Isolation and characterization of Flavobacterium columnare strains infecting fishes inhabiting the Laurentian Great Lakes basin.

Flavobacterium columnare, the aetiological agent of columnaris disease, causes significant losses in fish worldwide. In this study, the prevalence of F. columnare infection was assessed in representative Great Lakes fish species. Over 2000 wild, feral and hatchery-propagated salmonids, percids, centrarchids, esocids and cyprinids were examined for systemic F. columnare infections. Logistic regression analyses showed that the prevalence of F. columnare infection varied temporally and by the sex of the fish, whereby females had significantly higher prevalence of infection. A total of 305 isolates of F. columnare were recovered. Amplification of the near complete 16S rRNA gene from 34 representative isolates and subsequent restriction fragment length polymorphism analyses demonstrated that all belonged to F. columnare genomovar I. Phylogenetic analysis of near complete 16S rRNA gene sequences also placed the isolates in genomovar I, but revealed some intragenomovar heterogeneity. Together, these results suggest that F. columnare genomovar I is widespread in the Great Lakes Basin, where its presence may lead to mortality.

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.

Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment.

Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

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.

Comparative proteomic analysis of virulent and rifampicin-attenuated Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the aetiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study, we compared a wild-type strain (CSF 259-93) with a rifampicin-resistant strain and virulence-attenuated strain of F. psychrophilum (CSF 259-93B.17). The attenuated strain harboured a mutation in the rpoB gene consistent with resistance to rifampicin. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry demonstrated an altered proteome with eight proteins characteristic for the parent strain and six that were unique to the attenuated strain. Immunoblotting with a diagnostic monoclonal antibody (FL-43) identified a putative antigen (FP1493) that was subsequently cloned, expressed as a recombinant protein and confirmed as recognized by FL-43. 2D-PAGE, immunoblotting with rainbow trout, Oncorhynchus mykiss (Walbaum), convalescent antisera and mass spectrometry of bacterial whole-cell lysates revealed several uniquely expressed immunoreactive proteins including FP1493. An FP1493 recombinant subunit vaccine was tested, but did not provide protection against challenge with the CSF259-93 strain. While the exact mechanism responsible for altered protein synthesis and attenuation of CSF 259-93B.17 is still unknown, the differentially expressed immunoreactive proteins are a valuable resource to develop subunit vaccines and to identify proteins that are potentially involved in 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.

Characterization of susceptibility and carrier status of burbot, Lota lota (L.), to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida and Renibacterium salmoninarum.

In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species.

Protection against heterologous Streptococcus iniae isolates using a modified bacterin vaccine in Nile tilapia, Oreochromis niloticus (L.).

Streptococcus iniae is a significant pathogen impacting aquaculture production worldwide. The objectives of this study were to determine whether a developed modified S. iniae (ARS-98-60) bacterin vaccine is efficacious in Nile tilapia, Oreochromis niloticus (L.), against challenge with heterologous isolates from diverse geographical locations and to evaluate protein and antigenic variability among the isolates tested. Two groups of tilapia (approximately 5 g) were intraperitoneally (IP) vaccinated with 100 microL of the vaccine or sham vaccinated with 100 microL of sterile tryptic soy broth and held for 28 days. Fish were challenged with each isolate by IP injection of 2-3 x 10(7) CFU per fish using calcein to mark fish prior to cohabitation for challenge. The results demonstrated significant protection against all challenge isolates, and relative percent survivals ranged from 79% to 100%. SDS-PAGE analysis of whole-cell lysate proteins from the S. iniae isolates demonstrated similar protein profiles between 10 and 31 kDa and variation in profiles between 35 and 100 kDa. Western blot analysis using antiserum from vaccinated fish (ARS-98-60) demonstrated shared immunogenic proteins among all isolates in the molecular mass range of 22-35 kDa and high molecular mass material >150 kDa. The results suggest that the developed S. iniae vaccine has broad ranging protection among isolates exhibiting different protein profiles.

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.

Passive immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), against Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease and rainbow trout fry syndrome.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS), causes high mortality in cultured salmonids. The present study was designed to determine the role antibody plays in conferring protection to rainbow trout fry, Oncorhynchus mykiss (Walbaum), by passive immunization with convalescent serum or serum from adult rainbow trout immunized with F. psychrophilum, and goat anti-F. psychrophilum serum. In each experiment, rainbow trout fry were injected intraperitoneally with antiserum and challenged by subcutaneous injection with a virulent strain (CSF-259-93) of F. psychrophilum 24-h post-immunization. Relative percentage survival (RPS) ranged from 9-42% when rainbow trout fry (mean weight 1.3 g) were injected with a 1:2 dilution of 25 microL of convalescent serum ranging in enzyme-linked immunosorbent assay antibody titres from 1600-102400. Rainbow trout fry (mean weight 1.0 g) passively immunized with 25 microL of serum from immunized adult fish exhibited RPS values of up to 57%. In each of these experiments, RPS increased with increasing antibody titres against F. psychrophilum. Passive immunization with 25 or 50 microL goat anti-F. psychrophilum serum, however, did not confer protection to fry (mean weight 1.3 g). These results suggest that trout antibody plays a role in conferring protection to F. psychrophilum, but antibody alone is unable to provide complete protection.