Non-vaccinated Fish Research Articles

Fish Welfare – A Case Study: Reviling for the first-time side effects of vaccination in European sea bass (Dicentrarchus labrax) and barramundi (Lates calcarifer) in the Israeli fish farming

Vaccination is an effective way to control many infectious diseases in fish. Israeli fish farming has successfully used two vaccines over the last 30 years and has no problem with the side effects of vaccination. However, after introducing new species, a new problem emerged: these fish, after vaccination, demonstrated peritoneal lesions such as granulomas. At the same time, the fish did not show retarded growth or suffering during the fattening period. This study was conducted to establish the connection between vaccination and the appearance of granulomas. Evidence drawn from this research work and comparing vaccinated and non-vaccinated fish confirms that intraperitoneal granulomas do not impact the growth, performance, or fish fillet quality at harvest.

Protective efficacy and immune responses of largemouth bass (Micropterus salmoides) immunized with an inactivated vaccine against the viral hemorrhagic septicemia virus genotype IVa

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to the aquaculture industry, prompting the need for effective preventive measures. Here, we developed an inactivated VHSV and revealed the molecular mechanisms underlying the host's protective response against VHSV. The vaccine was created by treating VHSV with 0.05 % formalin at 16 °C for 48 h, which was determined to be the most effective inactivation method. Compared with nonvaccinated fish, vaccinated fish exhibited a remarkable increase in survival rate (99 %) and elevated levels of serum neutralizing antibodies, indicating strong immunization. To investigate the gene changes induced by vaccination, RNA sequencing was performed on spleen samples from control and vaccinated fish 14 days after vaccination. The analysis revealed 893 differentially expressed genes (DEGs), with notable up-regulation of immune-related genes such as annexin A1a, coxsackievirus and adenovirus receptor homolog, V-set domain-containing T-cell activation inhibitor 1-like, and heat shock protein 90 alpha class A member 1 tandem duplicate 2, indicating a vigorous innate immune response. Furthermore, KEGG enrichment analysis highlighted significant enrichment of DEGs in processes related to antigen processing and presentation, necroptosis, and viral carcinogenesis. GO enrichment analysis further revealed enrichment of DEGs related to the regulation of type I interferon (IFN) production, type I IFN production, and negative regulation of viral processes. Moreover, protein-protein interaction network analysis identified central hub genes, including IRF3 and HSP90AA1.2, suggesting their crucial roles in coordinating the immune response elicited by the vaccine. These findings not only confirm the effectiveness of our vaccine formulation but also offer valuable insights into the underlying immunological mechanisms, which can be valuable for future vaccine development and disease management in the aquaculture industry.

New Zealand rickettsia-like organism and Tenacibaculum maritimum vaccine efficacy study.

A cohort of Chinook salmon juveniles was vaccinated, with an autogenous bivalent vaccine against New Zealand RLOs (NZ-RLO1) and Tenacibaculum maritimum. A proportion of the cohort was not vaccinated to act as controls. At smoltification, the fish were challenged with NZ-RLO1, NZ-RLO2, or T. maritimum. We found that challenge with T. maritimum by immersion in (7.5 × 105 cfu/mL of water) did not yield any pathology. Challenge with RLOs produced clinical signs that were more or less severe depending on the challenge route, dose or vaccination status. Survival was significantly higher for vaccinated fish within the groups challenged with NZ-RLO1 by intraperitoneal injection with a relative percent survival (RPS) of 48.84%. Survival was not significantly different between vaccinated and non-vaccinated fish for groups challenged with NZ-RLO2 by intraperitoneal injection or by NZ-RLO1 by immersion. Yet, anecdotally the clinical disease presentation (manifesting as haemorrhagic, ulcerative skin lesions) was more severe for the non-vaccinated fish. This study demonstrates that autogenous vaccine against NZ-RLO is protective against severe disease and death by NZ-RLO1 challenge which warrants implementation and further evaluation under field conditions. Yet, this study also highlights the importance of the route of administration and dose when evaluating pathogenicity and vaccine efficacy.

Immune response of DNA vaccinated-gilthead seabream (Sparus aurata) against LCDV-Sa infection: relevance of the inflammatory process.

