**4. Origins and evolution of leprosy**

#### **4.1. Genomics of modern leprosy**

Whole genome sequencing has revealed large numbers of pseudogenes in both *M. leprae* and '*M. lepromatosis*' [7, 8, 98–100]. These genomic studies revealed that both *M. leprae* and '*M. lepromatosis*' have undergone a reductive evolution in which extensive recombination events have occurred between dispersed repetitive sequences, leading to less than half of their genomes containing functional genes. In a preliminary study [7], it was indicated that the genome of '*M. lepromatosis*' (~3.22 Mb) was 1.6% smaller than that (~3.27 Mb) of *M. leprae* [98, 99]. A comprehensive parallel study gave a similar genome size of ~3.21 for '*M. lepromatosis*' [8]. Functional comparisons revealed that whereas *M. leprae* has a defective *heme* pathway, '*M. lepromatosis*' lacked several genes needed for amino acid synthesis [8]. It is apparent that '*M. lepromatosis*' is the closest known mycobacterial taxon to the established species of *M. leprae*. Phylogenetic analysis indicates that '*M. lepromatosis*' and *M. leprae* diverged from a most recent common ancestor (MRCA) about 13.9 million years ago [8].

#### **4.2. Evolutionary origins of leprosy bacilli**

**Figure 7.** A phylogeny of selected *M. leprae* strains. The phylogeny was derived from an alignment of genomic SNPs [46]; ancient strains are denoted in bold. Phylogenies were generated in MEGA7 [105], using Maximum Likelihood methods. Phylogenies based on Neighbour Joining methods generated similar dendrograms. The scale represents the number of substitutions per site. Bootstrap values were determined from 500 replicates. '*M. lepromatosis*' was used as an out-group

**Methods and comments**

**PCR**: ML RLEP 129/99 bp; MTB 123 bp/92 bp

indicate relative disease load for Kiskundorozsma-

ML ketomycolates for Szombathely Grave 6

not tested; **Lipids**: mycolates, mycolipenate and mycocerosates for Bélmegyer-Csömöki domb Grave 22

**PCR**: ML RLEP 129/99 bp; 111 bp; 80 bp and probe; RepLep 66 bp and probe; SNP typing indicates migratory patterns into Europe. Coinfections suggest role of MTB in

Daruhalom dűlő II Grave KD517

decline of European leprosy

products sequenced

**(CE)**

4th–5th 10th–16th 1st 10th–13th

Matheson et al. [69] 2009 Israel 1st **PCR**: ML RLEP 129/99 bp; MTB IS6110 123/92 bp

Molnár et al. [74] 2015 Hungary 7th–9th **PCR**: MTB IS6110 123/92 bp; IS1081 113 bp; ML

Various 6th–11th

**Table 2.** aDNA and lipid biomarker detection of ancient *M*. *leprae* and *M*. *tuberculosis* complex co-infections.

Minnikin et al. [29] 2011 Hungary 7th **PCR**: Not re-tested; **Lipids**: mycolates and mycocerosates

Minnikin et al. [29] 2011 Hungary 15th **PCR**: Not re-tested; **Lipids**: MTB methoxymycolates and

Nuorala et al. [97] 2004 Sweden 10th–13th **PCR**: ML RLEP 129/99 bp; MTB 123 bp/92 bp. Nested

(not shown). Subtypes are indicated in brackets.

20 Hansen's Disease - The Forgotten and Neglected Disease

Donoghue et al. [72] 2005 Egypt

Donoghue et al. [30] 2015 Central and

Cases are listed according to year of study.

