Isolation,Identification And Some Biological Characteristics Of Riemerella Anatipestifer In Three Areas Of Chongqing

Riemerella anatipestifer(RA),one of the most common diseases in duck industry,is a bacterial pathogen that causes infectious serositis in ducklings.Equally,causes diseases in geese,turkeys,quails and other animals.Once the duck becomes sick,it often causes serious economic losses.The prevention and control of Duck infectious serositis is a difficult problem in duck industry.One is to prevent and control Riemerella anatipestifer by using antibiotics,but the drug resistance of Riemerella anatipestifer is increasingly prominent.And the other is to prevent and control Riemerella anatipestifer by using vaccine immunization,but the existing research shows that there are many serotypes of Riemerella anatipestifer,and lacking cross protection among serotypes,which leads to the vast majority of vaccines only having better protection against epidemic strains of the homologous serotype,but little or no protective effect on epidemic strains of the different serotypes.Therefore,in order to effectively prevent and control the disease caused by Riemerella anatipestifer,we need to further study the biological characteristics of Riemerella anatipestifer to provide theoretical basis for the prevention and control of Riemerella anatipestifer.In this study,the epidemic strains of Riemerella anatipestifer were isolated and identified from duck farm materials in three districts and counties of Chongqing.ERIC PCR was used for molecular typing,screening of sensitive antibiotics and detection of resistance genes.CAS(chrome azurol sulfonate)was used for the isolates,The expression of Tdsr(Ton B dependent siderophore receptor)gene was cloned and prokaryotically expressed.The results are as follows: 1.Isolation,identification and ERIC-PCR typing of Riemerella anatipestifer.In this experiment,30 strains of Riemerella anatipestifer were isolated from duck farms in Dazu,Yongchuan and Rongchang from 2018 to 2019.30 strains of Riemerella anatipestifer were identified by morphological observation,biochemical test,16 Sr RNA gene,specific omp A and Dna B gene identification.The isolates were typed by ERIC-PCR method to obtaine 8 types,mainly type 1,2 and 3,which were 14 strains of type 1(46.7%),4 strains of type 2(13.3%)and 4 strains of type 3(13.3%).2.Detection of siderophore of Riemerella anatipestifer isolates.In this experiment,30 isolates were tested for siderophore secretion ability by CAS detection method.The results showed that 30 strains of Riemerella anatipestifer grew well on the CAS detection plate,and orange circles appeared around the colonies on the CAS detection board.The diameter of the orange circle around the colony on the CAS detection plate was 13-22 mm,of which 50% were between 17 and 18mm;the diameters were below 17 mm,accounting for 26.7%;and above 18 mm,accounting for 23.3%.The results showed that there existed differences in the ability of isolates to secrete siderophores.3.Cloning and prokaryotic expression of Tdsr gene.The full length of Tdsr gene of the isolated strain(RA11)was successfully cloned by TA cloning method,which was 2346 bp.The predicted analysis of nucleotide and corresponding amino acid sequence showed that it was most likely to be the epitope of B antigen,located at 24-26,59-63,29-136,179-183 and other amino acid residues.The recombinant expression plasmid p ET32a-Tdsr1 was successfully constructed and the exogenous expression of the recombinant protein was carried out by prokaryotic expression system.The results of SDS-PAGE electrophoresis showed that the recombinant protein existed in the form of inclusion bodies with a size of about 75 KD.The optimized expression conditions were as follows:the final concentration of 0.6 m M IPTG,constant temperature at 37 ? for 4h,and a large number of inclusion body form target proteins were obtained.Using WB(Western blot)method to detect the his-tagged protein,the single band length is about 75 KD.4.Drug sensitivity test and drug resistance gene detection of Riemerella anatipestifer isolates.In this experiment,19 kinds of antimicrobial agents such as ?-lactam,aminoglycoside,tetracycline and quinolone were used to test the drug sensitivity of Riemerella anatipestifer isolates.at the same time,the resistance genes of ?-lactam,aminoglycoside and tetracycline were detected.The results of drug sensitivity test showed that the isolates were most sensitive to cefazolin,cefotaxime and florfenicol,and most resistant to kanamycin and clindamycin.Strain RA11 was resistant to 13 antibiotics,while strain RA14 was relatively sensitive to only four drugs.The results of drug resistance gene detection showed that each strain carried at least 3 kinds of drug resistance genes and up to 7 kinds of drug resistance genes.Genes of participating in the mediation of ?-lactam drug resistance were detected,bla-IMP ?bla CTX-M.Genes of involving in mediating tetracycline drug resistance were detected,tet C?tet E?gyr A.Genes of involving in mediating aminoglycoside drug resistance were detected,aac(3)-I?aph(3')-IIa?adeB?armA.