Lymphocystis disease is one of the main viral pathologies affecting cultured gilthead seabream (Sparus aurata) in the Mediterranean region. Recently, we have developed a DNA vaccine based on the major capsid protein (MCP) of the Lymphocystis disease virus 3 (LCDV-Sa). The immune response triggered by either LCDV-Sa infection or vaccination have been previously studied and seem to be highly related to the modulation of the inflammatory and the IFN response. However, a comprehensive evaluation of immune-related gene expression in vaccinated fish after viral infection to identify immunogenes involved in vaccine-induced protection have not been carried out to date. The present study aimed to fulfill this objective by analyzing samples of head-kidney, spleen, intestine, and caudal fin from fish using an OpenArray® platform containing targets related to the immune response of gilthead seabream. The results obtained showed an increase of deregulated genes in the hematopoietic organs between vaccinated and non-vaccinated fish. However, in the intestine and fin, the results showed the opposite trend. The global effect of fish vaccination was a significant decrease (p<0.05) of viral replication in groups of fish previously vaccinated, and the expression of the following immune genes related to viral recognition (tlr9), humoral and cellular response (rag1 and cd48), inflammation (csf1r, elam, il1β, and il6), antiviral response (isg15, mx1, mx2, mx3), cell-mediated cytotoxicity (nccrp1), and apoptosis (prf1). The exclusive modulation of the immune response provoked by the vaccination seems to control the progression of the infection in the experimentally challenged gilthead seabream.

Comparative evaluation of the antioxidant potential of rainbow trout exposed to Yersiniosis vaccine and non-vaccinated fish

The antioxidant capacity of liver in fish is a major property which could prevent the diminished quality and safety of fish resulted from oxidation reactions. The aim of this study was to compare the antioxidant capacity of the liver of rainbow trout exposed to the yersiniosis vaccine and nonexposed fish. The amounts of carotenoids, glutathione, cupric assay, and lipid peroxidation levels of liver were determined. Then, the samples were normalized by Bradford test. The results showed that significant differences may exist in lipid peroxidation level (p&lt;0.05). The total antioxidant capacity, glutathione, and carotenoids content showed no significant difference in both group (p≥0.05). The use of vaccine, in addition to increasing resistance to yersiniosis, could prevent the lipid oxidation of fish liver.

Field Efficacy of a Feed-Based Inactivated Vaccine against Vibriosis in Cage-Cultured Asian Seabass, Lates calcarifer, in Malaysia.

Vibrio spp. are important aquaculture pathogens that cause vibriosis, affecting large numbers of marine fish species. This study determines the field efficacy of a feed-based inactivated vaccine against vibriosis in cage-cultured Asian seabass. A total of 4800 Asian seabass, kept in a field environment, were separated equally into two groups (vaccinated and non-vaccinated) in duplicate. Fish of Group 1 were orally administered the feed-based vaccine on weeks 0 (prime vaccination), 2 (booster), and 6 (second booster) at 4% body weight, while the non-vaccinated fish of Group 2 were fed with a commercial formulated pellet without the vaccine. Fish gut, mucus, and serum were collected, the length and weight of the fish were noted, while the mortality was recorded at 2-week intervals for a period of 16 weeks. The non-specific lysozyme activities were significantly (p &lt; 0.05) higher in the fish of Group 1 than the non-vaccinated fish of Group 2. Similarly, the specific IgM antibody levels in serum and mucus were significantly (p &lt; 0.05) higher in Group 1 than in Group 2, as seen in the second week, with the highest level 8 weeks after primary immunization. At week 16, the growth performance was significantly (p &lt; 0.05) better in Group 1 and showed lower bacterial isolation in the gut than Group 2. Despite the statistical insignificance (p &gt; 0.05), the survival rate was slightly higher in Group 1 (71.3%) than Group 2 (67.7%). This study revealed that feed-based vaccination improves growth performance, stimulates innate and adaptive immune responses, and increases protection of cultured Asian seabass, L. calcarifer, against vibriosis.

