Isolation And Identification Of Riemerella Anatipestifer From Weifang City In Shandong Province

Duck infectious serositis,once called New ducks syndrom,nuekseptieemia,and Anatipestifer syndrome,etc.,is an acute,septic infectious disease caused by Riemerella anatipestifer(RA).Domestic ducks,wild ducks,goslings,turkeys,pheasants and many other birds are susceptible.The lesions are mainly characterized by fibrous pericarditis,fibrous perihepatitis,and fibrous pneumonia,commonly known as "Three Guarantees." In addition,symptoms such as air sac inflammation,arthritis,conjunctivitis,and meningitis are also common in the lesions.The disease is one of the major infectious diseases that endangers the duck farming industry,causing huge economic losses to the farming industry.At present,vaccine prevention and antibacterial treatment are the main means to prevent and treat the disease.Therefore,the isolation and identification of RAs,the detection of drug resistance,and the typing of pathogenic serotypes for clinically collected suspected cases have important guiding significance for the selection of high-sensitivity drugs and appropriate vaccines in the clinical breeding process.This study is mainly divided into the following three parts:Ⅰ.Preparation of RA single factor serumIn this test,seven whole-body immunogens were prepared using 7 strains of Riemerella anatipestifer,and healthy male rabbits of about 1 kg were immunized.Finally,blood was collected from the heart to prepare antiserum.The agglutination titer of antisera prepared from 7 serotype RA strains and the serotype bacterial can reach 1: 4800.The prepared corresponding antigens were cross-absorbed with antisera to obtain 7 types of single factor serums which is type 1,type 2,type 6,type 7,type 10,type 11,and type 13.The titers of the seven types of single factor serum were 1: 1600,1: 1600,1: 3200,1: 800,1: 3200,1: 800,1: 1600,respectively.All of the seven RA antisera prepared could only be visually aggregated by the same serotype RA bacterial antigen,and had no cross-reaction with other serotype RA bacterial antigens.23 strains of Riemerella anatipestifer were clinically isolated by 7 single-factor sera obtained from the test for serotype identification,and 10 strains of RA-1 type,11 strains of RA-2 type and 2 strains of RA-11 type were found.This study laid a foundation for further studying the difference of pathogenicity among different serotypes,understanding the biological characteristics of the main epidemic strains of Riemerella anatipestifer in Weifang area,and better understanding the pathogenic mechanism and immune mechanism of duck serositis.Ⅱ.Isolation and identification of RA clinical isolates in Weifang,Shandong ProvinceThe breeding industry in Weifang City occupies an important position in the breeding industry in Shandong Province.The study material was from sick and dead ducks with suspected serositis in Weifang City,Shandong Province.Tissue liver,brain and other tissues were aseptically collected,streaked and inoculated on a modified Martin agar medium,placed in a candle jar,and cultured in a 37 °C incubator for 18-24 hours.Pick the suspicious colonies for Gram staining and observe under a microscope.The morphology is negative,single or chain-shaped short bacillus;Biochemical test showed that the reaction of the bacteria with catalase and oxidase was consistent with the biochemical characteristics of RA.After purifying and culturing the suspected strain,the nucleic acid of the strain was extracted and detected by PCR.Only the nucleic acid extracted from the experimental group was amplified by PCR,and the target fragment length 809 bp was obtained.Ⅲ.Drug resistance test of 23 RA clinical isolatesDrug susceptibility test and drug resistance gene detection were performed on 23 RA strains.The results showed that 23 strains of RA isolated in Weifang were extremely resistant to aminoglycosides and quinolones,and only a few strains were sensitive to them;21 of 23 RAs were detected to mediate aminoglycosides drug-resistant ant(3")-Ia,aac(6’)-Ib,and aph(3’)-IIa three inactivating enzyme genes,armA of 16 S RNA methylation.In addition,RA plasmid-mediated Quinolone-resistant four qnr genes qnra,qnrB,qnrC,qnrD,and two efflux pump genes QepA and oqxAB were also detected.The results showed that only a few strains detected the qnrS gene,and other qnr genes and efflux pump genes were not detected.