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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., (© 2024 John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

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.

Aeromonas salmonicida , a Gram-negative bacterium, causes the disease furunculosis in multiple fish species. We present the complete genome sequence of the atypical A. salmonicida strain T30, which was isolated from furunculosis in sablefish in Manchester, WA, USA. Analyzing this genome will help to identify the bacterium's role in marine aquaculture., Competing Interests: The authors declare no conflict of interest.

Infectious hematopoietic necrosis (IHN) is a significant viral disease affecting salmonids, whereas Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD), remains one of the most significant bacterial pathogens of salmonids. We explored maternal immunity in the context of IHN and BCWD management in rainbow trout (Oncorhynchus mykiss) aquaculture. Two experimental trials were conducted where different groups of female broodstock were immunized prior to spawning with an IHNV DNA vaccine or a live attenuated F. psychrophilum (Fp B.17-ILM) vaccine alone, or in combination. Progeny were challenged with either a low or high dose of IHNV at 13 days post hatch (dph) and 32 dph or challenged with F. psychrophilum at 13 dph. Mortality following a low-dose IHNV challenge at 13 dph was significantly lower in progeny from vaccinated broodstock vs. unvaccinated broodstock, but no significant differences were observed at 32 dph. Mortality due to BCWD was also significantly reduced in 13 dph fry that originated from broodstock immunized with the Fp B.17-ILM vaccine. After vaccination broodstock developed specific or neutralizing antibodies respectively to F. psychrophilum and IHNV; however, antibody titers in eggs and fry were undetectable. In the eggs and fry mRNA transcripts of the complement components C3 and C5 were detected at much higher levels in progeny from vaccinated broodstock and showed a significantly increased and rapid response post-challenge compared with unvaccinated broodstock. After challenges pro-inflammatory cytokine expression was immediately and considerably elevated in the fry from vaccinated broodstock vs. unvaccinated broodstock, whereas adaptive immune genes were elevated to a lesser degree. Results suggest that maternal transfer of innate and adaptive factors at the transcript level occurred because development of lymphomyeloid organs is not complete in such young fry. In addition to documenting maternally derived immunity in teleosts, this study demonstrates that broodstock vaccination can confer some degree of protection to progeny against viral and bacterial pathogens., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Burbot (Lota lota) are an ideal candidate for cool or cold-water aquaculture and are gaining interest because of their high economic value, low temperature requirements, and fast growth rate. Limited information exists on the innate and adaptive immune systems of this species. This is partly due to the lack of species-specific tools to determine antibody responses following disease or vaccination or to characterize the immune response in general. An anti-IgM monoclonal antibody (mAb 27C) was developed and characterized via enzyme-linked immunosorbent assay (ELISA) and Western blot for species specificity, affinity to the heavy chain of burbot IgM, and cross-reactivity to other reagents used in the analysis. The 27C monoclonal antibody was further utilized to develop an ELISA protocol to measure the specific antibody response of burbot following exposure to two pathogenic strains of Aeromonas sp. (A141 and IR004). This ELISA confirmed that vaccinated burbot that survived the challenge with either strain developed statistically higher titers of anti-Aeromonas antibodies specific for the relative strain when compared to fish that were not vaccinated or challenged. Western blot analysis further demonstrated that burbot surviving challenge had serum IgM that recognized distinct antigens specific to the strain they were challenged with, A141 bound to antigens in the 50-250Kda range and IR004 bound to a distinct 150Kda antigen. Western blots further indicated that each strain shared antigenic regions regardless of experimental Aeromonas strain exposure. Finally, immunofluorescent staining confirmed that mAb 27C binds to membrane-bound IgM (presumably B cells) on burbot head kidney cells. Taken together, results from this study demonstrate that mAb 27C specifically recognized burbot IgM and will be an important tool to further characterize the adaptive and cellular immune responses of this fish species., (Copyright © 2023. Published by Elsevier Ltd.)

