Case Report: A Case of Hap/orchis pumilio Infection in Golden

iJ ;fl\\'IX'Ugz Taiwan Vet J 37 (4): 246-252, 2011
Case Report: A Case of Hap/orchis pumilio Infection in Golden
Severum, Heros severus var., in Taiwan
1
Shao-Wen HUNG, 2Pan-Chen L1U, 2Shiun-Long LIN, 2Kwong-Chung TUNG, 2Chen-Hsuan
CHANG, 2Chun-Yang CHAN, *2Way-Shyan WANG
1 Agricultural
2
Biotechnology Research Center, Academia Sinica, Taipei 715, Taiwan
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing
University, Taichung City 402, Taiwan
(Received: November 29, 2010. Accepted: June 28, 2011)
ABSTRACT
Outbr eak of Haplorchis pumilio infestation occurred in a cichlid breeding farm in Taiw an in April, 2009. The farm held about 7,000 golden severum (Heros severus var.), w eighing 50-60
g, in freshwater cult ure ponds. The average temperatu re of weekly changed cult ure w ater was
25-28°C . The fish were fed wi th a commercia l pulverized eel diet. App roximate ly 300 golde n severum
showed signs of poor appetit e, rotte n fins , abnormal pigmentation and rapid respiration rate as we ll
as numerous wh itish cysts, 0.5- 1 mm in diameter , on the surface of the caudal fin. There were an
average number of 40-60 cysts per infected fish. A significant pathogen ic parasite, metacercariae of
Haplorchis pumilio, was identif ied. However, microbiological studies revealed no significant findings.
The pond owner was advised to treat the w ater with 1 ppm benzalkonium chloride (BKC) and 25
ppm forma lin to kill other ectoparasites and bacteria twice a week for a month. We also recomme nded that snails as the host of this parasite should be eradicated artificially and disinfe cted the cultural
bonds using 10 ppm BKC before re-feeding fish. In addition, w e suggested that wild birds, cats and
dogs should be prevented from eating diseased or dead fish . The parasite w as eventually brought
under control. [Hung SW, Liu PC, Lin SL, Tung KC , Chang CH , Chan CY , * Wang WS. Case Report: A
Caseof Haplorchis pumilio Infection in Golden Severum, Heros se verus var., in Taiw an. Taiwan Yet J 37 (4):
246-252, 2011. * Corresponding author Tel : 886-4-22840894 ext 508, Fax: 886-4-22862073, E-mail :
wswang@dragon.nchu.edu.tw]
TAO
Key words: golden severum, Haplorchis pumilio, metacercariae, parasite
INTRODUCTION
cercarial infection can result in mass economic loss.
Some metacercariae in fi sheries and/or aquaculture
The Digenea is one of the three major taxa of
parasitic plat yhelminthes . Man y studies have shown
products (fi sh and shellfis h) may be a source of infection in human and domestic animals [6,23 ,26].
that their adult stage is parasitic to vertebrat es
Hap/orch is pumilio has six alternative names in
[4,18,21]. Aside from bein g hosts to adult digeneans,
current use: Monostomum pumilio, Monorchotrema
fishes may be also infected by the metacercarial larval
taihokui , Monorchitrema taihokui, Hap/orchis milvi ,
Kasr aini and Hap /orchis taihokui (Nishigori) [17].
Hap/orchis pumilio is a trematode and a member of
stage. Metacercariae infestation may decrease the
value of fish as a food or ornamental purpose. Meta-
Hap/o rchis pumilio Infection in Gold en Severum
247
the family Heterophyidae. The sub-family Haplore-
same time , thei r contents were extruded and fixed in
hinae belongs to Digenea, a parasitic worm, whose
larva and metacercarial cysts are found to occur commonly in fish of various sizes and ages in ponds and
tanks [2,5,27].
70% alcohol for 24h. Under microscopic observation,
the parasite was identified based on its morphology
and further investigated.
