Analysis On Phylogenetic Group Of Avian Pathogenic Escherichia Coli From Ducks In Eastern China And Characterizations Of Its Virulence-Associated Genes

Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) manifested as pericarditis, parahepatitis, peritonitis, septicemia and so on, meningitis are continuously increasing in present. For the various serotypes of APEC—176 O antigens,103 K antigens, 83 H antigens, and antibotic resistance of bacteria, the difference of areas and species, it was difficult to eradicated for animal husbandry to serious harm, and was also a zoonotic pathogens. The mechanisms involved in the pathogenesis were not completely understood. In this study, PCR, multiplex PCR and dot blot were used to investigate phylogenetic group and the virulence-associated genes (VAGs) of the APEC isolates from ducks in eastern China, and research of sequence analysis, deletion of invasion-associated gene ibeA of the isolate DE205B were also made.During the period of 2005—2007,501 isolates were isolated from clinical samples in dead/illed ducks with the suspected E. coli infection, which collected from different regions of Jiangsu, Anhui, Shandong etc. in eastern China. Biochemical experiments such as sugar fermentation, urease, IMViC and TSI agar culture, and chromogenic medium verified that 480 isolates were determined as E. coli. These showed escherichia infection existed widely in ducks in eastern China, moreover, APEC from ducks have become increasingly complex, the most frequent clinical symptoms were pericarditis, parahepatitis, peritonitis and arthritis etc., especially the percentages of isolates with neural symptoms were 74.17% (356/480); according to the isolated parts, the percentages of brain isolates were 32.08% (154/480) heart isolates 23.96% (115/480), liver isolates 20.21% (97/480), spleen isolates 0.83% (52/480), air sac isolates 4.79% (23/480) and kidney isolates(2.50%,12/480), some isolates also can be separated from other parts, all these have also great related with regions, species, hosts and so on. To determine the genetic origin and molecular characterizations, one simple and rapid phylogenetic grouping technique based on triplex PCR which used a comination of chuA and yiaA 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(56.25%), group B2 (19.17%), group D (16.46%), group B1 (8.13%) in 480 APEC from ducks isolated from eastern China, which have important reference value to the study on ecological distribution and genetic evolution of APEC in China.16 pairs of primers were designed and synthesized to detected the prevalence of 16 non-invasion associated virulence genes in 480 APEC isolates from ducks by PCR and multiplex PCR assays, namely afa/draB, iha, papC, tsh, mat, chuA,fyuA, irp2, iucD, iutA, iroN, iss, kpsMTII, traT, vat and malX. The results demonstrated that in addition to the kpsMTII was all negative, others have different degree distributions in APEC isolates from ducks, that is to say, iutA 85.83% (412/480), mat 83.13% (399/480), iss 83.13% (399/480), iroN 64.38% (309/480), iucD 63.96%(307/480), tsh 52.71% (253/480), traT 47.71% (229/480), irp2 32.50% (153/480),fyuA 27.92% (134/480), chuA 27.29% (132/480),iha 6.88% (33/480),vat 3.13% (15/480),papC 1.88% (9/480), malX 1.04% (5/480), afa/draB 0.21% (1/480); there are 89 different virulence gene distribution patterns found by comparing, which including 1 to 13 genes, and showed the distribution of VAGs of APEC isolates from ducks in eastern China was more complicated.To investigate the distribution of invasion-associated genes of 480 APEC isolated from ducks,11 pairs of primers were designed according to the published sequences of invasion-associated genes of E. coli, namely fimC, sfalfocCD, cvi/cva, neuC, ompA, cnf1/2, hlyA, gimB, ibeA, yijP and aslA. Then the distribution of 11 DNA fragments in these isolates were detected by PCR and mutiplex PCR assays. The results demonstrated that in addition to hlyA, cnf1/2 and sfalfocCD were all negative, others have different degree distributions in APEC isolates from ducks, that is to say, yijP 94.38% (453/480), ompA 91.25% (438/480), fimC 80.00% (384/480), cvi/cva 67.29% (323/480), aslA 41.25% (198/480), ibeA 3.96% (19/480), gimB and neuC both 1.67% (8/480); there are 30 different invsion-associated gene distribution patterns found by comparing, which including 1 