Modulation of the innate immune responses in Indian major carp, Catla catla following experimental infection with Flavobacterium columnare

Aquaculture 510 (2019) 22–31

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Modulation of the innate immune responses in Indian major carp, Catla catla following experimental infection with Flavobacterium columnare

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Ravindraa,b, Pravata K. Pradhana, , Veena Pandeb, Manoj K. Yadava, Dev K. Vermaa, ⁎ Neeraj Sooda, a b

ICAR-National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow 226002, Uttar Pradesh, India Department of Biotechnology, Kumaun University, Nainital 263136, Uttarakhand, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Catla catla Flavobacterium columnare Innate immunity Histopathology

Columnaris disease, caused by Flavobacterium columnare, is a serious bacterial disease responsible for causing devastating rates of mortality in numerous species of freshwater fish. This disease can cause mortalities of up to 100% within 24 h, leading to severe economic losses in the aquaculture industry. Notwithstanding the enormous impacts this disease can have, very little is known regarding the interaction between the host and bacterium. To gain a better understanding of the host immune response, an attempt has been made to study the sequential changes in innate immunity parameters of Catla catla, following bath challenge with F. columnare. The clinical signs in challenged fish included lethargy, respiratory distress, swimming near the water surface and accelerated opercular movements. Histopathological examination of the gills from infected fish revealed the presence of bacterial masses in the gills, fusion of secondary filaments and sloughing of epithelial cells with a consequential disruption to the gill architecture within 24–48 h. Importantly, some innate immunity parameters, namely the production of reactive oxygen species and nitric oxide as well as serum myeloperoxidase activity showed significant increases (p < .05) during the initial stages of infection i.e. up to 12 h post infection (hpi). The other measured parameters, such as alternative complement activity (ACH50) and total anti-protease activity were significantly decreased (p < .05) in the experimental group up to 12 and 24 hpi, respectively. Further, significant increases (p < .05) in lysozyme and ACH50 activity were observed from 12 and 48 hpi, respectively. Total serum protein and globulin level declined significantly (p < .05) in the later stages of infection, whereas the α-2 macroglobulin activity and serum albumin level were unchanged throughout the experiment. This study indicates that the immune response in catla is modulated following infection with F. columnare. Knowledge regarding changes in the immune response will help in developing strategies for improving resistance against columnaris disease.

1. Introduction Columnaris disease, caused by Flavobacterium columnare, is frequently encountered and is a devastating bacterial disease of freshwater fish, affecting numerous wild and cultured fish species (Decostere and Haesebrouck, 1999; Declercq et al., 2013; Dong et al., 2016). To date, the disease has been reported from > 60 fish species from Asia, Europe, North and South America, Africa and Australia, and the host range of F. columnare is expanding (Lafrentz et al., 2012; Declercq et al., 2013; Loch and Faisal, 2015). The disease is reported to cause acute mortalities of up to 100% within 24 h (Declercq et al., 2013) and is considered to be a major limiting factor in the growth of aquaculture (Verschuere et al., 2000; Balcazar et al., 2006; Dorsey and Robertson,

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2013). Importantly, catla (Catla catla), the fastest growing Indian major carp cultured throughout the Indian subcontinent (FAO, 2017), is highly susceptible to this disease (Dash et al., 2009; Verma and Rathore, 2013). Flavobacterium columnare, the aetiological agent of the disease (Amin et al., 1988; Bernardet, 1989), principally affects the skin and gills of fish (Declercq et al., 2013). To date, most of the studies on this pathogen have been carried out on the mode and dynamics of adhesion (Decostere et al., 1999; Olivares-Fuster et al., 2011), on the virulence of different genomovars (Arias et al., 2004; Thomas-Jinu and Goodwin, 2004; Darwish and Ismaiel, 2005; Shoemaker et al., 2008) and adaptive immunity (Grabowski et al., 2004; Bebak et al., 2009; Shoemaker et al., 2011). A study of the literature indicates that there is a paucity of

