See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/242188150

Jaundice in cultured hybrid catfish, Clarias betrachus x Clarias fuscusi

Article · January 2008

CITATION READS

1 163

7 authors, including:

Pen Heng Chang

National Taiwan University
33 PUBLICATIONS   433 CITATIONS   

SEE PROFILE

All content following this page was uploaded by Pen Heng Chang on 17 April 2014.

The user has requested enhancement of the downloaded file.

 Jaundice in cultured hybrid catfish, Clarias
betrachus x Clarias fuscusi
Pen-Heng Chang, DVM PhD; Shu-Ting Kuo, DVM MVSc; Min-Hsiu Chen, MS; Chien Tu, DVM
MVSc; Shu-Hua Huang, DVM MVSc; Tung-Ming Lai, DVM; Wen-Chun Yu, DVM MVSc

An outbreak of jaundice occurred in hybrid catfish, Clarias betrachus x Clarias fuscus farm in
Ilan County, Taiwan in September 2001. Approximately 90 percent mortality occurred in adult
fish, however, none in juvenile fish. The most consistent external signs of diseased fish were
yellow discoloration all over the body and distended abdomens. Internally, yellow discoloration
was noted in the visceral organs and abdominal tissues. The histopathology revealed diffuse
bilirubin depositions in the liver and kidney, and the bilirubin deposited as a cast formation in
renal tubule. Multiple melanomacrophage aggregate (MMA) depositions were found on the
spleen and posterior kidney, and hemosiderin was engulfed by melanomacrophages in MMA.
Hemolytic jaundice of hybrid catfish was concluded. All samples have been analysed for blood
parasites, bacterial and virus isolation without success.
Key Words: Jaundice, hybrid catfish, Clarias betrachus x Clarias fuscusi, pathology

Introduction has cause 5-20 percent mortality in cul-

T he catfish Clarias fuscus was an im-
portant value-added aquaculture spe-
cies in Taiwan. It reaches a mature size of
25- 30 cm, and average weight of 500-600
g after approximately 1.5 years. In an at-
tempt to improve production through the
development of a larger hybrid, the Thai
catfish, C. betrachus was imported in
1975.4
Most of the catfish ponds are located
in southern Taiwan. The Chinese catfish is
usually reared in polyculture with grass
carp and silver carp, but there probably are
several tens of ha of water surface for the
monoculture of this species in Taiwan.2
Production ponds are earthen, about 0.3
acres in size, with two m of water depth.
Ponds receive a frequent input of new wa-
ter. 2 The animal is feed with fresh offal
from poultry or pig abattoir.
Since 1980, jaundice characterized by
a yellow coloration of the skin and muscle
From the Department of Veterinary Medicine, College of
Bio-Resources and Agriculture, National Taiwan Universi-
ty, Taipei 106, Taiwan (Chang, Chen); the National Vete-
rinary Research Institute, Council of Agriculture, Executive
Yuan, Tansui 251, Taiwan (Kuo, Tu); I Lan Livestock Dis-
ease Control Center, Yilan 268, Taiwan (Huang, Lai, Yu);
Tzu Chi College of Technology, Hualien 970, Taiwan
(Chen).
Correspondence: Dr. Pen-Heng Chang
E-Mail: penheng@ntu.edu.tw
tured yellowtail, Seriola quinqeradiata, in
Japan; and an unknown pathogenic bacte-
ria was suspected as the aetiological
agent.7,15,16 A jaundice disease of cultured
hybrid catfish was also reported as catfish
jaundice or yellow catfish disease in Thai-
land by Pearson and Chinabut.8 This dis-
ease was characterized by clinical jaundice
and distended abdomens in fish. The mor-
tality can be up to one hundred percent in
affected ponds.8 Person et al. has con-
cluded that the disease was caused by
feeding fish with rancid chicken viscera.8
Infectious haemolytic anaemia causes
jaundice outbreaks in seawater-cultured
coho salmon, Oncorhynchus kisutch
(Walbaum), in Chile.14 Other pathogen as-
sociated with the disease has been re-
ported in jaundice of cultured coho
salmon.13
An outbreak of jaundice disease has oc-
curred in a commercial hybrid catfish farm
in northern Taiwan. This study describes
the epizootic and associated histopathol-
ogy.

