Fish immunization has been carried out for over 50 years and is generally accepted as an effective method for preventing a wide range of bacterial and viral diseases. Vaccination efforts contribute ...to environmental, social, and economic sustainability in global aquaculture. Most licensed fish vaccines have traditionally been inactivated microorganisms that were formulated with adjuvants and delivered through immersion or injection routes. Live vaccines are more efficacious, as they mimic natural pathogen infection and generate a strong antibody response, thus having a greater potential to be administered via oral or immersion routes. Modern vaccine technology has targeted specific pathogen components, and vaccines developed using such approaches may include subunit, or recombinant, DNA/RNA particle vaccines. These advanced technologies have been developed globally and appear to induce greater levels of immunity than traditional fish vaccines. Advanced technologies have shown great promise for the future of aquaculture vaccines and will provide health benefits and enhanced economic potential for producers. This review describes the use of conventional aquaculture vaccines and provides an overview of current molecular approaches and strategies that are promising for new aquaculture vaccine development.
Experiments were conducted to optimize triploid induction parameters, and assess triploid sterility, in burbot. Duration and timing of shocks were based on degree minutes, temperature multiplied by ...time, denoted as °C min. Hydrostatic shock experiments investigated the duration of shock using 7500 or 8500 psi at 180°C min post‐fertilization. Thermal shocks investigated duration of shock and post‐fertilization shock timing using a shock of 16°C. Sterility experiments investigated egg survival when diploids were crossed with triploids. Hydrostatic shock of 7500 psi for 10 or 20°C min can induce triploidy ≥90% and exhibits survival that is statistically similar, p ≤ 0.05, to controls. Hydrostatic shock of 8500 psi for 5 or 10°C min yielded triploid induction of 93% and 100%, respectively, with survival that is statistically similar to controls, p ≤ 0.05. Thermal induction experiments indicated shocks at 120°C min post‐fertilization, for durations between 350 and 450°C min, have potential to induce triploidy ≥90% while facilitating survival statistically similar to controls, p ≤ 0.05. Induction of tetraploidy was observed. Sterility experiments determined that triploid burbot are functionally sterile. These results may allow production of burbot where sterility is required.
Teleosts possess three immunoglobulin (Ig) heavy chain isotypes viz., IgM, IgT and IgD and all three isotypes are reported in rainbow trout. The expression of these Ig isotypes in response to ...different immunization routes was investigated and results provide a better understanding of the role these Igs in different tissues. Rainbow trout (Oncorhynchus mykiss) were immunized with an attenuated Flavobacterium psychrophilum strain, 259-93-B.17 grown under iron limiting conditions, by intraperitoneal, anal intubation and immersion routes. Serum, gill mucus, skin mucus and intestinal mucus samples were collected at 0, 3, 7, 14, 28, 42 and 56 days post immunization by sacrificing four fish from each treatment group and the unimmunized control group, and the IgM levels were estimated by an enzyme linked immunosorbent assay (ELISA). In addition, blood, gill, skin and intestinal tissue samples were collected for Ig gene expression studies. The secretory IgM, IgD and IgT gene expression levels in these tissues were estimated by reverse transcription quantitative real time PCR (RT-qPCR). Levels of IgM in serum, gill and skin mucus increased significantly by 28 days after immunization in the intraperitoneally immunized group, while no significant increase in IgM level was observed in fish groups immunized by other routes. Secretory IgD and IgT expression levels were significantly upregulated in gills of fish immunized by the immersion route. Similarly, secretory IgT and IgD were upregulated in intestines of fish immunized by anal intubation route. The results confirm mucosal association of IgT and suggest that IgD may also be specialized in mucosal immunity and contribute to immediate protection to the fish at mucosal surfaces.
•Intraperitoneal immunization results in significant increase in IgM secretion in serum, gill mucus and skin mucus of rainbow trout.•Tissues that were directly exposed to the bacterin, depending upon the immunization route, expressed the highest amount of IgM transcripts.•Upregulation of IgT and IgD genes in mucosal tissues suggest that these two antibody isotypes are specialized mucosal antibodies.•IgT and IgD offer early protection to the fish at mucosal surfaces against invading pathogens.
