ANITA KONI1*, SILVA TAFAJ2, DANIELA LODA3, M. BEATRICE BONIOTTI3, M. LUDOVICA PACCIARINI3, XHELIL KOLECI1
1Faculty of Veterinary Medicine, Agricultural University of Tirana-Albania
2 National Reference Laboratory for Tuberculosis- University Clinic ʺShefqet Ndroqiʺ, Tirana-Albania
3Istituto Zooprofilattico Sperimentale della Lombardia e dell´Emilia-Romagna: Centro Nazionale di Referenza per la Tuberculosi Bovina, Brescia- Italy
*Corresponding author e-mail: firstname.lastname@example.org
Bovine tuberculosis is a contagious, chronic bacterial disease caused by Mycobacterium bovis. Typically, it is a respiratory infectious disease, but any body organ can be affected, especially well-oxygenated tissues. A very large range of domestic and wild animals are susceptible to M. bovis. Lack of clinical signs in the infected animals from early stage, presence of reservoirs and limitation on diagnostic methods interfere with successful control and eradication program of the disease. Differentiation of Mycobacterium tuberculosis complex by bacteriological and biochemical classic methods are time consuming. The last years, there are available new molecular methods used in diagnose and research and they play a crucial role in control and eradication programs of bovine tuberculosis. The aim of this study was to characterize M. bovis isolates by using updated molecular methods. In this study we analyzed four DNA samples extracted from M. bovis bacteria isolates. The bacteria culture was identified as M. bovis by cultural and staining methods. Molecular identification of isolates was done by a multiplex PCR assay and PCR products were visualized by electrophoresis in 2% agarose gel. The molecular characterization of bacterial DNA was performed by gyrB – restriction fragment length polymorphism (gyrB-RFLP), variable – number of tandem repeat (VNTR) typing using 12 markers ETR-A-BC-D-E-QUBS 11a-11b-26-1895-15-3232-MIRU26 and spoligotyping.All extracted DNA samples were identified as Mycobacterium tuberculosis complex, species Mycobacterium bovis. The gyrB-RFLP method identified that isolates belong to M. bovis. Genotyping method VNTR showed 7-5-3-3-3-27/28-3-3-4-3-6-4 tandem repeaters for each specific marker in correspondence to the exact same order described previously in material and methods. In addition, results of spoligotyping assay showed that M. bovis isolates in Albania are characterized by SB0989 spoligotype described for the first time in Germany-Europe. This study indicates that all mycobacteria isolates belong to Mycobacterium bovis subsp. bovis. Further molecular study based on genotyping and spoligotyping methods are needed for molecular epidemiological study and to link human cases with source of infection in framework of “One Health” philosophy.
Keywords: Albania, bovine tuberculosis, Mycobacterium bovis, spoligotyping