Non-vaccinated Fish Research Articles

Identification and expression profile of multiple genes in Nile tilapia in response to formalin killed Streptococcus iniae vaccination

Twenty-eight expressed sequence tags (ESTs) were isolated from a Nile tilapia ( Oreochromis niloticus) vaccinated vs non-vaccinated subtractive library at 12-h post injection of a formalin killed Streptococcus iniae ARS-98-60 vaccine. The 28 ESTs were classified in terms of their putative functions. Half of the ESTs identified were unknown proteins. Of the remaining half ESTs, 17% have putative functions in protein biosynthesis and 11% have putative functions in immunity, energy production, and signal transduction, respectively. Immunity-related ESTs identified included high density lipoprotein-binding protein vigilin, immunoglobulin heavy chain, and QM-like protein. Quantitative PCR revealed that one EST (cytochrome c oxidase subunit II) was highly upregulated (1825 ± 336 fold) in vaccinated fish compared to that in non-vaccinated fish. Of the remaining 27 ESTs, nine were significantly ( P < 0.05) upregulated (<20 fold) in vaccinated fish. The nine significantly upregulated genes included five unknown or hypothetical proteins and four known proteins (high density lipoprotein-binding protein vigilin, QM-like protein, ribosomal protein S13, and ribosomal protein L5). The upregulation of these genes induced by killed S. iniae vaccines suggest that they might play important role in Nile tilapia defense against S. iniae infection.

Immune responses in Atlantic salmon (Salmo salar) following protective vaccination against Infectious salmon anemia (ISA) and subsequent ISA virus infection

Infectious salmon anemia (ISA) is an orthomyxoviral disease that has had devastating effects on farmed Atlantic salmon. ISA is still a disease resulting in continued loss of revenues and therefore development of effective vaccines is of great importance. Commercial vaccines against ISA are available, but the efficacy is poorly described. There is little information about vaccine-induced immune factors preventing ISA virus (ISAV) infection today. In this study we assessed the protective effects and immunogenicity of vaccines containing three different quantities of the inactivated ISAV antigen. Our findings indicated that immunization induced effective protection in Atlantic salmon with a relative percent survival (RPS) as high as 86. The level of protection was correlated to the amount of ISAV antigen in the vaccine, and fish immunized with high antigen amounts produced detectable ISAV-specific and neutralizing antibodies. While ISAV infection was detectable in non-vaccinated control fish challenged by cohabitation, no infection was detected in fish immunized with high antigen amounts. After challenge, transcriptional analysis of selected immune-related genes demonstrated activation of innate immune responses in ISAV-infected control fish, but not in vaccine protected fish. This study furthers the knowledge about vaccine efficacy and vaccine-induced immunity to ISAV challenge in Atlantic salmon.

Effect of parasitism on vaccine efficacy against Streptococcus iniae in Nile tilapia

Limited information is available on vaccine performance in parasitized fish. The objective of this study was to determine if parasitism of fish affected vaccine efficacy. Antibody level, hematology and survival of Nile tilapia vaccinated with a modified S. iniae bacterin were compared among non-parasitized fish, fish parasitized by Trichodina heterodentata and Gyrodactylus cichlidarum, and fish parasitized by T. heterodentata, G. cichlidarum and Ichthyophthirius multifiliis (Ich). Among vaccinated fish, fish free from parasites ( Trichodina, Gyrodactylus and Ich) had the highest antibody level (0.43, SE = 0.14). Significantly (p < 0.05) lower anti- S. iniae antibody was noted in parasitized vaccinated fish (0.30, SE = 0.08). Among the vaccinated treatments, fish parasitized by Trichodina, Gyrodactylus and Ich showed the lowest survival (80.0%, SE = 10.0), significantly (p < 0.05) lower than vaccinated fish free from parasites (97.5%, SE = 2.5) or parasitized by Trichodina and Gyrodactylus (95.0%, SE = 5.0). Following challenge with S. iniae, non-vaccinated fish free from parasites showed the higher survival (47.5%, SE = 2.5) than non-vaccinated fish parasitized by Trichodina and Gyrodactylus (37.5%, SE = 2.5). Non-vaccinated fish parasitized by all 3 parasites showed the lowest survival (27.5%, SE = 2.5) post challenge . Relative percent survival (RPS) demonstrated a decrease in vaccine performance for the group of fish that were parasitized with Trichodina and Gyrodactylus and Ich. RPS was 72% compared to 95 and 92%, respectively, in the other vaccinated treatments following challenge. This study demonstrated a reduction in vaccine performance in parasitized tilapia and highlights the importance of monitoring or controlling parasite levels in the aquaculture setting to optimize vaccine efficacy.

