Specific and natural antibody response of cod juveniles vaccinated against Vibrio anguillarum

The purpose of the present study was to study specific and natural antibody levels in individual cod juveniles before and after being vaccinated against Vibrio anguillarum. Different vaccine preparations and vaccination regimes, i.e. bathing, dipping, i.p. injection or combination of treatments were employed and the performance of different groups to bath challenge by the bacterium tested. Antibody responses to V. anguillarum antigens in groups vaccinated by bathing and/or dipping were negligible, while responses were observed in i.p. injected fish. Fish receiving i.p. injection in addition to bathing, showed significant antibody response. Both groups showed increased levels of natural antibodies while levels were low in other groups. Fish bathed or dipped showed higher mortality when challenged than untreated fish, while fish that received a second vaccination showed the best protection. It was not ascertained whether there is a long term difference between the effects of immersion versus i.p. injection as a booster method. Levels of antibodies against V. anguillarum antigens or natural antibodies in groups with the lowest mortalities show that neither could have been used to predict protection given by the vaccines tested.     

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.     

Susceptibility of juvenile and sub-adult Atlantic halibut (Hippoglossus hippoglossus L.) to infection by Vibrio anguillarum and efficacy of protection induced by vaccination

Experimental bath challenge of juvenile and sub-adult Atlantic halibut with Vibrio anguillarum induced severe mortalities of 47 and 80%, respectively. However, animals vaccinated with a commercial V. anguillarum vaccine demonstrated excellent protection against the disease (100% RPS). This study also describes the gross pathology and histological changes associated with this infection. A loss of coordination, haemorrhage at the fin base and splenomegaly were frequent findings. Serum agglutinating activity demonstrated a rise following vaccination, the mean log2 titre rising from 3.8 to 8.4. This was associated with a significant rise in antibody-mediated complement killing ability of immune serum when compared to non-immune serum.     

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.     

Immune and histopathologic responses of DNA-vaccinated hybrid striped bass Morone saxatilis x M. chrysops after acute Mycobacterium marinum infection

The post-challenge immune and histopathologic responses of hybrid striped bass vaccinated with a DNA vaccine encoding the Mycobacterium marinum Ag85A gene and subsequently challenged with M. marinum were investigated. Juvenile hybrid striped bass Morone saxatilis x M. chrysops were injected intramuscularly with 25 or 50 microg DNA plasmid and developed significant specific protective responses to live bacterial challenge 120 d post-vaccination. Both vaccine groups demonstrated increased survival, reduced splenic bacterial counts, and reduced granuloma formation compared to the control groups 14 d after challenge with approximately 8 x 10(5) cfu M. marinum g(-1) fish body wt. The vaccine groups also developed more rapidly and significantly increased antibody and lymphoproliferative responses post-challenge compared to control groups, and these post-challenge immune responses appear to be vital against M. marinum infection in vaccinated hybrid striped bass. No significant differences in immune responses were recognized between the 25 and 50 microg vaccination groups, and these groups eventually experienced mortalities, splenic bacterial counts, and granuloma formation 28 d post-challenge comparable to those of the control groups at 14 d post-challenge. Therefore, vaccination of hybrid striped bass with a DNA vaccine encoding the M. marinum Ag85A gene provided significant but limited duration of protection against an acute high-dose M. marinum challenge.     

Efficacy of a modified live Flavobacterium columnare vaccine in fish

Flavobacterium columnare is an aquatic bacterium that is responsible for columnaris disease. This aquatic pathogen has a worldwide distribution and is highly infectious to both warm and cold water fish. A modified live F. columnare vaccine was developed by repeated passage of a virulent strain on increasing concentrations of rifampicin that resulted in attenuation. Here we report vaccination/challenge trials to evaluate efficacy and safety. In separate laboratory trials, immersion vaccination of channel catfish (Ictalurus punctatus) fry between 10 to 48 days post hatch (DPH) with experimental vaccine or licensed product resulted in relative percent survival (RPS) between 57–94% following challenge. Similarly, a vaccination/challenge trial using largemouth bass (Micropterus salmoides) fry at 10 DPH was performed using various doses of licensed product under laboratory conditions. Results demonstrated safety of the vaccine and significant protection following challenge with RPS values between 74–94%, depending on vaccine dose. Together, these trials demonstrate the vaccine administered to early life-stage channel catfish and largemouth bass is safe and reduces mortality following challenge with F. columnare.     

