Molecular Epidemiological Analysis Of Major Virulence Factors Of Avian Escherichia Coli And Preparation Of Monoclonal Antibodies Against Avian E.Coli’s F1and P Pili

Avian colibacillosis is a common bacterial communicable disease caused by some certain serotypes of Escherichia coli(E. coli). Since the first report in1894, Avian colibacillosis had a world-wide spread, with the prevalence of infection and secondary infection, which has brought massive losses to the world poultry industry. In recent years, the existence of various pathogenicity islands(PAI) in many pathogens has been found, which is possible to be associated with the evolution of pathogenicity, making the pathogenicity of E. coli more complicated. According to this situation, it is very necessary for a better pathogen detection and analysis of chicken colibacillosis. The pili is a significant virulence factor of E. coli, thus the establishment of a fast and specific diagnosis method and the preparation of its monoclonal antibodies will be helpful to characteristic analysis of Avian colibacillosis.Eight sets of primers to detect F1pili,P pili,locus of enterocyte effacement(LEE),E. coli type three secretion system2(ETT2) and high-pathogenicity island (HPI) were synthesized referencing from the published work. A total of41samples were obtained from chickens from the north region of Jiangsu province and tested by the PCR methods,and four cases(9.76%)were LEE positive, twenty—two cases(53.7%) detected HPI positive; ten cases (24.4%) were Pap positive,twenty cases (48.8%) were F1positive,seventeen cases (41.5%) detected ETT2positive, after comparison,14different virulence gene types were found out of41cases of clinical samples.And different types of E. coli with similar epidemiological in different organs, in the lungs and duodenum, HPI+, ETT2+, Pap+, F1carrying rate significantly higher than that of the liver. After the primers were tested for their specificities by using reference E. coli strains, all the isolates were submitted to detection, and the data showed that56isolates (40.88%) were F1+,10isolates (7.3%) were Pap+,2isolates(1.5%) were LEE+,35isolates (25.55%) were ETT2+and34isolates (24.82%) were and identified that isolates have11different virulence factor genetypes.In order to establish a fast and specific diagnosis method of F1and P fimbriae,two sets of primers specific to PilA and papA were designed and synthetized according to the published work.Following the gene PilA and papA were amplified from isolate strains CLul223-3and CLu1220-6in avian Escherichia coli of study I by PCR.Two gene sequences were subcloned into expression vector pGEX-6p-I respectively to obtain two recombinant strains of pGEX-PilA and pGEX-papA. The sequencing results showed that the nucleotide homologies were97%and100%compared with type F1and P fimbriae from GenBank. The recombinant plasmids were transformed into E.coli BL21(DE3) and induced by IPTG. The result of SDS-PAGE analysis showed that PilA and papA antigen genes were successfully expressed in E.coli BL21(DE3). The molecular weights were44Ku and45Ku (named GST-PilA and GST-papA). The recombination proteins GST-PilA and GST-papA were expressed with the form of inclusion body. The Western-blot analysis showed that two kinds of protein could bind specifically with mouse Anti-GST Tag Monoclonal Antibody.Then the spleen cells of immunized mice were fused with SP2/0myeloma cells by a routine method,and the specific antibody was detected by indirect-ELISA method.The hybridoma cells strain D7,E6and F9,which could stably secrete monoclonal antibody. The antibody belongs to IgG1,IgG1and IgG2b subtype after subclass identification of the heavy chain while the light chain is Kappa type.All of them with ELISA titers of1:64000,1:128000and1:64000for ascites respectively. The result of Western-blot revealed that the three monoclonal antibody was detected and only reacted with a Fimbriae protein band specifically, and have no cross reactivity each other. Further more, direct agglutination test by using the monoclonal antibodies was performed to detect isolate strains in study Ⅰ, and made a basis for further setting up a fimbria-testing method which had high specificity and sensibility,moreover could be operated easily and quickly.