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 Japanese eel Anguilla japonica (Wakabayashi and Egusa, 1973), red
seabream Pagrus major and yellowtail Seriola quinqueradiata
Pangasius pangasius is one of the important farmed freshwater cat- (Yasunaga et al., 1982), channel catfish Ictalurus punctatus (Meyer and
fish species with high economic values. Diseases of farmed catfish Bullock, 1973) and turbot Scophthalmus maximus (Padros et al., 2006).
have been identified to be one of the key factors likely to impact future In addition to fish, mild to severe systemic infection also occur in rep-
development (Park et al., 2012; Kazuń and Siwicki, 2013; Nguyen, tiles, birds and mammals (Park et al., 2012). The virulence of E. tarda
2015). Edwardsiella tarda, a Gram-negative, motile rod shaped bacteri- is mainly demonstrated by its ability to invade and survive within
um ubiquitous to the aquatic environment, causes edwardsiellosis in 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
⁎ Corresponding author.
E-mail address: [email protected] (T.J. Abraham).
of health status of fish under different conditions as well as for accurate
1
Present address: Department of Marine Biotechnology, AMET University, 135 East diagnosis, vaccination and selective breeding for disease resistance in
Coast Road, Kanathur, Chennai 603112, Tamil Nadu. 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 25

earlier workers have shown that innate protective mechanisms in fish plastic (FRP) circular tanks containing 300 L of bore-well water. The fish
are the first line of defense and there is a clear relation between innate were acclimatized for 3 weeks with continuous aeration and fed a
immune defense factors and disease resistance of fish (Ellis, 2001; balanced basal dry pellet feed (CP9931, CP Pvt. Ltd., Andhra Pradesh,
Swain and Nayak, 2003; Mohanty and Sahoo, 2010; Devi et al., 2012). India) twice daily at the rate of 2% of their body weight. The wastes
Although the non-specific defense factors play a major role, specific and faecal matter were siphoned out and 50% water exchange was
defense factors are also important as many pathogens have developed done once in 4 days.
mechanisms to evade non-specific defense factors. Understanding the
dynamic changes in immunoreactivity (innate and specific) against a 2.3. Preparation of Edwardsiella tarda outer membrane protein (OMP)
specific pathogen in fish could reveal the underlying mechanism of antigen
host-pathogen interaction that could help early disease diagnosis.
Considerable progress has been made over the 50 years in describing The OMP antigen from E. tarda CGH9 was prepared by following Maji
and understanding the fish immune system (Muiswinkel and Nakao, et al. (2006) with some modification. In brief, the strain maintained in
2014). Harris and Bird (2000) opined that immunological responses of glycerol stock was revived on TSA. It was then transferred to TSB and
fish to stress are dependent on the actions of various hormones, their incubated at 30 °C for 24 h. The cells were harvested by centrifugation
interactions with each other, with immunocompetent cells as well as at 8000 rpm for 25 min at 25 °C. The cell pellets were washed thrice
with other endogenous factors such as cytokines. In fish, insulin-like using sterile phosphate buffer saline (PBS) and finally resuspended in
growth factor-I (IGF-1) is involved in the regulation of protein, lipid, 20 mL PBS. This suspension was treated with 2% sodium dodecyl
carbohydrate, and mineral metabolism in the cells, differentiation and sulphate (SDS) and 2% mercaptoethanol for 20 min at 60 °C for
proliferation of the cells, and ultimately body growth. It mediates solubilisation. The extract was then centrifuged at 8000 rpm for
many of the growth-promoting actions of growth hormone [GH] 30 min at 4 °C and the supernatant was dialysed for 48 h against sterile
(Moriyama et al., 2000). Insulin-like growth factor binding proteins PBS. After dialysis the supernatant was filter (φ 0.22 μ) sterilized and
(IGFBPs) are also documented in several finfish species (Kelley et al., stored at − 20 °C until further use. The protein concentration was
2001; Peterson and Small, 2005). Kelley et al. (2001) proposed that estimated by the method of Lowry et al. (1951).
