| Pasteurella multocida (Pm), a kind of capsular gram-negative bacilli, can cause bovine, pig, rabbit, chicken and other poultry and livestock, even human beings to hemorrhagic septicemia or respiratory diseases as the main characteristics of the important zoonotic pathogens, which widely distributed in the world, resulting in mutual infection in the same or different species of animal. According to different bacterial capsular antigens, P. multocida can be divided into A, B, D, E and F five capsular serotypes, in which to cattle with pathogenicity mainly is the capsular serotype A, B or E. Ever popular in the cattle mainly is capsular serotype B, which main cause hemorrhagic septicemia. However, recently popular mainly is capsular serotype A, which main result in pulmonary infection. At present, bovine respiratory diseases have caused a great threat to the cattle industry. The diseases often break out in cattle after importing them7～10d, and the main characters are clinical symptoms of dyspnea, cough, nasal flow foam purulent fluid and diarrhea; incidence rate reaches as high as50%～100%, and death rate10%～50%. Since2008, it has been reported the case that isolated P. multocida serotype A from cattle, which has caused diseases happened in several provinces and cities in our country, resulting in great economic losses to the cattle industry.Bacterial virulence factors play an important role in pathogenicity and the survival and reproductive abilities in hosts. Parts of virulence factors have immunogenicity. Research to the virulence factors of this kind will provide important theoretical basis for the prevention and treatment of Pasteurellosis. This study selected7major virulence factors from many factors induced with immunity:outer membrane protein A (OmpA), outer membrane protein H (OmpH), filamentous hemagglutinin protein (FhaB), outer membrane lipoprotein E (PlpE), Heme acquisition system receptor (HasR), PM0979and PM0442. We take6strains of different virulence of bovine P. multocida capsular serotype A as the research object. This thesis analyzed the genes of virulence factors from the nucleotide sequence level and transcription level, attempted to explore the key gene causing the virulence differences of P. multocida. Main research contents and results are as follows:1. Cloning and sequence analysis of virulence-related genes of6strains of bovine P. multocida capsular serotype AIn this study, we take the genomic DNA of6strains bovine P. multocida serotype A (CQ1, CQ2, CQ3, CQ4, CQ5, CQ6) as templates, amplified7virulence genes by PCR (ompA, ompH, pfhB2, plpE, hasR, pm0979, pm0442), and then cloned into the pMD19-T vector. After sequenced, we analysis virulence genes among6strains of bovine P. multocida serotype A, making homology analysis and respectively constructing phylogenetic tree to determine the genetic evolutionary distance. The results show that the difference among these7virulence genes in6strains is little, and the nucleotide sequence and the deduced amino acid sequence homology are very high. The homology of nucleotide sequence was greater than94.7%, and the homology of amino acid sequence was greater than87.9%. Those genes in different strains of P. multocida are highly conservative. It suggests that there has little correlation among6strains of different pathogenicity with nucleotide sequences and its deduced amino acid sequences of those genes. There may have other more virulent genes involved in this process, or there may be due to expression abundance of genes and cause by different regulations.2. The method establishment of SYBR Green I real-time quantitative PCR of virulence-related genes of P. multocidaIn this work, we refer to the ompA, ompH, pfhB2, plpE, hasR, pmO979, pmO442and housekeeping gene16S rRNA sequence of P. multocida in the GenBank, choosing the conservative region of gene, based on the strict principles of primer designing, using the software Oligo7.0design specific primers for genes. By experimenting with temperature gradient and establishing standard curves, we optimize and determine the annealing temperature and reaction performance of virulence genes for QRT-PCR, including linear standard curves, high amplification efficiency and consistent repetitive responses. The results show that the optimal annealing temperature of gene ompA, ompH, pfhB2, plpE, hasR, pm0979and pm0442respectively were57℃,55.7℃,57℃,60.1℃,64.5℃,64.5℃,59℃and the housekeeping gene16S rRNA is55.7℃. All genes’amplification efficiency E are90.8%-97.2%and feasible degree R2are0.994～1.000, the vast majority in the range of from0.998to1.000, with good repeatability. That laid a good basis for follow-up study in the methodology. 3. The study of difference in virulence-related genes expression of two strains bovine P. multocida capsular serotype A in vivo and in vitroIn the research, we take two strains bovine P. multocida serotype A CQ2and CQ6as object, studied7virulence genes (ompA, ompH, pfhB2, plpE, hasR, pm0979and pm0442) in the main organs of mice (liver, lung, spleen and kidney) at transcription level and combined with the analysis of the expression level in vitro. The results show that:(1) the expression of most virulence genes of PmCQ2and PmCQ6make a reversal from the vitro into the vivo, suggested that the survival environment of P. multocida is different in vivo and in vitro causing the different regulation with expression of these genes. There is an exception that hasR gene expression of PmCQ6in liver was markedly higher than that of PmCQ2, implied that hasR gene may not important to virulence.(2) From a single gene in the distribution of organs expression, majority of genes of PmCQ2&PmCQ6in liver, spleen and kidney expressed largely, relatively less in lung. However, exception for pm0442&pm0979of PmCQ2, they are expressed abundantly in lung. P. multocida is mainly caused lung infection and diseases. Therefore, we speculated that those two genes may critical to the virulence.(3) The expression of pm0442displays more prominent than any other gene. Whether it is PmCQ2or PmCQ6, the transcription level of pm0442much lower than ompA and ompH in vitro, but in vivo the situation is just at the opposite. That suggests pm0442may play a key role in virulence in vivo. This work laid a foundation for in-depth study of virulence genes of P. multocida. |
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