Naturally Lactococcus garvieae infection in rainbow trout

12
DIDINEN (B.I.) AND COLLABORATORS
Naturally Lactococcus garvieae infection in rainbow
trout (Oncorhyncus mykiss Walbaum, 1792): new
histopathological observations, phenotypic and
molecular identification
B.I. DIDINEN1, B. YARDIMCI2*, E.E. ONUK2, S. METIN1*, P. YILDIRIM1
Suleyman Demirel University, Egirdir Fisheries Faculty, Isparta, Turkey
Ondokuz Mayıs University, Faculty of Veterinary Medicine, Department of Diseases of Aquatic Animals, Samsun, Turkey
1
2
*Corresponding author: byardimci@omu.edu.tr or banuyardimci@gmail.com
SUMMARY
RESUME
The disease outbreak occurred in rainbow trout (15-107g) at an average water
temperature of 13.5°C during september-december 2012. The cumulative
mortality rate was approximately 15 % within 4 months. The most typical
symptoms in infected fish were marked mono and bilateral exophthalmos,
opacity and thickening, along with pseudomembrane formation in swimbladder and bulbus arteriosus walls described for the first time during an
epizootic outbreak of lactococcosis, petechial and focal haemorrhages in
viscera, enlarged liver and spleen. 25 bacterial isolates from sick fish were
identified as Lactococcus garvieae by morphological, physiological, and
biochemical features and confirmed by PCR using species-specific primers.
Further more, 16S rRNA gene of one isolate (KC4) was partially sequenced
and showed 100 % identity with the Genbank sequences of L. garvieae.
According to the histopathological examination, severe panophthalmitis,
peri and myocarditis and gastritis were described. Pathological findings were
also observed in kidney and liver. Subcapsular and sinuzoidal mononuclear
cell infiltration and necrosis of the hepatocytes has not been described
previously for lactococcosis. Moreover, this is the first report of a focal
mononuclear cell infiltration in the lamina propria of stomach in some fish.
In conclusion, this study presents interesting and original data on the
caracterization of L. garvieae isolated from rainbow trout, particularly new
histopathological observations.
Infection naturelle de truites arc en ciel (Oncorhynchus mykiss
Walbaum, 1792) par Lactococcus garvieae: Nouvelles observations
histopathologiques, phénotypiques et identification moléculaire
Key words : Lactococcus garvieae, rainbow trout
histopathology, molecular identification
L’épidémie est survenue chez la truite arc en ciel (15-107g) à une
température d’eau moyenne de 13,5 ° C en septembre-décembre 2012. Le
taux de mortalité cumulée a été d’environ 15% en 4 mois. Les symptômes
les plus typiques des poissons infectés ont été des exophthalmies mono et
bilatérales, une opacité et un épaississement, avec la formation de fausses
membranes, au niveau de la vessie natatoire et des parois du bulbe artériel,
des pétéchies et hémorragies localisées dans les viscères, le foie et la rate. Il
s’agit de la première description de la formation de pseudomembranes sur
la vessie natatoire et les parois du bulbe artériel au cours d’une épizootie de
lactococcose. 25 isolats bactériens obtenu à partir des poissons malades ont
été identifiés d’après leurs caractéristiques morphologiques, physiologiques
et biochimiques, comme étant des souches de Lactococcus garvieae ce qui a
été confirmé par PCR en utilisant des amorces spécifiques de cette espèce.
Le gène de l’ARNr 16S d’un iisolat (KC4), qui a été partiellementséquencé,
partage 100% d’identité avec les séquences de L. garvieae disponibles dans
GenBank. Selon l’examen histopathologique, des panophtalmies sévères,
des péricardites, des myocardites et des gastrites ont été décrites. Des
anomalies ont également été observées dans les reins et le foie. L’infiltration
sous-capsulaire et sinuzoïdale de cellules mononucléaires et la nécrose des
hépatocytes n’ont pas été décrites précédemment pour la lactococcose. En
outre, l’infiltration focale de cellules mononucléaires a été signalée dans la
lamina propria de l’estomac chez certains poissons.