the measurement of IGFBPs might provide an assessment of growth
status of fish. As the reports on pangas immune responses and 2.4. Experimental inoculation
endocrine regulatory mechanism against E. tarda infection are limited,
the present investigation was undertaken to assess the endocrine Thirty each of healthy P. pangasius from the acclimatized stock were
regulatory activities of P. pangasius, and the sequential changes in transferred to four FRP tanks - two each for control and test groups -
various immune effector activities and their significance in rendering containing 300 L of bore-well water. The fish of test group were
protection to pangas during experimental challenge with E. tarda. 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. Materials and methods 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,
2.1. Bacterial strain 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
The bacterial strain Edwardsiella tarda CGH9 used in this study was enhanced dose with 0.1 mL of diluted E. tarda CGH9 cell suspension
isolated from African catfish Clarias gariepinus (Burchell 1822) finger- was intramuscularly administered adjacent to the dorsal fin so as to
ling (5.0 g; 6 cm) with typical symptoms of dropsy in a grow-out achieve a dose of about 1.60 × 108 cells/fish. The fish were maintained
pond located in Deganga (Lat 22°41′01″N; Long 88°39′10″E), North 24 in their respective tanks and sampled for serum on 2, 6, 8, 12 and
Parganas district, West Bengal, India during the routine fish disease 15 days post-stimulation.
surveillance. Briefly, the morbid C. gariepinus fingerlings with typical
disease symptoms (n = 10) and also apparently healthy fingerlings 2.5. Sampling of blood and isolation of head kidney (HK) leucocytes
(n = 10) were brought to the laboratory in oxygen filled polythene
bags separately for bacteriological analysis. Prior to bacteriology, the After 5 days post bacterial inoculation, two fish from each group
morbid and healthy catfish juveniles (5 each) were first rinsed in sterile were randomly picked for the collection of blood and isolation of head
saline, wiped the adhering saline with sterile paper towels and dissect- kidney. The fish were first anesthetized with clove oil (50 μL/L water)
ed aseptically. Inocula from the kidney were aseptically collected, and were bled by caudal vein puncture using 2 mL sterile plastic syringe.
streaked on to tryptone soya agar (TSA) and Edwardsiella ictaluri agar An aliquot of blood was heparinized using 2.7% EDTA and processed for
[EIA] (Shotts and Waltman, 1990) plates, and incubated at 30 ± 2 °C measurement within an hour of collection. The non-heparinized blood
for 24–48 h. Based on dominance and definite colony morphology, was allowed to clot at room temperature (≈ 30 °C) by keeping the
representative green translucent colonies of 1.0 mm size from EIA syringe in slanting position and kept at 4 °C overnight. The serum
were picked, purified on TSA and maintained on TSA slants at room samples were collected by centrifugation at 2500 rpm for 15 min and
temperature (28 ± 2 °C). The strain CGH9, phenotypically identified stored at − 20 °C. After the collection of blood, the head kidney and
as E. tarda, was subjected to confirmative identification by molecular spleen were collected following the procedure described by Maji et al.
methods as described in Adikesavalu et al. (2015). The E. tarda strain (2006) with some modification. In brief, the head kidney and spleen
CGH9 with NCBI accession number KC914626.1 was then stored in were removed aseptically by dissecting the fish ventrally and pressed
5 mL aliquots at − 20 °C in tryptone soya broth (TSB) containing 20% through sterile stainless steel screens into PBS (pH 7.4) containing anti-
glycerol. biotics, viz. penicillin 100 IU/mL (HiMedia, India) and streptomycin
100 μg/mL (HiMedia, India). Isolation of leucocytes was done by
2.2. Collection and maintenance of fish layering the cell suspension on density gradient, Histopaque (Sigma,
USA) at 1:3 ratio and centrifuged at 1200 rpm for 30 min. The mononu-
Healthy experimental pangas catfish, Pangasius pangasius (61.71 ± clear cells present between plasma and density gradient were collected
2.36 g and 20.21 ± 0.76 cm) were brought from Kantipota, South 24 and washed with PBS. The cells were finally suspended in RPMI-1640
Parganas district, West Bengal, India (Lat. 22°27′49″ N; Long. 88°24′ medium with 2.05 mM L-glutamine (Hyclone, USA) supplemented
41″ E), and disinfected by placing in 5 ppm KMnO4 solution for with 10% fetal calf serum (HiMedia, India), 25 mM of HEPES free acid
15 min. The fish were stocked at the rate of 50 fish/fiberglass reinforced (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). 2.8. Determination of serum cortisol, insulin like growth factor-1 (IGF-1)
Enumeration and viability of the purified leucocytes was determined and insulin like growth factor binding protein-1(IGFBP-1) levels
by tryphan blue dye exclusion method using a haemocytometer.
