Animal tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium bovis, which has a wide host range affecting both domestic and wild animals, as well as humans. Cattle is the main host species for M. bovis. However, several wildlife hosts—i.e. badger, red deer, wild boar, brushtail possum—can carry and spread the pathogen to livestock (Palmer et al., 2012; Fitzgerald and Kaneene, 2013).

Infected animals with progressive disease shed the bacteria in mucus, saliva, aerosols, feces, milk, and sometimes in urine, vaginal secretions, or semen. TB can thus be transmitted by a number of routes, most commonly through inhalation, through contact with the excreta of infected animals (Phillips et al., 2003), as well as the ingestion of contaminated food and water (Allen et al., 2021). Mycobacterium bovis can survive in water and soil for long periods (Fine et al., 2011), raising the risk of infection.

Mycobacterium bovis represents a serious public health concern in both industrialized and non-industrialized countries, where the absence or inadequacy of TB control programs, immunodeficiency, malnutrition, or the adoption of social and cultural behaviors (e.g., close contact with animals and consumption of animal products, improper cooking practices and poor hygiene) increase the risk of human infection (Malama et al., 2013; Olea-Popelka et al., 2017; Mohamed, 2019). Mycobacterium bovis is also one of the biggest challenges facing rural communities and the farming industry, as it causes both direct and indirect economic losses. It increases morbidity and mortality, lowers milk and meat production, reduces fertility and hampers the commercial trade in animals (Pérez-Morote et al., 2020).

Molecular epidemiology based on genetic profiles of M. bovis, which consists of spoligotypes and mycobacterial interspersed repetitive unit-variable number tandem repeat codes (MIRU-VNTR), is recognized as a valuable tool for the identification of sources of infection, routes of transmission and host preference, and the understanding of M. bovis transmission dynamics within a population or an ecosystem (El-Sayed et al., 2016). In recent years, web technologies and geo-epidemiology based on Geographic Information System (GIS) technology, have emerged as useful tools in epidemiology to monitor several diseases in time and space, including human (Beiranvand et al., 2016; Gehlen et al., 2019) and animal (Zaragoza Bastida et al., 2012; Hauer et al., 2015; Proud Tembo et al., 2020; Reis et al., 2020; Skuce et al., 2020; Almaw et al., 2021; Reis et al., 2021) TB.

In Sicily, the largest island in the Mediterranean and one of Italy’s 20 regions, TB is still a major concern despite the implementation of state and local eradication programs for TB in cattle. Our group has recently conducted two molecular-epidemiological investigations based on the genotyping of M. bovis isolates from livestock and wild animals, first throughout the island (Amato et al., 2018), and subsequently in the province of Messina, in northeastern Sicily (Marianelli et al., 2019). A large variety of M. bovis strains, which were in several cases common to different species of livestock, were identified (Amato et al., 2018; Marianelli et al., 2019). Livestock reared in the district of Caronia, situated in the province of Messina, showed the highest share of TB both in the island as a whole, and in the province (Amato et al., 2018; Marianelli et al., 2019). More than 50% of TB cases in the province of Messina were isolated from Caronia-bred livestock (Marianelli et al., 2019).

To shed light on the potential risk factors affecting the persistence of M. bovis infection in livestock in the district of Caronia – we conducted an in-depth geo-epidemiological investigation, combining genotyping, geospatial (GIS), and phylogenetic analyses to investigate the distribution of TB and M. bovis genotypes in livestock, the genetic relationships among M. bovis isolates, the transmission dynamics of M. bovis infection and the role played by the ecosystem in the spread of the pathogen in livestock in this highly circumscribed, and at the same time geographically diverse, high-incidence area.

