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camelids and captive gibbons in Switzerland (unpublished results).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.