Development of formalin killed vaccine candidate against streptococcosis caused by Enterococcus sp. in Nile tilapia

Multiple bacteria under Streptococcus, Enterococcus and Lactococcus genera are considered to be associated with Streptococcosis. This study was conducted to develop and evaluate the effectiveness of whole cell formalin killed vaccine prepared from E. faecalis, E. hirae and E. faecium against the streptococcosis in Nile tilapia. To develop the candidate vaccine, efficacy of whole cell killed E. faecalis (strains BFF1B1 & BFTS22), E. hirae (strain BFTS29) and E. faecium (strain BFTS31) were evaluated. The vaccine prepared by mixing 3 % formalin and left for 24 h to kill the bacterial cells. Physiological saline (0.85 %) was mixed to the harvested formalin free killed cells to make final suspension for vaccine. This vaccine was administered to the fingerling tilapia (average weight 30 ± 5 g) by interperitoneal injection (IP) and bath immersion (BI) methods. The vaccinated group was monitored for 28 days to assess the developed immunity and vaccine safety into the fish. Post vaccination in vivo challenge test were performed against the pathogens. The hematological parameters (RBC, WBC, platelets and hemoglobin) and specific IgM were found significantly higher (p < 0.001) in the vaccinated fish compared to those in the non-vaccinated fish. The mean mortality in IP and BI method ranged from 10.0 ± 0.00 % to 23.3 ± 5.77 % and 18.33 ± 5.30 % to 40.0 ± 0.10 %, respectively. The relative percentage survival (RPS) in IP and BI varied from 68.78 % to 88.57 % and 59.52 % to 69.05%, respectively. Mortality was significantly lower in the immunized fish compared to those in non-immunized fish. The RPS were not different (p > 0.05) between IP and BI vaccination method. Formalin killed vaccine showed an excellent efficacy against Enterococcus sp. infection in Nile tilapia by IP method although bath immersion could also be an alternative.

Evaluation of Edwardsiella piscicida basS and basR mutants as vaccine candidates in catfish against edwardsiellosis.

Catfish farming is the largest aquaculture industry in the United States and an important economic driver in several southeastern states. Edwardsiella piscicida is a Gram-negative pathogen associated with significant losses in catfish aquaculture. Several Gram-negative bacteria use the BasS/BasR two-component system (TCS) to adapt to environmental changes and the host immune system. Currently, the role of BasS/BasR system in E.piscicida virulence has not been characterized. In the present study, two mutants were constructed by deleting the basS and basR genes in E.piscicida strain C07-087. Both mutant strains were characterized for virulence and immune protection in catfish hosts. The EpΔbasS and EpΔbasR mutants were more sensitive to acidic environments and produced significantly less biofilm than the wild-type. In vivo studies in channel catfish (Ictalurus punctatus) revealed that both EpΔbasS and EpΔbasR were significantly attenuated compared with the parental wild-type (3.57% and 4.17% vs. 49.16% mortalities). Moreover, there was significant protection, 95.2% and 92.3% relative percent survival (RPS), in channel catfish vaccinated with EpΔbasS and EpΔbasR against E.piscicida infection. Protection in channel catfish was associated with a significantly higher level of antibodies and upregulation of immune-related genes (IgM, IL-8 and CD8-α) in channel catfish vaccinated with EpΔbasS and EpΔbasR strains compared with non-vaccinated fish. Hybrid catfish (channel catfish ♀ × blue catfish ♂) challenges demonstrated long-term protection against subsequent challenges with E.piscicida and E.ictaluri. Our findings demonstrate BasS and BasR contribute to acid tolerance and biofilm formation, which may facilitate E.piscicida survival in harsh environments. Further, our results show that EpΔbasS and EpΔbasR mutants were safe and protective in channel catfish fingerlings, although their virulence and efficacy in hybrid catfish warrant further investigation. These data provide information regarding an important mechanism of E.piscicida virulence, and it suggests EpΔbasS and EpΔbasR strains have potential as vaccines against this emergent catfish pathogen.

