Table_1.xlsx

<p>Mycobacterium avium subsp. hominissuis (MAH) is an important zoonotic pathogen with raising global health concerns. In humans, MAH is one of the most widespread non-tuberculous mycobacterial species responsible for lung disease. In animals, MAH is frequently isolated from pigs; however, it is also an opportunistic pathogen for other mammals including cattle. To elucidate the genetic diversity of MAH in cattle, a molecular characterization of isolates (n = 26) derived from lymph nodes was performed. Fourteen isolates originated from slaughtered cattle with visible altered lymph nodes at meat inspection, whereas 12 isolates were from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Variable number of tandem repeat (VNTR) analysis was performed at 20 loci to examine genetic differences of isolates and to compare to previously reported VNTR data of human isolates from different countries. Genetic elements IS901, IS1245, IS1311, LSPA17, ITS1 sequevar, and hsp65 code were determined. Interestingly, two bovine MAH isolates harbored ISMav6 and hsp65 code 15, which so far has only been observed in human isolates. We supposed that VNTR data of Swiss samples would show clustering with European samples. Minimum spanning tree and unweighted pair group method using arithmetic averages analyses based on the VNTR data indicated a specific cluster of MAH isolates obtained from lymph nodes without any gross pathological changes of healthy slaughtered cattle. Comparing Swiss isolates with isolates from different other countries, no geographical clustering was observed; however, four Swiss isolates had an identical VNTR profile as human isolates from the Netherlands, the United States, and Japan. These findings indicate a possible public health issue.</p

Table_1_Strain diversity in Mycobacterium avium subsp. paratuberculosis-positive bovine fecal samples collected in Switzerland.XLSX

Paratuberculosis or Johne’s disease is a chronic intestinal disease in domestic and wild ruminants. It affects global dairy economy and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The objective of this study was to analyze strain diversity in MAP-positive fecal samples by using a particular single nucleotide polymorphism (SNP) distinguishing between cattle (C-) and sheep (S-) type MAP and analysis of SNPs within gyrA and gyrB genes differentiating between Types I, II, and III. Moreover, mycobacterial interspersed repetitive unit and variable-number tandem repeat (MIRU-VNTR) analysis using eight established loci was performed. A total of 90 fecal samples from diseased animals presenting diarrhea and/or weight loss, originating from 59 bovine herds across 16 cantons of Switzerland were screened by PCR for the MAP-specific F57 and IS900 genes and were further subtyped. 96.7% and 3.3% of the samples contained C- and S-type MAP, respectively. Ten INRA Nouzilly MIRU-VNTR (INMV) profiles, with a discriminatory index of 0.802, calculated based on 65 epidemiological independent genotypes, were detected: INMV 1 (33.8%), INMV 2 (23.1%), INMV 6 (16.9%), INMV 9 (9.2%), INMV 116 (4.6%), INMV 3 (3.1%), INMV 5 (3.1%) and INMV 72 (1.5%), including two novel INMV profiles, namely INMV 253 (3.1%; S-type III) and INMV 252 (1.5%; C-type). INMV 1, INMV 2, and INMV 6 comprised almost 75% of the F57- and IS900-positive samples. Typing data from 11 herds suggest that there are some herds with intra-herd diversity of genotypes. The results of this study indicate a heterogeneity of MAP in Switzerland.</p

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

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

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