diazinon
Fenoxycarb
Malathion
Tefluthrin
Imidacloprid
Indoxacarb
Phoxim
Pirimiphos methyl
Pyriproxifen
Thiamethoxam
Endosulphan alpha
MRM
Transitions
(m/z)
192.1
192.1
222.1
250.0
250.0
350.2
350.2
349.9
349.9
419.1
419.1
355.1
355.1
381.2
381.2
167.1
167.1
305.0
305.0
302.1
302.1
331.1
331.1
419.1
419.1
256.0
256.0
528.1
528.1
299.1
299.1
306.2
306.2
322.1
322.1
292.0
292.0
404.8
Produkt jon
m/z
Frag.
(V)
160.1
132.0
165.1
169.1
132.1
125.1
55.2
197.9
97.0
193.0
419.0
88.0
108.0
160.1
118.1
125.1
85.0
169.0
153.0
116.1
88.0
127.0
99.0
419.0
193.0
208.7
174.6
203.0
150.0
129.0
77.0
164.2
108.2
227.1
185.1
211.0
181.0
373.1
104
104
90
90
90
100
90
120
120
100
90
130
120
130
130
80
80
100
100
110
110
90
90
1100
130
100
100
120
120
80
80
150
150
120
120
150
150
120
303
CE
(V)
18
34
20
10
15
20
15
15
20
25
15
21
25
21
50
10
25
5
20
12
20
15
15
20
17
15
20
36
16
10
35
20
20
10
10
20
20
10
Rt
(min)
R2
Average
recovery
±RSD (%)
8.05
0.9995
98.8±8.9
14.00
10.50
0.9989
0.9994
86.24±3.1
103.21±2.2
20.05
0.9576
59.6±3.4
20.05
0.9989
55.43±8.9
18.77
0.9811
78.91±5.4
14.85
0.9918
97.02±1.0
17.34
0.9965
93.05±2.4
17.27
0.9947
75.35±5.8
17.45
0.9987
82.64±5.9
17.30
0.9998
92.25±2.9
15.18
0.9992
83.19±3.1
18.07
0.9966
73.03±5.5
10.30
0.9988
95.99±4.3
18.12
0.9990
73.55±2.9
17.95
0.9997
74.27±3.2
18.08
0.9918
86.72±4.3
19.46
0.9996
59.16±9.0
9.13
0.9923
64.02±5.7
16.33
0.9986
99.33±11.2
100
80
60
40
20
0
-20
-40
Chlorpyrifos
Fenpropathrin
Endosulphan
alpha
Thiamethoxam
Thiocarb
Tefluthrin
Pyriproxifen
Pirimiphos
methyl
Phoxim
Malathion
Indoxacarb
Imidacloprid
Fenoxycarb
Diazinon
Carbosulfan
Cyromazine
Clothianidin
Carbofuran
-80
-100
Acetamiprid
-60
Graph. 2. Matrix effects (%)
For all the studied pesticides the LOQ of 0.01 mg/kg, was reached while mathematically calculated
LOD was 0.005 mg/kg.
Based on the graphic of matrix influences (%) (Graph. 2.), it can be concluded that the fatty tissue
of a brown hare as a matrix manifests the greatest influence on the carbofuran analysis (86.92%). In
case of carbofuran, imidacloprid, thiocarb, thiametoxam, endosulphan alpha, fenproparthrin and
chlorpyrifos matrix brings about the increase in signal, whereas in case of all the other analysed
insecticides the matrix is the cause of the decrease in the signal. The influence of a brown hare
matrix on most insecticides overcoming 15%, which indicates, that for the determination of these
pesticides a matrix match calibration is necessary.
The evident public concern regarding food safety is manifested through a growing number of
literary available data dealing with this issue. Moller (2014) and Inthavong et al. (2014) used
QuEChERS method for the extraction of pesticides at validation LC-MS/MS and GC-MS/MS
method for the determination of pesticide residues in fatty tissue of animals.
In order to insure the food safety and human health and taking into consideraton the fact that
pesticides can be transferred from animals in a direct way or through food chain a regulation on
maximum amounts of pesticide residues in food was passed (Gazette 29/2014, EC Regulation
396/2005).
A validated LC-MS/MS multiresidual method was obtained which can be used as a routine method
for the determination of insecticide residues in fatty tissue of a brown hare:
 For all the studied pesticides the LOQs were set on 0.01 mg/kg, whereas the mathematically
calculated LODs were 0.005 mg/kg.
 Extraction recoveries for spiking levels of 0.05, 0.1 and 0.2 mg/kg were 55.4 - 103.2±1.0 11.2%.
 The linearity for the concentrations of 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 μg/mL, were with
R2>0.99 in organic solvent, while in the matrix R2 were lower but over 0.99.
304
Acknowledgement
The authors acknowledge the financial support of the Provincial Secretariat for Agriculture, Water
Management and Forestry, Project Ref. 104-401-3840/2014-07-262.
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305
THE UNUSUAL COLONY LOSSES IN VOJVODINA PROVINCE
Jelena Babić1*, Sara Savić1, Miroslav Ćirković1, Igor Stojanov1, Ivan Pihler2, Nada Plavša3
1
University of Novi Sad, Faculty of Agriculture, Department of animal science, Novi Sad, Serbia
3
University of Novi Sad, Faculty of Agriculture, Department of veterinary medicine, Novi Sad, Serbia
*Corresponding author: jelenababic@niv.ns.ac.rs
2
Abstract
The apiculture has been in decline in Serbia as well as in Europe and the USA over recent decades,
as is shown by the decreasing numbers of managed honey bee (Apis mellifera) colonies. Over the
past 10 years, beekeepers have been reporting unusual weakening of bee numbers and colony
losses. According to the European Food and Safety Authority (EFSA) no single cause of declining
bee numbers has been identified.
During a two-year study (December, 2012 – December, 2014) 210 bee hives were examined from
63 apiaries in Vojvodina province. All samples were examined in laboratories of Scientific
Veterinary Institute „Novi Sad“. Examination included detection of Varroa destructor, Aethina
tumida, Tropilaelaps spp, Nosema spp, Paenibacillus larvae. Nosema spp. were detected in 42.48%
of the samples, Varroa destructor in 21.43%, Paenibacillus larvae in 2 examined samples. There
were no evidence of Aethina tumida and Tropilaelaps spp from examined samples from Vojvodina
province. According to the results of our study, major detected problems at Vojvodina’s apiaries are
still Nosema spp. and Varroa destructor. Our further study will try to determinate others possible
causes of colony losses in this part of Serbia.
