Aquaculture 462 (2016) 24–29

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Aquaculture

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Edwardsiella tarda induces dynamic changes in immune effector activities

and endocrine network of Pangasius pangasius (Hamilton, 1822)
Harresh Adikesavalu a,1, Pradipta Paul a, Leesa Priyadarsani a, Sayani Banerjee a,
Siddhartha N. Joardar b, T. Jawahar Abraham a,⁎
a
Department of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria, Kolkata 700094, West Bengal, India
b
Department of Veterinary Microbiology, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, Belgachia, Kolkata 700037, West Bengal, India

a r t i c l e i n f o a b s t r a c t

Article history: Dynamic changes in immune effector and endocrine regulatory activities of pangas catfish, Pangasius pangasius
Received 23 March 2016 against Edwardsiella tarda infection were evaluated in this study. Healthy pangas were inoculated intramuscular-
Received in revised form 26 April 2016 ly with a dose of 2.17 × 107 cells/fish to induce edwardsiellosis. The challenged pangas exhibited excessive mucus
Accepted 27 April 2016
secretion, petechial hemorrhages and protruded hemorrhagic anus after 24 h post-inoculation. The non-specific
Available online 29 April 2016
humoral defense factors such as serum lysozyme and anti-protease activity showed a steady increase from 5 dpi.
Keywords:
A sustained ceruloplasmin activity against E. tarda infection was observed. The non-specific cellular defense fac-
Edwardsiellosis tor respiratory oxidative burst activity decreased on 5 dpi followed by an increase on 10 dpi. The
Pangasius pangasius myeloperoxidase activity and nitric oxide production showed an increase on 5 dpi followed by reduction. Anal-
Immune responses ysis of specific immune parameter in-vitro revealed suppression of lymphocyte proliferation, when stimulated
Outer membrane proteins using E. tarda outer membrane protein. The antibody production, as measured by indirect ELISA, appeared to
Cortisol be suppressed until 15 dpi and increased thereafter till the end of the experiment. A robust secondary immune
Endocrine regulation response was observed with an enhanced dose after 10 dpi. Further, the level of serum insulin-like growth
factor-1 (IGF-1) was reduced significantly when challenged with E. tarda and did not reach the normalcy even
after 22 dpi. The levels of serum cortisol and insulin-like growth factor binding protein-1 (IGFBP-1) increased sig-
nificantly on 5 dpi and reduced thereafter to near normal on and after 15 dpi. The results of this first kinetic study
suggest that E. tarda infection may influence the endocrine regulatory and immune effector activities of
P. pangasius significantly.
Statement of relevance:

➣ Infectious diseases in farmed catfish can result in 60–100% loss.

➣ Edwardsiella tarda infection in catfish negatively impacts the regulation of GH/IGF-I/IGFBP network and the
production.

© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Pangasius pangasius is one of the important farmed freshwater cat-
fish species with high economic values. Diseases of farmed catfish
have been identified to be one of the key factors likely to impact future
development (Park et al., 2012; Kazuń and Siwicki, 2013; Nguyen,
2015). Edwardsiella tarda, a Gram-negative, motile rod shaped bacteri-
um ubiquitous to the aquatic environment, causes edwardsiellosis in
⁎ Corresponding author.
E-mail address: abrahamtj1@gmail.com (T.J. Abraham).
1
Present address: Department of Marine Biotechnology, AMET University, 135 East
Coast Road, Kanathur, Chennai 603112, Tamil Nadu.
Japanese eel Anguilla japonica (Wakabayashi and Egusa, 1973), red
seabream Pagrus major and yellowtail Seriola quinqueradiata
(Yasunaga et al., 1982), channel catfish Ictalurus punctatus (Meyer and
Bullock, 1973) and turbot Scophthalmus maximus (Padros et al., 2006).
In addition to fish, mild to severe systemic infection also occur in rep-
tiles, birds and mammals (Park et al., 2012). The virulence of E. tarda
is mainly demonstrated by its ability to invade and survive within
host phagocytic cells, produce toxins and disseminate septicemia (Rao
et al., 2001, 2003), and multifactorial pathogenesis (Park et al., 2012).
The knowledge of fish immune system is essential for the evaluation
of health status of fish under different conditions as well as for accurate
diagnosis, vaccination and selective breeding for disease resistance in
aquaculture (Muiswinkel and Muiswinkel and Nakao, 2014). Several

