Veterinary Immunology and Immunopathology 117 (2007) 137–141 www.elsevier.com/locate/vetimm
Serum antibody response of Indian major carp, Labeo rohita to three species of pathogenic bacteria; Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens P. Swain *, A. Behura, S. Dash, S.K. Nayak Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga 751002, Bhubaneswar, India Received 19 October 2006; received in revised form 19 February 2007; accepted 21 February 2007
Abstract The immune response to mixed whole cell antigens of Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens, the common Gram negative bacterial pathogens associated with diseases of Indian major carps were evaluated for their efficacy in triggering antibody responses in rohu, Labeo rohita (Ham.). The rohu yearlings were either immunized with antigens from single bacterial strain, A. hydrophila, E. tarda and P. fluorescens or a combination of all three. An antibody response was detected at 1st week post immunization that rose significantly ( p < 0.05) at 4th week post immunization in all the immunized groups. The antibody level started declining after 8th week but persisted up to 10th week post immunization in all the immunized groups. Similarly, no significant difference ( p > 0.05) in the antibody level was found between groups immunized with single and mixed bacterial antigens. Moreover, the use of mixed bacterial antigens did not jeopardize the specific immune response to the vaccine components. Upon challenge with single pathogen, a high relative percent survival was recorded in the group immunized with mixed bacterial antigens and was comparable to those fish immunized with the single bacteria. # 2007 Elsevier B.V. All rights reserved. Keywords: Antibody response; Indian major carps; Labeo rohita; Mixed bacterial antigens; Rohu
1. Introduction Diseases are major bottlenecks in the development and sustainability of aquaculture practices throughout the world. Among different types of infectious agents bacterial pathogens are often responsible for severe mortalities in a wide range of fishes at different stages of growth (Grisez and Ollevier, 1995; Swain et al., 2002). Due to emergence of antibiotic resistant strain, residual effect and environmental degradation/pollution caused by regular use of antibiotics, much attention has been
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(P. Swain). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.02.010
paid to immunize the fish with vaccine(s) for better protection (Evelyn, 1997). During the past decades, the development of suitable vaccine(s) and vaccination strategy have helped in controlling infectious diseases in aquaculture with respect to type of pathogen and cultured species (Vinitnantharat et al., 1999; Hastein et al., 2005). However, till date only a few vaccines, i.e., against enteric red mouth, furunculosis, vibriosis, pasteurellosis, yersiniosis, etc., are available (Evelyn, 1997; Larsen and Pedersen, 1997; Vinitnantharat et al., 1999; Gudding and Evesen, 2005). These vaccines are mostly monovalent containing the whole cell antigen or components of a single pathogen. The major problem associated in immunizing individual fish with antigen is that it lives in an environment, where a wide range of
P. Swain et al. / Veterinary Immunology and Immunopathology 117 (2007) 137–141
2.3. Preparation of bacterial antigen
other pathogens and secondary invaders are present, and a specific immunization strategy may be able to protect a specific pathogen or disease only. Nevertheless, it is not possible to immunize the fish with all pathogens due to practical difficulties, stress factors and adverse reaction at injected area (Hastein et al., 2005). On the other hand, immunization strategy involving the multiple antigens can be useful to elicit immune responses thereby protecting against a wide range of pathogens and this may also reduce the cost of immunization and handling stress to fish. India ranks third among the world freshwater fish producers (FAO, 2003) with Indian major carps viz., Labeo rohita, Catla catla and Cirrhinus mrigala are the most preferred cultured species. During culture, significant losses due to a variety of infectious agents are reported. Among these, the bacterial pathogens belonging to the genus Aeromonas, Pseudomonas, Edwardsiella, Flavobacterium, etc., are mainly responsible for severe mortality and morbidity of Indian major carps (Bootsma et al., 1977; Kumar et al., 1986; Shome et al., 1996). Despite of sever economic loss, a limited research effort till date has been made to develop a monovalent vaccine (Karunasagar et al., 1997; Chandran et al., 2002) without any attempt on polyvalent or mixed vaccines. Therefore, the present investigation was carried out to evaluate the antibody response against the mixed bacterial antigens in order to possibly develop a vaccine for use in Indian major carps.