Vaccination of grouper broodfish (Epinephelus tukula) reduces the risk of vertical transmission by nervous necrosis virus

Nervous necrosis virus (NNV) has caused mass mortality in many species of cultured fish at larval stage. Strong evidence of vertical transmission of NNV has been reported in the carrier broodstock of striped jack and sea bass. An effective immunization program was developed and monitored in adult groupers (Epinephelus coioides) with average body weight of 1.35kg. The highest neutralizing antibody titers were found in the fish intramuscularly injected with adjuvanted NNV vaccine at 109TCID50kg−1, and the enhanced 2-fold neutralization activity could sustain up to 17 months post-vaccination (mpv). An immunization program was applied in the broodstocks of grouper (Epinephelus tukula) with body weight of 35–60kg. The levels of NNV-specific antibodies detected, from 1 to 5 mpi, in the homogenates of the eggs from the vaccinated broodfish were elevated than that from non-vaccinated fish. By nested RT-PCR, NNV was detectable in the eggs from the non-vaccinated fish at Month 5, but not in the eggs from vaccinated fish. It is therefore suggested that vaccination will be a potentially practical measure to reduce the risk of vertical transmission of NNV of grouper broodfish under stress of repeated spawning.

Hematological and immunological responses of Nile tilapia after polyvalent vaccine administration by different routes

The efficacy of a polyvalent bacterin vaccine against Aeromonas hydrophila, Pseudomonas aeroginosa and Enterococcus durans administered by different routes in Nile tilapia was assessed by analyzing hematological and immunological parameters 7 and 21 days after vaccination. Treatments consisted of: non-vaccinated tilapia; tilapia vaccinated by intraperitoneal injection with 2x10(8) formalin-inactivated bacteria·mL-1; tilapia vaccinated orally with 2x10(7) formalin-inactivated bacteria·g-1, feed for 5 days; tilapia vaccinated by immersion bath in 2x10(7) formalin-inactivated bacteria·mL-1, for 20 minutes. Vaccinated fish groups presented higher hematocrit, number of erythrocytes and leukocytes than the non-vaccinated group. Serum agglutination titer of intraperitoneally vaccinated fish was higher on both evaluation periods for the three bacteria strains. Only on day 21 post-vaccination fish from the oral and immersion vaccination groups presented higher serum agglutination titer than the non-vaccinated fish for A. hidrophyla and E. durans. Serum antimicrobial activity in vaccinated fish was higher for P. aeroginosa and E. coli than in non-vaccinated fish on both evaluation periods. The different vaccine administration routes stimulated hematological and immunological responses in Nile tilapia 21 days post-vaccination, but intraperitoneal vaccination presented higher total number of leukocytes, lymphocytes and serum agglutination titer.

In vitro and in vivo interaction of macrophages from vaccinated and non-vaccinated channel catfish ( Ictalurus punctatus) to Edwardsiella ictaluri

