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.

An experimental vaccine against Aeromonas hydrophila can induce protection in rainbow trout, Oncorhynchus mykiss (Walbaum).

A candidate vaccine against Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss, was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the intraperitoneal (IP) cavity and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a dose response that could be used to establish a standard challenge having an LD(50) of approximately 3 x 10(7) colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re-challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper-immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freund's complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.

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.