http://dx.doi.org/10.1016/j.aquaculture.2016.04.033
0044-8486/© 2016 Elsevier B.V. All rights reserved.
 H. Adikesavalu et al. / Aquaculture 462 (2016) 24–29
earlier workers have shown that innate protective mechanisms in fish
are the first line of defense and there is a clear relation between innate
immune defense factors and disease resistance of fish (Ellis, 2001;
Swain and Nayak, 2003; Mohanty and Sahoo, 2010; Devi et al., 2012).
Although the non-specific defense factors play a major role, specific
defense factors are also important as many pathogens have developed
mechanisms to evade non-specific defense factors. Understanding the
dynamic changes in immunoreactivity (innate and specific) against a
specific pathogen in fish could reveal the underlying mechanism of
host-pathogen interaction that could help early disease diagnosis.
Considerable progress has been made over the 50 years in describing
and understanding the fish immune system (Muiswinkel and Nakao,
2014). Harris and Bird (2000) opined that immunological responses of
fish to stress are dependent on the actions of various hormones, their
interactions with each other, with immunocompetent cells as well as
with other endogenous factors such as cytokines. In fish, insulin-like
growth factor-I (IGF-1) is involved in the regulation of protein, lipid,
carbohydrate, and mineral metabolism in the cells, differentiation and
proliferation of the cells, and ultimately body growth. It mediates
many of the growth-promoting actions of growth hormone [GH]
(Moriyama et al., 2000). Insulin-like growth factor binding proteins
(IGFBPs) are also documented in several finfish species (Kelley et al.,
2001; Peterson and Small, 2005). Kelley et al. (2001) proposed that
the measurement of IGFBPs might provide an assessment of growth
status of fish. As the reports on pangas immune responses and
endocrine regulatory mechanism against E. tarda infection are limited,
the present investigation was undertaken to assess the endocrine
regulatory activities of P. pangasius, and the sequential changes in
various immune effector activities and their significance in rendering
protection to pangas during experimental challenge with E. tarda.
2. Materials and methods
2.1. Bacterial strain
The bacterial strain Edwardsiella tarda CGH9 used in this study was
isolated from African catfish Clarias gariepinus (Burchell 1822) finger-
ling (5.0 g; 6 cm) with typical symptoms of dropsy in a grow-out
pond located in Deganga (Lat 22°41′01″N; Long 88°39′10″E), North 24
Parganas district, West Bengal, India during the routine fish disease
surveillance. Briefly, the morbid C. gariepinus fingerlings with typical
disease symptoms (n = 10) and also apparently healthy fingerlings
(n = 10) were brought to the laboratory in oxygen filled polythene
bags separately for bacteriological analysis. Prior to bacteriology, the
morbid and healthy catfish juveniles (5 each) were first rinsed in sterile
saline, wiped the adhering saline with sterile paper towels and dissect-
ed aseptically. Inocula from the kidney were aseptically collected,
streaked on to tryptone soya agar (TSA) and Edwardsiella ictaluri agar
[EIA] (Shotts and Waltman, 1990) plates, and incubated at 30 ± 2 °C
for 24–48 h. Based on dominance and definite colony morphology,
representative green translucent colonies of 1.0 mm size from EIA
were picked, purified on TSA and maintained on TSA slants at room
temperature (28 ± 2 °C). The strain CGH9, phenotypically identified
as E. tarda, was subjected to confirmative identification by molecular
methods as described in Adikesavalu et al. (2015). The E. tarda strain
CGH9 with NCBI accession number KC914626.1 was then stored in
5 mL aliquots at − 20 °C in tryptone soya broth (TSB) containing 20%
glycerol.
2.2. Collection and maintenance of fish
Healthy experimental pangas catfish, Pangasius pangasius (61.71 ±
2.36 g and 20.21 ± 0.76 cm) were brought from Kantipota, South 24
Parganas district, West Bengal, India (Lat. 22°27′49″ N; Long. 88°24′
41″ E), and disinfected by placing in 5 ppm KMnO4 solution for
15 min. The fish were stocked at the rate of 50 fish/fiberglass reinforced
25
plastic (FRP) circular tanks containing 300 L of bore-well water. The fish
were acclimatized for 3 weeks with continuous aeration and fed a
balanced basal dry pellet feed (CP9931, CP Pvt. Ltd., Andhra Pradesh,
India) twice daily at the rate of 2% of their body weight. The wastes
and faecal matter were siphoned out and 50% water exchange was
done once in 4 days.
2.3. Preparation of Edwardsiella tarda outer membrane protein (OMP)
antigen
The OMP antigen from E. tarda CGH9 was prepared by following Maji
et al. (2006) with some modification. In brief, the strain maintained in
glycerol stock was revived on TSA. It was then transferred to TSB and
incubated at 30 °C for 24 h. The cells were harvested by centrifugation
at 8000 rpm for 25 min at 25 °C. The cell pellets were washed thrice
using sterile phosphate buffer saline (PBS) and finally resuspended in
20 mL PBS. This suspension was treated with 2% sodium dodecyl
sulphate (SDS) and 2% mercaptoethanol for 20 min at 60 °C for
solubilisation. The extract was then centrifuged at 8000 rpm for
