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Development of a molecular tool for the differentiation of Mycobacterium bovis and molecular characterization of Mycobacterium bovis isolates in Malawi

Kapalamula, Thoko Flav 北海道大学

2021.03.25

概要

Bovine tuberculosis (bTB) caused by Mycobacterium bovis is a neglected tropical and zoonotic disease of animals and humans. BTB burden is high in developing countries because of the presence of multiple risk factors maintaining the circulation of the disease. The lack of surveillance tools and limited laboratory support has significantly contributed to the underestimation of the disease's burden. Hence there is a critical need for simplified and low-cost methods for the detection of M. bovis that can easily be integrated for use in developing countries. Additionally, molecular epidemiological studies become essential to provide information of circulating M. bovis strains necessary for formulating optimal bTB control approaches.

 In chapter I, a loop-mediated isothermal amplification (LAMP) assay was developed for specific detection of M. bovis by targeted the region of difference 4 (RD4), a 12.7kb locus that is deleted in all M. bovis strains but inserted in other Mycobacterium tuberculosis complex (MTBC) species. The assay's specificity was assessed by 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven MTBC reference strains, 22 non-tuberculous mycobacteria and five other bacteria. All M. bovis isolates were tested positive, while all other bacteria were negative. The LAMP assay detected 10 copies of M. bovis genomic DNA within 40 minutes.

 Considering that wet LAMP requires cold chain maintenance of reagents especially enzymes. In chapter II, dry LAMP assay was developed based on wet LAMP method established in previous study. Field evaluation was performed on cattle samples collected during routine post-mortem examination at the three regional abattoirs in Malawi. Additionally, clinical samples from the National TB reference laboratory in Lilongwe were also subjected to dry LAMP assay. A total of 1,547 cattle were examined, out of these 146 had tuberculosis - like lesions and samples were collected. Dry LAMP assay detected 82 (65.6%) as M. bovis while multiplex PCR performed on the same samples detected 78 (62.4%) as M. bovis. Out of 86 clinical isolates, only one was positive as M. bovis.

 In chapter III, the molecular epidemiology of M. bovis in central parts of Malawi was elucidated to gain insights into the transmission dynamics, sources, and circulating population of M. bovis strains. Samples were collected during the previous study. Molecular typing tools; deletion analysis, spoligotyping and MIRU-VNTR were performed. Our findings show that European 1 clonal complex (81%) and spoligotype SB0131 (56%) are dominant. We found a high genetic diversity of M. bovis in the area. The isolated strains showed genetic relationships with M. bovis isolates previously reported in neighbouring countries.

 The LAMP assay developed in this study provides a better option the detection of M. bovis in developing countries where the burden of bTB is high. The molecular findings of M. bovis in central parts of Malawi significantly increases the understanding of bTB in the area and suggesting the need for the development of new control strategies or enhance the current control programs to control the disease.

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