Vet Res Commun DOI 10.1007/s11259-014-9593-2
ORIGINAL ARTICLE
Effect of Edwardsiella tarda immunization on systemic immune response, mucosal immune response and protection in catla (Catla catla) Khriezhato Nakhro & Anushree Das & Dibyendu Kamilya
Accepted: 21 January 2014 # Springer Science+Business Media Dordrecht 2014
Abstract The effect of immunization on systemic and cutaneous mucosal immune responses of fish and their possible relation with protection has not been fully assessed. In this study, healthy catla (Catla catla) were immunized against Edwardsiella tarda using two antigenic preparations namely, whole cell bacterin (B) and bacterin mixed with Freund’s complete adjuvant in a 1:1 (v/v) ratio (B+A) followed by a booster dose after 3 weeks of first injection. Different systemic and cutaneous mucosal immune responses were measured at weekly interval upto 8th week post vaccination (pv). Fish were challenged 8 weeks pv with live E. tarda to study vaccine induced protection. The result showed that although there were strong systemic as well as mucosal immune responses, particularly after booster dose, the challenge produced low to moderate protection in terms of relative percent survival (RPS). The maximum RPS (50 %) was recorded in the adjuvanted bacterin group after 8 weeks pv. Low to moderate protection after challenge, which may be attributed to the intracellular nature of E. tarda and/or use of crude antigenic preparation, accounts for new strategy to be developed for immunization programme against such intracellular pathogen. The results collectively suggest possible involvement of systemic as well as mucosal immune responses in inducing protective immunity in catla.
Keywords Immunization . Edwardsiella tarda . Catla (Catla catla) . Immune response . Relative percent survival
K. Nakhro : A. Das : D. Kamilya (*) Department of Fish Health and Environment, College of Fisheries, Central Agricultural University, Lembucherra, Tripura (W) 799 210, Tripura, India e-mail: [email protected]
Introduction Vaccination has become an important immunoprophylactic measure against bacterial infections in farmed fish (Gudding et al. 1999). Several vaccine preparations have been explored for use in the aquaculture industry against a variety of bacterial diseases, including the bacterin against pathogenic vibriosis (Toranzo et al. 2009), furunculosis (Midtlyng 1997), enteric red-mouth disease (Stevenson 1997) and motile aeromonad septicemia (LaPatra et al. 2010), the live attenuated vaccine against enteric septicemia of catfish (Klesius and Shoemaker 1998) and edwardsiellosis (Yang et al. 2013). In addition to that, different novel vaccine preparations are also being developed (Kwon et al. 2006; Swain et al. 2010; Choi and Kim 2011; Sun et al. 2011). The effectiveness of a vaccine is generally established by challenge experiments. Eliciting antibody as well as cellmediated immune (CMI) response to protective antigen is also important in determining vaccine efficacy. Recently, there has been a trend to even measure innate immune components to evaluate vaccine efficacy (Saikia and Kamilya 2012; Behera and Swain 2012; Bharadwaj et al. 2013; Yang et al. 2013). However, involvement of cutaneous mucosal immunity in determining the vaccine efficacy has not been given much attention even though some reports describes the relation of mucosal antibody titres with vaccine induced protection. Edwardsiellosis, caused by the intracellular bacterium Edwardsiella tarda, is a common bacterial disease which occurs in a wide range of fishes, reptiles, birds and mammals including humans (Rao et al. 2001). This bacterium is associated with diseases of all the life stages of fish resulting in massive mortalities and large economic losses in fish farming worldwide (Plumb 1999; Mohanty and Sahoo 2007). Several vaccination trials against this bacterial disease have been employed to protect fish from edwardsiellosis. However, vaccine induced time-dependent alterations on different immune
Vet Res Commun
responses and their relation with protection has not been fully assessed. More specifically, information on the effect of vaccine on different parameters of cutaneous mucosal immunity of fish is meagre. Thus, the present study was undertaken to delineate the kinetics of systemic as well as cutaneous mucosal immune responses of catla (Catla catla) following immunization with E. tarda bacterin and to find out any relation between the immune responses and vaccine induced protection after homologous pathogen challenge.
