Detection Of The Yersinia High-pathogenicity Island And Relationship Between The Pathogenicity In The Avian Pathogenic Escherichia Coli Strains

Avian colibacillosis is a group of diseases collectively which is caused by some serotypes of E. coli. With the development of the domestic intensive poultry industry colibacillosis which has a significantly increased trend have became one of the most important poultry bacterial diseases. In order to investigate the distribution of avian pathogenic E. coli in Anhui Province (avian pathogenic Escherichia coli, APEC), detect the carry of High pathogenility island of Yersinia pestis in avian pathogenic Escherichia coli, and prove the relationship between the avian colibacillosis and the islands, all kides of methords have been established. In we have refered the related domestic and foreign research and used the microbiology, molecular biology, animal pathology methods.This experiment identified eight avian pathogenic Escherichia coli and detect all genes located on Yersinia HPI among avian pathogenic E. coli of Anhui Province by PCR according to the the Yersinia HPI gene sequence published in Genbank and the 12 pairs of specific primers.In addition, this experiment test LD50 of the strains which have been detected HPI positive with the purpose of explore the relationship between HPI carrying and the avian pathogenic.1 Isolation and identification of avian pathogenic Escherichia coli in AnhuiThe sources of this experiment derived from the dead or sick chickens in areas of Anhui Province. The disease material was taken on normal culture medium directly at aseptic environment. Observed modality and characteristics of E. coli which were isolated by macconkey agar and assay 10 index of physiolgical chemistry to ascertain the avian Escherichia coli. In consequence,8 strains of E. coli were isolated and identified. Intraperitoneal injection was been given to chicks of 2 days-old.Each group had 10 chicks and each chicks were inoculated 0.2ml clinical isolated bacterium.The results show that 12～72h after inoculation,the drug groups have the behaviors as gloomy, difficulty breathing, shortness of breath, vertical fin, vertical hair, drop food or waste.After, each group have different degrees of death. The major pathological lesion as the balloon turbidity, pericardial effusion, liver enlargementand congestion were been found by dead chickens autopsy.8 avian pathogenic Escherichia coli were isolation and identification.Determine 3 highly pathogenic strains and 5 moderate pathogenic strains,and E.coli O2 (CVCC1565) was highly pathogenic strain. 2. The detection forHPI genes of avian pathogenic E. coli strains in AnhuiExtracting the genomic DNA of bacterial with a small dose of bacterial genome DNA extraction kit.12 pairs of oligonucleotide primers were designed basing on the high conservative regions of the gene sequence in GeneBank, concluding the irp9, irp8, irp7, irp6, ybtA, irp2, irp1, irp3, irp4, irp5 and fyuA and AsnT-tRNA-intB gene of the Yersinia enterocolitica WA (O:8). The HPI genes of eight APEC strains in Anhui have been detected by the established PCR,and take the E.coli 02 (CVCC1565) standard strains as a positive control, X090405EC non-pathogenic strain as a negative control. The result showed that the positive rates of HPI genes were 100%. And the nucleotide acid sequences were detected, the homology were analyzed in NCBI Blast, comparing with the sequences logging in GeneBank. The results showed that the sequences of segments of each gene were consistent with the expected, and the homologies were more than 95%. The results show that the APEC strain detected HPI positive and escherichia coli fails to detect HPI positive, it can be concluded that the pathogenicity of strains may carry the relationship with HPI.3 Determination of LD50 of strains of avian Escherichia coli in AnhuiThe high-pathogenic strain of 22, low-pathogenic strain of 13, the standard strain CVCC1565and non-pathogenic strain X090405EC were configured to 5 dose groups as 104,105,106,107,108 cfu/ml.Each group of chicks have been given 0.2ml intraperitoneal injection,and the standard strains were used as positive control, non-pathogenic strains were used as a negative control. After continuous observation of 14d, the results showed that strain of 13,strain of 22, and standard strains of CVCC1565 had a performance of the typical E. coli poisoning disease symptoms and had different degrees of death. Autopsy of dead chickens have been given timely and lesions were saved in 10% formalin. Giving a detailed record of chicken deaths in each group, mortality and survival rates and calculating LD50 of strain of 13, strain of 22,and standard strains according to the modified Kerr's law.The results were respectively 106.7,105.9 and 104.7. It can be concluded from the experiment that the pathogenic escherichia coli didn't carry HPI and and did not appear death. Meanwhile, the E.coli (X0904EC13), E.coli X0904EC22 and E.coli (CVCC1565) carried HPI genes and had different levels of death.So that the pathogenicity of e. coli in a certain extent depends on HPI.However, the E.coli (X0904EC13), E.coli X0904EC22,E.coli (CVCC1565) carried HPI and the homology reached 95%, besides,different strains had different toxicity. This phenomenon show that pathogenic escherichia coli's toxicity didn't only depend on HPI. 4 Pathological morphology change of Avian colibacillosisDead chick tissues which fixed in 10% formalin solution in advance were respectively pass through washing, dehydration by different concentrations gradient alcohol, xylene, pouring wax, embedding,the tissue biopsy, staining by HE and other steps.Besides, slices were taken photographs. The results showed that evident lesions of the heart, liver and ileum from the experimental group could been detected, rather than the the negative control group failed to detect.Above all,8 APEC were separated from clinical avian pathogenic successfully and HPI genes were detected from the isolates and standard strains by using molecular biology techniques.In addition, the certain relationship between the carrying HPI and Avian E. coli disease has been given a preliminary research. Moreover, it might provide a basis for the further study about the molecular pathogenic mechanism of Avian colibacillosis.