The serum cortisol level was determined by using the cortisol test
EIA kit (AccuBind Elisa Microwells, Monobind Inc., Lake Forest, USA)
2.6. Non-specific immune responses 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,
The serum lysozyme content was assayed by turbidometric method San Diego, CA), respectively. Optical density was measured by microti-
modified to a microtitre plate (Sahoo et al., 2008; Devi et al., 2012). The tre plate reader at 450 nm.
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 2.9. Statistical analyses
myeloperoxidase, ceruloplasmin and anti-protease activities was as
per Sahoo et al. (2005, 2011). The respiratory oxidative burst activity Data were analyzed by one-way analysis of variance (ANOVA) using
by neutrophils was determined by the reduction of nitroblue tetrazoli- microsoft excel version 2007. Comparisons of mean values were
um (NBT) to formazan (Mohanty and Sahoo, 2010). The production of determined by Duncan's multiple range test (Duncan, 1955). Probability
nitric oxide (NO) by macrophages was assessed as described in Devi level of 0.05 was used to find out the significance in all cases.
et al. (2012).
3. Results

2.7. Specific immune responses 3.1. Clinical signs and symptoms

2.7.1. Lymphocyte proliferation assay The experimentally infected pangas showed typical signs of acute
The proliferative response of HK leucocytes was determined by septicemia with excessive mucus, petechial hemorrhages on the ventral
tetrazolium based colorimetric assay as described by Maji et al. (2006) sides and fins along with protruded hemorrhagic anus. Edematous
with modification. Briefly, the HK leucocytes were distributed into the swelling was observed at the injected site, which become ulcerated or
wells of a 96-well cell culture plate in such a way to get a final popula- necrotized as the disease progressed. Internally, the liver was
tion of 1.50 × 106 cells. Mitogen concanavalin A [ConA] (Genei, India) discoloured and kidney became enlarged with septicemic changes.
stock solution was prepared at the concentration of 80 μg/mL using Abdominal distension with yellowish ascites was observed rarely.
growth medium and 100 μL with the final concentration of 40 μg/mL Since the infective dose was sub-lethal, there were no mortalities in
was added to each well (positive control). Similarly, stock solution of test group and the cause of infection was confirmed by reisolating
crude OMP antigen of E. tarda CGH9 was also prepared and 100 μL E. tarda from the kidney of challenged pangas (N = 4) on EIA. No
with the final concentration of 40 μg/mL was added to each well (test death was recorded in unchallenged control group.
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- 3.2. Non-specific immune responses
yl)-2, 5-diphenyl tetrazolium bromide] (HiMedia, India) solution
dissolved in PBS (pH 7.2) at a concentration of 10 mg MTT/mL was A significant increase (p b 0.05) in serum lysozyme levels (U/mL) of
added to all the wells and incubated again at 30 °C for 6 h. After incuba- challenged pangas was observed till 15 dpi (451.12 ± 22.00 U/mL)
tion, 150 μL of the supernatant was carefully removed without compared to control (339.45 ± 17.54 U/mL) and reduced thereafter.
disturbing the cells or the formazan precipitate and 150 μL of dimethyl Significant differences were also observed on day 10 and day 15 when
sulphoxide (DMSO) was added in to all the wells followed by 20 μL of compared to 5 dpi and/or 22 dpi (p b 0.05). The difference in serum
glycine buffer (0.1M glycine, 0.1M NaCl, pH 10.5). The contents of the lysozyme activity between 10 and 15 dpi was insignificant (p N 0.05).