JVCS, Vol. 1, No. 2, April, 2008 47

 Chang et al
Materials and Methods
Fish
The hybrid catfish, Clarias betrachus x
Clarias fuscusi, farm in which a mortality
outbreak was reported in September 2001
has 4 small ponds of 0.3 acre each and 1
large pond of two acres. A population of
80,000 adult hybrid catfish, 200-300 g,
was raised in the large pond, and a total
population of 90,000 juvenile fish, 30-40 g,
was raised in the small ponds. Fish were
origined from natural spawning in the farm
from three year old animals sexually
mature. Chicken offal collected from a
processing plant was given twice daily as
the only feed.
The pond was supplied with clean
river water flowed through a small channel
in the nearby. Water beneath about 1 meter
in the pond was collected for analysis.
Water quality analysis was performed
using Merck Spectroquant test.
Pathogen examinations
Moribund fish were collected from the
farm and ten fish were subjected to stan-
dard necropsy procedures.1
Live fish was put on a bed of crushed
ice under dimmed light. A damp cloth was
used to cover the fish head. A sample of
one ml whole blood was drawn from the
caudal vein for parasite examination.
Blood smears were prepared and
Wright’s Stain was performed in order to
detect the parasite. Bacterial isolation was
performed from the kidney, liver, spleen
and heart of catfish. Five fish were col-
lected and the isolation was individually
carried out. By inoculating on Trypticase
Soya Agar (TSA, Difco) plates and incu-
bated at 28℃ for 48 hours. Virus isolation
was performed from the kidney, liver and
spleen. Tissues were prepared in five fish
pools, homogenized in 1x PBS, centri-
fuged, filtered through a 0.22 μm filter
membrane, inoculated onto monolayer
cultures of BF2 (bluegill fry), FHM (fat-
head minnow), TO2 (Tilapia ovary), EPC
(Epithelioma papillosum cyprini) cells and
primary catfish kidney cells. The cell cul-
48
tures were grown in L15 medium contain-
ing 10 % fetal calf serum with 100 IU/ml
penicillin and 100 μg/ml streptomycin.
Cell monolayers were grown in 25 cm2
flasks and inoculated with 0.1 ml of the
filtrated homogenate. Cell cultures were
incubated at 28℃ for 14 d, and daily ex-
amined for a cytopathic effect. A 0.1 ml
aliquot of culture fluid was inoculated
onto a second set of similar cell lines. This
blind passage was incubated for an addi-
tional 1 week, and observed daily for cy-
topathic effect.
Histopathology
Diseased fish were autopsied and tissue
samples were preserved in Bouin's solu-
tion for less than two days. Fixed tissues
were embedded in paraffin wax, sectioned
at five um, stained with haematoxylin and
eosin (H&E) and examined microscopical-
ly. Berlin blue stain and Stein’s iodine
staining were also used for hemosiderin
and bilirubin detection, respectively.
Histopathologic examination of the
collected fish was performed. Samples of
the integument and visceral organs were
fixed in 10% neutral formalin, embedded
in paraffin, sectioned at five um, and
stained with Mayer's hematoxylin and eo-
sin.
Result and Discussion
Only adult fish were affected. Diseased
fish ranged in size from 200g to 300g.
Adult fish began to show anorexia and
depression. Mortality rate then increased
rapidly and yellow discoloration on body
surface were observed. A thousand fish
died per day for more than 20 days and
this resulted in 90 percent mortality in
adult fish in September 2001. No juveniles
died.
Results revealed there was no
apparent correlation between the water
quality in the largest pond and mortality
(Table 1). No blood parasites were
detected on blood
JVCS, Vol. 1, No. 2, April, 2008
 Jaundice in Cultured Hybrid Catfish
Table 1. Water parameters monitored in ponds of the diseased hybrid catfish farm
Parameter Adult fish Pond
Dissolved oxygen (ppm) 5.3
Temperature (℃) 26.3
pH 6.97
+
NH4 (ppm) 0.14
NO2-N (ppm) 0.18
smears. No bacteria were isolated from
diseased fish and no cytopathic effects
were reported in any cell line tested.
The most consistent external signs of
diseased fish were more a yellow
discoloration all over the body and
