Arch. Pol. Fish.

(2015) 23: 141-148

DOI 10.1515/aopf-2015-0016
RESEARCH ARTICLE

Pathology of Edwardsiella tarda infection in African catfish,

Clarias gariepinus (Burchell 1822), fingerlings

Thangapalam Jawahar Abraham, Prakash Kumar Mallick, Harresh Adikesavalu,

Sayani Banerjee

Received – 04 July 2015/Accepted – 28 August 2015. Published online: 31 October 2015; ©Inland Fisheries Institute in Olsztyn, Poland
Citation: Abraham T.J., Mallick P.K., Adikesavalu H., Banerjee S. 2015 – Pathology of Edwardsiella tarda infection in African catfish, Clarias
gariepinus (Burchell 1822), fingerlings – Arch. Pol. Fish. 23: 141-148.

Abstract. Edwardsiella tarda is one of the serious fish Keywords: Edwardsiella tarda, Clarias gariepinus,
pathogens infecting both cultured and wild fish species. This pathogenicity, histopathology, necrosis, lymphocyte
study aimed to assess the phenotypic characterization and infiltration.
pathogenicity of E. tarda isolated from Clarias gariepinus
(Burchell) with dropsy and histopathological alterations. The
causative agent was identified with Vitek 2, and its
pathogenicity was determined by intramuscular injection. The Introduction
challenged catfish exhibited vertical hanging, frothing, excess
mucus production, listing, swollen abdomen, anorexia, fin
The African catfish, Clarias gariepinus (Burchell), is
and tail rot, and reddish operculum. The LD50 of E. tarda PBB
and PBP strains was found to be 8.52 x 106 and 1.68 x 107 considered widely to be one of the most important
cells fish-1, respectively. Histopathological observations on tropical catfish species for aquaculture. Not native to
catfish infected naturally revealed lymphocyte infiltration in Indian waters, this species had a clandestine entry
muscle and focal necrosis, hyperplasia, edema, and swelling into India, first into West Bengal, and later it spread
of the gill lamellar epithelium. The kidney of diseased fish to other states (Thakur 1998). The introduction of
exhibited ischemic type tubulopathy, necrosis of nephritic
this species has raised many concerns because of its
tubules, hyperplastic hematopoietic tissue, rupture of the
tubular basement membrane, hydropic dystrophy of nephritic negative impacts on native fish fauna through preda-
cells, neutrophil infiltration, fibrinoid necrosis of nephretic tion (Thakur 1998). The culture of C. gariepinus in
tubules, hemosiderin deposition, and edema. The liver rural ponds, tanks, cement cisterns, and even derelict
sections revealed lymphocyte infiltration, dilation of hepatic waters using chicken and slaughter house wastes as
sinusoids, expansion of space between hepatic sinusoids, and feed is very common in India. This catfish has be-
focal necrosis. The inflammatory responses observed in
come an excellent aquaculture species, not only be-
kidney and liver in the present study were presumably
suppuration and were attributed to the potential virulence cause of its tolerance of environmental extremes, but
factors of E. tarda. also for its high annual production, high growth rate,
and high feed conversion rate (Singh and Lakra
2011). Fish are susceptible to a wide variety of bacte-
T.J. Abraham [+], P.K. Mallick, H. Adikesavalu, S. Banerjee
Department of Aquatic Animal Health, Faculty of Fishery Sciences, rial pathogens especially when they are subjected to
West Bengal University of Animal and Fishery Sciences, Chakgaria, stressors, i.e., poor water quality and overstocking.
Kolkata – 700094, West Bengal, India Infectious diseases are the main cause of economic
e-mail: [email protected]
losses in the aquaculture industry, which is
© Copyright by Stanis³aw Sakowicz Inland Fisheries Institute in Olsztyn.
© 2015 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
142 Thangapalam Jawahar Abraham et al.

