Leprosy in wild chimpanzees

Humans are considered as the main host for Mycobacterium leprae1, the aetiological agent of leprosy, but spillover has occurred to other mammals that are now maintenance hosts, such as nine-banded armadillos and red squirrels2,3. Although naturally acquired leprosy has also been described in captive nonhuman primates4–7, the exact origins of infection remain unclear. Here we describe leprosy-like lesions in two wild populations of western chimpanzees (Pan troglodytes verus) in Cantanhez National Park, Guinea-Bissau and 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). These findings suggest that M. leprae may be circulating in more wild animals than suspected, either as a result of exposure to humans or other unknown environmental sources.


Statistics
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Software and code
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Data collection
No software was used Data analysis All raw reads were adapter-and quality-trimmed with Trimmomatic v0.35. The quality settings were "SLIDINGWINDOW:5:15 MINLEN:40". Paired-end (PE) data were additionally processed with SeqPrep (https://github.com/jstjohn/SeqPrep) to merge overlapping pairs. Preprocessed reads were mapped onto the M. leprae TN reference genome (GenBank AL450380.1) with Bowtie2 v2.2.5. SNP calling was done using VarScan v2.3.9. To avoid false-positive SNP calls the following cutoffs were applied: minimum overall coverage of five non-duplicated reads, minimum of three non-duplicated reads supporting the SNP, mapping quality score >8, base quality score >15, and a SNP frequency above 80%. InDel calling was done using Platypus v0.8.1 followed by manual curation. We used the Integrative Genomics Viewer v 2.8.13 and Basic Local Alignment Search Tool (BLAST) v 2.11.0+. Dating analyses were done using BEAST2 v2.5.2.
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Data
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Study description
Study description -We report on leprosy-like lesions in two wild populations of western chimpanzees in the Cantanhez National Park (CNP), Guinea-Bissau, and the Taï National Park (TNP), Côte d'Ivoire, West Africa. We screen chimpanzee faecal and necropsy samples for the presence of M. leprae and conduct phylogenomic comparisons with other strains from humans and other animals.

Research sample
The research sample is represented by two populations of wild chimpanzees (Pan troglodytes verus) in CNP and TNP. We conducted this study in these two populations in response to leprosy-like lesions observed during behavioural monitoring. We did not discriminate between age and sex classes, instead we collected data on as many individuals as possible for analysis of leprosy symptoms. Analyses in this paper focus on symptomatic individuals. These two chimpanzee populations include male and female individuals and age estimates range from newborn to adult (~40 years of age). There are a minimum of 12 chimpanzee communities at CNP, all unhabituated to researchers, with approximately 35-60 individuals per community (age and sex composition of all communities unknown). At one community (Caiquene-Cadique), we estimate at least 48 individuals, including 16 adult females, 13 adult males, 3 subadults and 16 immatures (juveniles and infants). At TNP, the three human-habituated chimpanzee communities include a total of 91 individually recognised chimpanzees.

Sampling strategy
We performed non-invasive sampling through the collection of faeces from symptomatic and asymptomatic chimpanzees at CNP and TNP. In CNP, where chimpanzees are not habituated to human observers, this is performed by collecting faecal material found under chimpanzee nests or in proximity to chimpanzee signs (e.g. food remains or knuckle prints). At the time of faecal collection, the identity of the chimpanzee was not known. At CNP, camera traps were deployed at 211 locations including across different habitat types within the home range of eight of the 12 putative chimpanzee communities. Targeted camera traps were deployed to record and monitor chimpanzee behaviour and disease occurrence. Systematic camera traps were deployed across central CNP at a minimum distance of 1km between sampling points. At TNP chimpanzees are followed by researchers on a daily basis and faeces are collected right after observing defecation. In both cases, faeces are collected with the aid of a plastic or wooden spatula and placed in 2ml or 15ml tubes dry or with RNAlater. For this study we analysed all available faecal samples from individuals which displayed clinical signs of leprosy and optimal sample sizes could not be determined beforehand. For TNP we included only samples from the South community since leprosy was observed only in members of this chimpanzee community. Necropsies on dead chimpanzees were performed by trained veterinarians at TNP as part of the health monitoring program. For this study, we tested all available chimpanzee necropsy samples in our collection.