MATERIALS AND METHODS
Fish and culture condition
Outbreak of a Haplorchis pumilio infestation occurred in a cichlid-breeding farm in Taiwan in April , 2009 . There were about
7,000 golden severum (Heros severus var.), weighing
50-60 g, held in a freshwater pond (depth: 50 ern; area:
330 rrr'). The average weekly water temperature was
25-28 °e . The fish were fed with a commercial pulverized eel diet.
Parasitological examination
The diseased fish
had many whitish cysts on the caudal fin and these
whitish cysts were collected and identifi ed. At the
Microorganism isolation The bacterial cultures
from the gill, liver, kidney and skin surface of ill golden severum were performed. The samples were inoculated onto TSA and blood agar at 20 e for 24-48h,
respectively.
0
Analysis of water quality
Items included water
temperature (T) , dissolved oxygen (DO), pH value, nitrate, ammonia and salinity.
RESULTS
There were about 300 diseased golden severum
(Fig. 1) which were found to have poor appetite, rotten
fins, abnormal pigmentation and increased respiration
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Fig . 1 Many whitish nodules on the surface of caudal fin of golden severum (Heros severus var.) following infection with
Hap/orchis pumilia (arrows).
248
Shao-Wen HUNG et al
rate . The total mortality rate was 4.29% (300/7 ,000) .
The sick fish had many whitish cysts, 0.5-1 mm in di-
developed to be mature. According above observation
results, the parasite was classified as metacercariae of
amet er, on the surfa ce of the caudal fin (Fig. 1). The
average num ber of cysts was 40-60 per infected fish .
A significant pathogenic parasite was identified based
on the armature of the ventro-genital complex. We observed that the cyst was oval with a fine and transparent wall. The cyst had body brown color metacercaria
covered with spines (Fig . 2A and Fig . 3B) . A well developed dige stive system and a small oral suck er were
observed und er a microscope (Fig . 2B, C and Fig . 3A) .
Its mouth open ed to a mid oral sucker. Its short prepharynx opened to a muscular pharynx and a short
esophagus (Fig. 3A , B). There were two arched intestinal ceaca to be found (Fig . 2B and Fig. 3B). Moreover, we found that some metacercariae owned a light
green di sc (or coin shaped disc) insid e the inte stinal
H ap/orchis pumilio . However, there were no signifi-
caeca. Testes were transversely elongated (Fig. 2D) .
The rest of the reproductive system was still not well
cant microbiological findings. In analysis of water
quality of the disease fish pond , low dissolved oxy gen
level and high nitrate and ammonia were found in culture water (Table I).
DISCUSSION
Mass eco nomic loss in the aq uaculture industry
may be attributed to one or more factors including
misuse of drugs, high density culture, environmental
polluti on and infectious/noninfectious diseases [7,20].
High mortality rates among cultured fishes are often
assoc iated with relatively crowded and confined co nditions. It has been well established that environmental factors such as dissolved oxygen level, organic materials and pat hogens play roles in the immune re-
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Fig. 2 Hap/orchis pumi/io metacercariae and cyst. (A) The longitudinal section of the cysts and Hap/orchis pumllio metace rcariae inside (scale bar = 0.3 mm). (8) Hap/orchis pumi/io metacerca riae shedding its cyst (scale bar = 0.3 mm). (e) The
anterior head of Hap/orchis pum illo metacercariae (scale bar = 0.1 mm). (D) The poste riortail of Hap/orchis pumi/io metacercar iae (scale bar = 0.1 mm). Abbreviations are as follows: t-testis; os-oral sucker; c-cyst.
Hap/orchis pumilio Infection in Golden Severum
249
Fig. 3 The hand-drawn figure of the Hap/orchis pumilio metacercariae and cyst. (A) The cyst. (8) The Hap/orchis pumilio
metacercariae. Abbreviations are as follows : m-mouth; os-oral sucker; pp-prepharynx; mp-muscular pharynx ; sp-spine; iintestinal ceaca; t-testis
sponse of cultured fish, and biologic density also appears to have a significant effect on fish immunology.