negatively impacted by various bacterial pathogens. dissected aseptically. Inocula from kidney and
The most important bacterial diseases in tropical fish ascites, and also from the kidney of healthy
culture systems are hemorrhagic septicemia caused fingerlings were streaked on to brain heart infusion
by Aeromonas spp. (Hidalgo and Figueras 2012), agar (BHIA) and incubated at 30±2°C for 24 h. Rep-
edwardsiellosis associated with E. tarda (Sahoo et al. resentative colonies based on dominance and dis-
2000, Mohanty and Sahoo 2007, Park et al. 2012), tinct colony morphology were picked randomly from
and columnaris disease caused by Flavobacterium the BHIA plates, purified by repeated streaking on
columnare (Declercq 2013). Edwardsiellosis is BHIA plates, and maintained on BHIA slants. A se-
a septicemic disease characterized by extensive le- ries of biochemical reactions were performed (Col-
lins 2004, Austin and Austin 2012) to identify the
sions in the skin, muscle, and internal organs that in-
bacterial strains isolated from kidney and ascites.
fects commercially important fish including eels,
Definite identification of two bacterial strains (PBB
channel catfish, mullet, chinook salmon, flounder,
and PBP) was done with an automated bacterial iden-
carp, tilapia, and striped bass (Park et al. 2012).
tification system (Vitek 2 – Compact, BioMerieux,
In recent years, catfish farming has been growing
France).
in importance in West Bengal. Catfish production in
India and West Bengal has been on the rise thanks to
the high economic returns that can be made from Determination of LD50 of Edwardsiella
modest investments. Following Andhra Pradesh, the tarda strains
state of West Bengal has held the second position in
catfish production since 2008. The contribution of Twenty 500 l capacity fiberglass reinforced plastic
West Bengal’s catfish production has been in the (FRP) tanks were selected, cleaned, disinfected, and
range of 16-20% of the total catfish production of In- dried. All the tanks were filled with clean bore-well
dia since 2007 (DAHDF 2012). Incidences of dis- water and were labeled as T1, T2, T3, and T4 for E.
eases in catfish aquaculture are increasing because tarda PBP strain and T5, T6, T7, and T8 for E. tarda
of the intensification of culture practices. The present PBB strain. Positive (injected with sterile saline, C+)
study recorded the phenotypic characteristics of E. and negative (no injection, C-) controls for each strain
tarda isolated from diseased C. gariepinus with were also maintained. All the tanks were covered
dropsy, its pathogenicity, and the histopathological with nylon netting for adequate protection. Clarias
caused by natural infection. gariepinus aged 45 days (length 110 ± 5 mm and
weight 11.40 ± 2.46 g) were procured from Naihati,
Bodtalla fish market, North 24 Parganas district,
Materials and Methods West Bengal, India. The fish (n=200) were brought to
the laboratory, disinfected with 5 ppm potassium
permanganate for 10 min and stocked in the 500 l
Bacteriology capacity FRP tanks at a density of 50 tank-1 contain-
ing 300 l clean bore-well water. The fish were accli-
Morbid African catfish, Clarias gariepinus, matized for about two weeks, and during this period
fingerlings with dropsy (n=15) and healthy individu- they were fed with Tubifex sp. and cooked chicken
als (n=15) from a disease-affected pond located in offal at a rate of 2% body weight. Accumulated
Naihati (22°88’81”N; 88°45’23”E), North 24 wastes and feces were removed once every three days
Parganas district, West Bengal were brought to the and 50% of the water was exchanged. Nine each of
laboratory in oxygen-filled polythene bags. At the the healthy fish were selected, released into the ex-
laboratory, the fish were first rinsed in sterile physio- perimental tanks, and acclimatized for three days.
logical saline, wiped with sterile paper towels, and Two E. tarda strains (PBP and PBB) isolated from the
 Pathology of Edwardsiella tarda infection in African catfish, Clarias gariepinus (Burchell 1822), fingerlings 143