Key words: apiculture, colony losses, Vojvodina, Varroa destructor, Nosema spp
Introduction
The importance of bees and beekeeping is demonstrated directly through the production of honey
and other food and feed supplies (EFSA, 2014). Furthermore, honey bees Apis mellifera are
critically important in sustaining biodiversity by providing essential pollination for a wide range of
plants (Bradbear, 2009) as the most economically valuable pollinators of crop monocultures
worldwide. FAO (The Food and Agriculture Organization of the United Nations) estimates that of
the 100 crop species that provide 90% of food worldwide, 71 are pollinated by bees (EFSA, 2014).
According to FAO and the European Union the value of that indirectly importance of bees is 20-30
times higher than value of honey production (Petrović et al, 2013). Also, 35% of the human diet is
thought to benefit from pollination (Stindl and Stindl, 2010).
The apiculture has been in decline in Serbia as well as in Europe and the USA over recent decades,
as is shown by the decreasing numbers of managed honey bee (Apis mellifera) colonies (Van
Engelsdorp et al, 2009; VanEngelsdorp and Meixner, 2010; Vejsnaes et al, 2010; Neumann and
Carreck, 2010; Nazzi et al, 2012; Meixner et al, 2014). Over the past 10 years, beekeepers have
been reporting unusual decreasing of bee numbers and colony losses. Mortality is the highest at the
end of winter and beginning of spring (UNEP, 2010; Genersch and Aubert, 2010).
Group of authors Potts et al. (2010) compiled data from 18 European countries to assess trends in
the number of honey bee colonies and beekeepers between 1965 and 2005. They found consistent
declines in honey bee colony numbers in central European countries, but some increases in
Mediterranean countries. Furthermore, beekeeper numbers have declined in all European countries
306
examined. In Canada, the average level of winter loss of honey bee colonies over the winter of
2013/14 was 25.0% (CAPA, 2014). According to the annual survey conducted by the Bee Informed
Partnership and the U.S. Department of Agriculture (USDA) the total loss of honey bee colonies
over the winter 2013/14 was 23.2 %, which is slightly less than in previous years when bee losses
over the winter had an average of 29.6 % (USDA, 2014). Beekeepers from apiaries in Chile in early
spring 2010 reported the massive death of bees (Rodriguez et al, 2012). The total colony losses that
occurred in South Africa in 2010-2011 were 46.2 % (Pirk et al., 2014). The average percentage of
winter losses in Germany was from 3.8% (2004/05) to 15.2% (2005/06), but regionally higher
losses were reported (Genersch et al, 2010). The recorded losses in the Republic of Serbia in the last
decade varied from 30 to 70% (Plavša et al, 2013). The decreasing numbers of managed honey bee
colonies in some parts of the world is caused by different pathogens, improper pesticide and
herbicide use, weather conditions, beekeeping technology, antimicrobial resistance due to
uncontrolled use of antibiotics, malnutrition, the environment, the failure by public administrations
to control cross-border trade in breeding stock, socio-economic factors, ageing of the beekeeper
population and lower market prices for their products and services (van Engelsdorp and Meixner,
2010; CAPA, 2014; Rose et al, 2014; OIE, 2014; Chen et al, 2014). According to the European
Food and Safety Authority (EFSA) no single cause of declining bee numbers has been identified.
Many studies have indicated that the losses were usually the result of a combination of different
causes (Riviere et al, 2013; Chen et al, 2014). Honey bees succumb to a variety of pathogens such
as Nosema spp, Ascarapis woodi, Paenibacillus larvae, Melissococcus plutonius, Aethina tumida,
Tropilaelaps spp, Varroa spp and viruses (Genersch et al, 2010; Antúnez et al, 2012). Ability of
honey bees to resist those infections is compromised if they are malnourished or exposed to
pesticides (Antunez et al, 2015). COLOSS (Prevention of Honeybee Colony Losses) and the US
Department of Agriculture's Area wide and Managed Pollinator CAP (Coordinated Agricultural
Project) made the first step in understanding and mitigating honey bees losses and objectively
discriminated among types of colony mortality occurring worldwide. This will enable a more
informed and appropriate allocation of research efforts into CCD and other causes of mortality in
general (Williams et al, 2010).
Vojvodina province has excellent natural conditions for beekeeping. However, unusual colony
losses that the beekeepers have encountered represent an important problem whose etiology hasn't
been sufficiently examined.
The aim of the present study was examination of the presence of different honey bees pathogens as
potential causes of unusual colony losses in Vojvodina province.
During a two-year study (December, 2012 – December, 2014) 210 bee hives were examined. The
samples of bees which were used as material for this study have been collected in 63 apiaries in the
area of Vojvodina province, Republic of Serbia. All apiaries were clinically examined in detail,
whereupon the brood sample size 10 x 10 cm (Regulations about establishment of Programme of
animal health protection measures, 2009) were taken, together with approximately 100 adults bees
from the back frames, approximately 200–250 bees removed from unsealed brood combs and
finally,samples from bottom board were taken for laboratory analysis.
All samples were examined in laboratories of Scientific Veterinary Institute „Novi Sad“.
Examination included detection of Varroa destructor, Aethina tumida, Tropilaelaps spp, Nosema
spp, Paenibacillus larvae.
Detections of Varroa destructor, Aethina tumida, and Tropilaelaps spp were done in according to
OIE Terrestrial Manual 2008 (OIE, 2008). Microscopic method of counting Nosema spores using
hemacytometer was done on the basis of the instructions Testing for Nosema Spores using
307
Hemacytometer, Instructional Poster # 167 (Reuter et al, 2010). Diagnosis of American foulbrood
(AFB) was based on the presence of clinical signs and identification of the pathogenic agentPaenibacillus larvae in according to OIE Terrestrial Manual 2008 (OIE, 2008; OIE, 2014).
Results
Varroa destructor was present in 21.43% of the samples, while the microscopy analysis of
macerated bee abdomens showed the presence of the spores Nosema spp. in 42.48% of the samples.
