Advertisement

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Reservoirs and transmission routes of leprosy; A systematic review

  • Thomas Ploemacher,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

    Affiliation Faculty of Science, Freudenthal Institute & Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, the Netherlands

    ⨯
  • William R. Faber,

    Roles Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Faculty of Medicine, Department of Dermatology, University of Amsterdam, Amsterdam, the Netherlands

    ⨯
  • Henk Menke,

    Roles Conceptualization, Investigation, Writing – original draft, Writing – review & editing

    Affiliation Faculty of Science, Freudenthal Institute & Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, the Netherlands

    ⨯
  • Victor Rutten,

    Roles Writing – review & editing

    Affiliations Faculty of Veterinary Medicine, Utrecht University, the Netherlands, Dept of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Republic of South Africa

    ⨯
  • Toine Pieters

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing

    t.pieters@uu.nl

    Affiliation Faculty of Science, Freudenthal Institute & Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, the Netherlands

    ⨯

Abstract

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae) and the more recently discovered Mycobacterium lepromatosis (M. lepromatosis). The two leprosy bacilli cause similar pathologic conditions. They primarily target the skin and the peripheral nervous system. Currently it is considered a Neglected Tropical Disease, being endemic in specific locations within countries of the Americas, Asia, and Africa, while in Europe it is only rarely reported. The reason for a spatial inequality in the prevalence of leprosy in so-called endemic pockets within a country is still largely unexplained. A systematic review was conducted targeting leprosy transmission research data, using PubMed and Scopus as sources. Publications between January 1, 1945 and July 1, 2019 were included. The transmission pathways of M. leprae are not fully understood. Solid evidence exists of an increased risk for individuals living in close contact with leprosy patients, most likely through infectious aerosols, created by coughing and sneezing, but possibly also through direct contact. However, this systematic review underscores that human-to-human transmission is not the only way leprosy can be acquired. The transmission of this disease is probably much more complicated than was thought before. In the Americas, the nine-banded armadillo (Dasypus novemcinctus) has been established as another natural host and reservoir of M. leprae. Anthroponotic and zoonotic transmission have both been proposed as modes of contracting the disease, based on data showing identical M. leprae strains shared between humans and armadillos. More recently, in red squirrels (Sciurus vulgaris) with leprosy-like lesions in the British Isles M. leprae and M. lepromatosis DNA was detected. This finding was unexpected, because leprosy is considered a disease of humans (with the exception of the armadillo), and because it was thought that leprosy (and M. leprae) had disappeared from the United Kingdom. Furthermore, animals can be affected by other leprosy-like diseases, caused by pathogens phylogenetically closely related to M. leprae. These mycobacteria have been proposed to be grouped as a M. leprae-complex. We argue that insights from the transmission and reservoirs of members of the M. leprae-complex might be relevant for leprosy research. A better understanding of possible animal or environmental reservoirs is needed, because transmission from such reservoirs may partly explain the steady global incidence of leprosy despite effective and widespread multidrug therapy. A reduction in transmission cannot be expected to be accomplished by actions or interventions from the human healthcare domain alone, as the mechanisms involved are complex. Therefore, to increase our understanding of the intricate picture of leprosy transmission, we propose a One Health transdisciplinary research approach.

Author summary

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae) and the more recently discovered Mycobacterium lepromatosis (M. lepromatosis). The two leprosy bacilli cause similar stigmatizing pathologic conditions. M. leprae primarily targets the skin and the peripheral nervous system. Currently it is considered a Neglected Tropical Disease. The transmission pathways of M. leprae are not fully understood. Solid evidence exists of an increased risk for individuals living in close contact with leprosy patients, most likely through infectious aerosols, created by coughing and sneezing, but possibly also through direct contact. However, this systematic review underscores that human-to-human transmission is not the only way leprosy can be acquired. Anthroponotic and zoonotic transmission have both been proposed as modes of contracting the disease, based on data showing identical M. leprae strains shared between humans and armadillos. A better understanding of possible animal or environmental reservoirs is needed, because transmission from such reservoirs may partly explain the steady global incidence of leprosy despite effective and widespread multidrug therapy. Reducing transmission cannot be expected from the human healthcare domain alone, as the mechanisms involved are complex. Therefore, we propose a One Health transdisciplinary research approach.

Citation: Ploemacher T, Faber WR, Menke H, Rutten V, Pieters T (2020) Reservoirs and transmission routes of leprosy; A systematic review. PLoS Negl Trop Dis 14(4): e0008276. https://doi.org/10.1371/journal.pntd.0008276

Editor: Carlos Franco-Paredes, Hospital Infantil de Mexico Federico Gomez, MEXICO

Received: November 21, 2019; Accepted: April 7, 2020; Published: April 27, 2020

Copyright: © 2020 Ploemacher et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Leprosy, also called Hansen’s disease, results from infection with Mycobacterium leprae (M. leprae) or Mycobacterium lepromatosis (M. lepromatosis). It is a chronic infectious disease which primarily affects the skin and peripheral nerves. It ranges from a localized to a systemic infection. Damage of peripheral nerves can lead to serious impairment and disability.[1] Transmission pathways of M. leprae are not fully understood. Solid evidence exists of an increased risk for individuals living in close contact with leprosy patients, most likely through infectious aerosols, created by coughing and sneezing, but possibly also through skin to skin contact.[2,3] Multidrug treatment (MDT) developed in the 1980s is an effective chemotherapy and has played a critical role in the worldwide reduction of the leprosy burden, by reducing human-to-human transmission.[4] However, despite widespread application of this therapy, the World Health Organization recorded 208,619 new leprosy cases globally in 2018, only slightly less than the 219,075 cases in 2011.[5] Even though leprosy has been eliminated in most countries, in certain areas endemic leprosy persists.

Hansen (1874) was the first to report rod-shaped bodies resembling bacteria in cells from leprosy patients using a light microscope.[6] Before that, leprosy was thought to be of environmental or hereditary nature.[7] After the discovery by Hansen, attempts were made to grow the pathogen on an array of artificial media and in numerous animals with the purpose of studying its characteristics, and potential treatments. With an improved tissue staining technique called Ziehl-Neelsen (ZN) it became possible to detect and visualize acid fast bacilli (AFB); and the Wade-Fite modification for the demonstration of M.leprae. In cases of leprosy, unspecified non-cultivable AFB were found.

M. leprae is an obligate intracellular pathogen that has never been cultured in vitro but can be cultivated in vivo in experimental animals. Shepard demonstrated in the early 1960s that the mouse footpad (MFP) could be infected with M. leprae.[8] Characteristic of this model are the local infection and the slow replication rate.[8,9] Armadillos had been used successfully in medical research, and their cool body temperature of 32–35°C attracted the attention of leprosy researchers. In 1971 Kirchheimer and Storrs, working in Louisiana (USA), reported the first successful experimental infection of the nine-banded armadillo with M. leprae.[10,11]

Until 2008, leprosy was thought to be caused exclusively by M. leprae. Analysis of leprosy patients in Mexico by Han et al. showed that a second species, M. lepromatosis, causes leprosy as well.[12]

M. leprae and M. lepromatosis diverged from a common ancestor about 13.9 million years ago (Mya). The two pathogens are similar in genome size (∼3,27 MB), and were subject to extensive reductive evolution.[12,13] Protein-coding genes share 93% nucleotide sequence identity.[14] Using genome scale comparison, the worldwide distribution and evolution of M. leprae have been studied. Phylogenetically, at least four SNP types (1–4) and five branches (0–4) and 16 SNP subtypes (A to P) have been distinguished. Global distribution is thought to have coincided with human migration.[15,16]

In medieval times, leprosy was endemic all over Eurasia.[17] Currently, this so-called Neglected Tropical Disease is endemic in regions within countries of the Americas, Asia, and Africa. In Europe, it is now only rarely reported, and infection is acquired outside Europe. The reasons for a spatial inequality of leprosy in endemic pockets within a country are still largely unexplained.

Further research is required to clarify transmission mechanisms and natural reservoirs of leprosy pathogens.[18] In the Americas, the nine-banded armadillo (Dasypus novemcinctus) has been established as a natural host and reservoir of M. leprae, and identical M. leprae strains are shared between humans and armadillos.[19,20] A reservoir can be defined with respect to a target population as ‘one or more epidemiologically connected populations or environments in which a pathogen can be permanently maintained and from which infection is transmitted to the target population’.[21] Hence, both anthroponotic and zoonotic transmission have been proposed as modes of transmission. However, Scollard argued recently that there is a general bias towards human-to-human transmission of leprosy.[22]

In 2016, both M. leprae and M. lepromatosis DNA was found in red squirrels (Sciurus vulgaris) with leprosy-like lesions in the British Isles.[23] This was highly unexpected, primarily because leprosy was considered to be a disease of humans (with the exception of the armadillo), and secondly, because M. leprae was thought to have disappeared from the United Kingdom. The M. leprae strain isolated from red squirrels is essentially the same as the one that circulated in humans in medieval England and Denmark, and is closely related to the strains carried by armadillos in the southern United States.[23] Transmission routes from animal reservoirs are possibly interrelated with the environment in an elaborate ecological cycle, which has yet to be unraveled. This may partly explain the steady global incidence of leprosy, as effective and widespread multidrug therapy reduces only human-to-human transmission.

This systematic review aims to provide an overview of worldwide research regarding non-human reservoirs and transmission. Potential natural reservoirs and additional transmission routes of leprosy are discussed.

Method

A systematic review was conducted targeting leprosy transmission research data using PubMed and Scopus as sources. The review protocol was not pre-registered at PROSPERO. The search strategy was composed of three topics: wildlife research, environmental research, and studies on zoonosis and vectors of leprosy. Search strategies have been listed in the PRISMA flowchart (Fig 1). PRISMA stands for Preferred Reporting Items for Systematic Reviews and Meta-Analyses. It is an evidence-based minimum set of items for reporting in systematic reviews and meta-analyses.[24]

thumbnail
Download:
Fig 1.

https://doi.org/10.1371/journal.pntd.0008276.g001

Wildlife research looked to find studies on known reservoirs, the armadillo and the red squirrel and prevalence, and on new reservoirs. Environmental research revolved around the possibility of survival of M. leprae or M. lepromatosis in extracellular conditions. Knowledge on the transmission of leprosy is limited, and any evidence for leprosy transmission by animals or insects was targeted through the search on zoonosis and vectors.

Publications between January 1, 1945 and July 1, 2019 were included. Articles that appeared with a title translated to English qualified for inclusion. Translations from Portuguese were obtained using an online translation service, and a Chinese article was translated with the help of a native colleague. This enabled an inclusion process as described in Fig 1. Three searches were performed, on leprosy in wildlife, environmental sources of leprosy, and leprosy zoonosis and vectors, using the following search strings.

Scopus, wildlife: TITLE-ABS-KEY (leprosy) OR TITLE-ABS-KEY (m.leprae) OR TITLE-ABS-KEY (m.lepromatosis) AND TITLE-ABS-KEY (wildlife) OR TITLE-ABS-KEY (animal).