Corresponding authors. E-mail addresses: [email protected] (P.K. Pradhan), sood_neeraj@rediffmail.com (N. Sood).

https://doi.org/10.1016/j.aquaculture.2019.05.015 Received 29 May 2018; Received in revised form 6 May 2019; Accepted 7 May 2019 Available online 08 May 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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Aldrich, St. Louis, MO, USA) and bled from the caudal vein. An aliquot of blood was heparinised (50 IU ml−1) for respiratory burst activity and the remaining blood was allowed to clot for serum collection. The serum samples were stored at −80 °C until analysis. Serum from individual fish was used to monitor the innate immunity parameters, whereas, serum from 5 fish (5 × 30 μl) was pooled to determine alternative complement activity. For histopathological examination, the gills from three fish in the control and a further three fish in the infected group were fixed in 10% neutral buffered formalin on each sampling day. The animal care and experimental challenge were approved by the Institutional Animal Ethics Committee of ICAR-NBFGR, Lucknow.

information regarding the mechanisms through which F. columnare modulates the innate immune response of the host to its advantage. The innate immunity is the fundamental defence mechanism which offers immediate and permanent protection against a wide array of pathogens (Magnadottir, 2006). The sequential changes in innate immunity parameters against any pathogen can reveal mechanisms of host-pathogen interaction and the information generated can be useful for developing future control strategies. Keeping the above in view, the present study has been undertaken to understand the innate immune response of C. catla following infection with F. columnare. 2. Material and methods

2.4. Histopathological analysis 2.1. Experimental animals and their maintenance The formalin-fixed tissues were processed routinely for preparing paraffin blocks, and 4 μm sections were cut. The tissue sections were stained with haematoxylin and eosin (H & E) and examined under a compound microscope (Olympus BX53, Tokyo, Japan).

Healthy catla juveniles (n = 400) (53.4 ± 2.41 g; 15.6 ± 1.1 cm) were procured from a fish farm belonging to the ICAR-National Bureau of Fish Genetic Resources, Lucknow. The fish were kept in fibre reinforced plastic (FRP) tanks (1000 l) with static water and acclimatized in the wet laboratory for 2 weeks. They were fed twice daily with a commercial diet (ABIS Exports India Pvt. Ltd., Chhattisgarh, India) at 2% body weight per day. About one-third of the water in the tanks was exchanged daily. Before initiation of the experiment, five fish were sampled randomly for bacterial and parasitological examination to ensure that fish were free of F. columnare and parasitic infection. During the experimental period, the temperature, dissolved oxygen and pH were 24.5 ± 1.4 °C, 6.8 ± 0.78 mg l−1, 8.2 ± 0.42, respectively as measured by a multiparameter water quality meter (Hanna Instruments, Romania), whereas, nitrite and ammonia were estimated following APHA (1998) and found to be 0.014 ± 0.008 mg l−1 and 0.107 ± 0.02 mg l−1, respectively.

2.5. Innate immunity parameters 2.5.1. Reactive oxygen species (ROS) production Production of ROS was determined as per Anderson and Siwicki (1995). Briefly, 100 μl of heparinised blood was mixed with an equal volume of 0.2% nitroblue tetrazolium (NBT) prepared in 1× phosphate buffer saline (PBS) and incubated for 30 min at 25 °C. Thereafter, 50 μl of the reaction suspension was taken in a glass tube and mixed with 1 ml of dimethyl formamide (Sigma–Aldrich) to solubilise the formazan product. The tubes were centrifuged at 3000 ×g for 5 min and the amount of reduced NBT was determined by measuring OD of the supernatant at 540 nm using a UV–Vis spectrophotometer (Hitachi, Pleasanton, CA).

2.2. Bacterial strain 2.5.2. Nitric oxide (NO) production Production of NO was measured using a Nitrite/Nitrate Assay Kit (Sigma Life Science, St. Louis, USA), following the manufacturer's instructions. The optical density was measured at 540 nm using a microplate reader (Synergy™ HTX). The molar concentration of nitrite in the sample was determined from a standard curve, generated prior to the test using known concentrations of sodium nitrate.