30 min at 4 °C and the supernatant was dialysed for 48 h against sterile
PBS. After dialysis the supernatant was filter (φ 0.22 μ) sterilized and
stored at − 20 °C until further use. The protein concentration was
estimated by the method of Lowry et al. (1951).
2.4. Experimental inoculation
Thirty each of healthy P. pangasius from the acclimatized stock were
transferred to four FRP tanks - two each for control and test groups -
containing 300 L of bore-well water. The fish of test group were
intramuscularly administered with 0.1 mL of diluted E. tarda CGH9 cell
suspension adjacent to the dorsal fin so as to get a sub-lethal dose of
2.17 × 107 cells/fish. The control fish received 0.1 mL of sterile saline.
The fish were maintained in their respective tanks and sampled on 5,
10, 15 and 22 day post-inoculation (dpi). On 11 dpi, ten fish each from
the test group tanks were transferred to new FRP tanks and an
enhanced dose with 0.1 mL of diluted E. tarda CGH9 cell suspension
was intramuscularly administered adjacent to the dorsal fin so as to
achieve a dose of about 1.60 × 108 cells/fish. The fish were maintained
in their respective tanks and sampled for serum on 2, 6, 8, 12 and
15 days post-stimulation.
2.5. Sampling of blood and isolation of head kidney (HK) leucocytes
After 5 days post bacterial inoculation, two fish from each group
were randomly picked for the collection of blood and isolation of head
kidney. The fish were first anesthetized with clove oil (50 μL/L water)
and were bled by caudal vein puncture using 2 mL sterile plastic syringe.
An aliquot of blood was heparinized using 2.7% EDTA and processed for
measurement within an hour of collection. The non-heparinized blood
was allowed to clot at room temperature (≈ 30 °C) by keeping the
syringe in slanting position and kept at 4 °C overnight. The serum
samples were collected by centrifugation at 2500 rpm for 15 min and
stored at − 20 °C. After the collection of blood, the head kidney and
spleen were collected following the procedure described by Maji et al.
(2006) with some modification. In brief, the head kidney and spleen
were removed aseptically by dissecting the fish ventrally and pressed
through sterile stainless steel screens into PBS (pH 7.4) containing anti-
biotics, viz. penicillin 100 IU/mL (HiMedia, India) and streptomycin
100 μg/mL (HiMedia, India). Isolation of leucocytes was done by
layering the cell suspension on density gradient, Histopaque (Sigma,
USA) at 1:3 ratio and centrifuged at 1200 rpm for 30 min. The mononu-
clear cells present between plasma and density gradient were collected
and washed with PBS. The cells were finally suspended in RPMI-1640
medium with 2.05 mM L-glutamine (Hyclone, USA) supplemented
with 10% fetal calf serum (HiMedia, India), 25 mM of HEPES free acid
(Amresco, USA), 25 mM of sodium bicarbonate (HiMedia, India) and
26 H. Adikesavalu et al. / Aquaculture 462 (2016) 24–29
antibiotics (penicillin 100 IU/mL and streptomycin 100 μg/mL).
Enumeration and viability of the purified leucocytes was determined
by tryphan blue dye exclusion method using a haemocytometer.
2.6. Non-specific immune responses
The serum lysozyme content was assayed by turbidometric method
modified to a microtitre plate (Sahoo et al., 2008; Devi et al., 2012). The
result was expressed as serum lysozyme (units/mL) and one unit of
lysozyme activity was defined as the amount of sample causing a
reduction in absorbance of 0.001/min. The determination of serum
myeloperoxidase, ceruloplasmin and anti-protease activities was as
per Sahoo et al. (2005, 2011). The respiratory oxidative burst activity
by neutrophils was determined by the reduction of nitroblue tetrazoli-
um (NBT) to formazan (Mohanty and Sahoo, 2010). The production of
nitric oxide (NO) by macrophages was assessed as described in Devi
et al. (2012).
2.7. Specific immune responses
2.7.1. Lymphocyte proliferation assay
The proliferative response of HK leucocytes was determined by
tetrazolium based colorimetric assay as described by Maji et al. (2006)
with modification. Briefly, the HK leucocytes were distributed into the
wells of a 96-well cell culture plate in such a way to get a final popula-
tion of 1.50 × 106 cells. Mitogen concanavalin A [ConA] (Genei, India)
stock solution was prepared at the concentration of 80 μg/mL using
growth medium and 100 μL with the final concentration of 40 μg/mL
was added to each well (positive control). Similarly, stock solution of
crude OMP antigen of E. tarda CGH9 was also prepared and 100 μL
with the final concentration of 40 μg/mL was added to each well (test
wells). The remaining wells were filled up to 200 μL with the medium
(negative control) and the plate was incubated for 24 h at 30 °C. After
incubation, 20 μL of filter sterilized MTT [3-(4, 5-dimethyl thiazol-2-
yl)-2, 5-diphenyl tetrazolium bromide] (HiMedia, India) solution
dissolved in PBS (pH 7.2) at a concentration of 10 mg MTT/mL was
added to all the wells and incubated again at 30 °C for 6 h. After incuba-
tion, 150 μL of the supernatant was carefully removed without
disturbing the cells or the formazan precipitate and 150 μL of dimethyl
sulphoxide (DMSO) was added in to all the wells followed by 20 μL of
