key: cord-0309314-7h8houjl
authors: Hockings, Kimberley J.; Mubemba, Benjamin; Avanzi, Charlotte; Pleh, Kamilla; Düx, Ariane; Bersacola, Elena; Bessa, Joana; Ramon, Marina; Metzger, Sonja; Patrono, Livia V.; Jaffe, Jenny E.; Benjak, Andrej; Bonneaud, Camille; Busso, Philippe; Couacy-Hymann, Emmanuel; Gado, Moussa; Gagneux, Sebastien; Johnson, Roch C.; Kodio, Mamoudou; Lynton-Jenkins, Joshua; Morozova, Irina; Mätz-Rensing, Kerstin; Regalla, Aissa; Said, Abílio R.; Schuenemann, Verena J.; Sow, Samba O.; Spencer, John S.; Ulrich, Markus; Zoubi, Hyacinthe; Cole, Stewart T.; Wittig, Roman M.; Calvignac-Spencer, Sebastien; Leendertz, Fabian H.
title: Leprosy in wild chimpanzees
date: 2020-11-11
journal: bioRxiv
DOI: 10.1101/2020.11.10.374371
sha: d9f92330406d948497f14ceed078726e9f0b5a68
doc_id: 309314
cord_uid: 7h8houjl

Humans are considered the main host for Mycobacterium leprae, the aetiologic agent of leprosy, but spill-over to other mammals such as nine-banded armadillos and red squirrels occurs. Although naturally acquired leprosy has also been described in captive nonhuman primates, the exact origins of infection remain unclear. Here, we report on leprosy-like lesions in two wild populations of western chimpanzees (Pan troglodytes verus) in the Cantanhez National Park, Guinea-Bissau, and the Taï National Park, Côte d’Ivoire, West Africa. Longitudinal monitoring of both populations revealed the progression of disease symptoms compatible with advanced leprosy. Screening of faecal and necropsy samples confirmed the presence of M. leprae as the causative agent at each site and phylogenomic comparisons with other strains from humans and other animals show that the chimpanzee strains belong to different and rare genotypes (4N/O and 2F). The independent evolutionary origin of M. leprae in two geographically distant populations of wild chimpanzees, with no prolonged direct contact with humans, suggests multiple introductions of M. leprae from an unknown animal or environmental source.

every year, of which 2.3% are located in West Africa 5 . Transmission is thought to occur primarily between individuals with prolonged and close contact via aerosolised nasal secretions and entry through nasal or respiratory mucosae, but the exact mechanism remains unclear 6, 7 .

The role of other routes, such as skin-to-skin contact, is unknown.

Leprosy-causing bacteria were once thought to be obligate human pathogens 8 . However, they can circulate in other animal hosts in the wild, such as in nine-banded armadillos (Dasypus novemcinctus) in the Americas and red squirrels (Sciurus vulgaris) in the United Kingdom 9,10 .

Although initial infection was most likely incidental and of human origin, secondary animal hosts can subsequently represent a source of infection to humans [10] [11] [12] [13] [14] . In captivity, nonhuman primates, such as chimpanzees (Pan troglodytes) 15 , sooty mangabeys (Cercocebus atys) 16, 17 and cynomolgus macaques (Macaca fascicularis) 18 , developed leprosy spontaneously (i.e. not through laboratory experiments). However, it is unknown whether these species also contract leprosy in the wild. Table 1 ), 31,044 (5.0%) contained chimpanzees.