Flavobacterium columnare isolate (BE), used in the present study, was isolated from bubble eye goldfish (Carassius auratus) and confirmed using species-specific PCR (Darwish et al., 2004) and 16S rRNA gene sequencing (accession no. MH246209). The strain was found to belong to genetic group 2, on the basis of phylogenetic analysis of 16S rDNA gene (LaFrentz et al., 2018) (Supplementary Fig. 1). For experimental infection, bacteria were streaked on cytophaga agar (Daskalov et al., 1999) containing 1 μg/ml tobramycin (Decostere et al., 1997). After 48 h, the colonies displaying the typical F. columnare morphology were inoculated in 30 ml of cytophaga broth for 2 days and thereafter, transferred to flasks containing 600 ml of cytophaga broth. The optical density (OD) of the broth was measured at 550 nm using a spectrophotometer (Synergy™ HTX, BioTek Instruments, Inc. Highland Park, USA) to determine colony forming units (cfu). 2.3. Experimental design

2.5.3. Myeloperoxidase (MPO) activity The MPO activity in serum samples was determined as per Quade and Roth (1997). Briefly, 10 μl of serum diluted in 90 μl of Hank's balanced salt solution (without Ca2+or Mg2+) was added to each well in a 96-well microtitre plate. Then 35 μl of freshly prepared 20 mM 3,3′,5,5′ tetramethyl benzidine hydrochloride (TMB) (Genei, Bangalore, India) was added to each well. After 2 min, the reaction was stopped by adding 35 μl of TMB stop solution and the OD was measured at 450 nm using a microplate reader (Synergy™ HTX).

One hundred and eighty fish (n = 180) were challenged by bath immersion for 60 min in 120 l of water containing a lethal dose (LD50) of F. columnare at 2.4 × 107 cfu/ml, calculated 7 days earlier using the same batch of fish (Reed and Muench, 1938). A further 180 fish were mock-challenged using a similar immersion method in 120 l of water containing sterile cytophaga broth in a volume equal to the amount of bacterial culture for 60 min. Out of 180 fish in each group, 160 fish were transferred to 2 tanks (1000 l), each containing 80 fish for studying innate immunity parameters, whereas, the remaining twenty fish from the control and infected groups were transferred to separate FRP tanks (300 l) to follow the subsequent patterns of mortality. For studying the immune parameters, ten fish from each tank, from the infected and the control tank, were randomly sampled on 3, 6, 12, 24, 48, 72 and 96 h post infection (hpi). Prior to the collection of blood, fish were euthanased with an overdose of MS-222 (200 mg l−1) (Sigma-

2.5.4. Total anti-protease activity For determination of the total anti-protease activity, the protocol of Zuo and Woo (1997) was followed with some minor modification. Briefly, 10 μl of test serum was mixed with 100 μl of trypsin (Bovine pancreas type I, Sigma–Aldrich) and incubated at 25 °C for 30 min. Ten μl of test serum mixed with 100 μl PBS served as the serum blank and 10 μl PBS mixed with 100 μl of trypsin served as the positive control, whereas, 110 μl PBS served as the negative control. Subsequently, 1 ml of casein (Sigma–Aldrich) dissolved in PBS (2.5 mg/ml) was added to the samples and incubated for 15 min at 25 °C. Thereafter, the reaction was stopped by adding 500 μl of 10% trichloroacetic acid and the tubes were centrifuged at 10,000 ×g for 5 min to remove the precipitating proteins. The OD of the supernatants was measured at 280 nm and the percent trypsin inhibition was calculated by comparing it to the test and positive samples using the following formula; 23

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Cleveland, USA) and serum lysozyme values were expressed as μg/ml equivalent of HEWL activity.

Reference value = (absorbance of positive control − absorbance of negative control)

2.5.8. Total serum protein, albumin and globulin Total serum protein was determined as per Bradford (1976) using a kit (AMRESCO, Solon, Ohio, USA) and a standard curve was prepared using a known concentration of bovine serum albumin (BSA) (AMRESCO). Serum albumin level was estimated by a bromocresol green (BCG) dye binding method (Doumas et al., 1971) using a kit (Siemens Ltd., Vadodara, India). Globulin level was calculated by subtracting the albumin value from total serum protein.

