Molecular Epidemiological Investigation Of Mycoplasma Synoviae Strains Isolated From Some Areas Of China During 2016 To 2019

Mycoplasma synoviae(MS)is one of the most important pathogens in chickens and turkeys,which can cause acute or chronic infectious synovitis or pneumocystitis.When it is co-infected with Escherichia coli,Newcastle disease virus or infectious bronchitis virus,it can cause synergistic pathogenic effect.In addition,MS infection can also result in reduced egg production and eggshell apex abnormalities.In recent years,MS infection is widely prevalent in chicken flocks in China,which has caused serious economic losses to the chicken industry.Therefore,strengthening the surveillance of MS in chicken flocks and elucidating the transmission dynamics are of great significance for the prevention and control of MS infection.The main purposes of this study are as follows:(1)MS isolates were genotyped by variable lipoprotein hemagglutinin A gene(vlhA)sequence analysis and Multilocus sequence typing in order to invesigate the dominant genotypes in China and reveal the genetic and evolutionary relationship among MS strains with different genotypes.The transmission and epidemic situation of MS in China were clarified by traceability analysis,which provides data support for effective prevention and control of MS infection.(2)The antibiotic resistance phenotype was identified by antibiotic susceptibility test in vitro,and then,according to the results of drug resistance phenotype identification,drug-resistant strains were selected to study the drug resistance at the gene level,so as to provide a basis for clinical drug use.1.Epidemiological investigation and genotyping of Mycoplasma synoviae in some areas of China during 2016 to 2019.In order to understand the latest prevalence of Mycoplasma synoviae infection in China,samples were collected from chicken farms suspected of infecting MS in 8 provinces including Jiangsu,Anhui,Shandong,Henan,Hebei,Ningxia,Heilongjiang and Hubei during 2016 to 2019.A total of 47 MS isolates were obtained.Sequencing and phylogenetic analysis of the 5'end of the vlhA gene of these isolates showed that 46 of them belonged to the K genotype,and the other one belonged to the E genotype,respectively.The results showed that the prevailing dominant genotype in China was K genotype,which was significantly different from the prevailing genotype in other countries and regions.VlhA typing has the advantages of simplicity and rapidity,but the degree of differentiation is slightly insufficient,while MLST typing is mainly based on the allelic polymorphism of 7 pairs of house-keeping genes(Adk,atpG,Efp,Gmk,nagC,Ppa and recA)to classify MS,and it has the ability to distinguish more subgroups,which is more conducive to the study of the transmission dynamics of pathogens.Therefore,the MLST typing was used to study the multi-locus sequence typing of 47 MS isolates.The results showed that 47 isolates were divided into 15 different new sequence types,all of which belonged to the same clone group.All 47 isolates had new allele sequences,and the number of new alleles were:Adk(n=2),atpG(n=2),nagC(n=10),recA(n=3),and there is no new allele sequence in Efp,Gmk and Ppa.The largest number of alleles was nagC(n=11),and the least was Gmk(n=1)and Ppa(n=1).Among the 15 new sequence types,there were 25 MS isolates belonging to ST34,accounting for 53.19%(25/47)of the total number of isolates.8 isolates belonged to ST93,accounting for 17.02%(8/47),and 2 isolates belonged to ST90,accounting for 4.26%(2/47).The other 12 sequence types each contained one isolate,accounting for 2.13%(1/47)of the total number of isolates.The results of MLST typing showed that ST34 was the dominant sequence type in China.Based on the BURST analysis,47 MS isolates and 185 reference strains were divided into 15 clonal groups and one singleton group.In this study,47 MS isolates,15 sequence types including 47 MS isolates belonged to group 11,independent of other 14 clonal groups and singletons.The results showed that the epidemic clone group in China is group 11,and it was not closely related to the rest of the clone groups in the world,that was,it had a strong regionality.Among the 185 international MS strains,the dominant groups were group 12(22.16%),group 9(13.51%)and group 6(9.19%).The strains belonging to these three groups accounted for 44.86%(83/185)of the total MS reference strains.Combined with the national distribution of reference strains,group 12 and group 9 could be the dominant clonal groups in Europe,and the prevalence of MS has a certain regionality.To sum up,46 K genotype MS strains and 1 E genotype MS strain in vlhA typing belonged to 15 sequence types in group 11 in MLST typing.The results of the two genotyping methods showed that the predominant genotypes of MS in China had not changed significantly,which reflected that the MS strains isolated in China had an independent origin.2.Antibiotic resistance research on MS isolatesAt present,the commonly used clinical antibiotic medicines for the treatment of mycoplasma disease were macrolides(tylosin,tilmicosin),Pleuromutilins(valnemulin,tiamulin),tetracyclines(doxycycline hydrochloride,oxytetracycline,doxycycline)and fluoroquinolones(enrofloxacin),as well as the combination of lincomycin and spectinomycin used to prevent and treat MS in recent years.In this study,32 representative MS isolates were tested for antibiotic susceptibility in vitro with the above 9 commonly used antibiotic medicines.The minimum inhibitory concentrations(MIC)of 32 representative MS isolates were<0.016?1?g/mL,<0.016?1 ?g/mL,<0.016?0.031 ?g/mL,<0.016?0.063 ?g/mL,0.25?8?g/mL,0.5?8 ?g/mL,0.125?4 ?g/mL,4?32 ?g/mL and 0.063?2 ?g/mL,respectively.The results of antibiotic susceptibility test showed that(1)All isolates were sensitive to the combination of lincomycin and spectinomycin(2)32 MS isolates were sensitive to pleuromutilins and macrolides.(3)The results of resistance to tetracyclines were not completely consistent,and all isolates were sensitive to doxycycline.For doxycycline hydrochloride,except for 3 moderately sensitive strains,the others were sensitive.For oxytetracycline,except one strain was moderately sensitive,the others were sensitive.(4)All isolates were resistant to enrofloxacin.In order to further investigate the mechanism of enrofloxacin resistance in MS,four resistance genes in the fluoroquinolone resistance determination region of 32 MS isolates were amplified by PCR and sequenced.The results showed that among the parC genes,32 MS isolates had the substitution of I(Ile)-T(Thr)at position 85,and 27 isolates of MS had the substitution of S(Ser)-P(Pro)at position 446 of the parE gene,which could be related to the decreased sensitivity of fluoroquinolones.This study provided a data reference for the drug prevention and control of MS.