Macrophages from catfish vaccinated with an Edwardsiella ictaluri vaccine and macrophages from non-vaccinated catfish were used in in vitro and in vivo studies with red-fluorescent E. ictaluri to assess phagocytic ability, reactive oxygen and nitric oxide production and bactericidal activity. In the in vitro experiment, macrophages were harvested from vaccinated and non-vaccinated fish and then exposed to red-fluorescent E. ictaluri. Results of this study showed that E. ictaluri can survive and replicate in macrophages from non-vaccinated catfish (relative percent killing, RPK, from 0.011 to 0.620 and from −0.904 to 0.042 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) even in the presence of reactive oxygen and nitrogen products. Macrophages from vaccinated fish were significantly ( p < 0.05) more efficient in killing E. ictaluri (RPK from 0.656 to 0.978 and from 0.011 to 0.620 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) and produced significantly ( p < 0.05) higher amounts of ROS (10-fold increase) and nitrogen oxide (about 10-fold increase) than macrophages from non-vaccinated fish. In the in vivo experiment, vaccinated and non-vaccinated catfish were injected with red-fluorescent E. ictaluri to allow the interaction between macrophages and other components of the immune system. After 6 h, macrophages were harvested from the fish and seeded in glass chamber slides and bactericidal activity was measured in vitro. Results showed in vivo interaction of other components of the immune system enhanced bactericidal activity of macrophages from vaccinated fish. In another set of experiments, catfish were intraperitoneally injected with fluorescent bacteria opsonized with immune serum or non-opsonized and necropsied in the first 48 h after bacterial challenge to observe localization of E. ictaluri between vaccinated and non-vaccinated catfish. Vaccinated fish were able to control the dispersion of E. ictaluri in the body and red-fluorescent bacteria were observed only in the spleen, anterior and trunk kidney. In non-vaccinated fish E. ictaluri was able to replicate and invade all organs with the exception of the brain. We further determined that macrophages seeded with E. ictaluri could cause infection in non-vaccinated fish upon reinoculation with in vitro infected-macrophages. Overall, the results indicated that macrophages from vaccinated fish are activated and responsible for rapid clearance of infection upon re-exposure to virulent E. ictaluri.

Transcription of immune genes upon challenge with viral hemorrhagic septicemia virus (VHSV) in DNA vaccinated rainbow trout ( Oncorhynchus mykiss)

Even though DNA vaccination has proven as one of the most effective methods in controlling fish rhabdoviruses, the immune mechanisms responsible for protection are still unknown. Many studies have focused on studying which cytokines and immune genes are triggered in response to the vaccine at different times post-vaccination. However, to elucidate the mechanism(s) responsible for protection, to our understanding it is also of great relevance to study the immune response to the virus in fish that have been previously vaccinated and compare it to the effects that the virus might have on non-vaccinated fish. This type of study has never been performed to date in fish. Thus, in the current work, we vaccinated rainbow trout ( Oncorhynchus mykiss) with a DNA vaccine against viral hemorrhagic septicemia virus (VHSV), and 30 days post-vaccination we challenged the fish with a virulent VHSV. It was then, that we studied the immune response to the virus at very early times post-infection in fish, in order to compare the effects of VHSV on vaccinated or non-vaccinated trout. We studied the levels of expression of interleukin 1β (IL-1β), major histocompatibility complex (MHC) class Iα and IIα genes, immunoglobulin M (IgM), CD8α, type I interferon (IFN), Mx, IFN-γ and natural killer enhancing factor (NKEF) in head kidney, spleen and blood. When we compared the effect that VHSV had on vaccinated fish to the effect that the virus produced in fish vaccinated with the empty plasmid, the genes that were significantly up-regulated were IL-1β and MHC IIα in the spleen at day 1 post-infection, MHC Iα in all organs at day 1 post-infection, and IFN and Mx in the spleen and blood at days 1 and 3 post-infection, respectively. Genes that correlate with an increased specific immune response were not significantly increased in response to VHSV in these vaccinated animals. The results suggest that DNA vaccination induces a memory state in fish that, on the contrary to what would occur in mammals, primes the non-specific immune responses upon a later encounter with the virus.

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-1β 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.

