Isolation And Identification Of Mycoplasma Bovis Guizhou Strains And Molecular Characterization Of Major Genes

Mycoplasma bovis(M.bovis)wasthe pathogeny which mainly caused calf pneumonia,adult bovine mastitis,arthritis,conjunctival keratitis,otitis media and genital tract inflammation.Since 2008,Mycoplasma bovis pneumonia outbroke in many provinces and cities of China.And there were many suspected cases in Guizhou province.Because of the lack of pathogenic and biological characteristics informationof local epidemic strains,there were difficulties for diagnosis and prevention of this disease.The study based on the serum epidemiological investigation data of mycoplasma bovis pneumonia and chose the positive samples by the established rapid diagnosis of pathogens.The research of biological characteristics of pathogen and characteristics of pathogenic genome was carried out to lay the foundation of diagnosis,prevention and control the disease.1.Establishment of the rapid detection method for Mycoplasma bovis:To achieve the goal that rapid diagnosis and quantitative analysis of pathogeny of mycoplasma bovis pneumonia,the common PCR method and TaqMan real-time fluorescence quantitative PCR were established to detect the Mycoplasma bovis.The PCR methods primers were designed based on TU gene and the condition of both meithods were optimizated.The results showed that 752 bp fragment was amplificated form the template DNA of Mycopalsma bovis strains PG45.The optimum annealing temperature was 60 ?.The Taqman real-time fluorescent quantitative PCR was established,based on plasmid standard sample of Mycoplasma bovis TU gene.The method's standard curve's slope,intercept and correlation coefficient were-3.24,46.20,-0.996 individually.Those two methods could distinguish pathogenic Mycoplasma mycoides subsp mycoides,Mycoplasma pneumoniae,and Mycoplasma bovis and show good specificity,clinical application.The results of sensitivity 1.034 �10-6g / L?1.91�1010copies/?L in the minimum DNA detectable concentration.2.Isolation and identification of Mycoplasma bovis Guizhou strains:The antibody of1074 serum samples was detected by ELISA and mycoplasma bovis pneumonia positiveclinical cases were selected based on the Clinical pathological observation and PCR detection.The mycoplasma bovis Guizhou strains were isolated and cultured by mycoplasma culture technique and identified by morphological,biochemical and molecular biological methods.The results showed that the antibody positive rates were 1.03%.The clinical symptoms of Mycoplasma bovis pneumonia cases were body weight loss,depression,nasal bleeding,salivation,orbital exudate,and suffering from arthritis symptoms.Two positive clinical case samples were tested by PCR method.Solid cultures of Mycoplasma bovis showed a typical "fry like" colony.The cultures of Mycoplasma bovis Guizhou strains were observed by Giemsa staining and the results showed blue purple culture in the form of irregular filamentous under the ordinary microscope.The electron microscope observation showed that Mycoplasma bovis was spherical without cell wall.Biochemical identification showed that the isolated strains were able to digest phosphatase and sensitive to the saponin.The OPPD/F gene similarity between the two isolates and Mycoplasma bovis standard strain over 99%.So we determined the two Mycoplasma strains were Mycoplasma bovis and named Mycoplasma bovis GZ1,Mycoplasma bovis GZ2.3.Whole gene sequencing of Mycoplasma bovis Guizhou strain GZ-2 : The Mycoplasma Mycoplasma bovis GZ-2 cultures were collected,and the total DNA genomic materials were extracted.The total DNA samples were sequenced by the novogene with the small genome sequencing technique.The gene was assembled by SOAP denovo 2.04,and the assembly result was evaluated by KMER analysis and GC-depth analysis.COG,KEGG and GO gene databases were used to analyze the function gene of Mycoplasma bovis Guizhou GZ2 strain.The results showed that the GC-depth scatter plots exhibited the shape of Poisson