PubMed, wildlife: (Leprosy OR M.leprae OR M.lepromatosis) AND (wildlife OR animal)

Scopus, environment: TITLE-ABS-KEY (m.leprae OR m.lepromatosis OR leprosy ) AND TITLE-ABS-KEY (environment OR ecology OR soil OR water ).

PubMed, environment: (leprosy OR m.leprae OR m.lepromatosis) AND (environment OR ecology OR soil OR water)

Scopus, vectors: TITLE-ABS-KEY (m.leprae OR m.leprae OR leprosy ) AND TITLE-ABS-KEY (zoonosis OR vector OR insect OR arthropod OR amoeba).

PubMed, vectors: (leprosy OR m.leprae OR m.lepromatosis) AND (zoonosis OR vector OR insect OR arthropod OR amoeba).

Articles from the search were included by TP (first author) based on title, then on abstract, and finally on a full-text assessment. Articles were included when any relevance to the primary subject of the search was assumed. Exclusion criteria were used to reduce the number of irrelevant studies. For the wildlife search subject, studies on experimental inoculation of animals, studies performed ex vivo, and drug animal studies were rejected. For the other search subjects, inclusion for relevance based on title and abstract was less strict to reduce loss of relevant abstracts. In vitro studies were accepted, for instance, because these studies could provide relevant evidence for the survival of M. leprae outside a host. Fig 1 shows the number of articles selected and rejected during the search process. 81 of the articles used in this article were selected in this way. To maximize finding eligible articles, the citations and references of included articles and related reviews were investigated (“snowballing”). This yielded an additional 64 articles.

Data extracted in the results were predominantly qualitative. Quantitative data were copied from the full texts. In some cases, when percentages were not available in the texts, they were calculated. Some prevalence data of armadillos were pooled. This was done to reduce the prevalence reporting in this systematic review, instead of mentioning prevalence in each region. No meta-analysis has been performed with quantitative data.

Methods of studies and result interpretation were considered, but risk of bias was not systematically scored. A discussion of the limitations of some studies is incorporated where these could have influenced the findings or conclusions made. It was not possible to formally assess publication bias due to the variation of study methods. For the PRISMA checklist accompanying the PRISMA flow diagram of the search we refer to the online supplementary data: S1 PRISMA Checklist.

Results

Wildlife reservoirs

The nine-banded armadillo in the southern United States (see Table 1).

In 1975, Walsh et al. reported seven nine-banded armadillos with leprosy-like clinical signs from different locations in Louisiana.[25] In a large scale follow-up study 1033 armadillos from different locations in Louisiana as well as the neighboring states Texas, Florida and Mississippi were examined.[26] A total of 50 animals (4.8%) were found with naturally acquired leprosy-like disease. The prevalence varied from 0% to 29.6%, depending on the location where armadillos were caught. On average, the prevalence in Louisiana was 7.1% (n = 691) and in Texas 1.1% (n = 88). The histopathological pattern and the presence of acid fast bacilli nine-banded armadillos resembled lepromatous leprosy, and suggested that it was caused by M. leprae.[27,28]

thumbnail
Download:
Table 1. Prevalence of M. leprae in wildlife 9-banded armadillos in the United States.

https://doi.org/10.1371/journal.pntd.0008276.t001

There was scientific controversy about the origin of leprosy in wild armadillos.[29] The possibility of laboratory spillover was considered, and the absence of mycobacterial infection in armadillos from the same region was reported.[30,31] Clinical and epidemiological investigations defined the disease in these armadillos, and confirmed its identity with human leprosy.[32,33] The concern of the possible wildlife contamination caused by experimentally infected armadillos addressed in 1986 by Truman et al. using a new technique. Phenolic glycolipid I(PGLI) is a M. leprae specific antigen, and by an enzyme-linked immunosorbent assay (ELISA) anti-PGL I antibodies can be detected in serum samples.[34] They tested stored blood samples from wild nine-banded armadillo which had been collected between 1960 and 1964, prior to the first reported experimental infection of armadillos in 1971. Positive samples confirmed that leprosy infection in armadillos predated the laboratory experiments.[35]

Gradually, the extent of natural infection of armadillos with M. leprae in the southern United States (Alabama, Arkansas, Florida, Georgia, Louisiana, Mississippi and Texas) became apparent with infection rates between 0% and 7,5%.[36–41] Truman et al. (1990) showed a discrepancy between histopathological findings and serological findings in an armadillo study population.[42] Histopathological presence of AFB in the ear was associated with late stage and disseminated disease, while early stages could be detected by anti-PGL I antibodies in serum. This was shown in 216 nine-banded armadillos captured in Louisiana: 12.3% of these animals were positive for anti-PGL I, while only 2.7% of the armadillos were positive on histopathological examination. Truman et al (1991) proceeded with the investigation of 565 nine-banded armadillos from three sites in Louisiana and one site in Texas. Of the 35 Texan armadillos, 17.1% had anti-PGL I antibodies, and 5.7% had AFB. Of the 530 Louisiana armadillos, 15.8% had anti-PGL I antibodies, and 3.4% had AFB.[43]

With the introduction of the Polymerase Chain Reaction (PCR) technique, new investigations were initiated. Job et al. compared PCR detection rates with other methods for detection of M. leprae.[44] Thirty armadillos were investigated. PCR of lymph nodes showed that 16 animals (53.3%) were infected with M. leprae, while a positive ELISA for anti-PGL1 and lepromatous granuloma were found in 2 (6.7%). Disease signs were found at autopsy in 13.3% of these armadillos. In 2002, Paige et al. studied the prevalence of and a possible relationship between Trypanosoma cruzi and M. leprae infection in 415 wild nine-banded armadillos from Louisiana.[45] Antibodies against pathogen specific antigens were detected using ELISA. The average prevalence of antibodies against M. leprae was 19% and against T. cruzi 3.9%. Morgan and Loughry (2009) studied nine-banded armadillos in western Mississippi and could not find clinical signs of leprosy in young or juvenile armadillos, suggesting no vertical transmission.[46] Loughry et al. (2009) studied the possible spread of M. leprae eastward from Mississippi, Alabama and Georgia into Florida.[47] Armadillo samples were examined for anti-PGL I antibodies and PCR for the detection of M. leprae. Infection prevalence ranged from 0–10%. In the eastern sampling sites that were considered M. leprae free, a few infected armadillos were found. They concluded that M. leprae was spreading eastward, although prevalence rates were still much lower than in the populations west of the Mississippi river. According to Williams and Loughry (2012) the prevalence of infected armadillos in the southern United States, measured by anti-PGLI antibodies ranged from 4.5% in 2005 to 14.9% in 2010, with an average of 9.3% over a six year period.[48] Perez-Heydrich et al. (2016) studied demographic and spatial risk factors for M. leprae exposure of nine-banded armadillos in Western Mississippi.[49] GPS locations of the armadillos were determined by tagging armadillos. Over a period of six years (2005–2010), 74 of 466 animals (15.9%) were seropositive for anti-PGL I. No spatial risk factors were found, and the study indicated that M. leprae infection is spatially homogeneous in the armadillo population in Western Mississippi.

Armadillos in other parts of the Americas (see Table 2).

The presence of M. leprae and leprosy in wild nine-banded armadillos was discovered in the southern United States, and most studies on its prevalence have been performed there. Armadillos are common throughout the southern United States nowadays, but their geographic range extends from northern Argentina up to Mexico. They migrated from Mexico into the United States.[20] Only the nine-banded armadillo is common in the southern United States, but other armadillo species are indigenous to South America. Whereas wildlife studies almost exclusively focused on the nine-banded armadillo, susceptibility for M. leprae infection by experimental inoculation was also observed for other armadillo species like the Venezuelan or Llanos long-nosed armadillo (Dasypus sabanicola) [50,51] and the seven-banded armadillo (Dasypus hybridus).[52,53] In 1984, Amezcua et al. reported the occurrence of a wild armadillo infected with M. leprae in Mexico.[54] They found acid-fast bacilli in ears, tongue and lymph nodes. Homogenates of infected nodules and lymph nodes did not grow on Löwenstein-Jensen and 7H10 media. Mouse footpad inoculation using skin and lymph nodule suspensions showed growth comparable to inoculation with M. leprae. Martinez and colleagues reported a captured nine-banded armadillo in Argentina with mycobacteriosis with nerve involvement.[55] Staining and biochemical analysis as well as mouse footpad inoculation strongly suggested M. leprae. Deps et al. (2007, 2008) studied the presence of anti-PGL-I antibodies in Brazilian armadillos from the state of Espirito Santo. In their 2007 study, 29.7% of animals (n = 37) were positive, and in their 2008 study, 10.6% of the animals (n = 47) were positive.[56,57] Frota et al. (2012) examined 27 nine-banded armadillos, and 2 six-banded armadillos in the state of Ceará, Northeast Brazil, a leprosy endemic area.[58] Samples from the ear, nose, liver and spleen were examined by a nested M. leprae-specific repetitive element (RLEP) PCR assay. Five of the nine-banded armadillos, and 1 six-banded armadillo were positive for M. leprae specific DNA.

thumbnail
Download:
Table 2. Prevalence of M. leprae in wildlife armadillos in other parts of the Americas.

https://doi.org/10.1371/journal.pntd.0008276.t002

In 2009, Deem et al. reported on three-banded (Tolypeutes matacus) and nine-banded armadillos in the Gran Chaco, Bolivia.[60] Three-banded (n = 2) and nine-banded (n = 8) armadillos were negative for anti-PGLI antibodies. Cardona-Castro et al. (2009) examined armadillos in the forest areas surrounding cities with leprosy patients in Colombia during a two year period.[61] Ear lobe biopsies from 22 nine-banded armadillos were PCR analyzed for the M. leprae-specific repetitive element (RLEP) region. PCR was positive in 40.9% (9 of 22) of the armadillos.

Pedrini et al. (2010) examined 44 armadillos, from the mid-Western area of the state of São Paulo, and the Pantanal Floodplain of the state of Mato Grosso do Sul in Brasil.[62] They examined 18 nine-banded armadillos (D. novemcinctus), 22 six-banded armadillos (Euphractus sexcinctus), two greater naked-tailed armadillos (Cabassous tatouay), and two Southern naked-tailed armadillos (Cabassous unicinctus). In addition, 10 road killed animals were also analyzed. None of the samples were positive for M. leprae by PCR.

Squirrels (see Table 3).