glycine buffer (0.1M glycine, 0.1M NaCl, pH 10.5). The contents of the
wells were mixed thoroughly by pipetting and the plate was incubated
for 10 min at 30 °C. The cell culture plate was read on a microtitre plate
reader at 595 nm.
2.7.2. Indirect enzyme linked immunosorbent assay (indirect ELISA)
ELISA was performed in a 96 well plate (Nest, USA) as per Mishra
and Sekhar (1997). Briefly, the plates were coated in duplicate with
coating buffer (carbonate bicarbonate buffer, pH 9.6) containing crude
OMP antigen in such a way that each well has 2.5 μg of antigen and
kept at 4 °C overnight. After discarding the coating buffer, blocking
solution (5% skimmed milk powder in PBS) was added and the plate
was incubated at 37 °C for 2 h. After incubation, the plate was washed
thoroughly four times with PBS containing 0.05% Tween-20 (PBST).
Then 100 μL of diluted (1:200 in PBS) test and control fish sera was
added in to the appropriate antigen coated wells. The plate was kept
at 37 °C for 2 h and washed as mentioned above. Then 100 μL of diluted
(1:100 in PBS) anti-pangas horse radish peroxidase conjugate devel-
oped in the laboratory following the protocol of Swain and Nayak
(2003) was added in to each well and left for 2 h at 37 °C. The plate
was then thoroughly washed and 100 μL of substrate solution (15 μL
6% H2O2, 0.025 g O-phenylene diamine dihydrochloride in 25 mL citrate
buffer) was added in dark. The colour development was read after
20 min at 492 nm using microtitre plate reader.
2.8. Determination of serum cortisol, insulin like growth factor-1 (IGF-1)
and insulin like growth factor binding protein-1(IGFBP-1) levels
The serum cortisol level was determined by using the cortisol test
EIA kit (AccuBind Elisa Microwells, Monobind Inc., Lake Forest, USA)
as per the manufacturer's instructions. The serum IGF-1 and IGFBP-1
were determined by ELISA based IGF-1 and IGFBP-1 kits (MyBiosource,
San Diego, CA), respectively. Optical density was measured by microti-
tre plate reader at 450 nm.
2.9. Statistical analyses
Data were analyzed by one-way analysis of variance (ANOVA) using
microsoft excel version 2007. Comparisons of mean values were
determined by Duncan's multiple range test (Duncan, 1955). Probability
level of 0.05 was used to find out the significance in all cases.
3. Results
3.1. Clinical signs and symptoms
The experimentally infected pangas showed typical signs of acute
septicemia with excessive mucus, petechial hemorrhages on the ventral
sides and fins along with protruded hemorrhagic anus. Edematous
swelling was observed at the injected site, which become ulcerated or
necrotized as the disease progressed. Internally, the liver was
discoloured and kidney became enlarged with septicemic changes.
Abdominal distension with yellowish ascites was observed rarely.
Since the infective dose was sub-lethal, there were no mortalities in
test group and the cause of infection was confirmed by reisolating
E. tarda from the kidney of challenged pangas (N = 4) on EIA. No
death was recorded in unchallenged control group.
3.2. Non-specific immune responses
A significant increase (p b 0.05) in serum lysozyme levels (U/mL) of
challenged pangas was observed till 15 dpi (451.12 ± 22.00 U/mL)
compared to control (339.45 ± 17.54 U/mL) and reduced thereafter.
Significant differences were also observed on day 10 and day 15 when
compared to 5 dpi and/or 22 dpi (p b 0.05). The difference in serum
lysozyme activity between 10 and 15 dpi was insignificant (p N 0.05).
The serum ceruloplasmin levels of challenged pangas, as expressed in
OD at 540 nm, increased significantly (p b 0.05) on 5 dpi (0.41 ± 0.11
OD) and 15 dpi (0.43 ± 0.02 OD) when compared to control (0.33 ±
0.02 OD). The difference in serum ceruloplasmin levels between control
and 22 dpi was insignificant (p N 0.05). An insignificant (p N 0.05)
decrease in serum anti-protease activity, as percent inhibition, was ob-
served between control (7.79 ± 0.88%) and 5 dpi (7.40 ± 0.45%). This
was followed by a significant (p b 0.05) increase to 29.33 ± 1.33% on
15 dpi. The activity was reduced thereafter to 13.72 ± 3.48% on
22 dpi. Myeloperoxidase activity, as expressed in OD at 450 nm, of chal-
lenged pangas serum increased significantly (p b 0.05) on 5 dpi (0.20 ±
0.014 OD) from day 0 (0.15 ± 0.008 OD). Subsequently, a drop was
noticed on 10 dpi (0.17 ± 0.005 OD) that almost reached the level of
control on and after 15 dpi. A significant reduction in serum superoxide
anion production, as expressed in OD at 540 nm, on 5 dpi (0.320 ±
0.064 OD) than that of control (0.420 ± 0.011 OD), followed by a signif-
icant increase (p b 0.05) on 10 dpi (0.530 ± 0.005 OD) was observed.
The levels were close to control on 15 dpi (0.380 ± 0.011) and 22 dpi
(0.394 ± 0.034) and the differences were insignificant (p N 0.05). The
NO production (μM) in HK leucocytes of challenged pangas increased
insignificantly (p N 0.05) on 5 dpi (385 ± 92 μM) from day 0 (302 ±
45 μM). Subsequently, a significant (p b 0.05) one fold reduction was ob-
served on 15 dpi (160 ± 71 μM) compared to control, which increased
insignificantly thereafter to 221 ± 61 μM (Table 1).
 H. Adikesavalu et al. / Aquaculture 462 (2016) 24–29 27

