Epidemiology Of Bovine Tuberculosis And Isolation And Identification Of Its Pathogens

Bovine tuberculosis is one of chronic consumption of infectious zoonoticdiseases caused by Mycobacterium bovis. It is classified as a must-be-reported diseaseby OIE, while listed it to the2nd kind of quarantive animal disease in China. In ourcountry, tuberculosis is an enormous disease burden, about half of the populationcarry latent tuberculosis (TB) infection, that much higher than the global averageandmore than10.6%of human tuberculosis are caused by M. bovis. It has been estimatedthat50million cattle worldwide have been infected by M. bovis, resulting in annualeconomic losses of approximately3billion dollars.This disease causes economiclosses in livestock farming and poses a serious threat to public health and food safety.So enhancing the epidemiology and etiology of bovine tuberculosis, in order toresearch the prevalence station, it is important to prevent and control bovinetuberculosis. This research investigated the prevalence of bovine tuberculosis in someprovinces, isolated60strains of Mycobacterium from Xinjiang Uygur AutonomousRegionusing Two level-multiplex PCR to identify isolatesand to analyse drugresistance of M. bovis.(1)1187whole blood samples were collected from97farms throughout12provinces such as Xinjiang, Hebei, Ningxia Autonomous Region. The results,1187samples were detected with IFN-γ method，the individual positive rate is24.1%, grou-p positive rate is54.6％.(2) In present study, modified Lowenstein-Jensen solid medium, Sodiumpyruvate solid medium, the BACTEC MGIT960system and the BacT/Alert3Dsystem were used to isolate Mycobacteria from cow’s lymph node and lung samples.Two level-multiplex PCR were optimized and used to identify isolates. The first levelPCR was a duplex PCR respectively targeting special regions corresponding to thegenus Mycobacterium and Mycobacterium tuberculosis complex (MTBC) on16SrRNA. The second level PCR was a hepta-plex PCR with7pairs of primers targetingdifferent genomic deletion regions of MTBC members, and species weredifferentiated by amplified profiles particularly corresponding to each species. As aresults, we got60isolates of Mycobacteria. The first level PCR showed that，all of the60isolates belonged to the genus Mycobacterium, and51of which were MTBCmembers. The51MTBC isolates produced the same profile in the second level PCR,which was identical to M. bovis. The60strains also were subjected to a16S rRNAgene sequencing identification method for further confirmation, The51MTBCisolates was identical to M.bovis, the9NTM isolates was identified as4non-chromogenic Mycobacterium strains,2M.intermedium strains,2M.senuensestrains and1M.paraffinicum strain.(3) Drug resistance features for4first-line anti-tuberculosis drugs (RFP、INH、EMB、SM) were determined using drug susceptibility tests on whole cell level andPCR-based DNA sequencing targeting drug resistance related genes, rpoB, katG,inhA, embB and rrs. As a result, all of the51M.bovis do not detecte drug resistanceby both drug susceptibility tests and PCR-based DNAsequencing.The results show that the prevalence of bovine tuberculosis infection is very serious, quarantine is imbalanced in different provinces and epidemiological surveydata is not comprehensive in china. This study is to optimize the two level-multiplexPCR for identify isolates and demonstrated that this modified PCR, as a new detectionmethod in China, are rapid, rliable and low-cost.