Experiments showed that ground squirrels (Ictidomys tridecemlineatus) were susceptible to infection with M. leprae. Galletti et al. (1982) observed AFB counts in these squirrels that indicated multiplication under hibernation inducing conditions.[63] Recent studies have shown that red squirrels (Sciurus vulgaris) in England and Scotland (where the endangered red squirrel is competing with the non-native gray squirrel from North America) are infected with M. lepromatosis or M. leprae. Meredith et al. (2014) reported a novel presentation of a dermatitis in 6 red squirrels from various locations throughout Scotland. All animals showed bilateral areas of variable alopecia and cutaneous swelling of the snout area, lips, eyelids, pinnae and the distal parts of all limbs.[64] Histological examination on three squirrels showed granulomatous dermatitis, and epithelioid macrophages forming sheets, in addition to large numbers of AFB. PCR of various dermatitis causing pathogens was performed. Sequencing of the hsp65 PCR amplicons from the squirrels revealed 99% sequence homology with M. lepromatosis (FJ924). The exact nature of the mycobacterium involved and the characterization required further research. In view of these findings, another British research group re-examined four stored red squirrels with epidermal hyperplasia originating from the Isle of Wight and Brownsea Island. The presence of M. lepromatosis was confirmed by PCR in all four samples.[65]

thumbnail
Download:
Table 3. Prevalence of M. leprae and M. lepromatosis in wild squirrels.

https://doi.org/10.1371/journal.pntd.0008276.t003

A follow-up study was conducted to map the magnitude of infection in the UK.[23] The study also first reported neural involvement in red squirrels infected with M. leprae (n = 8) and M. lepromatosis (n = 4). Samples from 13 squirrels with and 101 animals without leprosy-like symptoms were examined using differential PCR for M. leprae and M. lepromatosis. M. lepromatosis was found in red squirrels from Scotland (6/44), Ireland (2/39), and the Isle of Wight (1/1).[66] All squirrels from Brownsea Island (n = 25) were positive for M. leprae. 21 squirrels without clinical signs and all 13 animals with clinical signs were PCR positive. Anti-PGLI ELISA was positive in a significant number of the red squirrel samples (see Table 3). Gray squirrels were also tested by PCR (n = 3) and for anti-PGLI antibodies (n = 4), but all samples were negative. Phylogenetic comparison of British and Irish M. lepromatosis with two Mexican M. lepromatosis strains from humans showed that they diverged from a common ancestor around 27,000 years ago. The M. leprae strain appeared closest to the one that circulated in medieval England. It is therefore likely that the original introduction of M. leprae into red squirrels occurred when leprosy was still prevalent in the region. The medieval squirrel fur trade might have played a role in the transmission of M. leprae between red squirrels and humans.

Red squirrels were found to be a reservoir for M. leprae and M. lepromatosis in the British Isles, but the prevalence of M. leprae infection is variable and often low in the different UK red squirrel populations.[23,67] Between 2013 and 2016, a follow-up study on the Isle of Wight was done to determine the infection rate in red squirrels.[66] Samples from red squirrels (n = 92) found dead were examined by PCR for M. leprae and M. lepromatosis. One of the squirrels tested positive for M. lepromatosis, none for M. leprae.

No evidence has been found for infection with M. leprae in squirrels or wild rodents outside the UK. Schilling et al. (2019) studied the white-throated woodrat (Neotoma albigula, n = 72) from Mexico, and from Europe (England, France, Germany, Switzerland and Italy) the Siberian chipmunk (Tamias sibiricus, n = 35), Eurasian red squirrel (n = 96), Eastern gray squirrel (Sciurus carolinensis n = 67), and Pallas’s squirrel (Callosciurus erythraeus n = 103). All samples of all these animals tested with PCR for M. lepromatosis and M. leprae were negative.[67]

Tió-Coma et al. (2020) examined sixty-one red squirrels and one Japanese squirrel (Sciurus lis) from the Netherlands and fifty-three red squirrels from Flanders, Belgium for the presence of lesions and M. leprae and M. lepromatosis DNA.[68] No clinical signs of leprosy were observed. All samples were negative in PCR analysis.

Nonhuman primates.

Cases of nonhuman primates with leprosy have been reported sporadically. All case reports described here involve imported animals. These cases were observed in highly monitored animal research facilities, not in the wild.

In 1977, leprosy-like symptoms were reported in a captive chimpanzee (Pan troglodytes) by Donham and Leininger.[69] Follow-up of disease progression and post-mortem examination confirmed that the pathogen was either M. leprae or a pathogen indistinguishable from it.[70,71] Hubbard et al. (1991) reported natural infection with M. leprae of two chimpanzees.[72] The animals had been in a United States research facility for over 25 years. Gormus et al. (1991) tested sera of these chimpanzees that had been naturally infected with leprosy and of 160 other chimpanzees housed in two primate centers.[73] An ELISA antibody assay for PGL I and the non-specific mycobacterial antigen lipoarabinomannan (LAM) was used. Seven animals were positive for anti-PGLI, and five for anti-LAM antibodies. In 2010, Suzuki et al. reported leprosy in a chimpanzee imported from Sierra Leone used for medical research in Japan.[74] SNP type of the leprosy strain was determined. The strain was SNP type 4, a strain found in West Africa, in line with the origin of the chimpanzee.

In 1979, a sooty mangabey (Cerocebus atys) monkey imported into the USA from West-Africa was observed with leprosy-like symptoms in the face.[75] The etiologic agent was identified as M. leprae by the following criteria: invasion of nerves, staining properties, electron microscopic findings, non-cultivability, lepromin reactivity, infection patterns in mice and armadillos and sensitivity to sulfone. Supposedly, the animal acquired the disease from a patient with active leprosy.[76] A second captive sooty mangabey monkey with naturally acquired leprosy was reported, that had been in direct contact with the first sooty mangabey monkey, and leprosy was believed to have been transmitted between those two monkeys.[77]

Hagstad (1983) screened 5 rhesus monkeys (Macaca mulatta) and 21 bonnet monkeys (Macaca radiata) in Andhra Pradesh, India.[78] The monkeys were owned as pets (n = 3) or used for begging (n = 23). The 26 monkeys were in frequent contact with handlers in a leprosy endemic area. No AFB were observed in ear lobe tissue smear slides.

Valverde et al. (1998) reported the first case of leprosy in an Asian macaque (Macaca fascicularis) imported from a leprosy endemic area in the Philippines. Diagnosis of infection was obtained by a PCR specific for M. leprae. Clinical presentation, histopathological findings, and serology of anti-PGL-I were compatible with human borderline (BB) leprosy.[79]

Honap et al. (2018) sequenced M. leprae genomes from frozen tissue samples of 3 of the above infected nonhuman primates (the chimpanzee from Sierra Leone, the first sooty mangabey from West Africa and the cynomolgus macaque from The Philippines).[80] M. leprae strains from the chimpanzee and sooty mangabey monkey belong to the human M. leprae Branch 4 lineage commonly found in West Africa. Phylogenetic analysis also showed that the cynomolgus macaque M. leprae strain belongs to M. Leprae Branch 0 and is most closely related to the modern human M. leprae strain S9 from New Caledonia (southwestern Pacific). In addition, samples from wild ring-tailed lemurs (Lemur catta, n = 41) originating from Madagascar and wild chimpanzees (Pan troglodytes schweinfurthii, n = 22) from Kibale National Park, Uganda were examined. DNA was extracted from the samples for detection of M. leprae and M. tuberculosis, and a variety of mycobacteria pooled with one sequence DNA using qPCR. All samples were negative.

Screening for new animal species.

The detection of leprosy infection in new species has, until now, resulted from coincidental observations. Screening of new species has been performed to find potential wildlife reservoirs. Finding M. leprae or M. lepromatosis would justify further study. The above mentioned studies by Schilling et al. (2019), Hagstad (1983) and Honap et al. (2018) can also be considered screening studies.

Job and colleagues (1988) looked for leprosy infections in small wild animals in Louisiana, in view of the discovered high prevalence in wild armadillos.[81] Both ears from road killed rabbits (n = 51), nutria (n = 56), raccoons (n = 17), and opossums (n = 311) were examined with a modified Fite’s stain. All samples were negative.

In the armadillo study by Pedrini et al. (2010), road killed species were examined for M. leprae DNA by PCR: ring-tailed coati (Nasua nasua, n = 2), skunk (Didelphis albiventris, n = 1), hedgehog (Sphigurrus spinosus, n = 1), South-American raccoon (Procyon cancrivorus, n = 1), restless cavy (Cavia aperea, n = 1), greater grison (Gallictis vittata, n = 2), and crab-eating fox (Cerdocyon thous, n = 2). All samples were negative.

Maruyama et al. (2018) screened various wildlife and captive species in Brazil for the presence of M. leprae DNA.[82] Nasal swabs were collected from 69 captive (zoo animals) and free wild animals from the leprosy endemic Mato Grosso and Pantanal regions, independent of clinical signs. M. leprae DNA was detected in: Owl monkey (Aoutus trivirgatus; n = 1), Capuchin monkey (Sapajus paella; n = 2), Lowland tapirs (Tapirus terrestris; n = 2, 1 captive), Margay (Leopardus weidii; n = 1, captive).

Leprosy-like disease.

Leprosy-like disease, attributed to mycobacteria, has been reported in a variety of animals. Phylogenetic analysis compares the genetic sequence, or a fragment thereof, and determines the similarity between two strains. It can be visualized in a family tree, where the arms indicate the temporal distance to a previous common ancestor. Direct genetic comparison of genes can reveal overlap in sequences. This can explain overlap in characteristics of pathogens.

In 1926, a cutaneous mycobacteriosis called skin-tuberculosis was reported in Indonesian water buffaloes.[83] A large series of case studies was published under the disease name Lepra bubalorum by Lobel (1936).[84,85] In 1954, Kraneveld and Roza published the post-Second World War status of the disease. They introduced the name Lepra Bovina, as the disease had been observed in bovine species other than the water buffalo.[86] There had been no progression towards clarification of the pathogen. Similar to leprosy, the microorganism could not be cultured or grown in laboratory animals. Since a last report in 1961, no more cases have been published.[87,88] It is unclear whether the condition still exists in Indonesia. Clinical symptoms and histopathological findings indicating neural involvement were not observed.

Mycobacterium lepraemurium causes leprosy-like symptoms in rats. The given name is based on the symptoms, and rather misleading, as it suggests close relation to M. leprae. M. lepraemurium is part of the M. avium-complex, a mycobacterial sub-group different from M. leprae, as was shown by phylogenetic analysis.[89] M. lepraemurium is one of the pathogens causing feline leprosy.[90–94] Another feline leprosy causing pathogen was discovered, and named Candidatus “Mycobacterium lepraefelis”, and this pathogen is closely related to M. leprae.[95] Canine leprosy is a disease associated with members of the Mycobacterium simiae clade, and thus unrelated to M. leprae.[96,97] A thelitis-causing pathogen in dairy goats and cows appeared to be closely related to M. leprae, and was named M. uberis.[98–100]

Environmental reservoirs (see Table 4)

The environment is defined as any location outside the host, where M. leprae resides before either dying or infecting a new host. Indications for the presence of viable M. leprae in the environment can be found from several experimental studies portraying the possibility of M. leprae to survive extracellularly under a variety of conditions.[101–105] These studies are shown in Table 4, along with more recent studies. More recent experiments are described in this section.

thumbnail
Download:
Table 4. Environmental reservoirs of M. leprae and M. lepromatosis.

https://doi.org/10.1371/journal.pntd.0008276.t004

Lavania et al. (2006) took 18 soil samples in 15 villages near JALMA, the national leprosy research institute in India.[106] Six of these samples were positive for M. leprae DNA by PCR. DNA is very stable and can be preserved and detected long after deposition. Later studies incorporated the detection of M. leprae specific 16S rRNA. RNA is a copy of a selection of the DNA sequence needed for protein production, and has a very short half-life, often only a few hours. Detection of M. leprae specific RNA is therefore an indication of the pathogens’ viability. Lavania et al. (2008) collected soil samples in the city of Ghatampur, India.[107] A distinction was made between samples from around houses in leprosy patient areas (n = 40) and non-patient areas (n = 40). 24 samples from the patient area were positive by PCR. 22 of these samples were also positive for 16S rRNA. Six samples of the non-patient area were positive by PCR.; all six were also positive for 16S rRNA.

