Mucosal response in African catfish after administration of Vibrio anguillarum O2 antigens via different routes

The aim of this study was to investigate the possibility of mucosal vaccination in African catfish (Clarias gariepinus) with Vibrio anguillarum O2 bacterins. The antigen was administered via different routes: anal intubation, oral administration, intraperitoneal injection and immersion. To monitor the antigen uptake, a competitive ELISA was used. The antibody response was measured using an indirect ELISA. Increased antibody levels were found in bile and mucus upon anal intubation, which was not the case after intraperitoneal injection. The data indicate that oral vaccination of fish may be possible when antigens can reach the second gut segment in sufficient quantities and in the right form as confirmed by the recorded substantial induction of systemic and mucosal immunity. The results obtained are a strong indication for mucosal immune response and the two compartmental models for immune response in fish.     

Characterization of serum and mucosal antibody responses in white sturgeon (Acipenser transmontanus Richardson) following immunization with WSIV and a protein hapten antigen

Serum and cutaneous mucus antibodies were monitored in white sturgeon for 15 weeks following intraperitoneal immunization. Ten fish were immunized (50 microg) with white sturgeon iridovirus (WSIV) or white sturgeon gonad (WSGO) tissue culture cells emulsified with or without FCA. An additional group was immunized with FITC:KLH+FCA. Fish were booster immunized at 6 weeks. Fish immunized with FITC:KLH+FCA produced significant serum antibodies to FITC by 6 weeks and this response peaked at 12 weeks (average titer 31,000). Mucosal antibodies to FITC were first detected at 12 weeks and significantly elevated by 15 weeks (average titer 18). Anti-WSIV antibody titers were detected in the serum by 9 weeks in fish immunized with WSIV and WSIV+FCA, but only a small number responded to immunization. At 15 weeks, four fish immunized with WSIV produced serum antibodies (average titer 838) and one fish immunized with WSIV+FCA had a serum titer of 1600. Mucosal anti-WSIV antibody titers of 8 and 16 were observed in two fish from the WSIV group at 12 weeks while four different fish from this group responded at 15 weeks (average titer 4). Western Blot using a monoclonal antibody confirmed immunoglobulin in mucus, and specificity to WSIV was further demonstrated by immunocytochemistry using serum from fish immunized with WSIV. Specific antibody was not detected in mucus of fish immunized with WSIV+FCA, WSGO, or WSGO+FCA. Collectively, these experiments demonstrate that white sturgeon can generate a specific antibody response following immunization, and is the first report showing mucosal immunoglobulin is present in this species.     

Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla

Vibriosis due to Vibrio vulnificus serovar E (biotype 2) is one of the main causes of mortality in European eels cultured in Europe. The main objective of this study was to develop a vaccine and a vaccination procedure against this pathogen. With this aim, we tested several vaccine formulations (inactivated whole-cells with and without toxoids--inactivated extracellular products--from capsulated and uncapsulated strains, attenuated live vaccines and purified lipopolysaccharide [LPS]) on eels maintained under controlled laboratory conditions using different delivery routes (injection and immersion). To study the immune response we estimated antibody titers and bactericidal/bacteriostatic activity in mucus and serum. To evaluate protection, we calculated the relative percent survival (RPS) after intraperitoneal (i.p.) injection and bath challenge of the pathogen. The overall results indicate that: (1) capsular antigens seem to be essential for protective immunization; (2) vaccines confer the highest protection when administered by i.p. injection; (3) booster is needed to achieve good protection by immersion; (4) enriching the vaccine with toxoids enhances protection to optimal levels (RPS values around 70 to 100%, depending on the delivery route); and (5) the protective effect in serum and mucus depends on the route of administration and seems to be related to the production of specific antibodies.     