The formalin killed cells of A. hydrophila, E. tarda and P. fluorescens were separately sonicated at 40 Hz for 5 min, followed by centrifugation at 10,000 g for 15 min. After centrifugation, supernatant was collected and the protein content of each supernatant was estimated by the standard method of Lowry et al. (1951). Finally, the supernatant was stored at 20 8C till further use.
2. Materials and methods
2.6. Experimental design
Groups of rohu yearlings were immunized with antigen from single species of bacteria or with a mixture of the three bacterial antigens, separately. During the entire course of investigation, physico-chemical parameters like pH, temperature, dissolved oxygen and total hardness were found to vary from 7.2 to 7.6, 27 to 30 8C, 5.5 to 6.5 mg/ml and 90 to 110 ppm, respectively. No specific variations ( p > 0.05) could be recorded among any of these parameters and were within the permissible limit.
Rohu yearlings of average weight 100–150 gm, obtained from fish farms, were acclimatized in the wet laboratory of Fish Health Management Division of Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, India, 15 days prior to the start of the experiment. Ten rohu yearlings per tank were maintained in 1000 l-cemented tanks of the wet laboratory. The fish were fed with artificial carp diet with constant aeration and daily one-third water exchange.
All the three bacterial species were separately grown in brain heart infusion broth (Hi-media, India) for overnight at 30 8C. After incubation the total bacterial count was enumerated by standard plate count method. The bacterial culture was inactivated with 1% formalin at 4 8C overnight. The bacteria were then centrifuged at 10,000 g for 10 min and washed twice in phosphate buffer saline (PBS, pH 7.2). Finally, individual bacterial suspension containing 109 CFU ml 1 was made in same PBS. 2.4. Preparation of mixed bacterial antigen The mixed bacterial antigen was prepared by mixing the three bacteria, in equal concentration so that final bacterial concentration of the mixture was 109 CFU ml 1. 2.5. ELISA antigen
2.2. Bacterial pathogens
2.7. Immunization of rohu yearlings with single and mixed bacterial antigens
Three virulent bacterial pathogens, namely Aeromonas hydrophila (FHM01), Edwardsiella tarda (FHM98) and Pseudomonas fluorescens (FHM96), obtained from Fish Health Management Division of CIFA were used in the present investigation.
A group of 20 rohu yearlings each was separately immunized, intraperitoneally with 0.2 ml of single antigen containing 109 CFU ml 1 of formalin killed bacterial cells. Similarly, 80 rohu yearlings were immunized with 0.2 ml of mixed antigens containing
P. Swain et al. / Veterinary Immunology and Immunopathology 117 (2007) 137–141
109 CFU ml 1 of formalin killed combined bacterial cells. Blood was collected from immunized rohu through cardiac puncture from 1st week to 10th week post immunization. After bleeding, blood was allowed to clot and then serum was collected and stored at 20 8C till further use. 2.8. Preparation of anti-rohu-globulin-HRPO conjugate The rabbit anti-rohu globulin conjugate was prepared as per the method of Swain et al. (2002) using sera obtained from healthy adult rohu of average weight 250–300 gm. Briefly, serum was collected from healthy rohu and pooled to 10–15 ml. An equal volume of saturated ammonium sulphate solution was mixed with the pooled sera drop by drop and then placed on a magnetic stirrer for overnight at 4 8C. The sample mixture was centrifuged at 10,000 g for 10 min at 4 8C and the precipitate was dissolved with 5 ml carbonate–bicarbonate buffer (pH 9.6). The sample was then centrifuged at 10,000 g for 10 min at 4 8C. The pellet was collected and the volume was made to 2 ml with carbonate–bicarbonate buffer (pH 9.6). The globulin solution was dialyzed using dialysis membrane (Snakeskin, USA) against PBS (pH 7.2) for 72 h at 4 8C, after which the globulin was collected. The anti-rohu globulin sera were raised in rabbit (New Zealand white bred) as per the method of Lund et al. (1991). Finally, the HRPO conjugate was prepared with the hyper immune anti-rohu globulin sera by using HRPO conjugation kit (Genei, India).
