Detection And Analysis On The Pathogenicity Islands ETT2among Escherichia Coli

Pathogenic Escherichia coli (E. coli) are important agents responsible for economic losses both in humans and animals. The variety of newly discovered pathogenicity island (PAI) of E. coli virulence makes the problem more complex. E. coli type three secretion system2(ETT2) is an important pathogen in piglet colibacillosis, which size is about29.9kb. Study abroad is beinge on the ETT2.There is a dearth of recent information on the prevalence of ETT2pathogenicity island. In pursuit of these goals, E. coli isolates were obtained from weaned piglets and bovine mastitis from different regions of Jiangsu Province, PCR methods were established by using two pair of primers that derived from the published nucleotide sequences of ECs3703and ECs3737genome, deposited in GenBank, and the expected products were detected. These isolates were identified by the PCR. Epidemiology of ETT2was identified by this method. The results show105feces samples were ETT2-producing E.coli,including13strains from diarrheic piglets (32.50%),35strains from edematous piglets (92.11%),11strains from edematous and diarrheic piglets (91.67%) and46strains from Bovine Mastitis (38.98%).To determine the relationship between ETT2locus and other virulence factors of E. coli, the ETT2-positive samples were submitted for bacteriological isolation and culture、biochemical identification and PCR. The results show that the ETT2locus was identified not only in ETEC but also in various STEC. Furthermore, the data also revealed that the presence of ETT2among STEC and STEC/ETEC were higher than that of ETEC. Among the22ETEC isolates,17(77.27%) were ETT2-harboring E. coli, while40(86.96%) and20(83.33%) were ETT2-positive among the46STEC and24STEC/ETEC isolates respectively. But37.40%(46/123) in Bovine Mastitis isolates. Among the46ETT2mastitis-causing isolates, the presence of ST2, LT1, HPI, CNF2, F17a, Tra and Hly were67.39%(31/46),39.13%(18/46),36.96%(17/46),52.17%(24/46),4.35%(2/46),45.65%(21/46) and19.57%(9/46) respectively.To investigate the types of ETT2locus among the pathogenic Escherichia coli, Thirty-five sets primers used for PCR amplification were designed and analyzed with DNAStar software based on all the published ECs3703-ECs3737genes of ETT2locus deposited in GenBank. Among the123bacteria were detected by the PCR methods,11types ETT2were found in this research, including an entire type (designated as type A) and10deletion types ETT2(named as type B, C, D, E, F, G, H, I, J and K). There are5types (A, B, E, F and G) of ETT2locus prevails in the pathogenic E. coli isolates from pigs. Among the17ETT2islands in ETEC isolates,3(17.65%) were the complete ETT2form (type A),1(5.88%) were type B,9(52.94%) were type E,2(11.76%) were type F and2(11.76%) were type G. Among the20ETT2islands in STEC/ETEC isolates,3(15.00%) were type A,9(45.00%) were type B and8(40.00%) were type E. But among the40ETT2islands in STEC isolates, just only two types were found, including33(82.50%) were type A and7(17.50%) were type E. Further analysis revealed that the type were high associated with the presence of shigatoxin type2e (Stx2e). In contrast to those from pigs, there are8types (B, C, D, E, H, I, J and K) ETT2locus were found in the pathogenic E. coli isolates from cows, and6types (C, D, H, I, J and K) were new member of the family which were not present in the bacteria from pigs. There is no single isolates from cows carried type A, F and G ETT2islands.To determine the contribution of ETT2pathogenicity island to the virulence of E. coli, to assess their disease risk in animals, six isolates strains with varying degrees deletion of ETT2pathogenicity islands and one with complete ETT2genome were chosen to challenge mice. These strains were picked up to determine the50%lethal doses to evaluate their pathogenicity. The results show that the LD50of type B isolate is5.335×105(0.2mL), the LD50of type A,H and J isolate is9.486×107(0.2mL), the LD50of type D isolate is5.335×108(0.2mL), the LD50of type I isolate is9.486×108(0.2mL), the LD50of type K isolate is1.687×109(0.2mL).