| Mycoplasma synoviae(MS)is an important pathogen in poultry,which can cause respiratory diseases,airsacculitis,infectious synovitis,tenosynovitis,and pad swelling in chickens and turkeys.MS infection leads to growth retardation,reduced egg production,and decreased egg quality in infected chickens,causing considerable economic losses in the chicken and turkey industry.In recent years,it has been founded that infected chickens with MS have severe airsacculitis and oviduct embolism,which may be related to the air sac and oviduct tropism of MS.In this study,MS was isolated and identified,antimicrobial susceptibility testing,and biological characteristics were by collecting suspected MS infection airsacculitis and oviduct embolism chicken material samples.To study the pathogenicity of MS isolates by animal infection model.The specific test results are as follows:1.During 2021～2022,MS monitoring was conducted on 9 large-scale laying and breeding chicken farms in Shaanxi Province,Shanxi Province,and Ningxia Hui Autonomous Region.A total of 259 serum samples were collected from five breeding farms.ELISA antibody level detection found that the positive rate of serum in one breeding farm was 3.33%,and the positivity rates of MS antibody in the other four breeding farms were up to 90～97%.176 air sac and oviduct tissue samples of chicken suspected to be infected with MS were collected aseptically.The PCR positive samples were isolated and identified,MS was isolated using a modified Frey’s medium.Combined with PCR detection,colony morp Hology,Giemsa staining,serological tests,and sequence analysis of isolated strains vlh A gene.19 strains of MS were isolated successfully.2.In vitro antimicrobial susceptibility testing were performed on 19 strains of MS isolates,and10 commonly used antibiotics were selected.The results showed that MS isolates were highly sensitive to valnemulin hydrochloride and tiamulin.While showed strong resistance to roxithromycin and levofloxacin hydrochloride,which have been widely used clinically in recent years.Roxithromycin and levofloxacin belong to macrolides and fluoroquinolones.Therefore,amplification screening and sequence analysis of the macrolide and fluoroquinolone-related resistance genes of the MS isolates showed that the macrolide rrl allele had a single point mutation of A2058 G and A2059 G nucleotides.The substitution of a mino acids T(Thr)-I(Ile)and P(Pro)-S(Ser)in the par C gene and par E gene of fluoroquinolones is the main reason for antibiotic resistance in MS,which provides reference for clinical use of antibiotics to prevent and control MS.3.In order to deter mine the load and distribution of MS in different tissues of infected chickens,the primer MS-F/MS-R was designed according to the MS standard strain vlh A gene published by Gene Bank.Using MS XP01 as the template,the vlh A gene was specifically amplified 208 bp,and the recombinant plasmid was constructed using p MD-18 T vector.The plasmid concentration was measured and its copy number was calculated according to the formula.The reaction conditions of q PCR were optimized and the copy number was used as a positive standard to establish a standard curve,which was used as the MS q PCR detection method.MS was infected in three different ways of foot pads and aerosol inoculation,inoculation foot pads,and aerosol inoculation,and animal infection model was established.The results showed that the probability of airsacculitis was 80%(4/5)in animals inoculated by foot pads and aerosol,and the probability of airsacculitis was the same in animals inoculated by aerosol only.However,the animals inoculated through the foot pads route only showed swelling of the foot pads,but no cases of airsacculitis.No oviduct embolism was observed in all experimental groups,but MS distribution could be detected by q PCR in the oviduct of infected chickens.In conclusion,the epidemiological investigation of MS infection in chicken farms in Shaanxi Province,Shanxi Province and Ningxia Hui Autonomous region was carried out.MS was isolated and cultured with modified Frey’s medium,and the antimicrobial susceptibility of the MS isolates was tested.The animal infection model was established by inoculating MS in three different ways,and the MS q PCR detection method was used to deter mine the load and distribution of MS in different tissues of infected chickens.The test results provide reference for the study of MS pathogenicity. |