**Authors Year Region Century** 

Hungary Israel Sweden

Eastern Europe

The deep origins of mycobacterial disease remain to be clearly defined [3, 47, 98, 99]. In contrast to tuberculosis, which appears to stretch back hundreds of thousands of years [57, 58], the earliest manifestations of human leprosy are found in skeletal remains only about 4000 years old [101]. However, the older participation of animal hosts cannot be ruled out, as it is increasingly evident that Pleistocene megafauna may have had a major involvement in tuberculosis evolution [58]. A possible ancestral organism to the organisms that cause leprosy may have been more like modern *M. haemophilum*, an emerging pathogen with a variety of possible natural reservoirs. The first significant link identified between *M. leprae* and *M. haemophilum* was established a quarter of a century ago in a study of the so-called 'phenolic glycolipids' (PGLs) [13]. As shown in **Figure 3**, the similarity in the oligosaccharide composition of the PGLs was striking and the mycocerosate profile (**Figure 2**) almost identical. This early key observation was subsequently reinforced by taxonomic studies that showed a close association of *M. leprae* and *M. haemophilum* [14, 15, 102]. Again, in studies comparing *M. leprae* and '*M. lepromatosis'*, *M. haemophilum* was the nearest neighbour [8, 39], as illustrated in **Figure 8**. The recent determination of a full genome (~4.23 Mb) for *M. haemophilum* confirmed the close link [16], as shown in **Figure 8**. *M. haemophilum* is consistently placed outside of the *M. leprae*/*M. lepromatosis* group but between *M. leprae* and other mycobacteria such as the *M. tuberculosis* complex. It was suggested that the reductive evolution of *M. leprae* and '*M. lepromatosis*' was not shared with the most recent common ancestor but started after the divergence of *M. haemophilum* from both taxa [16]. The relatedness of *M. haemophilum, M. leprae,* '*M. lepromatosis*' and related taxa is shown in **Figure 8**.

#### **4.3. Animal and environmental sources of leprosy ancestors**

To assess the involvement of ancient relatives of *M. haemophilum* in the evolution of leprosy bacilli, it is necessary to consider the ecological, environmental and animal host preferences of

the diseases can have been contracted from human sources. A direct evolutionary pathway from ancient squirrel-like animals to humans is unlikely, but it is possible that squirrels are representative of other animals that may have acted as environmental reservoirs. In the case of '*M. lepromatosis*', a geographical association between patients and Mexican field rats (*Rattus* 

The Distribution and Origins of Ancient Leprosy http://dx.doi.org/10.5772/intechopen.75260 23

Cases of tuberculoid nodular thelitis in both cattle [116] and goats [117] appear to be caused by uncultivable acid-fast species related to *M. leprae* and '*M. lepromatosis*'. However, the interrelationships between these agents, infecting cattle and goats, need to be defined more precisely before the disease can be considered as a true variety of leprosy. A complex scenario is emerging regarding the status of infections categorised as 'feline leprosy' [118–121]. After many early reports of diverse manifestations of cat leprosy, a definitive study clarified the scene [122]. It was apparent that the rat leprosy bacillus, *M. lepraemurium*, made a contribution to disease, but the influence of a novel uncultivable *Mycobacterium*, whose closest relative was *M. malmoense*, was noted. In a follow-up study [123], it was observed that younger cats were susceptible to *M. lepraemurium*, but more mature felines typically harboured the novel uncultivable agent. In an interesting development, PCR amplification of 16S rRNA sequences, from the uncultured feline agent AJ294740-6, showed that the greatest nucleotide identity was shared with *M. leprae* and *M. haemophilum*, as well as *M. malmoense*; indeed a specific additional nucleotide correlated with only with *M. leprae* [124]. This particular taxon, expressed in cases from eastern Australia, New Zealand and possibly Canada, has been provisionally labelled '*M. lepraefelis*' [121]. Three North American feline infections appeared to be caused by another uncultivable agent with close 16S rRNA relatedness to *M. leprae* and more distant affinity to *M. haemophilum,* among other species [125]. Initially labelled '*M. visibilis*', but more properly '*M. visibile*', this taxon remains uncharacterised and unfortunately unavailable for further study [120]. In a limited area of southeast Australia, studies of feline leprosy have revealed the presence of *M. lepraemurium* and an uncultivable novel agent, labelled '*M. tarwinense*'. This agent was indicated to be a fastidious member of the *M. simiae* complex [120, 126] so it does not appear to have a direct relationship with *M. leprae*

The precise interrelationships between all the bacterial taxa causing leprosy-like diseases require further study. It is clear that *M. leprae* or '*M. lepromatosis*' cause human leprosy and the same agents can routinely infect armadillos and squirrels. The apparent affinities of the feline leprosy taxon, labelled '*M. lepraefelis*', with *M. leprae* and *M. haemophilum* must be explored. The agents causing tuberculoid nodular thelitis in cattle and goats appear to have an affinity with established leprosy bacilli and this should be thoroughly investigated. In view of present uncertainties, it is premature to consider any concept of an *M. leprae* complex, as has been

*rattus*) suggests a possible environmental reservoir [8].