Control value = (absorbance of test sample − absorbance of respective serum blank) Percent inhibition = [(reference value − control value)/(reference value)] × 100 2.5.5. Alpha-2 macroglobulin (α-2 M) activity The serum α-2 M activity was determined following the method detailed in Zuo and Woo (1997) with some modification. Briefly, 10 μl serum was mixed with 100 μl of trypsin and thereafter 90 μl of 0.1 M Tris–HCl, pH 8.2 was added to the above solution and incubated for 40 min at 25 °C along with serum blank which contained 10 μl of test serum mixed with 190 μl of Tris–HCl, whereas 100 μl of trypsin mixed with 100 μl of Tris–HCl served as the positive control and 200 μl TrisHCl served as a negative control. Then the reaction mixtures were incubated at 25 °C for 20 min with 2 ml of Na-benzoyl-DL-arginine-p-nitroanilide HCl (BAPNA, Sigma–Aldrich) (80 μg/μl) dissolved in Tris–HCl containing 20 mM CaCl2. The reaction was stopped by adding 500 μl of 30% (v/v) acetic acid and then OD of the above supernatants was taken at 410 nm using a microplate reader (Synergy™ HTX). Finally, the percent trypsin inhibition was calculated in a similar manner as the total anti-protease activity.

2.6. Statistical analysis All the data were represented as the arithmetic mean ± SE values. Prior to the analysis, data were checked for normality using a ShapiroWilk test. Statistical analysis of data was compared by a one-way ANOVA followed by Duncan's multiple range tests to determine significant differences at the 5% level (p < .05) using SPSS (23.0 version, USA). 3. Results 3.1. Clinical signs and mortality In the experimentally infected fish, no clinical signs were observed up to 6 hpi, whereas, by 12 hpi, fish started exhibiting lethargy and mild respiratory distress. By 24 hpi, the clinical signs increased in severity and included swimming near the water surface with accelerated opercular movements. The severity of lesions was found to decrease from 48 hpi. In the fish groups kept separately for observing mortality pattern, fish started dying from 24 hpi in the infected group and by 96 hpi, a cumulative mortality of 45% was observed (Fig. 1). No clinical signs and mortality were observed in fish in the control group. Further, in the experimentally infected fish kept for sequential sampling, a total of 12 fish died during the experimental period. Out of the 12 fish, 7 fish died by 24 hpi, whereas, four fish died during 24–48 hpi, and one more fish died during 48–72 hpi. Importantly, fish from the experimentally infected group sampled at each time interval showed differences in the severity of the clinical signs presented by individual fish.

2.5.6. Alternative complement activity (ACH 50) The alternative complement activity was assayed using rabbit red blood cells (RaRBCs) as per Ortuno et al. (1998) with some modification. Briefly, RaRBCs were washed three times in EGTA-Mg++-HBSS buffer (10 mM EGTA, 10 mM MgCl2 and 1× HBSS) and re-suspended to 2 × 108 cells/ml in the same buffer. Thereafter, an aliquot of 125 μl pooled sera from the control as well as from the infected group at each time interval was serially diluted two-fold in EGTA-Mg++-HBSS buffer. Then 50 μl of RaRBCs suspension was added to each tube, followed by incubation at 20 °C for 120 min. The haemolytic reaction was stopped by adding 1.575 ml EDTA-HBSS buffer (10 mM EDTA and 1× HBSS) and the tubes were centrifuged at 1600 ×g for 5 min. For obtaining values of maximum (100%) and minimum (0%) haemolysis, 1.7 ml of distilled water and EGTA-Mg++-HBSS buffer each were added to 50 μl RaRBCs, respectively. The OD of the supernatant was taken at 414 nm using a microplate reader (Synergy™ HTX). The value of Y/100 - Y and the serum volume were log-transformed and plotted on a pre-programmed Excel spreadsheet. The volume of serum required to lyse 50% of the RaRBCs was calculated using the following formula;

