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. 2025 May 27:27:101090.
doi: 10.1016/j.ijppaw.2025.101090. eCollection 2025 Aug.

Molecular detection of Leishmania and other vector-borne agents in free-ranging and captive herpetofauna from Costa Rica

Mario H Alves  1 , Jairo Alfonso Mendoza-Roldan  1 , Paula Alfaro-Segura  2 , Mariaelisa Carbonara  1 , Aarón Gómez  3 , Natalia Montero Leitón  3 , Jazmín Arias Ortega  3 , Alberto Solano-Barquero  2   4 , Alicia Rojas  2   4 , Domenico Otranto  1   5
Affiliations

Molecular detection of Leishmania and other vector-borne agents in free-ranging and captive herpetofauna from Costa Rica

Mario H Alves et al. Int J Parasitol Parasites Wildl. .

Abstract

Vector-borne pathogens in amphibians and reptiles represent an emerging concern in wildlife, with implications for ecosystem dynamics and potential zoonotic risks. In this study, we screened 108 animals from Costa Rica, including 46 captive snakes, 24 free-ranging reptiles, and 38 free-ranging amphibians, for the presence of Trypanosomatidae, Anaplasmataceae, Borrelia, Rickettsia, and Hepatozoon spp. Blood smear analysis revealed protozoa gametocytes in 3.7 % of the animals sampled, and 11.1 % of amphibians and reptiles were molecular positive for at least one pathogen. Specifically, 7.4 % of the samples tested positive for Leishmania spp., 1.85 % for Trypanosoma spp., 0.9 % for Anaplasma spp., and 1.85 % for Hepatozoon spp. Notably, this study reports the first molecular detection of Leishmania in an amphibian species (Rhinella horribilis) and confirms the presence of mammalian pathogenic Leishmania infantum in captive snakes in Central America. The presence of potential zoonotic agents in both captive and free-ranging herpetofauna underscores the importance of screening wildlife species, including understudied host groups such as amphibians, to better understand their role in disease ecology.

Keywords: Amphibians; Leishmania infantum; Protozoa; Reptiles; Wildlife.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Map of Costa Rica showing specific locations in the San José, Alajuela, and Puntarenas provinces where the samples were collected. The white circles indicate the collection sites for free-ranging amphibian and reptile samples, while the white square marks the location where samples from reptiles under human care were collected. The map was prepared using QGIS software version 3.36.1 — Maidenhead, with ESRI Satellite and ESA WorldCover land-use map 2021 imagery.
Fig. 2
Fig. 2
A. Gamonts in erythrocytes of American White Lipped Frog (Leptodactylus fragilis) molecularly positive for Hepatozoon sp. B. Gamonts in erythrocytes of Common Spiny-tailed Iguana (Ctenosaura similis). C. gamonts in erythrocytes of Eyelash viper (Bothriechis schelegelii) molecularly positive for Hepatozoon sp. Scale bars 50 μm.
Fig. 3
Fig. 3
Species of captive snakes molecularly positive for Leishmania infantum. A. Central American rattlesnake (Crotalus simus) B. Eyelash viper (Bothriechis schelegelii).
Fig. 4
Fig. 4
Phylogenetic analysis of Leishmania spp. detected in this study according to a fragment of the 18S rRNA. A. Bayesian inference phylogenetic tree showing a cluster with L. infantum and second one with other Leishmania spp. Each species is color-coded and sequences derived in this study are marked with a black diamond. Posterior probability (PP) values are indicated next to each node and size and color of node circles are proportional to the PP. B. Templeton Crandall Sing haplotype network of Leishmania spp. with 95 % connection limit. Sequences derived from the sampled animals are specified in each haplotype group. Circle size is proportional to the number of sequences grouped in the same haplotype. Hatch marks represent mutational steps between two haplotypes. C. Principal component analysis of genetic distances between Leishmania spp. Sequences derived from this study are shown in the graph.
Fig. 5
Fig. 5
Phylogenetic analysis of Trypanosoma spp. detected in this study according to a fragment of the 18S rRNA. A. Bayesian inference phylogenetic tree. Sequences derived in this study are marked with a black diamond. Posterior probability (PP) values are indicated next to each node and size and color of node circles are proportional to the PP. B. Templeton Crandall Sing haplotype network of Trypanosoma spp. with 95 % connection limit. Sequences derived from the sampled animals are marked with a black diamond and shadowed in the network. Circle size is proportional to the number of sequences grouped in the same haplotype. Hatch marks represent mutational steps between two haplotypes.
Fig. 6
Fig. 6
Bayesian inference phylogenetic analysis of Anaplasmataceae detected in this study according to a fragment of the 16S rRNA. The sequence derived from our samples is marked with a black diamond. Posterior probability (PP) values are indicated next to each node and size and color of node circles are proportional to the PP.
Fig. 7
Fig. 7
Bayesian inference phylogenetic analysis of Hepatozoon spp. detected in this study according to a fragment of the 18S rRNA. Each species is color-coded and sequences derived in this study are marked with a black diamond. Posterior probability (PP) values are indicated next to each node and size and color of node circles are proportional to the PP.
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