Table 1
Immune and endocrine regulatory responses of Edwardsiella tarda CGH9 challenged Pangasius pangasius.

Parameters Control Days post-inoculation

5 10 15 22

Non-specific immune responses

Lysozyme activity (U/mL) 339.45 ± 17.54aABC 387.23 ± 14.93bADE 430.00 ± 22.01cBDF 451.12 ± 22.00cCEG 363.69 ± 24.38abFG
Ceruloplasmin activity (OD at 540 nm) 0.33 ± 0.02aABC 0.41 ± 0.11bcdA 0.40 ± 0.01befB 0.43 ± 0.02ceCD 0.36 ± 0.01adfD
Anti-protease activity (% inhibition) 7.79 ± 0.88aABC 7.40 ± 0.45aDEF 20.25 ± 2.36ADGH 29.33 ± 1.33BEGI 13.72 ± 3.48CFHI
Myeloperoxidase activity (OD at 450 nm) 0.150 ± 0.008abAB 0.200 ± 0.014ACDE 0.170 ± 0.005BCFG 0.150 ± 0.006acDF 0.152 ± 0.01cbEG
Respiratory oxidative burst activity (OD at 540 nm) 0.420 ± 0.011abAB 0.320 ± 0.064ACDE 0.530 ± 0.005BCFG 0.380 ± 0.008acDF 0.394 ± 0.034cbEG
Nitric oxide production (μM) 302.00 ± 45.00abAB 385.00 ± 92.00aCDE 185.00 ± 92.00cdAC 160.00 ± 71.00ceBD 221.40 ± 60.95bdeE

Specific immune responses

Lymphocyte proliferation with non-specific stimulator, ConA 2.47 ± 0.01ABC 1.85 ± 0.21ADE 3.39 ± 0.13aBD 3.26 ± 0.05aCE ND
(OD at 595 nm)
Lymphocyte proliferation with specific antigen E. tarda 1.55 ± 0.07abA 1.61 ± 0.29acB 1.83 ± 0.09dAB 1.77 ± 0.02bcd ND
CGH9 OMP (OD at 595 nm)
Sero reactivity of E. tarda CGH9 inoculated P. pangasius 0.26 ± 0.041abAB 0.220 ± 0.042acCD 0.24 ± 0.035bcEF 0.35 ± 0.021ACEG 0.44 ± 0.021BDFG
sera to anti-pangas immunoconjugate
(OD at 492 nm)

Endocrine regulatory responses

Serum cortisol (μg/dL) 30.20 ± 2.04aA 47.06 ± 0.19A 39.27 ± 0.63A 35.96 ± 0.38A 31.61 ± 0.37aA
Serum insulin-like growth factor-1 (ng/mL) 8.33 ± 0.10AB 0.43 ± 0.10AC 1.40 ± 0.16AC 2.03 ± 0.10aA 2.25 ± 0.25aBC
Serum insulin-like growth factor binding protein-1 (ng/mL) 4.92 ± 0.16aA 7.77 ± 1.08AB 6.02 ± 0.04AB 5.05 ± 0.39aB 4.65 ± 0.10aB

A–I: Values sharing common superscripts within the row differ significantly (p b 0.05); a–f: Values sharing common superscripts within the row differ insignificantly (p N 0.05); ConA:
Concanavalin A; OD: Optical density; OMP: Outer membrane proteins; ND: Not done.

