Leishmaniasis is a vector-borne disease caused by parasitic protozoa of the genus Leishmania. These include various species of medical significance, responsible for approximately 1 million new cases and tens of thousands of deaths per annum (Burza et al., 2018). Ongoing research on the genus Leishmania has recently seen the emergence of a new subgenus of parasites, the Leishmania (Mundinia) (Espinosa et al., 2018; Cotton, 2017) to sit alongside the other 3 established subgenera, Leishmania (Leishmania), Leishmania (Viannia), and Leishmania (Sauroleishmania) (Lainson and Shaw, 1987; Akhoundi et al., 2016). The parasites in the subgenus L. (Sauroleishmania) infect reptiles and, although they are not of direct medical importance, they are of comparative interest, as they have been secondarily derived from mammal-infecting species (Croan et al., 1997; Noyes et al., 1998; Raymond et al., 2012; Coughlan et al., 2017). The majority of reported human infections arise from parasites in the subgenera L. (Leishmania) and L. (Viannia). However, the new subgenus L. (Mundinia) also contains human pathogens, Leishmania (Mundinia) martiniquensis (Desbois et al., 2014) and Leishmania (Mundinia) orientalis (Jariyapan et al., 2018), and another human pathogen is 1 of the new species described here, Leishmania (Mundinia) chancei. Species nonpathogenic to humans in subgenus Mundinia include Leishmania (Mundinia) enriettii (Muniz and Medina, 1948) found in guinea pigs and Leishmania (Mundinia) macropodum (Barratt et al., 2017) found in macropods, and the other new species described here, Leishmania (Mundinia) procaviensis. Phylogenetic analysis indicates that L. (Mundinia) sit at the base of the Leishmania clade and are the earliest branching subgenus (Butenko et al., 2019). This may partially explain their wide geographical distribution and variation in mammalian hosts, and possibly their transmission by non–sand fly vectors (Chanmol et al., 2019b, Bečvár et al., 2021). The subgenus L. (Mundinia) is therefore of both biological and medical significance within the genus Leishmania. Here we describe 2 new species in the subgenus, Leishmania (Mundinia) chancei n. sp. from Ghana and Leishmania (Mundinia) procaviensis n. sp. from Namibia.

MATERIALS AND METHODS

RESULTS

Sequences of 1,200 nucleotides for RNA PolII and 488 nucleotides for RPL23a from L. chancei and L. procaviensis were analyzed by BLAST searches, which showed the sequences differed from those for all other previously named Leishmania species deposited within the DDBJ/ENA/GenBank and TriTrypDB databases. The new RNA PolII sequences were most similar to those from Leishmania enriettii, with 98.4% and 98.5% identity for L. chancei and L. procaviensis, respectively, whereas the new RPL23a sequences were most similar to those from Leishmania orientalis, with 98.5% and 95.5% identity for L. chancei and L. procaviensis, respectively. These results indicated a probable classification within the subgenus L. (Mundinia).

The genetic distances revealed by the new sequence data were examined by making various pairwise comparisons. Regarding RNA PolII, the differences of 1.6% between L. chancei and L. enriettii, 1.5% between L. procaviensis and L. enriettii, and 1.6% between L. chancei and L. procaviensis are all greater than the differences of 0.6% between Leishmania donovani and Leishmania infantum and 0.6% between Leishmania amazonensis and Leishmania mexicana. The 4 species in these latter 2 pairwise comparisons are well-established existing species of Leishmania. Similarly, for RPL23a, the differences of 1.5% between L. chancei and L. orientalis, 4.7% between L. procaviensis and L. orientalis, and 4.7% between L. chancei and L. procaviensis are all greater than the differences of 0.4% between L. donovani and L. infantum and 1.1% between L. amazonensis and L. mexicana. These analyses support the designation of the isolates of L. chancei and L. procaviensis as new species.

To investigate this further, 4 different types of phylogenetic trees were generated, maximum likelihood (ML), neighbor-joining (NJ), minimum evolution (ME), and maximum parsimony (MP) trees, with both RNA PolII and RPL23a data sets, and in all 8 combinations the 2 new species clustered with the others in the subgenus L. (Mundinia) (Figs. 1, 2). In each case L. chancei and L. procaviensis clustered with L. enriettii and L. orientalis in a subgroup of these 4 species, in most trees with the other 2 L. (Mundinia) species, L. martiniquensis and L. macropodum, as a sister group, but always these 6 species were within a well-defined L. (Mundinia) clade with 91–100% bootstrap support (depending on the tree method used). The other 3 subgenera of Leishmania, L. (Leishmania), L. (Viannia), and L. (Sauroleishmania) were also resolved with high bootstrap support (87–100%), consistent with previous analyses (Butenko et al., 2019).

Figure 1.

Phylogenetic analysis of RNA PolII sequences from Leishmania species using different algorithms: maximum likelihood (ML), neighbor-joining (NJ), minimum evolution (ME), or maximum parsimony (MP). Numbers at nodes were derived by bootstrapping with 1,000 replicates. The 4 subgenera of Leishmania are indicated with numbered brackets: 1. L. (Leishmania); 2. Leishmania (Sauroleishmania); 3. Leishmania (Viannia); and 4. L. (Mundinia). The positions of the 2 new species are indicated with asterisks. Accession numbers for each species are given in Table I.

Figure 2.