During clinical trial a characteristic AFB image was identified (Picture1), whereupon the brood
samples size 10 x 10 cm were taken to identification of Paenibacillus larvae. Microbiological
characterization confirmed the presence of Paenibacillus larvae in both samples, one of them being
from the area of Novi Sad, and the other from Sremska Mitrovica. All colonies of the whole two
apiaries were eliminated and all beekeeping material was destroyed. Also, in areas of those two
apiaries all bee colonies were checked for visual symptoms of AFB. There were no evidence of
Aethina tumida and Tropilaelaps spp from examined samples from Vojvodina province.
Picture 1. Clinical AFB image in samples taken from/in the area of Novi Sad
The ANSES laboratory in France designated by the European Commission as the EU Reference
Laboratory and OIE Reference Laboratory for bee diseases has implemented an active
epidemiological surveillance programme on honey bee colony mortality in 17 EU Member States
during 2012 and 2013. Results of that surveillance programme confirmed that the major honey bee
diseases are varroosis, American foulbrood, European foulbrood, nosemosis and chronic paralysis
(Chauzat et al, 2014). The main focus of most recent studies about colony mortality has been on the
pathogens (Meixner et al, 2014).
The most serious of all pathogens is the parasitic mite, Varroa destructor, which contributes
significant losses to honeybee (Dietemann et al. 2012; Martin et al. 2012).
Many studies have confirmed substantial contribution of Varroa destructor to honey bee losses
across the Northern hemisphere (Dietemann et al, 2012).This ubiquitous parasite act as a vector for
viruses by facilitating their transfer between hosts (Nazzi et al, 2012; Chen et al, 2014). Also, the
308
mites have been reported to activate inapparent virus infections in honey bees probably by down
regulation of honey bee immune genes (Meixner et al, 2014).
Nosema spp has been reported to cause collapse of colonies in both spring and winter (Meixner et
al, 2014). Nosema ceranae as the new parasite has rapidly spread all over the world and the transfer
of this parasite is nearly impossible to avoid (Ritter, 2014). It was present in European Apis
mellifera from 1998 and perhaps from the mid-1990s in the USA and possibly elsewhere (Paxton,
2010).
American foulbrood is considered one of the most contagious and destructive infectious diseases
affecting the larval and pupal stages of honey bees that have been spread nearly all over the world
(Ritter, 2014; OIE, 2014).
During the US national survey of honey-bee pests and diseases, Varroa mites were detected in
approximately 90% of all sampled apiaries in the period from 2010 to 2012. In the same survey,
Nosema spores were found in 47 to 57 %.
The German bee monitoring project has shown the prevalence of Nosema infection 31% in 2005
and less than 14% in 2007. That project also demonstrated a statistically highly significant
difference between the Varroa infestation rate of surviving colonies and of colonies which
collapsed over winter (Genersch et al, 2010).
The results show that in our region there is still no evidence of Aethina tumida and Tropilaelaps,
although they do represent a threat. Aethina tumida was detected for the first time in Reggio
Calabria, South West Italy on 5th September 2014. Until March 2015, more than a thousand
apiaries have been inspected in Calabria and Sicily. Aethina tumida has been confirmed in 61
apiaries in Calabria region and in one apiary in Sicily. Because of that, in those regions
approximately 3,500 honeybee colonies have been destroyed (Chauzat et al, 2015).
During 2006 and 2007 in Spain 73 % of the samples had viral presence, but most (80%) had one
virus and only 20% had two different viruses which was lower than expected (Antunez et al, 2012).
According to the results of our study, major detected problems at Vojvodina’s apiaries are still
Nosema spp. and Varroa destructor. Our further study will try to determinate other possible causes
of colony losses in this part of Serbia. In the preserved samples of the adult bees, as well as in the
ones of Varroa mites, the analysis on the most important viruses that affect honeybees such as
Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), Sacbrood Virus (SBV), Chronic
Bee Paralysis Virus (CBPV), Acute Bee Paralysis Virus (ABPV), Kashmir Bee Virus (KBV), and
Israeli Acute Paralysis Virus (IAPV) will primarily be done (Antunez et al, 2012).
By regularly controlling the bees we get the real insight into this problem of unusual colony losses,
and based on this insight we can aim at the” source of the problem” and by doing so, preserve the
health of bees. A preserving the health of bees is an integral part of good environmental
management, food security, enhanced global agriculture and global economy.
Acknowledgments
This study was supported by Project TR 31084 of Ministry of Education, Science and
Technological Development, Republic of Serbia.
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311
________________________________________________________________________
Session № 5
AQUACULTURE
Full papers
_______________________________________________________________________
312
Plenary lecture:
CONVERTING WASTE LANDFILL IN POND AREAS
Miroslav Ćirković*1, Brankica Kartalović1, Miloš Pelić1, Nikolina Novakov1, Dragana Ljubojević1,
Sanja Jovanić2, Željko Mihaljev1
1
2
Scientific Veterinary Institute "Novi Sad", Novi Sad, Serbia
Institute of Chemistry, Technology and Metallurgy, Belgrade, Serbia
3
Faculty of agriculture Novi Sad, Novi Sad, Serbia
* Corresponding author: miroslavcirkovic@yahoo.com
Abstract
Fertile soil is a resource that is in the progress of civilization is increasingly reduced. The amount of
fertile soil can be successfully increased with remediation of landfills. The basis of remediation
makes the removal of contaminated soil layer in sufficient quantity. A real system represents a
construction of ponds area where one part of soil is removed out of pond area and the other part is
exploited for making embankments. Each embankment is covered with a layer of "healthy"
excavated of deeper layers of the soil. In order to neutralize the organic components and to achieve
buffering environment, we used hydrated and slaked lime in an amount of 2000 kg / ha. In further
production, we also use hydrated lime in quantities of 3,000 kg by the end of the growing season.
Thus prepared pond we were filling with water during thirty days before hatching with fish. We
used the polyculture of common carp and bighead. Successful production we were provided by
using different types of aerators and with addition of well water from the first aquifer capacity of
1000 L / ha per minute. For feeding the fish we used the complete nutrients and achieve production
of 2000 kg / ha.