Efficacy of anti-koi herpesvirus DNA vaccine in carp Cyprinus carpio fry by immersion method and hyperosmotic infiltration

Indonesian carp production has decreased due to mass mortality caused by herpesvirus koi infection. Safe prevention techniques can be applied en masse and are relatively inexpensive are needed. This study aimed to examine the effect of immunization using anti-koi herpesvirus (KHV) DNA vaccine at a dose of 1.3 × 108 cfu.mL-1 through immersion with different lengths of time after hyperosmotic infiltration for 2 minutes. There were six groups with three replications: vaccination with 60 minutes immersion (A), 90 minutes (B), 120 minutes (C), non-vaccinated fish but infected with KHV (positive control), non-vaccinated fish and not infected with KHV (negative control), control fishes soaked with whole bacteria (control bacteria without DNA vaccine). The results showed that vaccination for 60 minutes gave the highest relative survival (93.55%). Thus, vaccination by immersion and hyperosmotic infiltration effectively improves the survival and relative survival of fish, and this method can be an alternative in the early control of KHV infection in common carp and koi.

Don't Let It Get Under Your Skin! - Vaccination Protects the Skin Barrier of Common Carp From Disruption Caused by Cyprinid Herpesvirus 3.

Vaccination is the best form of protecting fish against viral diseases when the pathogen cannot be contained by biosecurity measures. Vaccines based on live attenuated viruses seem to be most effective for vaccination against challenging pathogens like Cyprinid herpesvirus 3. However, there are still knowledge gaps how these vaccines effectively protect fish from the deadly disease caused by the epitheliotropic CyHV-3, and which aspects of non-direct protection of skin or gill integrity and function are important in the aquatic environment. To elucidate some elements of protection, common carp were vaccinated against CyHV-3 using a double deletion vaccine virus KHV-T ΔDUT/TK in the absence or presence of a mix of common carp beta-defensins 1, 2 and 3 as adjuvants. Vaccination induced marginal clinical signs, low virus load and a minor upregulation of cd4, cd8 and igm gene expression in vaccinated fish, while neutralisation activity of blood serum rose from 14 days post vaccination (dpv). A challenge infection with CyHV-3 induced a severe disease with 80-100% mortality in non-vaccinated carp, while in vaccinated carp, no mortality was recorded and the virus load was >1,000-fold lower in the skin, gill and kidney. Histological analysis showed strongest pathological changes in the skin, with a complete destruction of the epidermis in non-vaccinated carp. In the skin of non-vaccinated fish, T and B cell responses were severely downregulated, inflammation and stress responses were increased upon challenge, whereas vaccinated fish had boosted neutrophil, T and B cell responses. A disruption of skin barrier elements (tight and adherence junction, desmosomes, mucins) led to an uncontrolled increase in skin bacteria load which most likely exacerbated the inflammation and the pathology. Using a live attenuated virus vaccine, we were able to show that increased neutrophil, T and B cell responses provide protection from CyHV-3 infection and lead to preservation of skin integrity, which supports successful protection against additional pathogens in the aquatic environment which foster disease development in non-vaccinated carp.

Histopathology of head kidney tissues in challenged rohu, Labeo rohita Hamilton after vaccinating with Aeromonas hydrophila antigens

The study was conducted to evaluate the vaccination effects on rohu, Labeo rohita head kidney tissues while assessing the vaccine efficacy of Aeromonas hydrophila antigens. Six acclimatized rohu groups were immunized with three antigenic formulations (outer membrane proteins, somatic and whole-cell antigen) @ 200 µg/fish and also with equal volume of Freund's incomplete adjuvant (FIA), separately. Simultaneously, two non-vaccinated groups, i.e., injected with FIA (100µl), normal saline solutions (0.85%) and one control without injection were maintained for 28 days. All rohu were challenged with median lethal dose of A. hydrophila (2.85 × 106 cells/rohu) intraperitoneally. After 7 days, highest cumulative mortality (%) of ˃88% was found for all non-vaccinated groups. During histopathological observations in head kidney tissues of all treatment and control groups, numerous histopathological changes in the nephritic cells like mild loss of typical tubular epithelial lining, necrosis, thickening of renal epithelial lining, haemorrhages, inflammation, distorted and widening of the lumen with vacuolated surrounding and the constricted lumen of nephritic tubules were noticed for vaccinated rohu in contrast to non vaccinated groups before A. hydrophila challenge. In case of all non-vaccinated fish, including control, extensive degenerated and necrotized head kidney tissues were observed, whereas it was least observed in vaccinated rohu after 7 days A. hydrophila challenge. Results suggest that OMPs antigen along with FIA was the premier vaccine approach for improving resistance to Aeromonas disease and reduce mortality in rohu. Similarly, vaccination with all three antigenic formulations, preferably when applied along with FIA, can effectively protect the head kidney against A. hydrophila infection.

