Isolation, Identification Of Riemerella Anatipestifer And Cloning, Sequencing Of Its Outer Membrane Protein A

Riemerella anatipestifer (RA) infection is a serious worldwide problem of natural waterfowl and domestic turkey, duck and goose populations. The infection typically exacts a severe economic cost, especially when poultry is also infected with other microorganisms. Mortality and morbidity rates are usually between 10 and 30% but mortality of as high as 75% has been recorded in infected duck farms. Once the diease invades duck and goose flocks, it can become endemic. Eradication can be difficult, with repeated infectious episodes possible.Infections caused by R. anatipestifer are characterized by the systemic development of exudative serositic lesions, and should be differentiated from fowl cholera, infections caused by Escherichia coli, Streptococcus faecium, and Salmonella. Identification and typing of R. anatipestifer involves phenotypic, biochemical and molecular characterization. Till now, at least 21 serotypes of R. anatipestifer have been isolated in the world.Twenty strains of R. anatipestifer were isolated and identified based on biochemical tests from suspected sick ducks with infectious serositis in Guangdong province and the results of animal experiment showed that the pathogenicity of the isolated R. anatipestifer strains is very high. The result of drug sensitivity test show twenty R. anatipestifer strains have had widely drug-resistance. The result of 20 R. anatipestifer serotyped by slide agglutination test showed the prevalence of serotype was 10 serotype which amount to 75% of the isolated R. anatipestifer.A pair of 809bp primers(primer 1 and 2) were designed and synthesized, which complemented to 208~227 and 997~1017 sites of the 42kDa major antigenic outer protein OmpA gene of R. anatipestifer serotype 15 strain CVL110/89. Another pair of 334bp primers(primer 3 and 4) were designed and synthesized, which complemented to 508~526 and 824～843 sites of the 16S- rDNA gene of R. anatipestifer. According to two pairs of published primers and using whole bacteria cells as the template, the duplex PCR has been established. The results showed that two fragments as they were expected were amplified from all detected R. anatipestifer strains, while Escherichia coli, Pasteurella multocida, Salmonella and Staphylococcus aureus strains isolated from duck gave negative results.Thus, this duplex PCR assay provides a rapid and accurate method for identification of R. anatipestifer and can be used to rapid identification and diagnosis of R. anatipestifer (infection).According to Outer Membrane Protein A (OmpA) gene sequences from R. anatipestifer strains in the GenBank, a pair of specific primers was designed. Eight R. anatipestifer from Fujian, Guangdong and Shandong were amplified in the whole open reading frame(ORF) by PCR and then cloned and sequenced. The comparative analysis of the sequences with twenty-six R. anatipestifer strain in GenBank indicated all OmpA gene had a long complete ORF composed of 1164 nucleotides, which encoded a protein of 387 amino acids. All initiation codon were ATG and end codon were TAA. The sequence of OmpA were relatively conservative which identity was 88.2%～100% and the identity of the amino acids was 88.9～100%. The results showed that the sequence of OmpA relatively conservative and the identity of the nucleotide and amino acid had no positive relation with the isolate serotype, the year or location of isolation.