En conclusion, l’identification moléculaire, phénotypique, et la sensibilité
aux antimicrobiens de L. garvieae isolé de la truite arc en ciel et de nouvelles
observations histopathologiques au cours épidémie de lactococcosis en
Turquie a été réalisée dans cette étude.
Mots clés : Lactococcus garvieae, truite arc en ciel,
histopathologie, identification moléculaire
Introduction
Lactococcus garvieae, the etiological agent of
lactococcosis, causes significant economic losses both
in marine and freshwater aquaculture all over the World
[20,37]. Lactococcosis in cultured fish have been reported
in several countries, such as United States, Australia, South
Africa, Japan, Korea, Taiwan, Iran, England, Italy, Spain,
Israel, France, Bulgaria, Greece and Portugal [8,9,10,13,1
4,18,20,21,25,30,32,34,37] and was regularly observed in
rainbow trout since 2001 in Turkey [3,6,12,16,24,29,35].
The streptococcal/lactococcal infections usually cause
highmorbidity and mortality and last a long period of time in
the farmed fish [5]. The losses from lactococcosis produced
can exceed approximately 50–80% of the total production
in rainbow trout [38]. Lactococosis regularly reoccurred,
especiallyduring the warm summer months. Therefore, L.
garvieae is now considered as one of the most important
pathogens at in the rainbow trout industry in Turkey [3].
The isolation of the disease in human showing its zoonotic
character is also important. The studies were showed the
Revue Méd. Vét., 2014, 165, 1-2, 12-19
HISTOPATHOLOGY OF LACTOCOCCUS GARVIEAE INFECTION
ability of L. garvieae to cause late prosthetic, heart bypass
grafting or joint prothesis, infection in patient [4,22,27] .
L. garvieae causes hemorrhagic septicaemia, enteritis,
ascites, bilateral exophthalmus with haemorrhage; darkening
of the skin; congestion of the intestine, liver, kidney, spleen, and
brain; and hemorrhagic enteritis in farmed trout [11,16,17,31].
There is a few published data on the histopathological lesions
associated with lactococcosis. The main histopathological
findings in infected fish are epicarditis, peritonitis, enteritis,
meningitis and panophthalmitis [6,14,15,19].
The identification of L. garvieae can be performed by
conventionally microbiological methods and API diagnostic
kits [2,11,13]. PCR technique in identification of L. garvieae
strains comes to the forefront [1,3,19,33,34,36,39].
The disease can be treated with chemotherapeutics,
but selection of efficient antimicrobial agents is sometimes
complex due to resistant strains. L. garvieae isolates in Turkey
are totally resistant to gentamycin, neomycin, lincomycin,
sulfamethoxazole-trimethoprim, which are very common
in Turkey; moreover, resistance is developed against
oxytetracycline, erythromycin, amoxicillin, florfenicol and
doxycycline to a great extent [3].
In this article, we describe the lactococcosis of rainbow
trout raised during september-december 2012 in Turkey. L.
garvieae was isolated as the causative agent of the disease
and identified. The morphological, biochemical and cultural
characteristics of L. garvieae, its identification by 16 S rRNA
sequence, and its histopathology are described.
Materials and Methods
FISH SAMPLING
A total of 20 rainbow trout (body weight 15-107g )
suspected of showing the clinical signs of the disease were
collected from a farm in the Mediterranean region of Turkey
during september-december 2012. The land-based farm has
hatchery unit and concrete ponds with a total production
capacity of 450 tons per year.
ISOLATION AND IDENTIFICATION OF BACTERIA
For bacterial isolation, samples were obtained from the
kidney, liver, spleen, brain and eye of each fish, streaked on
trypticase-soy agar plates (TSA, Merck), and incubated at
25°C for 48 h. Single colonies were restreaked on the same
media to obtain pure isolates. 25 isolates obtained from ten
sick fish were identified by morphological, physiological,
biochemical, and enzymatic characterization. For long-term
preservation, cultures were frozen at -70 oC in trypticase-soy
broth (TSB, Merck) with 15 % (v/v) glycerol.