Gene expression of leucocytes in vaccinated Japanese flounder ( Paralichthys olivaceus) during the course of experimental infection with Edwardsiella tarda

In this paper, we focused on the detection of differentially expressed genes in peripheral blood leucocytes (PBL) during the course of Edwardsiella tarda infection in vaccinated and non-vaccinated Japanese flounder ( Paralichthys olivaceus). cDNA microarray analysis was performed to compare the gene expression patterns of the PBL between the vaccinated and non-vaccinated fish in response to E. tarda inoculation. Fish were vaccinated twice, at a two-week interval and experimentally challenged with E. tarda two weeks after the second vaccination. Among the 1187 analyzed genes, 42 genes were up-regulated during the course of infection either in vaccinated or non-vaccinated fish. These genes included immune-related genes, such as MMP-9, MMP-13, CXC chemokine, CD20 receptor and hepcidin. Some immune-related genes were down-regulated after the E. tarda challenge, i.e. interferon inducible Mx protein, MHC class II-associated invariant chain, MHC class II alpha and MHC class II beta encoding genes, immunoglobulin light chain precursor, immunoglobulin light chain and IgM. These responses are thought to be a common reaction of Japanese flounder PBL in the course of edwardsiellosis, irrespective of immunized condition. Ten genes were significantly up-regulated only in vaccinated fish, and 11 genes were significantly up-regulated only in non-vaccinated fish. These genes may have a correlation with the efficacy of vaccination, although we have no evidence to link the different gene expression patterns and the efficacy of vaccination at present.

Dietary levamisole modulates the immune response and disease resistance of Asian catfish Clarias batrachus (Linnaeus)

In order to determine the immunomodulatory effect of dietary levamisole in Asian catfish (Clarias batrachus), fish were fed four different diets for 10 days: a formulated diet as control and the same diet supplemented with 50, 150 or 450 mg levamisole kg−1 feed. The serum bacterial agglutination titre against Aeromonas hydrophila as a measure of specific immunity, serum haemagglutination titre, natural haemolytic complement activity (ACH50), myeloperoxidase and lysozyme activities, total protein level and oxidative radical production by neutrophils as a measure of non-specific immunity as well as disease resistance against A. hydrophila challenge to separate vaccinated and non-vaccinated groups were evaluated at 0, 1, 2 and 3 weeks after last administration of levamisole. Levamisole supplement at the lowest level (50 mg kg−1) significantly enhanced oxidative radical production and serum myeloperoxidase (MPO) content immediately after 10 days of feeding, which reached peak values after 3 and 2 weeks of feeding respectively. Haemolytic complement and haemagglutination titre were significantly enhanced after 3 and 1 weeks respectively. Haemolytic complement activity and MPO activities were significantly raised to 150 mg kg−1 after 3 and 2 weeks, respectively. At the highest level of levamisole feeding (450 mg kg−1) significant decreases in superoxide production and complement activity were measured immediately after levamisole feeding, which returned to the normal level after 1 week post- feeding. Fish were challenged with a virulent strain of A. hydrophila at 0, 1, 2 and 3 weeks after levamisole feeding, and the cumulative per cent survival was recorded over 10 days. Feeding levamisole at 50, 150 or 450 mg kg−1 increased per cent survival in vaccinated fish immediately after levamisole feeding, and survival was significantly higher at 450 mg kg−1. There was no difference in mortality patterns in non-vaccinated fish. The results support the use of levamisole at 50 mg kg−1 feed for 10 days as an immunostimulant in Asian catfish farming.

Induction time for resistance to microsporidial gill disease caused by Loma salmonae following vaccination of rainbow trout ( Oncorhynchus mykiss) with a spore-based vaccine

Resistance to re-infection of rainbow trout to Loma salmonae, a microsporidian gill parasite has been previously documented and this study examined how rapidly this resistance develops. Naive rainbow trout were inoculated intraperitoneally (IP) with an inactivated spore-based vaccine and were then given an oral challenge with a high dose of L. salmonae spores at various weeks after being vaccinated. Non-vaccinated naive fish (exposed group) were challenged alongside of each group of vaccinated fish to ensure that the challenges were relatively standardised. In each group of fish, four weeks after the challenge, numbers of xenomas were counted on a gill arch for all fish. Vaccinated trout were completely resistant to a L. salmonae challenge six weeks after vaccination, although the onset of resistance began at approximately week 3, as observed with a reduction in the percent infected and xenoma intensity. The maximum percent infected for the vaccinated fish was 83% following a challenge two weeks following vaccination, whereas for the exposed group the maximum prevalence of 100% was reached several times. With continued research, a spore-based vaccine for L. salmonae has the potential to become the first commercially available parasite vaccine for fish.