diseased C. gariepinus were used in the bacterial petechial hemorrhages in the fins. Internally, mild
challenge test. bloody ascites and inflamed liver, spleen, and kidney
The bacterial strains E. tarda (PBP and PBB) were found. The bacterial isolates from the kidney
maintained on BHIA slants were streaked onto BHIA and ascites of diseased catfish were presumptively
plates separately and incubated at 30 ± 2°C for 24 h identified as E. tarda. No E. tarda and/or other bacte-
to obtain a young culture. One colony each of the ria could be isolated from the kidney of healthy cat-
strains were aseptically picked, transferred to 10 ml fish. The phenotypic characteristics of two bacterial
BHI broth (BHIB) separately, and incubated at 30 ± strains (PBB and PBP) isolated from the kidney of cat-
2°C for 24 h. Mass culture was done in 500 ml BHIB fish fingerlings with dropsy as assessed with conven-
tional tests and Vitek 2 – Compact (BioMerieux,
at 30 ± 2°C for 24 h for both the strains separately
France) are presented in Tables 1 and 2, respectively.
and centrifuged at 7500 rpm at 20°C for 10 min to
The bacterial strains were confirmed as E. tarda,
collect the cells. The pellets thus obtained were
though they exhibited minor variations in the bio-
washed three times with sterile physiological saline
chemical characteristics such as L-lactate
and suspended in 5 ml saline. The numbers of bacte-
alkalinization and succinate alkalinization (Table 2).
rial cells in the saline suspensions were determined
by spread plating on BHIA.
Table 1
All the experimental tanks (T1 - T8) and control Biochemical characterization of Edwardsiella tarda strains
tanks (C+ and C-) contained nine fish each in dupli- from Clarias gariepinus with dropsy. sR – short rod; w – weak
cate and 100 l of clean bore-well water. The cells of E. Bacterial strains
tarda strains from 10-1 to 10-4 dilutions were injected
E. tarda E. tarda
intramuscularly at 0.1 ml fish-1 at the dorsal fin base Biochemical reaction PBB PBP
in such a way so as to get 108-105 cells fish-1. Positive
Gram reaction - -
control fish received sterile saline and negative control Morphology sR sR
received no injection. The challenged fish were main- Oxidase - -
tained in their respective tanks for 22 days and fed O/F reaction (glucose) +/+ +/+
daily with Tubifex sp. and cooked chicken offal on de- Acid from glucose + +
Gas from glucose + +
mand. Observations of mortality, external signs of in-
Acid from mannitol - -
fections, cannibalism, and behavioral changes were Motility + +
recorded daily, and based on the mortality data LD50 Catalase + +
was determined (Reed and Muench 1938). Arginine dihydrolase - -
Lysine decarboxylase + +
Ornithine decarboxylase + +
Histopathology Indole production + +
Methyl red reaction + +
The gills, muscle, liver, and kidney tissues of natu- Voges Proskauer reaction - -
rally-infected catfish were fixed in Bouin’s solution for Protease - -
48 h. The fixed samples were prepared histologically Lipase - -
Amylase - -
using standard techniques, embedded in paraffin
Aesculin hydrolysis - -
wax, and 5 μm sections were prepared and stained Growth on MacConkey agar + +
with hematoxylin and eosin (Roberts 2001). Growth at 4°C - +W
Growth at 30°C + +
Growth at 37°C + +
Results Growth in 0% sodium chloride (w/v) + +
Pigmentation - -
Hydrogen sulphide production + +
The diseased C. gariepinus fingerlings showed loss of Nitrate reduction + +
pigmentation, swelling of the abdominal surface, and Sodium citrate utilization - -
144 Thangapalam Jawahar Abraham et al.

Table 2
Biochemical characteristics of Edwardsiella tarda strains from diseased Clarias gariepinus as assessed with the Vitek 2 Compact
system (Biomerieux, France)
Bacterial strains Bacterial strains
and reactions and reactions
Biochemical characteristics PBB PBP Biochemical characteristics PBB PBP
Adonitol (ADO) - - Ala-Phe-Pro-arylamidase (APPA) - -
Alpha-glucosidase (AGLU) - - Alpha-galactosidase (AGAL) - -
Beta-glucoronidase (BGUR) - - Beta-alanine arylamidase pNA (BAlap) - -
Beta-xylosidase (BXYL) - - Beta-galactosidase (BGAL) - -
Citrate (sodium) (CIT) - - Beta-glucosidase (BGLU) - -
D-Cellobiose (dCEL) - - b-N-Acetyl-glucosaminidase (BNAG) + -
D-Glucose (dGLU) + + Coumarate (CMT) + -
D-Maltose (dMAL) + + Ellman (ELLM) + -
D-Mannitol (dMAN) - - Gamma-glutamyl transferase (GGT) - -
D-Mannose (dMNE) + + Glu-Gly-Arg-arylamidase (GGAA) - -
D-Sorbitol (dSOR) - - Glutamyl arylamidase pNA (AGLTp) - -
D-Tagatose (dTAG) - - Glycine arylamidase (GlyA) - -
D-Trehalose (dTRE) - - L-Pyrrolydonyl-arylamidase (PyrA) - -
Fermentation/ glucose (OFF) + + L-Histidine assimilation (IHISa) - -
H2S production (H2S) + + L-Lactate alkalinisation (ILATk) - +
L-Arabitol (IARL) - - L-Lactate assimilation (ILATa) - -
Lipase (LIP) - - L-Proline arylamidase (ProA) - -
L-Malate assimilation (IMLTa) - - Malonate (MNT) - -
Lysine decarboxylase (LDC) + + O/129 Resistance (O129R) + +
Orinithine decarboxylase (ODC) + + Palatinose (PLE) - -
Phosphatase (PHOS) + + Succinate alkalinization (SUCT) - +
Saccharose/Sucrose (SAC) - - Tyrosine arylamidase (TyrA) - -
Urease (URE) - - b-N-acetyl-galactosaminidase (NAGA) - -
5-Keto D-gluconate (5KG) - -