A presumptive identification of the isolates was
performed using the following tests: Gram staining and
Revue Méd. Vét., 2014, 165, 1-2, 12-19
13
cell morphology, motility, oxidase and catalase activities,
oxidative and fermentative degradation of glucose with O/F
basal medium (Merck) supplemented with 1% glucose, H2S
production on triple-sugar iron medium (TSI, Merck), gas
from glucose in TSI, production of hemolysin on Columbia
sheep blood agar (bioMerieux), caseinase on TSA with skim
milk, amylase on TSA with starch and lipase on Tributyrin
Agar (Merck) with Glycerol tributyrate (Merck), citrat
utilization on Simmon Sitrat Agar (Merck), methyl red/ VP
on MR-VP broth, growth at different temperatures (4, 15, 37,
42 and 45°C) on TSB and growth on TSB broth containing
0,3,5 and 8 % NaCl (Merck) and MacConkey agar (Merck).
Additional tests were performed using API 20 E and API 50
CH systems (bioMerieux) [5,23].
DNA EXTRACTION, PCR AMPLIFICATION AND SEQUENCING
Genomic DNA of isolates was extracted from pure
subcultures using DNeasy Blood & Tissue kit (Qiagen,
GmbH) according to the manufacturer’s instructions.
Molecular identification of isolates was carried out
using the L. garvieae specific oligonucleotide primers
pLG-1 (5’-CATAACAATGAGAATCGC-3’) and pLG2 (5’-GCACCCTCGCGGGTTG-3’) [39]. The The PCR
reaction mixture containing, 1xPCR Buffer, 1.5 mM of MgCl2,
0.2 mM each dNTP, 1.0 U Taq polymerase (Fermentas),
1µM each primer and 5 µl template DNA in DEPC-treated
water (Sigma) to a final volume of 25 μl. Thirty five cycles of
amplification were performed in a Thermo PxE 0.2 thermal
cycler (Thermo Scientific) after an initial denaturation step at
95°C for 3 min. Each cycle consisted of a denaturation step at
94°C for 1 min, an annealing step at 55°C for 1 min, and an
extension step at 72°C for 1,5 min, with a final extension at
72°C for 10 min. The expected size of the PCR product was
1100 bp. All the PCR products were electrophoresed in 1,5 %
agarose gel containing ethidium bromide and then visualized
using an UV transilluminator. L. garvieae ATCC 43921 and
V. salmoninarum NCIMB 13133 were used as a positive and
negative controls, respectively.
The partial 16S rRNA gene of the isolate KC4
was amplified using the universal primers 518F
(5’-CCAGCAGCCGCGGTAATACG-3’)
and
800R
(5’-TACCAGGGTATCTAATCC-3’). The PCR products were
purified and sequencing on single strand using 518F primers
by a commercial sequencing company (Iontek Istanbul,
Turkey). The sequences were compared with reference
sequences published in the GenBank database using the
Basic Local Alignment Search Tool (BLAST).
ANTIMICROBIAL SENSITIVITY
Antimicrobial tests were performed on the kidney isolate
using disc diffusion assay on Mueller–Hinton agar (Oxoid).
National committee for clinical laboratory standards
(NCCLS) were used for the evaluation of the results [28].
14
HISTOPATHOLOGICAL EXAMINATION
Following the necropsy, tissue samples from skin, gill,
muscle, liver, kidney, spleen, heart, stomach, intestine, brain,
eye and gonads were taken for histopathological examination
and fixed in 10% neutral formaldehyde solution. Tissue
samples taken are routinely processed and embedded in
paraffin. Tissue sections of 4-6 µ in width were stained
with haematoxyline-eosin (HE) and examined under light
microscope (Eclipse E600, Nikon).
Results and Discussion
The disease outbreak occurred in rainbow trout weighing
53.75±40.77g in a farm at an average water temperature of
13.5°C during September-December 2012. Cumulative
mortality attributed to this pathogen was 20 % within 4
months. Monthly mortality rates were almost equal. The
outbreak was associated with under stressful conditions,
including overcrowding and poor water quality.
DIDINEN (B.I.) AND COLLABORATORS
previously described by other studies [14,16,18,26,32,34].