Materials and methods Experimental fish and rearing Healthy catla (weight 30–40 g) were obtained from a local fish farmer. The fish were acclimatized in 1,000 l circular tanks at ambient temperature with aeration and were fed twice daily with a pelleted diet @5 % of body weight. The physicochemical parameters of the water were monitored throughout the experimental period to maintain optimum condition. Periodical water exchange (upto 50 %) was done to remove the waste feed and fecal matter. Bacteria and preparation of antigen The E. tarda strain (ET-PG-29), known to be pathogenic to catla (Devi et al. 2012) was used in all the experiments. The whole-cell bacterin was prepared as previously described (Kamilya et al. 2006a). E. tarda was grown on tryptic soy broth for 48 h at 30 °C to a density of approximately 1010 viable cells ml-1. The bacterial cell suspension was treated with formalin to a final concentration of 1.0 % (v/v) and left overnight at 4 °C. The suspension was washed three times with phosphate-buffered saline (PBS, pH 7.2) and sterility was checked. The washed, formalin-killed bacterial cells were then resuspended in PBS and the protein content of the bacterin was measured by biuret method (Gornall et al. 1949). The bacterin was stored frozen at −20 °C until used. Immunization of experimental fish Catla were divided randomly in three groups with three replicates for each group (40 fish in each replicate tank). Before immunization, fish were monitored by bacteriological methods to confirm the absence of any interfering pathogen. Fish were anaesthetized using clove oil @ 100 μl L−1, and were immunized intraperitonially by two types of antigen preparations. The first group was injected with 200 μl of bacterin (designated as B). The second group received bacterin mixed with Fruend’s complete adjuvant (FCA, Bangalore Genei, India) in a 1:1 ratio (v/v) ratio (designated as B+A). The third group, comprised of control fish, injected with only
PBS. Each of the treatment groups received 200 μg of protein per fish. Booster dose was administered 3 weeks after the first injection using the same amount of antigen. However, for the second antigen group Fruend’s incomplete adjuvant was used instead of FCA. Sampling, serum collection, mucus collection and isolation of head kidney (HK) leukocytes Samples were collected from each replicate tank (3 fish for each group) at 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th week post vaccination (pv). Prior to sacrificing the fish for the separation of the HK, anaesthetized fish were bleed directly from the heart/caudal vein. Serum was collected from the clotted blood and stored at −20 °C in sterilized vial until further use. Mucus was collected by gentle scraping just posterior to the operculum (Cain et al. 2000). An equal volume of PBS containing 0·02 % azide was added to collected mucus, and samples were vortexed for 1 min and stored at 4 °C overnight. Mucus was then centrifuged at 1,200×g for 10 min and supernatant collected. The processed mucus samples were stored at −20 °C until needed for analysis. Isolation of HK leukocytes was done following the method described previously (Kamilya et al. 2006b). In brief, HK was removed aseptically from the immunized and control fish after blood collection and the HK cell suspension was obtained in complete RPMI-1640 medium. The leukocytes were isolated by density gradient centrifugation using the leukocyte isolation medium, HiSep (Hi-media). Purified leukocytes were counted using a haemocytometer and the cell viability was determined by the trypan blue exclusion test. CMI responses Antigen- specific leukocyte proliferation The antigen-specific leukocyte proliferation was determined by the 3-(4,5-dimethyl thiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Hi-media) colorimetric assay method (Mosmann 1983). After distributing HK leukocytes into wells (100 μl per well) of a 96-well microtitre plate, 100 μl of antigen (final concentration being 20 μg/ ml) was added to each well for sensitization of the leukocytes. Some wells were kept without the addition of antigen. The plate was incubated for 72 h at 25 °C. Twenty μl of MTT solution (5 mg MTT ml−1 of PBS and sterilized by filtration) was added to all the wells and the plate was incubated further at 25 °C for 4 h. After incubation, the media were removed after centrifugation of the plate and 200 μl of dimethyl sulphoxide (Hi-media) was added to all the wells and mixed for 2 min. The microtitre plate was read at 595 nm using a microreader. The results were expressed as stimulation
Vet Res Commun
index (SI), which was calculated by dividing the mean optical density (OD) of stimulated cultures by the OD of the non-stimulated cultures.
Antigen-specific macrophage activating factor (MAF) production and detection Antigen-specific MAF production and detection of MAF activity were examined following the method described previously (Kamilya et al. 2006a). Aliquots of leukocyte suspension (2×106 cells ml −1) were pulsed with 0.5 ml medium containing 1×108 formalin killed E. tarda. Leukocytes were then incubated for 3 h at 25 °C and the cultures were supplemented with 10 % fetal calf serum. After 48 h of culture, the supernatants were harvested, centrifuge at 10 000×g to remove bacteria and cell debris, aliquoted and stored at −20 °C prior to use. For detection of MAF activity, supernatants were diluted in RPMI medium (1:4) and added to macrophage monolayers in 96-well tissues culture plates, prepared from unimmunized fish as described previously (Secombes 1990). The macrophages were cultured in the presence of the supernatants for 48 h and then their ability to reduce nitroblue tetrazolium chloride (NBT, Hi-media) was determined by respiratory burst assay (Kamilya et al. 2006b).
