Epidemiological Investigation Of Riemerella Anatipestifer And Identification Of New Specie Of Riemerella

Riemerella anatipestifer（RA）disease is a poultry infectious disease in the world caused by the Riemerella anatipestifer,and it has a high incidence.There are at least 21 identified serotypes of RA,and there is basically no cross-protection between the different serotypes,making it difficult for existing vaccines to prevent the disease.There is no reliable and specific PCR test available for the detection of this disease.In this study,a specific PCR assay was established using genome-wide data of 92 strains of RA.It also investigates the nationwide prevalence of RA from 2019 to 2021 to provide a reference for disease prevention and control.During the survey,a new strain of the genus Riemerella was discovered.The strain number is RCAD0985.First,the ATCC 11845 genome was used as a query to screen conserved fragments by comparing it with 92 whole genome sequences of RA included in the NCBI refseq genome database.The non-specific fragments were then removed by comparison with non-Riemerella anatipestifer in the NT.Design primers in candidate sequences by oligo 7.The primers were evaluated for specificity,conservativeness,sensitivity and accuracy.The primer amplification product was 334 bp long and located in the J6M13＿RS09305 gene.Amplification of Riemerella anatipestifer DNA samples at different dilutions with a minimum detection of 1×10^-5 ng/μL and 100 CFU/m L.The accuracy of the specific detection method established in this study was 99.0%（510/515）for RA.The agreement with the bacterial isolation and identification method was good（kappa=0.884,P<0.001）.Isolation of pathogens from diseased ducks and identification of RA serotypes by agglutination test.A total of 450 strains of RA were isolated,including 77 strains of type 2（17.1%）,50 strains of type 5（11.1%）,59 strains of type 6（13.1%）and 87 strains of type 11（19.3%）.The survey results showed that the main prevalent serotypes in China in the past three years were 2,5,6 and 11,and the dominant serotype was 11.A strain with morphological characteristics and biochemical properties more similar to RA,with a full-length genome of 2.5 Mb and a G+C content of 39.57%.The strains are located in the same evolutionary branch in the 16S r DNA phylogenetic tree for strains of the genus Riemerella.The range of Ortho ANIu values obtained by genome-wide comparison with the intraspecific bacteria of the genus Riemerella was 72.72%to 75.48%and DNA-DNA hybridization values were 20.2 to 22.1%.Combined with morphological observations and physiological and biochemical identification,RCAD0985 was confirmed to be Riemerella anaschoana.sp.nov..