3.2. Gross and histopathology On post-mortem examination, no gross lesions were observed in the gills of the experimentally infected fish up to 6 hpi (Fig. 2b). Gills at 12 hpi, however, were affected and had a yellowish-white discoloration (Fig. 2c). With the progression of infection, at 24 hpi, the gills had large areas of a white-yellowish discoloration (Fig. 2d). By 48 hpi, there was

Percent RaRBCs lysis (Y) = [Relative absorbance of the sample dilution /Relative absorbance of the 100%lysis] × 100

Relative absorbance of the sample dilution or 100%lysis = Absorbance of the sample dilution or absorbance of the 100% lysis − absorbance of the 0%lysis 2.5.7. Lysozyme activity A turbidometric assay for determination of the lysozyme activity was carried out according to Sankaran and Gurnani (1972) with some modification. Briefly, 125 μl of Micrococcus lysodeikticus suspension (0.2 mg/ml in 0.02 M sodium acetate buffer, pH 5.5) was added to 25 μl of each serum sample in a 96-well U-bottom microtitre plate and the OD was measured immediately at 450 nm. Then the plates were incubated at 24 °C for 1 h, and the change in turbidity was measured as the final OD, using a microplate reader (Synergy™ HTX). A standard curve was prepared using hen egg-white lysozyme (HEWL, USB Corporation,

Fig. 1. Mortality pattern of Flavobacterium columnare infected Catla catla during experimental period. 24

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Fig. 2. Gross pathology of Flavobacterium columnare infected Catla catla. a) Normal gills of control C. catla. b) Gills of experimentally infected fish at 6 hpi. d) Gills of infected fish showing yellowish-white discoloration at the tip of the filament at 12 hpi. d) Large areas of white-yellowish discoloration in affected gills at 24 hpi. e) Decrease in severity of lesions at 48 hpi. f) Lesions in the gills appeared to have healed by 96 hpi.

addition, a large number of eosinophilic granular cells were observed in the inflammatory exudate. Further, large clumps of bacteria were seen in the desquamated mass of epithelial cells (Fig. 3d, e and f). By 48 hpi, a decrease in the severity of lesions was observed as evident by a decrease in inflammatory exudate (Fig. 3 g). Resolution of the lesions was observed at 72 hpi and by 96 hpi, the gills of infected fish were almost similar to those of fish in the control group (Fig. 3h). No lesions however, were observed in the gills of fish from the control group throughout the experimental period (Fig. 3a).

a decrease in the severity of lesions (Fig. 2e). At 72 and 96 hpi (Fig. 2f), the lesions in the gills appeared to heal, and the gills were almost similar to those of fish in the control group (Fig. 2a). No gross lesions were observed in the internal organs, namely the kidney, liver and spleen of fish from the infected as well as the control group. Microscopic observation of the gill sections at 3 hpi, revealed the presence of bacteria at the base of the secondary filaments along with a mild inflammatory exudate (Fig. 3b). At 6 hpi, there was a general increase in bacterial biomass within the gills with desquamation of epithelial cells. By 12 hpi, inflammatory exudate consisting primarily of mononuclear cells along with a large number of bacterial cells was observed between secondary filaments (Fig. 3c) and there was sloughing of epithelial cells in the secondary filaments. At 24 hpi, there was extensive necrosis with loss of architecture of the gill filaments. In

3.3. Innate immunity parameters Catla from the infected group showed significantly higher (p < .05) reactive oxygen species (Fig. 4a), nitric oxide production (Fig. 4b) and 25