Humoral responses The total myeloperoxidase content of serum and mucus was estimated as described by Mohanty and Sahoo (Mohanty and Sahoo 2010). The lysozyme activity of serum and mucus was determined as described by Ellis (Ellis 1990). Lysozyme activity was expressed as units ml−1 where one unit was defined as the reduction in absorbance of 0.001 min−1. Serum and mucosal antibody titre was determined by the microtitre agglutination method (Karunasagar et al. 1997). Antibody titre was expressed as the reciprocal of the last dilution giving visible agglutination. Haemagglutination assay was done as described by Kumari and Sahoo (2005). The protein content of serum was estimated by following the biuret method (Gornall et al. 1949). Homologous pathogen challenge Homologous pathogen challenge of the immunized fish was done 8 weeks after the immunization. Ten fish from each tank were transferred to another set of tanks and were challenged with E. tarda (1×106 CUF ml−1). Challenge was performed by injecting each fish intraperitonially with 0.2 ml of bacteria. The cause of death and pathological changes were verified by re-isolation of bacteria from dead/infected samples. The mortality was recorded for 2 weeks and the relative percent survival (RPS) was calculated using the following formula:
RPS ¼ ½1−% of mortality in immunized group=% of mortality in control group 100:
Statistical analysis
Antigen-specific MAF production
Statistical analysis of data was performed using SPSS-15.0 for windows software (SPSS Inc., Chicago, IL, USA). Results are presented as mean±standard error of mean (SEM). Data were analyzed by the student’s t-test, one-way analysis of variance (ANOVA) and Tukey’s range test. Probability levels of 0.05 were used to find out the significance in all cases.
Antigen-specific MAF production showed almost similar trend to that of leukocyte proliferation activity in all the sampling weeks for all the immunized groups. The maximum MAF activity was observed in case of adjuvanted bacterin after 5th sampling week following the booster dose (Fig. 2). Myeloperoxidase activity of serum and mucus
Results Antigen-specific leukocyte proliferation All the immunized groups showed significant leukocyte proliferation activity (p
ORIGINAL ARTICLE
Effect of Edwardsiella tarda immunization on systemic immune response, mucosal immune response and protection in catla (Catla catla) Khriezhato Nakhro & Anushree Das & Dibyendu Kamilya
Accepted: 21 January 2014 # Springer Science+Business Media Dordrecht 2014
Abstract The effect of immunization on systemic and cutaneous mucosal immune responses of fish and their possible relation with protection has not been fully assessed. In this study, healthy catla (Catla catla) were immunized against Edwardsiella tarda using two antigenic preparations namely, whole cell bacterin (B) and bacterin mixed with Freund’s complete adjuvant in a 1:1 (v/v) ratio (B+A) followed by a booster dose after 3 weeks of first injection. Different systemic and cutaneous mucosal immune responses were measured at weekly interval upto 8th week post vaccination (pv). Fish were challenged 8 weeks pv with live E. tarda to study vaccine induced protection. The result showed that although there were strong systemic as well as mucosal immune responses, particularly after booster dose, the challenge produced low to moderate protection in terms of relative percent survival (RPS). The maximum RPS (50 %) was recorded in the adjuvanted bacterin group after 8 weeks pv. Low to moderate protection after challenge, which may be attributed to the intracellular nature of E. tarda and/or use of crude antigenic preparation, accounts for new strategy to be developed for immunization programme against such intracellular pathogen. The results collectively suggest possible involvement of systemic as well as mucosal immune responses in inducing protective immunity in catla.