Fish with poor immune status are more vulnerable to
bacterial, viral and parasitic infections. In analysis of
water quality of the disease fish pond in this case , low
dissolved oxygen and high nitrate and ammonia were
found in culture water. Furthermore, the number of
Table 1. The data of the water quality of the disease fish
pond
items
values
normal range
T
25 ± 0.5°C
23°C
DO
4 ppm
> 5.0 ppm
pH
7.66
6.5-8.5
nitrate
0.38 ppm
ammonia
0.7 ppm
< 0.1 ppm
< 0.5 ppm
salinity
0%0
0%0
The abbreviated words: temperature (T); dissolved oxygen (DO)
Hap/orchis pumilio may be increased in culture ponds,
because its life cycle has already been established and
has persisted for a long time. In the present case, the
cause of Hap/orchis pumilio infection in the cultured
severum fishes may have been a combination of the
above mentioned factors .
Hrubec et al. [9,10] reported that biochemical
reference intervals were significantly different between sunshine bass in the various culture systems including high-density recirculating systems (80 giL),
low-density tanks (5 giL) with biofilters and cages (70
giL) in a fresh water pond . Moreover, hematologic reference intervals were also significantly different between sunshine bass in the two culture systems, including high-density recirculating systems and tanks with
biofilters, and between sunshine and palmetto bass.
Therefore, the proper raising density is very important. Although there was only 4.29% (300 /7,000) mortality rate in this case, change of raising condition may
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250
Shao-Wen HUNG et al
be interrupt or decrease the disease occurrence.
It is possible in a cultured farm for Haplorchis
pumilio to complete its entire life cycle. It may start
with the presence of the first intermediate host (snails)
in the ponds followed by the introduction of the definitive hosts, such as wild birds (herons and cormorants)
and resident piscivorous predators into the farm.
Metacercariae may develop in any of the three stages :
adult, encysted, and unencysted stages [1,27] . If there
are suitable hosts to ingest those metacercariae, the
parasites are able to achieve maturity. Otherwise, the
parasites remain at the larval stage [2,3,11]. Sommerville examined the intestinal contents of piscivorous
birds at fish farms where parasitic infections had occurred and reported that there were massive amounts
of Haplorchis pumilio in the small intestine of the birds [24]. Furthermore, they found that a lizard, Varanus
sp., which is known to be a regular predator of fish
from both tanks and ponds, was found to be heavily infected with the parasite [24]. In our case , there were
many wild birds , dogs and cats around this fish farm,
which may have been intermediate or definitive hosts
for Hap lorchis pumilio , Furthermore, we found a lot of
snails in the ponds . Therefore, we postulate that the
life cycle of Haplorchis pumilio had already been established for a period of time prior to the outbreak of
the disease.
Diaz et al. reported that some structures are observed around the metacercaria inside the cyst [8].
Metacercaria of Hap larch is pumilio in cysts were extracted from muscular tissues of the base of caudal fins
from the natural host Rivulus harti. The cyst is oval
with a fine and transparent wall and bearing metacercaria whose body brown in color and covered with spines. Small oral sucker and nervous system were clearly observable. According to digestive system , the
mouth opens to mid oral sucker, followed by a short .
prepharynx which opens to a muscular pharynx and
ends in a short esophagus and arched intestinal ceaca .
Testes transversely elongated. Ventrogenital sac contains several rows of spines in the adult. Thecharacteristics of our isolate were similar with the reported
worm structures of Haplorchis pumilio, According to .
the characteristics of our isolate , therefore, the parasite .
was classified as metacercariae of Haplorchis pumilio,
Haplorchis pumilio infection has been reported
in many countries including Korean, India, China, Japan, Egypt and Palestine [4,13 ,25]. Furthermore, Haplorchis pumilio has been reco rded in humans [19].