Immunogenicity in Oreochromis niloticus vaccinated with sonicated antigens against streptococcosis

Streptococcosis causes great economic losses in intensive culture of tilapia. Vaccination is the most effective and safest way to tackle infectious diseases. Thus, this study sought the more effective and safer antigenic fraction after sonication of Streptococcus agalactiae to elaborate a vaccine against streptococcosis in Nile tilapia. For this, twenty-one days after vaccination with different fractions (soluble and insoluble) of S. agalactiae, the fish were challenged with the homologous strain (LD50). Then, samples were taken at zero, 14, 28, 60 and 90 days post-vaccination (DPV, n = 7). Blood and organs (cranial kidney, spleen and liver) were collected from vaccinated and unvaccinated fish. Finally, insoluble fraction vaccine presented the best effect, resulting in a 100% relative percent of survival (RPS) and without clinical manifestations. In view of the results, it was to evaluate the role of the insoluble fraction of the antigen in the protective immunity against streptococcosis. The results indicate that the spleen might be the main organ in the vaccine response in Nile tilapia due to the great morphological and immunological differences in vaccinated fish, evidenced by the greater of melanomacrophage centers (MMC) and IgM + lymphocytes in relation to the non-vaccinated fish. At 60 DPV, it was observed the peak of the protective immunity related to the maximum concentration of proteins, circulating leukocytes, antibody titers in the serum and tissue changes with greater expression of IgM + and MMC number in the spleen and kidney of Oreochromis niloticus. Vaccination with insoluble fraction of S. agalactiae was safe and provided effective protection against streptococcosis with maximum protective response at 60 DPV.

Safety and efficacy test to immersion vaccine against Streptococcus agalactiae and Aeromonas hydrophila for Tilapia (Oreochromis niloticus)

Alternative prevention should be developed to control disease outbreaks in Tilapia culture caused by co-infections of Aeromonas hydrophila and Streptococcus agalactiae. Applications of vaccines with low cost and simple methods are highly recommended. This study was conducted to find out the safety and the efficacy of immersion vaccine in Tilapia used bivalent vaccines with formalin inactivated of S. agalactiae and A. hydrophila. Tilapia (Oreochromis niloticus) were divided into two groups, vaccinated and not vaccinated, with single and double doses immersion. The results showed that single and double doses of immersion vaccine were safe for fish (SR 100%) and effectively increased the antibody titer values compared to non-vaccinated fish. The immersion vaccine method can be recommended as an alternative to controlled fish diseases, especially for both Motile Aeromonads Septicaemia and Streptococcosis caused by A. hydrophila and S. agalactiae infections, respectively.

A Novel Subunit Vaccine Based on Outer Capsid Proteins of Grass Carp Reovirus (GCRV) Provides Protective Immunity against GCRV Infection in Rare Minnow (Gobiocypris rarus).

The grass carp hemorrhagic disease, caused by the grass carp reovirus (GCRV), has resulted in severe economic losses in the aquaculture industry in China. VP4 and VP35 are outer capsid proteins of GCRV and can induce an immune response in the host. Here, three recombinant baculoviruses, AcMNPV-VP35, AcMNPV-VP4, and AcMNPV-VP35-VP4, were generated to express recombinant VP4 and VP35 proteins from GCRV type II in insect cells by using the Bac-to-Bac baculovirus expression system to create a novel subunit vaccine. The expression of recombinant VP35, VP4, and VP35-VP4 proteins in Sf-9 cells were confirmed by Western blotting and immunofluorescence. Recombinant VP35, VP4, and VP35-VP4 were purified from baculovirus-infected cell lysates and injected intraperitoneally (3 μg/fish) into the model rare minnow, Gobiocypris rarus. After 21 days, the immunized fish were challenged with virulent GCRV. Liver, spleen, and kidney samples were collected at different time intervals to evaluate the protective efficacy of the subunit vaccines. The mRNA expression levels of some immune-related genes detected by using quantitative real-time PCR (qRT-PCR) were significantly upregulated in the liver, spleen, and kidney, with higher expression levels in the VP35-VP4 group. The nonvaccinated fish group showed 100% mortality, whereas the VP35-VP4, VP4, and VP35 groups exhibited 67%, 60%, and 33% survival, respectively. In conclusion, our results revealed that recombinant VP35 and VP4 can induce immunity and protect against GCRV infection, with their combined use providing the best effect. Therefore, VP35 and VP4 proteins can be used as a novel subunit vaccine against GCRV infection.