3.3. Specific immune responses
3.3.1. Lymphocyte proliferation assay
Lymphocyte proliferation of HK leucocytes of E. tarda CGH9
sensitized pangas when tested with a non-specific stimulator, ConA
showed an initial significant decrease (p b 0.05) on 5 dpi (1.850 ±
0.212 OD) from the initial OD of 2.470 ± 0.005 at 595 nm. However,
the proliferative response was increased significantly (p b 0.05) on 10
(3.390 ± 0.125 OD) and 15 dpi (3.260 ± 0.050 OD) compared to
control. The proliferation of HK leucocytes of E. tarda CGH9 sensitized
fish, when tested with specific E. tarda CGH9 OMP antigen, displayed a
slight increase on different dpi when compared to control (Table 1).
The proliferative response was high on 10 dpi (1.83 ± 0.09 OD;
p b 0.05) when compared to control (1.55 ± 0.07 OD) and 5 dpi
(1.61 ± 0.29 OD). However, the difference in the proliferative response
of HK leucocytes of sensitized pangas between 10 and 15 dpi was insig-
nificant (p N 0.05).
3.3.2. Indirect enzyme linked immunosorbent assay (indirect ELISA)
Indirect ELISA was performed to assess the antibody production in
fish immune serum collected on different dpi and the values, expressed
in OD at 492 nm, are shown in Table 1. Initially on 5 dpi (0.22 ± 0.04
OD) and 10 dpi (0.24 ± 0.04 OD), the antibody levels of E. tarda sensi-
tized pangas serum decreased insignificantly (p N 0.05) than control
(0.26 ± 0.04 OD). From 15 dpi, a significant (p b 0.05) increase in the
production of antibody was observed till 22 dpi (0.44 ± 0.02 OD) in
sensitized pangas serum when compared to control. There existed sig-
nificant differences in the production of antibody in sensitized pangas
among the dpi (p b 0.05). The serum antibody production in stimulated
fish increased significantly (p b 0.05) to 0.43 ± 0.01 OD on day 2 post
stimulation and then to 0.48 ± 0.005 OD on 15 days post-stimulation,
when compared to 10 dpi (0.24 ± 0.04 OD). The highest antibody
production upon stimulation was observed on day 8 (0.49 ± 0.01 OD)
and the increment was statistically significant (p b 0.05) when
compared with all other days except 15 day post-stimulation.
3.4. Serum IGF-1, IGFBP-1 and cortisol levels
The levels of serum IGF-1 and IGFBP-1 in healthy P. pangasius were
observed to be 8.33 ± 0.12 ng/mL and 4.92 ± 0.16 ng/mL, respectively.
When challenged with E. tarda CGH9, the level of serum IGF-1 was
reduced significantly (p b 0.05) and drastically to 0.43 ± 0.10 ng/mL
on 5 dpi and increased gradually to 2.25 ± 0.25 ng/mL on 22 dpi. The
differences in IGF-1 levels on different dpi were significant (p b 0.05),
except between 15 dpi and 22 dpi. The level of IGFBP-1 increased signif-
icantly (p b 0.05) to 7.77 ± 1.08 ng/mL on 5 dpi (Table 1), which then
reduced significantly (p b 0.05) to near normal on 15 dpi (5.05 ±
0.39 ng/mL) and on 22 dpi (4.65 ± 0.10 ng/mL). Significant differences
existed in the levels of IGFBP-1 on different dpi (p b 0.05). The
differences in the level of IGFBP-1 between control and 15 dpi or
22 dpi as well as 15 dpi and 22 dpi were insignificant (p N 0.05). A
similar trend was observed in serum cortisol levels as in IGFBP-1. Signif-
icant differences (p b 0.05) existed in serum cortisol on different dpi.
4. Discussion
The present work describes a time dependent kinetic study on alter-
ations in immune effector activities and endocrine network of
P. pangasius administered with a sub-lethal dose of E. tarda that induced
edwardsiellosis, which is essential for effective health management
strategy. Many earlier studies described changes in non-specific and
specific immune parameters of Catla catla (Devi et al., 2012), Labeo
rohita (Mohanty and Sahoo, 2010), Indian major carps (Swain and
Nayak, 2003) and Clarias batrachus (Kumari and Sahoo, 2005) exposed
to E. tarda. Edwardsiella tarda challenged pangas exhibited clinical
signs similar to those described in C. catla (Devi et al., 2012) and
L. rohita (Mohanty and Sahoo, 2010). The significant increase in serum
lysozyme levels in challenged pangas till 15 dpi, so also in sheatfish,
Silurus glanis (Caruso et al., 2002), C. catla (Devi et al., 2012) and
L. rohita (Mohanty and Sahoo, 2010) when infected with E. tarda,
confirms the importance of lysozyme that acts against pathogenic infec-
tion. However, the magnitude of increase in those studies was 2–4 folds
compared to 1.3 folds of the present study. This could be attributed to
the intrinsic characteristics of the experimental catfish, which had a
high initial lysozyme level. These results suggest that the lysozyme is
secreted as first line of defense by the pangas granulocytes during
non-specific oxygen independent responses against E. tarda infection.
The serum ceruloplasmin levels of challenged P. pangasius increased
significantly till 15 dpi when compared to control and reached near
normalcy on 22 dpi. The differences within dpi, however, insignificant,
thereby indicating a sustained activity of ceruloplasmin against
28 H. Adikesavalu et al. / Aquaculture 462 (2016) 24–29
E. tarda infection in pangas and to deprive them of essential nutrients as
it is reportedly form a complex with copper and other divalent metal
cations and decrease the availability of ferrous, which is necessary for