polystyrene plates (Nunc, Denmark). The wells were separately coated with 50 ml of A. hydrophila, P. fluorescens and E. tarda ELISA antigen (2–4 mg/well) diluted in carbonate–bicarbonate buffer (pH 9.6) for overnight at 4 8C. The plates were then washed in phosphate buffer saline (pH 7.2) containing Tween-20 (PBS-T) and blocked with 100 ml of 3% skim milk powder for 2 h at 37 8C. The wells were further washed in PBS-T. The fish sera raised against single bacterial antigen was diluted to 1:50 with PBS (pH, 7.2) and added to homologous antigen-coated wells in duplicate per serum dilution. The plates were incubated at 37 8C for 1 h and washed thrice in PBS-T. Rabbit anti-rohu HRPO conjugate at a dilution of 1:200 was added to each well and incubated at 37 8C for 45 min. The wells were then thoroughly washed and added with 50 ml of substrate solution (5 mg of O-phenylenediamine tetrahydrochloride and 10 ml of H2O2 (38%, v/v) in 5 ml of acetate buffer, pH 5.0). The plates were incubated at 37 8C for 5 min in a dark chamber and finally the optical density (OD) was recorded at 450/655 nm in a microplate reader (BIO-RAD, USA). Similarly, indirect ELISA was done for serum samples raised against mixed bacterial antigens with single bacteria. The ELISA antigen of single bacteria was separately coated to polystyrene plate and titrated against the sera raised against mixed bacterial antigens using the same protocol as done above. The antibody level was expressed in terms of OD values at 1:50 dilution of serum after deducting the respective OD values obtained with control unimmunised rohu serum against respective single bacteria.
2.9. Agglutination 2.11. Challenge study The agglutination test was conducted in ‘U’ shaped microtitre plates. The sera raised against different bacterial antigen were serially two-fold diluted in PBS (pH 7.2) and then equal amount of homologous formalin-killed bacteria (107 CFU ml 1) were added separately to each well. The plates were kept overnight at room temperature and agglutination titre was calculated. The titre was calculated as the reciprocal of the highest dilution of serum showing complete agglutination of the bacteria. Similarly, the agglutination titre of sera raised against mixed bacterial antigens to single bacteria was also calculated. 2.10. Indirect ELISA The indirect ELISA was conducted as per the method of Swain et al. (2002) using 96 well microtitre
Fish immunized with mixed bacterial antigens were divided into four sub-groups (20 nos. per group) and challenged intraperitoneally with 0.2 ml of virulent A. hydrophila, P. fluorescens, E. tarda pathogen, separately and their combination containing 108 cells/ml at 8th week post immunization. Similarly fish immunized with single bacterial antigen and control unimmunized fish (20 nos. per pathogen) were challenged with the homologous bacterial pathogen in a similar manner as mentioned above. The mortality was recorded up to 7 days post challenge and the relative percent survival in each group was calculated as per the following formulae. Relative percent survival = [1-% of mortality in immunized group/% of mortality in unimmunized group] 100.
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2.12. Statistical analysis Two-way analysis of variance (ANOVA) was performed to analyse the data of agglutination test and indirect ELISA results. Duncan multiple range test (DMRT) was used to test the significant difference between the antibody level in the single and mixed bacterial antigens immunized groups using SAS Version 6.12 (SAS System for regression). 3. Results and discussion During past 1–2 decades, much attention has been given to design and develop suitable monovalent and polyvalent vaccines based on the antigenic nature of the used strains, dose, route of administration for controlling diseases in aquaculture (Busch, 1997; Evelyn, 1997; Collado et al., 2000; Romalde et al., 2005). In this investigation, whole cell antigens of A. hydrophila, E. tarda and P. fluorescens, in single and combined forms were evaluated for antibody response in rohu yearlings. The mean natural agglutinin titre (log 2) against A. hydrophila, E. tarda and P. fluorescens in healthy fish were recorded to be 2, 1 and 1, respectively. In both single and mixed antigen immunized groups, the antibody level was detected at 1st week post immunization and its level was highest ( p < 0.05) at 4th week post immunization (Fig. 1). The mean agglutination titre of 5, 4 and 2.7 for A. hydrophila, E. tarda and P. fluorescens, respectively, was recorded in groups immunized with single bacterial antigen where as that of 5, 3.7 and 2.7 for A. hydrophila, E. tarda and P. fluorescens, respectively was recorded in groups immunized with mixed bacterial antigens. The antibody level declined after 8th week but persisted up to 10th week post immunization. In indirect ELISA, the mean (S.D.) antibody level with OD value of 0.029, 0.037 and 0.029 was recorded for A. hydrophila, E. tarda and P. fluorescens,
Fig. 1. The serum antibody response to different bacteria in rohu yearlings immunized with single homologous bacterial antigen (continous line) and mixed bacterial antigens (dotted line).
