Tag Archives: Rabbit Polyclonal to MNT

In order to gain a better understanding of the molecular epidemiology

In order to gain a better understanding of the molecular epidemiology of isolates in Cameroon, 75 isolates of collected in three provinces of northern Cameroon were studied by spoligotyping. study. Bovine tuberculosis (TB) is endemic in many African countries, but economic constraints preclude the use of skin test and slaughter control strategies, which have proved effective in the developed world. In Cameroon, the majority of cattle herds are concentrated in the north (13), which is surrounded by Nigeria, Chad, and the Central African Republic. From visible lesion data obtained in the main slaughterhouses, it would appear that the prevalence of bovine TB in Cameroon is high (7). In addition, frequent cattle movement across the different areas of the country and across frontiers favors strain dissemination. In order to reduce the transmission of bovine TB, a bill from the Ministry of Livestock, Fisheries, and Animal Industries of Cameroon (no. 76/420) was introduced in 1976 to prevent the circulation of cattle GYKI-52466 dihydrochloride IC50 between Adamaoua and the other two areas of northern Cameroon, i.e., Extreme North and North. This action resulted in the isolation of cattle within Adamaoua. To date, few studies have been performed to determine the correct prevalence of infection at local and regional GYKI-52466 dihydrochloride IC50 levels (3, 16, 17, 19), and there are no available data regarding the variability of isolates within Cameroon. The aim of this study was to apply a number of molecular typing techniques to isolates from different slaughterhouses located in three different provinces of northern CameroonNorth, Extreme North, and Adamaouain order gain a better understanding of the geographical distribution of strains. The typing techniques used in this study were spoligotyping (11), pulsed-field gel electrophoresis (PFGE) (14) and, for some isolates, restriction fragment length polymorphism (RFLP) analysis with probe IS(1, 4, 18, 23). MATERIALS AND METHODS Mycobacterial strains. (i) isolates. Samples were collected in 1989C1990 and 1995C1996 from cattle in different slaughterhouses. These slaughterhouses were located in different provinces of northern CameroonNorth, Extreme North, and Adamaoua. This sampling regimen allowed the isolation of 123 isolates of which had classical cultural and biochemical properties (6). A total of 75 isolates were available for DNA typing. All 75 were subjected to spoligotyping, 65 were subjected to PFGE, and only 18 were subjected to RFLP analysis with probe ISstrains collected in northern Cameroon according to the DNA typing technique used (ii) Reference strains. DNA from H37Rv was used in order to obtain probe ISBCG (BCG Pasteur P3) was also used as a control in spoligotyping. Spoligotyping. For amplification of the direct repeat (DR) locus, we used either genomic DNA extracted by the method of Wilson (22) or cell lysates obtained by heat treatment. Spoligotyping was performed according to the technique of Kamerbeek et al. (11), as described for by Aranaz et al. (2). PFGE. Bacteria were grown in 40 ml of 7H9 broth (Difco, Detroit, Mich.) at 37C to the early exponential phase of growth. The cells were harvested by centrifugation, and PFGE was carried out with polymerase buffer (Roche/Boehringer), 100 M (each) deoxynucleoside triphosphate (Roche/Boehringer), 20 ng of H37Rv DNA, and 0.1 U Rabbit Polyclonal to MNT of polymerase (Roche/Boehringer). Fifty microliters of mineral oil (Sigma Aldrich, St. Louis, Mo.) was added. DNA amplification was performed using a Programmable Thermal GYKI-52466 dihydrochloride IC50 Controller thermocycler (MJ Research, Inc). Two series of cycles were performed: fives cycles at 94C for 1 min, 65C for 1.5 min, and 72C for 2 min and 35 cycles at 94C for 1 min, 60C for 1 min, and 72C for 2 min. These cycles were followed by a final elongation for 10 min at 72C. The amplified DNA was purified by extraction from a 1.2% agarose gel (Eurobio, les Ullis, France) in Tris-acetate-EDTA using a Geneclean II R kit (Bio 101 Inc., Vista, Calif.) according to supplier instructions. The.