distribution,so there were no bias of GC in the sequenced data.The whole genome sequence of the isolated strain showed that the genome size was 934881 bp,the coding region was655149 bp and the ratio of genome was 70.08%,GC% is 30.01%.The total length of noncoding region was 279732 bp,and the content of GC% was 27.7% accounting for 29.92%of the total genome length.There were 67 tandem repeats with a total length of 5807 bp,which accounted for 0.6211% of the total genome.There were 53 small satellite sequences and 5 microsatellite sequences.Genome NR database analysis of Mycoplasma bovis Guizhoustrain showed 510 genes were homologous with Mycoplasma bovis in 616 genes.COG,KEGG and GO gene databases analysis showed he relative pathway of Mycoplasma bovis carbon source in this strain was relatively complete,and glycerol instead of carbohydrate was used as its carbon source.Sequencing showed that Mycoplasma bovis strain GZ has 7 defense mechanisms proteins,4 VSP family proteins and several adhesion related protein.4.Major function genes analysis of Mycoplasma bovis strain GZ-2:The molecular characteristics of 3 defense mechanisms proteins,3 VSP family proteins and 3 adhesion related protein were analyzed by Blast,DNA Star,Mega 5m,ExPASY,IMED software.The results showed that the VSP family protein gene was highly conserved,and the homology of 3VSP family genes was 100% in different Mycoplasma bovis strains.It was found that the vspY protein of Mycoplasma bovis Guizhou strain may contain 7 antigenic epitopes,among which the 20-31 amino acid was a dominant epitope with high antigenic index.There were 22 repeats of vspC gene family between Mycoplasma bovis GZ-2 vspC gene and Mycoplasma bovis strain PG45.VspC protein may contain 2 epitopes,which advantage epitope of the 4-25 amino acids in high antigen index.The assumed VSP protein may contain 4 epitopes in which the dominant epitope is high by the 13-37 amino acid antigen index.defense mechanisms proteins multidrug ABC transporter ATP-binding protein,type I restriction modification system methyltransferase subunit gene protein homology of deference Mycoplasma bovis strains were 100%,while abortive phage infection protein gene homology of the Guizhou Mycoplasma bovis strain GZ-2 and Mycoplasma bovis strain PG45 was 99.9%.Mycoplasma bovis strain GZ-2 abortive phage infection protein gene had a mutation in the forty-seventh base sequences and caused the amino acid isoleucine into glutamine.Adhesion related protein analysis showed that P81 gene homology of two Mycoplasma bovis Guizhou strains GZ1 and GZ2 was 100%,92.1%~97.4% with other strainsand and 92.1% with strain PG45.Phylogenetic tree analysis based on the P81 gene showed that Mycoplasma bovis Guizhou strains were nearly related to Mycoplasma bovis trains isolated from domestic and had some evolutionary distance with PG45.Antigenic analysis showed that its 560-602 amino acid was an advantageous epitope with high antigenic index.The TU gene homology between Mycoplasma bovis Guizhou strain GZ1,GZ2 andstandard strain PG45 were 99.1% and 99.9%,and 99.3-99.7% with Hubei,Chongqing,Inner Mongolia strains.Phylogenetic tree analysis based on TU genes showed that Mycoplasma bovis strain GZ1 and Hubei,Chongqing,Inner Mongolia strains were located in the same branch,while Mycoplasma bovis strain GZ2 and PG45 were in the same branch.The amino acid of 101-108 was a dominant epitope with high antigenic index.The P48 gene homology of two Mycoplasma bovis Guizhou strains was 99%.The homology between Mycoplasma bovis Guizhou GZ1,GZ-2 strains and standard strain PG45 were 99.6% and 99.9%respectively and 99.6%~99.9% with other strains.Phylogenetic analysis based on the TU gene showed that the Mycoplasma bovis Guizhou strain GZ1 and PG45 were located in the same clade and located in different clades with Mycoplasma bovis strain GZ2 and other domestic strains.The average antigen tropism of P48 gene was 1.0220,among which the 4-27 amino acid was the dominant epitope with high antigen index.