Isolation And Identification Of Nontuberculous Mycobacteria From Cattle And Pig And Analysis Its Major Gene

Mycobacterium Including Nontuberculous mycobacterium and Mycobacterium tuberculosis and so on. Because of Nontuberculous mycobacterium's similar symptoms with tuberculosis, it is named. Nontuberculous mycobacterium is a Gram-positive bacteria,its Acid-fast staining is positive. To 2003, there are 95 kinds Nontuberculous mycobacterium have been found, most of them are pathogenic or opportunistic pathogen. In addition lung disease, it can cause a variety of animal other tissues disease, the clinical diagnosises are often reported misdiagnosed as tuberculosis. Although NTM and Mycobacterium tuberculosis are Mycobacterium, but the drug resistance are different, so can not be cured after the misdiagnosis as Mycobacterium tuberculosis. Meanwhile, most of the NTM on the PPD was negative, they are difficult to be found, that cause great losses to the livestock industry, so isolation and further research on the animal's NTM is necessary.NTM reports on the research, domestic and foreign literature, mostly published reports on human NTM, NTM few reports of animal. In this study,the research on animals NTM experiment is divided into four parts.1. Isolation and identification of non-tuberculosis mycobacteria:Collected 2,100 submandibular lymph nodes and mesenteric lymph nodes from pigs and cattle whose PPD test is negative in many parts of Jilin Province.These samples are grinded into a homogenized after treated with acid and are inoculated in L-J culture medium which is fresh, each sample was inoculated with three, cultured at 37℃, observed once every 3 days, continued 8 weeks. Acid-fast staining tests bacterials after growthing. Amplificed bacterias with L-J medium which are positive by acid-fast stain. Classified bacteria which are amplificed in several ways. The approaches taken have laboratory conventional methods, Biochemical methods, growth characteristics experiment and Molecular biology methods. Laboratory conventional methods:Bacteria are cultured at 28℃, 37℃,45℃on L-J medium and observe the growth of its. Biochemical methods:Heat-resistant catalase, nitrate reduction, Tween 80 hydrolysis, urease, aromatic sulfatase, iron uptake, tellurite reduction test bacteria isolated. Growth characteristics experiment:Application PNB medium, TCH medium, sodium chloride medium, medium picric acid, sodium glutamate glucose medium, MacConkey medium for identificating of bacteria isolated. Molecular biology methods:Application of heat shock protein hsp65 gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) Identification of bacteria isolated. Synthesis and analysis of the results of various methods, make the final identification. A total of 28 strains has been obtained. These mycobacteria were identified as follow:4 strains as Mycobacterium gilvum,2 strains as Mycobacterium scrofulaceum,2 strains as Mycobacterium intracellulare,6 strains as Mycobacterium senopi,1 strains as Mycobacterium bovis,3 strains as Mycobacterium fortuitum,2 strains as Mycobacterium szulgai,4 strains as Mycobacterium neoaurum.1 strains as Mycobacterium testudinis,1 strains as Mycobacterium gordonae,2 strains as Mycobacterium vaccae. The results show that although the samples were tested by PPD, but there are still non-tuberculous mycobacteria were isolated. Non-tuberculosis mycobacteria's epidemic situation in pigs, cattle is not optimistic. It shows the strong adaptability of non-tuberculosis mycobacteria that non-tuberculous mycobacteria have a higher epidemic undering the premise of feeding management and monitoring of the implementation and improvement of control measures,In recent years. In order to prevent a big impact by widespreading, we should be strengthening the monitoring of non-tuberculous mycobacteria, and prevention and control when preventionning and controlling Mycobacterium tuberculosis.2, Non-tuberculosis mycobacteria serologily group and antigenic analysis:Grouped non-tuberculosis serologily. Non-tuberculosis is inoculated on the Middlebrook 7H10 antigen medium. After growning of bacterial, using 0.3%formaldehyde in sterile saline preparars of immune preparations with dead bacteria and prepared immunization with viable preparations with sterile saline. Immune health rabbits in fourth, one week intervals, the first and second immunization with dead bacteria preparations, three or four times immunization with viable preparations. In the week after the forth immunization, antiserum with heart blood. Using phosphate buffer Containing carbolic acid washed non-tuberculosis on the antigen medium. After processing, is prepared antigen with PBS buffer. Make agglutination and agglutinin absorption test With anti-serum and antigen. Ultimately makeing non-tuberculosis serological grouping and analysis of antigens. The results is that non-tuberculosis were divided into five groups of serotype:NH1,NH2,NH5,ZH1,ZC4,ZC14 are one group,ZC2,ZC3,ZH9,ZH10 are one group,NH9,NC11,NC12,NC13,NC14,ZC11,ZC12 are one group,NH3,NH6,NC4,NC7,NC10,ZH5,ZH6,ZH13,ZC7,ZC8 are one group,NH8 are one group. Shows the distribution of serotypes of non-tuberculosis in Jilin Province is more specific classification structure.There is the value of Classification of further development and making control measures. Construction of DNA Library of Mycobacterium fortuitum that is a Nontuberculous Mycobacteria:Select Mycobacterium fortuitum from non-tuberculosis which has been recognized. Extracte whole genome by improving method and digeste genome with the restriction enzyme Sau3A I randomly. Through regulateing and controlling concentration of enzyme in system of gene fragments digested to digest gene in between 1000bp to 7000bp. The fragments are connected by way of random connections into pUC18 vector digested by BamHⅠ,then transform the prouduct in to DH5a made by ourself. The blue-white screening, PCR, restriction enzyme were used to detect the result of connection and get the positive connection efficiency. Scraping the positive bacteria from the surface of the medium. Stored at 80℃with glycerol.There are 4×104 positive have been gain in experiment.The genomic library is sucessful according to statistical calculation. The establishment of the library establish a good basis for the study of strain specific genes and gene structure and regulation. At the same time, to lay a foundation for the further study of other NTM.4 Application of genomic library and analysis of the hsp65 gene:Cloned the hsp65 gene fragment length of 413bp from Mycobacterium fortuitum's genomic library which has been prepared. After sequencing, using Blast, DNAStar software to analyze hsp65 gene to get the gene and evolutionary characteristics of Mycobacterium fortuitum. In this study,from the isolation and identification of the non-tuberculosis to serotyping.and to the construction of whole genome library and the application of genomic library, analysis of gene sequence.This study sets a processes for laboratory research on non-tuberculosis, lays the foundation for further research on non-tuberculosis in laboratory. At the same time,the date catched from the experiment provides the scientific basis for obtainning and controlling the latest epidemic of non-tuberculosis and make a rdasonable and effective control measures of non-tuberculosis.