to 7 genes, one isolate has not any. The results showed the distribution of invasion-associated genes of APEC isolates from ducks in eastern China was rather complicated.The method of dot blot was developed to detect largely the VAGs of avian colibacillosis. The DNA of the conservative fragment 342bp of ibeA as the template was amplified by PCR, sequenced and labeled with digoxigenin-dUTP as a probe by using random primers. Bacteria samples lysed directly on nylon membrane positively charged were hybridized and detected by dot blot hybridization with CSPD chemiluminescence. Specificity tests showed that only the nucleic acid of target gene was positive. Sensitivity tests showed that the minimum detective limit was 1.0×103 CFU of E. coli/ml on the membrane. The 19 isolates of 480 APEC isolates from ducks were positive by PCR, then by the DIG-labeled probe for three times, the positive rate was both 3.96%. It suggested that the DIG-labeled probe was so specific, sensitive and repeatable, that may be applied to the initial screening for a large number of clinical samples or detection directly of bacteria, especially detection of APEC infection without obviously clinical symptoms.On the basis of same classification in the main phylogenetic groups and similar VAGs compared with Newborn meningitis-causing E.coli(NMEC) isolate RS218, the isolate DE205B of APEC from ducks was detected about haemolytic reaction, pathogenicity experiment and infection in swine umbilical microvascular endothelial cells (SUMECs) in vitro. The results showed beta haemolytic activity on 5% sheep blood agar plates. Study of the pathogenicity experiment showed there were the common symptoms of avian colibacillosis, the LD50 to the mice and ducks were respectively 3.97 × 106 and 1.05 × 107 CFU/ml. DE205B could carried out the productive replication in SUMECs:a ladder band of small molecular weight fragments of DNA in DE205B-infected cells were observed in agarose gel electrophoresis; under the light microscopic level, the morphological changes of DE205B-infected cells occurred, including cells shrinkage, surface microvilli disappear, cytoplasm vacuolization, chromatin margination and nuclear condensation; and apoptosis of DE205B-infected cells can be found by flow cytometry; the invasive rate was 3.36%.The invasion-associated gene ibeA of APEC was cloned and analysized to seek the relationship between its variation and evolution. According to the published nucleotide sequences of the ibeA gene in NMEC RS218, one pairs of primer was designed and synthesized. The complete nucleotide sequence of ibeA was amplified from the genomic DNA of the isolate DE205B of APEC from ducks by PCR, then the recombinant plasmid verified by restriction enzyme analysis was sequenced. The results revealed that the gene ibeA’s region in coding was 1371bp, protein encoded by ibeA was 456 amino acides and molecular weight was 49544.33dal. Sequence comparision with other published the ibeA genes revealed that the homologies of nucleotide acids were higher (over 98.9%), genetic evolution analysis showed that there were very close relationships with NMEC.By electroporation pKD46 plasmid was transformed into the APEC isolate DE205B, and amplified the kanamycin resistance gene fragments whose flanks were homology with the target gene ibeA by PCR, transferred them into DE205B/pKD46 strains by electroporation methods. Under the help of reorganization function of Red system, the ibeA gene in DE205B genome was replaced. Moreover, plasmid pCP20 which coding FLP site-specific recombinase, was transferred into the positive transformants screened to remove the resistance gene by the FRT sites flanked. The study of the mainly biological characterizations of recombinant bacteria indicated that the capacity growth of the ibeA-mutant strain was slightly slower than that of the wild strain, the difference was not obvious in the first 6h, obvious increase after 7h; beta haemolytic activity on 5% sheep blood agar plates; the capacity of invasion in SUMECs decreased, the invasive rate decreased to 0.57%; and its virulence in mice decreased significantly, the LD50 was over 108CFU/ml. The E. coli whose invasion-associated gene ibeA was completely knocked out but without resistance gene, had been acquired by Red system, provided basis for further study of E. coli attenuated live vaccine.