Immune responses of zebrafish (Danio rerio) induced by bath-vaccination with a live attenuated Vibrio anguillarum vaccine candidate

A fish vaccine candidate, live attenuated Vibrio anguillarum, which can protect fish from vibriosis, was established in our laboratory. In this study, the protective immunological mechanism of live attenuated V. anguillarum was investigated in zebrafish as a model animal. After bath-vaccinated with the live attenuated strain, zebrafish were challenged with wild pathogenic strain to test the immunoprotection of the live attenuated strain. As the results, specific antibody response of fish against V. anguillarum was found to gradually increase during 28 days post-vaccination, and remarkable protection was showed with a high relative protection survival (RPS) of about 90%. Moreover, the vaccination changed the expressions of several immune-related genes in the spleens and livers of zebrafish. Among them, the expressions of pro-inflammatory factors such as IL-1 and IL-8 were tenderly up-regulated with about 3-4 fold in 1-7 days post-vaccination, while MHC II rose to a peak level of 4-fold in 7th day post-vaccination. These results gave some important messages about the mechanism of specific protection induced by live attenuated V. anguillarum and showed the availability of zebrafish model in the evaluation of the vaccine candidate.     

Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Vibriosis vaccines based on various sero-subgroups of Vibrio anguillarum O2 induce specific protection in Atlantic cod (Gadus morhua L.) juveniles

The purpose of this study was to investigate the efficacy of three monovalent and a trivalent vibriosis dip vaccines in juvenile Atlantic cod (Gadus morhua L.), examine whether the responses were specific and study the expression of selected immune genes after dip vaccination. In addition, the study addressed whether the deviating isolates of Vibrio anguillarum serotype O2 belongs to another sero-subgroup than the previously established sero-subgroups O2a, O2b and O2c. Rabbit V. anguillarum serotype O2 antiserum adsorbed with V. anguillarum O2a O-antigen was shown, by both ELISA and immunoblotting, to still contain serotype O2 specific antibodies. Cod V. anguillarum serotype O2 antiserum reacted only with isolate of homologous serotype and not with heterologous sero-subgroups. This indicates that the deviating V. anguillarum O2 isolates represent a new sero-subgroup differing from sero-subgroup O2a. The monovalent vaccines included formalin inactivated cultures of V. anguillarum sero-subgroup O2a, O2b or serotype O2, while the trivalent vaccine contained all three sero-subgroups. Cod mounted high protection 7 weeks post dip vaccination with monovalent vaccines when challenged with homologous isolates and significantly lower when challenged with heterologous isolates, regardless of sero-subgroups. The trivalent vaccine resulted in efficient protection against all sero-subgroups tested. Dip vaccination of cod juveniles did not result in detectable antibody production or alteration in gene expression of the heavy chain of IgM and IgD. In the trivalent vaccine group expression of IFNγ and IL-12p40 were significantly up-regulated 3 days post vaccination. However, in groups vaccinated against V. anguillarum sero-subgroups O2b or O2, IL-12p40 and IFNγ gene expression were slightly increased 3 and 55 days post vaccination, respectively.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Efficacy of an infectious hematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhynchus tshawytscha and sockeye O. nerka salmon

The level of protective immunity was determined for Chinook Oncorhynchus tshawytscha and sockeye/kokanee salmon (anadromous and landlocked) O. nerka following intramuscular vaccination with a DNA vaccine against the aquatic rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A DNA vaccine containing the glycoprotein gene of IHNV protected Chinook and sockeye/kokanee salmon against waterborne or injection challenge with IHNV, and relative percent survival (RPS) values of 23 to 86% were obtained under a variety of lethal challenge conditions. Although this is significant protection, it is less than RPS values obtained in previous studies with rainbow trout (O. mykiss). In addition to the variability in the severity of the challenge and inherent host susceptibility differences, it appears that use of a cross-genogroup challenge virus strain may lead to reduced efficacy of the DNA vaccine. Neutralizing antibody titers were detected in both Chinook and sockeye that had been vaccinated with 1.0 and 0.1 pg doses of the DNA vaccine, and vaccinated fish responded to viral challenges with higher antibody titers than mock-vaccinated control fish.     

