Flavobacterium columnare is a bacterial pathogen for many freshwater fish species. It is responsible for outbreaks in fish farms worldwide, causing high mortality rates. Fish vaccination is a potential approach for prevention and control of disease, with oral vaccines suitable for fish because of their easier application, low cost and minimum stress to fish. Alginate microparticles have been widely used as controlled release systems, including for fish vaccination. The aim of this study was to evaluate the capacity of oral and parenteral vaccines against F. columnare to induce a humoral response, as well as the in vivo efficiency in Nile tilapia fingerlings. The fingerlings were immunized with bacterin by intraperitoneal (i.p.), intramuscular (i.m.), oral and immersion routes, as well as orally with alginate microparticles containing formalin-killed bacteria. A sandwich ELISA was developed to detect specific antibodies against F. columnare. The animals were challenged with pathogenic strain BZ-1 to determine the relative percentage of survival. A significant humoral response was induced by bacterin administered by i.p. and i.m. routes (P \ 0.05). However, none of the vaccine preparations were effective in protecting fish against F. columnare infection (P \ 0.05). In spite of high antibody levels, there was no relation between immunoglobulin titers and resistance to columnaris for Nile tilapia fingerlings. These data suggest that use of serological analysis as the only method to determine vaccine efficiency against F. columnare infection in Nile tilapia can lead to imprecise results for the usefulness of these products in vivo.

Plasmid and chromosomal genes encode determinants of virulence for (Perry & Fetherston 1997, Parkhill et al. 2001, Deng et al. 2002. A 102-kb, unstable chromosomal area (locus pgm) is essential for Y. pestis virulence (Fetherston et al. 1992, Hinnebusch et al. 1996, Buchrieser et al. 1998.Typical strains of Y. pestis harbour three plasmids: pPst (9.5kb), encoding a plasminogen activator protease (Pla) (Sodeinde & Goguen, 1988; pFra (90kb), encoding a capsular protein Fraction 1 (F1) with antiphagocytic activities (Du et al. 2002) and murine toxin (Ymt), required for survival in the flea (Hinnebusch et al. 2002); pYV (70kb), encoding the Yop virulon which comprises both the Yop effectors proteins and the proteins necessary for injecting them into host cells. The Yop virulon enables the bacteria to survive and multiply in the lymphoid tissues of the host (Cornelis 2002). Several insertion sequences (IS100, IS200, IS285) present in the three plasmids favour recombination events and genetic plasticity (Filippov et al. 1995, Parkhill et al. 2001, Deng et al. 2002.However, atypical strains lacking some plasmids have been found in several foci around the world. On the other hand, strains containing extra DNA bands or additional cryptic plasmids have also been found (Filippov et al. 1990, Chu et al. 1998. In vitro, Y. pestis genome is very plastic and several changes have been described: emergence of additional DNA-bands, increasing of plasmid molecular mass, and integration of plasmids into the bacterial chromosome with or without loss of functions (Zsigray et al. 1985, Protsenko et al. 1991. Furthermore, the typical plasmids may be eliminated spontaneously at a high frequency during storage in the laboratory or through successive subcultures (Protsenko et al. 1991).The study of the plasmid content of Y. pestis isolates from the plague foci of Northeast Brazil, stored in the laboratory for several decades, showed that some of them displayed an atypical plasmid profile characterized by the absence of some plasmids or by the presence of extra DNA bands (Leal et al. 1997a, Leal & Almeida 1999, Cavalcanti et al. 2002. The absence of plasmids and the emergence of extra-DNA bands in the Brazilian isolates could also have been produced during storage or handling in the laboratory.To observe possible alterations in plasmids of the cultures in vitro and the impact of the alterations to their pathogenicity, three low subcultured and highly pathogenic Y. pestis isolates were submitted to serial subcultures. Colonies selected after subcultures were analyzed for their plasmid content and the presence of some characteristic genes of each plasmid. LD 50 in mice of the parental strains and derived cultures displaying different plasmid contents were compared. MATERIALS AND METHODSBacteria and culture conditions -The study involved two Brazilian isolates: one old (P. Exu 340, originating from a finger bone marrow in a fatal human case in 1969), and the most recent Brazilian isolate (P. CE 882, originating from a hemoculture from a plagu...

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