The number of independent events (i.e. images separated by at least 60 minutes) totalled 4,336, and of these, 241 (5.6%) contained chimpanzees with severe leprosy-like lesions, including four clearly identifiable individuals (two adult females and two adult males) across three communities (Fig 1b; Extended Data Figs 1-2; Supplementary Information Note 2). As with humans, paucibacillary cases in chimpanzees may be present but easily go undetected. Since minor physical manifestations of leprosy are difficult to observe, they are not reported in our observations. All symptomatic chimpanzees showed hair loss and facial skin hypopigmentation, as well as plaques and nodules that covered different areas of their body (limbs, trunk and genitals), facial disfigurement and ulcerated and deformed hands (claw hand) and feet (Fig 2a- leprae, we collected faecal samples and tested them with two nested polymerase chain reaction (PCR) assays targeting the M. leprae specific RLEP repetitive element and 18kDa antigen gene. One out of 208 DNA-extracts from CNP was positive in both assays and a second was positive only in the more sensitive RLEP PCR 19 (Fig 1c; Extended Data Fig 3) . Longitudinal observations through to 2020 showed that the initial small nodules seen on the ears, lips and under the eye became more prominent and were followed by several nodules on the eyebrows, eyelids, nostrils, ears, lips and face. Clinical manifestations later developed to include hypopigmentation of the skin on facial nodules, hands, feet and testicles, as well as the loss and abnormal growth of nails (Fig 2d-g Fig 4) . Formalin-fixed skin samples (hands and feet) were examined using hematoxylin and eosin, as well as Fite-Faraco, stains. In the histopathological examination, the skin presented typical signs of lepromatous leprosy characterised by a diffuse cutaneous cell infiltration in the dermis and the subcutis clearly separated from the basal layer of the epidermis (Extended Data Fig 5a) . We detected moderate numbers of acid-fast bacilli, single or in clumps, within histiocytes, indicative of M. leprae (Extended Data Fig 5b) . Since antibodies against the M. leprae-specific antigen phenolic glycolipid-I (PGL-I) are a hallmark of M. leprae infection in humans 20 , we also performed a PGL-I lateral flow rapid test 21 on a blood sample from this individual, which showed strong seropositivity (Extended Data Fig 6) The strain that infected Zora in Côte d'Ivoire, designated TNP-418, belongs to branch 2F, within which branching order was also mostly unresolved (Fig 3a-b) . The branch is currently composed of human strains from medieval Europe (n=7) and modern Ethiopia (n=2) (Fig. 3b) , and this genotype has thus far never been reported in West Africa (Fig 3b) . Bayesian The finding of M. leprae-induced leprosy in wild chimpanzee populations raises the question of the origin(s) of these infections. M. leprae is considered a human-adapted pathogen and earlier cases of leprosy affecting wildlife were compatible with anthroponosis. Therefore, the prime hypothesis would be a human-to-chimpanzee transmission. Potential routes of transmission include direct (e.g. skin-to-skin) contact and inhalation of respiratory droplets and/or fomites, with the assumption that, in all cases, prolonged and/or repeated exposure is required for transmission 4 . Chimpanzees at CNP are not habituated to human presence and are not approached at distances that would allow for transmission via respiratory droplets.

Although chimpanzees at CNP inhabit an agroforest landscape and share access to natural and cultivated resources with humans 28 , present-day human-chimpanzee direct contact is uncommon. The exact nature of historic human-chimpanzee interactions at CNP remains, however, unknown. At TNP, direct human contact with wild chimpanzees has not been reported, with the South community distant from human settlements and agricultural areas.

Human-to-animal transmission of pathogens has been shown at TNP 29,30 but involved respiratory pathogens (pneumoviruses and human coronavirus OC43) that transmit easily and do not require prolonged exposure. In addition, M. leprae is thought to be transmitted from symptomatic humans 31 and no leprosy cases have been reported among researchers or local research assistants. Combined with the rarity of the M. leprae genotypes detected in chimpanzees among human populations in West Africa, this suggests that recent human-tochimpanzee transmission is unlikely.