distended abdomens. The abdomen of
affected fish contained large amount of
yellowish ascitic fluid. Internally, yellow
coloration was also noted in the liver,
spleen, kidney, mesentery, body fat, and
serosa of gastroenteric tract. The spleen
(Fig. 1) was enlarged.
Figure 1 - Photograph of diseased hybrid catfish
showed enlarged spleen (arrow), and more a yel-
low coloration of body fat (arrow head).
On histological sections of affected
fish granules or brown pigment were ob-
served in the hepatocytes. Bile canaliculi
were plugged with bile pigment in the liver
(Fig. 2A). There was local swelling of he-
patocytes accompanied with cytoplasmic
vacuole formation (Fig. 2A). The spleen of
affected fish demonstrated diffuse lym-
phocyte depletion accompanied with mul-
tiple melanomacrophage aggregate
(MMA) formation in the parenchyma. In
the MMA, shiny, golden brown pigments
were observed and interpreted as large de-
posits of hemosiderin (Fig. 2B, C). The
posterior kidney showed brownish precipi-
JVCS, Vol. 1, No. 2, April, 2008
Juvenile fish pond Reference Value
5.3 >5.0
26.9 15-25
6.95 7 -8.2
0.12 <0.5
0.15 <1.0
tates in the tubule lumen and hyaline drop-
lets d in renal tubule epithelia (Fig. 2C).
Depositions of MMA in the parenchyma
of posterior kidney was evident. Hemosi-
derin deposits were demonstrated as a
bright greenish blue color by Berlin blue
staining (Fig. 3). Bilirubin deposits were
demonstrated as an emerald green color by
Stein’s iodine staining (Fig. 4).
The yellow coloration of tissues and
an excess of bilirubin in the liver and kid-
ney indicated that jaundice was occurring
in fish.9 Because the concentration of bili-
rubin in the circulating blood is high,
brownish precipitates of bile-stained al-
bumin in the lumen of the renal tubules is
evident.5 In contrast to mammals, fish
seem to have complex and species-specific
bile pigment compositions. Bilirubin con-
jugates were detected in the bile of almost
all fish studied.10 Bilirubin is a breakdown
product of hemoglobin. In hemolytic dis-
eases, large amounts of bilirubin present in
the liver for excretion may overload both
the hepatocellular bile uptake as well as
the intracellular and canalicular transport
process thus resulting in jaundice in ani-
mals.3,6
The formation of hemosiderin is
caused by the destruction or lysis of eryt-
hrocytes to an extensive degree.5 The pres-
ence of large amounts of hemosiderin in
the spleen is the sign of hemolytic ane-
mia.5,6 In the outbreak, the deposition of
hemosiderin and bilirubin in tissues con-
tributed to the diagnosis of hemolytic
jaundice in catfish. Clinically, catfish
jaundice or yellow catfish disease was
used to describe the disease.8
49
 Chang et al
A
C ruses. All assays carried out failed in iden-
Figure 2 - Histological section of a diseased hybr-
id catfish. (A) Local swelling of hepatocytes ac-
companied with cytoplasmic vacuole formations
(arrow) was noted in the liver (H&E, x100). (B)
Multiple melanomacrophage aggregate formation
and discrete melanomacrophages deposited in the
parenchyma (arrow) and in the blood vessel of
spleen (H&E, x400). (C) Brownish precipitates in
the tubule lumen(arrow), and hyaline droplets in
renal tubule epithelia (arrow head) (H&E, x400).
The discoloration of tissues noticed in
the present case is very similar to those of
jaundice disease in the farmed hybrid cat-
fish in Thailand,9 and the discoloration of
tissues is due to the presence of the pig-
ment bilirubin, a breakdown product of
50
Figure 3 - Hemosiderin deposits (arrow) were
demonstrated as a bright greenish blue color by
Berlin blue staining in the parenchyma of spleen.
Figure 4 - Bilirubin deposits (arrow) were demon-
strated as an emerald green color by Stein’s
iodine staining in the renal lumens of kidney.
haemoglobin. It may be concluded that the
fish are suffering from a haemolytic anae-
mia associated with lipoid degeneration
Analyses were conducted on affected fish
in order to detect pathogen organisms in-
cluding blood parasites, bacteria and vi-
tifying a pathogen agent. We also traced
back to the culture history and found that
