Molecular characterization of mycobacterium bovis isolates with high copy number of IS6110 by restriction fragment length polymorphism

Mycobacterium bovis is the causative agent of bovine tuberculosis and belongs to the Mycobacterium tuberculosis complex. M. bovis usually carries only one or a few copies of the insertion sequence IS6110 in its genome. The aim of this study was evaluation of copy number of IS6110 in M. bovis isolates by restriction fragment length polymorphism [RFLP] method. In this experimental study, 25 lymph node specimens of tuberculin-positive cattle were collected and cultured by standard methods, afterward genomic DNA was extracted by chloroform-isoamyl alcohol. Genetic studies were conducted by Pvull and DNA hybridization with IS6110. Two isolates displayed more than of 4 copies of IS6110 by RFLP [IS6110-RFLP] method. The results of this study are unique and specific in Iran but reported in the world rarely. Therefore the new strains of M. bovis imported to Iran from other countries of the world

Isolation and purification of low molecular weight proteins from liquid culture for mycobacterium tuberculosis by chromatography

Tuberculosis [TB] is a disease caused by a bacterium called Mycobacterium tuberculosis. M. tuberculosis has different molecular weight secreted antigens. Low molecular weightproteinssecreted into the culture medium by M. tuberculosisare thought to play an important role in the development new TB diagnostic tests and new vaccines against tuberculosis. In this report, we describe isolation and purification of low-molecular-weight proteinssecreted by M. tuberculosis. Initially by biphasic medium, bacteria from Lowenstein-Jensen solid medium transferred to a Dorset-Henley liquid medium and After 6 weeks of growth, the bacteria with a 0.22 micron filters of liquid medium containing secreted proteins were isolated and the secreted proteins was precipitated by ammonium sulfate. Protein concentrations were determined by using the lowry protein assay. Then low molecular weight proteins were purified by Sephadex-G75 gel chromatography and we studied purification of low molecular weight proteins by Coomassie-Blue stained SDS-PAGE. The results showed that low molecular weight secreted proteins purified from M. tuberculosis strain DT. Also, low molecular weight proteins made up approximately 65.3% of total proteins. This study demonstrated that without break down of bacteria bodies can be purified low molecular weight secreted proteins from M. tuberculosisliquid medium by Sephadex-G75 gel chromatography

Genetic structure change in harvard vaccine strain of clostridium tetani for the period of 1990 to 2010 by pulsed-field gel electrophoresis

PFGE facilitates the differential migration of large DNA fragments through agarose gel by constantly changing the direction of the electrical field during electrophoresis. Possibility of high difference between strains and repeatability make PFGE one of the strong molecular methods in study of bacterial strains in epidemiology. To identifying and DNA fingerprinting of vaccine strain of Clostridium tetani by PFGE technique. Also, possibility of genotyping profile changes in frequency of vaccine strain of C. tetani during the period of 1990 to 2011. The vaccine strain of C. tetani was provided by Razi Vaccine and Serum Research Institute in Karaj. The seeds were inoculated into Columbia blood agar and grown for 72 h. The cultures were incubated at 35[degree]C in anaerobic conditions. The PFGE analyses were performed using genomic DNA digested with the restriction enzyme SmaI. The electrophoresis analyses were carried out on a CHEF DR III apparatus [Bio Rad] and band patterns obtained were then analyzed. The PFGE profile obtained from vaccine strain during a period of more than two decades revealed no remarkable genetic changes and mutations. This type of analysis provides detailed data useful for surveillance of vaccine strains and isolates as well as for the selection of certain predominant profiles for further investigation. This study showed no considerable change in chromosomal genome of Harvard, the vaccine strain. It is therefore concluded that the vaccine produced by Razi Institute had evidently no alteration or modification in accordance to PFGE profile analysis during a period of more than two decades

Evaluation of relative potency of human tuberculin skin test in the guinea pigs sensitized with mycobacterium tuberculosis, M. bovis BCG and M. avium

The tuberculin skin test is the most commonly used test for diagnosing tuberculosis [TB] infection. The basis of tuberculin testing is the induction of a delayed hypersensitivity reaction to the intradermal injection of tuberculin. Unfortunately, this test is incapable of distinguishing Mycobacterium tuberculosis infection from Bacille Calmette-Guerin [BCG] vaccination or infection with non-tuberculous mycobacteria. The aim of this study is to evaluate the relative potency of human tuberculin skin test] produced by Razi Vaccine and Serum Research Institute [in the guinea pigs sensitized with M. tuberculosis, M. bovis BCG and M. avium. For skin test, different groups of guinea pigs were sensitized with M. tuberculosis, M. avium and M. bovis BCG. Guinea pigs were injected intradermally with 0.1 ml of 0.4, 2 and 10 micro g/ml of tuberculin. Skin reactions [diameters of erythema, in millimeters] were independently measured 24 h after injection and results were calculated. The results showed that the specificity index of human tuberculin test for guinea pigs sensitized with M. bovis BCG in compare of guinea pigs sensitized with M. tuberculosis was equal and for guinea pigs sensitized with M. avium was not equal. This study demonstrated that human tuberculin test produced by Razi Institute for diagnosis of latent infection to M. tuberculosis has lower specificity for M. bovis in comparison with M. avium