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Fig. 3. Sequential pathology of Flavobacterium columnare infected Catla catla. a) Normal gill tissue of C. catla from the control group. b) Presence of bacterial masses at the base of the secondary filaments (arrows) along with mild inflammatory exudate at 6 hpi. c) Inflammatory exudate consisting of mononuclear cells along with long bacterial cells (arrows) in gill secondary filaments at 12 hpi. d–f), Gills showing extensive necrosis with architectural loss of the gill filaments (arrowheads), a large number of eosinophilic granular cells (white arrows) in the inflammatory exudate and large clumps of bacterial cells (arrow) embedded in the desquamated mass of epithelial cells at 24 hpi. g) Decrease in inflammatory exudate along with fusion of secondary filaments at their base (arrows) at 48 hpi. h) Resolution of the lesions at 96 hpi.

findings from the current study suggest that enhanced production of ROS by the host during the initial stages of infection might be one strategy to overcome the infection by phagocytosis (Mohanty et al., 2007). As with the increase in ROS production, there was also an increase in production of nitric oxide during the early stages of infection. The increase in NO production following infection with F. columnare could be attributed to lipopolysaccharide, which is a component of cell walls of Gram-negative bacteria and is reported to stimulate NO production in fish (Yin et al., 1997; Tafalla and Novoa, 2000). Nitric oxide, produced by activated phagocytic cells i.e. monocytes and macrophages, plays an important role in innate immunity as it has antimicrobial activities (Laing et al., 1999) and exert cytostatic as well as cytotoxic effects (Guoyao and Morris, 1998). Besides the protective role it can confer, NO can result in tissue injury in the host, particularly during acute inflammation (Thiemermann, 1997; Kengatharan et al., 1998). The results presented here are in accordance with those reported earlier following bacterial infection (Rodriguez et al., 2008; Nakhro et al., 2013). In contrast to the findings of the present study, inhibition of NO production has been reported previously against F. columnare infection (Zahran et al., 2014) and this discrepancy could be due to late sampling in the study missing the main window of activity, i.e. 14 days following experimental infection. It is to be noted that NO is produced during acute infection, which is usually the case with F. columnare infection. The induction of ROS and NO has been shown to play a crucial role in limiting the growth of fish pathogens. From these results, it can be inferred that F. columnare infection would have enhanced the production of ROS and NO as an immediate protective mechanism thereby, contributing in containing the infection. Myeloperoxidase is an important enzyme produced by neutrophils during respiratory burst (Klebanoff, 2005). The enzyme utilizes hydrogen peroxide generated during respiratory burst to produce hypochlorous acid with a potent microbicidal action (Dalmo et al., 1997). In this study, MPO activity was observed to be higher up to 12 hpi. After 24 hpi, no significant differences were recorded in the infected group in comparison to the control group. Similar results have been reported following infection with other bacterial pathogens, namely A. hydrophila (see Das et al., 2011) and Vibrio alginolyticus (see Wang et al., 2016). Increased production of MPO, as observed in the current study, might be due to an inflammatory response in the early stages of infection to protect from bacterial damage. Several proteases are secreted by pathogens to annihilate host's proteins for obtaining nutrients (Kumru et al., 2017). On the other hand, anti-proteases, produced by the host, can modulate the protease activities of pathogens and control a variety of protease-mediated processes including resistance to assault by infectious agents (Ellis, 1987; Zuo and Woo, 1997). These anti-proteases consist mainly of α-2 anti-protease, α-2 anti-plasmin and α-2 macroglobulin (α-2 M). In the present study, there was a decrease in total anti-protease activity of the infected catla till 24 hpi. The decrease in host anti-protease activity could be due to proteases secreted by F. columnare. The strong induction of protease concentration in the growth medium within 24 h of incubation with F. columnare (Newton et al., 1997) also supports the present assumption. Importantly, the proteases secreted by the bacterial pathogen have been reported to cause the tissue damage (Dalsgaard, 1993). Therefore, it could be assumed that protease activity of the F. columnare would have contributed to severe necrosis in the gills of experimentally infected catla in the present study. Similar to the observations in the current study, a decrease in anti-protease activity in

