| Mycoplasma bovis prefers to colonize respiratory tract and reproductive tract in cattle,causing mastitis,arthritis,calf pneumonia,otitis media and other diseases.Usually cows suffered co-infection with M.bovis and other pathogen,resulting in serious economic losses.In recent years,the disease of M.bovis with large-scale pasture has been increasing gradually,accompanied by the demand of etiology and diagnostic techniques in M.bovis.In this study,genome sequencing,pathogen characteristics,growth characteristics,biochemical characteristics and animal experiments were carried out based on clinical isolates from different samples.The results showed that the detection rate of M.bovis is 9～17% by using PCR method.1 Isolation and identification of M.bovisIn this experiment,10 strains of M.bovis were identified from the positive samples detected by PCR,colony morphology and biochemical characteristics experiments combined with PCR.Gram staining is not easy to stain,but Jimsa staining is blue and purple.The colonies are typical fried egg shape,and the morphology of bacteria is mostly sphere.No glucose is used and no arginine is hydrolyzed.OD value and p H value were taken as the ordinate and sampling time as the abscissa to draw the growth curve.The results showed that the OD value ranged from 0.011 to 0.174,the rapid growth stage lasted from 24 to 36h,and the plateau phase began after 42 h.The p H value showed a decreasing trend with the increase of time.The p H value decreased sharply within 24h～42h,and was relatively flat after 42 h.The mice virulence test of M.bovis strain was carried out.Some of the mice had fibrous encapsulation in the heart and inflammatory changes in the HE staining sections of the lungs.2 Epidemiological investigation of M.bovisThis study investigated the detection rate of M.bovis in samples submitted by farms in Anhui,Fujian and other provinces and cities from 2018 to 2020.A total of 2556 samples including milk,nose swab,lung,cartilage,etc.were detected,among which 327 samples were positive for M.bovis,with a detection rate of 12.8%.The number of samples from Hebei,Ningxia,Heilongjiang,Jiangsu and Inner Mongolia was higher,with the detection rate ranging from 9 to 17%,suggesting that M.bovis infection was widespread in local farms.In the cows with positive samples,it was found that most of them were infected by stealth,and some cows’ milk yield and milk quality decreased.Occasionally typical clinical symptoms such as calf pneumonia,and other pathogenic bacteria mixed infection,leading to a variety of cattle diseases,seriously hindered the development of local animal husbandry.3 Total Genome Sequencing of MycoplasmaIn this study,the whole genome of 14 clinical isolates was sequenced,and the bioinformatics software was used to conduct multi-locus sequence analysis with 12 endemic strains of M.bovis in China,and the minimum spanning tree was constructed.The genome-wide evolutionary map was constructed by pan-genomic analysis,which indicated the dominant epidemic strains in China,and laid a theoretical foundation for the epidemiology and prevention and control measures of M.bovis disease.In conclusion,this study investigated the epidemiology of M.bovis in some provinces and cities from 2018 to 2020,which can provide reference for the prevention and control measures of M.bovis.In terms of laboratory diagnosis of bovine mycoplasma,we can refer to the improved mycoplasma culture medium and PCR identification procedure in this experiment.In terms of the growth characteristics of M.bovis,it can be referred to that the strain entered the rapid growth phase between 24 and 36 h in this experiment,and the physiological activity of the strain was strong,which provided the basic theoretical basis for vaccine research and antigen coating.In terms of pathogenicity of M.bovis,the BALB/c mice model needs further study.Whole genome sequencing results have expanded theoretical data on genetic characteristics and pathogenesis of M.bovis. |
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