Keywords: soil, remediation, aeration, hydrated lime
Introduction
In the literature until now there has not been a lot of data relating to the construction of a pond on
the land which served as a landfill and where remediation was performed. This is one of the first
papers dealing with this issue. One of the first things that must be done is to remove the surface
layer of soil to reduce the amount of organic matter (Pelić, 2014). Location for fish pond
construction is a very important and sometimes has crucial importance for the financial success of
companies (Ćirković et al., 2002). It is particularly important that the water used for the pond has
optimal physical and chemical characteristics for fish breeding and that there is enough water
during the whole growing season (Ćirković et al., 2002). Zooplankton and phytoplankton are also
very valuable indicators of water quality (Attayde and Bozelli, 1998; Hakkari, 1972; Pontin and
Langley, 1993; Zakaria et al., 2007; Wu, 1984; Weber and Weber 1998). For successful carp
production it is necessary to provide adequate environmental conditions, density of population,
balanced diet and adequate handling with fish (Ćirković et al., 2012). Serbia is a country with
traditional production of common carp, and as such occupies third place in the production of carp
per capita in the world, behind the Czech Republic and China, with a production of 1.3 kg per
capita (Ljubojević et al., 2014). Adequate health care and appropriate intervention aims to preserve
and improve fish production (Ćirković and Novakov, 2013).
The aim of this paper is to show that it is possible to produce fish on land where remediation has
been done, which is a significant contribution to environmental protection and rural development.
313
The experiment was conducted within the meat industry. The land where pond was built was used
as a village landfill. Construction of the fish pond started by removing a soil layer with a thickness
of 1 m. 12000 m3 of excavated land was transported and disposed of, 12000 m3 was dug up and
used, and 6000 m3 was brought from the site outside of the landfill. Production took place in an
area under the water of 5 ha.
Before starting the fish production, agricultural limestone was applied to the bottom of empty pond
in an amount of 2000 kg/ha. Filling of the fish pond with water was completed from two wells with
a depth range of 75 to 95 meters, using a pumps capacity of 1000 l / min for each well. Aeration
was conducted using aerators (mud pumps) with a capacity of 2000 l/min spraying the water with
“wentury aerator“. The flow of the water was 12 sec L/ha.
Production of carp was carried out during 2014 growing season from 15 of April to 20 of October.
One-and two-year old common carp were stocked in the fish pond and the total stocking density
was 500 kg/ha, with a ratio of 300 kg/ha for two-year old and 200 kg/ha for one-year old carp. The
average weight of an one year old carp was 80 g and a two-year old carp was 800 g.
During the production, commercial extruded and pelleted fish feed, with 32% of proteins and 10%
of fat, were given to fish. Feeding was carried out twice a day in 8 am, and the 3 pm.
The flow of the water was 12 sec L/ha. During the production season continuous monitoring of the
basic environmental conditions was undertaken twice a week, particularly the amount of dissolved
oxygen, biological oxygen demand, chemical oxygen demand, quantification of consumption of
permanganate, amount of nitrite, nitrate, chloride, total phosphorus, ammonium ions and ammonia
and pH.
The use of hydrated lime was 1000kg/ha in April, May and June, while in July, August, September
and October was 2000 kg/ha.
During the growing season the health status and condition of the carp was regularly controlled at
least twice a month.
Quality control of the pond water was tested with standard methods (SRPS, SRPS EN ISO, EPA).
The pH of a solution is measured as negative logarithm of hydrogen ion concentration. At a given
temperature, the intensity of the acidic or basic character of a solution is indicated by pH or
hydrogen ion concentration.
The Chemical Oxygen Demand (COD) test determines the oxygen requirements equivalent of
organic matter that is susceptible to oxidation with the help of a strong chemical oxidant. COD is
determinate by titration method by using dichromate.
The Biochemical Oxygen Demand (BOD) is an empirical standardized laboratory test which
measures oxygen requirements for aerobic oxidation of decomposable organic matter and certain
inorganic materials in water, polluted waters and wastewater under controlled conditions of
temperature and incubation period. The quantity of oxygen required for the above oxidation
processes is a measure of the test.
KMnO4 indicates organic pollution. It is determinate by titration in the acid medium.
Dissolved Oxygen- All living organisms are dependent upon oxygen in one form or another to
maintain the metabolic processes that produce energy for growth and reproduction. Dissolved
oxygen was measured by portable meter WTW OXY 300.
314
Ammonia is produced by the microbiological degradation of organic nitrogenous matter. Ammonia
produces a yellow coloured compound when it reacts with alkaline Nessler reagent, provided the
sample is clarified properly. Ammonia is determined by measuring absorbance with UV/VIS
spectrophotometer.
Nitrogen (Nitrate) (NO3-) Nitrate is the most highly oxidised form of nitrogen compounds
commonly present in natural waters. Significant sources of nitrate are chemical fertilizers, decayed
vegetable and animal matter, domestic effluents, sewage sludge disposal to land, industrial
discharge, leachates from refuse dumps and atmospheric washout. Nitrates are determined by
measuring absorbance with UV/VIS spectrophotometer.
Nitrogen (Nitrite) (NO2-) Nitrite in water is either due to oxidation of ammonium compounds or
due to reduction of nitrate. As an intermediate stage in the nitrogen cycle, it is unstable. Nitrite was
determined by measuring absorbance with UV/VIS spectrophotometer.
Chloride (Cl-) The presence of chloride in natural waters can be attributed to dissolution of salt
deposits or discharges of effluents from chemical industries. They are determined by titration.
Phosphate and orthophosphate are determined by spectrophotometric measurement.
Metals – all metals are determined by AAS.
Chemical analysis: Water content of fish fillets was determined after drying the samples at 105 °C
to a constant weight for 24 hours (SRPS ISO 1442:1997). Crude protein content was determined by
Kjeldahl and ash was determined after burning. Crude lipid in fish tissue was also analyzed using
the Soxhlet System with ether as a solvent (SRPS ISO 1443:1997).
Extraction of lipids was done using Spirić et al. (2010) method for extraction of lipids from fish
muscle by ASE. Fatty acid Methyl esters are determined by Gas Chromatography.
Results
At the end of the growing season, the average weight of the two-year old carp was 1000 g, and 2.5
kg for a three-year old carp. Harvesting density was about 3000 kg/ha. Conversion was 1.6 for
formulated feed in the dry matter. Mortality ranged from 5% in two-year old carps, and 10% in oneyear old carps.