Flavobacterial diseases, caused by bacteria in the order Flavobacteriales, are responsible for devastating losses in farmed and wild fish populations worldwide. The genera Flavobacterium (Family Flavobacteriaceae ) and Chryseobacterium ( Weeksellaceae ) encompass the most well-known agents of fish disease in the order, but the full extent of piscine-pathogenic species within these diverse groups is unresolved, and likely underappreciated. To identify emerging agents of flavobacterial disease in US aquaculture, 183 presumptive Flavobacterium and Chryseobacterium isolates were collected from clinically affected fish representing 19 host types, from across six western states. Isolates were characterized by 16S rRNA gene sequencing and phylogenetic analysis using the gyrB gene. Antimicrobial susceptibility profiles were compared between representatives from each major phylogenetic clade. Of the isolates, 52 were identified as Chryseobacterium species and 131 as Flavobacterium . The majority of Chryseobacterium isolates fell into six clades (A-F) consisting of ≥ 5 fish isolates with ≥ 70% bootstrap support, and Flavobacterium into nine (A-I). Phylogenetic clades showed distinct patterns in antimicrobial susceptibility. Two Chryseobacterium clades (F & G), and four Flavobacterium clades (B, G-I) had comparably high minimal inhibitory concentrations (MICs) for 11/18 antimicrobials tested. Multiple clades in both genera exhibited MICs surpassing the established F. psychrophilum breakpoints for oxytetracycline and florfenicol, indicating potential resistance to two of the three antimicrobials approved for use in finfish aquaculture. Further work to investigate the virulence and antigenic diversity of these genetic groups will improve our understanding of flavobacterial disease, with applications for treatment and vaccination strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Heckman, Yazdi, Pomaranski, Sebastião, Mukkatira, Vuglar, Cain, Loch and Soto.)

Objective: Renibacterium salmoninarum is a pathogenic gram-positive bacterium and is the causative agent of bacterial kidney disease (BKD), a malady that mainly impacts salmonid species. Experimental challenges were conducted to assess the virulence and challenge route for select R. salmoninarum strains (CK-90 and ATCC 33739) in Rainbow Trout Oncorhynchus mykiss., Methods: The CK-90 strain was intracoelomically injected (100 μL) at a high dose containing 4.80 × 10 6  CFU/g of fish (optical density at 525 nm [OD 525 ] = 1.779) and a low dose containing 6.86 × 10 5  CFU/g of fish (OD 525  = 1.077); alternatively, fish were immersed in a solution containing 4.5 × 10 7  CFU/mL of fish (OD 525  = 0.886). The ATCC 33739 strain (originating from Brook Trout Salvelinus fontinalis) was also included and intracoelomically injected at 3.58 × 10 5  CFU/g of fish (OD 525  = 1.431) to discern differences in virulence between the strains., Result: Clinical signs of BKD manifested at approximately 10 d postchallenge, and mortalities began at 19 days postchallenge. To confirm infection and quantify R. salmoninarum antigen load, an enzyme-linked immunosorbent assay (ELISA) was conducted using kidney tissue collected after the challenge. Rainbow Trout that were challenged with CK-90 by injection (both high- and low-dose groups) exhibited significantly higher mortality than fish that were injected with ATCC 33739 or those that were exposed to CK-90 via immersion challenge. The R. salmoninarum p57 (57-kDa protein) antigen was confirmed via ELISA. Antigen load for fish injected with CK-90 (high dose: OD 405  = 0.71; low dose: OD 405  = 0.66) was significantly higher than that for fish injected with ATCC 33739 (OD 405  = 0.34). The CK-90 strain (both high and low doses) was more virulent than ATCC 33739, which caused no mortalities over the 28-days trial. Although there were no mortalities among ATCC 33739 fish, the ELISA confirmed that the R. salmoninarum antigen infiltrated kidney tissue in those fish., Conclusion: The immersion challenge methodology for R. salmoninarum CK-90 was ineffective for inducing mortalities at the examined dose., (© 2022 American Fisheries Society.)

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., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Sablefish (Anoplopoma fimbria) are an emerging aquaculture species native to the continental shelf of the northern Pacific Ocean. There is limited information on both innate and adaptive immunity for this species and new tools are needed to determine antibody response following vaccination or disease outbreaks. In this paper, a monoclonal antibody, UI-25A, specific to sablefish IgM was produced in mice. Western blotting confirmed UI-25A recognizes the heavy chain of IgM and does not cross react to proteins or carbohydrates in serum of four other teleost species. An ELISA was developed to measure Aeromonas salmonicida specific IgM in the plasma of sablefish from a previous experiment where fish were immunized with a proprietary A. salmonicida vaccine. UI-25A was used in Western blot analyses to identify immunogenic regions of A. salmonicida recognized by this specific IgM from vaccinated sablefish. Immunofluorescent staining also demonstrated the ability of UI-25A to recognize membrane-bound IgM and identify IgM + cells in the head kidney. These results demonstrate the usefulness of UI-25A as a tool to improve the understanding of antibody-mediated immunity in sablefish as well as to provide valuable information for vaccine development and expansion of aquaculture efforts for this fish species., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2021 John Wiley & Sons Ltd.)

