| Pullorum disease is an infectious disease in poultry which is caused by Salmonella enterica serovar Gallinarum biovar Pullorum (Salmonella Pullorum) mainly in young chickens within2-3weeks. Infections with Salmonella Pullorum usually caused very high morbidity and mortality, potentially approaching100%because of acute septicemia. Adult chickens infected by Salmonella Pullorum might be carried subclinically or lead to reduced egg production and hatchability plus a range of atypical signs.The pullorum disease could cause great economic losses to the poultry industry. The main sources of infection come from sick chicken and carriers because of its horizontal or vertical transmission. It is nessary to understand the function gene related to vertical transmission and find the novel targets for drugs or vaccines in contributing to prevention and treatment of this disease.Transposition mutagenesis is an effective method to investigate the function genes. After virulence genes related to pathogenicity of Salmonella Pullorum were inserted by a random transposon, the bacteria could not be isolated in the host because the function of these inserted genes would be lost to cause no ablity of persistent infection of Salmonella Pullorum.In this study, the model of chicken embryo infected by Salmonella Pullorum was established and12pUTmini-Tn5Km2(Cm)(tagF) vectors were used to construct of Salmonella Pullorum S06004transposon mutant libraries which were applied to negativly screen attenuated mutants. After the inserted genes of obtained mutants were identified, their phenotype, growth characteristics and virulence of these mutants were further determined. 1. Establish the model of chicken embryo infected by Salmonella PullorumTo screen the function genes related to virulence or vertical transmission, the model of chicken embryo infected by Salmonella Pullorum was established, the inoculation method, dose, age of chicken embryo and bacterial colonization of organs were studied. The result showed that16-day-old chicken embryos were inoculated by allantoic cavity with100CFU and200CFU as input pools. After chickens were hatched, bacteria isolated from their livers and spleens were taken as a resource of output pools in which mutants were negatively screened.2. Screening and identification of attenuated mutants in the model of chickenembryo infected by Salmonella PullorumTransposon which contains specific signal label tagF sequence and the antibiotic resistance gene (KmR and CmR) in pUTmini-Tn5Km2(Cm)(tagF) vector can randomly insert into Salmonella Pullorum genome.12mutant libraries containing1,200strains were constucted.15mutants were screened out in the model of embryos infected by Salmonella Pullorum. After the flanking sequences of transposon were amplified by ligation with specific adaptor, transposon-disrupted genes were identified by sequencing and comparison with the bacterial genome in NCBI. Inserted genes of11mutants had been identified including genes involved in invasion (uvrY::Tn5), multiplication (folA::Tn5, miaA::Tn5, ftsN::Tn5), metabolism (citC::Tn5, yabF::Tn5, rfbG::Tn5, untitled1::Tn5), defense (wza::Tn5) and unknown fuctions (untitled2::Tn5, untitled3::Tn5). The transposons were found to be located in virulence plasmid (1/11) gene and in non-SPI-encoded gene (10/11). The growth characteristics and biochemical characteristics of9mutants were determinated. The results indicated that the disruption of genes could not significantly influence the phenotypic characteristics and growth speed of mutants. The in vivo competition index indicated that mutants could not compete with wild type Salmonella Pullorum, which demonstrated that pathogenity of mutants are weak and could not cause disease. This study should contribute to a better understanding of the pathogenic mechanisms involved in progression of disease caused by Salmonella Pullorum, and identification of novel live vaccine candidates and new potential antibiotic targets. |
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