Keywords Immunization . Edwardsiella tarda . Catla (Catla catla) . Immune response . Relative percent survival
K. Nakhro : A. Das : D. Kamilya (*) Department of Fish Health and Environment, College of Fisheries, Central Agricultural University, Lembucherra, Tripura (W) 799 210, Tripura, India e-mail: [email protected]
Introduction Vaccination has become an important immunoprophylactic measure against bacterial infections in farmed fish (Gudding et al. 1999). Several vaccine preparations have been explored for use in the aquaculture industry against a variety of bacterial diseases, including the bacterin against pathogenic vibriosis (Toranzo et al. 2009), furunculosis (Midtlyng 1997), enteric red-mouth disease (Stevenson 1997) and motile aeromonad septicemia (LaPatra et al. 2010), the live attenuated vaccine against enteric septicemia of catfish (Klesius and Shoemaker 1998) and edwardsiellosis (Yang et al. 2013). In addition to that, different novel vaccine preparations are also being developed (Kwon et al. 2006; Swain et al. 2010; Choi and Kim 2011; Sun et al. 2011). The effectiveness of a vaccine is generally established by challenge experiments. Eliciting antibody as well as cellmediated immune (CMI) response to protective antigen is also important in determining vaccine efficacy. Recently, there has been a trend to even measure innate immune components to evaluate vaccine efficacy (Saikia and Kamilya 2012; Behera and Swain 2012; Bharadwaj et al. 2013; Yang et al. 2013). However, involvement of cutaneous mucosal immunity in determining the vaccine efficacy has not been given much attention even though some reports describes the relation of mucosal antibody titres with vaccine induced protection. Edwardsiellosis, caused by the intracellular bacterium Edwardsiella tarda, is a common bacterial disease which occurs in a wide range of fishes, reptiles, birds and mammals including humans (Rao et al. 2001). This bacterium is associated with diseases of all the life stages of fish resulting in massive mortalities and large economic losses in fish farming worldwide (Plumb 1999; Mohanty and Sahoo 2007). Several vaccination trials against this bacterial disease have been employed to protect fish from edwardsiellosis. However, vaccine induced time-dependent alterations on different immune
Vet Res Commun
responses and their relation with protection has not been fully assessed. More specifically, information on the effect of vaccine on different parameters of cutaneous mucosal immunity of fish is meagre. Thus, the present study was undertaken to delineate the kinetics of systemic as well as cutaneous mucosal immune responses of catla (Catla catla) following immunization with E. tarda bacterin and to find out any relation between the immune responses and vaccine induced protection after homologous pathogen challenge.
Materials and methods Experimental fish and rearing Healthy catla (weight 30–40 g) were obtained from a local fish farmer. The fish were acclimatized in 1,000 l circular tanks at ambient temperature with aeration and were fed twice daily with a pelleted diet @5 % of body weight. The physicochemical parameters of the water were monitored throughout the experimental period to maintain optimum condition. Periodical water exchange (upto 50 %) was done to remove the waste feed and fecal matter. Bacteria and preparation of antigen The E. tarda strain (ET-PG-29), known to be pathogenic to catla (Devi et al. 2012) was used in all the experiments. The whole-cell bacterin was prepared as previously described (Kamilya et al. 2006a). E. tarda was grown on tryptic soy broth for 48 h at 30 °C to a density of approximately 1010 viable cells ml-1. The bacterial cell suspension was treated with formalin to a final concentration of 1.0 % (v/v) and left overnight at 4 °C. The suspension was washed three times with phosphate-buffered saline (PBS, pH 7.2) and sterility was checked. The washed, formalin-killed bacterial cells were then resuspended in PBS and the protein content of the bacterin was measured by biuret method (Gornall et al. 1949). The bacterin was stored frozen at −20 °C until used. Immunization of experimental fish Catla were divided randomly in three groups with three replicates for each group (40 fish in each replicate tank). Before immunization, fish were monitored by bacteriological methods to confirm the absence of any interfering pathogen. Fish were anaesthetized using clove oil @ 100 μl L−1, and were immunized intraperitonially by two types of antigen preparations. The first group was injected with 200 μl of bacterin (designated as B). The second group received bacterin mixed with Fruend’s complete adjuvant (FCA, Bangalore Genei, India) in a 1:1 ratio (v/v) ratio (designated as B+A). The third group, comprised of control fish, injected with only