Efficacy of a bivalent vaccine against eel diseases caused by Vibrio vulnificus after its administration by four different routes

Vulnivaccine, a vaccine against vibriosis caused by Vibrio vulnificus serovar E (formerly biotype 2), confers acceptable levels of protection to eels after its administration by prolonged immersion in three doses. Recently, a new pathogenic serovar, named serovar A, has been isolated from vaccinated eels in a Spanish freshwater eel farm. The main objective of this work was to design a bivalent vaccine, and to study its effectiveness against the two pathogenic serovars. With this aim, eels weighing around 20 g were immunised with the bivalent vaccine by oral and anal intubation, intraperitoneal injection (i.p.) and prolonged immersion. The overall results indicated that: (i) the new vaccine delivered by oral and anal intubation induced protection levels higher than 80%, to that achieved after i.p. vaccination; (ii) oral and anal vaccination induced a significant systemic and mucosal immune response; (iii) the protection after vaccination by whichever routes was related to antibody titres in plasma; (iv) mucosal and systemic compartments showed different kinetics of antibody production; (v) evidence for passive transfer of antibodies from plasma to gut mucus were found after i.p. and anal vaccination, and finally, (vi) vaccination did not enhance the production of lysozyme, in plasma or mucus. In conclusion, this new vaccine is effective in protecting eels against vibriosis caused by the two eel-pathogenic serovars of V. vulnificus, the oral delivery system is a promising way which may be used in intensive culture facilities during the whole growth period of eels.     

Immunological importance of the second gut segment of carp. III. Systemic and/or mucosal immune responses after immunization with soluble or particulate antigen

Mucosal and systemic (serum) immune responses were studied after oral, anal or intramuscular (i.m.) immunization with particulate ( Vibrio anguillarum ) or soluble (ferritin) antigen. Antigen specific antibodies were found by ELISA in skin mucus after repeated oral or anal administration of bacteria, but not after immunization with ferritin. Daily feeding with bacteria did not give detectable antibodies in serum, while regular oral administration of ferritin resulted in an increase of specific antibodies during the first 3 weeks. From that time immunosuppression was observed, as the antibody titre decreased despite the continued ferritin feeding. Immunosuppression was also found after a second anal intubation or i.m. injection with ferritin, independent of the route of priming (i.m. or anal). On the contrary, a second anal intubation of bacteria resulted in a secondary serum response. These results combined with those reported in Parts I and II of the study indicate an important immunological role for the second gut segment. Because mucosal as well as serum responses can be obtained by anal immunization with bacteria, the significance for oral vaccination is discussed.     

Fish DNA vaccine against infectious hematopoietic necrosis virus: efficacy of various routes of immunisation

The DNA vaccine, pIHNVw-G, contains the gene for the glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV), a major pathogen of salmon and trout. The relative efficacy of various routes of immunisation with pIHNVw-G was evaluated using 1.8 g rainbow trout fry vaccinated via intramuscular injection, scarification of the skin, intraperitoneal injection, intrabuccal administration, cutaneous particle bombardment using a gene gun, or immersion in water containing DNA vaccine-coated beads. Twenty-seven days after vaccination neutralising antibody titres were determined, and 2 days later groups of vaccinated and control unvaccinated fish were subjected to an IHNV immersion challenge. Results of the virus challenge showed that the intramuscular injection and the gene gun immunisation induced protective immunity in fry, while intraperitoneal injection provided partial protection. Neutralising antibodies were not detected in sera of vaccinated fish regardless of the route of immunisation used, suggesting that cell mediated immunity may be at least partially responsible for the observed protection.     

Protective immunity of Nile tilapia against Ichthyophthirius multifiliis post-immunization with live theronts and sonicated trophonts

Two immunization trials were conducted to evaluate host protection of Nile tilapia, Oreochromis niloticus against Ichthyophthirius multifiliis (Ich). Immunizations were done with live theronts or sonicated trophonts by bath immersion and intraperitoneal (IP) injection. The immunized fish were challenged with theronts 21 days post-immunization in trial I and 180 days post-immunization in trial II. The serum anti-Ich antibody and cumulative mortalities of tilapia were determined after theront challenge. Serum anti-Ich antibody was significantly higher (P<0.05) in tilapia immunized with live theronts by immersion or IP injection or with sonicated trophonts administered by IP injection than tilapia immunized with sonicated trophonts by immersion, with bovine serum albumin by IP injection, or non-immunized controls. Host protection was acquired in fish immunized with live theronts by immersion or IP injection. Tilapia immunized with sonicated trophonts by IP injection were partially protected with a 57-77% survival in both trials. At 180 days post-immunization, serum antibody titers had declined in immunized fish yet they were still able to survive challenge. The protection appears not to be solely depending on serum antibody response against Ich.     