Turankar et al. (2012) took 207 soil samples of areas where leprosy patients reside in the Purulia district, West Bengal, India.[108] Seventy-one samples were positive for M. leprae DNA, and 28 contained viable M. leprae based on rRNA PCR. It was attempted to compare patient and environment samples using SNP and VNTR methods, but only SNP type comparison succeeded. SNP type 1 was detected, but subtypes varied among soil samples and patients, even when patients were related. Turankar et al. (2016) repeated their earlier study in the same district.[109] Of 160 soil samples, 52 contained M. leprae DNA. M. leprae specific RNA was detected in 16 of these positive samples.

Mohanty et al. (2016) took soil and water samples in India from leprosy endemic areas close to patients, and control samples from areas considered leprosy-free.[110] 25.4% of the soil samples and 24.2% of the water samples from the leprosy endemic area were positive for M. leprae 16S rRNA, while all the control samples were negative.

Holanda et al. (2017) found M. leprae DNA in water sources in five municipalities of Ceará, Brazil.[111] Arraes et al. (2017) continued in these districts and analyzed newly acquired samples for viable leprosy specific gyrA mRNA.[112] Samples were taken from environmental water surfaces, of which 76.7% contained gyrA mRNA. Tió-Coma et al. (2018) expanded studies of M. leprae in soil.[113] Soil samples from 2–8 cm. depth were collected from 3 geographically different areas: Bangladesh, England, and Suriname. In Bangladesh, samples were collected close to leprosy patients’ bedrooms. In Suriname, samples were taken from areas inhabited by the nine-banded armadillo: the villages Pikin Slee and Gujaba, and the former leprosy colonies Batavia and Groot Chatillon. In England and Scotland, samples were taken from Brownsea Island (M. leprae infected red squirrel area), and the Isle of Arran (M. lepromatosis infected red squirrel area). M. leprae specific DNA was found in samples from Brownsea Island, Suriname, and Bangladesh, but not the Isle of Arran. M. lepromatosis DNA could not be detected in soil samples from the Isle of Arran.[114]

Turankar et al. (2019) studied the association between non-tuberculous mycobacteria and M. leprae in the environment of leprosy endemic regions in India. They analysed soil (n = 388) and water (n = 250) samples for RLEP DNA and 16S rRNA. RLEP DNA was detected in 118 soil samples (30%) and 48 water samples (19%). 16S rRNA was detected in 53 soil samples (14%) and 30 water samples (12%).[115]

Zoonotic and anthroponotic transmission

Filice et al. (1977) tried to assess, in a limited study, whether armadillo exposure is a risk factor for developing leprosy. Leprosy patients in Louisiana (n = 19) did not have more exposure to armadillos than neighborhood matched controls (n = 19).[116]

Deps et al. (2008) carried out the same type of study in Brazilian leprosy patients and a control group of patients with chronic diseases other than leprosy.[117] Exposure to armadillos was categorized as non-existent, indirect or direct. Physical contact was considered direct contact and living in an armadillo habitat was considered indirect contact. Direct exposure was significantly higher in leprosy patients (68%, n = 506) than in the control group (48%, n = 594) with OR 2.01 (95%CI 1.36–2.99, p = 0.0001).

Schmitt et al. (2010) studied the relation between armadillo meat consumption and leprosy in a case-control study of Brazilian leprosy patients (n = 121) and patients with other skin diseases (n = 242).[118] No significant difference was observed in armadillo meat consumption between the two groups: OR 0.77–1.90 (p = 0.44). The study supported, however, an association with unfavourable socioeconomic indicators as leprosy patients differed significantly (p<0.05) from controls in access to treated water (90% vs. 96%), lower family income based on minimum wage, and more contact with other leprosy patients (35% vs. 7%)

Truman et al. (2011) used whole-genome sequencing of M. leprae on isolated DNA from human skin biopsies of 50 American leprosy patients and armadillo liver, spleen or lymph-nodes of 33 Louisiana captured nine-banded armadillos.[119] Seven SNP subtypes were found in all samples, but selection of patients without travel history (n = 29) reduced the number of SNP subtypes to four. SNP subtype 3I, generally associated with European-American populations was present in all armadillos and 26 of 29 American patients without travel history. Further analysis of subtypes using variable number tandem repeat (VNTR) profiles showed that one specific M. leprae genotype (3I-2-v1) was present in 28 of 33 wild armadillos (85%) and 25 of 39 patients (64%) who resided in areas where exposure to armadillos was possible. They concluded that human leprosy in Louisiana and Texas may result from contact with infected armadillos.

Sharma et al. (2015) tested for M. leprae subtypes in the southeastern USA using VNTR SNP (sub-)type identification.[19] They found serologic and PCR evidence for M. leprae infection in 106 (16.4%, n = 645) nine-banded armadillos from eight locations in Alabama, Florida, Georgia, and Mississippi. Human M. leprae was isolated from frozen skin biopsies of 52 leprosy patients from the same geographic region as the sampled armadillos. Lymph node tissue for PCR analysis was obtained from 95 of the 106 infected armadillos. 42 of 95 samples contained enough DNA for genotyping. SNP subtype 3I-2-v1 was present in 35 of 42 armadillos (83%) and 12 of 52 humans (23%). The newly identified SNP subtype 3I-2-v15 was found in 7 of 42 armadillos (17%) and 10 of 52 humans (19%). SNP subtype 3I-2-v15 has not been reported outside the state of Florida. The leprosy patients with this genotype all lived in Florida. These data show that the M. leprae strains currently circulating in this part of the USA are closely related to medieval European M. leprae strains and suggest that leprosy was brought to the southern United States by the early European settlers since the sixteenth century. Armadillos must have acquired M. leprae from humans in the past 400 years.

Da Silva et al. (2018) have reportedly found evidence for zoonotic leprosy in Brazil, but actually studied leprosy occurrence in armadillos, and armadillo related risk factors for human leprosy and anti-PGL I titers separately.[120] They determined infection in 10/16 armadillos (62%) by PCR of liver and spleen samples. Blood samples from the local population (n = 146) were analyzed for PGL I. Stratification was performed based on hunting or eating armadillos, and analyzed further based on frequency. Of 146 individuals, over the previous year, 27 (18.5%) hunted armadillos, 96 (65.8%) handled or prepared the meat for consumption, 91 (62.3%) ate armadillo meat at least once, and 27 (18.5%) ate them more than once per month. The number of leprosy patients among armadillo hunters (4 of 27, 14.8%) was significantly higher than among non-hunters (3 of 119, 1.8%) OR 6.73 (95%CI 1.41–32.09 p = 0.02). Anti-PGL I titers were different when stratifying for exposure frequency. Eating armadillo meat more than 12 times per year was associated with an insignificant increased risk of PGL I positive antibody titers (OR 1.77 95%CI 0.64–4.89). The median anti-PGL I titer was significantly increased in this group (n = 27), compared to never eating (n = 55, p = 0,03) and eating 1–12 times per year (n = 64, p = 0.01).

Stefani et al. (2019) studied nine-banded armadillos (n = 12) and local volunteers (n = 176) in the rural endemic area of Mamiá Lake of the Coari municipality, Brazil.[121] The armadillos were supplied locally. Volunteers were examined for clinical signs of leprosy by a dermatologist. Armadillo skin, spleen, liver, lymph, adrenal glands, ovary and fallopian tubes and human skin lesions suspect of leprosy were biopsied and examined histopathologically. All tissues from armadillos were tested with qPCR for the M. leprae specific RLEP sequence. Six new leprosy patients were identified among the local volunteers. None of the armadillo samples showed M. leprae DNA or AFB.

Potential vectors (see Table 5)

Attempts at discovering vectors for M. leprae have been reported throughout the twentieth century. In older studies, leprosy bacilli viability could not be shown by culture or the use of animal models. These studies are not discussed in depth, because of their limited impact. Three studies contained only a description of the theoretical role of arthropods in the transmission of leprosy.[122–124] In several studies, leprosy patients were used to test the uptake of leprosy bacilli from lesions.[125–131] In others, leprous material from patients was used to feed insects.[132–134] Insects were also collected from leprosy patient’s homes.[131,135–137] None of these studies were able to show possible transmission by insects. At most, insects would take up the bacteria or transmit a small number of bacilli to a subsequent feeding target, but this would not be considered a valid transmission route.

thumbnail
Download:
Table 5. Potential vectors of M. leprae.

https://doi.org/10.1371/journal.pntd.0008276.t005

M. leprae is an obligate intracellular pathogen. Intracellular microorganisms have been associated with free living amoebae (FLA) without a known benefit for either. It is currently proposed to define these microorganisms and FLA as “endocytobionts”. Such a relationship was studied for M. leprae. Phagocytosis of fluorescent M. leprae by amoebae was observed by Lahiri and Krahenbuhl (2008) and Wheat et al. (2014). Inside the amoebae M. leprae was seen flocking to the more acidic regions of the cell.[138,139] Lahiri and Krahenbuhl (2008) showed that M. leprae remained viable for at least three days as assessed by MFP.[138] Wheat et al. (2014) found that M. leprae remained viable for 35 days in Acanthamoebae lenticulata, A. castellanii, A. polyphaga, Hartmannella vermiformis, and H. vermiformis, and up to 8 months within amoebic cysts.[139] The degree of M. leprae phagocytosis by Acanthamoeba sp.S-11 was shown by Paling et al. (2018) to be nutrition driven.[140] Amoebae would phagocytose co-cultured M. leprae increasingly in poor nutrient conditions.

As amoebae are present worldwide in the environment (e.g., soil, water and air), they may potentially play a role in the survival of M. leprae outside the mammalian host. Exposure of mammals to infected amoebae could be a risk factor for transmission. In this form of transmission, the amoebae would function as a vector. Turankar et al. (2019) found an association between M. leprae DNA and Acanthamoeba DNA in environmental samples.[115] Viable M. leprae could be found using 16S rRNA detection in 106 of 700 soil samples (15%), and 34 of 400 water samples (8%). Acanthamoeba DNA was associated with viable M. leprae. Of the 106 soil samples positive for 16S rRNA, 30 were also positive for Acanthamoeba DNA (28%). Of the 112 negative soil samples, 10 were positive for Acanthamoeba DNA (9%). Water samples showed the same association. Acanthamoeba DNA was detected in 14 out of 34 16S rRNA positive samples (41%) and 1 out of 39 M. leprae DNA positive 16S rRNA negative soil samples (2.6%). The association between viable M. leprae and Acanthamoeba DNA was significant for soil (p = 0.002) and water (p = 0.009).