Haplorchis pumilio infections have been recorded in
many kinds offish, such as Cyprin idea, Siluridae, Cottidae, Cichlidae and Mugilidae [14,22]. Many studies
have indicated that this worm could infect piscivorous
birds, dogs, cats, foxes, lizards and shrews in the wild,
as well as pigeons, rats, mice, guinea-p igs, hamsters,
cats and rhesus monkeys in the laboratory [11,12]. Although we did not capture wild birds around the farm
to investigate their role in the disease, we strongly suspect that wild birds were associated with the worm infection in the severum ponds .
Parasitic infections of Haplorchis pumilio are becoming an increasingly important public health concern so there is an urgent need to improve methods of
identification and control of this disease. The best preventative strategy for Haplorchis pumilio infections is
to radically reduce the vector, snails, and to protect
fish stocks in cultured farms from outside predation.
Previous reports have demonstrated that formalin
causes detachment of a parasitic feeding stage (trophont) of fish dinoflagellida parasites but does not inhibit division. Furthermore, formal in also could interrupt the life cycle of the worm such as snail elimination [15,16,28]. Therefore, we sugge sted that the pond
owner first eliminated adult snails artificially and disinfected the cultural ponds using 10 ppm BKC before
re-feeding fish. Then , to treat the golden severum fish
with 1 ppm benzalkonium chloride (BKC) and 25 ppm
formalin to kill other ectoparasites and bacteria. We
further recommended that the parasitic hosts should be
removed. These measures brought the parasite under
control.
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ACKNOWLEDGEMENTS
We would like to thank Taiwan 's Council of Agriculture (Executive Yuan) for supporting this study
(98AS-9.2.4-BQ-B1(21) and (99AS-9.2.2-BQ-B 1(28)).
REFERENCES
1.
Asanji MF. Williams MO. Studies on the excystment of
trematode metacercariae in vitro . Z Par asit enl<d 47:
Hap/orchis pumilio Infection in Go lden Severum
an d their zoonotic significance. Indian J An im Sci 42:
151 -163. 1975.
2.
Chi
rr.
Dalsgaard A . Turnbull JF . Tua n PA . Murrell KD.
Prevalence of zoonotic t r emat odes in f ish f rom a Vi etnamese
fi sh- f ar ming
commu nity . J Parasltot 94:
423 -428 . 2 0 0 8 .
3.
in
by
parasites
in
the
16. Pap ern a
I.
Reproduction
cycle
and
tolerance
to
t emperature and salinity of Amy/oodinium ocettetum
M. First report of human infection with Gynaecotyla
17 . Pe arson JC . A r ev ision of the sub fami ly Hap/orchinae
squa t arolae and fi r st I<orean record of Hap /or chis
Looss 1899 (Trematoda : Heteropbyidea) I. The Hap /orchis
Coulibaly NO. Salembere S. Bessin R. Larval C/inos tomum
group. Par asitolog y 5 4 : 6 01-6 76. 18 9 9 .
18 . Phan VT. Ersb011 AK. Bui TQ. Nguyen HT. Murrel l D.
Dalsgaa rd A . Fish- Borne Zoonotic Tr emat odes in Cultured
infection of c ichlid fish in the lake of I<ompienga in
an d Wild- Cau ght Fre shwat er Fish from the Red Rive r
Bur k ina Faso: a menace to haleutic exp loitation and
Delta . Vietnam. Vec tor Borne Zoonotic Dis. 20 10 . (ac-
Dech r uk sa W. I<railas D. Ukong S. Inkapat anak ul W.
cepted)
19. Radomyos P. Bunna g D. Har inasu ta T. Hap/orchis pumilio
Koonchornboon T. Trematode inf ect ions of the freshwat er
(t.ooss) infection in man in northeastern Thailand . Sou -
snai l fam ily Thiaridae in the Khek River . Thail and .
theast Asian J Trop Med Public Healt h 14: 223-227. 1983.
Southeast As ian J Trop Med Public Health 38: 10 16 -1 0 28 .