The relatively old age of the lineages leading to the chimpanzee strains nevertheless raises the possibility of an ancient human-to-chimpanzee transmission. This hypothesis is, however, also unlikely for three reasons. First, the density of human populations at CNP and TNP 1,500-2,000 ya was even lower than it is currently, therefore reducing the likelihood of an ancient human-to-chimpanzee transmission. Second, two such human-to-chimpanzee transmission events would be required to explain our findings since M. leprae strains in CNP and TNP have distinct evolutionarily origins. Third, we can assume that if such transmission had occurred and the bacterium had persisted in chimpanzees, it should have spread more broadly as observed in M. leprae-infected squirrels and armadillos 10, 12, 13 . Therefore, ancient human-to-chimpanzee transmission is not a plausible mechanism to explain the presence of M. Red outline represents chimpanzee communities with at least one individual with clinical manifestations of leprosy, confirmed using molecular analysis; orange outline represents chimpanzee communities with at least one individual with clinical manifestations of leprosy; yellow colour represents monitored communities where clinical manifestations of leprosy have not been observed nor confirmed through molecular analysis. c, Location of the three habituated chimpanzee communities monitored at TNP (N: North; S: South; E: East). Estimated home ranges of chimpanzee communities at TNP are shown by 100% Minimum Convex Polygons of direct chimpanzee follows from December 2013 to October 2016. Red outline represents the community with individuals with clinical manifestations of leprosy, confirmed using molecular analysis and serological tests; blue colour represents communities where leprosy has not been recorded. CNP imagery is from Sentinel-2 (available at Sentinel Hub), and home range estimates were calculated in R using the package 'adehabitatHR' 39 . Table 2 ), including the two new chimpanzee strains (in bold red) and 21 new genomes from West Africa (in bold), 500 bootstrap replicates and M. lepromatosis as outgroup. Sites with missing data were partially deleted (80% coverage cut-off), resulting in 4470 variable sites used for the tree calculation. Subtrees corresponding to branches were retrieved in MEGA7 40 . Corresponding genotypes are indicated on the side of each subtree. Samples are binned according to geographical origin as given in the legend. Animal silhouettes were available under Public Domain license at phylopic (http://phylopic.org/)

Observational study and sample collections were performed at the Cantanhez National Park (CNP) in southern Guinea-Bissau and the Taï National Park (TNP) in western Côte d'Ivoire The TNP (5082 km 2 ) consists of an evergreen lowland rainforest and is the largest remaining primary forest fragment in West Africa. It is home to a wide range of mammals that include 11 different nonhuman primate species 48, 49 . There are no settlements or agricultural areas inside the National Park. As of October 2020, the three habituated communities, North, South and East, comprise 25, 39 and 35 individuals, respectively, although community sizes have varied over time. Systematic health monitoring of these communities has been ongoing since 2000 23 .

At CNP, camera traps (Bushnell Trophy Cam models 119774, 119877 and 119875) were deployed at 211 locations including across different habitat types (forest, mangrove-forest edge, orchards) within the home range of eight of the 12 putative chimpanzee communities (Fig 1b) . Camera traps were set up over six data collection periods ranging from 2015 to 2019 (Extended Data Table 1 ). Targeted camera traps were deployed to record and monitor chimpanzee behaviour and disease occurrence. To maximise the chances of recording specific behaviours and identify leprosy-like symptoms in individuals, targeted camera traps were set up in locations that chimpanzees were known to use most often, sometimes in clusters, precluding uniform survey designs. Targeted camera traps were set up in video mode and active 24h per day. When triggered, targeted cameras recorded 10 to 60s of video with a minimum interval of 0.6s or 2s, depending on the camera trap model. Furthermore, systematically placed camera traps were used to obtain measures of wildlife occurrence and habitat use across the heterogeneous landscape 41 . Systematic camera traps were deployed across central CNP, at a minimum distance of 1km between sampling points, as well as within the home range of one chimpanzee community (Caiquene-Cadique) and were spaced at least 500m from one another. Abnormalities in behaviour or clinical signs of disease are immediately reported and followed by detailed observation by the on-site veterinarian. In order to reduce the risk of transmission of human diseases to the chimpanzees, stringent hygiene measures have been put in place, including an initial five days quarantine for observers, keeping a distance of at least 7 meters, obligatory wearing of masks, with only healthy observers allowed to work in the forest 50, 51 .

