Typing And Identification Of Mycoplasma Bovis In Ningxia And Its Effect On The Function Of Bovine Peripheral Blood Mononuclear Macrophages

Mycoplasma bovis(M.bovis)is a highly contagious bovine pathogenic disease.Due to the lack of effective prevention and control measures,it is currently circulating worldwide and has become a common bovine disease in China.Ningxia Hui Autonomous Region is the main area for cattle breeding in China,and its cattle breeding level and production performance rank among the best.However,the occurrence of M.bovis has seriously affected the healthy development of cattle breeding in Ningxia.In this study,we performed an epidemiological investigation of M.bovis,the genetic diversity evaluation of epidemic strains of M.bovis,and the infection mechanism of M.bovis to BMDMS.The results are as follows:1.In this study,ELISA was used to detect 1980 serum samples from all counties in Ningxia from 2016 to 2018.The results showed that the detection rate of M.bovis in 22 counties or districts was 100%.The seroprevalence of M.bovis in the three years showed an increasing trend,and the annual difference was significant(P<0.05).Wuzhong City had the highest positive rate.and the prevalence were differences among different counties.Epidemiological investigations had proved that M.bovis infection in Ningxia is widespread and the infection rate is increasing year by year.2.In order to investigate the structure and genetic diversity of M.bovis in Ningxia,the multi-site sequence typing(MLST)method was used to type 65 isolates from different areas in Ningxia from 2009 to 2018,which was analyzed and compared with domestic different ST strains and international strain PG45.The results show that there were only two types in Ningxia,including ST 10 and ST 134.The evolutionary tree revealed three different lineages:the Hubei-1 strain of ST26,the HBHS01 and Shananxi04 strains of ST172,the NHD0966 and EZ-2 strains of ST43 were all in the same lineage as the ST10 strain;the PG45 strain(ST17)and NMH7 strain(ST173)are in the same lineage;the strains of ST 134(43.1%)was an independent lineage.ST 134 strain is a new ST strain,first discovered in 2009 and currently only widely distributed in Ningxia.The ST10 strain has been widely spread in many provinces in China,and has been widely spread in Ningxia since 2010.3.In order to study the effects of different M.bovis of ST on the apoptosis and immune function of BMDMS.In this study,3 different ST-type M.bovis were used to infect BMDMS using different multiples of infection(MO1).The apoptosis,reactive oxygen species and different immune factors were tested at four different time points.The results showed that the apoptosis was inhibited for BMDMS infected with different ST-type M.bovis,and there was no difference among different ST isolates.M.bovis can inhibit the release of ROS.BMDMS can cause inflammation and immune killing by releasing cytokines such as IL-1β,1L-6,IL-12,IL-17,IL-23 and INF-α factors,and the antibody content was increases at 6h and 1h after infection,and then decreased at 12h and 24h.4.In this study,proteomics and transcriptomics were used to detect intracellular protein changes after infection of BMDMS with M.bovis,and differential protein screening,functional analysis,signaling pathway analysis,and apoptosis and antigen processing delivery were performed according to the 1.3-fold difference.The results showed that the infection of BMDMS with M.bovis could inhibit the down-regulation of the apoptotic protein BAX and promote the up-regulation of apoptosis inhibitory protein BCL-2,inhibiting the apoptosis of BMDMS and promoting the survival of the cells;the significant up-regulation of CDK9 and down-regulation of NELF promoted the continuous transcription and translation of BCL-2;M.bovis could inhibit the apoptosis of BMDMS by inhibiting the Caspase-dependent apoptosis pathway;there is no difference in the changes of calcium levels and cytochrome C,which once again proves that M.bovis could pass inhibits BAX and Caspase8-mediated endoplasmic reticulum pathway and mitochondrial communication,inhibiting apoptosis;the significant down-regulation of AIF inhibited DNA fragmentation.The CapG protein was significantly down-regulated,which affected the movement of BMDMS,which affected BMDMS secretion of MHC Ⅱ.The amount of BOLA-DR protein was down-regulated,the amount of HLA-DM protein was down-regulated,and the content of CD74/Ii chain was significantly down-regulated,which indicated that M.bovis could inhibit the processing and presentation of BMDMS on M.bovis and escaped the killing of the host cells,so that M.bovis can survive in the host cell.The Nramp1 protein was significantly down-regulated,which affects the killing effect of macrophages to pathogenic microorganisms and allows the M.bovis to survive in its cells for a long time.