Nasal immunity is an ancient and conserved arm of the mucosal immune system in vertebrates. In teleost fish, we previously reported the presence of a nasopharynx-associated lymphoid tissue (NALT) ...characterized by scattered immune cells located in the trout olfactory lamellae. This diffuse NALT mounts innate and adaptive immune responses to nasal infection or vaccination. In mammals, lymphoid structures such as adenoids and tonsils support affinity maturation of the adaptive immune response in the nasopharyngeal cavity. These structures, known as organized NALT (O-NALT), have not been identified in teleost fish to date, but their evolutionary forerunners exist in sarcopterygian fish. In this study, we report that the rainbow trout nasal cavity is lined with a lymphoepithelium that extends from the most dorsal opening of the nares to the ventral nasal cavity. Within the nasal lymphoepithelium we found lymphocyte aggregates called O-NALT in this study that are composed of ∼ 56% CD4+, 24% IgM+, 16% CD8α+, and 4% IgT+ lymphocytes and that have high constitutive aicda mRNA expression. Intranasal (i.n.) vaccination with live attenuated infectious hematopoietic necrosis virus triggers expansions of B and T cells and aicda expression in response to primary i.n. vaccination. IgM+ B cells undergo proliferation and apoptosis within O-NALT upon prime but not boost i.n. vaccination. Our results suggest that novel mucosal microenvironments such as O-NALT may be involved in the affinity maturation of the adaptive immune response in early vertebrates.
•Long-term protection following nasal vaccination in teleosts has not previously been evaluated.•Intranasal (I.N.) vaccination was shown to be safe to at least 12 mo post-vaccination in rainbow ...trout”•I.P. but not I.N. delivery of ERM bacterin induced significant protection 6 mo post-vaccination.•I.N.-vaccinated fish with live attenuated IHNV vaccine showed the lowest mortality 6 mo post-vaccination.•Spleen repertoire analyses confirmed unique expansions of VH-JH rearrangements depending on the vaccine and delivery route.
Previous research demonstrated that bacterial and viral vaccines delivered via the nasal route in rainbow trout (Oncorhynchus mykiss) at 7 and 28 days post-vaccination are highly protective (>95% protection). Long-term protection following nasal vaccination in teleosts has not been evaluated. The goal of this study was to assess efficacy and immune responses at 6 months (mo) post-vaccination (mpv), and long-lasting immune responses at 12 mpv of two different vaccines: an inactivated enteric red mouth disease (ERM) Yersinia ruckeri bacterin and a live attenuated infectious hematopoietic necrosis virus (IHNV) vaccine. Juvenile rainbow trout were vaccinated for Y. ruckeri via intraperitoneal (I.P.) and intranasal (I.N.) routes, and for IHNV by intramuscular (I.M.) and I.N. routes, then challenged at 6 mpv. Immune responses were determined at 6 and 12 mpv. ERM vaccine I.P. delivery elicited significantly higher serum IgM-specific titers that remained elevated compared to mock-vaccinated fish at 6 mpv. By 12 mpv, antibody titers to Y. ruckeri were not significantly different across all treatments. Following Y. ruckeri challenge at 6 mpv, a significant difference in cumulative percent mortality (CPM) was found for I.P.-vaccinated fish but not I.N.-vaccinated fish. I.M. and I.N. vaccination with live attenuated IHNV did not result in significant specific serum IgM titers at 6 or 12 mpv. Yet, I.N.-vaccinated fish showed the lowest CPM 6 mpv indicating long-term protection that does not correlate with systemic IgM responses. Repertoire analyses confirmed unique expansions of VH-JH rearrangements in the spleen of rainbow trout 12 mpv that varied with the type of vaccine and route of vaccination. Combined, these data demonstrate that I.N. vaccination with a live attenuated viral vaccine confers long lasting protection, but I.N. ERM vaccination does not and booster before 6 mpv is recommended.