Salmonid diseases caused by infections of Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, remain difficult to manage as novel, pathogenic strains continue to emerge in aquaculture settings globally. To date, much of the research regarding treatment options and vaccine development has focused on rainbow trout (Oncorhynchus mykiss), but other inland-reared salmonids are also impacted by this Gram-negative bacterium. As such, Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis) were injection-challenged with a variety of previously reported F. psychrophilum strains isolated from disease diagnostic cases in salmonids, as well as a standard and well-studied F. psychrophilum strain (CSF 259-93) known to be virulent in rainbow trout. In three separate virulence assessments (Trials A, B and C), strains US063 (isolated from lake trout; Salvelinus namaycush) and US149 (isolated from Atlantic salmon) caused a significantly higher cumulative per cent mortality (CPM) relative to other strains in Atlantic salmon (p <.001 for all trials), with US149 causing significantly greater mortality than US063 in Trials A (CPM 97% vs. 65%, p =.008) and B (CPM 96% ± 2.3% vs. 81.33% ± 4.8%, p =.014). Trial C used a lower dose (1.86 × 10 8  CFU/mL) for US149, resulting in a lower mortality (78.67% ± 9.33%) relative to Trials A and B. CSF259-93 did not cause significant mortality in any trials. In brook trout, the strain 03-179 (originally isolated from steelhead trout; Oncorhynchus mykiss) was significantly more virulent than any other (CPM 100% ± 0%, p <.001), followed by US063 (73% ± 3.8%) and US149 (40% ± 6.1%,) respectively. Again, CSF259-93 did not cause significant mortality relative to a mock challenge treatment. Results provide information about the applicability of strain selection in F. psychrophilum virulence testing in Atlantic salmon and brook trout, demonstrating the high virulence of US063 and US149 for these salmonid species. This information is applicable for the development of therapeutics and vaccines against F. psychrophilum infections and demonstrates the reproducibility of the experimental challenge model., (© 2021 John Wiley & Sons Ltd.)

Burbot (Lota lota maculosa) are a potential new species for commercial aquaculture. As burbot culture expands, there is a need to further define pathogen susceptibility and characterize aspects of the burbot immune response in an effort to assess fish health. A recent clinical diagnostic case from juvenile burbot reared at a commercial production facility resulted in the isolation and identification of Flavobacterium columnare along with several Aeromonas spp. The F. columnare isolate was assigned to genetic group 1 via multiplex PCR, a genetic group commonly associated with columnaris disease cases in rainbow trout (Oncorhynchus mykiss). Virulence of the F. columnare isolate was assessed in vivo in both juvenile burbot and rainbow trout. Additionally, several of the Aeromonas sp. case isolates were identified via sequencing (16S rRNA, gyrB and rpoD) and a putative A. sobria isolate (BI-3) was used to challenge burbot, along with a known virulent Aeromonas sp. (A141), but BI-3 was not found to be virulent. Burbot were refractory to F. columnare when challenged by immersion, and it is likely that this is a secondary pathogen for burbot. Although refractory in burbot, the identified F. columnare isolate (BI-1) was found to be virulent in rainbow trout., (© 2020 John Wiley & Sons Ltd.)