Protection and antibody reactivity in lumpsucker (Cyclopterus lumpus L.) following vaccination against Pasteurella sp.

Two monovalent vaccines against pasteurellosis were developed and tested for efficacy using a previously established bath challenge model. High levels of specific antibodies were detected following vaccination. While the vaccine efficacy trial indicated that some level of protection was obtained, high mortality was still observed. qPCR analysis of head kidney tissues from surviving fish post challenge showed no difference in bacterial numbers in vaccinated and non-vaccinated fish. Clinical symptoms observed in moribund and diseased fish included white spots on the skin and around the eyes, frayed fins and redness around the mouth and fin bases. Despite production of specific antibodies, the protection against experimental challenge was relatively weak. A reason for this could potentially be that the specific antibodies produced are not alone enough to provide complete protection against pasteurellosis in lumpsuckers. Confocal and scanning electron microscopy of head kidney leucocytes exposed to Pasteurella sp. in vitro gave indications of the interactions between the pathogen and leucocytes. The results indicate that parts of the immune system other than humoral antibodies could be important for protection against pasteurellosis. Our combined results highlight the need for further work on host-pathogen interaction between Pasteurella sp. and lumpsuckers.

Phagocytic and Bactericidal Properties of Channel Catfish Peritoneal Macrophages Exposed to Edwardsiella ictaluri Live Attenuated Vaccine and Wild-Type Strains.

Edwardsiella ictaluri (E. ictaluri), a Gram-negative, intracellular, facultative bacterium, is the causative agent of enteric septicemia of catfish (ESC), which is one of the most significant diseases of farmed channel catfish. Macrophages have a critical role in major defense mechanisms against bacterial infections by migrating to the site of infection, engulfing and killing pathogens, and priming adaptive immune responses. Vaccination of catfish with E. ictaluri live attenuated vaccine (LAV) strains increased the efficiency of phagocytosis and bacterial killing in catfish peritoneal macrophages compared in vitro with macrophages from non-vaccinated fish. Recently, our group developed several protective LAV strains from E. ictaluri. However, their effects on the antigen uptake and bacterial killing in catfish macrophages have not been evaluated. In this study, we assessed the phagocytic and bactericidal activity of peritoneal macrophages in the uptake of E. ictaluri wild-type (WT) and two LAV strains. We found that phagocytosis of LAV strains was significantly higher compared to their WT counterpart in peritoneal macrophages. Moreover, the uptake of E. ictaluri opsonized with sera from vaccinated catfish was more efficient than when opsonized with sera from sham-vaccinated fish. Notably, catfish macrophages did not lose their phagocytic properties at 4°C, as described previously in mammalian and zebrafish models. Also, opsonization of E. ictaluri with inactivated sera from vaccinated and sham-vaccinated catfish decreased significantly phagocytic uptake of bacteria at 32°C, and virtually suppressed endocytosis at 4°C, suggesting the important role of complement-dependent mechanisms in catfish macrophage phagocytosis. In conclusion, our data on enhanced phagocytic capacity and effective killing ability in macrophages of vaccine strains suggested the LAVs’ advantage if processed and presented in the form of peptides to specific lymphocytes of an adaptive immune system and emphasize the importance of macrophage-mediated immunity against ESC. Furthermore, we showed the role of complement-dependent mechanisms in the phagocytic uptakes of E. ictaluri in catfish peritoneal macrophages at 4 and 32°C. Finally, LAV vaccine-induced bacterial phagocytosis and killing properties of peritoneal macrophages emphasized the importance of the innate immune responses in ESC.