Dietary immunostimulants influence specific immune response and resistance of healthy and immunocompromised Asian catfish Clarias batrachus to Aeromonas hydrophila infection

In order to determine the efficacy and immunoreversal effect of the 4 dietary immunomodulators, viz. lactoferrin, beta-1,3 glucan, levamisole and vitamin C, on disease resistance of a commercially important catfish, Clarias batrachus, fish were fed diets supplemented with various levels of these substances in 2 subgroups, healthy and immunocompromised, during a 30 d trial. An artificial immunosuppressive state was induced by giving 3 intraperitoneal (i.p.) injections of cyclophosphamide (CYP) at a dose level of 200 mg kg(-1) body weight at 1 wk intervals in the immunocompromised vaccinated subgroup and 3 consecutive injections 3 d before challenge in the immunocompromised non-vaccinated subgroup. On the first day of the experiment, the fish were vaccinated against a formalin-killed Aeromonas hydrophila bacterin. After 30 d, antibody titre (as measured through bacterial agglutination titre) and disease resistance against A. hydrophila were determined. The results demonstrate that all 4 immunomodulators were capable of significantly (p < 0.05) enhancing the specific immune response; this was evident through raised antibody titre and protection against A. hydrophila in both healthy and immunocompromised vaccinated subgroups compared to their respective controls. Similarly, all 4 substances significantly raised the survival rates in immunocompromised and healthy non-vaccinated fish. Thus, these substances were capable of reducing the immunosuppression induced by CYP injections in both vaccinated and non-vaccinated fish compared to their respective controls. Among the 4 substances studied, beta-1,3 glucan was found to be the most effective immunomodulator, followed by levamisole, lactoferrin and vitamin C in Asian catfish. Therefore, the results support the introduction of these substances into the diet of fish grown in farms under immunosuppressive/stressful conditions in order to enhance protection against infection and offer economic benefits.

Efficacy of injection vaccines against Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum)

Efficacy of mineral oil-based experimental injection vaccines against Flavobacterium psychrophilum were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), under laboratory and field conditions. The vaccines consisted of formalin- or heat-inactivated whole bacterium cell preparations of two different serotypes (Fd and Th) or a combination of serologically different F. psychrophilum (Fd and/or Th and/or Fp(T);Th). Specific antibody responses against the bacterium in plasma and skin mucus were evaluated post-vaccination with enzyme-linked immunosorbent assay. Efficacy of the vaccinations was determined by challenge trials to F. psychrophilum with the vaccinated rainbow trout. Significantly higher antibody levels in plasma were detected in vaccinated fish compared with mock-vaccinated fish. Injection vaccination did not trigger specific antibody production in the skin mucus. Significantly higher survival of i.p. vaccinated fish compared with non-vaccinated fish was observed during the challenge. The results suggest that mineral oil-based injectable vaccines containing formalin- or heat-inactivated virulent cells of F. psychrophilum effectively triggered specific antibody production and protected the fish against bacterial cold water disease.

Biological effect of vaccination can be assessed directly from diluted whole blood of rainbow trout using homologous blood phagocytes as immunosensors

A rapid and simple method is presented for determining antibody activity following vaccination, directly from diluted fish blood. The proposed method evaluates the effects of specific antibodies on ingestion by blood phagocytes, and may be used for measuring antibody levels following vaccination. The enhancing effect of trout IgM on ingestion was measured by luminol-amplified chemiluminescence (CL) emission of blood phagocytes. Respiratory burst (RB) activity of blood phagocytes was induced with the strain MT004 of bacterial fish pathogen Aeromonas salmonicida. To determine the boosting level of specific IgM on ingestion, various volumes of purified trout IgM containing specific antibodies against A. salmonicida were added to blood samples collected from non-vaccinated fish, and the RB activity of blood phagocytes was measured. The presence of antibodies in plasma of artificially prepared immune blood (AIB) was confirmed using enzyme-linked immunosorbent assay (ELISA). At a final blood dilution of 1:250, the mean RB activity of blood samples boosted with IgM was more than seven times higher, compared to other tested blood dilutions boosted with equal amount of IgM. Accordingly, a dilution of 1:250 was employed in the field study of vaccinated and non-vaccinated fish. The levels of A. salmonicida-specific antibodies in plasma samples of vaccinated and non-vaccinated fish were additionally confirmed with the ELISA assay. Based on these results, it is proposed that the biological activity of elicited antibodies can be assessed directly from diluted fish blood, using homologous blood neutrophils as immune sensors.