For salmonid producers, a common threat is Flavobacterium psychrophilum. Recent advancements in bacterial coldwater disease (BCWD) management include the development of a live-attenuated immersion vaccine that cross-protects against an array of F. psychrophilum strains. Emerging family Flavobacteriaceae cases associated with clinical disease have been increasing, including pathogenic isolates of Flavobacterium spp. and Chryseobacterium spp. The cross-protective ability of a live-attenuated F. psychrophilum vaccine was determined against three virulent Flavobacteriaceae isolates. Juvenile rainbow trout were vaccinated, developed high F. psychrophilum-specific antibody titres and were challenged with Chryseobacterium spp. isolates (S25 and T28), a Flavobacterium sp. (S21) isolate, a mixed combination of S21:S25:T28, and a standard virulent F. psychrophilum CSF259-93 strain. Results demonstrated strong protection in the CSF259-93 vaccinated group (relative per cent survival (RPS)=94.44%) when compared to the relevant CSF259-93 controls (p < .001). Protection was also observed for vaccinated fish challenged with the S21:S25:T28 mix (RPS = 85.18%; p < .001). However, protection was not observed with the S21, S25 or T28 isolates alone. Analysis of whole-cell lysates revealed differences in protein banding by SDS-PAGE, but conserved antigenic regions by Western blot in S25 and T28. Results demonstrate that this live-attenuated vaccine provided protection against mixed flavobacterial infection and suggest further benefits against flavobacteriosis., (© 2020 John Wiley & Sons Ltd.)

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.

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., (© 2019 John Wiley & Sons Ltd.)

Flavobacterium psychrophilum , the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout ( Oncorhynchus mykiss ). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within F. psychrophilum At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North American F. psychrophilum isolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from O. mykiss experiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification of F. psychrophilum in North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species. IMPORTANCE Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred F. psychrophilum isolates that were recovered from diseased fish across North America. Results highlighted multiple F. psychrophilum genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques., (Copyright © 2019 American Society for Microbiology.)

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, remains one of the most significant bacterial diseases of salmonids worldwide. A previously developed and reported live-attenuated immersion vaccine (F. psychrophilum; B.17-ILM) has been shown to confer significant protection to salmonids. To further characterize this vaccine, a series of experiments were carried out to determine the cross-protective efficacy of this B.17-ILM vaccine against 9 F. psychrophilum isolates (representing seven sequence types/three clonal complexes as determined by multilocus sequence typing) in comparison with a wild-type virulent strain, CSF-259-93. To assess protection, 28-day experimental challenges of rainbow trout (Oncorhynchus mykiss) fry were conducted following immersion vaccinations with the B.17-ILM vaccine. F. psychrophilum strains used in challenge trials were isolated from several fish species across the globe; however, all were found to be virulent in rainbow trout. The B.17-ILM vaccine provided significant protection against all strains, with relative percent survival values ranging from 51% to 72%. All vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum-specific antibodies) that increased out to the time of challenge (8 weeks postimmunization). Previous studies have confirmed that antibody plays an important role in protection against F. psychrophilum challenge; therefore, specific antibodies to the B.17-ILM vaccine strain appear to contribute to the cross-protection observed to heterologous strain. The ability of such antibodies to bind to similar antigenic regions for all strains was confirmed by western blot analyses. Results presented here support the practical application of this live-attenuated vaccine, and suggest that it will be efficacious even in aquaculture operations affected by diverse strains of F. psychrophilum., (© 2018 John Wiley & Sons Ltd.)

Advances in technology are making it easier for rapid field detection of microbes in aquaculture. Specifically, real-time quantitative PCR (qPCR) analysis, which has traditionally been confined to laboratory-based protocols, is now available in a handheld, field-portable system. The feasibility of using the Biomeme handheld qPCR system for rapid (<50 min) on-site detection and monitoring of Flavobacterium psychrophilum from filtered water samples was evaluated. Paired water samples were collected over a 23-d period from microcosm tanks that housed fish injected with known levels of F. psychrophilum. Water samples were filtered through 0.45-μm nitrocellulose filters and were analyzed with both the Biomeme qPCR platform and a traditional bench qPCR protocol. The two methods identified similar fluctuations in F. psychrophilum DNA throughout the study. Standard curves relating quantification cycles to the number of F. psychrophilum colony-forming units (CFU) were constructed and analyzed; results indicated that CFU increased rapidly between days 6 and 8 of the trial and then progressively decreased during the remaining 15 d. Average calculated log 10 (CFU/mL) values were significantly correlated between the two platforms. Rapid, field-based qPCR can be incorporated into daily water quality monitoring protocols to help detect and monitor microbes in aquaculture systems., (© 2018 American Fisheries Society.)