Pseudomembrane like formation of the intestines, kidney and
spleen for lactococcosis was reported by Eldar and Ghittino
(1999) and Chen et al. (2002). However, to our knowledge,
this is the first observation of a pseudomembrane formation
on swim-bladder and bulbus arteriosus walls during an
epizootic outbreak of lactococcosis.
Phenotypic characteristics of isolated L. garvieae strains
were found highly homogeneous. However, differences were
observed in hippurate hydrolysis and acid production from
melibiose, ribose and mannitol among L. garvieae isolates
(Table 1). Different results were reported about utilization
of hippurate, β-glucuronidase, citrat, VP, pyrrolidonyl
arylamidase, urease, acid from D-mannose, D-lactose,
D-ribose, sorbitol and raffinose by L. garvieae strains
[3,12,34].
In PCR assay targeting the 16S rRNA gene, PCR products
of the expected sized (1100 bp) were observed in all L.
garvieae isolates obtained from diseased fish but also for the
reference strain (figure 2).
Infected trout exhibited lethargy, anorexia, dark
coloration, marked mono and bilateral exophthalmos with
periocular haemorrhages (figure 1.A) and eyeball disruption.
The lesions of eyes were similar with findings of Avcı et al.
2013 in experimental immersion administration study.
Figure 2: L. garvieae specific PCR, 1100 bp. M; Marker (100-3000 bp), 1; L.
garvieae ATCC 43921, 2-11; L. garvieae field isolates, 12; V. salmoninarum
NCIMB 13133.
Figure 1: Rainbow trout naturally infected with L. garvieae A. Marked
exophthalmos. B. Focal haemorrhages in liver. C,D. Opacity and thickening,
along with pseudomembrane formation in swim-bladder and bulbus
arterious walls
In the macroscopic examination, transparent fluid
accumulation in the body cavity were found. Skeletal
muscle and liver were seen to be anemic and revealed some
linear congestion (figure 1.B). Opacity and thickening were
observed along with pseudomembrane formation on swimbladder and bulbus arteriosus walls (figure 1.C,D). Liver
and spleen were enlarged. Petechial and focal haemorrhages
were seen in liver, adipose tissue, pyloric seca and muscle.
Bloody gelatinous exudate in the lumens of intestine were
observed. These clinical findings were similar to those
The partial 16S rRNA gene region sequence of isolate
KC4 showed 100 % identity with various L. garvieae
isolates avalaible in Genbank (GenBank accession number
KC333889.1, HF562964.1 and JX975402.1).
Altun et al., (2012) noted that L. garvieae strains in Turkey
developed resistance to oxytetracycline, erythromycin,
amoxicillin and florfenicol. Similary, some of our isolated L.
garvieae strains harbored resistance to oxytetracycline but
displayed sensitivity to amoxicillin, florfenicol and moderate
sensitivity to erythromycin (Table 2).
According to the histopathological examination major
lesions were found to be located in eyes, kidney, heart
Revue Méd. Vét., 2014, 165, 1-2, 12-19
HISTOPATHOLOGY OF LACTOCOCCUS GARVIEAE INFECTION
Characteristics
Colony size
Gram
Shape
Motility
Hemolysis
Oxidase
Catalase
O/F
Urease
Gelatinase
Caseinase
Lipase
Hippurate hydrolysis
Pyrrolidonyl arylamidase
Lysine decarboxylase
Ornithine decarboxylase
α-Galactosidase
β-Galactosidase
β-Glucuronidase
Alkaline phosphatase
Leucine arylamidase
Arginine dihydrolase
Citrat utilization
Indole
Voges-Proskauer
Methyl Red
Gas from glucose
NO3 reduction
Aesculin hydrolysis
H2S production on TSI
Growth in:
at +4˚C
at 15˚C
at 37˚C
at 42˚C
at 45 ˚C
Growth at:
% 1 NaCl
% 2.5 NaCl
% 6.5 NaCl
% 8 NaCl
Growth on McConkey agar
Acid production from:
Ribose
Mannitol
Sorbitol
<2 mm
+
coccus
α
F
+
v
+
+
+
+
+
+
+
+
+
+
+
+
+
+
v
v
-
Characteristics
Acid production from:
Lactose
Trehalose
Inulin
Raffinose
Starch
Glycogen
Glycerol
Erythritol
d-Arabinose
l-Arabinose
d-Xylose
l-Xylose
Adonitol
β-Methylxylidose
Galactose
d-Glucose
d-Fructose
d-Mannose
l-Sorbose
Rhamnose
Dulcitol
Inositol
α-Methylxy-d-mannoside
α-Methylxy-d-glucoside
N-Acetylglucosamine
Amygladin
Arbutin
Salicin
Cellobiose
Maltose
Melibiose
Saccharose
Melezitose
Xylitol
β-Gentiobiose
d-Turanose
d-Lyxose
d-Tagatose
d-Fucose
l-Fucose
d-Arabitol
l-Arabitol
Potassium gluconate
Potassium 2-ketogluconate
Potassium 5-ketogluconate
15
+
+
+
+
+
+
+
+
+
+
+
v
+
+
+
+
-
v, variable character
Table I: Phenotypic characteristics of L. garvieae isolates (n=25) from rainbow trout
and liver. A significant fibroplasia with inflamatory cell
infiltration and vascularization were found in oculer area.