In previous studies, the district of Caronia—a small area of about 230 km²—emerged as having the highest number of TB cases among livestock in endemic Sicily (Amato et al., 2018; Marianelli et al., 2019). An intensive TB surveillance program implemented in Caronia between 2014 and 2018 offered us a rare opportunity to conduct an in-depth geo-epidemiological investigation of TB employing genotyping, geospatial (GIS), and phylogenetic analyses to investigate the transmission dynamics of M. bovis infection in the district and the role played by the ecosystem in the spread of the pathogen in livestock.

A large number of farms and animals were screened for TB infection. A total of 107 infected farms were found, and 589 M. bovis isolates were collected from livestock (cattle and black pigs).

Geospatial analysis of TB cases confirmed that the disease was spread widely across the district of Caronia. However, M. bovis-infected herds, many of which showed multiple and recurrent cases, were not randomly distributed but rather concentrated in specific areas or niches, according to the landscape structure. The highest number of TB cases mapped in a large hilly area in north-central Caronia, crossed by the Buzza stream. The geographic distribution of TB in cattle was similarly found not to be random in other TB-endemic regions, such as Zambia (Proud Tembo et al., 2020) and Mexico (Bastida et al., 2017), suggesting that both biotic and abiotic ecological factors play a role in the spatial distribution of TB in high-prevalence areas.

Combined genetic profiles have been widely used in epidemiological studies of M. bovis infection across the globe (Hauer et al., 2015; Carvalho et al., 2016; Egbe et al., 2017; Machado et al., 2018; Reis et al., 2020), and have proven useful in boosting the discriminatory power of M. bovis typing, thus providing more detailed data for the molecular-epidemiological analysis of this pathogen. In our 589 M. bovis isolates, we identified 62 different genetic profiles by combining 11 spoligotypes and 50 MIRU-VNTR codes.

Sicily has a long history of TB infection in cattle, especially in the province of Messina, according to the Epidemiological Veterinary Bulletins of Sicily. ¹ Our group has previously genotyped M. bovis isolates from livestock and wild animals throughout Sicily (Amato et al., 2018) and, more recently, from livestock in the province of Messina (Marianelli et al., 2019). We found a large number of genetically different M. bovis strains causing the disease. Numerous common genetic patterns were identified in farm animals from neighboring and even relatively distant districts of the province of Messina (Marianelli et al., 2019). A high number of different genetic profiles (10 major profiles and 52 minor profiles) were even found analyzing a single district of the province, namely the district of Caronia. It is probably that the high level of M. bovis heterogeneity we observed in both cattle and black pigs reared in Sicily is due to factors involving the geography of the territory, farming traditions and livestock trade that led to the failure of the national eradication program based on “test and cull” method in cattle and susceptible animals on the island.

Spoligotypes SB0120, SB0134, and SB0841 were the most prevalent in Caronia and affected both cattle and black pigs, in agreement with previous work by our group showing these spoligotypes to be the commonest in Sicily (Amato et al., 2018; Marianelli et al., 2019). The largest variety of spoligotypes was found in cattle. This is likely due both to the fact that cattle are the main host species for M. bovis, and to the fact that many more isolates from cattle were analyzed in this study. A larger number of black pigs should be studied to better understand the host susceptibility to M. bovis infection in our setting.

As expected, spatial analysis of the 10 most frequent genotypes (M1–M10) revealed that identical profiles were mostly found in livestock reared in the same or neighboring herds. Moreover, cattle and black pigs shared identical genetic profiles, confirming an intense intra- and inter-species M. bovis transmission in the area under study, in accordance with our previous study (Marianelli et al., 2019). Major profiles also showed geographic specificities in that they occupied specific spatial niches, as previously documented in Portugal (Reis et al., 2020), Northern Ireland (Hauer et al., 2015), Great Britan ² and France (Skuce et al., 2020).