wells were mixed thoroughly by pipetting and the plate was incubated The serum ceruloplasmin levels of challenged pangas, as expressed in
for 10 min at 30 °C. The cell culture plate was read on a microtitre plate OD at 540 nm, increased significantly (p b 0.05) on 5 dpi (0.41 ± 0.11
reader at 595 nm. 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)
2.7.2. Indirect enzyme linked immunosorbent assay (indirect ELISA) decrease in serum anti-protease activity, as percent inhibition, was ob-
ELISA was performed in a 96 well plate (Nest, USA) as per Mishra served between control (7.79 ± 0.88%) and 5 dpi (7.40 ± 0.45%). This
and Sekhar (1997). Briefly, the plates were coated in duplicate with was followed by a significant (p b 0.05) increase to 29.33 ± 1.33% on
coating buffer (carbonate bicarbonate buffer, pH 9.6) containing crude 15 dpi. The activity was reduced thereafter to 13.72 ± 3.48% on
OMP antigen in such a way that each well has 2.5 μg of antigen and 22 dpi. Myeloperoxidase activity, as expressed in OD at 450 nm, of chal-
kept at 4 °C overnight. After discarding the coating buffer, blocking lenged pangas serum increased significantly (p b 0.05) on 5 dpi (0.20 ±
solution (5% skimmed milk powder in PBS) was added and the plate 0.014 OD) from day 0 (0.15 ± 0.008 OD). Subsequently, a drop was
was incubated at 37 °C for 2 h. After incubation, the plate was washed noticed on 10 dpi (0.17 ± 0.005 OD) that almost reached the level of
thoroughly four times with PBS containing 0.05% Tween-20 (PBST). control on and after 15 dpi. A significant reduction in serum superoxide
Then 100 μL of diluted (1:200 in PBS) test and control fish sera was anion production, as expressed in OD at 540 nm, on 5 dpi (0.320 ±
added in to the appropriate antigen coated wells. The plate was kept 0.064 OD) than that of control (0.420 ± 0.011 OD), followed by a signif-
at 37 °C for 2 h and washed as mentioned above. Then 100 μL of diluted icant increase (p b 0.05) on 10 dpi (0.530 ± 0.005 OD) was observed.
(1:100 in PBS) anti-pangas horse radish peroxidase conjugate devel- The levels were close to control on 15 dpi (0.380 ± 0.011) and 22 dpi
oped in the laboratory following the protocol of Swain and Nayak (0.394 ± 0.034) and the differences were insignificant (p N 0.05). The
(2003) was added in to each well and left for 2 h at 37 °C. The plate NO production (μM) in HK leucocytes of challenged pangas increased
was then thoroughly washed and 100 μL of substrate solution (15 μL insignificantly (p N 0.05) on 5 dpi (385 ± 92 μM) from day 0 (302 ±
6% H2O2, 0.025 g O-phenylene diamine dihydrochloride in 25 mL citrate 45 μM). Subsequently, a significant (p b 0.05) one fold reduction was ob-
buffer) was added in dark. The colour development was read after served on 15 dpi (160 ± 71 μM) compared to control, which increased
20 min at 492 nm using microtitre plate reader. 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 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
3.3.1. Lymphocyte proliferation assay on 5 dpi and increased gradually to 2.25 ± 0.25 ng/mL on 22 dpi. The
Lymphocyte proliferation of HK leucocytes of E. tarda CGH9 differences in IGF-1 levels on different dpi were significant (p b 0.05),
sensitized pangas when tested with a non-specific stimulator, ConA except between 15 dpi and 22 dpi. The level of IGFBP-1 increased signif-
showed an initial significant decrease (p b 0.05) on 5 dpi (1.850 ± icantly (p b 0.05) to 7.77 ± 1.08 ng/mL on 5 dpi (Table 1), which then
0.212 OD) from the initial OD of 2.470 ± 0.005 at 595 nm. However, reduced significantly (p b 0.05) to near normal on 15 dpi (5.05 ±
the proliferative response was increased significantly (p b 0.05) on 10 0.39 ng/mL) and on 22 dpi (4.65 ± 0.10 ng/mL). Significant differences
(3.390 ± 0.125 OD) and 15 dpi (3.260 ± 0.050 OD) compared to existed in the levels of IGFBP-1 on different dpi (p b 0.05). The
control. The proliferation of HK leucocytes of E. tarda CGH9 sensitized differences in the level of IGFBP-1 between control and 15 dpi or
fish, when tested with specific E. tarda CGH9 OMP antigen, displayed a 22 dpi as well as 15 dpi and 22 dpi were insignificant (p N 0.05). A
slight increase on different dpi when compared to control (Table 1). similar trend was observed in serum cortisol levels as in IGFBP-1. Signif-
The proliferative response was high on 10 dpi (1.83 ± 0.09 OD; icant differences (p b 0.05) existed in serum cortisol on different dpi.