**4.4. Animal diseases resembling leprosy**

or '*M. lepromatosis*'.

discussed [6, 8, 118, 127].

**4.5. Overall interrelationships of leprosy affiliates**

**Figure 8.** A phylogeny of *M. leprae* strains and other mycobacterial species. Genomic sequence coding for DnaN [103] from illustrative mycobacterial species was aligned with Clustal Omega [104] and their phylogeny inferred with MEGA7 [105] using the Maximum Likelihood methods and the Hasegawa-Kishino-Yano model with possible invariant sites. Phylogenies consistent with this interpretation were obtained with Neighbour-joining methods and when concatenated amino acid sequence of conserved proteins was used in the alignment. Bootstrap values are derived from 500 replicates.

this taxon. *M. haemophilum* is slow growing, requires iron supplementation and prefers a low growth temperature of 30°C. The first description of *M. haemophilum* was as a pathogen causing skin infections, particularly not only in immunocompromised patients [106, 107], but also in healthy children [108]. In a range of children, a variety of other clinical manifestations were encountered [15]. In two instances, *M. haemophilum* infections mimicked the appearance of leprosy [109, 110] and a co-infection of *M. leprae* and *M. haemophilum* has been reported [111]. Also, animal infections are common, with zebra fish (*Danio rerio*) being particularly susceptible [15]. More recently, a heavily infected leatherback sea turtle (*Dermochelys coriacea*) was found [112]. Infection of a haemophiliac with *M. haemophilum* was linked to contact with raw shrimp [113]. This suggests that *M. haemophilum* can move freely in a variety of environments, but it does not give a clear indication whether there is a particular zoonotic host in which the evolution of *M. haemophilum* may have occurred.

As noted previously, both *M. leprae* and '*M. lepromatosis*' can cause disease in squirrels [11, 12, 43, 44]. The presence of leprosy in armadillos is long established [9, 10, 114, 115] and, indeed, the armadillo was a prime source of material for early studies of the leprosy bacillus [79–81, 83, 84]. It is apparent that infected armadillos can spread leprosy to the human population [9, 10]. However, the leprosy introduced into the Americas by human migration was passed on to indigenous armadillos [46] so they can be eliminated as an environmental evolutionary source. The involvement of squirrels in the UK is more intriguing as it is difficult to envisage how the diseases can have been contracted from human sources. A direct evolutionary pathway from ancient squirrel-like animals to humans is unlikely, but it is possible that squirrels are representative of other animals that may have acted as environmental reservoirs. In the case of '*M. lepromatosis*', a geographical association between patients and Mexican field rats (*Rattus rattus*) suggests a possible environmental reservoir [8].

#### **4.4. Animal diseases resembling leprosy**

this taxon. *M. haemophilum* is slow growing, requires iron supplementation and prefers a low growth temperature of 30°C. The first description of *M. haemophilum* was as a pathogen causing skin infections, particularly not only in immunocompromised patients [106, 107], but also in healthy children [108]. In a range of children, a variety of other clinical manifestations were encountered [15]. In two instances, *M. haemophilum* infections mimicked the appearance of leprosy [109, 110] and a co-infection of *M. leprae* and *M. haemophilum* has been reported [111]. Also, animal infections are common, with zebra fish (*Danio rerio*) being particularly susceptible [15]. More recently, a heavily infected leatherback sea turtle (*Dermochelys coriacea*) was found [112]. Infection of a haemophiliac with *M. haemophilum* was linked to contact with raw shrimp [113]. This suggests that *M. haemophilum* can move freely in a variety of environments, but it does not give a clear indication whether there is a particular zoonotic host in which the

**Figure 8.** A phylogeny of *M. leprae* strains and other mycobacterial species. Genomic sequence coding for DnaN [103] from illustrative mycobacterial species was aligned with Clustal Omega [104] and their phylogeny inferred with MEGA7 [105] using the Maximum Likelihood methods and the Hasegawa-Kishino-Yano model with possible invariant sites. Phylogenies consistent with this interpretation were obtained with Neighbour-joining methods and when concatenated amino acid sequence of conserved proteins was used in the alignment. Bootstrap values are derived from 500 replicates.

