Evolution of the MTC in Africa

The MTC is believed to have had its origins between 40,000 and 70,000 years ago as a pathogen of humans occupying parts of Northeast Africa (Comas et al. 2013; Wirth et al. 2008).

6.2.1 Mycobacterium africanum

Mycobacterium africanum is the cause of up to 50% of all human TB cases in parts of West Africa but is uncommonly diagnosed elsewhere in the world despite human movement across the globe (Bentley et al. 2012; de Jong et al. 2010). For this reason, it has been postulated that a natural animal reservoir of this organism might exist in West Africa (Bentley et al. 2012; de Jong et al. 2010). However, to date, there is no evidence for this hypothesis, and M. africanum was not detected in an extensive survey of rodents and shrews in Benin (Durnez et al. 2010).

Moreover, despite widespread surveys of mycobacterial disease in livestock in West Africa, the isolation of M. africanum from domesticated animals has rarely been documented. In Ghana, the isolation of M. africanum from tissues of a skin­test-positive cow has been reported on a single occasion (Asante-Poku et al. 2014), while there are three reports of the isolation of this organism from tissues (Cadmus et al. 2006) and milk (Cadmus et al. 2010; Ofukwu et al. 2008) from cattle in Nigeria. It seems probable that these infections represent the transmission of this pathogen from human handlers that were in close contact with these animals (Asante-Poku et al. 2014). Mycobacterium africanum has also been isolated from three chimpan­zees (Pan troglodytes) and a guenon (genus Cercopithecus) (Thorel 1980). How­ever, as these animals were captive at the time of diagnosis, humans may again have been the source of these infections. Nonetheless, while M. africanum rarely appears to be associated with animal disease, such cases should probably be regarded as potential sources for reinfection of humans (Asante-Poku et al. 2014; Cadmus et al. 2010).

Mycobacterium africanum is a genetically diverse group of organisms comprising two major sub-lineages, i.e., M. africanum West African 1 (WAF1) and West African 2 (WAF2) (de Jong et al. 2010). The former sub-lineage is genetically characterized by the genomic deletion of RD711 but does not have deletions of RD7, RD8, or RD10 (de Jong et al. 2010). The WAF2 sub-lineage shares the deletions RD7, RD8, and RD10 with animal-associated members of the RD9-deleted clade and can be distinguished from these in harboring the unique deletion RD702 (Bentley et al. 2012). Mycobacterium africanum WAF2 displays a diversity of spoligotype patterns (de Jong et al. 2009) (Tables 6.1 and 6.2).

6.2.2 The Chimpanzee Bacillus

The chimpanzee bacillus has provisionally been named following its isolation, on a single occasion, from a chimpanzee with multi-organ TB from the Tai' National Forest, Cote d’Ivoire (Coscolla et al.

Table 6.1 Host species and geographic origin of selected African isolates of the MTC

Table 6.2 Spoligotype patterns of selected African isolates of the MTC

Isolate

M. orygis

Spoligotype pattern

—■ Il I I I I I Il I ■ I I Il I I I — ——

M. orygis

■■■ I I I I I ■■■■ I I I ■ I I I I I I ■■ ■■■■

SIT181

SIT326

WAF1

Reference

Gey van Pittius et al. (2012) Mostowy et al. (2005) van Ingen et al. (2012) de Jong et al. (2009) de Jong et al. (2009) Cadmus et al. (2006) Asante-Poku

et al. (2014)

Dassie bacillus

■■■■■■ ■ ■■■ I ■■■■■ I I I I I I I I I I I I I I I ■■■ I ■■■

Parsons et al.

Dassie bacillus

68/7171

■■■■■■ ■ ■■■■■ I ■■■ I I I I I I I I I I I I I I I ■■■ ■■■■

M. microti-like (dassie bacillus)

■■■■■■■■■■■■■■■■■ I ' I I I I ' I I I I I I I ■■■ ■■■■

M. mungi

M. suricattae

ill ιrrrrmι ιιιιιrιιιιι ιrrrι Iiiiiiiiiiiiiiii rm

van Soolingen et al. (1998) Lutze-Wal- lace et al. (2006) Alexander et al. (2010) Parsons et al.

(2013) Chimpanzee ■■■ I I I I I I I I I I I I I I I I I I I I I I I I I I I ■■■■■■■ ■ I ■ Coscolla bacillus et al. (2013)

This suggests that this species might have an as-yet unidentified preferred animal host. Chimpanzees are known to hunt and feed on a variety of animal species, and in the case described by Coscolla et al. (2013), transmission of the pathogen may have resulted from the ingestion of infected prey. Unlike other animal-adapted members of the African RD9-deleted clade, the RD1 locus has not been deleted in the chimpanzee bacillus, and this strain may represent an ancient lineage of the animal-adapted species. Its occurrence in Cote d’Ivoire provides further support for the assumed West African origin of the animal-associated lineage.