myeloperoxidase activity (Fig. 4c) upto 12 hpi in comparison to fish in the control group. All the above parameters in the infected group were comparable to fish in the control group from 24 to 96 hpi. The total anti-protease activity in the infected group was significantly lower (p < .05) from 3 to 24 hpi in comparison to control group. Further, total anti-protease activity was also significantly lower (p < .05) at 3 and 6 hpi in the infected group compared to those examined at 12 and 24 hpi and thereafter, no difference was observed (Fig. 4d). α-2 macroglobulin activity in the infected group (Fig. 4e), however, did not show any difference to that of the control group throughout the experimental period. In the infected group, ACH50 activity at the initial stages of infection i.e. from 3 to 12 hpi was significantly lower (p < .05) than that in the control group. Thereafter, at 24 hpi, ACH50 of the infected group was comparable to the control group, whereas, at 48 and 72 hpi, the complement activity was significantly higher (p < .05) than the control group. The complement activity at 96 hpi, however, was similar to that in control group (Fig. 4f). Lysozyme activity of the infected catla was similar to fish in the control group at early stages of infection i.e. 3 and 6 hpi. With the progression of infection i.e. 12 hpi onwards, lysozyme activity was significantly higher (p < .05) in the infected group compared to the control group as well as those fish examined in the initial stages of infection i.e. 3 and 6 hpi (Fig. 4g). The total serum protein in the infected group was lower (p < .05) from 48 to 96 hpi (Fig. 4h), whereas, the serum globulin level was significantly lower (p < .05) from 24 hpi onwards up to 96 hpi (Fig. 4i). Serum albumin level (Fig. 4j) in the infected group was comparable to that of control group throughout the experimental period.

4. Discussion In the present study, the clinical signs presented were typically those of lethargy, swimming near the water surface and accelerated opercular movements, which were suggestive of respiratory distress. Further, the gross lesions were of a yellowish-white discoloration in the gills along with fraying of gill lamellae. The clinical signs and gross lesions in gills of catla are in conformity with previous reports of F. columnare infection in channel catfish Ictalurus punctatus and zebrafish Danio rerio (Olivares-Fuster et al., 2011), common carp Cyprinus carpio (see Declercq et al., 2015) and catla (Verma and Rathore, 2013). The histopathological lesions included bacterial masses attached to primary lamellae and secondary gill filaments, inflammatory exudate comprising of mononuclear cells and eosinophilic granular cells between secondary gill filaments along with sloughing of epithelial cells and fusion of gill filaments. It was assumed that the lesions in the gills in this study would have been responsible for respiratory distress leading to mortality in the infected group. These results are similar to those reported previously in Nile tilapia Oreochromis niloticus, koi carp Cyprinus carpio koi, common carp and rainbow trout Oncorhynchus mykiss following infection with F. columnare (see Amin et al., 1988; Decostere et al., 2002; Declercq et al., 2015). The antimicrobial defence mechanisms in teleosts mostly rely on reactive oxygen species (ROS) produced by the phagocytes (Laing et al., 1999; Das et al., 2011). In the present study, fish in the infected group showed significantly higher ROS at the initial stages of infection i.e. until 12 hpi. Similar results have been observed following infection with Aeromonas hydrophila (see Rodriguez et al., 2008; Das et al., 2011; Jothi et al., 2012) and Edwardsiella tarda (see Nakhro et al., 2013). The 27

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Fig. 4. Effect of Flavobacterium columnare infection on various non-specific immune parameters in Catla catla at different hours post infection: a) Respiratory burst activity; b) Nitric oxide production; c) Myeloperoxidase activity; d) Total anti-protease activity; e) α-2 macroglobulin activity; f) ACH50 activity; g) Lysozyme activity; h) Total serum protein level; i) Serum albumin level; and, j) Serum globulin level. Data are presented as mean ± S.E. Significant differences (p < .05) are indicated by different letters (a, b, c, d) over the bars.