The time of first mortality among the catfish challenged with E. tarda increased with each passing
fingerlings injected with bacterial suspension of 1 × day post-challenge.
108 cells fish-1 was 8 h 49 min for E. tarda PBB and The histopathological alterations of ulcerated
11 h 30 min for E. tarda PBP. All the catfish which re- muscle area, gills with reddish operculum, liver and
ceived 1x108 cells fish-1 died within seven days. The kidney of C. gariepinus with dropsy are documented
challenged catfish fingerlings were observed to be in Figs. 1-6. Infiltration of lymphocytes was seen in
under stress showing symptoms like petechial hem- the muscle of ulcerated C. gariepinus (Fig. 1). The
orrhages, inflammation or ulceration at the site of in- gills had focal necrosis in the filament, hyperplasia,
jection, vertical hanging from the water surface, edema, and swelling of the lamellar epithelium (Fig.
frothing on the water surface due to excess mucus 2). The kidney of diseased C. gariepinus exhibited
production, listing, swollen abdomen, anorexia, fin ischemic type tubulopathy, necrosis of nephritic tu-
and tail rot, and reddish operculum. The LD50 of E. bules, hyperplastic hematopoietic tissue, partial rup-
tarda PBB and PBP strains were found to be 8.52 × ture of tubular basement membrane, hydropic
106 cells fish-1 and 1.68 × 107 cells fish-1, respec- dystrophy of individual nephritic cells (Fig. 3),
tively. The severity of fin and tail rot on catfish when neutrophil infiltration, fibrinoid necrosis of nephretic
 Pathology of Edwardsiella tarda infection in African catfish, Clarias gariepinus (Burchell 1822), fingerlings 145

Figure 2. Photomicrograph of gill showing focal necrosis in the

Figure 1. Photomicrograph of muscle showing lymphocytic infil-
gill filament (NF), hyperplasia (H), edema (E) and swelling (S) of
tration (LI) (hematoxylin and eosin stain; x200).
lamellar epithelium (hematoxylin and eosin stain; x200).

Figure 4. Photomicrograph of kidney showing neutrophil infiltra-

tion (NI), hemosiderin deposition (HS), fibrinoid necrosis of
Figure 3. Photomicrograph of kidney showing ischemic type
nephretic tubules (FNN), and edema (E) (hematoxylin and eosin
tubulopathy (TI), necrosis of nephritic tubules (N), rupture of tu-
stain; x200).
bular basement membrane (R) and hydropic dystrophy of
nephretic cells (HD) (hematoxylin and eosin stain; x200).