Neumann et al. (2016) investigated the possibility of transmission of M. leprae by the kissing bug (Rhodnius prolixus) and mosquitoes (Aedes aegypti and Culex quinquefasciatus).[141] Both are known to draw blood from armadillos. The mosquitoes, however, did not meet the essential criterion for a vector: the ability to maintain an infectious load of the pathogen until the next feed. But M. leprae remained viable in kissing bugs for 20 days. Inoculation of MFP with feces of kissing bugs proved that M. leprae had remained viable. This is a well-known transmission pathway for Trypanosoma cruzi, causing the zoonotic, vector transmitted Chagas disease. T. cruzi remains viable during the kissing bug’s gastrointestinal tract passage. Kissing bugs transmit disease, not by biting, but by defecating near the bite wound. Rubbing or scratching infects the wound.

Da Silva Ferreira et al. (2018) achieved similar results with the adult female tick (Amblyomma sculptum).[142] Ticks were fed on rabbit blood containing M. leprae. Detection of M. leprae RNA in the midgut and ovaries of the ticks by qPCR analysis proved viability of the M. leprae until at least 2 days after feeding. M. leprae was visualized in gut samples with LAM-antibody immune-localization. In addition, M. leprae would, in some cases, spread to the ovaries of female ticks and subsequently infect tick eggs and larvae. Infected larvae were attached to rabbit skin for a five day maturation feeding period. Afterwards, up to 103 viable M. leprae could be isolated from the skin samples around the bite-mark. The study showed an ecological cycle of M. leprae in an experimental setting. The research group also performed the first successful cell culture of M. leprae in tick-IDE8 cells.

Discussion

This systematic review assessed publications of possible non-human environmental reservoirs and transmission pathways of M. leprae and M. lepromatosis. The results show a wildlife reservoir of M. leprae in armadillo species in the southern United States and a wildlife reservoir of M. leprae and M. lepromatosis in British and Irish red squirrels. The diagnosis of leprosy in captive non-human primates is limited to case-reports, and it is unknown to what degree species are infected. Screening for the presence of these mycobacteriae in other species has been negative, except for the finding in 2018 of genetic material of M. leprae in nasal swabs from a variety of animal species in Brazil.

Possible routes of transmission are schematically visualized in Fig 2. It is justified to ask whether or not we can speak of an environmental reservoir. RNA indicating the presence of viable M. leprae was found in environmental soil and water samples in Brazil and India.[143] M. lepromatosis has not yet been found in such samples, but this pathogen has only been discovered recently. In addition, it was found that amoebae are capable of taking up M. leprae by phagocytosis. Inside amoebae, M. leprae remained viable for days to weeks. This mechanism might contribute to environmental survival in the absence of a mammalian host. Moreover, M. leprae can also accumulate in amoebae. This gives amoebae a possible role as a vector in transmission. The only studies on vegetation reservoirs were on sphagnum species by Kazda (1980) (Table 4).[103,104] There are no recent studies using PCR techniques on the role of vegetation as an environmental reservoir.

thumbnail
Download:
Fig 2.

https://doi.org/10.1371/journal.pntd.0008276.g002

Humans and armadillos in the Southern United States share a specific M. leprae strain (SNP subtype 3I-2-v15). This finding is highly suggestive of a zoonotic and/or an anthroponotic transmission pathway between humans and armadillos. Exposure of humans to armadillos can eventually lead to leprosy and/or increased anti-PGL I antibody levels. Exposure to an animal or to animals’ excreta is influenced by behavior. For example, hunting and/or preparing armadillos for eating can each be expected to have a different risk of transmission, based on differences in exposure intensity and frequency. It is unclear whether transmission risk by rate of exposure to infected mammals is confounded by rate of exposure to an infected environment. This could be caused by shedding of leprosy bacteria by infected mammals.

At this moment, there is no evidence for a role for vectors in transmission. However, recent laboratory studies have shown a potential role for insects. M. leprae remains viable in the gastrointestinal tract of kissing bugs and is experimentally transmittable to the MFP through their feces, similarly to the transmission mechanism of T. cruzi. Kissing bugs can defecate after biting, and defecation near the bitten area transmits viable T. cruzi when the itchy bitten area is scratched, and feces is rubbed into the wound. In the same study, it was found that M. leprae does not remain viable within the gut of mosquitoes. It also has been shown that M. leprae can remain viable and reproduce in ticks, and spread to tick ovaria and eggs. Larvae from hatched eggs in turn are able to transmit viable M. leprae into a host during skin-bound maturation.

Animals can be affected by leprosy-like diseases, caused by pathogens phylogenetically closely related to M. leprae. Feline leprosy is caused by several pathogens, of which Candidatus “Mycobacterium lepraefelis” is closely related to M. leprae. M. uberis, the causative agent of nodular thelitis, is also closely related to M. leprae. M. haemophilum, which has been reported to infect the skin of immunocompromised human patients, was also found to be closely related to M. leprae.[144] The pathogen causing a mycobacteriosis called Lepra Bubalorum in Indonesian water buffaloes could also be related to M. leprae, but further research is needed to confirm this. These mycobacteria have been proposed to be grouped as a M. leprae-complex.[145] Such a classification connects the related mycobacteria, similar to species of the M. avium-complex. Insights from the transmission and reservoirs of members of the M. leprae-complex could be relevant for other pathogens in the complex. Currently, knowledge on the exact transmission mechanisms of M. leprae is poor. Using insights on related pathogens is a potentially efficient way to progress.

The studies included in this review differ in methods to determine the causative microorganism of leprosy. Techniques have developed over time from identifying unspecified non-cultivable acid-fast bacteria to specific DNA sequencing. The ability to detect M. leprae DNA with PCR techniques drastically changed the methods of modern studies. The variety of and changes in analysis methods for detection of M. leprae have resulted in poor standardization of methods, each with their particular strengths and weaknesses. This is also relevant for studies on M.leprae and M.lepromatosis in epidemiological settings. Anti-PGL I antibody detection in blood or sera indicates past or present exposure to M. leprae but provides little information on current infection status. Studies on zoonosis and prevalence should be performed with sample sizes determined by the population sizes. Results of these studies have to be reported with confidence intervals, as this would substantiate the extrapolation value of the studies. Many studies in this review lack this method of sample size determination. Geographic origins of the animals are described clearly in most armadillo prevalence studies. It is seen that prevalence fluctuates strongly between neighboring geographic regions. This limits the value of spatially and temporally randomly acquired (frozen) samples in screening studies, which often do not represent a wildlife (sub)population. Seldom are internal control standards like those normally used in clinical laboratories incorporated. This lack of rigor contributes to the anecdotal assessment sometimes given to molecular studies reporting M. leprae in non-human sources. M.leprae rRNA has been used as a viability marker in environmental studies.[108–111] Accuracy of this method was suggested to be inadequate.[146] A reliable standard for viability is needed. Standardization in environmental studies should be increased by first developing appropriate definitions and sample criteria.

This systematic review underscores that human-to-human transmission is not the only way leprosy can be acquired. The transmission of this disease is probably much more complicated than was thought before, as indicated in Fig 2. Transmission of M. leprae involves several factors and pathways. Fig 2 shows the animal and environmental factors that might play a role in the persisting prevalence of leprosy. The proposed factors and mechanisms transgress the disciplines of human healthcare. A reduction in transmission cannot be expected to be accomplished by actions or interventions from the human healthcare domain alone, as the mechanisms involved are complex. Therefore, to increase our understanding of the intricate picture of leprosy transmission, a One Health transdisciplinary research approach is required.[138] This entails integrating human, animal, and environmental health aspects to further elucidate the transmission mechanisms and patterns of M. leprae and M. lepromatosis. In addition, geographically tailored methods–combining epidemiological, laboratory, and anthropologic data–are needed to better understand the ecological differences between leprosy pockets.

Acknowledgments

The authors would like to thank Marja Kik for her feedback on an earlier version of this paper and Toon van der Gronde for his feedback on the methodological section. Furthermore, we would like to thank Nathalie Kuijpers for her English manuscript correction services. In addition, we would like to thank Frank-Jan van Lunteren for his comprehensive graphics (See, Fig 2).