As noted previously, both *M. leprae* and '*M. lepromatosis*' can cause disease in squirrels [11, 12, 43, 44]. The presence of leprosy in armadillos is long established [9, 10, 114, 115] and, indeed, the armadillo was a prime source of material for early studies of the leprosy bacillus [79–81, 83, 84]. It is apparent that infected armadillos can spread leprosy to the human population [9, 10]. However, the leprosy introduced into the Americas by human migration was passed on to indigenous armadillos [46] so they can be eliminated as an environmental evolutionary source. The involvement of squirrels in the UK is more intriguing as it is difficult to envisage how

evolution of *M. haemophilum* may have occurred.

22 Hansen's Disease - The Forgotten and Neglected Disease

Cases of tuberculoid nodular thelitis in both cattle [116] and goats [117] appear to be caused by uncultivable acid-fast species related to *M. leprae* and '*M. lepromatosis*'. However, the interrelationships between these agents, infecting cattle and goats, need to be defined more precisely before the disease can be considered as a true variety of leprosy. A complex scenario is emerging regarding the status of infections categorised as 'feline leprosy' [118–121]. After many early reports of diverse manifestations of cat leprosy, a definitive study clarified the scene [122]. It was apparent that the rat leprosy bacillus, *M. lepraemurium*, made a contribution to disease, but the influence of a novel uncultivable *Mycobacterium*, whose closest relative was *M. malmoense*, was noted. In a follow-up study [123], it was observed that younger cats were susceptible to *M. lepraemurium*, but more mature felines typically harboured the novel uncultivable agent. In an interesting development, PCR amplification of 16S rRNA sequences, from the uncultured feline agent AJ294740-6, showed that the greatest nucleotide identity was shared with *M. leprae* and *M. haemophilum*, as well as *M. malmoense*; indeed a specific additional nucleotide correlated with only with *M. leprae* [124]. This particular taxon, expressed in cases from eastern Australia, New Zealand and possibly Canada, has been provisionally labelled '*M. lepraefelis*' [121]. Three North American feline infections appeared to be caused by another uncultivable agent with close 16S rRNA relatedness to *M. leprae* and more distant affinity to *M. haemophilum,* among other species [125]. Initially labelled '*M. visibilis*', but more properly '*M. visibile*', this taxon remains uncharacterised and unfortunately unavailable for further study [120]. In a limited area of southeast Australia, studies of feline leprosy have revealed the presence of *M. lepraemurium* and an uncultivable novel agent, labelled '*M. tarwinense*'. This agent was indicated to be a fastidious member of the *M. simiae* complex [120, 126] so it does not appear to have a direct relationship with *M. leprae* or '*M. lepromatosis*'.

#### **4.5. Overall interrelationships of leprosy affiliates**

The precise interrelationships between all the bacterial taxa causing leprosy-like diseases require further study. It is clear that *M. leprae* or '*M. lepromatosis*' cause human leprosy and the same agents can routinely infect armadillos and squirrels. The apparent affinities of the feline leprosy taxon, labelled '*M. lepraefelis*', with *M. leprae* and *M. haemophilum* must be explored. The agents causing tuberculoid nodular thelitis in cattle and goats appear to have an affinity with established leprosy bacilli and this should be thoroughly investigated. In view of present uncertainties, it is premature to consider any concept of an *M. leprae* complex, as has been discussed [6, 8, 118, 127].

The possible origins and interrelationships of all agents causing leprosy-like disease are summarised in **Figure 9**. The phylogeny of *M. haemophilum* with *M. leprae* and '*M. lepromatosis*' indicates a deep common ancestor for all three taxa [16]; this ancestor is provisionally labelled