In response to population declines of North American Burbot Lota lota maculosa (hereafter, Burbot), conservation aquaculture methods have been developed for this species. In general, Burbot are relatively resistant to many salmonid pathogens; however, cultured juvenile Burbot have experienced periodic epizootic disease outbreaks during production. A series of trials was conducted to determine the virulence of select bacteria isolated from juvenile Burbot after outbreaks that occurred in 2012 and 2013 at the University of Idaho's Aquaculture Research Institute. Initial clinical diagnostics and sampling resulted in the isolation of numerous putative bacterial pathogens. To determine which bacteria were the most likely causative agents contributing to these epizootics, juvenile Burbot received intraperitoneal (IP) injections of select bacteria in log-phase growth. Mortality associated with specific isolates was recorded, and more comprehensive challenges followed this initial screening. These challenges used side-by-side IP and immersion methods to expose Burbot to potential pathogens. The challenges resulted in significantly higher mortalities in fish after IP injection with two Aeromonas sp. isolates compared to controls, but no significant difference in mortality for immersion-challenged groups was observed. Results demonstrate that two Aeromonas sp. isolates cultured from the epizootics are virulent to Burbot., (© 2018 American Fisheries Society.)

Purpose: This study describes the design, operation and evaluation of a community-based research (CBR) consult service within the setting of a Clinical and Translational Science Award (CTSA) institution. To our knowledge, there are no published evaluations of a CBR consult service at a CTSA hub., Methods: A community-based research Consult Service was created to support faculty, health care providers/research coordinators, trainees, community-based organizations and community members. A framework was developed to assess the stages of client engagement and to foster clear articulation of client needs and challenges. A developmental evaluation system was integrated with the framework to track progress, store documents, continuously improve the consult service and assess research outcomes., Results: This framework provides information on client numbers, types, services used and successful outreach methods. Tracking progress reveals reasons that prevent clients from completing projects and facilitates learning outcomes relevant to clients and funding agencies. Clients benefit from the expert knowledge, community connections and project guidance provided by the Consult Service team, increasing the likelihood of study completion and achieving research outcomes., Conclusion: This study offers a framework by which CTSA institutions can expand their capacity to conduct and evaluate community-based research while addressing challenges that inhibit community engagement., Competing Interests: Conflicts: The authors have no conflicts of interest.

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 ∼10(10) and 10(8) 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 (∼10(6) and 10(5) 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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD), which has a major impact on salmonid aquaculture globally. An Enterobacter species, C6-6, isolated from the gut of rainbow trout, Oncorhynchus mykiss (Walbaum), has been identified as a potential probiotic species providing protection against BCWD. This study examined the effects of alginate microencapsulation on the protective efficacy of C6-6 against BCWD in vivo when administered to rainbow trout fry orally or by intraperitoneal (IP) injection. Viable C6-6 bacteria were microencapsulated successfully, and this process (microencapsulation) did not significantly deteriorate its protective properties as compared to the administration of non-microencapsulated C6-6 bacteria. Both oral and IP delivery of C6-6 achieved significantly better protection than control treatments that did not contain C6-6 bacteria. The highest relative percent survival (RPS) resulted from IP delivery (71.4%) and was significantly greater than the highest oral RPS (38.6%). Successful intestinal colonization was not critical to protective effects of C6-6. The study showed that C6-6 administration, with or without encapsulation, was a viable choice for protecting fry from BCWD especially when administered intraperitoneally., (© 2014 John Wiley & Sons Ltd.)