20. Roberts RJ. Pearson MD. Infectious pancr eat ic necrosis
Dia z JH. Is f ish c onsum pt ion safe ? The Journal of the
Lou is iana State Medica l Society: offic ial organ of the
9.
caused
(Brown. 1931) (Oinoglagegida) . An n Parasito! Humaine
2007 .
8.
Diseases
Comparee 59 : 7 - 3 0 . 1984 .
pub lic health. Sante 5: 189 -193 . 1995.
7.
I.
aquaculture of warm water fi sh . Ann Rev Fish Dis 1:
Chung OS. Lee HJ. Kim YM. Sohn WM. Kwak SJ. Seo
(accept ed)
6.
15 . Paperna
Kor ea. Kor ean J Parasitol 36 : 1-6. 1998.
pum ilio in a pa tient . Par asitol ogy international. 20 10 .
5.
9 7 1- 9 7 8 . 1972 .
155-194. 19 91 .
Chung 0 1. Kong HH. Joo CY. Radix aur icularia corean a:
nat ur al sna il host of C/inostomum comp/anatum
4.
251
in Atl an ti c salmon. Sa/mo seter L. J Fish Dis 28 :
383-390 . 2005 .
2 1. Sayasone S. Vonghajack Y. Vanmany M. Rasphone O.
Lou is iana State Medical Soc iet y 156: 44-49 . 2004 .
Tesana S. UtZinger
Diaz M. Her nandez LE. Bash iru llah A . Studies on the
Div ersity of human intestinal he lminthiasi s in Lao PDR.
J.
Akkhavong K. Oder matt
P.
life cyc le of Hap/orchis pumilio (t.ooss. 1896) (Tremat oda:
Tra ns R Soc Trop Med Hyg 103 : 2 4 7-25 4 . 2009 .
Heterophyidae) in Venezuela. Rev ista Cient i f ic a 18 :
2 2 . She n NX. Notes on t he morphology and life his tory of
35 -42. 2008.
Hap/orchis pumilio
Hrub ec TC. Smith SA. Robe rtson JL. Feldman B. Veit
Zoo l Sin 11: 4 7 0-481. 1959 .
(Trem at oda: Heterophyidae) . Acta
HP. Libey G. Tinke r MK. Blood bio chemical r eference
23. Sohn WM . Eom KS. Min DY. Rim HJ. Hoang EH. Yan g
intervals for suns hine bass (Mor one chrysops x Morone
Y. Li X. Fishborne tremat ode met acerc ar iae in freshwater
sa xatilis) in three cu lture systems. Am J Vet Res 57:
fish from Guangxi Zhuang Au t onomou s Region. China.
624 -627 . 19 9 6 .
Kor ean J Parasitol 47 : 249-257. 2009 .
10. Hru bec TC. Smit h SA . Rober t son JL. Feld man B. Veit
2 4 . Sommer ville C. The life history of Hap /orc his pumilio
HP. Libey G. Tinke r MK. Comparison of hemat ologic
(t.ooss. 1896) from c ultured
r ef er enc e interva ls between culture system and type
233-241. 1982.
of hybr id st riped bass . Am J Vet Res 57: 618 -62 3. 1996 .
t ilapias. J Fis h Dis 5:
TAO
25 . Sri sa wangwon k T. Kania P. Tesana S. Arunyanart C.
11. Kay H. Murrell KD. Hansen AK. Madsen H. Trang NT.
Scan ning electron microscopy of the tegumentai surface
Hung NM. Dalsgaard A. Optimization of an experi ment al
of ad ult Hap/orchis pumitio (Loess ) . J Helrnint hol 63:
model for the r ec overy of adult Hap/orchis p umilio
141-147. 19 8 9.
(He terophyidae: Dige nea ). J Par asit ol 9 5: 629-633. 2009 .
