VIRULENCE FACTORS OF RIEMERELLA ANATIPESTIFER

Abstract

Riemerella anatipestifer is a gram-negative bacterium associated with epizootic infections mainly in domestic ducks, occurring as an acute or chronic exudative septicaemia. Twenty-one serotypes of R. anatipestifer have been identified. Compared to serotyping, molecular typing methods allow comparison of bacteria at the genotypic level. In this study, 35 R. anatipestifer isolates were characterized by repetitive sequence based-PCR (rep-PCR) with outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence by using either extracted genomic DNA or preparation of whole bacterial cells. The rep-PCR discriminated the 35 isolates into 19 electrophoretic types. DNA fingerprints obtained from genomic DNA or whole cells yielded comparable patterns. Substantial variation was seen among the rep-PCR fingerprints of different serotypes. Moreover, different polymorphisms of the rep-PCR fingerprints were evident among epidemiologically unrelated isolates of the same serotype. These results suggest the presence of repetitive extragenic palindromic-like elements within the genome of R. anatipestifer that can be used in some isolates to discriminate between different strains belonging to the same serotype. This approach may serve as a useful molecular tool for subtyping R. anatipestifer isolates for epidemiologic investigations. The whole cell procedure offers the advantage of ease of performance requiring only small quantities of cells. The ompA gene, encoding the 42kDa major outer membrane protein (OmpA) of R. anatipestifer and another partial gene p41, encoding a putative 41kDa protein (P4 l) showing certain identity with reovirus viral attachment protein sigma 1 are present in the type, serotype reference and field strains examined. Both genes showed strong antigenic characteristics and thus are potential vaccine candidates and diagnostic markers. The generation of protective immunity against R. anatipestifer in ducks was investigated by immunization with recombinant proteins and plasmid DNA constructs derived from ompA and p41 genes. Immunoblotting and ELISA results demonstrated that the purified recombinant proteins induced the production of humoral antibodies in immunized ducks. Neither recombinant proteins nor the corresponding DNA vaccines afforded protection against challenge with the virulent strain CVL34/90. In the development of the recombinant 4lkDa protein (rP41) based-ELISA to detect R. anatipestifer infection in ducks, the method successfully discriminated between ducks experimentally infected with different strains of R. anatipestifer and negative controls. The protein did not react with sera against many other gram­ negative bacteria in Western blot analysis. Furthermore, 25% of296 field sera samples analysed by this ELISA were positive. This indicates that the P4l may be a specific antigen to R. anatipestifer and a potential candidate antigen in ELISA for screening of R. anatipestifer infection.

Presently, no particular virnlence attributes at the molecular level have been well described for R. anatipestifer. Plasmids are frequently highly significant in bacterial virulence. The plasmid profile of 19 R. anatipestifer strains was reported in this study. Eighteen of them possess one or two plasmids. Of interest are the two plasmids isolated from the virulent strain CVL34/90. At least nine ORFs/coding regions have been identified on these plasmids, such as the ATP-binding protein homolog, virulence-associated protein, transposase, resolvase and bctA gene necessary for conjugal transfer in Bacteroides spp. It is conceivable these two plasmids may play an important role in the pathogenicity, replication or conjugation of R. anatipestifer.