Isolation And Identification Of Duck Avian Pathogenic E. Coli And Establishment Of Multiplex PCR Detection Methods

Nowadays,avian pathogenic Escherichia coli(APEC)are responsible for avian colibacillosis and causes significant economic losses in the poultry industry worldwide.Rapid and precise diagnosis is important precondition for treatment and control of the disease.In this study,7 APEC strains isolated from duck-farms located in Xuzhou city of Jiangsu Province,studied the phylogenetic groups,drug resistance and virulence genes,established multiplex PCR detection of APEC from duck.The research provide support for clinical prevention and control and mechanism research.The main research contents and results are as follow:1.Pathogens isolated for hepar from diseased cherry valley ducks in Xuzhou city of Jiangsu Province during 2015.Identified seven strains as Escherichia.coli based on its colony morphology,microscopic characteristics,biochemical test,16 S rRNA molecular biological experiments.Seven isolated strains were assigned as phylogenetic group B2 by phylogenetic groups assay.Pathogenicity test for two strains named E17 and E4,indicating that two isolated Escherichia coli strains are pathogenicity.To determine the different drug sensitivity of Escherichia.coli,Kieby-Bauer paper method was adopted to make susceptibility test to 7 isolated strains with 29 kinds of susceptibility test paper slip.The results of susceptibility tests showed that seven strains were multiple drug resistance(MDR)strains.The least resistant to 5 drug,the most resistant to 21 drug.The result showed that the bacteria only showed high sensitivity to IPM,F and FZD,which would be used to instruct the administration procedure.The isolates were detected for 12 virulence genes by polymerase chain reaction(PCR),except E14,E17 and EB didn't check out stx2,were all amplified out.2.According to the APEC iss,cvaC,iucD,irp-2,iroN and tsh gene sequences published in GenBank,6 pairs of specific primers were designed by Primer 5.0,established multiplex PCR detection of APEC from duck.Detection the specificity and sensitivity of multiplex PCR,the results showd that: the minimum detectable amount of nucleic acid in DNA were 100 pg/?L,minimal limited colonies were 105 CFU.To verify the feasibility of the multiplex PCR,determined 25 APEC(assigned as phylogenetic group B2 by PCR assay)isolates and 79 non-pathogenic E.coli(assigned as phylogenetic group A(31.65%),B1(50.63%),B2(1.27%),D(16.46%)by PCR assay)isolates using the multiplex PCR.An isolate was considered to be pathogenic if it harbored more than three of iss,cvaC,iucD,irp-2,iroN and tsh genes.The accuracy for this hypotheses was 92.0%,while the error was 8.0%.The result suggest that multiplex PCR developed in this study could efficiently detect APEC.The research lay a foundation for further research of APEC from Xuzhou city of Jiangsu Province.