Discussion

In five out of 176 analyzed wild boars (2.8%; CI95%1.2 - 6.5) DNA appertaining to a member of the MTBC was detected by RT-PCR. The observed prevalence is in accordance with a previous study in wild boar originating from three different Swiss cantons, where the observed prevalence was 3.6% (Schoning et al., 2013). The present five samples were further analyzed by spoligotyping and molecular characterization based on 24 loci, revealing a common source of infection or a transmission chain of M. microti (Supplementary material). Based on these findings and the available information from literature, a transboundary presence in the Swiss and the Italian border region along the Province of Como is shown (Boniotti et al., 2014). The identical spoligotyping and MIRU-VNTR profile (ETRs loci) was observed by Boniotti and colleagues in five out of 26 isolates analyzed between 2007 and 2009. Over this period, a slightly higher prevalence of 5.8% was described in Italy. This suggests a persistent presence of one M. microti strain affecting the local wild boar population over at least a decade. The spoligotyping profile SB0118, shared by all isolates, is the only profile detected in Switzerland so far, independently from the host species and showing a large host range. Additionally to wild boars, this spologotype has been isolated from domestic cats, South American camelids and captive gibbons in Switzerland (unpublished results). Interestingly, different wild rodent species originating from hot spots areas examined for MTBC were all tested negative by molecular and cultural methods (manuscript in preparation). The predominant species identified in this study belonged to the MAC (Table 1). This complex comprises several clinically important mycobacterial species (van Ingen et al., 2018). M. avium (n=25) was the most prevalent member of the complex, followed byM. colombiense (n=3) , M. chimaera/intracelullare group(n=2) and M. vulneris(n=1) (Table 1). M. avium is a thermophilic slowly growing mycobacterium and comprises four subspecies, namely M. avium subsp. avium (Maa ), M. aviumsubsp. silvaticum (Mas ), M. avium subsp.hominissuis (Mah ) and M. avium subsp.paratuberculosis (Map ). Sequencing of the 3’ region of thehsp65 gene can unambiguously distinguish between these subspecies (Turenne et al., 2006). Mah has the broadest host range compared to the other members of the MAC, nevertheless, a clear differentiation between environmental and host-specific members of the MAC is necessary to better understand its distribution, host-adaptation and clinical implications (Turenne et al., 2006). Contrary to the observations from a Spanish study (Garcia-Jimenez et al., 2015), where MAC was isolated more often from subadults, no correlation between MAC and age of the animals was observed in the present survey. Overall, a significant increase of mycobacterial infection correlated with an increase of age (Figure 2), suggesting that the isolated mycobacteria are probably the results of a prolonged infection or colonization more than a transient presence. This is corroborated by the observation that co-infections, including one animal infected by M. microti , were mostly in adult animals and none of the wild boars from the juvenile group was infected by multiple species. Co-infections with MTBC/NTM or two different NTM have been described in animals and humans (Garcia-Jimenez et al., 2015, Gcebe and Hlokwe, 2017, Lim et al., 2011, Stepanyan et al., 2019, Yilmaz et al., 2017). In such cases, it is mostly unclear which agent infected first, and the host protective effect of the first infection from a second one remains unknown. A possible explanation for this increased number of co-infection in older individuals may be a cumulative effect with time and exposure. Overall no effect from NTM isolation and MTBC infection could be observed due to the small number of MTBC positive animals and one animal being co-infected with M. microti and M. neoaurum . In the past decades, various research articles attempted to describe MAC reservoirs and infection sources for humans and animals through molecular analyses. These included environmental surveillances, e.g. drinking water, bathrooms and hot tubs (Eisenberg et al., 2012, Falkinham et al., 2008, Hilborn et al., 2008). Noteworthy, certain studies reported a close genetic relatedness between human and swine isolates (Johansen et al., 2007, Mobius et al., 2006). In most cases, however, the source of infection remained unclear. The discrimination ofMap with the MALDI-TOF technique has been assessed in previous studies (Ravva et al., 2017, Ricchi et al., 2017). The 25 MAC isolates tested in the present study were all confirmed to be Mah by sequence analysis, and the first subspecies suggestion provided by MALDI-TOF was generally correct (80%). However, differentiation at MAC subspecies level with MALDI-TOF has to be performed with carefulness and might require additional gene sequence analysis. Wild boars (Sus scrofa ) are among the most widely distributed large mammals worldwide. Their natural range extends from Western Europe and the Mediterranean basin to Eastern Russia, Japan and South‐east Asia (Massei et al., 2015). The high mobility associated with the highest reproductive capacity among ungulates, enable an annual population growth rate of 250% under favorable food and weather conditions (Ebert et al., 2012, Gethoffer et al., 2007). Supplemental feeding of wild ungulates is prohibited by law in several Swiss Cantons, including Ticino. Despite this order and the annual population reduction by hunters, the population in the study area is rising, augmenting the animal-to-animal as well as the animal-to-human contact probabilities. Since the hunted animals of the present study were judged to be in an overall healthy condition suitable for human