Intraperitoneal vaccination of Atlantic cod, Gadus morhua with heat-killed Listonella anguillarum enhances serum antibacterial activity and expression of immune response genes

Serum-mediated reduction in bacterial count and expression of a number of immune response genes in the blood of Atlantic cod, Gadus morhua were investigated following intraperitoneal vaccination with heat-killed Listonella (Vibrio) anguillarum. Blood was collected from the caudal vein of both vaccinated and non-vaccinated (PBS-injected) fish at 0, 1, 3, 7 and 10 days post-vaccination (dpv). Serum protein concentration and antibacterial activity of the serum samples were determined. Whole blood was used for semi-quantitative RT-PCR of immune-related genes. Total serum protein was not significantly different between the vaccinated and non-vaccinated groups. Sera from the vaccinated fish significantly reduced L. anguillarum count on 3 dpv, with reductions of at least 2 log colony forming units per ml (CFU/ml) relative to the non-vaccinated fish. Expression of antibacterial genes, bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme and transferrin was significantly upregulated in the vaccinated fish, with maximum expression within 7 dpv. Cytotoxic-related and cell-mediated immunity genes such as, apolipoprotein A-I and the non-specific cytotoxic cell receptor protein (NCCRP-1) had maximum expression at 3 and 7 dpv, respectively. Significant upregulation in expression of pro-inflammatory cytokines, IL-1 beta and IL-8 was also observed in the vaccinated fish at 1 dpv. The upregulation of immune response genes following vaccination provides valuable information in the understanding of immune mechanisms against vibriosis in Atlantic cod particularly on the acute phase response during bacterial infection.     

Evaluation of a whole cell, p57- vaccine against Renibacterium salmoninarum.

A whole cell Renibacterium salmoninarum vaccine was developed using 37 degrees C heat treated cells that were subsequently formalin fixed; this treatment reduced bacterial hydrophobicity and cell associated p57. Coho salmon Oncorhynchus kisutch were immunized with the p57- vaccine by either a combination of intraperitoneal (i.p.) and intramuscular (i.m.) injections or per os. In the first experiment, i.p./i.m. vaccination of coho salmon with p57- cells in Freund's Incomplete Adjuvant (FIA) conferred a statistically significant increase in mean time to death after the salmon were i.p. challenged with 4.1 x 10(6) colony forming units (cfu) of R. salmoninarum. There was no significant difference in response between fish immunized with R. salmoninarum cell surface extract in FIA and those immunized with extracellular protein (ECP) concentrated from culture supernatant in FIA. The i.p. challenge dose resulted in complete mortality of all fish by Day 43. In a second experiment, fish were orally vaccinated with p57- R. salmoninarum cells encased in a pH protected, enteric-coated antigen microsphere (ECAM). Fish were bath challenged with 4.2 x 10(6) cfu ml-1 on Day 0 and sampled at time points of 0 (pre-challenge), 50, 90, or 150 d immersion challenge. Vaccine efficacy was determined by monitoring the elaboration of p57 in the kidneys of vaccinated and control fish. Fish vaccinated orally demonstrated a significantly lower concentration of p57 (p < 0.01) at Day 150 post challenge compared to fish receiving ECAMs alone. Fish receiving p57 cells without ECAM coating also showed a significantly lower p57 level (p < 0.03) versus control. In contrast, fish injected intraperitoneally with the p57- cells or fish fed p57+ R. salmoninarum cells in ECAMs demonstrated no significant difference (p > 0.05) versus controls. In summary, these studies suggest the preliminary efficacy of 37 degrees C treatment of R. salmoninarum cells as an oral bacterial kidney disease vaccine.     

Kinetics of juvenile sea bass (Dicentrarchus labrax, L.) systemic and mucosal antibody secreting cell response to different antigens (Photobacterium damselae spp. piscicida, Vibrio anguillarum and DNP).

The ELISPOT assay was used to measure the number of specific antibody secreting cells (ASC) induced during the primary and secondary immune responses in the spleen, head kidney and gut of juvenile (5 g) sea bass (Dicentrarchus labrax) to bacterial (Vibrio anguillarum and Photobacterium damselae ssp. piscicida) and hapten dinitrophenyl-conjugated to keyhole limpet haemocyanin (DNP-KLH) antigens administered intraperitoneally. High variability among individuals was observed at each sampling day. All fish were bath vaccinated to V. anguillarum at an earlier stage (2 g) in the farm of origin prior to the development of the experiments, and therefore only secondary and tertiary responses were measured in the group immunised with this bacterium. Significant differences to the controls were observed in the primary responses of the head kidney and the spleen to P. damselae ssp. piscicida and DNP, respectively. Frequency analysis of the production of ASC suggests that significant responses in the gut might be masked by the high error variance. The peak of the primary response was observed 4 days earlier to DNP (18-20 days post-immunisation) and it was significantly higher than the response to P. damselae ssp. piscicida. Higher numbers of ASC were observed in the secondary responses of the head kidney and spleen, although they were not statistically significantly different from the primary levels, probably due to the high error variance as supported by the frequency analysis. Nevertheless, together with a faster response (peak at 7 days post-immunisation), the data suggest that memory formation had occurred. Additionally, the data suggest that some suppression of the secondary immune response in the gut might have occurred. The head kidney appears to produce the highest number of specific ASC of the organs tested. It appears that sea bass show a relatively fast but short duration antibody response.     