Vaccines continue to play an enormous role in the progression of aquaculture industries worldwide. Though preventable diseases cause massive economic losses, injection-based vaccine delivery is ...cost-prohibitive or otherwise impractical for many producers. Most oral vaccines, which are much cheaper to administer, do not provide adequate protection relative to traditional injection or even immersion formulas. Research has focused on determining why there appears to be a lack of protection afforded by oral vaccines. Here, we review the basic immunological principles associated with oral vaccination before discussing the recent progress and current status of oral vaccine research. This knowledge is critical for the development and advancement of efficacious oral vaccines for the aquaculture industry.
Co‐infection of rainbow trout with infections haematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum is known to occur, and it has been speculated that a combined infection can result ...in dramatic losses. Both pathogens can persist in fish in an asymptomatic carrier state, but the impact of co‐infection has not been well characterized or documented. In this study, it was hypothesized that fish co‐infected with F. psychrophilum and IHNV would exhibit greater mortality than fish infected with either pathogen alone. To test this, juvenile rainbow trout were co‐infected with low doses of either IHNV or F. psychrophilum, and at 2 days post‐initial challenge, they were given a low dose of the reciprocal pathogen. This combined infection caused high mortality (76.2%–100%), while mortality from a single pathogen infection with the same respective dose was low (5%–20%). The onset of mortality was earlier in the co‐infected group (3–4 days) when compared with fish infected with F. psychrophilum alone (6 days) or IHNV (5 days), confirming the synergistic interaction between both pathogens. Co‐infection led to a significant increase in the number of F. psychrophilum colony‐forming units and IHNV plaque‐forming units within tissues. This finding confirms that when present together in co‐infected fish, both pathogens are more efficiently recovered from tissues. Furthermore, pathogen genes were significantly increased in co‐infected groups, which parallel the findings of increased systemic pathogen load. Extensive tissue necrosis and abundant pathogen present intracellularly and extracellularly in haematopoietic tissue. This was pronounced in co‐infected fish and likely contributed to the exacerbated clinical signs and higher mortality. This study provides novel insight into host–pathogen interactions related to co‐infection by aquatic bacterial and viral pathogens and supports our hypothesis. Such findings confirm that mortality in fish exposed to both pathogens is greatly elevated compared to a single pathogen infection.
Abstract A mouse monoclonal antibody (mAb FL100A) previously prepared against Flavobacterium psychrophilum ( Fp ) CSF259‐93 has now been examined for binding to lipopolysaccharides (LPS) of this ...strain and Fp 950106‐1/1. The corresponding O‐polysaccharides (O‐PS) of these strains are formed by identical trisaccharide repeats composed of l ‐Rhamnose ( l ‐Rha), 2‐acetamido‐2‐deoxy‐ l ‐fucose ( l ‐FucNAc) and 2‐acetamido‐4‐R 1 ‐2,4‐dideoxy‐ d ‐quinovose ( d ‐Qui2NAc4NR 1 ) where R 1 represents a dihydroxyhexanamido moiety. The O‐PS loci of these strains are also identical except for the gene ( wzy1 or wzy2 ) that encodes the polysaccharide polymerase. Accordingly, adjacent O‐PS repeats are joined through d ‐Qui2NAc4NR 1 and l ‐Rha by wzy2 ‐dependent α(1–2) linkages in Fp CSF259‐93 versus wzy1 ‐dependent β(1–3) linkages in Fp 950106‐1/1. mAb FL100A reacted strongly with Fp CSF259‐93 O‐PS and LPS but weakly or not at all with Fp 950106‐1/1 LPS and O‐PS. Importantly, it also labelled cell surface blebs on the former but not the latter strain. Additionally, mAb binding was approximately 5‐times stronger to homologous Fp CSF259‐93 LPS than to LPS from a strain with a different R‐group gene. A conformational epitope for mAb FL100A binding was suggested from molecular dynamic simulations of each O‐PS. Thus, Fp CSF259‐93 O‐PS formed a stable well‐defined compact helix in which the R 1 groups were displayed in a regular pattern on the helix exterior while unreactive Fp 950106‐1/1 O‐PS adopted a flexible extended linear conformation. Taken together, the findings establish the specificity of mAb FL100A for Wzy2‐linked F. psychrophilum O‐PS and LPS.