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 4. Discussion
of HK leucocytes of sensitized pangas between 10 and 15 dpi was insig-
nificant (p N 0.05). 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
3.3.2. Indirect enzyme linked immunosorbent assay (indirect ELISA)
edwardsiellosis, which is essential for effective health management
Indirect ELISA was performed to assess the antibody production in
strategy. Many earlier studies described changes in non-specific and
fish immune serum collected on different dpi and the values, expressed
specific immune parameters of Catla catla (Devi et al., 2012), Labeo
in OD at 492 nm, are shown in Table 1. Initially on 5 dpi (0.22 ± 0.04
rohita (Mohanty and Sahoo, 2010), Indian major carps (Swain and
OD) and 10 dpi (0.24 ± 0.04 OD), the antibody levels of E. tarda sensi-
Nayak, 2003) and Clarias batrachus (Kumari and Sahoo, 2005) exposed
tized pangas serum decreased insignificantly (p N 0.05) than control
to E. tarda. Edwardsiella tarda challenged pangas exhibited clinical
(0.26 ± 0.04 OD). From 15 dpi, a significant (p b 0.05) increase in the
signs similar to those described in C. catla (Devi et al., 2012) and
production of antibody was observed till 22 dpi (0.44 ± 0.02 OD) in
L. rohita (Mohanty and Sahoo, 2010). The significant increase in serum
sensitized pangas serum when compared to control. There existed sig-
lysozyme levels in challenged pangas till 15 dpi, so also in sheatfish,
nificant differences in the production of antibody in sensitized pangas
Silurus glanis (Caruso et al., 2002), C. catla (Devi et al., 2012) and
among the dpi (p b 0.05). The serum antibody production in stimulated
L. rohita (Mohanty and Sahoo, 2010) when infected with E. tarda,
fish increased significantly (p b 0.05) to 0.43 ± 0.01 OD on day 2 post
confirms the importance of lysozyme that acts against pathogenic infec-
stimulation and then to 0.48 ± 0.005 OD on 15 days post-stimulation,
tion. However, the magnitude of increase in those studies was 2–4 folds
when compared to 10 dpi (0.24 ± 0.04 OD). The highest antibody
compared to 1.3 folds of the present study. This could be attributed to
production upon stimulation was observed on day 8 (0.49 ± 0.01 OD)
the intrinsic characteristics of the experimental catfish, which had a
and the increment was statistically significant (p b 0.05) when
high initial lysozyme level. These results suggest that the lysozyme is
compared with all other days except 15 day post-stimulation.
secreted as first line of defense by the pangas granulocytes during
non-specific oxygen independent responses against E. tarda infection.