Phylogenetic analysis of RPL23a sequences from Leishmania species using different algorithms: maximum likelihood (ML), neighbor-joining (NJ), minimum evolution (ME), or maximum parsimony (MP). Numbers at nodes were derived by bootstrapping with 1,000 replicates. The 4 subgenera of Leishmania are indicated with numbered brackets: 1. L. (Leishmania); 2. L. (Sauroleishmania); 3. L. (Viannia); and 4. L. (Mundinia). The positions of the 2 new species are indicated with asterisks. Accession numbers for each species are given in Table I.

DESCRIPTIONS

Table I

List of species used for phylogenetic analysis together with the World Health Organization codes of the isolates and their Genbank/ENA/DDJB accession numbers.

DISCUSSION

Based on the genetic and phylogenetic analyses described here we conclude that the 2 new species described are both members of the subgenus L. (Mundinia). Ghana has been long suspected as endemic for leishmaniasis as the north of the country is close to the semiarid Sahel belt, an ecotope in which Leishmania major cases have been reported in other parts of West Africa (Boakye et al., 2005). However, when the first Ghanaian cases were reported in 1999 they were found in the Ho District of the Volta region in the south of Ghana (Kweku et al., 2011), in areas with luxuriant vegetation that were originally primary rain forest. Biopsy samples from this new focus of leishmaniasis were initially identified as L. major (Fryauff et al., 2006), but a follow-up study instead indicated a new species (Villinski et al., 2008). This conclusion was confirmed by the first isolation of parasites in culture and their initial characterization (Kwakye-Nuako et al., 2015), and here we name this parasite Leishmania (Mundinia) chancei.

There have been limited studies to determine the prevalence of leishmaniasis in Ghana, but where these have been conducted clinical signs have indicated 3–4% of the general population and 12–30% of schoolchildren to have ulcers and/or scars suggestive of leishmaniasis (Kweku et al., 2011). More recent studies in 3 communities in the Oti region (formerly part of the Volta region) showed that 32% of the skin ulcers examined were Leishmania positive by PCR (Akuffo et al., 2021a). In a parallel study Leishmania skin test positivity indicated that 42% of individuals in these communities had been exposed to Leishmania (Akuffo et al., 2021b). Therefore, although the exact prevalence and incidence in the population remain uncertain, it is certain that cutaneous leishmaniasis is a frequent infection in the Oti and Volta regions of Ghana. From the existing data, it is not possible to conclude that all of these are due to infection with L. chancei but given the normally focal nature of the disease this may well be the case, and this remains the only parasite conclusively identified from these regions.

Animal reservoirs of L. (M.) chancei are presumed to exist based on the zoonotic nature of most of the leishmaniases, but remain unknown to date. Experimental studies have shown that L. (M.) chancei is unable to infect guinea pigs (Bečvár et al., 2020) and that the African rodents Arvicanthus niloticus and Mastomys natalensis are unlikely to be reservoir hosts (Sadlova et al., 2020). The vectors are also unknown and, whilst these would normally be expected to be phlebotomine sand flies, there is increasing evidence for midges acting as vectors for Leishmania (Mundinia) species (Dougall et al., 2011; Seblova et al., 2015; Chanmol et al., 2019a; Bečvár et al., 2021). This has not been conclusively proven, but if it was confirmed that some or even all of the subgenus L. (Mundinia) had midge vectors, this would then require consideration of the definition of the genus Leishmania itself. In this circumstance, an option would be to take the Leishmania (Mundinia) into a new genus based on their different vectors. However, given the clinical and molecular similarity of the L. (Mundinia) species to other Leishmania, a preferable option would be to revise the definition of the genus Leishmania, allowing for both sand fly and midge vectors. Either way, this awaits definitive conclusions regarding the natural vectors of Leishmania (Mundinia) species.

There are only a few reports of leishmaniasis from southern Africa, and this region is not usually considered a high risk for the acquisition of infection by either travelers or residents. Nevertheless, Grové and colleagues conducted some investigations in the 1970s in the area surrounding Keetmanshoop in Namibia (Grové, 1989). Several isolates of Leishmania were collected, some from human cases, some from sand flies, and some from rock hyraxes. These were characterized using the methods available at the time, including multilocus isoenzyme analysis, and it is interesting to note that some of the rock hyrax isolates were recognized as “distinctly different” to those from the human cases and sand flies (Grové, 1989). One of these isolates survived in the Lancaster cryobank, LV425, and upon DNA sequence analysis was found to represent a new species, here named Leishmania (Mundinia) procaviensis. Further work is required to characterize the biological properties of this parasite, including its pathogenicity. Based on its difference from all other currently known human-infective parasites, its presence in rock hyraxes but an apparent absence of human infection, our working assumption is this new species is nonpathogenic to humans, but this has not been conclusively proven. If true, in this respect it appears to be like L. (M.) enriettii and L. (M.) macropodum within L. (Mundinia).

The results described here now bring the total number of named species in L. (Mundinia) to 6. One interesting feature of the L. (Mundinia) is their wide geographical distribution compared to other subgenera. Various analyses (Noyes et al., 2002; Dougall et al., 2011; Pothirat et al., 2014; Kwakye-Nuako et al., 2015; Harkins et al., 2016; Butenko et al., 2019) have shown them to typically be the earliest branching clade within the genus Leishmania, including the analyses described here. The explanation for this may be that they evolved when the continents were together in Gondwana, with the other subgenera evolving after the breakup of the supercontinent. Thus, further study of the L. (Mundinia) will help us to understand the evolution of the genus Leishmania and how some of these species came to be important human pathogens.