Molecular epidemiology, diagnosis, pathogenesis, and control of Salmonella enterica serovar enteritidis infection in poultry.

摘要

The research on Enteritidis in this thesis involved three aspects: molecular epidemiology, serological diagnosis, and pathogenesis. In order to understand the molecular epidemiology of Enteritidis, a repetitive sequence based PCR technique was used. A novel repetitive sequence element, Salmonella Enteritidis repeat element (SERE) was identified in the Enteritidis genome. A SERE based PCR was standardized to fingerprint Enteritidis isolates of diverse origin representing various phage types. SERE-PCR identified 5 distinct but closely related SERE-PCR patterns among 54 Enteritidis isolates. In addition, 34 Enteritidis isolates of phage type 4 were grouped into 4 distinct closely related SERE-PCR types. Moreover, analysis of 54 strains of other Salmonella serovars including 12 O-serogroup D serovars revealed a unique fingerprint pattern for most of the strains. These results suggest that SERE-PCR may be utilized to develop species and serogroup-specific fingerprint patterns for isolates of Salmonella serovars.; A latex agglutination test (LAT), an enzyme linked immunosorbent assay (ELISA), and a rapid strip immunoblot assay (RSIA) were developed using a recombinant SEF14 fimbrial antigen of Enteritidis to specifically identify Enteritidis infected chickens. rSEF14 reacted only with serum from Enteritidis infected chickens and no positive reactions were observed with serum obtained from birds infected with several Salmonella and other avian pathogens suggesting that rSEF14 antigen is specific for Enteritidis. The rSEF14-ELISA and rSEF14-RSIA identified antibodies in serum from more than 80% of experimentally infected birds during first two weeks of infection and 100% of the birds subsequently. In addition, Enteritidis specific antibodies were also detected in egg yolks of infected hens as early as 6 days post-infection using rSEF14-ELISA and rSEF14 RSIA. The results of our investigation suggest that while rSEF14-LAT is simple, specific and rapid, it is less sensitive than ELISA and RSIA, and that these assays can be valuable tools for serological monitoring of Enteritidis infection in the field.; The role of SEF14, SEF17, and SEF21 fimbrial antigens in Enteritidis pathogenesis was evaluated. Stable, single, defined, sefA (SEF14), agfA (SEF17) and fimA (SEF21) insertionally inactivated fimbrial gene mutants of Enteritidis were constructed through homologous recombination of DNA material between wild type fimbrial genes and 5′, 3′ truncated fimbrial genes in the suicide plasmid vector pKNOCK-Km. In vitro studies using human enterocyte cell lines and chickens macrophage cell lines did not indicate a major role for these fimbrial antigens in modulating either the invasion of enterocytes or uptake by the macrophages. Similarly, in vivo studies in chickens also revealed no major differences in the ability of these mutant strains to colonize chicken ceca or to invade liver and spleen. These results suggest that SEF14, SEF17, and SEF21 fimbriae may not play a major role in Enteritidis pathogenesis.; The recombinant SEF14, SEF17, and SEF21 antigens were evaluated for protection of chickens against Enteritidis colonization of various tissues. No apparent differences were observed in recovery of Enteritidis from cecum, liver, or spleen of chickens vaccinated with different fimbrial antigens. These results supported our earlier data that the SEF14, SEF17, and SEF21 fimbriae may not play a major role in Enteritidis, pathogenesis.