'PROTOLEP' in **Figure 9**. This hypothetical taxon is considered to incorporate characteristic cell envelope lipids, such as the C34 mycocerosates found in *M. leprae* and *M. haemophilum* (**Figure 2**). Sensitive lipid biomarker analysis has the potential to help identify the uncultivable agents causing feline leprosy ('*M. lepraefelis*', '*M. visibile*') and tuberculoid nodular thelitis in cattle and goats (**Figure 9**). It is an open question whether these agents have any affinity with *M. leprae*, '*M. lepromatosis*' or *M. haemophilum*, but it seems likely that the feline cases that are associated with both *M. lepraemurium* and '*M. tarwinense*' [119, 120, 126] (**Figure 9**) are distinct. '*M. tarwinense*' appears to be an affiliate of the *M. simiae* complex, which appeared to have little phylogeny with *M. leprae* and related taxa until detailed genomic characterisation of nontuberculous mycobacteria indicated that particular *M. simiae* complex members (*M. florentinum*, *M. interjectum*, *M. sherrissii*, *M. triplex*) apparently have genes for PDIM synthesis (**Figure 9**) [128]. It would be of interest to discover if there is any similarity between the proven PDIMs of *M. leprae* and those suggested to be expressed by these members of the

The Distribution and Origins of Ancient Leprosy http://dx.doi.org/10.5772/intechopen.75260 25

An understanding of the origins and spread of leprosy depends on establishing detailed knowledge of the ancient genotypes and their correlation with modern disease. The overall scenario has been expanded by the recent characterisation of the distinct modern clade, currently labelled '*M. lepromatosis*'. The availability of a full genome for '*M. lepromatosis*' is allowing specific probes to be developed to search for ancient expression of this biotype. Ongoing research is demonstrating that subtle lipid biomarker differences may be of value in distinguishing '*M. lepromatosis*' from *M. leprae*. The overall picture for the global development of leprosy suggests that the ancient disease evolved into a number of recognisable clades in Africa/Eurasia. It is clear that leprosy was introduced into the Americas by human migration, and the disease was passed on to indigenous armadillos. The deeper origins of leprosy appear to be inextricably linked to relatives of the environmental taxon *M. haemophilum*. Diseases in cats, cattle and goats, with affiliations and resemblances to leprosy, require

For basic studies on leprosy and related mycobacteria, DEM and GSB are grateful for support from LEPRA (www.lepra.org.uk), in the form of Studentships: Gary Dobson 1980–1983, Gurdyal S. Besra 1987–1990, Susanne Hartmann 1991–1994. The Leverhulme Trust Project Grant F/00 094/BL supported the development of lipid biomarker detection of ancient leprosy (OY-CL, DEM, GSB). GSB acknowledges support in the form of a Personal Research Chair from Mr. James Bardrick and the UK Medical Research Council (grant MR/K012118/1) and

*M. simiae* complex.

**5. Conclusions**

detailed investigation.

**Acknowledgements**

Wellcome Trust (grant 081569/Z/06/Z).

**Figure 9.** Origins and interrelationships of agents causing leprosy-like disease. Proposed relationships requiring further study are indicated (?). 'PROTOLEP' represents a prototype taxon with the specific type of outer membrane lipids expressed in *M. haemophilum, M. leprae* and possibly '*M. lepromatosis*'. *M. simiae* complex (**1**) represents species (*M. florentinum*, *M. interjectum*, *M. sherrissii*, *M. triplex*) apparently expressing genes for PDIM synthesis; *M. simiae* complex (**2**) includes the remaining species [128].

'PROTOLEP' in **Figure 9**. This hypothetical taxon is considered to incorporate characteristic cell envelope lipids, such as the C34 mycocerosates found in *M. leprae* and *M. haemophilum* (**Figure 2**). Sensitive lipid biomarker analysis has the potential to help identify the uncultivable agents causing feline leprosy ('*M. lepraefelis*', '*M. visibile*') and tuberculoid nodular thelitis in cattle and goats (**Figure 9**). It is an open question whether these agents have any affinity with *M. leprae*, '*M. lepromatosis*' or *M. haemophilum*, but it seems likely that the feline cases that are associated with both *M. lepraemurium* and '*M. tarwinense*' [119, 120, 126] (**Figure 9**) are distinct. '*M. tarwinense*' appears to be an affiliate of the *M. simiae* complex, which appeared to have little phylogeny with *M. leprae* and related taxa until detailed genomic characterisation of nontuberculous mycobacteria indicated that particular *M. simiae* complex members (*M. florentinum*, *M. interjectum*, *M. sherrissii*, *M. triplex*) apparently have genes for PDIM synthesis (**Figure 9**) [128]. It would be of interest to discover if there is any similarity between the proven PDIMs of *M. leprae* and those suggested to be expressed by these members of the *M. simiae* complex.
