Kenneth D. Cain

Antibody-antigen kinetics following immunization of rainbow trout (Oncorhynchus mykiss) with a T-cell dependent antigen.

Enhancement of the immune response through affinity maturation of the antibody response is a feature of the mammalian immune system and has important implications with respect to development of vaccination strategies. However, an absence of germinal centres and apparent lack of somatic hypermutation of immunoglobulin V genes suggests that this phenomenon does not occur in fish. We investigated the question of affinity maturation in rainbow trout (Oncorhynchus mykiss) by measuring antibody-antigen binding kinetics using a BIAcore biosensor. Following immunization with a T-cell dependent antigen (FITC-KLH), relative binding affinities of serum and mucosal antibodies were assessed based on their dissociation rate constants (k(diss.)). A detectable serum anti-FITC response developed by 4 weeks post-immunization, and a consistent shift to higher affinity antibody production (i.e. a decrease in k(diss.)) was observed over the ensuing course of the immune response. An average k(diss.) of 3.5 x 10(-4)+/-0.27 x 10(-4)sec(-1) was observed during early stages of the response (4 weeks), while by 6 weeks this decreased significantly (p<0.05). Further reduction in k(diss.) was observed, with a low of 1.2 x 10(-4)+/-0.06 x 10(-4)sec(-1) being observed by week 12. Analysis of the anti-FITC response in skin-derived mucus revealed a similar pattern of decreasing k(diss.) as the immune response progressed. While these data clearly demonstrate a 2-3 fold increase in antibody-antigen binding during the course of the immune response in trout, the magnitude of this increase is much less than that seen in the mammalian immune response. This may reflect differences in the mechanisms underpinning this phenomenon in divergent species.

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.

Vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), with recombinant and DNA vaccines produced to Flavobacterium psychrophilum heat shock proteins 60 and 70

Flavobacterium psychrophilum heat shock proteins (Hsp) 60 and 70 are highly immunogenic and were therefore investigated as potential vaccine candidates. Recombinant Hsps were purified from Escherichia coli and rainbow trout (Oncorhynchus mykiss) were intraperitoneally injected with phosphate buffered saline/Freunds complete adjuvant (FCA), 8 microg of rHsp60/FCA, rHsp70/FCA or a combination of 4 microg each of rHsp60 and rHsp70/FCA. Antibody responses against recombinant Hsp60 and Hsp70 8 weeks post-immunization were observed, but only fish immunized with rHsp70 exhibited highly elevated antibody levels against F. psychrophilum whole cell lysate. Some cross reactivity occurred, which may have been due to the V5 tag common to both proteins. Protection against F. psychrophilum challenge was not observed in any treatments at 8 weeks post-immunization. To further investigate any protective effect of these proteins, hsps were polymerase chain reaction amplified and cloned into pVAX1. Rainbow trout were intramuscularly injected with 8 microg of pVAX1hsp60, pVAX1hsp70 or a combination of 4 microg each of pVAX1hsp60 and pVAX1hsp70. Antibody responses at 4 weeks post-immunization were low and protection was not observed following challenge at 6 or 10 weeks post-immunization. Although Hsps of F. psychrophilum have been shown to be immunodominant, these antigens do not appear to be good vaccine candidates when delivered alone or in combination.

Combining Suppression Subtractive Hybridization and Microarrays To Map the Intraspecies Phylogeny of Flavobacterium psychrophilum

ABSTRACT Reciprocal subtractive libraries were prepared for two strains of Flavobacteriumpsychrophilum, one virulent and the other avirulent in a trout challenge model. Unique clones were sequenced and their distribution assessed among 34 strains. The analysis showed that F. psychrophilum is composed of two genetic lineages, possibly reflecting host specificity.

3 - Barrier function and immunology

Barrier and immunological functions of the gut in fish are discussed in this chapter. The gut is known to be an important site often targeted by invading pathogenic microbes, and constant exposure to aquatic microbes requires defense mechanisms to be present in the gut that act rapidly to limit infection. These include many physical, chemical, and cellular components that prevent infection by presenting a barrier to invasion; however, innate and adaptive immune responses can be initiated in the gastrointestinal tract as part of a mucosal immune response. The mucosal immune system in fish includes not only the gut but also mucosal tissues associated with the gills and skin. In mammals, this system is well developed and exposure to pathogens or immunization with antigens delivered to one mucosal site results in antigen-specific antibody production at other mucosal sites. Fish are capable of mounting similar responses via the gut and skin, but the mucosal system in fish functions in a more primitive manner than in mammals. Protective functions of the gut of fish involve inhibition of colonization by microbes at mucosal sites, rapid elimination of pathogens through innate responses, and development of adaptive immunity following antigen uptake and processing.