References

  1. 1. Sarno EN, Duppre NC, Sales AM, Hacker MA, Nery JA, de Matos HJ. Leprosy exposure, infection and disease: a 25-year surveillance study of leprosy patient contacts. Mem Inst Oswaldo Cruz. 2012 Dec;107(8):1054–9. pmid:23295758
  2. 2. Araujo S, Freitas LO, Goulart LR, Goulart IMB. Molecular Evidence for the Aerial Route of Infection of Mycobacterium leprae and the Role of Asymptomatic Carriers in the Persistence of Leprosy. Clin Infect Dis. 2016 Dec 1;63(11):1412–20. pmid:27558564
  3. 3. Gama RS, Gomides TAR, Gama CFM, Moreira SJM, de Neves Manta FS, de Oliveira LBP, et al. High frequency of M. leprae DNA detection in asymptomatic household contacts. BMC Infect Dis. 2018 Dec 2;18(1):153. pmid:29609530
  4. 4. Rodrigues LC, Lockwood DN. Leprosy now: epidemiology, progress, challenges, and research gaps. Lancet Infect Dis. 2011 Jun;11(6):464–70. pmid:21616456
  5. 5. World Health Organisation Weekly Epidemiological Record. 2019;94:389–412.
    • 6. Hansen GA. [Studies on the causes of leprosy]. Nor Mag Laegervidenskaben. 1874;4(SUPPL):1–88.
    • 7. Hansen GA. On the Etiology of Leprosy. Br Foreign Medico-chirurgical Rev. 1875 Apr;55(110):459–89.
    • 8. Shepard CC. The experimental disease that follows the injection of human leprosy bacilli into foot-pads of mice. J Exp Med. 1960;112(3):445–54. pmid:19867175
    • 9. Shepard CC. Multiplication of Mycobacterium leprae in the foot-pad of the mouse. Int J Lepr. 1962;30(3):291–306.
    • 10. Kirchheimer WF, Storrs EE, Binford CH. Attempts to establish the Armadillo (Dasypus novemcinctus linn.) as a model for the study of leprosy. II. Histopathologic and bacteriologic post-mortem findings in lepromatoid leprosy in the Armadillo. Int J Lepr Other Mycobact Dis. 1972;40(3):229–42. pmid:4574269
    • 11. Storrs EE, Walsh GP, Burchfield HP, Binford CH, Walsh GP, Meyers W, et al. Leprosy in the armadillo: new model for biomedical research. Science. 1974;183(4127):851–2. pmid:4589901
    • 12. Han XY, Seo YH, Sizer KC, Schoberle T, May GS, Spencer JS, et al. A new Mycobacterium species causing diffuse lepromatous leprosy. Am J Clin Pathol. 2008;130(6):856–64. pmid:19019760
    • 13. Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR, Wheeler PR, et al. Massive gene decay in the leprosy bacillus. Nature. 2001 Feb;409(6823):1007–11. pmid:11234002
    • 14. Garbino JA, Marques W, Barreto JA, Heise CO, Rodrigues MMJ, Antunes SL, et al. Primary neural leprosy: systematic review. Arq Neuropsiquiatr. 2013;71(6):397–404. pmid:23828524
    • 15. Monot M, Honoré N, Garnier T, Araoz R, Coppée J-Y, Lacroix C, et al. On the origin of leprosy. Science. 2005 May 13;308(5724):1040–2. pmid:15894530
    • 16. Benjak A, Avanzi C, Singh P, Loiseau C, Girma S, Busso P, et al. Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae. Nat Commun. 2018;9(1):352. pmid:29367657
    • 17. Schuenemann VJ, Avanzi C, Krause-Kyora B, Seitz A, Herbig A, Inskip S, et al. Ancient genomes reveal a high diversity of Mycobacterium leprae in medieval Europe. PLOS Pathog. 2018;14(5):e1006997. pmid:29746563
    • 18. Bratschi MW, Steinmann P, Wickenden A, Gillis TP. Current knowledge on Mycobacterium leprae transmission: a systematic literature review. Lepr Rev. 2015;86(2):142–55. pmid:26502685
    • 19. Sharma R, Singh P, Loughry WJJ, Lockhart JM, Inman WB, Duthie MS, et al. Zoonotic Leprosy in the Southeastern United States. Emerg Infect Dis. 2015;21(12):2127–34. pmid:26583204
    • 20. Loughry WJ, McDonough CM. The Nine-Banded Armadillo: A Natural History. Oklahoma: University of Oklahoma Press; 2013.
      • 21. Viana M, Mancy R, Biek R, Cleaveland S, Cross PC, Lloyd-Smith JO, et al. Assembling evidence for identifying reservoirs of infection. Trends Ecol Evol. 2014 May;29(5):270–9. pmid:24726345
      • 22. Scollard DM. Editorial: Infection with Mycobacterium lepromatosis. Am J Trop Med Hyg. 2016;95(3):500–1. pmid:27430540
      • 23. Avanzi C, Del-Pozo J, Benjak A, Stevenson K, Simpson VR, Busso P, et al. Red squirrels in the British Isles are infected with leprosy bacilli. Science. 2016 Nov;354(6313):744–7. pmid:27846605
      • 24. PRISMA [Internet]. [cited 2020 Mar 18]. Available from: http://www.prisma-statement.org/
        • 25. Walsh GP, Storrs EE, Burchfield HP, Cotrell EH, Vidrine MF, Binford CH. Leprosy-like disease occurring naturally in armadillos. J Reticuloendothel Soc. 1975;18(6):347–51. pmid:1214263
        • 26. Walsh GP, Storrs EE, Meyers W, Binford CH. Naturally acquired leprosy-like disease in the nine-banded armadillo (Dasypus novemcinctus): recent epizootiologic findings. J Reticuloendothel Soc. 1977;22(4):363–7. pmid:926090
        • 27. Binford CH, Meyers WM, Walsh GP, Storrs EE, Brown HL. Naturally acquired leprosy-like disease in the nine-banded armadillo (Dasypus novemcinctus): histopathologic and microbiologic studies of tissues. J Reticuloendothel Soc. 1977;22(4):377–88. pmid:411927
        • 28. Meyers WM, Walsh GP, Brown HL, Rees RJ, Convit J. Naturally acquired leprosy-like disease in the nine-banded armadillo (Dasypus novemcinctus): reactions in leprosy patients to lepromins prepared from naturally infected armadillos. J Reticuloendothel Soc. 1977;22(4):369–75. pmid:336883
        • 29. Mcfadzean JA, Ridley DS. Studies on the inoculation of mycobacterium leprae into monkeys. Trans R Soc Trop Med Hyg. 1961;55(3):235–8.
        • 30. Skinsnes OK. “Leprosy” in wild armadillos. Int J Lepr Other Mycobact Dis. 1976;44(3):376–7. pmid:789264
        • 31. Binford CH, Storrs EE, Walsh GP. Disseminated infection in the nine-banded armadillo (Dasypus novemcinctus) resulting from inoculation with M. leprae. Observations made on 15 animals studied at autopsy. Int J Lepr Other Mycobact Dis. 1976;44(1–2):80–3. pmid:945242
        • 32. Folse DS, Smith JH. Leprosy in wild armadillos (Dasypus novemcinctus) on the Texas Gulf Coast: anatomic pathology. J Reticuloendothel Soc. 1983;34(5):341–57. pmid:6644691
        • 33. Marchiondo AA, Smith JH, File SK. Naturally occurring leprosy-like disease of wild armadillos: ultrastructure of lepromatous lesions. J Reticuloendothel Soc. 1980;27(3):311–25. pmid:6988587
        • 34. Truman RW. Host responses to the phenolic-glycolipid-1 antigen of Mycobacterium leprae. Louisiana State University; 1985.
          • 35. Truman RW, Shannon EJ, Hagstad H V., Hugh-Jones ME, Wolff A, Hastings RC. Evaluation of the origin of Mycobacterium leprae infections in the wild armadillo, Dasypus novemcinctus. Am J Trop Med Hyg. 1986 May;35(3):588–93. pmid:3518509
          • 36. Smith JH, Folse DS, Long EG, Christie JD, Crouse DT, Tewes ME, et al. Leprosy in wild armadillos (Dasypus novemcinctus) of the Texas Gulf Coast: epidemiology and mycobacteriology. J Reticuloendothel Soc. 1983;34(2):75–88. pmid:6350581
          • 37. Job CK, Harris EB, Allen JL, Hastings RC. A random survey of leprosy in wild nine-banded armadillos in Louisiana. Int J Lepr Other Mycobact Dis. 1986;54(3):453–7. pmid:3528348
          • 38. Clark KA, Kim SH, Boening LF, Taylor MJ, Betz TG, Mccasland F V. Leprosy in armadillos (Dasypus novemcinctus) from Texas. J Wildl Dis. 1987;23(2):220–4. pmid:3586200
          • 39. Stallknecht DE, Truman RW, Hugh-Jones ME, Job CK. Surveillance of Naturally Acquired Leprosy in a Nine-banded Armadillo Population. J Wildl. 1987;23(2):308–10.
          • 40. Howerth EW, Stallknecht DE, Davidson WR, Wentworth EJ. Survey for leprosy in nine-banded armadillos (Dasypus novemcinctus) from the southeastern United States. J Wildl Dis. 1990 Jan;26(1):112–5. pmid:2406467
          • 41. Smith JH, File SK, Nagy BA, Folse DS, Buckner JA, Webb LJ, et al. Leprosy-like disease of wild armadillos in French Acadiana, Louisiana. J Reticuloendothel Soc. 1978;24(6):705–19. pmid:376833
          • 42. Truman RW, Job CK, Hastings RC. Antibodies to the phenolic glycolipid-1 antigen for epidemiologic investigations of enzootic leprosy in armadillos (Dasypus novemcinctus). Lep Rev. 1990;61(1):19–24.
          • 43. Truman RW, Job CK, Hastings RC, Kumaresan JA, Mcdonough CM. Seasonal and spatial trends in the detectability of leprosy in wild armadillos. Epidemiol Infect. 1991;106(3):549–60. pmid:2050208
          • 44. Job CK, Drain V, Williams DL, Gillis TP, Truman RW, Sanchez RM, et al. Comparison of polymerase chain reaction technique with other methods for detection of Mycobacterium leprae in tissues of wild nine-banded armadillos. Lepr Rev. 1991;62(4):362–73. pmid:1784151
          • 45. Paige CF, Scholl DT, Truman RW. Prevalence and incidence density of Mycobacterium leprae and Trypanosoma cruzi infections within a population of wild nine-banded armadillos. Am J Trop Med Hyg. 2002;67(5):528–32. pmid:12479557
          • 46. Morgan RE, Loughry WJ. Consequences of Exposure to Leprosy in a Population of Wild Nine-banded Armadillos. J Mammal. 2009;90(6):1363–9.
          • 47. Loughry WJ, Truman RW, McDonough CM, Tilak M-K, Garnier S, Delsuc F. Is leprosy spreading among nine-banded armadillos in the southeastern United States? J Wildl Dis. 2009 Jan;45(1):144–52. pmid:19204343
          • 48. Williams Andrew J, Loughry WJ. Temporal Aspects of Leprosy Infection in a Wild Population of Nine-Banded Armadillos. Southeast Nat. 2012;11(2):173–82.
          • 49. Perez-Heydrich C, Loughry WJ, Anderson CD, Oli MK. Patterns of Mycobacterium leprae infection in wild nine-banded armadillos (Dasypus novemcinctus) in Mississippi, USA. J Wildl Dis. 2016 Jul;52(3):524–32. pmid:27195687
          • 50. Convit J, Pinardi M. Inoculation del M. leprae en dos especies de armadillos: D. sabanicola y D. novemcinctus. Acta científica Venez. 1974;25:51–4.
          • 51. Convit J, Pinardi ME, Aranzazu N. The armadillo as an Experimental Model in Biomedical Research. Pan Am Heal Organ Sci Publ. 1978;366:41–6.
