Establishment Of Lamp Detection Method For Mycoplasma Bovis And Characterization Of The Attenuated Strain By Culture In Vitro

Mycoplasma bovis(M.bovis) is an important pathogen causing a variety of diseases in dairy and feedlot cattle. M.bovis is often overlooked, Since no typical symptoms of the disease and often mixed infection with other pathogens. Scholars were increasingly aware of the dangers of M. bovis, as technology advances and ongoing research of the disease. M.bovis was firstly identified in 1961 from a case of mastitis. M.bovis-related diseases occur globally. So far, there are no effective antibiotics to treat pneumonia, mastitis, arthritis and other related diseases caused by M.bovis. No commercial vaccine is available either. Our group first reported outbreak of M.bovis disease which caused enormous economic losses in 2008 in China.This paper was divided into two parts. The first part, established loop-mediated isothermal amplification method beased on a specific gene uvrC for rapid deagnosis of M.bovis. The second part attenuated M.bovis by continuous passage in vitro method, and evaluated the virulence of the strain. This assay may contribute to research the virulence factors and attenuated vaccine. The detail of Studied as follows.1. A Loop-mediated isothermal amplification (LAMP) targeting UvrC of Mycoplasma bovis (M. bovis) was developed and evaluated. The assay specificity amplified only M. bovis, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The sensitivity of the LAMP assay in pure cultures was 10-fold more sensitive than that of the PCR assay, with a detection limit of 3.4×10 CFU. The accuracy of the LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The assay was applied to 74 specimens collected from cattle with respiratory disease or from healthy individuals, and compared to PCR assay. The sensitivity and specificity of the LAMP assay with regard to PCR were 100% and 73%. In conclusion, this study developed the rapid, more specific, and sensitive LAMP test for M. bovis detection in the clinical setting.2. Attenuated M.bovis used continuous passage method in vitro. MbovHB0801 strain as the primary subculture.This strain had strong pathogenicity by previous experiments proved. Among strains in different passages, no significant difference in the growth curve,but there were significant differences in protein by SDS-PAGE and Western blot assay. We used four different passages of strains numbered Fl,F115,F150.1,F150.2 infect animals to evaluate the virulence between strains. The results showed that the virulent of passgaes 150 times M.bovis had been reduced.In the four strains,F 150.2 infection groups had significantyl increased in body weight compared with Fl infection proups(P<0.01). F150.2 infection groups had lower score of lung lesions,no significant difference with control group (P>0.05),but Significantly reduced compared with F1 infection groups(P<0.01), F150.2 infection groups could induce good humoral and cellular immunity. Comprehensive comparison,this generation of strain had less virulent and could induce a good immune response.Therefore,we got a good attenuated M.bovis,the strain...