PBS. Each of the treatment groups received 200 μg of protein per fish. Booster dose was administered 3 weeks after the first injection using the same amount of antigen. However, for the second antigen group Fruend’s incomplete adjuvant was used instead of FCA. Sampling, serum collection, mucus collection and isolation of head kidney (HK) leukocytes Samples were collected from each replicate tank (3 fish for each group) at 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th week post vaccination (pv). Prior to sacrificing the fish for the separation of the HK, anaesthetized fish were bleed directly from the heart/caudal vein. Serum was collected from the clotted blood and stored at −20 °C in sterilized vial until further use. Mucus was collected by gentle scraping just posterior to the operculum (Cain et al. 2000). An equal volume of PBS containing 0·02 % azide was added to collected mucus, and samples were vortexed for 1 min and stored at 4 °C overnight. Mucus was then centrifuged at 1,200×g for 10 min and supernatant collected. The processed mucus samples were stored at −20 °C until needed for analysis. Isolation of HK leukocytes was done following the method described previously (Kamilya et al. 2006b). In brief, HK was removed aseptically from the immunized and control fish after blood collection and the HK cell suspension was obtained in complete RPMI-1640 medium. The leukocytes were isolated by density gradient centrifugation using the leukocyte isolation medium, HiSep (Hi-media). Purified leukocytes were counted using a haemocytometer and the cell viability was determined by the trypan blue exclusion test. CMI responses Antigen- specific leukocyte proliferation The antigen-specific leukocyte proliferation was determined by the 3-(4,5-dimethyl thiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Hi-media) colorimetric assay method (Mosmann 1983). After distributing HK leukocytes into wells (100 μl per well) of a 96-well microtitre plate, 100 μl of antigen (final concentration being 20 μg/ ml) was added to each well for sensitization of the leukocytes. Some wells were kept without the addition of antigen. The plate was incubated for 72 h at 25 °C. Twenty μl of MTT solution (5 mg MTT ml−1 of PBS and sterilized by filtration) was added to all the wells and the plate was incubated further at 25 °C for 4 h. After incubation, the media were removed after centrifugation of the plate and 200 μl of dimethyl sulphoxide (Hi-media) was added to all the wells and mixed for 2 min. The microtitre plate was read at 595 nm using a microreader. The results were expressed as stimulation
Vet Res Commun
index (SI), which was calculated by dividing the mean optical density (OD) of stimulated cultures by the OD of the non-stimulated cultures.
Antigen-specific macrophage activating factor (MAF) production and detection Antigen-specific MAF production and detection of MAF activity were examined following the method described previously (Kamilya et al. 2006a). Aliquots of leukocyte suspension (2×106 cells ml −1) were pulsed with 0.5 ml medium containing 1×108 formalin killed E. tarda. Leukocytes were then incubated for 3 h at 25 °C and the cultures were supplemented with 10 % fetal calf serum. After 48 h of culture, the supernatants were harvested, centrifuge at 10 000×g to remove bacteria and cell debris, aliquoted and stored at −20 °C prior to use. For detection of MAF activity, supernatants were diluted in RPMI medium (1:4) and added to macrophage monolayers in 96-well tissues culture plates, prepared from unimmunized fish as described previously (Secombes 1990). The macrophages were cultured in the presence of the supernatants for 48 h and then their ability to reduce nitroblue tetrazolium chloride (NBT, Hi-media) was determined by respiratory burst assay (Kamilya et al. 2006b).
Humoral responses The total myeloperoxidase content of serum and mucus was estimated as described by Mohanty and Sahoo (Mohanty and Sahoo 2010). The lysozyme activity of serum and mucus was determined as described by Ellis (Ellis 1990). Lysozyme activity was expressed as units ml−1 where one unit was defined as the reduction in absorbance of 0.001 min−1. Serum and mucosal antibody titre was determined by the microtitre agglutination method (Karunasagar et al. 1997). Antibody titre was expressed as the reciprocal of the last dilution giving visible agglutination. Haemagglutination assay was done as described by Kumari and Sahoo (2005). The protein content of serum was estimated by following the biuret method (Gornall et al. 1949). Homologous pathogen challenge Homologous pathogen challenge of the immunized fish was done 8 weeks after the immunization. Ten fish from each tank were transferred to another set of tanks and were challenged with E. tarda (1×106 CUF ml−1). Challenge was performed by injecting each fish intraperitonially with 0.2 ml of bacteria. The cause of death and pathological changes were verified by re-isolation of bacteria from dead/infected samples. The mortality was recorded for 2 weeks and the relative percent survival (RPS) was calculated using the following formula:
RPS ¼ ½1−% of mortality in immunized group=% of mortality in control group 100:
Statistical analysis
Antigen-specific MAF production
Statistical analysis of data was performed using SPSS-15.0 for windows software (SPSS Inc., Chicago, IL, USA). Results are presented as mean±standard error of mean (SEM). Data were analyzed by the student’s t-test, one-way analysis of variance (ANOVA) and Tukey’s range test. Probability levels of 0.05 were used to find out the significance in all cases.
Antigen-specific MAF production showed almost similar trend to that of leukocyte proliferation activity in all the sampling weeks for all the immunized groups. The maximum MAF activity was observed in case of adjuvanted bacterin after 5th sampling week following the booster dose (Fig. 2). Myeloperoxidase activity of serum and mucus
Results Antigen-specific leukocyte proliferation All the immunized groups showed significant leukocyte proliferation activity (p