3.4. Serum IGF-1, IGFBP-1 and cortisol levels The serum ceruloplasmin levels of challenged P. pangasius increased
significantly till 15 dpi when compared to control and reached near
The levels of serum IGF-1 and IGFBP-1 in healthy P. pangasius were normalcy on 22 dpi. The differences within dpi, however, insignificant,
observed to be 8.33 ± 0.12 ng/mL and 4.92 ± 0.16 ng/mL, respectively. 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 on 15 dpi. The ability of intracellular survival of E. tarda (Rao et al.,
it is reportedly form a complex with copper and other divalent metal 2001, 2003) may be the reason for the observed lymphocyte
cations and decrease the availability of ferrous, which is necessary for suppression.
microbial replication (Ellis, 2001). Contrarily, Mohanty and Sahoo The antibody production, as assessed by indirect ELISA, in
(2010) reported insignificant difference in ceruloplasmin levels experimentally challenged P. pangasius was low upto 10 dpi possibly
between control and E. tarda injected L. rohita. The serum anti- due to decrease in the overall lymphocyte proliferation and blastogenic
protease levels, in terms of percent inhibition, in challenged effect. It is likely that use of sub-optimal dose of antigens may lead to the
P. pangasius had an insignificant reduction on 5 dpi when compared to consumption and substantial decrease of natural antibodies that may
control. The subsequent increase in anti-protease activity by 3 folds on appear at the first stage as false suppression (Sinyakov and Avtalion,
10 dpi and 4 folds on 15 dpi in infected fish compared to control possibly 2009). The antibody production increased thereafter due to the
indicated the production of protease inhibitors such as α1-anti- synthesis of acquired antibodies that takes place in the later stage.
protease, α2-anti-plasmin and α2-macroglobulin (Ellis, 2001; Sahoo High antibody response on and after 15 dpi was also documented in
et al., 2005) and production of effector protein molecules like afimbrial I. punctatus infected with E. ictaluri (Camp et al., 2000) and in C. catla
hemagglutinin, type III and VI secretion systems, other exotoxins and infected with E. tarda (Devi et al., 2012). A second enhanced dose of
exoenzymes by E. tarda (Leung et al., 2012). The results of the present E. tarda CGH9 was administered 10 days after first dose, which evoked
study, however, contradict to that of Mohanty and Sahoo (2010) in a significant robust antibody production within 2 days post-
E. tarda challenged L. rohita, who reported a steady increase, not beyond stimulation. A significant increase in antibody production compared to
the level of control, after a large initial reduction. The decrease in 10 dpi (first dose) was observed till the end of stimulation experiment,
bacterial load from the circulation may also be a reason for increased indicating the presence of a robust secondary immune response in
anti-protease activity. The increase in acute phase proteins (APP) such pangas similar to higher mammals (Goldsby et al., 2003) and olive
as lysozyme, ceruloplasmin, antiproteases during the course of infection flounder fish, Paralichthys olivaceus (Sun et al., 2010).
is an indication of the survivors' immune response to accomplish Our study also demonstrated that E. tarda challenge can influence
homeostasis (Steel and Whitehead, 1994; Ellis, 2001). These findings the endocrine network of P. pangasius significantly. Only few reports
suggested that immune responses of P. pangasius against E. tarda are available on the IGF-1 levels of catfish. Nguyen (2015) noted an
infection depend primarily on non-specific immune defense factors. IGF-1 level of 19.07 ± 4.48 ng/mL in tra catfish Pangasianodon
The respiratory burst activity of infected pangas reduced significant- hypophthalmus, which was about 3–5 times higher than that reported
ly on 5 dpi, whereas myeloperoxidase activity increased significantly in channel catfish, I. punctatus, 4.19 ± 0.36 ng/mL at 21.7 °C and
when compared to control. However, the activities of respiratory burst 5.39 ± 0.28 ng/mL at 26.0 °C (Silverstein et al., 2000) and about above
increased and myeloperoxidase reduced significantly on 10 dpi. Both 2 times higher than that of the present study at 28.0 °C. In challenged
respiratory burst and myeloperoxidase activities reduced significantly pangas, the IGF-1 levels decreased significantly on 5 dpi, and never
on and after 15 dpi when compared to 10 dpi, which indicated that reached the normal even after 22 days of challenge. On the other
the surviving fish are becoming normal. Decrement in respiratory hand, the levels of IGFBP-1 increased significantly on 5 dpi and reached
burst activity immediately after infection with E. tarda was also near normal on 15 dpi. The physiological stress as determined by the
observed in L. rohita (Mohanty and Sahoo, 2010) and C. catla (Devi serum cortisol levels from 30.20 ± 2.04 μg/100 mL on day 0 to
et al., 2012). The initial reduction in super oxide anion production 47.06 ± 0.19 μg/100 mL on 5 dpi and the immune responses evident
may be due to the ability of E. tarda to survive within the host phagocyt- in the treatment can explain the observed low IGF-1 and high IGFBP-1
ic cells by producing enzymes like superoxide dismutase and catalase to levels. Similar results were recorded in Peterson et al. (2007) in
neutralize the effects of reactive oxygen species (Rao et al., 2001, 2003). E. ictaluri challenged channel catfish during the acute infection phase.