At CNP, chimpanzee faecal samples were collected between July 2017 and December 2018.

The date and putative chimpanzee community were recorded for each faecal sample. As defecation was rarely observed and to prevent the collection of redundant samples from the same individual, we avoided multiple samples found under the same chimpanzee nest and paid special attention if multiple samples were found in proximity on trails 45, 52, 53 . All samples were collected with the aid of a wooden spatula and stored at ambient temperature in 15ml tubes containing NAP buffer 54 

To determine whether faecal samples positive for M. leprae belonged to one or two individuals of CNP, we amplified chimpanzee DNA at 11 microsatellite loci and one sexing marker 55 . Due to the small quantity of starting DNA, not all loci were amplified and in some cases the amplification quality was low, impacting our ability to confidently interpret allele peak profiles (e.g. sample GB-CC064 failed to amplify for 5 out of the 11 loci) (Supplementary Information Note 4).

M. leprae DNA was searched for using two nested PCR systems targeting the distinct but conservative repetitive element RLEP and the 18-kDa antigen gene as previously described (Extended Data Table 5 ). As several copies of RLEP are present in the M. leprae genome, this assay is considered to be more sensitive than 18 kDa, for which there is only a single copy. To prevent contamination at the laboratory at RKI and to enable us to identify if it occurs, we followed the following procedures: (1) denaturation at 95°C for 3 min, followed by 50 cycles of 95°C for 30 sec, 55°C (18kDa primers) or 58°C (RLEP primers) for 30 sec, and 72°C for 1 min as well as an elongation step at 72°C for 10 min. For the nested PCRs, 2µL of a 1:20 dilution of the primary PCR product was used as a template. Molecular grade water was used as a non-template control. PCR products were visualized on a 1.5% agarose gel stained with GelRed® (Biotium, CA, USA). Bands of the expected size were purified using the Purelink Gel extraction kit (Thermo Fisher Scientific, MA, USA). Both RLEP and 18-kDa nested PCR products are too short for direct Sanger sequencing. Therefore, fusion primers (primary PCR primers coupled with M13F and M13R primers) (Extended Data Table 5 ) were used for further amplification of the cleaned PCR products, applying the same conditions as in the primary PCR, but running only for 25 cycles.

The resulting extended PCR products were then enzymatically cleaned using the ExoSAP-IT™ PCR Product Cleanup assay (Thermo Fisher, MA, USA) and Sanger sequenced using M13 primers. Resulting sequences were compared to publicly available nucleotide sequences using the Basic Local Alignment Search Tool (BLAST) 56 .

To further confirm the infection, skin samples were sent to the German Primate Center in Göttingen, Germany for histopathological analyses. Samples were immersion-fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with standard hematoxylin & eosin (HE) using the Varistain Gemini staining automat (Thermo Fisher Scientific, MA, USA).

Samples were also stained with Fite-Faraco stain for the identification of acid-fast bacilli. Information Table 1) were converted into dual-indexed libraries using the NEBNext® Ultra™ II DNA Library Prep kit (New England Biolabs, MA, USA) 57, 58 . To reconstruct whole genomes, libraries were target-enriched for M. leprae DNA using in-solution hybridisation capture with 80 nt RNA baits designed to cover the whole M. leprae genome (2-fold tiling; design can be shared upon request to the corresponding authors) and following the myBaits protocol as previously DNA was extracted from skin biopsies using the total DNA extraction method as described previously 60 . DNA was quantified with a Qubit fluorometer using the Qubit™ dsDNA BR Assay kit (Thermo Fisher Scientific, MA, USA) prior library preparation. DNA libraries were prepared using the Kapa Hyper Prep kit (Roche, Switzerland) as per the manufacturer's recommendation using Kapa Dual Indexed Adapter (Roche, Switzerland) followed by in-solution capture enrichment with 80nt RNA baits with 2x tiling density for 48h at 65°C as described recently 60 . Post-capture amplification was performed with seven cycles.

