Isolation And Identification Of Avian Pathogenic Escherichia Coli From Chicken And Characterizations Of Its Virulence-Associated Genes

Avian conlibacillosis caused by avian pathogenic Escherichia coli (APEC) manifested as pericarditis, parahepatitis, peritonitis, omphalitis and so on. Sometimes APEC caused acute septicemia also. The number of reports about APEC was continuously increasing in present. APEC had many serotypes. The distribution of different regions was very different. There were also significant differences in drug resistance of isolates in different areas. So, Isolation and identification of APEC, analysis of molecular epidemiological characteristic, detection of drug sensitivity had an important guiding significance for controlling avian conlibacillosis. In this study, APEC isolates were isolated and identified from a chicken farm on the Shanghai, and their major virulence-associated genes were detected. To find the pertinency between virulence-associated genes that APEC isolates carried and pathogenicity of APEC isolates, one simple and rapid phylogenetic grouping technique was adopted to analysis main phylogenetic groups in APEC isolates. This study offered some references for revealing the pathogenic mechanism of APEC.During the period of December 2009 to May 2010,80 Escherichia coli isolates were isolated from clinical samples in dead/illed chicken with the suspected Escherichia coli infection. Biochemical experiments such as MacConkey agar medium culture, eosin methylene blue agar medium culture, and using PCR by amplifying 16S rRNA gene of Escherichia coli were determined as Escherichia coli.52 APEC isolates of 80 Escherichia coli isolates were isolated from dead/illed chicken's tissue. The other 28 Escherichia coli isolates that were isolated from avian fecal and avian Intestinal contents are avian fecal commensal Escherichia coli (AFEC). In 52 APEC isolates,31 were isolated from hearts (38.75%),13 were isolated from kidneys (16.25%),7 were isolated from livers(8.75%), 1 were isolated from lungs (1.25%).5 pairs of primers were designed and synthesized to detect the prevalence of 5 virulence genes (iroN, ompT, hlyF, iss and iutA) in 80 Escherichia coli isolates from chicken by PCR assays. The results demonstrated that 59 isolates carried all of the five virulence genes, and 52 APEC isolates that were isolated from tissue on average possessed 4.7 of the 5 genes. AFEC isolates on average possessed 2.1 of the 5 genes.49 APEC isolates (49/52) and 10 AFEC isolates (10/28) possessed all of the 5 genes. In this study, The accuracy rate of distinguished between APEC and AFEC is 83.75%(67/80). On this basis, this study also established a detect method basing on multiplex PCR which used a combination of hlyF, iss, iutA and 16S rRNA genes to detect APEC.To determine the genetic origin and molecular characterization, one simple and rapid phylogenetic grouping technique based on PCR which used a combination of chuA and yjaA genes as well as an anonymous DNA fragment TspE4.C2, was adopted to analysis main phylogenetic groups in the APEC isolates. There were group A 25.00%, group B1 59.61%, group B2 5.77%, group D 9.62% in 52 APEC isolates, and group A 32.14%, group B1 53.57%, group B2 10.71%, group D 7.50% in 28 AFEC isolates, which have important reference value to the study on ecological distribution and genetic evolution of APEC.Another 11 pairs primers were designed and synthesized to detect the prevalence of 11 virulenece genes in 80 Escherichia coli isolates.4 genes (tsh, mat, iucD, traT) were non-invasion associated virulence genes, and 7 genes (fimC, cvi/cva, ompA, gimB, ibeA, yijP, aslA) were invasion associated virulence genes. The results demonstrated that in addition to the gimB and ibeA were all negative, others had different degree distributions in 80 Escherichia coli isolates. That is to say, tsh 21.15%(11/52), mat 100%(52/52), iucD 92.21%(28/52), traT 57.69%(30/52),fimC 98.08%(51/52), cvi/cva 25.00%(13/52), ompA 90.04%(47/52) of 52 APEC isolates, and tsh 17.86%(5/28), mat 96.43%(27/28), iucD 57.14%(16/28),traT46.42%(13/28),fimC25.00%(7/28), cvi/cva 17.86%(5/28), ompA 100%(28/28) of 28 AFEC isolates. By comparison, iucD gene and aslA gene had significant difference between APEC isolates and AFEC isolates.To determine the different drug sensitivity of Escherichia coli, sensitivity of 15 commonly used drugs for 80 Escherichia coli isolates were detected using disc diffusion method. The results was that the proliferation of Escherichia coli isolates over 50%were inhibited when used ceftriaxone, amikacin or levofloxacin, and ceftriaxone was the most effective one of 15 drugs. The result demonstrated that no significant differences between APEC and AFEC in drug sensitivity.This was a focus that the pertinence between distribution of virulence-genes in APEC isolates and pathogenicity of APEC isolates. In this study, The pathogenicity of the isolates A31 and A74 was compared to demonstrate the influence of different distribution of virulence-genes in them. The results was that the LD50 to the mice of isolates A31 and A74 were 6.46×108CFU/ml and 8.62×105CFU/ml, respectively. This result showed that isolate A74 had stronger pathogenicity than isolate A31. These will provide a theoretical basis for further study of the pathogenic mechanisms of APEC.