Some genotypes (i.e., major profiles M3 and M5) were isolated mostly from farms situated west of the Caronia stream. It is likely that this mountainous area, characterized by steep slopes, has created physical barriers that prevented these profiles from spreading east. The sporadic TB cases due to M3 and M5 recorded in the eastern part of the district may be attributable to local livestock trading. Other profiles (i.e., major profiles M1, M2, M4, M7, and M10) were isolated from numerous herds grouped in a large hilly area ranging from the north to the center of the district, along the Buzza stream. The high density of herds both along the Buzza and in the surrounding hills may have promoted contact between infected and uninfected animals, especially in the dry season, at watering points along the stream, and may thus explain the high prevalence of M. bovis infection recorded in that area and the co-circulation of a high number of different major genotypes. Interestingly, profiles M6 and M8 were both found only in the eastern part of the district, east of a long rocky ridge, and grouped mostly on a hilly plateau where livestock share pastures. The physical barrier of the long rocky ridge may have prevented indirectly the spread of these two profiles to western Caronia, by restricting the movement of infected animals—both livestock and wildlife. The lack of the livestock trade between the eastern and western part of Caronia may also explain the above results.

The geographical distribution of major M. bovis genotypes observed in the Caronia district strongly suggests that geography and landscape morphology play a key role in the spread of this pathogen. Mountains with steep slopes and rocky ridges constitute physical barriers to animal moving and grazing, and consequently to the spread of infection. Conversely, pastures, plateaus and streams favor animal aggregation and thus increase the risk of inter- and intra-species contagion. A recent study conducted in Zambia identified the sharing of grazing grounds and drinking water as the most important risk factors in the spread of TB (Proud Tembo et al., 2020). The importance of water resources as indirect routes of cross-species TB transmission was highlighted also by others (Barasona et al., 2017).

Farming methods can significantly contribute to increase the risk of inter- and intra-herd transmission of TB. In Sicily, livestock is raised in small-scale farms, where multiple animal species are often reared together. Cattle and black pigs are the most common species of livestock in the area. Extensive agricultural practices are used for both cattle and black pigs throughout the year. Cattle are reared in grasslands and natural pastures, whereas black pigs are raised in free or semi-free roaming conditions in the woods and natural pastures of the Nebrodi Park. Cattle and black pigs may therefore come into contact with each other at shared pastures, waterholes and feeding sites, where infected animals may transmit the infection to healthy animals, be it within or between farms and/or species.

Major profiles, such as M1 and M3–M5, were isolated from non-neighboring herds as well, some of which quite distant from one another. These data point to the presence of multiple sources of infection. Presumably, beyond the above-mentioned extensive and multi-species livestock rearing systems, local livestock trade, livestock auctions and the exchange of stud animals between farms, which reflect local culture, may explain cross-contamination across the district. These are all factors known to have significant impacts on M. bovis transmission (Green et al., 2008; Martínez-López et al., 2014; Palisson et al., 2016; Fielding et al., 2020).

Contact with wildlife is regarded an additional factor driving the spread of TB infection (Proud Tembo et al., 2020; Reis et al., 2021). The district of Caronia is part of the Nebrodi Park. Wild mammals living in the park are likely to play an important role in the epidemiology of M. bovis infection. Caronia is mostly mountainous and covered in thick vegetation which provides habitat for hedgehogs, wildcats, martens and dormice. Unfortunately, the literature offers little information on the potential role of these wild mammals in M. bovis transmission to livestock. Delahay et al. (2002) reviewed the role of several wild mammals—i.e. badgers, foxes, stoats, weasels, otters, deer, lagomorphs and other small mammals—in the transmission of M. bovis to cattle in the United Kingdom. Although they documented M. bovis infection in several mustelids (i.e., stoats and weasels), they found no wild mammal, apart from the badger, to represent a significant, self-maintaining reservoir of TB infection (Delahay et al., 2002). In New Zealand, Lugton et al. (1995) isolated this pathogen from European hedgehogs with lung lesions, concluding that these animals might constitute a reservoir for the disease. In France, Michelet et al. (2018) described M. bovis infection in red foxes. Ultimately, however, the lack of sufficient data and proper surveillance systems on many wild mammals hampers the understanding of their epidemiological role in TB infection, and further studies are therefore needed. For the time being, not having considered wildlife in the present study, we are unable to draw any conclusions on the possible involvement of wildlife in the transmission of TB infection in the district of Caronia. We are planning a study to address this matter.

