Isolation And Identification Of One Strain Of Riemerella Anatipestifer And Analysis Of Its Resistance Gene Cluster

Riemerella anatipestifer is also called infectious serositis of ducks.It is a contact acute septic infectious disease of waterfowl caused by Riemerella anatipestifer.Ducklings of 1 to 8weeks of age are susceptible and have a very high mortality rate.Adult ducks are generally not infected with this disease.Riemerella anatipestifer has many ways of transmission,and it can be spread through drinking water,litter,etc.Adult ducks carrying the pathogen have also become important spreaders of the disease.With the continuous development of the duck industry,more and more attention is paid to the prevention and treatment of Riemerella anatipestifer.Vaccines and drugs are important methods to prevent and control this disease.Due to the numerous serotypes of Riemerella anatipestifer,and there is no cross-protection between the various serotypes,vaccine prevention and control of the disease has certain limitations,so antimicrobials are the main way to prevent and control the disease.However,it also brings serious antimicrobial resistance problems,which not only affects the duck industry,but also poses a potential threat to human public health.In this study,more than 300 samples including blood samples,heart,liver,brain and other tissues were collected from some duck farms located in Hebei,Hubei and Zhejiang provinces,and used for isolation and identification of Riemerella anatipestifer by biochemical identification,PCR,antimicrobial resistance testing.Meanwhile,the isolated strains were counted on the plate,and the animal test was designed to determine the median lethal dose(LD₅₀).Finally,the animal regression test was carried out according to the LD₅₀.The pathological changes of the liver,spleen,brain,and bursa tissues of experimental animals were examined by H.E staining.The activity of serum transaminase was determined.Moreover,the genome of the isolated strains was sequenced and analysed to screeng drug resistance genes,thereby explore its possible drug resistance mechanism.Results indicated that one strain of Riemerella anatipestifer was isolated and identified,and designated as RBT-1 strain.The LD₅₀ was 9.759×10¹⁰CFU/m L.After challenging,samples were collected at 1dpi(Day Post Infection,dpi),3dpi,5dpi,7dpi.Different degrees of pathological damage which including local necrosis of liver cells,a small amountof inflammatory cell infiltration,spleen hemorrhage,and epithelial cells in the bursa cavity deformed and shed was observed in the liver,spleen,bursa and brain tissues from the infected group.In contrast,no obvious tissue lesion was observed in control group.Importantly,the Riemerella anatipestifer was isolated from the brain tissue of ducklings in the infected group.The results of antimicrobial resistance analysis showed that the RBT-1 strain was resistant to ampicillin,streptomycin,polymyxin B and compound trimethoprim,and to cefoperazone,amikacin,and highly sensitive to mycin,doxycycline,norfloxacin,ciprofloxacin.Whole genome sequencing results showed that the RBT-1 strain carried ATP binding cassette(ABC)antibiotic efflux pump,fus E family,ABC-F ATP binding cassette ribosome protective protein,and antibiotic resistant isoleucine-t RNA Synthetase(ile S),Lpx gene,anti-aminosalicylic acid thymidine synthase,fol P gene,MFS efflux system family,and quinolone drug-resistant gene gyr B.In summary,this study obtained one strain of Riemerella anatipestifer RBT-1 through isolation and identification.The animal regression test showed that the strain is pathogenic and can damage to liver and other tissues.The drug resistance testing and whole-genome sequencing revealed that there was a certain correlation between its carried resistance gene or cluster and drug resistance phenotype.It suggests that there are irrational drug useage in duck farms located in Hebei,Hubei and Zhejiang provinces.This study might provide a help for the prevention and control of Riemerella anatipestifer in duck farms.