Isolation And Identification Of Avian Pathogenic Escherichia Coli And Construction Of Iucb Gene Deletion Mutant

E. coli is a commonly occurring zoontic pathogen. According to the common clinical classification, E.coli can be divided into three categories entitled as:intestinal pathogens, extraintestinal pathogenic E. coli (ExPEC) and commensal bacteria. In case of poultry, E. coli can be found as a commensal bacterium in the normal micro flora of the intestinal tract along with other host mucosal surfaces. Only few strains possessing specific virulence factors have been identified as avian pathogenic E. coli (APEC). APEC is one of the most common pathogen of poultry, causing respiratory tract or systemic infection leading to serious economic losses in China. With the improvement and the intensification of the poultry industry in China, Colibacillosis caused by avian pathogenic E.coli and/or its co-infection with other pathogens have been emerged as a major problem.In order to investigate and analyze epidemiology of chicken pathogenic E.coli,45isolates were isolated from poultry farms covering8areas surrounding Wuhan. The study put emphasis on the biology characteristics, drug resistance, and serotypes and determined the strain ACN003.Then delected virulence gene iucB of ACN003to construct mutant strain△iucB. The main contents of the study are as follows:1. Isolation and identification of chicken E. coli and detection of characteristic45strains were isolated from the chicken with colibacillosis. Bacterial morphology, cultural characteristics, biochemical characteristics and PCR were applied to identify the isolates as E. coli.The results of biochemical tests showed that all of the isolates belonged to E.coli.5pairs of primers were designed and synthesized to detect the prevalence of5virulence genes (tsh, iucD, papC, iss and irpl) in45isolates from chicken by PCR assays. The result showed that these were significantly distributed among the isolates; covering almost80%of the total isolates. There were64.44%of the isolates harboring iucD. The proportion of the irp2, tsh and papC was33.33%,28.88%and4.44%, respectively.From45strains, there were21strains identified with available serogroups. They were O78、02、04、O11、O20、018、038and0128. The main serogroup was078.To determine the genetic origin and moleculer characterization, one simple and rapid method based on PCR which used a combination of chuA and yjaA genes as well as an anonymous DNAfragment TspE4.C2was adopted to analysis main phylogenetic groups. There were36strains belonged to group Bl, accounting for80%(36/45) of the share of strains,8strains belonged to group D, accounting for17.78%(8/45),1strain belonged to group A, accounting for2.22%(1/45).Kirby-Bauer paper method was used to identify the drug resistance for the isolates with22kinds of commonly used test paper slip. The results of the test showed that the drug resistance of the isolates were becoming stronger,37.78%of the strains were resisted to more than15kinds of antibiotics. The isolates were highly sensitive to amikacin, neomycin, polymyxin B and nitrofurantoin.2. Construction of gene deletion strain ACN003△iucBXiaoGan (ACN003) strain was selected because it had the strongest virulence. Homologous recombination was applied to construct the iucB deletion strain (△iucB-A3). The virulence of mutant strain was detemined by mice infection experiment. The result showed that the virulence of the mutant strain was not significantly attenuated compared to the wild strain.