Background: Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease in salmonids. Earlier research showed that a rifampicin-passaged strain of F. psychrophilum (CSF 259-93B.17) caused no disease in rainbow trout (Oncorhynchus mykiss, Walbaum) while inducing a protective immune response against challenge with the virulent CSF 259-93 strain. We hypothesized that rifampicin passage leads to an accumulation of genomic mutations that, by chance, reduce virulence. To assess the pattern of phenotypic and genotypic changes associated with passage, we examined proteomic, LPS and single-nucleotide polymorphism (SNP) differences for two F. psychrophilum strains (CSF 259-93 and THC 02-90) that were passaged with and without rifampicin selection., Results: Rifampicin resistance was conveyed by expected mutations in rpoB, although affecting different DNA bases depending on the strain. One rifampicin-passaged CSF 259-93 strain (CR) was attenuated (4 % mortality) in challenged fish, but only accumulated eight nonsynonymous SNPs compared to the parent strain. A CSF 259-93 strain passaged without rifampicin (CN) accumulated five nonsynonymous SNPs and was partially attenuated (28 % mortality) compared to the parent strain (54.5 % mortality). In contrast, there were no significant change in fish mortalities among THC 02-90 wild-type and passaged strains, despite numerous SNPs accumulated during passage with (n = 174) and without rifampicin (n = 126). While only three missense SNPs were associated with attenuation, a Ser492Phe rpoB mutation in the CR strain may contribute to further attenuation. All strains except CR retained a gliding motility phenotype. Few proteomic differences were observed by 2D SDS-PAGE and there were no apparent changes in LPS between strains. Comparative methylome analysis of two strains (CR and TR) identified no shared methylation motifs for these two strains., Conclusion: Multiple genomic changes arose during passage experiments with rifampicin selection pressure. Consistent with our hypothesis, unique strain-specific mutations were detected for the fully attenuated (CR), partially attenuated (CN) and another fully attenuated strain (B17).

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Flavobacterium psychrophilum causes bacterial cold-water disease in multiple fish species, including salmonids. An autochthonous Enterobacter strain (C6-6) inhibits the in vitro growth of F. psychrophilum, and when ingested as a putative probiotic, it provides protection against injection challenge with F. psychrophilum in rainbow trout. In this study, low-molecular-mass (≤3 kDa) fractions from both Enterobacter C6-6 and Escherichia coli K-12 culture supernatants inhibited the growth of F. psychrophilum. The ≤3-kDa fraction from Enterobacter C6-6 was analyzed by SDS-PAGE, and subsequent tandem mass spectroscopy identified EcnB, which is a small membrane lipoprotein that is a putative pore-forming toxin. Agar plate diffusion assays demonstrated that ecnAB knockout strains of both Enterobacter C6-6 and E. coli K-12 no longer inhibited F. psychrophilum (P < 0.001), while ecnAB-complemented knockout strains recovered the inhibitory phenotype (P < 0.001). In fish experiments, the engineered strains (C6-6 ΔecnAB and C6-6 ΔecnAB) and the wild-type strain (C6-6) were added to the fish diet every day for 38 days. On day 11, the fish were challenged by injection with a virulent strain of F. psychrophilum (CSF 259-93). Fish that were fed C6-6 had significantly longer survival than fish fed the ecnAB knockout strain (P < 0.0001), while fish fed the complemented knockout strain recovered the probiotic phenotype (P = 0.61). This entericidin is responsible for the probiotic activity of Enterobacter C6-6, and it may present new opportunities for therapeutic and prophylactic treatments against similarly susceptible pathogens., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease and can cause significant mortality in salmonid aquaculture. To better evaluate disease prevention or treatment methods for F. psychrophilum in the laboratory, a waterborne challenge model that mimics a natural outbreak is needed. Here we report on the development of a waterborne challenge model for F. psychrophilum in which we incorporated variables that may influence challenge success: specifically, scarification prior to bacterial exposure and culture of F. psychrophilum under iron-limited culture conditions to potentially increase the probability of establishing disease. Additionally, two F. psychrophilum strains, CSF 259-93 and THC 02-90, were used in this model to test whether there were virulence differences between strains. Mortality was significantly higher in scarred fish than unscarred fish (81.5 vs. 19.4%), supporting the hypothesis that disruptions in the dermal layer enhance mortality in F. psychrophilum waterborne challenges. Although mortality differences were not significant between iron-replete and iron-limited treatments, mortality was high overall (> 30%). There was a significant difference in mortality between CSF 259-93 and THC 02-90 treatments, although both strains caused high mortality in injection challenges. In conclusion, this waterborne challenge model can be used to evaluate potential disease prevention and treatment methods., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The genome sequence of Flavobacterium psychrophilum strain CSF259-93, isolated from rainbow trout (Oncorhynchus mykiss), consists of a single circular genome of 2,900,735 bp and 2,701 predicted open reading frames (ORFs). Strain CSF259-93 has been used to select a line of rainbow trout with increased genetic resistance against bacterial cold water disease., (Copyright © 2014 Wiens et al.)