Oral vaccination of African catfish with Vibrio anguillarum O2: effect on antigen uptake and immune response by absorption enhancers in lag time coated pellets

The impact on antigen uptake and antibody response by the addition of absorption enhancers to Vibrio anguillarum O2 antigen was studied in oral vaccination trials of African catfish (Clarias gariepinus). Oral vaccination was achieved by feeding lag time coated pellets. The lag time coat prevents premature release of the encapsulated vaccine in the tank, before ingestion of the pellets by the fish. To monitor the antigen uptake, a competitive ELISA was used. The antibody response was measured using an indirect ELISA. Feeding of bacterin-layered pellets without absorption enhancers resulted in a rather low antigen uptake and antibody levels. The addition of absorption enhancers such as sodium salicylate, sodium caprate and vitamin E TPGS increased the serum antigen levels and specific antibody levels in the systemic circulation. Skin mucus antibody levels were higher after oral vaccination compared to the IP and control group. The addition of absorption enhancers in the oral groups further increased the antibody levels obtained in the skin mucus.     

斑点叉尾鲖源海豚链球菌Srr蛋白海藻酸钠-壳聚糖口服疫苗的制备及其免疫效果研究

制备海藻酸钠-壳聚糖-海豚链球菌Srr蛋白微球疫苗, 并检测其对斑点叉尾鲙的免疫效果。采用乳化法利用海藻酸钠-壳聚糖包被Srr蛋白, 测定其包封率、载药率及包被蛋白的抗原性; 通过拌饲投喂免疫斑点叉尾鲙, 分为Srr组、Srr微球组、空微球组以及对照组, 间接ELISA法检测免疫后斑点叉尾鲙的血清抗体水平, 试剂盒检测多项血清非特异性指标; 于免疫后第4周利用海豚链球菌攻毒, 计算各组相对保护率, 并通过实时荧光定量PCR检测相关基因的表达量。结果显示, 通过乳化法制得形态为圆形或椭圆形、大小较为均一的微球疫苗, 粒径为(4.26±1.13) μm, 包封率为92.38%, 载药率为19.41%, Western-blot分析表明Srr蛋白微球具有较好的抗原性; Srr微球组的抗体效价峰值出现在第4周, 明显高于其他组, 血清总蛋白、T-SOD以及溶菌酶活力均显著或极显著高于其他实验组, 并获得60%的相对保护率。荧光定量分析结果显示, Srr微球组攻毒后24h和48h各免疫基因表达量均有所上调。Srr蛋白微球疫苗能够提高斑点叉尾鲙抵抗海豚链球菌的能力, 对海豚链球菌起到了一定的预防作用。     

Characterisation of mucosal and systemic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance

Mucosal and systemic antibody production in rainbow trout, Oncorhynchus mykiss (Walbaum), was evaluated following different antigen delivery routes. A BIAcore instrument (Pharmacia) allowed direct detection of antibody-antigen interactions by surface plasmon resonance changes. These interactions were measured in real-time without secondary reagents or extraneous labels. Groups of rainbow trout were immunised with a hapten-carrier antigen consisting of fluorescein isothiocyanate (FITC) conjugated to keyhole limpet haemocyanin (KLH) or phosphate buffered saline (PBS) pH 7.2. Antigens were administered intraperitoneally (i.p.) with or without Freund's complete adjuvant (FCA) or peranally (p.a.) directly to the gastrointestinal (GI) tract. Serum and mucosal anti-FITC responses were significantly (P<0.05) higher in FITC-KLH/FCA groups, clearly showing that adjuvant incorporation enhances mucosal as well as sytemic immunity. Antigen uptake and processing in fish immunised p.a. and i.p. without adjuvant was much less efficient and resulted in relatively low levels of serum and mucosal antibody production. Interestingly, mucosal responses in these groups peaked prior to serum responses suggesting possible early stimulation of mucosal defences. Mucosal antibody production in fish receiving FITC-KLH/FCA correlated more closely with serum responses, indicating possible transfer of serum derived antibody to mucosal sites. Mucosal and serum responses were confirmed as immunoglobulin (Ig) by subsequent reactivity with an anti-trout serum IgM monoclonal antibody (1.14) passed over flow cells containing anti-FITC antibodies. Further analysis showed significantly lower (P<0.05) reactivity of early mucus anti-FITC components (4 weeks post-immunisation) to 1.14. Purified serum and mucus Ig from non-immunised fish showed different protein banding patterns by SDS-PAGE under reducing conditions. Immunoblotting with 1.14 also showed weak reactivity to mucus Ig in control fish while reacting strongly to mucus Ig from immunised fish. These data suggest that early mucosal responses in trout may consist of heterogeneous forms of Ig differing in characteristics to serum Ig. BIAcore analysis in this context and as a means of measuring antibody response proved useful, and has the potential to become a valuable new tool in the study of fish immunology.     