          • 52. Storrs EE, Walsh GP, Burchfield HP. Development of leprosy in another species of armadillo Dasypus hybridus (L): genetic and immunologic implications. J Trop Med Hyg. 1975;78(10–11):216–8. pmid:765488
          • 53. Balina LM, Valdez RP, de Herrera M, Costa Cordova H, Bellocq J, Garcia N, et al. Experimental reproduction of leprosy in seven-banded armadillos (Dasypus hybridus). Int J Lepr Other Mycobact Dis. 1985;53(4):595–9. pmid:3910749
          • 54. Amezcua ME, Escobar-Gutiérrez A, Storrs EE, Dhople AM, Burchfield HP. Wild Mexican armadillo with leprosy-like infection. Int J Lepr Other Mycobact Dis. 1984;52(2):254–5. pmid:6539311
          • 55. Martinez AR, Resoagli EH, De Millan SG. [‘Wild’ leprosy in Dasypus novemcinctus (Linn. 1758)]. Arch Argent Dermatol. 1984;34(1):21–30.
          • 56. Deps PD, Antunes JMA de P, Tomimori-Yamashita J. Detection of Mycobacterium leprae infection in wild nine-banded armadillos (Dasypus novemcinctus) using the rapid ML Flow test. Rev Soc Bras Med Trop. 2007;40(1):86–7. pmid:17486263
          • 57. Deps PD, Marcelo J, Antunes AP, Faria C, Bührer-Sékula S, Camargo ZP, et al. Research regarding anti-PGL-I antibodies by ELISA in wild armadillos from Brazil Pesquisa de anticorpos anti PGL-I através de ELISA em tatus selvagens do Brasil. Rev Soc Bras Med Trop. 2008;41(Suplemento II):73–6.
          • 58. Frota CC, Lima LNC, Rocha A da S, Suffys PN, Rolim BN, Rodrigues LC, et al. Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil. Mem Inst Oswaldo Cruz. 2012;107(suppl 1):209–13.
          • 59. Deps PD, Antunes JMAP, Faria C, Bührer-Sékula S, Camargo ZP, Opromola D V., et al. Pesquisa de anticorpos anti PGL-I através de ELISA em tatus selvagens do Brasil. Rev Soc Bras Med Trop. 2008;41(SUPPL. 2):73–6.
          • 60. Deem SL, Noss AJ, Fiorello C V., Manharth AL, Robbins RG, Karesh , William B.rmadillos in the Gran Chaco B. Health Assessment of Free-Ranging Three-Banded (Tolypeutes matacus) and Nine-Banded (Dasypus novemcinctus) Armadillos in the Gran Chaco, Bolivia. J Zoo Wildl Med. 2009;40(2):245–56. pmid:19569470
          • 61. Cardona-Castro N, Beltrán-Alzate JC, Romero-Montoya IM, Li W, Brennan PJ, Vissa V. Mycobacterium leprae in Colombia described by SNP7614 in gyrA, two minisatellites and geography. Infect Genet Evol. 2013;14:375–82. pmid:23291420
          • 62. Pedrini SCB, Rosa PS, Medri ÍM, Mourão G, Bagagli E, de Magalhães Lopes CA. Search for Mycobacterium leprae in wild mammals. Brazilian J Infect Dis. 2010;14(1):47–53.
          • 63. Galletti G, Cavicchi G, Ussia G. Replication of Mycobacterium leprae in hibernating ground squirrels (Citellus tridecemlineatus). Acta Leprol. 1982;52(88):23–31.
          • 64. Meredith A, Del Pozo J, Smith S, Milne E, Stevenson K, McLuckie J. Leprosy in red squirrels in Scotland. Vet Rec. 2014;175(11):285–6. pmid:25234460
          • 65. Simpson V, Hargreaves J, Butler H, Blackett T, Stevenson K, McLuckie J. Leprosy in red squirrels on the Isle of Wight and Brownsea Island. Vet Rec. 2015;177(8):206–7.
          • 66. Butler HM, Stevenson K, McLuckie J, Simpson VR. Further evidence of leprosy in Isle of Wight red squirrels. Vet Rec. 2017;180(16):407.1–407.
          • 67. Schilling A-K, Avanzi C, Ulrich RG, Busso P, Pisanu B, Ferrari N, et al. British Red Squirrels Remain the Only Known Wild Rodent Host for Leprosy Bacilli. Front Vet Sci. 2019;6:8. pmid:30775369
          • 68. Tió‐Coma M, Sprong H, Kik M, Dissel JT, Han X, Pieters T, et al. Lack of evidence for the presence of leprosy bacilli in red squirrels from North‐West Europe. Transbound Emerg Dis. 2020 Mar 2;67(2):1032–4.
          • 69. Donham KJ, Leininger JR. Spontaneous Leprosy-Like Disease in a Chimpanzee. J Infect Dis. 1977;136(1):132–6. pmid:886203
          • 70. Leininger JR, Donham KJ, Rubino MJ. Leprosy in a Chimpanzee: Morphology of the Skin Lesions and Characterization of the Organism. Vet Pathol. 1978;15(3):339–46. pmid:356407
          • 71. Leininger JR, Donham KJ, Meyers WM. Leprosy in a chimpanzee. Postmortem lesions. IntJLeprOther MycobactDis. 1980;48(4):414–21.
          • 72. Hubbard GB, Lee DR, Eichberg JW, Gormus BJ, Xu K, Meyers WM. Spontaneous Leprosy in a Chimpanzee (Pan troglodytes ). Vet Pathol. 1991;28(6):546–8. pmid:1771747
          • 73. Gormus BJ, Xu KY, Alford PL, Lee DR, Hubbard GB, Eichberg JW, et al. A serologic study of naturally acquired leprosy in chimpanzees. Int J Lepr Other Mycobact Dis. 1991;59(3):450–7. pmid:1890369
          • 74. Suzuki K, Udono T, Fujisawa M, Tanigawa K, Ichi Idani G’, Ishii N. Infection during Infancy and Long Incubation Period of Leprosy Suggested in a Case of a Chimpanzee Used for Medical Research. J Clin Microbiol. 2010;48(9):3432–4. pmid:20631101
          • 75. Gerone PJ. Hansen’s disease in the sooty mangabey monkey. Star. 1982;41(4):1.
          • 76. Meyers WM, Walsh GP, Brown HL, Binford CH, Imes GD, Hadfield TL, et al. Leprosy in a mangabey monkey—naturally acquired infection. Int J Lepr Other Mycobact Dis. 1985;53(1):1–14. pmid:3889184
          • 77. Gormus BJ, Wolf RH, Baskin GB, Ohkawa S, Gerone PJ, Walsh GP, et al. A second sooty mangabey monkey with naturally acquired leprosy: first reported possible monkey-to-monkey transmission. Int J Lepr Other Mycobact Dis. 1988;56(1):61–5. pmid:3373087
          • 78. Hagstad H V. Leprosy in sub-human primates: potential risk for transfer of Mycobacterium leprae to humans. Lepr Rev. 1983;54(4):353–6. pmid:6668987
          • 79. Valverde CR, Canfield D, Tarara R, Esteves MI, Gormus BJ. Spontaneous leprosy in a wild-caught cynomolgus macaque. Int J Lepr Other Mycobact Dis. 1998;66(2):140–8. pmid:9728446
          • 80. Honap TP, Pfister L-A, Housman G, Mills S, Tarara RP, Suzuki K, et al. Mycobacterium leprae genomes from naturally infected nonhuman primates. PLoS Negl Trop Dis. 2018;12(1):e0006190. pmid:29381722
          • 81. Job CK, Allen JL, Hastings RC. Search for leprous infection in some small wild animals of Louisiana. Int J Lepr Other Mycobact Dis. 1988;56(4):630–1. pmid:3065423
          • 82. Maruyama FH, Morgado TO, Pacheco RC, Nakazato L, Dutra V. Molecular detection of Mycobacterium leprae by Polymerase Chain Reaction in captive and free-ranging wild animals. Brazilian J Infect Dis. 2018;22(5):445–7.
          • 83. Kok J, Roesli M. Huid-tuberculose (?) bij buffels. Ned Bl voor Diergeneeskd. 1926;38(6):465–72.
          • 84. Lobel L. Lepra Bubalorum. Int J Lepr. 1936;82:72–96.
          • 85. Lobel L. Thesis: Lepra Bubalorum. Universiteit Utrecht; 1934.
            • 86. Kraneveld FC, Roza M. Lepra bubalorum and lepra bovina in Indonesia. Doc Med Geogr Trop. 1954 Dec;6(4):303–14. pmid:13231773
            • 87. Ressang AA, Sutarjo . Leprae Bubalorum. Part I. Report on transmissional experiments. Commun Vet. 1961;5:89–101.
            • 88. Ressang AA. Lepra Bubalorum. Part II. Case report of an extraordinary distribution of leprosy granuloma nodules in a buffalo cow. Commun Vet. 1961;5:103–6.
            • 89. Benjak A, Honap TP, Avanzi C, Becerril-Villanueva E, Estrada-García I, Rojas-Espinosa O, et al. Insights from the Genome Sequence of Mycobacterium lepraemurium: Massive Gene Decay and Reductive Evolution. MBio. 2017;8(5):e01283–17. pmid:29042494
            • 90. Poelma FG, Leiker DL. Cat leprosy in the Netherlands. Int J Lepr Other Mycobact Dis. 1974;42(3):307–11. pmid:4477560
            • 91. Malik R, Hughes M, James G, Martin P, Wigney D, Canfield P, et al. Case Report Feline leprosy: two different clinical syndromes. J Feline Med Surg. 2002;4:43–59. pmid:11869054
            • 92. Courtin F, Huerre M, Fyfe J, Dumas P, Boschiroli ML. A case of feline leprosy caused by Mycobacterium lepraemurium originating from the island of Kythira (Greece): diagnosis and treatment. J Feline Med Surg. 2007;9(3):238–41. pmid:17239641
            • 93. O’Brien CR, Malik R, Globan M, Reppas G, McCowan C, Fyfe JA. Feline leprosy due to Mycobacterium lepraemurium. J Feline Med Surg. 2017 Jul;19(7):737–46. pmid:28592223
            • 94. O’Brien CR, Malik R, Globan M, Reppas G, McCowan C, Fyfe JA. Feline leprosy due to Mycobacterium lepraemurium: Further clinical and molecular characterisation of 23 previously reported cases and an additional 42 cases. J Feline Med Surg. 2017;19(7):737–46. pmid:28592223
            • 95. O’Brien CR, Malik R, Globan M, Reppas G, McCowan C, Fyfe JA, et al. Feline leprosy due to Candidatus ‘Mycobacterium lepraefelis’: Further clinical and molecular characterisation of eight previously reported cases and an additional 30 cases. J Feline Med Surg. 2017 Sep;19(9):919–32. pmid:28838294
            • 96. Dedola C, Zobba R, Pinna Parpaglia ML, Chessa B, Antuofermo E, Polinas M, et al. First report of canine leprosy in Europe: molecular and clinical traits. Vet Rec. 2014 Feb 1;174(5):120.
            • 97. Foley JE, Borjesson D, Gross TL, Rand C, Needham M, Poland A. Clinical, Microscopic, and Molecular Aspects of Canine Leproid Granuloma in the United States. Vet Pathol. 2002;39(2):234–9. pmid:12009061
            • 98. Pin D, Guérin-Faublée V, Garreau V, Breysse F, Dumitrescu O, Flandrois J-P, et al. Mycobacterium Species Related to M. leprae and M. lepromatosis from Cows with Bovine Nodular Thelitis. Emerg Infect Dis. 2014;20(12):2111–4. pmid:25417797
            • 99. Chartier C, Albaric O, Cesbron N, Despres J, Hoogveld C, Michelet L, et al. Tuberculoid nodular thelitis in a dairy goat flock. Vet J. 2016;209:199–200. pmid:26831157
            • 100. Benjak A, Avanzi C, Benito Y, Breysse F, Chartier C, Boschiroli M-L, et al. Highly Reduced Genome of the New Species Mycobacterium uberis, the Causative Agent of Nodular Thelitis and Tuberculoid Scrotitis in Livestock and a Close Relative of the Leprosy Bacilli. mSphere. 2018;3(5):e00405–18. pmid:30282756
            • 101. Desikan K V. Viability of Mycobacterium leprae outside the human body. Lepr Rev. 1977;48(4):231–5. pmid:400807