Table 1 shows the range of the maximum and minimum concentration of tested parameters in well
water and the water from the fish pond before and after the addition of lime. The Results of the
analysis of the well water indicate that it has a very good quality which corresponds to the quality of
drinking water. In the pond water the highest values reach level of ammonia, which decreases after
using lime and descends into the acceptable values as shown in Graph 1.
Table 2 summarizes the chemical content and fatty acid composition of the sampled carps. It should
be noted that 12 specimens were taken for analysis. The amount of protein was 16.21 % and fat
content 4.42%. A lipid analysis enabled the classification and quantitative determination of fatty
acids and besides that the sum of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA),
polyunsaturated fatty acids (PUFA), n-3 acids, n-6 acids, n-3/n-6 ratio, n-6/n-3 ratio and ratio of
PUFA/SFA, as well as the ratio of USFA/ SFA which represent the indicators of lipid quality. The
Total amount of SFA was (27%), the amount of MUFA was (28%) and PUFA (44%) content was
the highest. The ratio of n-3/n-6 was 0.92, while the ratio PUFA/SFA was 1.62.
During the health status of fish monitoring erythrodermatitis was diagnosed. The disease appeared
from the middle of August with prevalence of 25%. After treatment with flumequin in pelleted feed,
the symptoms of disease disappeared.
315
Table1. Quality of well water and pond water before and after addition of lime, the range of
maximum and minimum concentration of tested parameters
The
tested parameter
pH
Chemical Oxygen
Demand (COD)
Biochemical Oxygen
Demand (BOD)
Unit
Method
mgO2/L
SRPS H.Z1.111: 1987
SRPS ISO
6060:1994*
mg O2/L
H1.002*
KMnO4
mg/L
Dissolved Oxygen
mg/L
Total N
mgN/L
Ammonia
mgN/L
Nitrate
mgN/L
Nitrite
mgN/L
Chloride
mg/L
Phosphate
mgP/L
Ortophosphate
mgP/L
Metals
Iron
Zink
Arsenic
mg/L
mg/L
µg/L
SRPS EN ISO
8467:2007*
SRPS ISO
5814:1994
Computing
SRPS ISO
H.Z1.184:1974*
SRPS ISO 78903:1994*
SRPS EN
26777:2009*
SRPS ISO 9297:1997
SRPS ISO 92971:2007
SRPS EN ISO
6878:2008*
SRPS EN ISO
6878:2008*
EPA 7000b*
EPA 7000b*
EPA 7010*
The measured
value (before
addition of
lime)
7.4-7.8
The measured
value
(after addition
of lime)
7.8-8.5
The
measured
value
(well water)
7.5-7.7
20-26
5-11
2.83-3.82
6-9
<4
<4
30.8-35.8
21-25
1.53-1.81
5.8-6.1
7.56-7.95
5.0-5.67
2.24-2.94
1.25-1.75
2.07-2.721
0.6-1.51
0.41-0.524
<0.02
0.37-0.42
0.43-0.46
1.60-1.83
0.04-0.050
0.03-0.054
0.003
21-24
17-23
8-10
0.031-0.040
0.012-0.015
0.026-0.032
0.025-0.027
0.006-0.013
0.015-0.022
<0.068
<0.011
2.04-2.65
<0.067
<0.011
<1.37
<0.068
0.021-0.026
<1.37
Ammonia -NH3
before addition of lime
after addition of lime
well water
Graph 1. Level of ammonia in well water and in the pond water before and after addition of lime
316
Table2. Chemical and fatty acid composition of produced carp
Parameter
Water content (%)
Protein content (%)
Fat content (%)
Ash content (%)
Fatty acid, %
C12:0
C14:0
C15:0
C16:0
C17:0
C18:0
C20:0
SFA
C16:1, n-9
C18:1cis-9, n-9
C18:1cis-11, n-7
C20:1, n-9
MUFA
C18:2, n-6
C18:3, n-6
C18:3, n-3
C20:2, n-6
C20:3, n-6
C20:3, n-3
C20:4, n-6
C20:5, n-3
C22:5, n-3
C22:6, n-3
PUFA
n-6
n-3
n-3/n-6
n-6/n-3
PUFA/SFA
USFA/SFA
PUFA/MUFA
Carp
78.36 ±0.24
16.21 ±0.12
4.42 ±0.17
1.02 ±0.02
0.06 ±0.01
1.53 ±0.25
1.11 ±0.08
18.35 ±0.28
1.32 ±0.09
4.51 ±0.12
0.26 ±0.03
27.15 ±0.38
5.73 ±0.28
19.39 ±0.21
2.33 ±0.27
1.35 ±0.12
28.79 ±0.48
10.29 ±0.11
5.22 ±0.06
5.96 ±0.14
0.33 ±0.07
0.91 ±0.06
0.89 ±0.05
6.21 ±0.11
4.05 ±0.08
4.47 ±0.24
5.75 ±0.48
44.08 ±0.55
22.96 ±0.20
21.12 ±0.48
0.92 ±0.02
1.09 ±0.02
1.62 ±0.04
2.68 ±0.05
1.53 ±0.04
Discussion
The results of the growth indicate that it is possible to breed carps in ponds where remediation has
been performed and where there is a lot of organic material, which could not be completely
eliminate by removing the surface layers of soil. The favourable weight of the carp at harvesting is
the result of good environmental conditions that were based on the use of well water, adequate flow,
aeration, use of hydrated lime, quality of complete feed.
317
The accumulation of contaminants in the fish tissues depends on their concentration in the water,
the lifetime of the fish, the age and the amount of body fat of fish (Bordajandi et al., 2006). Direct
transfer of pollutants is most common from the environment in the sediment and from the sediment
into the aquatic environment and aquatic organisms. State of the ecosystem, water and sediment
directly affects the quality and safety of fish meat (Zoumis et al., 2001). There is a possibility of
mobilization of pollutants in meat and organs of fish (Balter and Lecuyer, 2010), which in the pond
can reach as a result of pollution of the immediate surroundings. The degree of contamination of
water and harvested fish from an ecosystem can indirectly serve as a bioindicator of the degree of
contamination of the ecosystem (Jankovic et al., 2011). Despite the removed soil, organic
production was high as well as the amount of ammonium, which was balanced with the constantly
adding of hydrated lime (the amount which is higher than usual). This helped to keep environmental
conditions in the optimum level for the cultivation of fish, so we had no problem with the smaller
intake of food, lack of oxygen level and higher mortality of fish. The results of the analysis of the
water from fish pond explain the reasons for it being built on remediated soil.