The immune response of brown trout (Salmo trutta L.) to injection with soluble antigens.

The immune response of brown trout (Salmo trutta) to horse serum and keyhole limpet haemocyanin was studied. Intraperitoneal and intramuscular injections were used, with and without adjuvant, in 209 fish. Complement-fixing antibodies (CFA) and precipitins were produced to both antigens. CFA were detected after 8 days to haemocyanin and after 13 days to horse serum. Maximum CFA titres to a single intraperitoneal injection of horse serum or haemocyanin were reached at 44 and 43-46 days respectively. Precipitins to a single injection of haemocyanin given intraperitoneally were detected after 19 days using gel diffusion. Similarly using the intramuscular route they were detected after 22 days. However, using counter-current electrophoresis, precipitins were detected after 8 days by the intraperitoneal route and after 9 days by the intramuscular. Precipitins to horse serum given intraperitoneally were demonstrated after 22 days by both gel diffusion and counter-current electrophoresis. Fish given 2 intraperitoneal injections of haemocyanin in adjuvant reached maximum CFA titres after 55 days; a 3rd injection on day 56 did not produce a marked increase in titre. Fish given intramuscular injections of haemocyanin in adjuvant showed maximum CFA titres at day 43. After a 3rd injection on day 56, maximum CFA titres were reached between days 92 and 106. Intramuscular injections gave significantly higher titres than those given by the intraperitoneal route. Some fish which showed no precipitins by gel diffusion were positive by counter-current electrophoresis. Precipitating antibodies to haemocyanin migrating in the beta2-gamma1 region were detected by immuno-electrophoresis.     

卵形鲳鲹对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫（Cryptocaryon irritans）的幼虫对卵形鲳鲹（Trachinotus ovatus）进行腹腔注射和体表感染,然后每隔1周用阻动试验（Immobilization assay）检测免疫鱼的抗血清和皮肤培养液对激刺隐核虫幼虫的阻动效价,在第14周,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示：两种免疫方法都能让卵形鲳鲹的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异：两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤粘膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是粘膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。     

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.     

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.     

Intragastric and intraperitoneal administration of Cry1Ac protoxin from Bacillus thuringiensis induces systemic and mucosal antibody responses in mice.

The spore-forming soil bacterium Bacillus thuringiensis produces parasporal inclusion bodies composed by delta-endotoxins also known as Cry proteins, whose resistance to proteolysis, stability in highly alkaline pH and innocuity to vertebrates make them an interesting candidate to carrier of relevant epitopes in vaccines. The purpose of this study was to determine the mucosal and systemic immunogenicity in mice of Cry1Ac protoxin from B. thuringiensis HD73. Crystalline and soluble forms of the protoxin were administered by intraperitoneal or intragastric route and anti-Cry1Ac antibodies of the major isotypes were determined in serum and intestinal fluids. The two forms of Cry1Ac protoxin administered by intraperitoneal route induced a high systemic antibody response, however, only soluble Cry1Ac induced a mucosal response via intragastric. Serum antibody levels were higher than those induced by cholera toxin. Systemic immune responses were attained with doses of soluble Cry1Ac ranging from 0.1 to 100 microg by both routes, and the maximal effect was obtained with the highest doses. High anti-Cry1Ac IgG antibody levels were detected in the large and small intestine fluids from mice receiving the antigen via i.p. These data indicate that Cry1Ac is a potent systemic and mucosal immunogen.     

Protective immunity in grouper (Epinephelus coioides) following exposure to or injection with Cryptocaryon irritans

The protective immunity of grouper (Epinephelus coioides) against Cryptocaryon irritans was determined after immunisation by surface exposure or intraperitoneal injection. Specific antibody titres of immunised fish serum and skin culture supernatant were determined by enzyme-linked immunosorbent assay and immobilisation assays. Specific antibody can be detected in some immunised fish at Week 1 and in all immunised fish at Week 2, and the peaks were between Weeks 4-6. Specific antibody was still evident in the serum and skin of immunised fish at Week 8, and provided good protection against challenge with C. irritans. These findings indicated that humoral and skin mucosal immunity play important roles in fish against C. irritans infection.     

