Isolation, Identification Of Mycoplasma Capricolum Subsp. Capripneumoniae And Establishment Of An Indirect Haemagglutination Test For Antibody Detection

M.capricolum subsp.capripneumoniae(Mccp)was firstly isolated from goats in Kenya in 1976,and it had been proven to be the pathogen of contagious caprine pleuropneumonia (CCPP) which is a severe disease of goats occurred in Africa and Asia.It is one of important diseases listed by the Office International Des Epizooties (OIE).Up to now,Mccp had only been isolated from 14 countires due to its fastidious nutrition requirement in vitro.CCPP was initially discribled in 1920's in China,but only one strain of Mccp was isolated by Xin Jiuqing et al from Shandong province in 2007.In this study,one Mycoplasma strain named as M1601 was isolated successfully from lung tissue of a sick goat suffered from resembling CCPP in a farm located in Gansu province.The strain M1601 were subjected to biochemistry test,PCR and PCR restriction enzyme digestion as well as animal infection test. The results showed that the characteristic of strain M1601 is consisitent with Mccp.Two rRNA operons(rrnB and rrnA) of 16s rRNA gene and five housekeeping genes(fusA, glpQ,gyrB,lepA and rpoB) were amplifid from the strain M1601 and submitted to the GenBank database.Their GenBank accession numbers were assigned as HM155915,GU736670,GU736671, GU736672, GU736673,GU736674 and GU736675,respectively.Homology analysis based on rrnB and rrnA gene indicated that M1601 shows 99.8% homology with Mccp reference strain F38 and field strain 4/2LC,99.1% with M.capricolum subsp.capri(Mcc)strain California kid,99.0% withM.Bovine group 7(MBG7)strain PG50 and M.mycoides subsp.Capri(Mmc) strain PG3, 98.8% and 98.9% with M.mycoides.subsp.mycoidesSC(MmmSC)strain PGI and M.mycoides.subsp.mycoide LC(MmmLC),97.1% with M. putrefaciens (Mput) strain KS-I,respectively.The phylogenetic tree constructed using rrnB gene showed that M1601,4/2LC and F38 were in the same branch(bootstrap value as 100%).Another phylogenetic tree was constructed based on a set of concatenated gene sequences from five housekeeping genes including fusA,glpQ,gyrB,lepA and rpoB gene.Two distinguished subclusters were observed.The M.mycoides subcluster comprised of MmmSC,MmmLC and Mmc.The M.capricolum subcluster included Mcc,MBG7,M1601,95043 and 9231Abosma.The latter three strains were present in the same branch (bootstap value is 93%).The results of molecular identification verified the strain M1601 as Mccp.Capsular polysaccharide (CPS) of Mccp was extracted and purified from the strain M1601 culture supernatant.And then an Indirect hemagglutination test (IHAT) for detection of Mccp antibody was established by employing sheep erythrocytes which sensitized by CPS as antigen.The IHAT shows good specificity characterized by negative reaction with positive sera against Mmc,M.ovipneumoniae (MO),M. hyopneumoniae and Mannheimia haemolytica.The seroconversion were detected in 14 days after the goats were infected with Mccp strain M1601.817 serum collected from different regions were detected by the IHAT and the positive rate was 14.1%.The results showed that the IHAT could be an useful tool for diagnosis and seroepidemiology investigation of CCPP.