Panophthalmitis was observed in which posterior chamber
(the back section of the eye’s interior), iris and optic nerve
papilla were more significantly involved. Cornea and ciliary
body were thickened more distinctly (figure 3.A,B). Eldar &
Revue Méd. Vét., 2014, 165, 1-2, 12-19
Ghittino (1999) reported that ocular alterations resulted from
a severe haemorrhagic panophthalmitis with destruction of
the anterior and posterior chambers, the optic nerve papilla
was heavily affected and inflammation progressed into the
retrobulbar fat and striated muscle in rainbow trout infected
with L. garvieae and Streptococcus iniae. Chang et al. (2002)
16
DIDINEN (B.I.) AND COLLABORATORS
Antibiotics
Ampicillin (10 µg)
Amoxycillin (25 µg)
Chloramphenicol (30 µg)
Clindamycin (2 µg)
Ciprofloxacin (5 µg)
Enrofloxacin (5 µg)
Erythromycin (15 µg)
Florfenicol (30 µg)
Gentamycin(10 µg)
Kanamycin (30 µg)
Neomycin (10 µg )
Oxytetracycline (30 µg)
Oxacillin (1 µg)
Penicillin G (10 IU)
Rifampicin (5 µg)
Streptomycin (10 µg )
Sulphadiazine (300 µg)
Sulphafurazole (300 µg)
Tetracycline (30 µg)
Tobramycin (10 µg)
Trimethoprim (5 µg)
Trimethoprim(1.25 µg) /sulfamethoxazole(23.75 µg)
L. garvieae isolates (n=25)
Sensitivity * (Zone sizes: mm)
S (17-27)
S (21-23)
R(14-19)
R (0)
R (5-14)
R (11-14)
I (14-20)
S (10-20)
S(10-14)
R (10-12)
R (0)
R (14-15)
R (0)
I (20-25)
R (0)
R (0)
R (0)
R (0)
R (12-15)
R (0)
R (0)
R (0)
*Sensitivity (S: susceptible; R: Resistant; I: Intermediate)
IU: International Unit
Table II: Sensitivities to antibiotics of the L. garvieae strains isolated
noted that lesions were found in periorbital tissue in the
eyes showing extensive fibroplasias with inflammatory cell
infiltration and hemorrhage. In this study, the lesions were
characterized by panophtalmitis, similar with aforementioned
reports. In addition to the ocular findings show similarity
with a group of immersion in the Avcı et al. 2013 study, the
agent, the natural infection through contaminated water was
connected to the skin or taken orally.
melanomacrophage centres in some fish. Chang et al. (2002)
determined that the renal tubules in the trunk kidney had
hyaline droplet deposition in the epithelia and hyaline
casts in the lumens. Apart from these findings, Eldar &
Ghittino (1999) noted a marked reactive hyperplasia of
the haematopoietic tissue in rainbow trout infected with L.
garvieae. We observed similar lesions in this study, however,
tubular necrosis was much more significant.