26. Tran TK. Murrell KD. Madsen H. Nguyen VK. Dalsg aard
12 . Kuntz RE. Chandler AC . Studies on Egyptian trematodes
A. Fishborne zoonotic t rematodes in raw f ish dishe s
with special refere nce to t he het er ophyids of mammals .
served in restaurants in Nam Dinh Province and Hano i.
I. Adu lt f lukes with descript ions of Phagieo/a /ongieollis
Vietnam . J Food Prot 7 2 : 2394 -2399 . 2009 .
n. sp.. Cynodip/ostomtim namrui n. sp. and a Stephanoprora
from cats . J Par as it ol 42 : 4 4 5 - 4 5 9 . 1956 .
13 . Looss A . Rech erches su r la f aune parasitaire de I
TEgypte . I. Memoires Ins t it ut Egyp t III. 1- 25 2 . 1896.
14 . Pande B. Shukla RP. Metace rcarial cyst of Hap/orehis
pumttto, it s developme nt in experimental mammals an d
t wo ot her heterophyid in f ec t ions of f reshwater f ishes
27 . Umadevi K. Madhavi R. The life cycle of Hap/orchis
pumilio (Trematoda: Heterophy idae ) f rom t he Ind ian
r egion . J Hel minthol 8 0 : 327-332. 2006.
28 . Ventura MT. Paperna I. Hist opath ology of Zchthyophthirius
muttititits , ntection in fishes . J Fish Bio/ 27: 185-203.
19 85.
Shao-Wen H UNG et
252
at
病例報告:金波蘿之單罣吸蟲戚染症
1 洪紹 文
2 劉邦成
2 林茍龍
2 董光中
2 張鎮璿
2 詹鈞 陽
1 中 央 研 究 院 農 業 生 物 科技 研 究 中 心
*2 王 清 賢
台 北市
2 國 立 中 興 大 學獸 醫 學 院獸 醫 學 系
台 中市
( 收 件 日 期 : 99 年11 月 29 日 。 接 受 日 期 : 100 年6 月 28 日 )
;高要
2 00 9 年 4 月 間在 台 灣 的 慈 綱 繁殖場爆發 單畢吸鑫 (Hap/orchis pumilia) 成染症 。 此飼 養場以市售 鰻 魚粉料與
地下水飼養平均 體重為 5 0 -6 0克，約 7 ， 0 0 0 隻金波蘿 (Heros
severus va r.) ， 每週平均 飼 養水 溫 為 2 5 -2 8 °C 。四 月問大
約 3 00隻金波蘿呈現食慾不佳、爛籍 、異常色素 沉著、快速呼吸及可見眾多直徑 0 . 5- 1 mm 白 色 囊腫於尾緒表面 。 每
隻病魚、成染 40 -6 0個囊腫 ，經檢查與鑑定為單辜吸蟲 (Hap/orchis pumilia ) 的 囊蝴成染 。 在微 生 物 學檢查結果則 無 明
顯 異常 。因此 ，建議飼養者以 1
ppm benzalkonium chloride
( B KC ) 和 2 5
ppm
細 菌 ， 每週 兩 次 持續治 療一 個 月 。 同 時建議先以 人工清 除 池 中 所有蝸 類並以 1 0
formalin 清 除 魚 龍 表 其 他類寄生蟲 或
ppm BKC 於 空 池進行消 毒 ， 並避免野
生 鳥 類與貓狗 食用 病死魚 。 經處置治 療後 ，疫情獲 得控制 。 [ 洪 紹 文、 劉 邦 成 、 林 茍 龍 、 董 光 中、 張 鎮 1毒 、 是去 鈞 陽、 *
王 i育 賢 。 病 例 報 告 : 金 i皮 護 之 單 莘 吸 蟲 感 染 症 。 台 灣 獸 醫 誌
37
(4): 246-252 . 2011 ° *通
886-4-22840894-508 . FAX : 886-4-22862073 ' E-mail : wswang@dragon.nchu.edu.tw]
局穿晶宮訝..i:金波蘿 ，車車鹿蟲 ，嘉錫 ，寄生蟲
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訊作者
TE L :