Epidemiological Investigation On LEE Pathogenicity Island Of E.coli Strains And A Multiplex PCR For Detection Of Rabbit Diarrhea Disease

Along with the development of rabbit industry, the rabbit diarrhea disease was also serious increasingly, especially young rabbit precede-weaned or post- weaned had a high death rate, leading to a heavy economy lose, thus was seriously restricting the development of rabbit industry. This research was based on the isolation and identification of E.coli from young diarrhea rabbit of 20-80 day age, and then a Molecular Epidemiological Investigation on LEE Pathogenicity Island of E.coli Strains was made in order to investigate the prevalence of EPEC strains with LEE pathogenicity island PAI,which provided theory basis for studying falling ill mechanism and the control measure of this disease. At the same time a multiplex PCR method was studied on the diagnosis of E.coli and clostridium perfrigens at the same time, which provide a quick and accurately diagnosis for rabbit diarrhea. This research consists of four parts.PartⅠInvestigation of young rabbit Escherichia coli disease This study made an investigation on the Escherichia coli of young rabbit around ablactation, and special emphasis was layed on the etiology. The major content included epidemiology, clinical manifestation, pathology morphology, pathogeny form and physics and chemistry characteristic and drug sensitivity test etc. From there some law and characteristics was found, and filled in the content about the rabbit Escherichia coli disease. This research had a guidance meaning to effectively diagnose and to prevent and cure the young rabbit Escherichia coli disease.PartⅡMolecular Epidemiological Investigation on LEE Pathogenicity Island of E.coli Strains Isolated from Diarrheic rabbits. In order to investigate the prevalence of EPEC strains with LEE pathogenicity island PAI positive strains, the virulence gene eaeA on LEE PAI were detected by PCR in E.coli isolated from diarrheic and healthy rabbits in Weifang, Qufu, Hengtai, Yantai, Laiwu, Tai`an in Shandong province. The results showed that the positive rate of eaeA in different diarrhea samples collected in 20-30 day age, 30-55 day age, 55-80 day age were 00/5, 72.7%, 23.5% , respectively. And no eaeA gene could be checked in isolates from healthy rabbit of the same day age. It was concluded that the EPEC possess the eaeA gene were a main pathogen of diarrhea in rabbit farms in Shandong province and the detection of LEE PAI should be a main method for EPEC molecular diagnosis and epidemiological investigation.PartⅢ A Multiplex PCR for detection of rabbit diarrhea diseaseA multiplex polymerase chain reaction was optimized to simultaneously detect two toxinal gene of REPEC and clostridium perfringen in this research. Four sets of specific primers were designed according to the sequences of eae and α-toxin gene in the gene bank. It was showed that all samples which contained eae and α-toxin could be amplified by the multiplex PCR using these two sets primers, yielding two specific bands of toxin gene of REPEC 833 bp and clostridium α-toxin 324 bp. But no specific band amplified from three other bacteria. As little as 10cfu of REPEC, 10cfu of clostridium perfringen were detected using electrophoresis in this multiplex PCR.