Leprosy is a mutilating disease caused by the intracellular bacilli Mycobacteria leprae (M. leprae) and/or lepromatosis (1). Despite the World Health Organization (WHO) removing leprosy from its list of public health concerns in 2001, the lack of significant reduction in new cases and the detection of leprosy in children indicate that transmission of the disease is still ongoing (2). This is evident in regions where active measures are taken to identify cases, such as door-to-door screenings, which consistently uncover new leprosy patients. Additionally, the prevalence of severe disabilities at the time of diagnosis in many countries suggests delayed detection and diagnosis (3). As a result, it is becoming increasingly apparent that we only see the tip of the iceberg of the global leprosy burden.

The exact transmission route of leprosy has not been fully elucidated yet. Different sites have been identified as potential entry and exit for M. leprae bacilli to the human body, namely the nose, mouth and skin (4). The highest bacillary burden is found in the epidermis of leprosy patients (5). The most probable transmission route of leprosy is via the aerial route (6), caused by the prolonged close contact to leprosy patients. Especially multibacillary patients are considered to drive leprosy transmission, given the high bacterial load. However, the nine-banded armadillo (7, 8), red squirrels (9), and chimps (10) have been confirmed as animal reservoirs and zoonotic transmission of M. leprae has been confirmed by genotyping (8) infected armadillos and leprosy patients in the US. Thus, the question as to whether the transmission pathway is direct or (partially) vector-driven remains unresolved (4).

The vector competence of Amblyomma sculptum from the family of hard ticks (Ixodidae) for M. leprae was demonstrated by Ferreira et al. (11) by artificially feeding adult females with M. leprae Thai-53 infected rabbit blood. Transovarial transmission of M. leprae was confirmed by the M. leprae specific RLEP qPCR. These findings are supported by results of Tongluan et al. (12) who injected Amblyomma maculatum ticks at adult and nymph stage with an M. leprae Thai-53 suspension derived from infected nude mice footpads. They confirmed the presence of M. leprae DNA in F₁ larvae and F₁ nymphs via RLEP qPCR. Both studies were able to grow M. leprae in cell lines derived from ticks, viability was confirmed by examination of the normalized expression levels of the M. leprae esxA gene (12) or 16S rRNA RT-qPCR (11). Transmission of M. leprae to a vertebrate host followed by an infection was only shown for M. leprae cultivated in and isolated from tick-derived cell lines. When inoculated directly into the footpad, these bacilli are able to establish a prolonged infection in mice (11, 12). Blood-feeding tick larvae were able to transfer viable M. leprae to a rabbit model. However, rabbit skin was analysed already 5 days after inoculation, a time frame too short to confirm a stable infection of the vertebrate host (11). Even though these studies were mainly aiming towards being able to grow M. leprae bacilli in vitro in a cell line, the experimental data suggests that there is a possibility for a transmission route of leprosy via ticks after taking their blood meal on a person with leprosy.

The Union of the Comoros has the highest per capita incidence of leprosy in Africa [as high as seven cases per 10,000 individuals on Anjouan (13, 14)], making it the only country of the African continent that did not reach the elimination target of less than 1 patient/10,000 population postulated by the WHO (3, 15). Despite the persistent efforts of the National Tuberculosis and Leprosy Control Programme, including intensified screenings since 2008 and the administration of post-exposure prophylaxis within the framework of the PEOPLE and BE-PEOPLE studies (Clinicaltrials.gov: NCT03662022 and NCT05597280), leprosy, a poverty-related disease, remains endemic on the islands Anjouan and Mohéli. In contrast, the wealthiest of the three islands, Grande Comore, is not considered a leprosy endemic region. Leprosy has a long incubation period of several months to decades, with an average of 2–4 years (16), which implies ongoing transmission of the disease by the high proportion of affected children on Anjouan and Mohéli (2). The potential contribution of non-human animal and environmental reservoirs to the transmission of leprosy represents a knowledge gap towards interrupting leprosy transmission. Further, the role of ticks as biological or mechanical vector has not been confirmed by epidemiological studies yet. Therefore, this study sought to investigate the presence of M. leprae DNA in a tick collection obtained from the Union of the Comoros as a means of further elucidating the potential involvement of ticks as a vector in leprosy transmission.

This study is the first to use molecular tools to screen wild, animal-derived ticks from a leprosy endemic country for the presence of M. leprae. The absence of M. leprae DNA was confirmed in all tested specimens from the Comoros. Next to M. leprae, M. lepromatosis can also cause leprosy disease in humans (1). We have tested the leprosy patient cohort in the Comoros for the presence of M. lepromatosis DNA by qPCR assay, with results suggesting that M. leprae is the only causative agent for leprosy on the Comoros (manuscript in preparation). Therefore, in this study ticks were only screened for the presence of M. leprae DNA.