DNA vaccine encoding myristoylated membrane protein (MMP) of rock bream iridovirus (RBIV) induces protective immunity in rock bream (Oplegnathus fasciatus)

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream (Oplegnathus fasciatus) in Korea. In this study, we investigated the potential of viral membrane protein to induce antiviral status protecting rock bream against RBIV infection. We found that fish administered with ORF008L (myristoylated membrane protein, MMP) vaccine exhibited significantly higher levels of survival compared to ORF007L (major capsid protein, MCP). Moreover, ORF008L-based DNA vaccinated fish showed significant protection at 4 and 8 weeks post vaccination (wpv) than non-vaccinated fish after infected with RBIV (6.7 × 105) at 23 °C, with relative percent survival (RPS) of 73.36% and 46.72%, respectively. All of the survivors from the first RBIV infection were strongly protected (100% RPS) from re-infected with RBIV (1.1 × 107) at 100 dpi. In addition, the MMP (ORF008L)-based DNA vaccine significantly induced the gene expression of TLR3 (14.2-fold), MyD88 (11.6-fold), Mx (84.7-fold), ISG15 (8.7-fold), PKR (25.6-fold), MHC class I (13.3-fold), Fas (6.7-fold), Fas ligand (6.7-fold), caspase9 (17.0-fold) and caspase3 (15.3-fold) at 7 days post vaccination in the muscle (vaccine injection site). Our results showed the induction of immune responses and suggest the possibility of developing preventive measures against RBIV using myristoylated membrane protein-based DNA vaccine.

Effect of oral booster vaccination of rainbow trout against Yersinia ruckeri depends on type of primary immunization

Vaccination of rainbow trout against Enteric Redmouth Disease (ERM) caused by Yersinia ruckeri can be successfully performed by administering vaccine (a bacterin consisting of formalin killed bacteria) by immersion, bath or injection. Booster immunization is known to increase the protection of fish already primed by one of these vaccination methods. Oral vaccination of trout (administering vaccine in feed) is an even more convenient way of presenting antigen to the fish but the effect of an oral booster has not previously been described in detail. The present work describes to what extent protection may be enhanced by oral boostering following priming with different administration methods. The study confirms that vaccination by 30 s dip into a bacterin (diluted 1:10) may confer a significant protection compared to non-vaccinated fish. The immunity may be optimized by booster immunization either provided as dip (most effective), bath (less effective) or orally (least effective). Oral immunization may be used as booster after dip but applied as a single oral application it induced merely a slight and statistically non-significant response. It is noteworthy that primary oral immunization followed by an oral booster vaccination showed a trend for an even weaker response. It should be investigated if continued exposure to a low antigen concentration – as performed by two oral immunizations - may induce tolerance to the pathogen and thereby leave the fish more vulnerable.

Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish: Implication for the robust innate defense mechanisms of teleosts

rag1−/− zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1−/− zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1−/− fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1−/− fish possess innate protection equivalent to that of rag1+/− fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.

RNA-Seq transcriptome analysis of the olive flounder (Paralichthys olivaceus) kidney response to vaccination with heat-inactivated viral hemorrhagic septicemia virus

Viral hemorrhagic septicemia (VHS) is a highly contagious disease of cultured flounder caused by VHS virus (VHSV). To develop effective VHSV vaccines, it is essential to understand the molecular mechanisms underlying the host's protective response against VHSV. The purpose of this study is to clarify which genes are involved in the protective response of olive flounder after VHSV vaccination. We first injected olive flounder intraperitoneally with 107 TCID50 heat-inactivated VHSV vaccine and evaluated the vaccine efficacy at 20 °C. Fish vaccinated with heat-inactivated VHSV were significantly protected compared to non-vaccinated fish, with a relative percentage survival of 83%. To analyze the vaccination-induced changes in the expression profiles of genes, kidneys were collected from control and vaccinated fish at days 1, 3, and 7 after vaccination and global gene expression profiling was carried out by RNA sequencing. The analysis revealed that 15,001 genes were differentially expressed by at least 2-fold between vaccinated fish and non-vaccinated controls. Of these, 58 genes clustered into the acute phase response, Toll-like receptor, interferon-inducible/regulatory proteins, and apoptosis pathways. These data provided insights into the molecular mechanisms underlying the protective immune response of olive flounder against heat-inactivated VHSV vaccine and might aid future studies to develop a highly immunogenic vaccine against VHSV in flounder.