Genotype Distribution Of Chinese Mycoplasma Bovis Isolates And Their Evolutionary Relationship To Strains From Other Countries

Mycoplasma bovis(M.bovis)is an emerging agent of cattle worldwide,causing pneumonia,mastitis and arthritis.The first reported case of M.bovis pneumonia in China was in 2008 and prevalent all over the country since then.The disease is difficult to control with chemotherapy,and vaccination would be the best alternative approach.An insight of the genetic diversity and population structure of M.bovis would assist in the development of novel vaccines,prevention as well as gaining an insight into evolutionary trends.Therefore,this study was undertaken to determine the genotypic distribution of Chinese strains and their similarity and evolution to isolates from other countries.Two different multilocus sequence typing(MLST)schemes of M.bovis(MLST-1 and MLST-2)and a conventional pulsed field gel electrophoresis(PFGE)were used to compare 44 Chinese strains and the M.bovis reference strain PG45 originally from the USA.Additionally,MLST-2 scheme was used to analyze the evolutionary relationship between isolates from China(n = 44),Australia,(n=8),and Israel(n=8)and compare ST types of these strains with those of 206 isolates retrieved from pubmlst database.The main results are as follows:1.The results showed a high genetic homogeneity of Chinese isolates;43 of 44(97.7%)Chinese isolates were identified as ST-10 and as ST-34 by MLST-1,while for MLST-2 42 of 44(95.5%)were identified as ST-10 with the two remaining isolates of ST-32 and ST43.PFGE clustered 42 of 44(95.5%)of the Chinese isolates into PT-I.The overall agreement rate between the three typing methods was 97.8%(95% CI:86.8-99.9%)The type strain PG45 was identified as a unique type by all three methods.2.Comparative analysis of Chinese isolates with Australian and Israeli isolates.When the MLST-2 scheme was further used to analyze 8 isolates of Australian and 8isolates of Israeli origin ST-10 was more dominant among Australian isolates(7/8),compared with those from Israel(3/8).3.Comparing of genotyped strains with database strains.The evolutionary relationship of the 60 isolates typed in this study assessed together with 206 additionalisolates retrieved from pubmlst/mbovis database analysed by geo BURST Minimum spanning tree(MST)confirmed that the Chinese,Israeli and Australian M.bovis isolates typed in this study that were predominantly ST-10,were clustered in CC3 with isolates originating from the USA.Thus,we hypothesized that the widespread distribution of ST-10 is a result of global livestock movements.These findings will help further the understanding of the global evolution of M.bovis and development of novel vaccines against M.bovis associated diseases.4.Using cg MLST to study the genotype of M.bovis strains from different country.Whole genome sequencing now allow high resolution for tracing of clonal population,here we have developed whole genome based a core genome multilocus sequence typing(cg MLST)scheme and applied onto 107 genome of M.bovis isolates of clonal and non-clonal isolates from China,Australia,Denmark and USA.We used the complete genome of M.bovis PG45 as the reference genome.Using all genome pairwise comparison of these 107 genome sequences resulted in 524 cg MLST targets present in all genomes with 100% overlap and >90% sequence similarity.Overall,>99.0% of the cg MLST targets were presented in all clonal and non-clonal isolates.Total of 524 core genes were retained as cg MLST target genes of which an average >90.4% to 99.0% were called successfully from 106 M.bovis genomes,while M.agalactiae(PG2)had 17.0%,M.mycoides sub.sp capri(PG3),M.ovipneumoniae(Y98)and M.arginine resulted in 0.00%,good targets,respectively.When tested against the clonal and non-clonal strains,we found cg MLST clusters were congruent with MLST-defined clonal group,which had various degree of diversity at the whole-genome level.Notably,cg MLST could distinguish among clonal and epidemiologically unrelated strians of the same clonal group,which could not be achieved using classical MLST schemes.Seventy-two M.bovis gnome form clonal group isolates had > 10 alleles differences and unambiguous differentiate form unrelated outgroup strians.Moreover,cg MLST revealed that there may be several sub-clones of emerging ST10 clone.In conclusion,the novel cg MLST scheme not only showed highly discrimination resolution as compared with PFGE and MLST insub-typing but also indicated capability to reveal more population structure characteristics than MLST.In summary,this study finds that only one dominant genotype M.bovis prevalent in China,which is coincident with Australian,American and Israel.Besides,this study confirmed that cg MLST is a more sensitive genotyping technique compared with PFGE and MLST.The results are meaningful for the control of M.bovis disease all over the world and the evolution of this pathogen.