In recent years, increased attention has been paid to the fact that the ponds create a large amount of
nitrogen, phosphorus and organic matter in the water. Nutrients which may cause eutrophication of
the water, coming either directly from the food, or coming from fish excrement (Watanabe et al.
1999). The easiest way to reduce the intake of these substances in the water, is usage of sufficiently
stable food in the water which contains nutritional components that fish can use maximum (Cho and
Bureau, 2001). All these facts are taken into account during the breeding of fish, so the diet was
carried out using a complete feed for carp. The results indicate that this type of diet achieved more
than good production results and the amount of pollutants in the analyzed water was minimal. By
analyzing the economic parameters it can be seen that they are favourable due to good conversion.
The meat quality of carp fish is very variable and changes under the influence of age, breeding
systems and nutrition. The fat content in the carp generally ranges from 2.3 to 16.8%, while the
protein content is less variable and generally is in the range of 14 to 18% (Vladau et al., 2008;
Trbović et al., 2009; Ćirkovic et al., 2011). Protein and fat content (16.2; 4.4) of analyzed carp
move within the following limits and is very favourable. The results of SFA and USFA obtained in
this study are comparable with the results of other authors. The ratio of n-3/n-6 ranged from 0.8 to
2.4 according to Steffens and Wirth (2007). The ratio of PUFA/SFA which is an indicator of the
quality of lipids was also beneficial (1.62). As the favourable fatty acid composition of carp
affected the entire mixture and mix of grains and liver, spleen and lung, It has been found that the
complete mixture has a very favourable effect on the quality of the fish meat (Ljubojević et al.,
2013).
Conclusions
It is possible to produce fish on land where remediation has been conducted.
Continuously monitoring of the environmental conditions during the entire process of production is
necessary.
Using well water has an advantage over the water from open water courses, due to its favorable
chemical composition. Water flow and aeration are required in a system with a higher organic load
of water. The use of hydrated lime is a limiting factor in fishery production in organic loaded water.
High production is the result of good nutrition and good environmental conditions. Favorable
conversion per kg was the result of quality balanced meals.
The results of chemical composition of carp meat shows that a very high-quality food is produced.
318
In monitoring the fish’s health status and adequate therapeutic measures, implementation is very
important in intensive production, especially in the ponds where a high level of organic matter is
expected.
Remediation contributes to environmental protection and rational usage of land area.
Acknowledgments
This work was supported by grants from the Ministry of Education, Science, and Technological
Development of the Republic of Serbia (project no. TR31011).
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Invited lecture
RAPID DETECTION OF IMPORTANT CARP VIRUSES BY LOOP-MEDIATED
ISOTHERMAL AMPLIFICATION (LAMP)
Vladimir Radosavljevic1*, Dragana Ljubojevic2, Vesna Milicevic1, Miroslav Cirkovic2, Dobrila
Jakić-Dimić1, Jelena Maksimovic-Zoric1, Jadranka Zutic1
1. Institute of Veterinary Medicine of Serbia, Belgrade, Serbia
2. Scientific Veterinary Institute, “Novi Sad”, Novi Sad, Serbia
* Corresponding author: vladimiradosavljevic@yahoo.co.uk
Abstract
Nucleic acid amplification is a valuable tool for the diagnosis of fish diseases. Several amplification
methods are available. Among these, PCR is the most widely used in various forms such as reverse
transcription PCR (RT-PCR), nested PCR and multiplex PCR. These PCR-based methods require
either high precision instruments for amplification or elaborate methods for detection of the
amplified products. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid
diagnostic method that can rapidly amplify a target region under isothermal conditions. The LAMP
reaction employs a Bst DNA polymerase and a set of four specific primers that recognize a total of
six distinct sequences on the target DNA. The most significant advantage of LAMP is the ability to
amplify specific sequences under isothermal conditions between 63°C to 65°C, thereby obviating
the need for a thermal cycler. Therefore, there is no time loss in thermal changes, and the
amplification efficiency of the LAMP method is extremely high. In this study, we used a LAMP
method for the detection of koi herpesvirus and spring viremia of carp virus, and evaluated its
sensitivity, specificity, and convenience. For the detection of koi herpesvirus we used a LAMP
method published by Gunimaladevi et al., and RT-LAMP protocol targeting the G-protein of the
virus for detection SVCV developed by Shivappa et al. was used. LAMP was carried out and the
reaction products are observed directly, after the addition of SYBR Green I. LAMP protocol is
rapid, sensitive, specific and convenient for detection of KHV DNA. The test requires only a heat
source and is completed in one hour. Considerably less time is required to obtain a result using
SYBR Green I stain, compared with traditional gel electrophoresis.
Keywords: LAMP, carp, virus detection
Introduction
Fish diseases are a constant threat to the sustainability and economic viability of aquaculture. Early
diagnosis plays a vital role in management of fish diseases. Traditionally, various biochemical and
serological tests have been used for fish disease diagnosis. However, the time and expertise required
for such diagnoses makes it difficult to easily adopt under production conditions. Traditional
methods for identifying pathogens are mainly based on phenotypic characteristics. Nucleic acidbased detection assays have also been used for the detection of pathogens. Various techniques,
including restriction digestion of nucleic acid, nucleic acid hybridization and DNA amplification
technology are currently being used. Polymerase chain reaction (PCR) and real-time PCR have
gained a wide popularity as a diagnostic tool. The advantage of PCR over other nucleic acid
techniques is the ease of development of the detection system, its high sensitivity and rapidity.
The recently designated cyprinid herpesvirus 3 (CyHV-3) is an emerging agent that causes fatal
disease in common and koi carp. This highly contagious pathogen has caused severe financial
losses in common and koi carp culture industries worldwide (Gilad et al. 2002; Perelberg et al.2003,
Hedrick et al. 2000). CyHV-3 has the largest genome among viruses in the order Herpesvirales and
321
serves as a model for mutagenesis of large DNA viruses. Spring viraemia of carp (SVC) is a
rhabdovirus infection capable of inducing an acute haemorrhagic and contagious viraemia in several
carp species and of some other cyprinid and ictalurid fish species. The aetiological agent of SVC is
spring viraemia of carp virus (SVCV), a member of the virus family Rhabdoviridae, and tentatively
placed in the genus Vesiculovirus (Fauquet et al. 2005). A variety of assays have been developed to
detect the presence of SVCV. The World Organization for Animal Health (OIE) recommended
diagnostic tests for SVCV include virus culture, reverse transcriptase (RT) PCR, enzyme-linked
immunosorbent assay (ELISA), immunofluorescent antibody technique (IFAT) and in situ
hybridization. However, detection of SVCV by PCR or serological detection must be confirmed by
virus culture.