In heart, especially subpericardial inflamatory cell
infiltration, fibroplasias and free eritrocytes among
myocardial muscles were seen (figure 3.C,D). The epicardial
lesions consisted mainly of inflammatory cells and varying
amounts of fibrin and cellular debris. These infiltrations of
lymphoid cell and activation of the ventricular endothelial
macrophages in myocardium, observed in this study, were
already described by Avcı et al. (2010). In additon, the lesions
characterized by pericarditis were in accordance with Eldar
& Ghittino (1999) and Chang et al. (2002).
In liver, Remak cordons were seen to be irregular and
epitelial cells were dissociated. Parenchymal or vacuolar
degeneration and extensive necrosis of the hepatocytes were
observed. Mononuclear cell infiltration was seen especially in
the subcapsular area, between hepatocytes and perivascular
area (figure 3.G,H). Histopathological, perivascular and
periductular mononuclear cell infiltration and degeneration
of hepatocytes, findings of the liver manifested in this case
was similar with these already described [2,6,19]. In addition,
subcapsular and sinuzoidal mononuclear cell infiltration and
necrosis of the hepatocytes has not been described previously
for lactococcosis.
In kidney, intersititial area was seen to be enlarged with
dense inflamatory cell infiltration and melanomacrophages
were found to be high in point of numbers. Parenchymal
or vacuolar degeneration of tubular epithelial cells and
tubular necrosis was observed. Hyalin drops were also seen
in tubular lumens (figure 3.E). Avcı et al. (2010) already
found degeneration and hyaline droplets in epithelium
of the proximal tubuli in the kidney and increases in
Pathological findings of the stomach reported here was
different from previous observations made by Altun et al.
(2005), especially with the lack of dissociation of the mucosal
epithelium of glands in the stomach. Moreover, we showed
for the first time a focal mononuclear cell infiltration in the
lamina propria of stomach in some fish (figure 3.F.).
Revue Méd. Vét., 2014, 165, 1-2, 12-19
HISTOPATHOLOGY OF LACTOCOCCUS GARVIEAE INFECTION
17
determined for lactococosis by Avcı et al. (2010). In contrast,
histopathological findings of skin, gill, muscle, intestine,
brain and gonads were not significant in this case.
In conclusion, this work presents phenotypic and
molecular identification, antimicrobial susceptibility
of L. garvieae isolated from rainbow trout and new
histopathological observations during lactococcosis outbreak
in Turkey.
References
Figure 3: A. Corneal thickening (asterisk) and general appearance of eye
(HE, 10x). B. New vascular formation (black arrow), dense inflamatory
cell infiltration (white arrows) in the eye and exudation in the anterior
chamber of eye (asterisk); (HE, 30x). C. Subpericardial inflamatory
cell infiltration in heart (black arrow); (HE, 40x). D. Haemorrhages
among myocardial muscles of heart (black arrows); (HE, 40x). E.
Tubular necrosis (black arrows), melanomacrophages (white arrow)
and mononuclear cell infiltration (asterisks) in kidney (HE, 100x). F.
Focal mononuclear cell infiltration in the lamina propria of stomach
(black arrows); (HE, 100x). G. Subcapsular haemorrhage and mononuclear cell infiltration (black arrow) and necrosis of the hepatocytes
(arrow heads); (HE, 100x). H. Mononuclear cell infiltration between
hepatocytes, perivascular and periductular area (black arrows) and
necrosis of the hepatocytes (arrow heads) in liver (HE, 100x).
A few focal necrotic area was also observed in spleen
Altun et al. (2005) also found necrosis and haemorrhagic
foci in the spleen. In contrast, Chang et al. (2002) determined
that diseased fish showing serositis consisting of fibroplasias,
and macrophage and lymphocyte infiltrations in spleen.
Fibrinous meningitis associated with the lactococcosis
have also been described in fish [6,14,19]. Altun et al.
(2005) noted epithelial hypertrophy in the gills with partialy
fusion of the gill lamellae. Avcı et al. (2010) determined
vascular changes including edema, hemorrhage and
telangiectasia, swelling of epithelial cells in gills. Severe
hyperemia, hemorrhages in the villus of the intestines and
infiltration of inflammatory cells in the lamina propria were
Revue Méd. Vét., 2014, 165, 1-2, 12-19
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