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Primary Study on the Pathogenicity and Outer Membrane Protein of Escherichia Coli O157 Isolated from Domestic Animals in Chongqing

Author: ShuaiLing
Tutor: WangHaoJu
School: Southwestern University
Course: Preventive Veterinary Medicine
Keywords: Escherichia coli O157 Pathogenicity PCR Omp SDS-PAGE Western blot(Wb)
CLC: R378.11
Type: Master's thesis
Year: 2008
Downloads: 113
Quote: 1
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Abstract

Infectious diarrhea has become a kind of extremely severe enteric infectious which caused by Entero-hemorrhagic Escherichia coli(EHEC)O157:H7 in recent years.EHEC O157:H7 could cause infectious diarrhea、hemorrhagic enteritis、uremic syndrome hemolytic(HUS)and thrombotic thrombocytopenic purpura(TIP)and so on.EHEC O157:H7 has broken out and spread many times after isolated.It was reported that this bacteria caused HUS and death to human even in 2006 and 2007.It is also widespread in China,furthermore,it has the tendency to spread with multiple factor. Therefore our country has listed EHEC as the 12 kinds of pathogenic microorganism which have major impact to people heath.So it is urgent to enhance the research of diagnosis、prevention and treatment and the development of vaccine for the outbreak and spread and helplessness in treatment. Outer membrane protein(Omp)related to pathogeny and immunologic mechanism is an important virulence factor.It is shown that drop has a wide perspective for prevention and treatment.This research established the foundation to the diagnosis、prevention and treatment of Escherichia coli O157 with the initial investigation on the pathogenicity and heritage and outer membrane protein of Escherichia coli O157 strains isolated from domestic animals in Chongqing.1.the pathogenicity of E.coli O157 analysisObjective To acquire and compare with the pathogenicity of E.coli O157 isolated from domestic animals in Chongqing.Methods Mouse were infected with 11 E.coli O157 strains isolated from nurseries of 6 area in Chongqing.Pathological sections prepared with mouse liver、spleen、kidney、intestine were observed with light microscope.Result All strains were susceptible and fatal to mouse,and caused tissue affections such as thinningz and hyperemia of intestinal wall、necrosis and exfoliation of intestinal mucosa、congestion and necrosis of liver and kidney、splenorrhagia、congestion of lung,and so on.Conclusion All strains isolated from Chongqing had the ability to damage mouse organs and cause mouse death,and the pathological changes had fairly unity although some difference in content.2.Detection of stx2/stx2c virulence genes of E.coli O157Objective To identify stx2/stx2c virulence genes whether exist in E.coli O157.Methods Polymerase chain reaction(PCR)was apply to amplify the stx2/stx2c B subunit,the gene was cloned into plasmid pMD18-T Vector and sequenced.The nucleotide sequence compared with other sequences in NCBI.Result Only the RY8 strain isloated from Rongchang/Yongchuan had the amplification.Nucleotide sequence analysis showed that the homology between cloned gene and other similar genes(stx2,stx2c,stx2d,stx2e,stx2f B subunit gene)were 99.3%、94.8%、86.3%、84.5%and 80%.Conclusion It was the first time we had examined the E.coli strain which presented stx2/stx2c B subunit,so it is necessary to enhance the detection of E.coli O157 in Chongqing.3 Omp of E.coli O157 by SDS-PAGE analysisObjective To acquire the genetic correlation of E.coli O157 isolated strains and Omp immunogenicity.Methods Omp was extracted by supersonic quassation、SLS and ultracen-trifugation,and typed by Omp typing.Antiserum was prepared by immuning rabbits with the major Omp,the Omp immunogenicity was analyzed by western blot.Result Isolated strains were typed for three Omps by SDS-PAGE:YB2 strain is Omp-A;YB1、YB3、YB4、YB5、YB6、YB8、YB9、YB10、YB11 strains are Omp-B;YY7 strain is Omp-C.All Omp of three Omps represent strains could generate antibody to be integrated with them,and there is the common antigenic fragments between Sakai type strain and isolated strains.Conclusion E.coli O157 strains isolated from Chongqing had single transmissibility,but appeared little differentiation.The major Omp have immunogenicity in strains,and there are some common immunogenic protein fragments between Sakai type strain and isolated strains.

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