While the whole genome sequence of the chimpanzee bacillus is publicly available, only a limited number of defined genetic markers have been described for this organism. These include deletions of RD7, RD8, RD9, RD10, and RD900. Moreover, the organism shares a single nucleotide polymorphism (SNP) in the gene Rv1510 with other members of the African RD9-deleted clade (Coscolla et al. 2013).

6.2.3 The Dassie Bacillus

The dassie bacillus was first isolated in 1954 from a rock hyrax (Procavia capensis) in South Africa (Wagner et al.

In rock hyraxes, infection with the dassie bacillus results in lesions typical of TB in many other species, and the respiratory tract appears to be the primary site of infection and disease (Parsons et al. 2008; Wagner et al. 1958). However, in severe cases, granulomas can occur in multiple organs (Cousins et al. 1994). In contrast, experimental infection of various laboratory animals with the dassie bacillus typi­cally results in limited lesions suggesting that the organism is attenuated in these species (Cousins et al. 1994; Smith 1965). In part, this attenuated phenotype has been attributed to the deletion of RD1^das, which includes the genes Rv3874 and Rv3875 that encode the proteins culture filtrate protein 10 kDa (CFP-10) and early secretory antigenic target 6 kDa (ESAT-6), respectively (Mostowy et al. 2004).

There is only a single report of a natural infection with the dassie bacillus in a species other than the rock hyrax. This occurred in a suricate, or meerkat (Suricata suricatta), which was captured in South Africa and maintained in captivity with possible exposure to infected hyraxes (Mostowy et al. 2004; Parsons et al. 2008). Transmission of the pathogen to novel hosts is therefore possible, but it is currently unclear if the organism is confined to hyrax populations because of its attenuated phenotype or because of ecological isolation. While frank granulomatous disease has been detected in free-living hyraxes, there is currently no sound evidence that the pathogen might affect the population ecology of this species. Nonetheless, rock hyraxes can display significant fluctuations in population size (Barry et al. 2015), and TB in this species could conceivably contribute to this phenomenon.

The dassie bacillus can be genetically distinguished from other MTC members as having genomic deletions of RD1^das, RD5^das, RDVirS^das, and N-RD25^das (Mostowy et al. 2004). Furthermore it displays a characteristic SNP in Rv1510 (Rv1510¹¹²⁹), a single nucleotide deletion in Rv0911 (Rv0911³⁸⁹) (Huard et al. 2006), and typical spoligotype patterns (Parsons et al. 2008; van Soolingen et al. 1994) (Tables 6.1 and 6.2).

6.2.4 Mycobacterium mungi

Tuberculosis in free-living banded mongooses (Mungos mungo) was first diagnosed in 1999 during an epidemic affecting populations along the Chobe River in northern Botswana (Alexander et al. 2002). Following the investigation of ongoing outbreaks of TB in mongooses between 2000 and 2010 in this region, the causative pathogen was described as a novel member of the MTC and named Mycobacterium mungi (Alexander et al. 2010). Surprisingly, despite the organism displaying a deletion within the RD1 region, it is highly pathogenic in mongooses, and causes severe morbidity and mortality in infected populations (Alexander et al. 2002, 2010). Uniquely, transmission of this infection appears to be via injuries and abrasions of the skin and the nasal planum (Alexander et al. 2010, 2015, 2016b; Flint et al. 2016). Clinical disease may first present as anorexia, cachexia, and distortion and erosion of the nasal planum (Alexander et al. 2002, 2010). Affected animals may show a lack of fear of humans (Alexander et al. 2002), and advanced stages of the disease may be associated with increased fecal glucocorticoid metabolite levels (Laver et al. 2012). Notably, once clinical signs are observed, progression to death usually occurs within 2-3 months (Alexander et al. 2010). At necropsy, granulomatous lesions may be found in all organs, but they are consistently present in the liver, spleen, lymph nodes, and lungs (Alexander et al. 2002).

It is unclear if the severity of the disease in mongooses is primarily a function of the pathogenicity of the organism or the susceptibility of the host. Whichever the case, TB in mongooses can lead to the collapse of infected populations (Alexander et al. 2010). There are no reports of M. mungi infection in species other than banded mongooses.

