Results

Of the 176 investigated wild boars collected during 2017 and 2018, mandibular lymph nodes from 101 (57.4%; CI95% 50.0 – 64.5) animals showed typical growth of Mycobacterium spp. (111 isolates in total) and AFBs by ZN staining were observed. The sequenced genes (16S rRNA, rpoB , hsp65 ) assigned 108 of 111 isolates to 24 described Mycobacterium species (Table 1). Three single isolates could not be classified to any known mycobacterial species and may represent new species. M. avium subsp. hominissuis(Mah ) predominated with 22.5% of the isolates, followed byM. nonchromogenicum with 21.6% of the isolates.
Five samples derived from three juvenile and two adult wild boars were positive by MTBC real-time PCR. Since no growth of mycobacteria over a 12 months incubation period could be achieved, species identification by direct spoligotyping using extracted DNA from the lymph nodes was performed. All five samples presented the same spoligotype signature SB0118 characterized by the presence of spacers 37-38 (www.Mbovis.org). The same signature is also known in the international spoligotyping database SpolDB4 as ST 539 and is indicative for M. microti(Brudey et al., 2006). Mycobacterial co-infections were detected in nine samples (5.1%), originating from adults (n=7) and juveniles (n=2) and comprising one M. microtipositive lymph node. Seven animals presented co-infections with two NTM (M. avium /M. vaccae , twice M. avium / M. nonchromogenicum , M. phlei /M. nonchromogenicum , M. nonchromogenicum /M. chimaera intracelullare group, M. scrofulaceum /M. florentinum , M. vaccae /Mycobacterium sp.) one animal presented co-infection with three NTM (M. fortuitum complex/M. engbaekii/M. vaccae ) and one animal M. microti/M. neoaurum . Molecular characterization based on 24 MIRU-VNTR loci was performed on four out of five M. microti containing samples and showed an identical code, suggesting a common source of infection or a transmission chain (Supplementary material S1). The geographical localization of the wild boars infected with M. microti is displayed in Figure 1A.
Adult wild boars were more prone to be infected with mycobacteria in comparison to juvenile animals or yearlings (Fig. 2). Overall, 74.4% of the analysed lymph nodes originating from adult animals showed growth of mycobacteria. Only 54.9% and 45.8% of the juveniles and yearlings presented viable NTM respectively (P -value < 0.05). A correlation between infected animals and their sex was not found, nor a significant association between the isolation of Mah and the three age groups (juveniles versus yearlings P = 0.151; juveniles versus adults P = 0.804; yearlings versus adults P = 0.384).
The distribution of the different NTM circulating among the analyzed animals was investigated based on the geographical data collected. The hunted wild pig population was divided into seven districts of the study area and, with the exception of the three northern districts where a low number of samples were obtained; a homogeneous distribution of the NTM cultured is shown in the remaining four districts (Fig 1B).