microbial replication (Ellis, 2001). Contrarily, Mohanty and Sahoo
(2010) reported insignificant difference in ceruloplasmin levels
between control and E. tarda injected L. rohita. The serum anti-
protease levels, in terms of percent inhibition, in challenged
P. pangasius had an insignificant reduction on 5 dpi when compared to
control. The subsequent increase in anti-protease activity by 3 folds on
10 dpi and 4 folds on 15 dpi in infected fish compared to control possibly
indicated the production of protease inhibitors such as α1-anti-
protease, α2-anti-plasmin and α2-macroglobulin (Ellis, 2001; Sahoo
et al., 2005) and production of effector protein molecules like afimbrial
hemagglutinin, type III and VI secretion systems, other exotoxins and
exoenzymes by E. tarda (Leung et al., 2012). The results of the present
study, however, contradict to that of Mohanty and Sahoo (2010) in
E. tarda challenged L. rohita, who reported a steady increase, not beyond
the level of control, after a large initial reduction. The decrease in
bacterial load from the circulation may also be a reason for increased
anti-protease activity. The increase in acute phase proteins (APP) such
as lysozyme, ceruloplasmin, antiproteases during the course of infection
is an indication of the survivors' immune response to accomplish
homeostasis (Steel and Whitehead, 1994; Ellis, 2001). These findings
suggested that immune responses of P. pangasius against E. tarda
infection depend primarily on non-specific immune defense factors.
The respiratory burst activity of infected pangas reduced significant-
ly on 5 dpi, whereas myeloperoxidase activity increased significantly
when compared to control. However, the activities of respiratory burst
increased and myeloperoxidase reduced significantly on 10 dpi. Both
respiratory burst and myeloperoxidase activities reduced significantly
on and after 15 dpi when compared to 10 dpi, which indicated that
the surviving fish are becoming normal. Decrement in respiratory
burst activity immediately after infection with E. tarda was also
observed in L. rohita (Mohanty and Sahoo, 2010) and C. catla (Devi
et al., 2012). The initial reduction in super oxide anion production
may be due to the ability of E. tarda to survive within the host phagocyt-
ic cells by producing enzymes like superoxide dismutase and catalase to
neutralize the effects of reactive oxygen species (Rao et al., 2001, 2003).
The present study, however, contradicts with the work of Mohanty and
Sahoo (2010), who observed decrease in superoxide anion production
and myeloperoxidase activity in E. tarda challenged carp, L. rohita. As
the production of myeloperoxidase was high on 5 dpi, it might have
utilized the reactive oxygen species, thereby reducing its level on
5 dpi in E. tarda sensitized P. pangasius. Similar immune trend was
also found by Kumari et al. (2006) in which seasonal variation in innate
immune parameters of C. batrachus was monitored. Nitric oxide
production followed a similar trend as that of myeloperoxidase activity.
However, the initial increase on 5 dpi was insignificant. The reduced
level of nitric oxide on subsequent dpi may be the result of microbicidal
activity to eliminate the invading pathogen. The lack of significant
differences between control and 5 dpi may probably be due to the
variability in immune response among individual fish and different
fish species (Campos-Perez et al., 2000). The level of nitric oxide started
to increase on 22 dpi, which indicated the recovery of challenged fish
immune system.
In the present study, the HK leucocytes from control and experimen-
tally infected fish were stimulated by T-cell mitogen, ConA and E. tarda
CGH9 OMP antigen. There was a significant initial reduction in mitogen
induced leucocytes on 5 dpi, which corroborate the observations of
Kazuń and Siwicki (2013), who observed a reduction in optical density
(OD) on ConA induced catfish C. gariepinus leucocytes after an
experimental infection with iridovirus. After the initial reduction, the
OD increased significantly on 10 dpi in mitogen induced leucocytes
compared to control. Interestingly, the response of leucocyte prolifera-
tion when induced by E. tarda OMP antigen had no change on 5 dpi. A
significant increase was observed on 10 dpi, which returned to normal
on 15 dpi. The ability of intracellular survival of E. tarda (Rao et al.,
2001, 2003) may be the reason for the observed lymphocyte
suppression.
The antibody production, as assessed by indirect ELISA, in
experimentally challenged P. pangasius was low upto 10 dpi possibly
due to decrease in the overall lymphocyte proliferation and blastogenic
effect. It is likely that use of sub-optimal dose of antigens may lead to the
consumption and substantial decrease of natural antibodies that may
appear at the first stage as false suppression (Sinyakov and Avtalion,
2009). The antibody production increased thereafter due to the
synthesis of acquired antibodies that takes place in the later stage.
High antibody response on and after 15 dpi was also documented in
I. punctatus infected with E. ictaluri (Camp et al., 2000) and in C. catla
infected with E. tarda (Devi et al., 2012). A second enhanced dose of