Mycobacterium mungi shares the deletion N-RD25^das with the dassie bacillus (Alexander et al. 2010). However, these organisms can be genetically distinguished from one another as the M. mungi genome contains a unique RD1 deletion (RD1^mon), which has to date been poorly characterized (Alexander et al. 2010). It also does not contain the SNP Rv0911³⁸⁹ and displays a characteristic spoligotype pattern (Alexander et al. 2010) (Tables 6.1 and 6.2).

6.2.5 Mycobacterium suricattae

Since the 1990s, TB has been known to occur in a population of well-studied free- living meerkats in the Kalahari Desert, South Africa (Alexander et al. 2002). Initially believed to have been caused by either M. tuberculosis or M. bovis (Alexander et al. 2002; Drewe et al. 2009a), the causative organism has since been genotyped as a distinct member of the MTC and named M. suricattae (Parsons et al. 2013). Transmission of the infection is primarily via the respiratory route (Drewe et al. 2009a); however, grooming and aggression are also associated with increased spread of TB in this species (Drewe 2010; Drewe et al. 2011). The infection in meerkats results in severe, generalized disease, but it is commonly first detected by the presence of distinctly enlarged submandibular lymph nodes (Alexander et al. 2002; Drewe et al. 2009a). Following the onset of lymph node involvement, progression of the disease is rapid, and clinical signs such as cachexia and lethargy precede death (Alexander et al. 2002). Consequently, the disease can have a devas­tating impact on meerkat colonies and can cause local extinctions of affected populations (Alexander et al. 2002). On postmortal examination, granulomas can be found in all organs, but they are especially prominent in the spleen, lungs, and lymph nodes of the head and neck (Drewe et al. 2009a). Immunodiagnostic tests of M. suricattae infection have been described for meerkats and include both serolog­ical assays (Drewe et al. 2009b) and an assay of cell-mediated immunity (Clarke et al. 2017).

Mycobacterium suricattae shares numerous genetic markers with the dassie bacillus, including the genomic deletions RD1^das, N-RD25^das, RDVirS^das, and the SNP Rv1510¹¹²⁹ and SND Rv0911³⁸⁹ (Parsons et al. 2013). However, because of a major deletion of the genomic region analyzed by spoligotyping, M. suricattae is exceptional in having no spoligotype pattern (Parsons et al. 2013; Dippenaar et al. 2015). Similarly, the 16S ribosomal DNA sequence of this pathogen differs from all other MTC members (Parsons et al. 2013).

6.2.6 Mycobacterium orygis

Mycobacterium orygis has been isolated from various captive antelopes in the Netherlands and an oryx in Saudi Arabia (van Soolingen et al. 1994), dromedaries (Camelus dromedarius) in the United Arab Emirates (Wernery et al. 2007), cattle and a rhesus macaque (Macaca mulatta) in Bangladesh (Rahim et al. 2007; van Ingen et al. 2012), and at least ten human patients originating from the South Asian subcontinent (Gey van Pittius et al. 2012; van Ingen et al. 2012). The pathogen is therefore similar to M. bovis in its ability to cause clinical TB in a wide variety of species. Isolation of this organism in Africa has been reported on three occasions, all from animals in South Africa. In two instances, the source of the isolate was described as a captive oryx (Mostowy et al. 2005) and an antelope from a zoo (van Ingen et al. 2012); however, it is unclear if these were indigenous animals (i.e., the gemsbok, Oryx gazella) or an imported species (e.g., an Arabian oryx, Oryx leucoryx). In the third case, isolation was from a semi-free-ranging African buffalo (Syncerus caffer) (Gey van Pittius et al. 2012). However, although this animal was born in South Africa, it originated from a herd that had been partly established with buffaloes imported from a Portuguese zoo (Gey van Pittius et al. 2012). As such, this finding may reflect the importation of this organism rather than its natural occurrence in Africa.

Given the small number of TB cases caused by M. orygis, and the diversity of species that it infects, it is currently unclear whether this organism should be regarded as primarily a pathogen of animals or of humans. Moreover, the geograph­ical distribution of M. orygis may indicate the importance of global animal and human movement in its epidemiology. The organism shares the deletions RD7, RD8, and RD10 with the African RD9-deleted species; however, it is unique in having the deletions RD12oryx, RDoryx_wag22, RDoryx_1, and RDoryx4 (Mostowy et al. 2005) and a characteristic set of spoligotype patterns (Gey van Pittius et al. 2012; Rahim et al. 2007; van Ingen et al. 2012) (Tables 6.1 and 6.2).