This study was aimed at optimizing the efficacy of a recently developed live attenuated immersion vaccine (B.17-ILM) as a promising vaccine against bacterial coldwater disease (BCWD) caused by ...Flavobacterium psychrophilum in salmonids. Rainbow trout (RBT) fry were vaccinated by immersion, and different parameters affecting vaccination such as fish size, vaccine delivery time, dose, duration of protection, booster regimes and vaccine growth incubation time were optimized. Specific anti-F. psychrophilum immune response was determined by ELISA. Protective efficacy was determined by challenging with a virulent strain of F. psychrophilum (CSF-259-93) and calculating cumulative percent mortality (CPM) and relative percent survival (RPS). All vaccinated fish developed significantly higher levels of serum antibody titers by week 8 when compared to their respective controls. Immersion vaccination for 3, 6 and 30 min produced significant protection with comparable RPS values of 47%, 53% and 52%, respectively. This vaccine provided significant protection for fish as small as 0.5 g with an RPS of 55%; larger fish of 1 g and 2 g yielded slightly higher RPS values of 59% and 60%, respectively. Fish vaccinated with higher vaccine doses of ∼1010 and 108 colony forming units mL−1 (cfu ml−1) were strongly protected out to at least 24 weeks with RPS values up to 70%. Fish vaccinated with lower doses (∼106 and 105 cfu mL−1) had good protection out to 12 weeks, but RPS values dropped to 36% and 34%, respectively by 24 weeks. Vaccine efficacy was optimum when the primary vaccination was followed by a single booster (week 12 challenge RPS = 61%) rather than two boosters (week 12 challenge RPS = 48%). Vaccination without a booster resulted in a lower RPS (13%) indicating the necessity of a single booster vaccination to maximize efficacy. This study presents key findings that demonstrate the efficacy and commercial potential for this live attenuated BCWD vaccine.
•B.17-ILM vaccine can provide strong protection from bacterial coldwater disease (BCWD).•Rainbow trout as small as 0.5 g can be effectively protected against BCWD.•Immersion vaccination with the B.17-ILM vaccine provides protection out to at least 24 weeks post initial immunization.•A vaccine dose as low as ∼105 cfu mL−1 can provide significant protection for fish.•A booster dose is needed to provide optimum protection.
Iron is essential for growth and virulence in most pathogenic bacterial strains. In some cases, the hosts for these pathogenic bacteria develop specialized strategies to sequester iron and limit ...infectivity. This in turn may result in the invading pathogens utilizing high‐affinity iron transport mechanisms, such as the use of iron‐chelating siderophores, to extend beyond the host defences. Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD) in salmonids, relies on iron metabolism for infectivity, and the genome of the model CSF‐259‐93 strain has recently been made available. Further, this strain serves as a parent strain for a live‐attenuated vaccine strain, B.17, which has been shown to provide rainbow trout with protection against BCWD. To elucidate specific gene expression responses to iron metabolism and compare strain differences, both F. psychrophilum strains were grown under iron‐limiting conditions and 26 genes related to iron metabolism were mapped for 96 hr in culture via qPCR analyses. Results indicate increased production of the ferrous iron transport protein B (FITB; p =.008), and ferric receptor CfrA (FR 1; p =.012) in the wild‐type CSF‐259‐93 strain at 72 hr and 96 hr post‐exposure to iron‐limiting media. In the B.17 vaccine strain, siderophore synthase (SS) expression was found to be downregulated at 72 hr, in comparison with 0h (p =.018). When strains were compared, FITB (p =.021), FR1 (p =.009) and SS (p =.016) were also elevated in B.17 at 0 hr and TonB outer protein membrane receptor 1 (TBomr1; p =.005) had a lower expression at 96 hr. Overall, this study demonstrated strain‐related gene expression changes in only a fraction of the iron metabolism genes tested; however, results provide insight on potential virulence mechanisms and clarification on iron‐related gene expression for F. psychrophilum.
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