Data collection
Data collection was performed by local field assistants, researchers and veterinarians working at CNP and TNP. At CNP, clinical data on unhabituated chimpanzees were collected using camera traps and faecal samples were collected with the aid of a wooden spatula and stored at ambient temperature in 15ml tubes containing NAP buffer. At TNP, data were collected by research assistants both on paper sheets and using the Cybertracker app, and by veterinarians who documented via pictures and videos. At TNP, the long-term health monitoring program includes continuous collection of faecal and urine samples from known adult chimpanzees. Faeces are transferred in 2ml cryotubes with the aid of a plastic spatula and frozen in liquid nitrogen. A full necropsy is systematically performed on chimpanzees found dead by the on-site veterinarian. Tissue samples of several internal organs are taken if the state of carcass decomposition allows.
Timing and spatial scale Camera traps were set up over six data collection periods ranging from 2015 to 2019 across CNP (1067 km2

Data exclusions
No specifica data were excluded from the study.

Reproducibility
To confirm our results of leprosy infection we used two PCR systems in parallel and tested several samples for each individual/ community. Positives were then further confirmed via next generation sequencing. For this purpose several individual libraries were generated to confirm M. leprae DNA presence in the samples.

Randomization
Randomization is not relevant for this type of study, which is based on investigating infectious causes of illness in wildlife. To maximize our chances of pathogen detection we sampled all individuals, whenever possible.

nature research | reporting summary
October 2018 Blinding Not applicable to this study since this is a study on a naturally occurring disease in wild animals.
Did the study involve field work?

Yes No
Field work, collection and transport Field conditions 125 km2), West Africa, lies within the Guinean forest-savannah mosaics, a biodiverse ecoregion buffering the Guinean moist forests in the south and the West Sudanian savannah in the north. The climate in Guinea-Bissau is characterized by a rainy season from mid-May to the end of October and a long dry season from November to mid-May. Cantanhez NP (N11° 14.287ʹ W15° 02.281ʹ) comprises the Cubucaré peninsula in the Tombali Region bordering Guinea-Conakry. The landscape in Cantanhez NP consists of a mosaic of coastal sub-humid forest patches, mangroves, savannah grassland, woodland and agriculture including mostly cashew orchards, shifting cultivation fields and mangrove swamp rice fields. Approximately 24,000 people across 200 villages and settlements are present inside the park. The TNP (5,082 km 2), located in the south-west of Ivory Coast bordering Liberia (N5° 38 56 W7° 05 43), 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. There are no settlements or agricultural areas inside the National Park. The climate in TNP is characterized by a rainy season from March/April to the end of October and a dry season from November to February/March.

Wild animals
At CNP, chimpanzees are not habituated to human observers and all data are collected remotely using camera traps. The age and sex distribution of chimpanzees within this population have not be calculated (as this requires accurately identifying all individuals). At one community (Caiquene-Cadique), we estimate at least 48 individuals, including 16 adult females, 13 adult males, 3 subadults and 16 immatures (juveniles and infants). At TNP, wild chimpanzee communities have been habituated by researchers since 1979. A team of field assistants and researchers follow the animals on a daily basis from a 7-meter distance, recording behavioural data and collecting faeces and urine samples whenever possible. In normal situations, each assistant or researcher has one focal individual per day to collect data and samples from. In disease outbreak situations, monitoring efforts are reinforced and sampling is attempted from all symptomatic and asymptomatic individuals. These populations include male and female individuals and estimation of age range is from newborn to adult (~40 years of age). As of March 2021, there are 91 individuals (40 males and 51 females), including 43 adults (14 males and 29 females), 5 adolescents (4 males, and 1 female), 19 juveniles (6 males and 13 females), and 24 infants (16 males and 8 females).

Field-collected samples
At CNP, chimpanzee faecal samples are collected by visiting chimpanzee nesting and feeding sites. Faecal samples were stored at room temperature in 15ml tubes containing NAP buffer, and shipped to Robert Koch Institute in Germany. At TNP, samples are