Loop-mediated isothermal amplification (LAMP) amplifies nucleic acid with high sensitivity,
specificity and rapidity under isothermal conditions (Mori et al. 2001). The LAMP reaction
employs a Bst DNA polymerase and a set of four specific primers that recognize a total of six
distinct sequences on the target DNA. The most significant advantage of LAMP is the ability to
amplify specific sequences under isothermal conditions between 63°C to 65°C, thereby obviating
the need for a thermal cycler (Mori et al. 2001). Therefore, there is no time loss in thermal changes,
and the amplification efficiency of the LAMP method is extremely high (Pillai et al. 2006). Several
reports on LAMP-mediated diagnostic methods have been developed for viral pathogens in
aquaculture. In this study, we used a LAMP method for the detection of koi herpesvirus and spring
viremia of carp virus, and evaluated its sensitivity, specificity, and convenience.
Virus strains and cell lines
SVCV reference strain, infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic
septicemia virus (VHSV), PFRV and infectious pancreatic necrosis virus (IPNV) were obtained
from EURL for Fish Diseases, Copenhagen, Denmark. Other SVCV strains were isolated and
preserved in our laboratory. The koi herpes virus was isolated from a koi carp by Hedrick et al.
(2000), generously provided by Dr S. M. Bergmann (Friedrich Loeffler Institute, Germany). The
CCB (Common carp brain) and EPC (Epithelioma papulosum cyprini) cell lines were used to
propagate the aforementioned viruses.
DNA/RNA extraction from cell culture
Viral RNA was extracted from cell cultures infected with different viruses using QIAamp RNA
Mini Kit according to the manufacturer’s recommendation. The viral DNA was isolated by QIAamp
DNA Mini Kit according to the manufacturer’s recommendation.
SVCV RT-LAMP reaction conditions
The RT-LAMP assay was carried out in a total mixture volume of 25 μL, using primers by
Shivappa et al. (2008). The reaction mixture contained 40 nmol l-1 each of inner primers SVCVFIP and SVCV BIP, 5 nmol l-1 each of outer primers SVCV-F3 and SVCV-B3, 35 μL of 20 μmol
l-1 dNTP mix, 5 μl of 5 mol l-1 betaine (Sigma-Aldrich, St Louis, MO, USA), 0,6 μl of 20 μmol l-1
MgSO4, 5 μl of 8 U Bst (Bacillus stearothermophilus) DNA polymerase (large fragment; New
England Biolabs Inc., Beverly, MA, USA), 25 μL of 10 x PCR buffer, 1 μL of 40 U of AMV
Reverse transcriptase (Promega, Madison, WI, USA) and 25 μL template RNA.The mixture was
incubated at 64°C for 60 min, and then the reaction was terminated by heating at 80°C for 2 min.
2μl LAMP products were electrophoresed on 2.0% agarose gel.
Aliquots of 6 μL LAMP reaction products were subjected to electrophoresis with 2% agarose gel
and analysed under a gel ultraviolet bioimage analyser after the gel was stained by ethidium
322
bromide. Alternatively, the reaction products were analysed by the colour change upon treatment
with a SYBR Green I (Invitrogen, CA, USA).
KHV LAMP reaction conditions
The LAMP was carried out in a 25 μL reaction volume, using primers by Gunimaladevi et al.
(2004). The reaction mixture contained 2 μL (40 pmol) of each KHV- FIP and -BIP, 1.0 μL (5
pmol) of KHV-F3 and -B3, 12.5 μL of 2X reaction mixture (40 mm Tris-HCl, 20 mm KCl, 16 mm
MgSO4, 20mm (NH4)2SO4, 0.2% Tween-20, 1.6 m betaine and 2.8 mm dNTPs each), 1.0 μL of
target DNA and 3.5 μL of distilled water. The mixture was heated at 95oC for 5 min, then chilled on
ice and 1.0 μL (8 U) of Bst (Bacillus stearothermophilus) DNA polymerase (large fragment; New
England Biolabs Inc., Beverly, MA, USA), was added. The mixture was incubated at 65°C for 60
min, and then the reaction was terminated by heating at 80°C for 2 min. Aliquots of 6 μL LAMP
reaction products were subjected to electrophoresis with 2% agarose gel and analysed under a gel
ultraviolet bioimage analyser after the gel was stained by ethidium bromide. Alternatively, the
reaction products were analysed by the colour change upon treatment with a SYBR Green I
(Invitrogen, CA, USA).
Specificity test of SVCV RT-LAMP
Total RNA was extracted from different virus infected and mock infected cells (SVCV, IHNV,
VHSV, IPNV and PFRV). These samples were used to evaluate specificity of the RT-LAMP assay.
Specificity test of KHV LAMP
Total RNA was extracted from different virus infected and mock infected cells (CyHV-1, CyHV-2,
CyHV-3). These samples were used to evaluate specificity of the RT-LAMP assay.
Sensitivity test of SVCV RT-LAMP
Ten-fold serial dilutions (10-1 to 10-7 diluted) of RNA extracted from SVCV-infected EPC cells
were used as templates for RT-LAMP following optimized conditions. The same diluted RNA
samples were also detected using nested RT-PCR as a control method to evaluate the sensitivity
compared with the RT-LAMP assay. The first step PCR was performed using an outer primer set
5’-TCT-TGG-AGC-CAA-ATA-GCT-CAR*-R*TC-3’ (SVCV F1) and 5’-AGA-TGG-TAT-GGACCC-CAA-TAC-ATH*-ACN*-CAY*-3’ SVCV R2), yielding a PCR product with 714 bp. The
second step PCR was carried out with an inner primer set (5’-TCT-TGG-AGC-CAA-ATA-GCTCAR*-R*TC-3’ (SVCV F1) and 5’-CTG-GGG-TTT-CCN*-CCTCAA-AGY*-TGY*-3’ (SVC R4),
yielding a PCR product with 606 bp.