E. tarda CGH9 was administered 10 days after first dose, which evoked
a significant robust antibody production within 2 days post-
stimulation. A significant increase in antibody production compared to
10 dpi (first dose) was observed till the end of stimulation experiment,
indicating the presence of a robust secondary immune response in
pangas similar to higher mammals (Goldsby et al., 2003) and olive
flounder fish, Paralichthys olivaceus (Sun et al., 2010).
Our study also demonstrated that E. tarda challenge can influence
the endocrine network of P. pangasius significantly. Only few reports
are available on the IGF-1 levels of catfish. Nguyen (2015) noted an
IGF-1 level of 19.07 ± 4.48 ng/mL in tra catfish Pangasianodon
hypophthalmus, which was about 3–5 times higher than that reported
in channel catfish, I. punctatus, 4.19 ± 0.36 ng/mL at 21.7 °C and
5.39 ± 0.28 ng/mL at 26.0 °C (Silverstein et al., 2000) and about above
2 times higher than that of the present study at 28.0 °C. In challenged
pangas, the IGF-1 levels decreased significantly on 5 dpi, and never
reached the normal even after 22 days of challenge. On the other
hand, the levels of IGFBP-1 increased significantly on 5 dpi and reached
near normal on 15 dpi. The physiological stress as determined by the
serum cortisol levels from 30.20 ± 2.04 μg/100 mL on day 0 to
47.06 ± 0.19 μg/100 mL on 5 dpi and the immune responses evident
in the treatment can explain the observed low IGF-1 and high IGFBP-1
levels. Similar results were recorded in Peterson et al. (2007) in
E. ictaluri challenged channel catfish during the acute infection phase.
Alzaid et al. (2016) postulated that the IGF axis acts as a central
governor of vertebrate growth and the expression of IGF axis is
repressed during infection to facilitate appropriate allocation of
resources towards immunity. IGF-I also seem to modify several aspects
of inflammation by influencing the actions of cytokines (Smith, 2010).
Recombinant interleukin IL-1β induced down regulation of IGF signal-
ling by up-regulating IGFBPs in Atlantic salmon Salmo salar (Pooley
et al., 2013), and transcriptional induction of IGFBP-1 and IGFBP-6
subtypes in rainbow trout primary immune tissues during immune
responses to viral and bacterial pathogens (Alzaid et al., 2016) were
documented. The results of the present study as well as the earlier ob-
servations (Pooley et al., 2013; Alzaid et al., 2016) suggest that the
host inflammatory response against disease down regulates IGF axis
signalling. Possibly, the low levels of circulatory serum IGF-1 could be
a reason for low antibody production upto 10 dpi. The in-vivo adminis-
tration of IGF-I reportedly increased the proliferation and enhanced
population of intrasplenic B cells (Jardieu et al., 1994). Our results
suggest that the pangas catfish can respond to the stress caused by
E. tarda challenge by secreting significantly higher levels of cortisol
and IGFBP-1, and impede the endocrine network by decreasing the
IGF-l level. Cortisol has depressive effects on a number of immune re-
sponses in fish (Harris and Bird, 2000) so also in our study. Although
the role of IGF-I in catfish is not known, other fish studies have reported
positive correlations between somatic growth and circulating IGF-I
levels (Moriyama et al., 2000; Dyer et al., 2004; Peterson and Small,
2005).
In general, most of the non-specific immune responses achieved ho-
meostasis by 15 dpi except serum lysozyme, antiprotease activity and
 H. Adikesavalu et al. / Aquaculture 462 (2016) 24–29
ceruloplasmin, which were high upto 15 dpi. Nevertheless, their levels
reached near normal on 22 dpi. The non-specific immune responses,
though lasted for only two weeks, cleared most of the invading
pathogens. The recovering fish showed normal movement, improved
food intake and reduced hyperaemic skin. During the 22 day post-
inoculation, the lymphocyte proliferation was suppressed and the
production of antibody was observed only after 15 dpi. This is the first
study that sheds light on the immune and endocrine responses of
catfish P. pangasius against E. tarda infection. Also, our results suggest
that E. tarda infection may impact the catfish production indirectly
through impacts on relative disease levels by regulating the GH/IGF-I/
IGFBP network. Expression studies on E. tarda virulence and immune
genes as well as IGF-I and GH genes in P. pangasius are important to
have a proper understanding on the initial reduction in respiratory
burst activity, lymphocyte suppression and negative impact on GH/
IGF-I/IGFBP network during E. tarda infection.
Conflicts of interest
The authors declare that they have no competing interests.
Acknowledgements
The research work was supported by the Indian Council of
Agricultural Research (Grant F. 10(12)/ 2012 – EPD dated 23.3.12), Gov-
ernment of India, New Delhi under the Niche Area of Excellence pro-
gramme. The authors thank the Vice Chancellor, West Bengal
University of Animal and Fishery Sciences, Kolkata for providing neces-
sary infrastructure facilities to carry out the work. All the experimental
protocols were approved by the University Ethical Committee.
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