            • 102. Kaur S, Kumar B, Ganguly NK, Chakravarti RN, Nk G, Rn C. Viability of M. leprae under normal and adverse atmospheric conditions. Lepr India. 1982;54(2):228–33. pmid:6752575
            • 103. Desikan K V., Sreevatsa . Extended studies on the viability of Mycobacterium leprae outside the human body. Lepr Rev. 1995;66(4):287–95. pmid:8637382
            • 104. Kazda J, Irgens LM, Müller K. Isolation of non-cultivable acid-fast bacilli in sphagnum and moss vegetation by foot pad technique in mice. Int J Lepr Other Mycobact Dis. 1980;48(1):1–6. pmid:6988344
            • 105. Kazda J. Occurrence of non-cultivable acid-fast bacilli in the environment and their relationship to M. leprae. Lepr Rev. 1981;52(Suppl 1):85–91.
            • 106. Lavania M, Katoch K, Sachan P, Dubey A, Kapoor S, Kashyap M, et al. Detection of Mycobacterium leprae DNA from soil samples by PCR targeting RLEP sequences. J Commun Dis. 2006;38(3):269–73. pmid:17373359
            • 107. Lavania M, Katoch K, Katoch VM, Gupta AK, Chauhan DS, Sharma R, et al. Detection of viable Mycobacterium leprae in soil samples: Insights into possible sources of transmission of leprosy. Infect Genet Evol. 2008;8(5):627–31. pmid:18599381
            • 108. Turankar RP, Lavania M, Singh M, Siva Sai KSRR, Jadhav RS. Dynamics of Mycobacterium leprae transmission in environmental context: Deciphering the role of environment as a potential reservoir. Infect Genet Evol. 2012;12(1):121–6. pmid:22101333
            • 109. Turankar RP, Lavania M, Singh M, Sengupta U, Siva Sai K, Jadhav RS. Presence of viable Mycobacterium leprae in environmental specimens around houses of leprosy patients. Indian J Med Microbiol. 2016;34(3):315–21. pmid:27514953
            • 110. Mohanty PS, Naaz F, Katara D, Misba L, Kumar D, Dwivedi DK, et al. Viability of Mycobacterium leprae in the environment and its role in leprosy dissemination. Indian J Dermatol Venereol Leprol. 2016;82(1):23–7. pmid:26728806
            • 111. Holanda MV de Marques LEC, Macedo MLB de, Pontes MA de A, Sabadia JAB, Kerr LRFS, et al. Presence of Mycobacterium leprae genotype 4 in environmental waters in Northeast Brazil. Rev Soc Bras Med Trop. 2017;50(2):216–22. pmid:28562758
            • 112. Arraes MLB de M, De Holanda MV, Lima LNGC, Sabadia JAB, Duarte CR, Almeida RLF, et al. Natural environmental water sources in endemic regions of northeastern Brazil are potential reservoirs of viable Mycobacterium leprae. Mem Inst Oswaldo Cruz. 2017;112(12):805–11. pmid:29211240
            • 113. Tió-Coma M, Wijnands T, Pierneef L, Schilling AK, Alam K, Roy JC, et al. Detection of Mycobacterium leprae DNA in soil: multiple needles in the haystack. Sci Rep. 2019 Dec 28;9(1):3165. pmid:30816338
            • 114. Kazda J, Irgens LM, Kolk AH. Acid-fast bacilli found in sphagnum vegetation of coastal Norway containing Mycobacterium leprae-specific phenolic glycolipid-I. Int J Lepr Other Mycobact Dis. 1990 Jun;58(2):353–7. pmid:2142948
            • 115. Turankar RP, Lavania M, Darlong J, Siva Sai KSR, Sengupta U, Jadhav RS. Survival of Mycobacterium leprae and association with Acanthamoeba from environmental samples in the inhabitant areas of active leprosy cases: A cross sectional study from endemic pockets of Purulia, West Bengal. Infect Genet Evol. 2019;72:199–204. pmid:30658215
            • 116. Filice GA, Greenberg RN, Fraser DW, Greenberg RN, Fraser DW. Lack of observed association between armadillo contact and leprosy in humans. Am J Trop Med Hyg. 1977;26(1):137–9. pmid:557294
            • 117. Deps PD, Alves BL, Gripp CG, Aragao RL, Guedes B, Filho JB, et al. Contact with armadillos increases the risk of leprosy in Brazil: A case control study. Indian J Dermatol Venereol Leprol. 2008;74(4):338–42. pmid:18797053
            • 118. Schmitt JV, Dechandt IT, Dopke G, Ribas ML, Cerci FB, Viesi JMZ, et al. Armadillo meat intake was not associated with leprosy in a case control study, Curitiba (Brazil). Mem Inst Oswaldo Cruz. 2010;105(7):857–62. pmid:21120353
            • 119. Truman RW, Singh P, Sharma R, Busso P, Rougemont J, Paniz-Mondolfi A, et al. Probable Zoonotic Leprosy in the Southern United States. N Engl J Med. 2011;364(17):1626–33. pmid:21524213
            • 120. da Silva MB, Portela JM, Li W, Jackson M, Gonzalez-Juarrero M, Hidalgo AS, et al. Evidence of zoonotic leprosy in Pará, Brazilian Amazon, and risks associated with human contact or consumption of armadillos. PLoS Negl Trop Dis. 2018;12(6):e0006532. pmid:29953440
            • 121. Stefani MMA, Rosa PS, Costa MB, Schetinni APM, Manhães I, Pontes MAA, et al. Leprosy survey among rural communities and wild armadillos from Amazonas state, Northern Brazil. PLoS One. 2019;14(1):e0209491. pmid:30629624
            • 122. Benchimol JL, Sá MR, Benchimol JL, Sá R, Romero Sa M. Adolpho Lutz and controversies over the transmission of leprosy by mosquitoes. Hist Cienc Saude Manguinhos. 2003;10(Suppl 1):49–93. pmid:14650407
            • 123. Dungal N. Is leprosy transmitted by insects? Lepr Rev. 1960;31(1):25–34.
            • 124. Dungal N. Is leprosy transmitted by arthropods? Lepr Rev. 1961;31(1):28–35.
            • 125. McFadzean JA, Macdonald WW. An investigation of the possible role of mosquitoes and bed bugs in the transmission of leprosy. Trans R Soc Trop Med Hyg. 1961;55(3):232–4.
            • 126. Narayanan E, Manja KS, Bedi BM, Kirchheimer WF, Balasubrahmanyan M. Arthropod feeding experiments in lepromatous leprosy. Lepr Rev. 1972;43(4):188–93. pmid:4666137
            • 127. Narayanan E, Sreevatsa , Kirchheimer WF, Bedi BM. Transfer of leprosy bacilli from patients to mouse footpads by Aedes aegypti. Lepr India. 1977;49(2):181–6. pmid:333183
            • 128. Narayanan E, Sreevatsa , Raj ADD, Kirchheimer WFF, Bedi BMM. Persistence and distribution of Mycobacterium leprae in Aedes aegypti and Culex fatigans experimentally fed on leprosy patients. Lepr India. 1978;50(1):26–37. pmid:349262
            • 129. Saha K, Jain M, Mukherjee MK, Chawla NM, Chaudhary DS, Prakash N. Viability of Mycobacterium leprae within the gut of Aedes aegypti after they feed on multibacillary lepromatous patients: a study by fluorescent and electron microscopes. Lepr Rev. 1985;56(4):279–90. pmid:3908862
            • 130. Banerjee R, Banerjee BD, Chaudhury S, Hati AK. Transmission of viable Mycobacterium leprae by Aedes aegypti from lepromatous leprosy patients to the skin of mice through interrupted feeding. Lepr Rev. 1991;62(1):21–6. pmid:2034021
            • 131. Sreevatsa , Girdhar BK, Ipe IM, Desikan K V. Can sandflies be the vector for leprosy? Int J Lepr. 1992;60(1):94–6.
            • 132. Geater JG. The fly as potential vector in the transmission of leprosy. Lepr Rev. 1975 Dec;46(4):279–86. pmid:1107727
            • 133. Moiser B. Hansen’s disease (leprosy) and cockroaches. East Afr Med J. 1947;24(6):230–6. pmid:20261291
            • 134. Dharmendra , Mukerjee N. Cockroach not responsible for transmission of leprosy. Int J Lepr. 1948;16(2):305.
            • 135. Shankara Manja K, Narayanan E, Kasturi G, et al. Non cultivable mycobacteria in some field collected arthropods. Lepr India. 1973;45(4):231–4.
            • 136. Narayanan E, Manja KS, Kirchheimer WF, Balasubrahmanyan M. Occurrence of Mycobacterium leprae in arthropods. Lepr Rev. 1972;43(4):194–8. pmid:4146908
            • 137. Blake LA, West BC, Lary CH, Fowler MER, Todd JR. Earthworms near leprosy patients’ homes are negative for acid-fast bacilli by fite stain, providing no link between leprous armadillos (Dasypus novemcinctus) and human leprosy. Microb Ecol. 1989;17(1):105–10. pmid:24197127
            • 138. Lahiri R, Krahenbuhl JL. The role of free-living pathogenic amoeba in the transmission of leprosy: a proof of principle. Lepr Rev. 2008;79(4):401–9. pmid:19274986
            • 139. Wheat WH, Casali AL, Thomas V, Spencer JS, Lahiri R, Williams DL, et al. Long-term Survival and Virulence of Mycobacterium leprae in Amoebal Cysts. Vinetz JM, editor. PLoS Negl Trop Dis. 2014 Dec 18;8(12):e3405. pmid:25521850
            • 140. Paling S, Wahyuni R, Winarni D, Astari L, Adriaty D, Agusni I, et al. Acanthamoeba SP.S-11 phagocytotic activity on Mycobacterium leprae in different nutrient conditions. African J Infect Dis. 2018;12(S):44–8.
            • 141. Neumann A da S, Dias F de A, Ferreira J da S, Fontes ANB, Rosa PS, Macedo RE, et al. Experimental Infection of Rhodnius prolixus (Hemiptera, Triatominae) with Mycobacterium leprae Indicates Potential for Leprosy Transmission. Lanz-Mendoza H, editor. PLoS One. 2016 May 20;11(5):e0156037. pmid:27203082
            • 142. da Silva Ferreira J, Souza Oliveira DA, Santos JP, Ribeiro CCDU, Baêta BA, Teixeira RC, et al. Ticks as potential vectors of Mycobacterium leprae: Use of tick cell lines to culture the bacilli and generate transgenic strains. PLoS Negl Trop Dis. 2018;12(12):e0007001. pmid:30566440
            • 143. Franco-Paredes C, Rodriguez-Morales AJ. Unsolved matters in leprosy: a descriptive review and call for further research. Ann Clin Microbiol Antimicrob. 2016;15:33. pmid:27209077
            • 144. Tufariello JM, Kerantzas CA, Vilchèze C, Calder RB, Nordberg EK, Fischer JA, et al. The Complete Genome Sequence of the Emerging Pathogen Mycobacterium haemophilum Explains Its Unique Culture Requirements. MBio. 2015;6(6):e01313–15. pmid:26578674
            • 145. Leprosy–we’ve much left to learn, but are looking to squirrels, cows and cats for insights. J Feline Med Surg. 2017;19(9):977–8. pmid:28838296
            • 146. Davis GL, Ray NA, Lahiri R, Gillis TP, Krahenbuhl JL, Williams DL, et al. Molecular Assays for Determining Mycobacterium leprae Viability in Tissues of Experimentally Infected Mice. Johnson C, editor. PLoS Negl Trop Dis. 2013 Aug 22;7(8):e2404. pmid:24179562