Influence of vaccination on the nitric oxide response of gilthead seabream following infection with Photobacterium damselae subsp. piscicida

The nitric oxide (NO) response of vaccinated and non-vaccinated juvenile gilthead seabream was studied in vivo and the NO response of isolated kidney macrophages of fish was studied in vitro. Fish were vaccinated with formalin-killed Photobacterium damselae subsp. piscicida ( Pdp) with or without Freund's incomplete adjuvant (FIA) and control fish received phosphate buffered saline (PBS). Thirty days later, fish were injected with a sublethal dose of Pdp and 3 fish/group were bled at time periods thereafter and serum nitrite and citrulline levels were determined as a measure of the NO response. All infected groups showed an increase in NO metabolites from 6 h to 27 days, with peak levels at 24 h. However, the response in bacterin-vaccinated fish was significantly higher than in the non-vaccinated group and the bacterin plus FIA resulted in a further significant enhancement. Similarly enhanced NO responses were produced in vitro by isolated macrophages obtained from vaccinated compared with non-vaccinated fish 30 days after vaccination following infection, with the response in macrophages from fish vaccinated with the bacterin plus FIA being significantly higher than those from fish vaccinated with the bacterin alone. Thus, vaccination resulted in an enhanced NO response to infection with Pdp in vivo and in vitro. Furthermore, the level of protection of fish to experimental challenge with virulent Pdp correlated with the level of the NO responses in the different groups.

Development of a DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination

Intramuscular injection of Japanese flounder, Paralichthys olivaceus (average weight approximately 2 g) with 1 and 10 μg of a plasmid DNA vaccine encoding the hirame rhabdovirus (HIRRV) glycoprotein gene (pCMV-HRVg) was found to provide strong protection against HIRRV. We also conducted a real-time PCR analysis to quantify immune-related genes, e.g. MHC class Iα, IIα, IIβ, TCR-α, β1, β2 and δ, to characterize the immune response at 1 and 7 days after DNA vaccination. In general, the copy numbers were at least 2-fold higher than those of the non-vaccinated fish. Interestingly, the gene expression of TCR β1 and β2 increased 1 day post-DNA vaccination, after which their copy numbers returned to levels similar to those before vaccination. These results suggest that the immune system of Japanese flounder was activated immediately after DNA immunization.

Vaccination and immune responses against atypical Aeromonas salmonicida in spotted wolffish ( Anarhichas minor Olafsen) juveniles

To study the effect of early vaccination, wolffish juveniles of size 50 and 90 mm, respectively, were vaccinated with an oil-adjuvanted atypical A. salmonicida bacterin. Vaccination resulted in significant protection after challenge with the homologous bacterial strain and specific antibody responses were demonstrated against whole bacteria as well as purified A-layer protein and LPS by ELISA and Western blotting but individual variation in immune responses was apparent. The A-protein was the most immunogenic bacterial component. In addition, higher numbers of immunoglobulin producing cells were detected by in situ hybridisation in kidney and spleen of vaccinated fish compared to non-vaccinated fish. Plasma cells were also present in gut and gills in equal numbers irrespective of treatment. No plasma cells were found in the skin. Finally, the frequencies of expressed V Hfamilies and C Lisotypes of wolffish immunoglobulins were shown by PCR. The relative expression of the three variable regions of the Ig heavy chain and the three isotypes of the Ig light chain in the spotted wolffish spleen seemed to be unaffected by immunisation with a complex antigen like the A. salmonicida bacterin.