the farm had been established since 1984.
The farmer was well experienced in cultur-
ing hybrid catfish and he had used fresh
chicken offal in the past as the only feed
without particular problem. Prior to the
outbreak fish had been feed with frozen
chicken offal transferred from another
nearby catfish farm that was also suffering
severe mortality. Despite the mortality of
the first farm the farmer fed the offal to
fish regardless. Field data collected from
these two farms would show similar symp-
toms and mortality in adult fish.
JVCS, Vol. 1, No. 2, April, 2008
 Jaundice in Cultured Hybrid Catfish
During the outbreak, the farmer was
advised to stop feeding the fish and im-
prove water quality by changing water.
Within a month the mortality rate de-
creased and the farmer returned to using
fresh chicken offal to feed the fish without
incident in adult and juvenile fish.
In this study we report the first out-
break of jaundice in cultured hybrid catfish
in Taiwan. Although the cause of hemolyt-
ic jaundice was not identified in this study,
feed additive of uncertain origin in the
chicken feed may not be excluded. The
References
1. Amos K. Procedures for the detection and identifi-
cation of certain fish pathogens. 3rd ed. Bethesda,
MD. Am Fish Soc; 1985; 114.
2. Chen LC. Culture of the Chinese catfish, Aquacul-
ture in Taiwan, Fishing News Books, 1990.
3. Ferguson HW. Liver. In Ferguson HW ed. Systemic
pathology of fish. Iowa State University Press,
Ames; 1989; 146-157.
4. Huang CF, Chen JD. Evaluation of Genetic Varia-
bility in Taiwan Endemic Catfish. J Fish Soc Tai-
wan 2003; 30: 253-262.
5. Jones TC, Hunt RD and King NW. Mineral deposits
and pigments. In: Veterinary Pathology, 6th ed.
Williams and Wilkins, Baltimore; 1997: 57-79.
6. Kelly WR. The liver and biliary system. In: Jubb
KVF, Kennedy PC and Palmer N ed. Pathology of
domestic animals, Vol. 2, 4th ed. Academic Press,
San Diego, California; 1993; 319-406.
7. Maeno Y, Nakajima K, Sorimachi M, Inui Y. Pa-
thophysiological studies of jaundiced yellowtail, Se-
riola quinqueradiata. Fish Pathology 1995; 30: 7-14.
8. Pearson M, Chinabut S. Jaundice disease in the hy-
brid catfish. Aquat Anim Health Res Inst newsletter
Article Vol. 2, No. 2, 1993.
9. Pearson MD, Chinabut S, Karnchanakharn S, et al.
Jaundice disease in the farmed catfish hybrid, Cla-
rias macrocephalus (Gunther) × C. gariepinus
(Burchell) in Thailand. J Fish Dis 1994; 17: 325-336.
10. Reichenbach-Klinke HH. Fish disorders of non-
JVCS, Vol. 1, No. 2, April, 2008
View publication stats
disease is thought to be due to the feeding
of fish with rancid chicken viscera.9 Fur-
ther research will be necessary to identify
the cause.
Acknowledgement
This work was supported by Bureau of
Animal and Plant Inspection and Quaran-
tine, ROC.
parasitic origin (except tumors). In: Reichenbach-
Klinke's Fish Pathology. TFH Publications, Hong
Kong; 1973; 349-405.
11. Saki T, Tabata N, Watanabe K. Bile pigments in the
bile of fresh water fish, rainbow trout, tilapia, and
pejerry. Agric Biol Chem 1988; 52: 3051-3056.
12. Saki T, Murata H, Endo M, et al. 2-Thiobarbituric
acid value and contents of α-tocopherol and bile
pigments in the liver and muscle of jaundiced yel-
lowtail, Seriola quinqueradiata. Agric Biol Chem
1989; 53: 1739-1740.
13. Smith PA, Larenas J, Contreras J, et al. Infectious
haemolytic anaemia of salmon: an emerging disease
occurring in seawater coho salmon (Oncorhynchus
kisutch) in Chile. In Proceeding: Proceedings of the
4th International Symposium of Aquatic Animal
Health. New Orleans, LA; 2002: 180.
14. Smith PA, Larenas J, Contreras J, et al. Infectious
haemolytic anaemia causes jaundice outbreaks in
seawater-cultured coho salmon, Oncorhynchus ki-
sutch (Walbaum), in Chile. J Fish Dis 2006; 29:
709–715.
15. Sorimachi M, Maeno Y, Nakajima K, et al. Causa-
tive agent of jaundice of yellowtail, Seriola quin-
queradiata. Fish pathol 1993; 28: 119-124.
16. Takaji I, Sorimachi M. Cultural characteristics of
the bacterium causing jaundice of yellowtail, Seriola
quinqueradiata. Fish Pathol 1994; 29: 25-28.
51