Benjamin R. LaFrentz

Isolation of rifampicin resistant Flavobacterium psychrophilum strains and their potential as live attenuated vaccine candidates

Previous studies have demonstrated that passage of pathogenic bacteria on increasing concentrations of the antibiotic rifampicin leads to the attenuation of virulence and these resistant strains may serve as live attenuated vaccines. Two rifampicin resistant strains of Flavobacterium psychrophilum, 259-93A.16 and 259-93B.17, were generated by passage on TYES plates containing increasing concentrations of rifampicin. Electrophoretic analysis of whole-cell lysates prepared from the parent and resistant strains identified five differentially expressed proteins between the 259-93B.17 strain and parent strain, while there were no differences identified between the 259-93A.16 and parent strain. The LPS banding patterns were identical between all three strains. Bacterial challenges of rainbow trout (Oncorhynchus mykiss Walbaum) with the resistant strains demonstrated that the 259-93B.17 strain was highly attenuated and the 259-93A.16 strain was modestly attenuated at the challenge doses tested. Immunization of rainbow trout with the live attenuated 259-93B.17 strain by intraperitoneal injection resulted in significant protection against challenge with the virulent parent F. psychrophilum strain at 8 and 15 weeks post-immunization and fish exhibited elevated specific antibody titers. Importantly, immersion delivery of the 259-93B.17 strain stimulated protective immune responses in fish at 10 weeks post-immunization. The results demonstrate that the rifampicin resistant 259-93B.17 strain may serve as an effective live attenuated vaccine for the prevention of F. psychrophilum infections.

Reproducible challenge model to investigate the virulence of Flavobacterium columnare genomovars in rainbow trout Oncorhynchus mykiss.

Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease and has significant economic impacts on aquaculture production worldwide. Molecular analyses have demonstrated that there is genetic diversity among F. columnare isolates. A review of the published literature that used restriction fragment length polymorphism analysis of the 16S rRNA gene revealed that all isolates typed from salmonids were Genomovar I. Our objective was to develop a laboratory challenge model for F. columnare in rainbow trout Oncorhynchus mykiss (Walbaum) and use the model to determine the virulence of Genomovar I and II isolates. Six F. columnare isolates were obtained from rainbow trout experiencing losses due to columnaris disease and were determined to be Genomovar I. Three of these were chosen for a preliminary assessment of virulence, and isolate 051-10-S5 was chosen for additional experiments to determine the reproducibility of the waterborne challenge model. In 2 independent experiments, cumulative percent mortalities (CPM) were 49 ± 10% and 50 ± 19%. Challenge of rainbow trout with Genomovar I and II isolates demonstrated a difference in the CPM, with the Genomovar II isolates inducing significantly higher CPM. This reproducible waterborne challenge model for columnaris disease in rainbow trout will be useful to investigate host-pathogen interactions, vaccine development, and other potential control strategies. This research also provides a basis for further defining the molecular diversity and virulence associated with F. columnare genomovars in rainbow trout and other salmonid species.

Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media

Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease, but the pathogenic mechanisms of this important fish pathogen are not fully understood. Identifying bacterial genes of F. psychrophilum differentially expressed in vivo may lead to a better understanding of pathogenesis and provide targets for vaccine development. Therefore, the present study used a proteomic approach to identify and quantify proteins of F. psychrophilum following growth in vivo and under iron-limited growth conditions. As determined by 2D polyacrylamide gel electrophoresis (2D-PAGE), numerous proteins exhibited different spot intensities following culture of the bacterium in vivo, and of these, 20 were selected and identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis and Mascot searches of the F. psychrophilum genome. Eighteen proteins exhibited increased spot intensities in vivo, and these included: several chaperone and stress proteins, gliding motility protein GldN, outer membrane protein OmpH, 2 probable outer membrane proteins (OmpA family), probable aminopeptidase precursor, probable lipoprotein precursor, 3-oxoacyl-[acyl-carrier-protein]-reductase, and several proteins with unknown function. Two proteins exhibited decreased spot intensities in vivo and were identified as ferritin FtnA and outer membrane protein OmpA (P60). Culture of F. psychrophilum in iron-limited media resulted in similar protein spot intensity changes for 6 of the 20 proteins identified following growth in vivo. Results from the present study suggest a role of upregulated proteins in the pathogenesis of F. psychrophilum and these may represent potential vaccine candidate antigens.

