Sequencing of hsp70 for discernment of species from the Leishmania (Viannia) guyanensis complex from endemic areas in Colombia

Abstract

Background Colombia is ranked very high among countries with the highest numbers of endemic Leishmania species (n = 9) causing human disease. Although much effort has been devoted to generating simple and specific tools for Leishmania species identification, challenges remain in the discrimination of species belonging to the Leishmania (Viannia) guyanensis complex: L. (V.) guyanensis and L. (V.) panamensis. Methods A set of seven reference strains of species belonging to the L. (Leishmania) and L. (Viannia) subgenera, clinical strains from human cases of cutaneous leishmaniasis (CL; n = 26) and samples collected from sylvatic mammals and sand flies (n = 7) from endemic areas in Colombia were analyzed in this study. The heat-shock protein 70 gene (hsp70) was amplified by PCR from DNA extracted from logarithmic-phase promastigotes or tissue samples, and the PCR products were sequenced. Sequence alignment was performed against a set of previously published and curated sequences, and phylogenetic analysis based on the maximum-likelihood and Bayesian inference approaches was conducted. Haplotype diversity among strains and species of the L. (V.) guyanensis complex was explored using a median-joining network. Results Sequencing of the hsp70 gene for L. (Viannia) spp. typing was comparable to species identification using isoenzyme electrophoresis or monoclonal antibodies. Complete species matching was found, except for one sylvatic sample with an identity yet unsolved. Among the L. (V.) panamensis clinical strains, two distinctive phylogenetic clusters were found to correlate with two different zymodemes: L. (V.) panamensis Z2.2 and Z2.3. Analysis of samples from sylvatic environments identified novel records of naturally infected wild mammal and sand fly species. Conclusions Our results support the adequacy of hsp70 gene sequencing as a single-locus approach for discrimination of L. (Viannia) spp., as well as for exploring the genetic diversity within the L. (V.) guyanensis complex. Graphical Abstract

Pathogens, reservoirs, and vectors involved in the transmission of vector-borne and zoonotic diseases in a Colombian region

“…In this study, Leishmania ( Viannia ) panamensis was detected in the rodent genus Oecomys , as was previously reported in a study about Leishmania detection using hsp70 gene [ 24 ]. The genus Oecomys belongs to the family Cricetidae, and its distribution includes Panama, Bolivia, Colombia, Venezuela, and Trinidad Island [ 45 , 46 ]).…”

“…Leishmania panamensis was detected in the tissues of a rodent of the species Oecomys sp. (ANDES-M 2578) collected during the sampling, as was reported in a study where the performance of the hsp70 gene sequencing was evaluated as an alternative for genotyping of Leishmania species [ 24 ]. On the other hand, ten leaf-nosed bats (Phyllostomidae) were captured (Table 2 ), and none of them was infected.…”

“… 8 2566, 2570, 2571, 2575, 2576, 2579, 2583, 2584 Didelphimorphia Didelphidae Gracilinanus sp. 1 2577 Didelphimorphia Didelphidae Didelphis marsupialis 4 Not collected Chiroptera Phyllostomidae Artibeus lituratus 5 2555, 2558, 2559, 2560, 2561 Chiroptera Phyllostomidae Carollia perspicillata 4 2556, 2562, 2563, 2564 Chiroptera Phyllostomidae Sturnira giannae 1 2557 Carnivora Canidae Canis lupus familiaris 30 Not collected a A specimen ANDES-M 2578 infected with Leishmania ( Viannia ) panamensis [ 24 ] …”

“…For Leishmania detection in mammal samples and phlebotomine sand flies, PCR was performed with the primers LITSR and L5.8S, which specifically amplify a fragment of 350-bp of ITS gene [ 22 ]. The positive samples by ITS were typing for hsp70 gene, following the protocol and sequencing algorithm designed by Van der Auwera et al (2013) [ 23 ] and modifications described by Hoyos et al (2022) [ 24 ]. Assays for detection of Plasmodium were performed in Anopheles mosquitoes.…”

Genetic diversity and comparative genomics across Leishmania (Viannia) species

“…In addition, when multilocus assessments have resulted in concordance with individual loci (Boité et al, 2012;Figueiredo et al, 2019), some species were underrepresented or not considered in the analyses. Therefore, in the absence of a genomewide analysis encompassing most species of the group coming from diverse geographic origins, it is unclear whether some markers lack resolution at the specific level (Schönian et al, 2010;Hoyos et al, 2022) or if taxonomy needs revision.…”