Epidemiological tracing of bovine tuberculosis in Switzerland, multilocus variable number of tandem repeat analysis of <i>Mycobacterium bovis</i> and <i>Mycobacterium caprae</i>

<div><p>Background</p><p>After 15 years of absence, in 2013 bovine tuberculosis (bTB), caused by <i>Mycobacterium</i> (<i>M</i>.) <i>bovis</i> and <i>M</i>. <i>caprae</i>, reemerged in the Swiss dairy cattle population. In order to identify the sources of infection as well as the spread of the agents, molecular-epidemiologic tracing by MIRU-VNTR analysis in combination with spoligotyping was performed. A total of 17 <i>M</i>. <i>bovis</i> and 7 <i>M</i>. <i>caprae</i> isolates were cultured from tuberculous bovine lymph nodes and analyzed with a set of 49 genetic markers by using automated capillary electrophoresis.</p><p>Results</p><p>The outbreak in the western part of Switzerland was caused by <i>M</i>. <i>bovis</i> spoligotype SB0120. With the exception of four single-locus variations observed in MIRU 20, the MIRU-VNTR profiles of the 17 <i>M</i>. <i>bovis</i> isolates were identical, indicating a single source of infection. <i>M</i>. <i>bovis</i> detected in one archival bovine specimen from the outbreak region showed an identical MIRU-VNTR profile, suggesting persistence of the agent in a dairy herd for nearly fifteen years. The outbreak in the eastern part of Switzerland was caused by <i>M</i>. <i>caprae</i> spoligotype SB0418. All Swiss <i>M</i>. <i>caprae</i> isolates showed the Lechtal-type MIRU-VNTR profile, described as endemic in wild ruminants and in dairy cattle in Austrian bordering regions. This suggests the agent was most likely introduced by Swiss dairy cattle summering on Austrian pastures.</p><p>Conclusions</p><p>The present study is the first MIRU-VNTR analysis of Swiss bTB mycobacterial isolates. The genotyping assay was found to be highly discriminating and suitable for the epidemiological tracing of further outbreaks. These findings will contribute to the development of an international MIRU-VNTR database aiming to improve bTB surveillance.</p></div

Allelic ladder specific for <i>locus</i> VNTR 3232 (A) and VNTR 2163a (B).

<p>The commercial size marker provided by the manufacturer was replaced with band ladders generated by different repeat numbers of <i>locus</i> VNTR 3232 (A) and VNTR 2163a (B). Allelic ladders represent a satisfactory solution for the problem of overestimation error due to the different nucleotide sequences between the size marker and analyzed fragment.</p

Map of Switzerland showing the geographical origin of the positive samples.

<p>Green stars indicate <i>M</i>. <i>caprae</i>, red stars <i>M</i>. <i>bovis</i>, both positive by RT-PCR testing and successively in culture; yellow stars indicate samples positive by direct RT-PCR though mycobacteria were not isolated in culture. Swiss Cantons where cases of bTB were detected are highlighted. The free software QGIS was used for map design (Source of layers: Swiss Federal Office of Topography).</p

Probable bTB transmission by which the infection spread between cattle of different farms in the outbreak caused by <i>M</i>. <i>bovis</i> SB0120.

<p>Arrows indicate movements of confirmed infected animals. Since cattle from two farms (only RT-PCR positive samples) spent the summer months on pasture together with animals originating from both farm A and farm B, unequivocal epidemiological contact tracing at single animal level was not demonstrable. These two premises are therefore not displayed.</p

Allele profiles of Swiss <i>M</i>. <i>bovis</i> and <i>M</i>. <i>caprae</i> isolates compared with three reference strains in the 24 MIRU-VNTR standard panel (A) and in the 25 MIRU-VNTR additional <i>loci</i> (B).

<p>Allele profiles of Swiss <i>M</i>. <i>bovis</i> and <i>M</i>. <i>caprae</i> isolates compared with three reference strains in the 24 MIRU-VNTR standard panel (A) and in the 25 MIRU-VNTR additional <i>loci</i> (B).</p