The present study, however, contradicts with the work of Mohanty and Alzaid et al. (2016) postulated that the IGF axis acts as a central
Sahoo (2010), who observed decrease in superoxide anion production governor of vertebrate growth and the expression of IGF axis is
and myeloperoxidase activity in E. tarda challenged carp, L. rohita. As repressed during infection to facilitate appropriate allocation of
the production of myeloperoxidase was high on 5 dpi, it might have resources towards immunity. IGF-I also seem to modify several aspects
utilized the reactive oxygen species, thereby reducing its level on of inflammation by influencing the actions of cytokines (Smith, 2010).
5 dpi in E. tarda sensitized P. pangasius. Similar immune trend was Recombinant interleukin IL-1β induced down regulation of IGF signal-
also found by Kumari et al. (2006) in which seasonal variation in innate ling by up-regulating IGFBPs in Atlantic salmon Salmo salar (Pooley
immune parameters of C. batrachus was monitored. Nitric oxide et al., 2013), and transcriptional induction of IGFBP-1 and IGFBP-6
production followed a similar trend as that of myeloperoxidase activity. subtypes in rainbow trout primary immune tissues during immune
However, the initial increase on 5 dpi was insignificant. The reduced responses to viral and bacterial pathogens (Alzaid et al., 2016) were
level of nitric oxide on subsequent dpi may be the result of microbicidal documented. The results of the present study as well as the earlier ob-
activity to eliminate the invading pathogen. The lack of significant servations (Pooley et al., 2013; Alzaid et al., 2016) suggest that the
differences between control and 5 dpi may probably be due to the host inflammatory response against disease down regulates IGF axis
variability in immune response among individual fish and different signalling. Possibly, the low levels of circulatory serum IGF-1 could be
fish species (Campos-Perez et al., 2000). The level of nitric oxide started a reason for low antibody production upto 10 dpi. The in-vivo adminis-
to increase on 22 dpi, which indicated the recovery of challenged fish tration of IGF-I reportedly increased the proliferation and enhanced
immune system. population of intrasplenic B cells (Jardieu et al., 1994). Our results
In the present study, the HK leucocytes from control and experimen- suggest that the pangas catfish can respond to the stress caused by
tally infected fish were stimulated by T-cell mitogen, ConA and E. tarda E. tarda challenge by secreting significantly higher levels of cortisol
CGH9 OMP antigen. There was a significant initial reduction in mitogen and IGFBP-1, and impede the endocrine network by decreasing the
induced leucocytes on 5 dpi, which corroborate the observations of IGF-l level. Cortisol has depressive effects on a number of immune re-
Kazuń and Siwicki (2013), who observed a reduction in optical density sponses in fish (Harris and Bird, 2000) so also in our study. Although
(OD) on ConA induced catfish C. gariepinus leucocytes after an the role of IGF-I in catfish is not known, other fish studies have reported
experimental infection with iridovirus. After the initial reduction, the positive correlations between somatic growth and circulating IGF-I
OD increased significantly on 10 dpi in mitogen induced leucocytes levels (Moriyama et al., 2000; Dyer et al., 2004; Peterson and Small,
compared to control. Interestingly, the response of leucocyte prolifera- 2005).
tion when induced by E. tarda OMP antigen had no change on 5 dpi. A In general, most of the non-specific immune responses achieved ho-
significant increase was observed on 10 dpi, which returned to normal meostasis by 15 dpi except serum lysozyme, antiprotease activity and
 H. Adikesavalu et al. / Aquaculture 462 (2016) 24–29 29

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