infected group as compared to fish analysed from the control group. The findings are in agreement with previous reports in F. columnare (Tripathi et al., 2005) and acute erythrodermatitis infection (Stosik et al., 2001). In conclusion, a bath challenge with F. columnare resulted in an acute infection and severe necrotic changes in the gills of affected catla juveniles. Further, some of the innate immunity parameters, namely production of reactive oxygen and nitrogen species and myeloperoxidase activity were higher in the challenged fish during the early stages of infection in comparison to fish in the control group. On the other hand, some of the immune parameters such as ACH50 and lysozyme activity were higher during the later stages of infection. It can be concluded, therefore, that F. columnare infection modulated the innate immune responses of the host, and higher activity of different innate immunity parameters would have helped in containment of the infection. The information generated in our study would be useful for developing strategies in improving resistance to F. columnare infection by stimulating the innate immunity through immunomodulation. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.05.015.

rohu has also been reported following infection with other bacterial pathogens, namely E. tarda (see Mohanty and Sahoo, 2010) and A. hydrophila (see Das et al., 2011). The results of the present study revealed that α-2 M activity remained unchanged throughout the experiment. Therefore, it was assumed that the observed decrease in total anti-protease activity would be due to a decrease in activity of antiproteases other than α-2 M. The results are in accordance with previous study (Freedman, 1991) in which no changes in α-2 M activity were observed in brook trout Salvelinus fontinalis following infection with A. salmonicida. The complement system plays a crucial role in the innate defence mechanism against common pathogens. Activation of complement leads to robust and efficient proteolytic cascades, which terminate in opsonization and lysis of the pathogen as well as in the generation of the classical inflammatory response (Dunkelberger and Song, 2010). There are three pathways of complement activation, out of which, the alternative complement pathway is more active in fish than classic and lectin pathways (Ellis, 2001; Holland and Lambris, 2002). In this study, ACH50 activity varied with the progression of infection. There was a strong inhibition of ACH50 activity up to 12 hpi. Thereafter, it returned to normal levels from 24 hpi and afterwards, it continuously increased from 48 to 72 hpi and again returned to normal levels at 96 hpi. These results are in agreement with an earlier report of F. columnare infection in channel catfish (Ourth and Bachinski, 1987). The authors suggested that sialic acid produced by Gram negative bacteria including F. columnare, might inhibit the alternate complement activity. Furthermore, destruction of C3 convertase enzyme by bacteria has also been reported to be responsible for decrease in complement activity (Tambourgi et al., 1993). Subsequent increases in ACH50 activity during the later stages of infection could have contributed in containing the infection. Lysozyme, secreted by leucocytes, plays an important role in innate immunity against microbes through opsonization, activation of complement system and phagocytosis (Ellis, 1990; Saurabh and Sahoo, 2008; Davis and Weiser, 2011). In the present study, lysozyme activity in infected catla was comparable to the control group until 6 hpi, whereas, from 12 to 96 hpi, it was significantly higher than those fish sampled from the control group. Hence, it could be assumed that an increase in lysozyme activity in experimentally infected catla might have played an important role in containing F. columnare infection in later stages. Previously, higher expression of lysozyme genes in fish has been reported to play an important role in increased survival against F. columnare infection (Yazawa et al., 2006; Peatman et al., 2013; Ren et al., 2015). Results similar to the present study have also been reported in other bacterial diseases such as infection with E. ictaluri (see Wang et al., 2013), E. tarda (see Caruso et al., 2002; Mohanty and Sahoo, 2010; Devi et al., 2012; Li et al., 2013) and A. hydrophila (see Jothi et al., 2012). In the present study, the total serum protein level was found to be comparable with that of the control group until 12 hpi, and from 48 hpi onwards, it was significantly lower until 96 hpi. The results are similar to those observed following infection with F. columnare in koi carp and brook trout (Tripathi et al., 2005; Rehulka and Minarik, 2007), A. hydrophila in common carp (Maqsood et al., 2009) and E. tarda in rohu and Nile tilapia (Mohanty and Sahoo, 2010; Benli and Yildiz, 2004). A decreased level of serum protein in infected group might be due to decreased synthesis, reduced absorption or higher protein loss. The serum albumin level did not show a significant difference between the control and experimentally infected group. In case of serum globulin, there was no change until 12 hpi, whereas, from 24 hpi onwards, a significant decrease in the level of serum globulin was observed in the

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