Isolation And Identification Of Mycoplasma Synoviae In Hubei Province And Improvement Of Avian Mycoplasma Culture Medium

Mycoplasma synoviae（MS）is one of the most important pathogens of poultry and is a serious threat to the healthy development of the poultry industry.To understand the epidemiological status of MS in Hubei Province,this study conducted pathogenic testing of arthropathy cases collected by the laboratory from 2019 to 2020,and performed molecular epidemiological surveys and systematic analysis of etiology based on the vlh A gene.At the same time,in response to the current situation in which it is difficult to culture MS using Mycoplasma gallisepticum（MG）culture medium,we developed a low-cost,high-efficiency culture medium to improve MS proliferation efficiency.The following results were obtained.1.Mycoplasma synoviae vlh A genetic variation analysisA total of 354 disease samples were collected from 19 localities in Hubei Province,and 21 MS strains were isolated by using the modified Frey’s medium.The isolation rate of the samples was 5.93%.The vlh A gene of the isolated strains were cloned and determined,bioinformatics analysis of the obtained sequences was carried out.The results showed that the full length of vlh A gene was 3000-3100 bp.The results showed that the homology of vlh A gene differed significantly among different isolates based on Geneioust software analysis;the phylogenetic tree constructed by alignment using the MEGA 11 software showed that the isolates in Hubei province mainly concentrated in two branches.BLAST analysis revealed that even the hypervariable MSPA region has a highly conserved sequence of 26 bp.Specific contiguous hypervariable and highly conserved regions were present in corresponding nucleotide sequences of both MSPB and MSPA.Based on PRR repeat sequence analysis a total of 2 genotypes were identified,the The K type（24/25）,which is unique to China,was predominant,followed by the D type（1/25）.This suggests that the existing epidemiological status of MS in Hubei province is complex.The genetic relationship between the isolates in Hubei Province and foreign strains is far which suggests that the source of infection is mainly of domestic origin,while the genetic relationship between the Jiangxia isolated strains and the Polish strain 1.2-146-12PL,the Yi Chang-1 strain and the French strain M2008.13/France is closely suggests that cross-border importation may also be present.2.Optimization of culture media for Mycoplasma gallisepticum and Mycoplasma synoviaeThe tissues of different animals were selected as raw materials,and the infusions were prepared by compound enzymatic hydrolysis method,acid hydrolysis method,steaming method and trypsin plus hydrochloric acid method,etc.The MG and MS growth promoters were preferentially selected by screening the animal tissues,screening the preparation methods,optimizing the amounts of additives and combining the cost of obtaining the materials.The results showed that trypsin combined with hydrochloric acid method for preparing chicken lung and pig lung infusion had significant growth promoting effect on MG,and the addition of 5%-10%could enhance the growth efficiency by 10¹²times and significantly shorten the MG culture cycle compare with modified Frey’s medium.10%chicken lung infusion was the most suitable additive for MS,which had significant growth promoting effect and could significantly improve the growth efficiency of MS by about 10¹¹times compare with modified Frey’s medium.In this study,we conducted a molecular epidemiological survey of MS in Hubei Province based on vlh A gene and confirmed that the genotype of MS prevalent in Hubei Province is type K,which provides a reference for understanding the current situation and MS prevalence in China.At the same time,the avian mycoplasma growth promoter developed could help to increase the proliferation efficiency for mycoplasma,decrease the costs of producing mycoplasma vaccines,and has good application prospects.