Enriched libraries were purified using a 1X ratio of KAPA Pure beads (Roche, Switzerland) followed by quantification with the KAPA library quantification kit (Roche, Switzerland) and quality control of the fragment with the Agilent 2200 TapeStation (Agilent Technologies, CA, USA). Libraries were then normalized and pooled across sequencing lanes on an Illumina NextSeq 500 on a high output kit v2; 75 cycles (Illumina, CA, USA).

Raw reads were processed as described elsewhere 24 . Putative unique variants of GB-CC064 and TNP-418 strains were manually checked and visualized using the Integrative Genomics Viewer 61 .

SNPs of the two newly sequenced genomes from chimpanzees were compared to the 263 publicly available M. leprae genomes (Supplementary Table 2 Sites with missing data were partially deleted (80% coverage cut-off), resulting in 4470 variable sites used for the tree calculation.

Dating analyses were done using BEAST2 (v2.5.2) 66 as described previously with 278 genomes and an increased chain length from 50 to 100 million 24 . Briefly, the concatenated SNPs for each sample were used for tip dating analysis (Supplementary Information Table 4 ). Hypermutated strains and highly mutated genes associated with drug resistance (in yellow, Table 3 ) were omitted 24,60 , manual curation of the MP and BEAST input file was done at the positions described in Supplementary Information Table 5 for GB-CC064 and TNP-418. Sites with missing data as well as constant sites were included in the analysis, as previously described 24 . Only unambiguous constant sites, i.e., loci where the reference base was called in all samples, were included.

The genome coverage for the strain infecting Woodstock was low. To be able to determine the genotype, we identified specific variants from the genome-wide comparison of TNP-418 (the strain infecting Zora, an individual from the same social group) with other strains from branch 2F (Supplementary Table 6 ). Variants were manually checked and visualized in the partially covered genome from the strain infecting Woodstock using IGV software (Supplementary Table 6 ). Two variants not covered by high throughput sequencing data were also selected for specific PCR screening. Primers were designed using the Primer3 web tool (http://bioinfo.ut.ee/primer3-0.4.0/) based on the Mycobrowser sequences 67 and are described in Extended Data Table 5 . All PCR conditions were the same as in the M. leprae screening PCRs except for the primer sets and associated annealing temperatures (Extended Data Table   5 ).

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International Primatological Society Conservation, Conservation International/Global Wildlife Conservation Primate Action Fund

The work at TNP was supported by the Max Planck Society, which has provided core funding for the Taï Chimpanzee Project since 1997; the work at TNP and all analyses performed on nonhuman primate samples were supported by the German

The work on human specimens was supported by the Fondation Raoul Follereau (STC, CRJ), the Heiser Program of the New York Community Trust for Research in Leprosy (JSS and CA: grant numbers P18-000250), and the Association de Chimiothérapie Anti-Infectieuse of the Société Française de Microbiologie (CA). CA was also supported by a nonstipendiary European Molecular Biology Organization (EMBO) long-term fellowship

Project and the Centre Suisse de Recherches Scientifiques for their constant support of our work in TNP. Thanks to Sylvain Lemoine for providing shapefiles on chimpanzee home ranges at Taï National Park. We would like to thank Prof

Bastien Mangeat and the Gene expression Core Facility from the Ecole Polytechnique Fédérale de Lausanne for support. The authors would like to thank Prof. Anne Stone from Arizona State University for her constructive comments on the manuscript. Finally, the authors are grateful to all the patients and clinical staff who participated in the study

Biosample codes for all samples used in this study are given in the Supplementary Data. Other relevant data supporting the findings of the study are available in this published article and its Supplementary Information files

HZ DNA extraction, library preparation, enrichment and whole genome sequencing: BM, CA, PB PCR and SNP confirmation: BM, LVP, CA Chimpanzee microsatellite analysis: CB, JL-J Computational analysis: CA

Supplementary Information is available for this paper.Correspondence and requests for materials should be addressed to FHL Extended data (see corresponding file)