The Flavobacterium psychrophilum gliding motility N (GldN) protein was investigated to determine its ability to elicit antibody responses and provide protective immunity in rainbow trout Oncorhynchus mykiss (Walbaum). GldN was PCR-amplified, cloned into pET102/D-TOPO, and expressed in Escherichia coli. Bacteria expressing recombinant GldN (rGldN) were formalin-inactivated and injected intraperitoneally (i.p.) into rainbow trout with Freund's complete adjuvant (FCA) in four separate studies that used two different immunization protocols followed by challenge evaluations. Fish injected with E. coli only in FCA served as the control. Antibody responses to F. psychrophilum whole-cell lysates measured by ELISA were low in all four studies. Protection against F. psychrophilum challenge was observed in the first study, but not in the three following studies. The discrepancies in results obtained in the later studies are unclear but may relate to formalin treatment of the antigen preparations. Overall, it appeared that rGldN delivered i.p. as a crude formalin-killed preparation is not a consistent vaccine candidate, and more work is required. Additionally, this study illustrates the importance of conducting multiple in vivo evaluations on potential vaccine(s) before any conclusions are drawn., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The etiological agent of bacterial coldwater disease (BCWD), Flavobacterium psychrophilum, can be transmitted both vertically and horizontally. Outbreaks of BCWD can result in significant losses in salmonid aquaculture. Reduction of outbreaks in fry may be possible through implementation of a management strategy in which progeny of heavily infected broodstock are culled from the general population. Diagnostic assays to quantify F. psychrophilum concentrations in tissue samples and confirm presence of the bacterium in ovarian fluid have been previously validated. In the current study, these assays were used to screen 60 female Rainbow Trout Oncorhynchus mykiss and 60 female Coho Salmon O. kisutch broodstock at two aquaculture facilities. Eyed eggs from 10 female broodstock (five fish from each facility) exhibiting graded levels of infection were transferred to the University of Idaho and monitored through early life stages for the presence of F. psychrophilum. Female Rainbow Trout broodstock were not positive for F. psychrophilum by enzyme-linked immunosorbent assay (ELISA) and prevalence was low in these progeny. However, ELISA optical density values for kidney correlated to F. psychrophilum prevalence in progeny (r = 0.938, P < 0.05) of Coho Salmon. Nested PCR on ovarian fluid was not a reliable indicator of vertical transmission in either species as broodstock ovarian fluid results did not correlate to F. psychrophilum prevalence in eyed eggs. Further research with these assays is necessary; however, results from this study indicate that broodstock screening may be a potential tool for evaluating F. psychrophilum infection levels, which could become an important component for disease management.

Objectives: To determine how grant funds are shared between academic institutions and community partners in community-based participatory research (CBPR)., Methods: Review of all 62 investigator-initiated R01 CBPR grants funded by the National Institutes of Health from January 2005 to August 2012. Using prespecified criteria, two reviewers independently categorized each budget item as being for an academic institution or a community partner. A third reviewer helped resolve any discrepancies., Results: Among 49 evaluable grants, 68% of all grant funds were for academic institutions and 30% were for community partners. For 2% of funds, it was unclear whether they were for academic institutions or for community partners. Community partners' share of funds was highest in the categories of other direct costs (62%) and other personnel (48%) and lowest in the categories of equipment (1%) and indirect costs (7%)., Conclusions: A majority of CBPR grant funds are allocated to academic institutions. In order to enhance the share that community partners receive, funders may wish to specify a minimum proportion of grant funds that should be allocated to community partners in CBPR projects., (© 2014 Wiley Periodicals, Inc.)