In the search for drivers for leprosy transmission, two previous studies (11, 12) identified ticks from the genus Amblyomma as potential competent vectors for M. leprae. More specifically, under experimental conditions the transovarial transmission and the survival of M. leprae in female ticks and tick-derived cells was confirmed. The majority of the wild tick collection analysed in our study were adult females, which are able to harbour and transmit M. leprae under experimental conditions. The small proportion of nymphs, which is the developmental stage most likely to parasitize humans and transmit other tick-borne diseases such as lyme disease (22) and ehrlichiosis (23), could explain our inability to detect M. leprae DNA in the tick collection that was studied.

Further, the tick collection consisted of a small ratio of Amblyomma ticks, the species with proven capacity to harbour M. leprae (11, 12), compared to R. microplus. Only 10 out of 263 (3.8%) ticks from the endemic islands Anjouan and Mohéli were A. variegatum while Yssouf et al. (17) classified 73 out of 742 (9.8%) ticks as A. variegatum. The reason for the different species distribution is that only a subset of the original collection was available for analyses at ITM, Antwerp. The selected number of ticks from Grande Comore, used as non-endemic controls, was matched to the species distribution found for the endemic islands in this study. Accordingly, the percentage of A. variegatum was smaller than the one found by Yssouf et al. on this island. However, both Rhipicephalus and Amblyomma ticks belong to the family of Ixodidae (or hard ticks). In their previous studies Tongluan et al. and Ferreira et al. were able to maintain M. leprae in Ixodes-derived cell lines which suggests a similar potential of all members of the Ixodidae family as a vector for M. leprae.

Even though the ticks analysed in our study were collected from cattle and goats and not from humans, feeding of cattle ticks on humans seems probable in situations where humans and livestock live closely together. For both R. microplus and A. variegatum which mainly feed on cattle and other large animals (24), such cross-over events have been reported (25–27). A recent publication by Faber et al. (28) is raising the hypothesis that a skin disease in water buffaloes described as lepra bubalorum could be caused by M. leprae and therefore act as animal reservoir. However, evidence for cases in Indonesia is only historical as there were no further reports for lepra bubalorum in cattle since 1961 (29) and there is no water buffalo population described in the Union of the Comoros (30).

Different other vectors have been suggested for the transmission of M. leprae, e.g., arthropods such as mosquitos (Aedes, Culex, Rhodnius) (31–33), flies (Musea, Calliphora and Stomoxys) (34), and sand flies (Phlebotomus, Sergentomyia). The latter are unlikely vectors as they cannot maintain viable M. leprae bacilli (35). Early studies on mosquitos confirmed the presence of acid-fast bacilli in the proboscis of mosquitos (A. aegypti and C. fatigans) after experimentally feeding on untreated leprosy patients (31, 32). However, viability determined by fluorescence microscopy reduced within seven days after feeding (32). Da Silva Neumann et al. have investigated R. prolixus, A. aegypti, and C. quinquefasciatus as possible vector, with the result that only R. prolixus has the ability to defecate infective M. leprae up to 20 days after infection with M. leprae Thai-53 infected rabbit blood (33). Additionally, amoeba have been found to have vector potential as they can phagocytose M. leprae. In vitro experiments showed that M. leprae can survive up to 72 h within the Acanthamoeba and up to 8 months in amoebal cysts while retaining infectivity for a nude mouse model (36, 37). However, for none of these vector candidates a clear correlation with leprosy infections in humans was identified.

Even though Ixodes ticks are potential competent vectors for M. leprae in vitro and pathogen transmission from livestock to humans via ticks is probable, all ticks from Anjouan, Mohéli, and Grande Comore that were investigated tested negative for M. leprae DNA. This finding lessens the chance that leprosy is a tick-borne zoonosis in the Union of the Comoros, rather than spread by human-to-human transmission.

Our results support the hypothesis that most leprosy infections are caused by human-to-human interactions rather than by a non-human animal or environmental reservoir of M. leprae and that close contact to a leprosy patient is the driving force of transmission. For the definitive exclusion of the role of ticks in the transmission of leprosy disease, a larger number of ticks also from other leprosy endemic regions should be analysed. The exploration of human-derived ticks and particularly ticks parasitising leprosy patients should be the focus of such studies. Further, qualitative case control studies investigating daily activities of leprosy patients and healthy controls will be useful for the generation of new hypotheses on the driving factors of leprosy transmission.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

All ticks were manually sampled in the presence of cattle owners. No suffering was imposed to sampled animals and the disturbance associated with sampling was underneath the regulatory threshold requiring the submission of the protocol to an institutional ethics committee, as described on European Union Directive 2010/63/UE. For the study of ticks, ethical approval was not required in accordance with the national legislation and the institutional requirements.

LK, EC, BJ, and SB contributed to conception and design of the study. AY and PT were involved with the tick sampling and provided the tick collection. EC and MD-L were involved in the methodology, investigation, and validation. LK, EC, and SB performed the formal analysis and statistics. SB was the project administrator and together with BJ supervised the study. LK wrote the original manuscript draft. All authors contributed to the article and approved the submitted version.