consumption, the Mah infection rate of 14% observed in the mandibular lymph node is noteworthy and may represent a veterinary public health concern. Only few publications extensively investigated the presence of NTM in wild boar (Garcia-Jimenez et al., 2015, Gortazar et al., 2011, Pate et al., 2016, Trcka et al., 2006). M. chelonaewas the most frequent NTM species isolated by García-Jiménez et al ., whilst Gortazar and colleagues did not detect M. chelonaein 124 wild boars tested. In the present study M. chelonae was not detected, suggesting a diversity of NTM species at geographical level. Members of the MAC were also irregularly detected: M. intracellulare , Maa , Mah and M. colombiense are nowadays classified as MAC members implicated as relevant pathogens in human and veterinary medicine. The dissection of the MAC at species or subspecies level is not always trivial (Turenne et al., 2006). It is therefore fundamental to implement advanced approaches in order to identify the exact species involved and estimate their relevance as potential infection source for consumers. Previous reports described NTM isolation rates in wild pigs varying from 8.9% (Trcka et al., 2006), 16.1% (Gortazar et al., 2011), 16.8% (Garcia-Jimenez et al., 2015) to 18.2% (Pate et al., 2016). In addition to the above mentioned NTM, M. peregrinum, M. nebraskense, M. lentiflavum, M. nonchromogenicum, M. engbaeki and M. septicumwere isolated from wild boar in recent studies from Brazil (Lara et al., 2011), Czech Republic (Trcka et al., 2006), Italy (Boniotti et al., 2014), Slovenia (Pate et al., 2016) and Spain (Garcia-Jimenez et al., 2015, Gortazar et al., 2011). Among the remaining species isolated in the present study for the first time,M. florentinum is noteworthy regarding the granulomatous lesions observed in the three affected animals. Wild boars are generally organized in social packs grouped around a nucleus of two or three sexually mature breeding sows. The rest of the group consists of newborn piglets and juvenile individuals (< 20 months of age) from the previous litter. Males are expelled from the pack by the time they reach sexual maturity. The average pack size can vary between 5 and 10 animals (Briedermann, 2009). The territory covered by packs may vary widely between geographical areas, strongly dependent from the landscape, presence of water and human influence. Packs covered 8-30 km2 in the Jura-region of Switzerland (Baettig, 1993), 2-40 km2 in southern France (Spitz, 1992) and 1-4 km2 in Italy (Boitani et al., 1994). A single group of animals tends to defends its core area: 1-3 km2 (Spitz, 1992), <1 km2 (Boitani et al., 1994) while the rest of the territory may overlap with home ranges of neighboring packs (Leuenberger, 2004). In contrast to packs, which tend to use only a small portion of their territory and move to another range at regular intervals, the adult male has a single territory that can range up to 50 km2. These individuals are able to move across the entire territory at daily basis, playing a crucial role in the spread of animal and zoonotic pathogens (Nugent et al., 2015, Schulz et al., 2019, Spitz, 1992). This high mobility may also be one of the possible explanations for the homogenous spread of the NTM species isolated across Canton of Ticino. Because of their rooting behavior and eating habits, wild boars are often in contact with environmental NTM species. Moreover the occasional consumption of dead small rodents and other carrion can enable direct transmission of pathogenic mycobacteria. However, this does not seem to cause generalization and clinical disease in all individuals, presumably because of the previously suggested genetic resistance of wild boars against bTB causing agents and possibly other mycobacterial species (Acevedo-Whitehouse et al., 2005, Dondo et al., 2007). An interesting finding was the presence of granulomatous lesions compatible with tuberculosis observed in a subset of lymph nodes analyzed. In particular, samples in which M. microti and M. florentinumwere detected, showed granulomatous lymphadenitis characterized by focal-extensive necrosis and in some cases dystrophic calcifications. These calcifications and the paucibacillary nature of the lesions suggests, however, a circumscribed and chronic process. Because of the design of the present study, it was not possible to assess the presence of further lesions in the wild boar carcass. Mandibular lymph nodes have been described to be the preferred entry point for mycobacteria in wild boars (Queiros et al., 2019). This is probably due to its eating habits allowing the intake of environmental contaminations and infected feed sources. After oro-nasal infection, viable microorganisms often concentrate in mandibular lymph nodes and from there are eliminated, persist or eventually disseminate throughout other organ systems. The mentioned lymph nodes are the most likely organ for visible lesion caused by MTBC in adult animals and in a relevant proportion of cases this is the only organ affected (Dondo et al., 2007, Martin-Hernando et al., 2007). The occurrence of visible lesions caused by M. microti in wild boars has been described previously (Boniotti et al., 2014). M. microti , although believed to be less pathogenic, has been described to cause extensive lesions, indistinguishable from those caused by other MTBC members in immunocompetent individuals (Frank et al., 2009, Geiss et al., 2005, Niemann et al., 2000, van Soolingen et al., 1998, Emmanuel et al., 2007). To the authors’ knowledge, this is the first time that similar lesions caused byM. florentinum are described in veterinary medicine. Classified as a slow growing Mycobacterium , M. florentinum is an opportunistic human pathogen isolated from immunocompetent and immunocompromised patients with various pulmonary disorders and