Spleen Index and Mannose-Binding Lectin Levels in Four Channel Catfish Families Exhibiting Different Susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.

Development of a culture independent method to characterize the chemotactic response of Flavobacterium columnare to fish mucus.

Flavobacterium columnare, the causative agent of columnaris disease, is a significant pathogen of many freshwater fish species worldwide and is considered one of the most important pathogens impacting the channel catfish (Ictalurus punctatus Rafinesque) industry in the United States. Recent research has demonstrated that F. columnare exhibits a chemotactic response to mucus from the skin of catfish and this response may be important for pathogenesis. In this study, a culture independent method was developed to quantify the chemotactic response of F. columnare to skin mucus. The method employs the use of blind-well chemotaxis chambers which overcomes difficulties using the traditional capillary tube assay and uses a cell proliferation assay to quantify viable cells which reduces the time and labor associated with culturing the bacterium. Application of the method to two sets of catfish skin mucus samples demonstrated that there is variation in the chemotactic response of F. columnare to individual catfish mucus samples, and similar results were obtained to previously published research using the traditional capillary tube method. The use of this method will enhance the ability to further characterize the chemotactic response of F. columnare to catfish skin mucus in order to increase the understanding of this important host-pathogen interaction.

Lack of association between Flavobacterium columnare genomovar and virulence in hybrid tilapia Oreochromis niloticus (L.) × Oreochromis aureus (Steindachner)

Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex-reversed hybrid tilapia, Oreochromis niloticus (L.)×O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established genomovars of F. columnare: I, II, II-B, III and I/II. The mean per cent mortality of fish challenged with genomovar I, II and III isolates ranged from 0 to 100, 3.3-78 and 3.3-75%, respectively. The mean per cent mortality of fish challenged with genomovar II-B ranged from 35 to 96.7%, and the only genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.

Complete Genome Sequence of Flavobacterium psychrophilum Strain CSF259-93, Used To Select Rainbow Trout for Increased Genetic Resistance against Bacterial Cold Water Disease

ABSTRACT 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.

Identification of immunogenic proteins within distinct molecular mass fractions of Flavobacterium psychrophilum

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.

Enhanced efficacy of an attenuated Flavobacterium psychrophilum strain cultured under iron-limited conditions

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.

Growth and survival of the fish pathogenic bacterium, Flavobacterium columnare, in tilapia mucus and porcine gastric mucin

Flavobacterium columnare, an economically important Gram-negative bacterium of freshwater farmed fish, colonizes the skin and gills in the initial steps of pathogenesis. The surface of fish is coated with mucus made up of high molecular weight glycoproteins. Limited studies have described the ability of bacterial pathogens to grow in fish mucus. Our objective was to determine if F. columnare isolates could grow and survive in formulated water (FW) containing autoclaved tilapia mucus or porcine gastric mucin. We demonstrated the ability of F. columnare genomovars I, II, II-B and III to replicate (2-3 logs) and survive (21 to >100 days) in FW containing tilapia mucus. In a second experiment, genomovar I and II isolates were found to replicate in FW containing tilapia mucus or porcine mucin but not in FW only. From a practical standpoint, fish handling and/or hauling results in stress that leads to mucus sloughing often with subsequent F. columnare infection. Flavobacterium columnare utilizes fish mucus as a nutrient source, and studies are underway to determine if growth in mucus or mucin results in differential protein expression and/or increased virulence of F. columnare towards fish.