Effectiveness of a vaccine against red sea bream iridoviral disease in a field trial test.

Since 1990, red sea bream iridovirus (RSIV) has caused high mortalities in the summertime in cultured red sea bream Pagrus major in southwest Japan. To establish control measures for red sea bream iridoviral disease (RSIVD), the effectiveness of a formalin-killed viral vaccine was evaluated in a field trial. Two groups each consisting of 1000 juvenile red sea bream were either intraperitoneally inoculated with vaccine (vaccinated group) or were not vaccinated (non-vaccinated group). After vaccination, the fish were held for 1 wk, then transferred to a marine net pen and observed for 12 wk. The cumulative mortalities caused by RSIVD in the vaccinated group or control group were 19.2 and 68.5%, respectively. Additionally, the presence of virus antigen in the spleen was investigated and body weight was measured 6 and 12 wk post vaccination. In the vaccinated group, viral antigen was not detected. The increase in body weight of vaccinated fish was significantly (p < 0.05) greater than that of control fish. These results suggest that the vaccine against RSIVD was effective in 1 field trial.     

Protection, immune responses and side effects in Atlantic salmon (Salmo salarL.) vaccinated against furunculosis by different procedures

In an experimental trial lasting approximately 6 months, 10 different vaccination regimes against furunculosis were studied in Atlantic salmon pre-smolts. Single and repeated administration of vaccine by the intraperitoneal (i.p.), immersion or oral route, and revaccination by combinations of these methods, were tested. In challenge assays initiated 8 and 16 weeks after vaccination, fish injected once with a trivalent vaccine, and fish injected twice with a monovalent vaccine, both containing aluminium phosphate as adjuvant, were moderately protected. Non-injection vaccination protocols consistently failed to protect the fish. Compared with unvaccinated fish, protected groups showed elevated antibody responses toAeromonas salmonicidaantigens throughout the study. Increasedin vitroproliferation of head kidney leucocytes from i.p. vaccinated fish was found 16 weeks after vaccination. The use of a polyvalent vaccine preparation, and revaccination by injection or the oral route improved both immune responses and survival, compared to a single inoculation of monovalent vaccine. In all groups subjected to i.p. administration of vaccine, minor to moderate intraperitoneal lesions were found. In conclusion, i.p. administration of adjuvanted vaccine, preferably in a polyvalent formulation, is the optimal method of inducing anti-furunculosis immunity in Atlantic salmon, and is apparently necessary for an effective immunoprophylaxis of salmonid fish against furunculosis.     

Inflammatory responses of Nile tilapia Oreochromis niloticus to Streptococcus agalactiae: effects of vaccination and yeast diet supplement

This study evaluated the effects of dietary supplementation with 0.3% Saccharomyces cerevisiae yeast cell wall and of vaccination against Streptococcus agalactiae on the cellular component of acute inflammation induced in the coelomic cavity of Nile tilapia Oreochromis niloticus and on survival of the fish after challenge. A total of 84 tilapia of mean (±SD) weight 125.0 ± 1.5 g were distributed among twelve 310 l fiberglass tanks according to a 2 × 2 × 3 factorial design in the following manner: with and without supplementation; 2 stimulations (oily solution without S. agalactiae vaccine and vaccination); 15 d later all fish were intracoelomically challenged with 108 CFU ml-1 of a homologous strain of S. agalactiae, and evaluated after 6, 24 and 48 h, with 7 replicates. The fish received the non-supplemented or supplemented diet for a total of 77 d. The vaccination was performed on the 60th day, intracoelomically, as a single injection of 0.5 ml of the vaccine containing 108 CFU ml-1. Fifteen days later, all the fish were challenged with S. agalactiae by means of an intracoelomic inoculation of 108 CFU ml-1. No mortality was observed among the supplemented fish. The fish that were fed the non-supplemented diet and immunized with the bacterium presented a mortality rate of 28.5%. Among the non-supplemented and non-immunized fish, the mortality rate was 38.09%. Supplementation, in both vaccinated and non-vaccinated fish, induced larger accumulations of thrombocytes, lymphocytes and macrophages at the inflammatory focus. The results suggest that supplementation with 0.3% yeast cell wall, in both vaccinated and non-vaccinated fish, improved the inflammatory response of the fish and protected against the challenge. Vaccination increased the defense response, but the effect was stronger when associated with supplementation with S. cerevisiae.