An attenuated strain of Flavobacterium psychrophilum (CSF259-93B.17) has shown potential as a vaccine for prevention of bacterial coldwater disease (BCWD) in rainbow trout, Oncorhynchus mykiss (Walbaum). Because BCWD outbreaks can result in high mortality in other salmonid species, specifically coho salmon, Oncorhynchus kisutch (Walbaum), the live-attenuated strain was tested as a vaccine in this species. Additionally, we hypothesized that culture of the vaccine strain under iron-limited conditions would lead to improved protection against BCWD. To test this hypothesis, coho salmon were either injection or immersion immunized with CSF259-93B.17 cultured in iron-replete or iron-limited medium. Resultant antibody titers were low and not significantly different between the two treatments regardless of vaccine delivery method (P > 0.05). Following injection challenge with a virulent F. psychrophilum strain, mortality for injection vaccinated fish was significantly reduced compared to the control but did not differ by treatment (P > 0.05). Relative percent survival (RPS) was high in both treatments (90% in iron-replete, 98% in iron-limited medium). Fish immunized by immersion with CSF259-93B.17 grown in iron-replete medium exhibited lower mortality (29.3%; RPS 46%) when compared to mock immunized fish, but this was not significant. However, mortality was significantly lower in fish immunized with CSF259-93B.17 grown in iron-limited medium (14.7%; RPS 73%) when compared to mock immunized fish. The results demonstrate that the live-attenuated F. psychrophilum strain can confer protection to coho salmon and vaccine efficacy is enhanced by culturing the strain under iron-limited conditions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens - dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) - administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study, 318 bacterial strains were isolated from the gastrointestinal (GI) tracts of 29 rainbow trout, Oncorhynchus mykiss (Walbaum). These bacteria were screened in vitro for their ability to inhibit growth of Flavobacterium psychrophilum, the causative agent of coldwater disease. Bacteria observed to inhibit F. psychrophilum growth were further screened against rainbow trout bile, as an indicator of their ability to survive in the GI tract. This screening resulted in narrowing the pool to 24 bacterial isolates. Those 24 isolates were then tested for pathogenicity in rainbow trout by intraperitoneal injection. Following a 28-day challenge, eight isolates were shown to cause direct mortality and were eliminated from further study. As a result, 16 bacterial isolates were identified as probiotic candidates with the potential to control or reduce disease caused by F. psychrophilum., (© 2012 Blackwell Publishing Ltd.)

It is hypothesized that the frequency of bacterial coldwater disease outbreaks can be reduced through the detection of the aetiologic agent, Flavobacterium psychrophilum, in broodstock followed by culling of eggs from heavily infected broodstock. Before a culling programme can be instituted, however, it is necessary to determine the sensitivity and specificity of existing assays for the detection of F. psychrophilum. In this study, tissue and ovarian fluid samples were collected from 224 fish at five hatcheries and screened using an enzyme-linked immunosorbent assay (ELISA), a membrane-filtration fluorescent antibody test (MF-FAT), bacteriological culture and nested PCR. Latent class analysis was used to estimate sensitivity and specificity of kidney culture, kidney ELISA, nested PCR and MF-FAT. Analytical sensitivity of the ELISA varied but was greatest when bacteria were cultured under iron-limiting conditions. Diagnostic sensitivity estimates ranged from 0.02 (kidney culture) to 0.97 (kidney ELISA). Specificity estimates ranged from 0.02 (MF-FAT) to 0.98 (kidney ELISA). In a separate challenge experiment, the ELISA confirmed the presence of F. psychrophilum in sub-clinically infected fish. Results from this study demonstrate that the ELISA is an appropriate tool to screen broodstock and provides an indication of infection severity, which is crucial for implementation of a screening/culling programme., (© 2012 Blackwell Publishing Ltd.)

Flavobacterium psychrophilum is the aetiological agent of bacterial coldwater disease (CWD), and this pathogen has large economic impacts on salmonid aquaculture worldwide. Previously, it was demonstrated that high levels of protection against F. psychrophilum challenge were conferred to rainbow trout, Oncorhynchus mykiss (Walbaum), by immunization with distinct molecular mass fractions of the bacterium, and specific antibodies were correlated with protection. In this study, an immunoproteomic analysis of F. psychrophilum was performed using two-dimensional polyacrylamide gel electrophoresis and Western blotting with serum from fish immunized with high- and mid-molecular mass fractions of the bacterium. Mass spectrometry was used to determine the protein identity, and 15 immunogenic proteins were positively identified following Mascot searches of the F. psychrophilum genome. Based on known function and immunogenicity of homologous proteins in other bacterial pathogens, antibodies specific for several of the identified proteins may be important for protective immunity from CWD. These include outer membrane protein OmpA (P60), trigger factor, ClpB, elongation factor G, gliding motility protein GldN and a conserved hypothetical protein. This work increases the understanding of the protective humoral immune response of rainbow trout against these distinct molecular mass fractions of F. psychrophilum and provides new potential targets for recombinant protein vaccine development., (© 2011 Blackwell Publishing Ltd.)