Development of a new vaccine delivery method for fish: percutaneous administration by immersion with application of a multiple puncture instrument

A new administration method was developed for vaccination of juvenile rainbow trout against β-haemolytic Streptococcus. This simple and convenient method was equal in effectiveness to intra-peritoneal injection. Small skin lesions were produced using a multiple puncture instrument while fish were immersed in a vaccine suspension containing formalin-killed Streptococcus iniae. Upon challenge 2 weeks after vaccination, mortality of fish vaccinated by this method was 40%, equal to that by intra-peritoneal injection, while non-vaccinated control fish and fish vaccinated by immersion (without multiple puncture) each experienced 80% mortality. High efficacy was obtained with the multiple puncture/immersion method even when vaccine was diluted 10-fold. Quantitative analysis using fluorescent microspheres revealed that both antigen uptake by skin and delivery to the kidney and spleen were more effective with this method than with immersion alone. Microspheres were found in the skin within the pinpoint lesions and pressure mark caused by multiple puncture instrument. The greater protection gained by the present method can be attributed to higher numbers of particulate antigens taken up by fish and delivered to the lymphoid tissues.

Protection, immune response and side-effects in European whitefish ( Coregonus lavaretus L.) vaccinated against vibriosis and furunculosis

As indicated by market demand and cultivation prospects, the whitefish ( Coregonus lavaretus L.) has the potential to become an important alternative to rainbow trout in fish farming in Finland. The fish processing industry has called for a fish species having non-pigmented flesh, and there is a long tradition of whitefish farming for stocking purposes in Finland. However, cultivation in net cages in the brackish water of the coastal area exposes fish to vibriosis ( Listonella ( Vibrio) anguillarum) and furunculosis ( Aeromonas salmonicida salmonicida). Hence, profitable whitefish farming requires the efficient control of both of these diseases. The efficacy of vaccination (Apoject 1800®) was studied in the laboratory using challenge tests and by monitoring specific antibody production (ELISA) and the blood leucocyte pattern. The fish (22 g) were vaccinated at 15°C and the immunity developed during 8 weeks prior to challenge with vibriosis (1.8×10 5 cfu) and 12 weeks prior to challenge with furunculosis (4.9×10 2 cfu). The challenge was performed by i.p. injection. Side-effects around the injection site were studied 16 weeks post-vaccination. The immune system of the whitefish responded well and vaccination gave good protection against vibriosis and furunculosis. The RPS was 100% following the Vibrio challenge and 99% following the Aeromonas challenge. The antibody levels continued increasing in the vaccinated group throughout the study period. Lymphocyte and neutrophil counts were elevated in vaccinated compared to non-vaccinated fish at 8 weeks post-vaccination, but not after 16 weeks. Vaccination produced weak adhesions around the injection site in 42% and minor pigmentation in 9% of the 65 fish examined.

The Protective Effect of Recombinant Glycoprotein Vaccine Against HIRRV Infection.

Hirame rhabdovirus (HIRRV) was first reported from flounder (Paralichthys olivaceus) in Japan, and has recently been isolated from an aquaculture farm in the Tongyoung area of Korea. Total RNA isolated from RTG-2 cells infected with HIRRV was used in RT-PCR to obtain a cDNA coding for the glycoprotein of HIRRV Korean strain, CA-9703. Around half sized G protein in the C-terminal region was expressed as glutathione-S-transferase fusion protein, and used as viral vaccine against HIRRV. Flounder fry were vaccinated with low (50μg/mL) and high (300μg/mL) doses of vaccine by immersing them for 7 min in vaccine solution. After 1 month, the fish were challenged with HIRRV either by immersion or intraperitoneal injection. Specific losses of fish treated with low and high vaccine doses by intraperitoneal injection challenge were both 6.7%, in contrast to 43.3% in non-vaccinated fish. Losses of vaccinated fish by immersion challenge were 1.7% and 0% for low and high vaccine doses, respectively, and 15% for the non-vaccinated control.