Figure 5. Photomicrograph of liver showing necrosis (N) and

Figure 6. Photomicrograph of liver showing expansion of space
lymphocytic infiltration (LI) (hematoxylin and eosin stain; x200).
between hepatic sinusoids (ES), dilation of hepatic sinusoids
(DH), and focal necrosis (FN) (hematoxylin and eosin stain;
x200).
146 Thangapalam Jawahar Abraham et al.

tubules, hemosiderin deposition, and edema (Fig. 4). gaseous necrosis in body musculature of Labeo
The liver sections showed focal necrosis and lympho- rohita, infected with E. tarda, leading to ulcer forma-
cyte infiltration (Fig. 5), dilation of hepatic sinusoids, tion. The gills of C. gariepinus were found to have fil-
expansion of space between hepatic sinusoids, and ament necrosis, hyperplasia, edema, and swelling of
focal necrosis (Fig. 6). lamellar epithelium. Mohanty et al. (2007) also re-
ported hyperplastic changes in the gills of L. rohita
infected with E. tarda. These changes could reduce
Discussion the surface area for effective respiration, which se-
verely stresses fish, or can even lead to death from
In the present study, the isolation and identification lack of oxygen. Therefore, a negative impact on respi-
of E. tarda from the kidney of diseased C. gariepinus ratory and physiological functions can generally be
fingerlings indicated edwardsiellosis. Edwardsiella assumed.
tarda infection in fish usually occurs under The histopathological alterations such as
imbalanced environmental conditions such as high ischemic type tubulopathy, partial rupture of tubular
water temperature, poor water quality, and high or- epithelium and hydropic dystrophy of individual ne-
ganic content (Park et al. 2012). Both E. tarda PBB phritic cells on the kidney of C. gariepinus fingerlings
and PBP strains were moderately virulent as per the indicated acute renal failure. This was further proved
degree of virulence (Pu et al. 2007) and the observed by the presence of fibrinoid necrosis of nephritic tu-
LD50 values (8.52 × 106 cells fish-1 and 1.68 × 107 bules, thereby indicating the severity of disease pro-
cells fish-1) on C. gariepinus fingerlings by intramus- cesses induced by E. tarda infection. Neutrophil
cular injection. These moderately virulent E. tarda infiltration combined with the proliferation of endo-
strains caused swollen abdomen when challenged in thelial and intraglomerular mesangial cells were also
healthy fish. Besides, the challenged catfish noted in the glomerular capillaries. Hemosiderin de-
fingerlings exhibited hemorrhage spots, vertical position was an indication of defense responses.
hanging, frothing, excess mucus production, listing, These irreversible changes in the kidney of diseased
anorexia, fin and tail rot, and reddish operculum. fish possibly led to mortalities and a production loss
The above results are, more or less, similar to the ob- of about 22% in the affected pond. The expansion of
servation (107.8 cells ml-1) reported for space between hepatic sinusoids, dilation of hepatic
intraperitonially injected Anabas testudineus (Sahoo sinusoids, focal necrosis, and lymphocyte infiltration
et al. 2000). Contrary to the present study, LD50 val- in the liver reflected the course of inflammatory pro-
ues of 4.0 × 105 cells fish-1 for intramuscularly in- cesses involving macrophages against the E. tarda in-
jected Ictalurus punctatus (Amandi et al. 1982) and vasion. The hepatocytes were either hypertrophoid or
7.1×101 cells fish-1 for intramuscularly injected necrotic, which is in accordance with Blazer et al.
Paralichthys olivaceus (Mekuchi et al. 1995) were re- (2007) as noted in the liver of Ameiurus nebulosus.
ported. The histopathological alterations of the present study
Infiltration of lymphocytes in the muscle fibers of are in agreement with those observed in C. gariepinus
ulcerated C. gariepinus indicated the activation of the (Ibrahem et al. 2010, 2011), P. olivaceus (Miwa and
first line of defense to ward off the invading bacterial Mano 2000), Scophthalmus maximus (Padros et al.
pathogen. Likewise, lymphocytic infiltration in the 2006) and I. punctatus (Raidal et al. 2004) infected
musculature of Oreochromis niloticus with with E. tarda. The inflammatory responses observed
edwardsiellosis was reported (Nagla et al. 2005). in kidney and liver of the present study were presum-
Meyer and Bullock (1973) observed the development ably suppuration. However, some authors described
of abscesses in the muscle of I. punctatus; while these responses as granulomatous in Pagrus major
Mohanty et al. (2007) reported liquefaction and (Miyazaki and Kaige 1985) and O. niloticus (Pirarat
et al. 2007).
 Pathology of Edwardsiella tarda infection in African catfish, Clarias gariepinus (Burchell 1822), fingerlings 147

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