lymphadenitis (Tortoli et al., 2005). Macroscopically and histologically the lesions caused by the two above mentioned species were indistinguishable. These findings highlight the importance of molecular characterization methods that allows a rapid and reliable differentiation of MTBC members from other NTM. The geographical distribution of M. florentinum appears to be widespread since human cases have been described in Italy, Finland, Japan and the US (Nukui et al., 2014, Syed et al., 2010, Tortoli et al., 2005). Interestingly, a significant proportion of Mycobacterium spp. other than M. microti and M. florentinum , were isolated from tissue samples that did not present granulomatous lesions or pathological finding in general (Table 2). Similar findings were recently described by two studies focused on lymph nodes from slaughter pigs (Mann et al., 2014, Muwonge et al., 2012). The presence of viable mycobacteria without evidence of histopathological granulomatous lesions might represent an early stage of the infection, which is not yet morphologically identifiable and in most cases probably results in the elimination of the microorganism (Mann et al., 2014). On the other hand, a certain contamination degree with environmental mycobacteria is not to be excluded. Because of the small size of the samples, disinfection of the mandibular lymph nodes superficial area was not possible. Based on the high sensitivity of the MTBC specific RT-PCR adopted, cross-contamination of the samples can be excluded. M. microti DNA was identified exclusively in lymph nodes presenting visible lesions and from animals processed on different days. Even though an infection was not proven with histology in all lymph nodes showing growth of Mycobacterium sp., the technique and instruments used for samples collection were above the ordinary hygiene standards adopted for meat processing. This indicates that an alarming high number of viable mycobacteria from different species are present in raw meat from hunted wild boars. Overall, MALDI-TOF MS showed to be reliable for the identification of NTM derived from veterinary specimens. Although the threshold recommended by the manufacturer enable the correct identification of only 72.1%, different authors evaluated lower cut-off values for mycobacteria with satisfactory results (Alcolea-Medina et al., 2019, Buchan et al., 2014, Mediavilla-Gradolph et al., 2015, Saleeb et al., 2011). In the authors’ opinion, an important drawback of the MALDI-TOF MS technology in comparison with sequence analysis of housekeeping genes like e.g. 16S rRNA, is that yet undescribed species will go undetected, either without any identification or as misidentification. In this study, three NTM species: M. colombiense (3/3 isolates) M. scrofulaceum(2/2 isolates) and M. monacense (one isolate) could not be identified although present in the MBT Mycobacteria Library 4.0. The most probable explanation for this is the restricted number of MSP (main spectrum profiles) present in the library used as reference. Moreover, one isolate of M. vulneris was misidentified as M. colombiense with a LSV of 2.08, demonstrating that closely related species still represent a challenge to be unambiguously identified and require sequence analysis for accurate assignment. An interesting aspect noticed during the present study, was the erroneous classification by BLAST analysis of the rpoB gene sequences derived from threeM. diernhoferi isolates tested. According to NCBI BLAST the closest species was M. aurum with a percentage identity score of 97%. The MALDI-TOF MS analysis suggested M. diernhoferi with an unequivocal LSV ≥ 2.0 for all three strains. Hence, a NCBI nucleotide search for M. diernhoferi rpoB sequences allowed the finding of an identical sequence, namely whole genome shotgun sequence derived from strain IP141170001 BioProject PRJNA354248. In our experience, this is a rare case, where a submitted sequence could not be found by similarity search, potentially leading to a misidentification of the analysed samples. Because NTM infections are not notifiable to public health authorities in most countries, data regarding the incidence and prevalence of diseases caused by these agents are lacking or difficult to compare between different countries. Switzerland is faced, as any other country, with NTM infections in humans (Kuznetcova et al., 2012, Latshang et al., 2011, Taillard et al., 2003), and identification of local potential infection sources is therefore of great relevance. Regarding the two species of NTM that have been most frequently isolated in the present study (M. avium subsp.hominissuis and M. nonchromogenicum ), MAC members have been described as the most common cause of NTM diseases in human in Northern Europe (Hoefsloot et al., 2013), Japan (Nishiuchi et al., 2017), Korea(Ko et al., 2018) and North America (Boyle et al., 2015). The classical localization affected by MAC is the respiratory system and in most cases, the source and route of infection remains unknown.
In conclusion, a remarkable number of mandibular lymph nodes collected from wild boars presented viable mycobacteria. Although the zoonotic risk of the isolated NTM remains unclear, it must be emphasized that, the hunted animals were intended for human consumption and among the isolated species (n=24), the large majority (n=18) has been described as human pathogens. In order to assess the possible role played by wild animals into the spread of mycobacteria, further epidemiological investigations including isolates from different sources are required. Moreover, the present findings show that, MALDI-TOF MS has a high concordance rate to the reference method and because of his rapidness, cost-effectiveness and high throughput, represent a valid diagnostic tool for identification of NTM species in veterinary medicine.