Sensitivity test of KHV LAMP
Ten-fold serial dilutions (10-1 to 10-7 diluted) of DNA extracted from KHV-infected CCB cells were
used as templates for LAMP following optimized conditions. The same diluted DNA samples were
also detected using PCR as a control method to evaluate the sensitivity compared with the LAMP
assay. The PCR was performed using an primer set 5’-GGG-TTA-CCT-GTA-CGA-G-3’ (KHV
TKf) and 5’-CAC-CCA-GTA-GAT-TAT-GC-3’ KHV TKr), yielding a PCR product with 409 bp.
Results
Specificity of SVCV RT-LAMP assay
To confirm the specific amplification, the RT-LAMP was first used to check the five different
SVCV strains. A characteristic of ladder pattern was detected from the amplified products of both
323
SVCV reference strain and virus strains isolated in Serbia. The cross-amplification of 200 ng each
of RNA of other fish disease viruses (IHNV, VHSV, IPNV and PFRV) was also evaluated to
determine the specificity of RT-LAMP method. It is shown that neither of these RNA extracted
from corresponding virus infected cells was positive by this method. This result indicates that RTLAMP assay is excellently specific to SVCV.
Specificity of KHV LAMP assay
To confirm the specific amplification, the RT-LAMP was first used to check the three different
KHV strains. A characteristic of ladder pattern was detected from the amplified products of both
KHV reference strain and virus strains isolated in Germany. The cross-amplification of 200 ng each
of DNA of other fish disease viruses (CyHV-1, and CyHV-2) was also evaluated to determine the
specificity of LAMP method. It is shown that neither of these DNA extracted from corresponding
virus infected cells was positive by this method. This result indicates that LAMP assay is
excellently specific to KHV.
Sensitivity of SVCV RT-LAMP assay
To compare the sensitivity of detection limit, we carried out RT-LAMP and semi-nested RT-PCR
using the same 10-fold serial dilutions of SVCV RNA. RT-LAMP was able to detect template at
10-6 dilution, same as the semi-nested RT-PCR. Therefore, the detection sensitivity of RT-LAMP
was equal to the semi-nested RT-PCR.
Sensitivity of KHV LAMP assay
To compare the sensitivity of detection limit, we carried out LAMP and PCR using the same 10fold serial dilutions of KHV DNA. LAMP was able to detect template at 10-7 dilution, while PCR
could only detect the presence of the virus up to the 10-5 dilution. Therefore, the detection
sensitivity of RT-LAMP was 100-fold higher than the PCR.
(a)
(b)
Figure 1. Sensitivity test for loop-mediated isothermal amplification (LAMP) and PCR using the same
tenfold series dilutions of koi herpes virus (KHV) DNA as templates. (a) Result of agarose gel
electrophoresis of LAMP products. (b) Result of SYBR green I dye of LAMP products. M: molecular
marker; lane 1: nondiluted DNA extracted from KHV-infected cells; lanes 2–8: 10-1 to 10-7 dilutions of DNA
extract from KHV-infected cells.
324
Spring viremia of carp and koi herpesvirus disease are the most important epidemic diseases for the
carp industry. At present, the various diagnostic techniques used for the detection of SVCV and
KHV include sell isolation on cell culture, histopathology, viral isolation, in situ hybridization,
electron microscopy, ELISA, nested PCR and real-time PCR. The PCR approach has been found to
be a useful technique for SVCV and KHV diagnosis. LAMP is a novel method that facilitates rapid
nucleic acid amplification using only simple equipment. LAMP is also a sensitive method which
can amplify a few DNA copies in less than 1 hour under isothermal condition (Shivappa et al 2008).
The amplification efficiency of the LAMP method is high because there is no time loss for thermal
change due to its isothermal reaction. The LAMP reaction has been successfully established in
diagnosing viral infection in several species of mammals, plants, fish and shellfish (Soliman and ElMatbouli, 2006). In this study, the LAMP diagnostic protocols were carried out for the detection of
SVCV and KHV. The LAMP reaction conditions were optimized by incubation of the target DNA
with the specific four primers at 65°C for 60 min. The amplified products appeared as a ladder-like
pattern on the gel due to the cauliflower-like structures (Notomi et al. 2000).
In conclusion, the LAMP protocols are rapid, sensitive, specific and convenient for detection of two
important carp viruses, SVCV and KHV. The test requires only a heat source reactor and is
completed in 1 h compared with 3-4 h for PCR. Considerably less time is required to obtain a result
using SYBR Green I stain, compared with traditional gel electrophoresis.
Acknowledgments
This research was funded within the project, TR 31011, TR 31075 by the Ministry of Education,
Science and Technological Development, Republic of Serbia.
References
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Fauquet C.M., Mayo M.A., Maniloff J., Desselberger U. & Ball L.A.: Virus Taxonomy. Eighth Report
of the International Committee on Taxonomy of Viruses Academic Press, London, UK, 2005.
Gilad O., Yun S., Andree K.B., Adkison M.A., Zlotkin A., Bercovier H., Eldar A., Hedrick R.P.: Initial
characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the
virus in koi, Cyprinus carpio koi. Diseases of Aquatic Organisms, 48, 101–108, 2002
Gunimaladevi I., Kono T., Venugopal M.N., Sakai M.: Detection of koi herpesvirus in common carp,
Cyprinus carpio L., by loop-mediated isothermal amplification. Journal of Fish Diseases, 27, 583–589,
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Hedrick R.P., Gilad O., Yun S., Spangenberg J.V., Marty G.D., Nordhausen R.W., Kebus M.J.,
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amplification reaction by turbidity derived from magnesium pyrophosphate formation” Biochemical and
Biophysical Research Communications, 289, 150-154, 2001
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T. “Loopmediated isothermal amplification of DNA”Nucleic Acids Research, 28, 63, 2000
Perelberg A., Smirnov M., Hutoran M., Diamant A., Bejerano Y., Kotler M.: Epidemiological description of a new viral disease afflicting cultured Cyprinus carpio in Israel. Israeli Journal of AquacultureBamidgeh, 55, 5–12, 2003
Pillai, D., Bonami, J. R.,

First International Symposium of Veterinary Medicine – ISVM2015

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