Genetic diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow trout, Oncorhynchus mykiss (Walbaum), and spawning coho salmon, O. kisutch (Walbaum)

Flavobacterium psychrophilum is the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. This study examined the genetic diversity of F. psychrophilum isolates retrieved from multiple epizootics at rainbow trout, Oncorhynchus mykiss, rearing facilities and from spawning coho salmon, O. kisutch. A total of 139 isolates were confirmed as F. psychrophilum by PCR assay and were further typed using pulsed-field gel electrophoresis (PFGE). Multiple epizootics at three proximally located rainbow trout rearing facilities were numerically dominated by three PFGE profiles, which accounted for 76% of all trout isolates. In coho salmon, 19 PFGE profiles were differentiated by PFGE and four numerically dominant PFGE profiles represented 56% of all coho salmon isolates. PFGE analysis also indicated that the average similarity of macrorestriction patterns of F. psychrophilum isolates was greater in rainbow trout than in coho salmon (88% vs. 70%). Furthermore, it was not unusual to isolate multiple PFGE profiles from a single coho salmon sample whereas only two PFGE profiles were shared between two sample dates separated by 1 month. It is clear that the domestic rainbow trout aquaculture facilities studied here were primarily affected by a complex of genetically related strains whereas spawning coho salmon supported a much more genetically diverse collection of F. psychrophilum.

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.

Evaluation of Egg Incubation Methods and Larval Feeding Regimes for North American Burbot

Abstract Incubation methods and larval feeding regimes were investigated for North American burbot Lota lota maculosa over 2 years. Three upwelling incubators were tested: 6.0-L McDonald-type jars, 2.0-L pelagic egg jars, and 1.2-L Imhoff cones. Larvae were allocated to five feeding regimes in year 1 (trial 1) and three feeding regimes in year 2 (trial 2). In trial 1, a live diet (marine rotifers Brachionus plicatilis and brine shrimp Artemia spp.) was administered from 11 d posthatch (dph) until introduction of a commercial diet at 21, 31, or 41 dph; the fourth treatment applied the commercial diet exclusively starting at 11 dph, and the fifth treatment used only the live diet. Trial 2 examined (1) exclusive use of live feed beginning at 16 dph; (2) use of live feed at 16–50 dph, which was combined with commercial feed at 31–50 dph, and use of only the commercial diet at 51–76 dph; and (3) use of the live diet at 16–50 dph, the addition of frozen brine shrimp at 31–50 dph, and use of the commercial diet ...

Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar)

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.

A Quantitative Enzyme-Linked Immunosorbent Assay and Filtration-Based Fluorescent Antibody Test as Potential Tools to Screen Broodstock for Infection with Flavobacterium psychrophilum

There is strong evidence that Flavobacterium psychrophilum, the etiologic agent of coldwater disease, is transmitted vertically; it has been hypothesized that disease management at hatchery facilities can be improved through broodstock screening and implementation of culling programs. This paper describes the development of two assays used to screen broodstock tissues (kidney and ovarian fluid) for the presence of F. psychrophilum. Four monoclonal antibodies (MAbs) were generated against outer membrane preparations of F. psychrophilum strain CSF (Clear Springs Foods) 259-93. Of these, MAb FL43 was selected for assay development; this MAb reacted with 67 isolates of F. psychrophilum but exhibited no reaction with two strains of F. columnare or single strains of F. pectinovorum, F. aquatile, F. branchiophilum, and F. saccharophilum. An enzyme-linked immunosorbent assay (ELISA) was developed using MAb FL43 as the capture antibody and MAb FL43 conjugated to horseradish peroxidase (enzyme number 1.11.1.7; IUBMB 1992) as the secondary detection antibody. The ELISA had a lower F. psychrophilum detection boundary of approximately 1.6 X 10(3) colony-forming units (CFU)/mL in kidney tissue homogenates spiked with known bacterial concentrations. Asymptomatic broodstock of coho salmon Oncorhynchus kisutch (n = 50 fish) were sampled, and 100% tested positive for infection by ELISA analysis of kidney tissue; bacterial load was estimated at 2.0 x 10(3) to 9.4 x 10(3) CFU/mL. Ovarian fluid was also collected from these same coho salmon as well as from broodstock of rainbow trout O. mykiss; however, the ELISA proved to be unsuitable for use with ovarian fluid. A filtration-based fluorescent antibody test (FAT) was subsequently developed by conjugating MAb FL43 to Alexa Fluor 488. This FAT was able to detect F. psychrophilum in 74% of ovarian fluid samples collected from coho salmon and 42% of ovarian fluid samples from rainbow trout. Interestingly, yellow-pigmented bacteria were isolated on culture plates from 100% of kidney and ovarian fluid samples. All yellow-pigmented colonies were tested by polymerase chain reaction, and 100% of the coho salmon and rainbow trout were confirmed positive for infection with F. psychrophilum.