Contrary to the 10 major profiles, most of the remaining 52 minor profiles affected only one or two animals each. The minor profiles were genetically related to the major profiles, mainly to M2, and in most cases presented genetic differences at only one or two loci. It has been suggested that the presence of single or double locus variations is consistent with a clonal expansion of a founder strain (Reis et al., 2020). It is therefore possible that the minor profiles belonging to spoligotypes SB0120, SB0841, SB0134 are derived from M2, M4 and M6/M7, respectively. Yet, since multi-locus VNTR analysis and similar typing methods are often confounded by homoplasy (evolutionary reversals, convergences, parallelisms and lateral gene transfer), which complicates the ability to trace patterns of descent (Pearson et al., 2009), the precision and accuracy of the above results may be affected. Homoplasy can be mitigated by the use of a large number of loci (e.g., 24) (Reyes et al., 2012). In addition, integration of sub-genomic genotyping methods with whole genome sequencing (WGS) will enable highly accurate phylogeny estimations (Pearson et al., 2009). MIRU-VNTR analysis can therefore be used as first-order analysis to characterize M. bovis from a specific area. WGS can subsequently be used to study the transmission dynamics of observed epidemics and to quantify the extent and direction of transmission at landscape level (Biek et al., 2012). This landscape genetics approach will enable researchers to assess how environmental variables contribute to the distribution of M. bovis infection and influence microevolutionary processes of M. bovis, as previously discussed by Biek and Real (2010).

As far as the geographical distribution of minor profiles is concerned, the only minor profiles to form groups—m49 and m50—affected 11 and 10 animals, respectively (as the others numbered a couple of cases each). These profiles were localized mainly in the central and eastern parts of Caronia. Conceivably, the analysis of a larger number of TB cases caused by minor profiles would reveal the formation of groups and link them to the landscape, as observed for the major profiles. Future investigations and regular updates will provide a better understanding of their clustering behavior.

The low number of cases due to minor profiles, coupled with their scattered geographical distribution, may suggest that the minor profiles originated more recently than the more firmly established major profiles, or indicate a reduced transmissibility compared to the major profiles.

In conclusion, this study paints a complex picture of the epidemiological dynamics of TB in this very limited geographical area. Numerous M. bovis genotypes, transmitted within and between neighboring and non-neighboring herds have been identified, and several risk factors providing insights into TB transmission and persistence have been described. The landscape structures—i.e. slopes, rocky ridges, hills, steams, plateaus, shared watering points and pastures, local livestock trading, extensive farming practices in multispecies herds, may all have contributed to the spread and maintenance of M. bovis infection in Caronia. The large number of different M. bovis genotypes here described makes the epidemiological situation of TB in Caronia unique. Geospatial analysis coupled with genetic analysis has proven useful in understanding the variations in the occurrence of TB and the distribution of M. bovis genotypes across Caronia, as well as in identifying vulnerable animal populations and at-risk geographic niches in the district. Our results can help in the planning of effective, tailor-made TB surveillance plans in the at-risk subareas of the district, especially those crossed by streams and characterized by a high density of farms. A study of TB among the wildlife of the Nebrodi Park will be conducted as a next step for a more comprehensive understanding of the transmission dynamics of M. bovis in livestock in this setting.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

Ethical review and approval was not required for the animal study because sample were collected during routine abattoir inspections within official contexts based on the national legislation for cattle and research projects funded by the Italian Ministry of health for the Sicilian black pigs. No animals were slaughtered for the purposes of this study and no permission was needed.

CM conceived and designed the study and wrote the paper. FP, DI, MP, and VM collected the data. CM and VV performed the analysis and interpretation of data. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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