Establishment Of Molecular Detection Techniques And Control Methods Of Tuberculosis In Dogs And Cats

Tuberculosis (abbreviated as TB for tubercle bacillus) is a common and often deadly infectious disease caused by mycobacteria, in humans mainly Mycobacterium tuberculosis. As far, TB in the human and livestock has been paid more attention rather than that in pets. There are two primary bacteria responsible for tuberculosis in dogs and cats, Mycobacterium tuberculosis and Mycobacterium bovis. Dogs can be infected with either type of bacteria but cats have been found to be quite resistant to M. tuberculosis and are primarily infected by M. bovis. Tuberculosis can be spread by inhalation of the bacteria or by ingestion of infected animal products. Pets infected with TB have subclinical symptom, easily confused with other respiratory diseases. Therefore, dogs and cats suffering from tuberculosis may be the most and the closest source of TB in human being. For the importance and emergency of tuberculosis study in pets, in our study, a molecular epidemiological survey of canine and feline TB in Shanghai was carried out by fluorescence quantitative PCR analysis. We are also actively exploring new ways in drug treatment and immunotherapy of TB in dogs and cats.1 Establishment of SYBR Green Quantitative PCR assay to detect tuberculosis in dogs and catsInsertion sequence 1081 (abbreviated as IS1081)of Mycobacterium tuberculosis was amplified and subcloned into TA vector.The gene was served as template DNA in Real-time Polymerase Chain Reaction (PCR) with SYBR Green I. The result showed that no primer dimer appeared from the melting curve, which showed annealing temperature was suitable. The optional reaction condition by Real-time PCR assay was determined with highly specificity and a broad linear range (1×107-1 copies/μL, and R=0.9995). The reaction efficiency is 1.09. The sensitivity of this method is lower than PCR assay, which approached 10 copies (DNA amount to about 31.4fg). In the sputum culture samples with TB positive, 19 out of 20 could be detected by this method, coincidence rate with about 95 %. The results showed the established real-time PCR assay was specific and highly sensitive with an extensive quantification range. 2 Molecular Epidemiological Survey of Mycobacterium Infection in Dogs and Cats352 samples of rhinorrhea, sputum, urine and pet food were collected from several districts in Shanghai.To the characterization of TB or Mycobacterium infection, total genomic DNAs were extracted and detected by PCR analysis. Several interest genes were chosen as below: 16S rRNA, IS6110, Rv3878, IS1081, ESAT-6 and CFP-10. The results showed that, in unhealthy pets, ESAT-6 and CFP-10 genes were more easily detected rather than that in healthy ones, with a ratio of 43% and 38.6%, respectively.However,16S rRNA positive samples in those ESAT-6 or CFP-10 PCR-positive ones was very low with a ratio of 7%～8%. What's more, Rv3878 (TB27.4), IS6110 or IS1081 could not be detected.The results of the primary molecular epidemiological survey indicated that unhealthy pets might have a higher chance to infect with nontuberculosis Mycobacterium (NTB).3 Evaluation on IFN-γlevel induced by the fusion protein CFP-10/ESAT-6 by ELISOPT assayCFP-10 and ESAT-6 genes were amplified from M. bovis, and fused with a (Gly4Ser)3 hydrophobic linker or protein transduction domain of HIV-1 TAT. And then the fusion genes were subcloned into pET32a (+) vector, which were designated as pCE106, and pCET, respectively. Transforming the plasmids into BL21 (DE3) and then induction with 0.1mM IPTG, the soluble fusion proteins were successfully expressed. Polyclonal antibodies were obtained from mice immunized with the protein. By immunoflorescence assay (IFA), the sera could specially react with the fusion proteins expressed by Bactobac eukaryotic expression system. Km mice immunized with the fusion proteins with 4 times. And then spleen cells were collected and separated the lymphocytes. Stimulated of CFP-10 (1-18aa) and ESAT-6 (1-16aa) peptides, the lymphocytes were detected and the level of specific IFN-γwas determined by ELISPOT assay. The results showed that protein transduction domain of TAT might have little effect on enhancing the immune response.4 Expression and purification of pantothenate kinaseCoaA gene, encoding pantothenate kinase (abbreviated as PanK), is a key regulatory enzyme for CoA synthesis in Mycobacterium. In this study, coaA gene was chosen and amplified from M. bovis and M. avium, respectively. The genes were subcloned into pET32a (+) vector. Induced with IPTG, PanK protein was successfully expressed. Of those proteins, a soluble peptide from M. avium PanK was purified by His-bind purification kit. In the future, activity of PanK and mutation in the enzymatic activity sites will be carried out to develop anti-TB drugs.