respectively in healthy and unimmunised control rohu yearlings. In groups immunized with single bacterial antigen, the highest antibody level expressed in terms of OD values of 0.083 (0.04), 0.114 (0.01) and 0.106 (0.04) for A. hydrophila, E. tarda and P. fluorescens, respectively was recorded at 4th week post immunization. While the values were 0.074(0.03), 0.108 (0.09), and 0.102 (0.05) for A. hydrophila, E. tarda and P. fluorescens, respectively in groups immunized with mixed bacterial antigens. No significant difference ( p > 0.05) in the antibody level in groups immunized with single bacterial antigen to that of mixed bacterial antigens immunized groups could be recorded from 4th to 8th week post immunization. In earlier reports, similar type of antibody response has also been recoded in different fishes including Indian major carps (Karunasagar et al., 1997; Chandran et al., 2002; Madetoja et al., 2006). The success of polyvalent vaccines often regulated by the concentration of individual antigens, cross reactivity and competition among different antigens (Amend and Johnson, 1984; Busch, 1997; Swain et al., 2003; Nikoskelainen et al., 2006). The antibody response was par ( p > 0.05) at all stages of post immunization in both single and mixed bacterial antigens immunized groups (Fig. 2). The study further suggested the existence of antigenic relation and lack of antigenic competition among these three Gram ve bacterial pathogens which we had also recorded earlier (Swain et al., 2003). Moreover, on challenge with single and mixed pathogens high protection was recorded in groups immunized with mixed bacterial antigen compared to 100% mortality in control groups for all the pathogens.
Fig. 2. The mean (S.D.) OD values of antibody level at 1:50 dilution of rohu sera in groups immunized with individual and mixed bacterial antigens.
P. Swain et al. / Veterinary Immunology and Immunopathology 117 (2007) 137–141 Table 1 The percentage of survivability of immunized rohu yearlings challenged intraperitoneally after 8th week post immunization with individual and mixed bacterial pathogens Pathogen challenged
A. hydrophila P. fluorescens E. tarda Mixed pathogen
Relative percentage of survivability in groups immunized with Individual bacterial antigen
Mixed bacterial antigen
80 80 80 Not done
80 70 80 70
The relative percent survival was found to be 80 in both single bacterial and mixed bacterial antigens immunized groups when challenged with single pathogen except 70% in case of P. fluorescens in mixed bacterial antigens immunized group. In combined challenge study with mixed bacterial immunized group, the relative percentage of survival was 70 (Table 1). The present study suggested the increase in antibody response to mixed bacterial antigens without jeopardizing the specific immune response to vaccine components which might be due to high cross reactivity and non-antigenic competition among these three bacterial pathogens. Therefore, strategies may be made to develop a mixed vaccine for protecting Indian major carps, so that they can be saved from these common infections and the cost of vaccine and vaccination can be reduced significantly with less stress to cultured fish. Acknowledgement The authors are thankful to the Director of Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Orissa, India for providing necessary facilities to carry out the present investigation. References Amend, D.R., Johnson, K.A., 1984. Evidence for lack of antigenic competition among various combinations of Vibrio anguillarum, Yersinia ruckeri, Aeromonas salmonicida and Renibacterium salmoninarum bacterins when administered to salmonid fishes. J. Fish Dis. 7, 293–299. Bootsma, R., Fijan, N., Blommaert, J., 1977. Isolation and preliminary identification of the causative agent for carp erythrodermatitis. Vet. Arch. 47 (6), 232–291. Busch, R.A., 1997. Polyvalent vaccines in fish: the interactive effects of multiple antigens. Dev. Biol. Stand 90, 245–256. Chandran, M.R., Aruna, B.V., Logambal, S.M., Dinakaran, M.R., 2002. Immunisation of Indian major carps against Aeromonas hydrophila by intraperitoneal injection. Fish Shellfish Immunol. 13, 1–9.
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