卵形鲳鯵对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳鯵(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示:两种免疫方法都能让卵形鲳鯵的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。       

Different antiamebic antibody isotype patterns in the large and small intestine after local and systemic immunization of mice with glutaraldehyde fixed Entamoeba histolytica trophozoites

We have determined the major immunoglobulin isotypes (IgG, IgA, IgM) of antiamebic antibodies induced in the serum and in the large and small intestine after local (oral and rectal) or systemic (intraperitoneal and intramuscular) immunization of mice with glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). IgA predominated in the small intestine after immunization through all routes, whereas in the large intestine similar antibody levels of the major isotypes were induced by rectal, intraperitoneal and intramuscular immunization. The intramuscular route elicited intestinal responses lower than those induced by the rectal and intraperitoneal routes, but higher than the slight IgA antibody increase observed after oral immunization. The differences in antiamebic antibody response patterns at the large and small intestine suggest that there are different mucosal effector compartments. They also indicate that isotype analysis of mucosal antibodies from the sites where an infectious agent resides is needed to evaluate whether a vaccine candidate induces responses of higher protective value in the appropriate site, and that the study of antibody responses must not be limited to sampling the serum or mucosal sites distant to the relevant one.     

Bacterial kidney disease as a model for studies of cell mediated immunity in rainbow trout (Oncorhynchus mykiss)

A cell mediated immune (CMI) response was measured in vitro to heat-killed and to paraformaldehyde fixed Renibacterium salmoninarum (Rs) in rainbow trout (Oncorhynchus mykiss) experimentally challenged with live Rs. The mitogenic response to the T lymphocyte mitogen Concanavalin A (Con A) was reduced during samplings 4 to 6 weeks after immersion, but no effect of the response to the B lymphocyte mitogen lipopolysaccharide (LPS) was detected. The subpopulation of lymphocytes, detected by the monoclonal antibody 1C2, was decreased from the 4th week to the 5th week of infection, and remained at the decreased level up to 10 weeks post immersion. The proportion of Immunoglobulin (Ig) bearing lymphocytes was not affected during the Rs infection period. The humoral antibody level to heat-stable Rs-antigens was increased up to 10 weeks after immersion but after 27 weeks was reduced to a level similar to that of the non-challenged fish. An anamnestic response was demonstrated in challenged fish, as intraperitoneal injection of heat-treated Rs bacteria into Rs challenged fish elicited a stronger humoral antibody response compared with injection into non-challenged fish.     

Determining Vaccination Frequency in Farmed Rainbow Trout Using Vibrio anguillarum O1 Specific Serum Antibody Measurements

Background

Despite vaccination with a commercial vaccine with a documented protective effect against Vibrio anguillarum O1 disease outbreaks caused by this bacterium have been registered among rainbow trout at Danish fish farms. The present study examined specific serum antibody levels as a valid marker for assessing vaccination status in a fish population. For this purpose a highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate sera from farmed rainbow trout vaccinated against V. anguillarum O1.

Study Design

Immune sera from rainbow trout immunised with an experimental vaccine based on inactivated V. anguillarum O1 bacterin in Freund’s incomplete adjuvant were used for ELISA optimisation. Subsequently, sera from farmed rainbow trout vaccinated with a commercial vaccine against V. anguillarum were analysed with the ELISA. The measured serum antibody levels were compared with the vaccine status of the fish (vaccinated/unvaccinated) as evaluated through visual examination.

Results

Repeated immunisation with the experimental vaccine lead to increasing levels of specific serum antibodies in the vaccinated rainbow trout. The farmed rainbow trout responded with high antibody levels to a single injection with the commercial vaccine. However, the diversity in responses was more pronounced in the farmed fish. Primary visual examinations for vaccine status in rainbow trout from the commercial farm revealed a large pool of unvaccinated specimens (vaccination failure rate = 20%) among the otherwise vaccinated fish. Through serum analyses using the ELISA in a blinded set-up it was possible to separate samples collected from the farmed rainbow trout into vaccinated and unvaccinated fish.

Conclusions

Much attention has been devoted to development of new and more effective vaccines. Here we present a case from a Danish rainbow trout farm indicating that attention should also be directed to the vaccination procedure in order to secure high vaccination frequencies necessary for optimal protection with a reported effective vaccine.