Abstract
There are few reports of Trypanosoma in snakes, as well as little information about its pathogenicity in these animals. Thus, the present study aimed to characterize Trypanosoma found in Boa constrictor snakes, to verify the influence of the parasitism on hematological and clinical biochemistry parameters, and to perform a phylogenetic study of the isolates. Blood samples from sixty-one boas were analyzed for the presence of trypanosomatids and by hematological and clinical biochemistry assays. The flagellates that were found in this analysis were used for cell culture, morphometry, and molecular analysis. Later, molecular typing phylogenetic studies were performed. Nine positive animals (14.75%) were identified by microscopy analysis. The hematological results showed that parasitized animals presented significantly lower levels of packed cell volume, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin. In the leukogram, eosinophils and heterophils counts were higher in parasitized animals. Considering the molecular analyses, the isolates presented a higher identity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the 18S small subunit ribosomal RNA (SSU rRNA) gene fragments with Trypanosoma serpentis. The phylogenetic tree, using the GAPDH, clustered all isolates with T. serpentis and Trypanosoma cascavelli. This is the first description of T. serpentis parasitizing boas and of the clinical changes caused by trypanosomatid infection in snakes.
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Acknowledgements
The authors are grateful to Francisca Soares (Laboratory of Immunology and Molecular Biology, Federal University of Bahia), Leane Gondim (Veterinary Hospital, Federal University of Bahia), and Paulo Roberto Bahiano (Salvador Zoo) for technical assistance. We are also grateful to Dr. Claudia D’Avila-Levy for the Trypanosoma reference isolates.
Funding
This work was supported by the Fundação de Apoio à Pesquisa e Extensão (FAPEX), through continuous resources obtained in extension projects. Maisa S. Fonseca is a pos-PhD fellow from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES). Marcos A. Mendonça is a PhD fellow from Fundação de Apoio à Pesquisa e Extensão (FAPESB). Rejâne Maria Lira-da-Silva is a research fellow from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). RWP is a technical development fellow from CNPq (Proc. 310058/2022-8). The funding agencies had no influence on the development of the studies and in the results presented herein.
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MSF: molecular and phylogenetic analysis, manuscript writing and editing. AJS: sample obtaining and microscopic, hematological, and clinical biochemistry analysis. MAM: microscopic, hematological, and clinical biochemistry analysis. GMR: sample obtaining. FSM: clinical biochemistry analysis. IB: sample obtaining. RML-d-S: sample obtaining. FFA: phylogenetic analysis. ARS: microscopic analysis, manuscript writing and editing. RPS: experimental design, critical revision of the manuscript. RM: fund obtaining, critical revision of the manuscript. RWP: conceptualization, fund obtaining, supervision of the project, critical revision of the manuscript.
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This work was approved by the Ethics Committee for the Use of Animals, of the School of Veterinary Medicine of the Federal University of Bahia (CEUA/UFBA 94/2018), by the Biodiversity Authorization and Information System (SISBIO 63053-3), and was registered at the National System for Management of Genetic Heritage (SISGEN A789EC8).
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Supplementary Figure 1.
Comparison of clinical biochemistry values between Trypanosoma sp. negative and positive boas. Parameters evaluated: total proteins (A), albumin (B), aspartate alanine transferase (ALT) (C), glucose (D), serum iron (E), creatinine (F), uric acid (G) and total cholesterol (H). 61 animals were evaluated, 52 negatives and nine positives. No statistical differences were observed in the Mann-Whitney test (p < 0.05). (PNG 156 kb)
Supplementary Figure 2
PCR results for the amplification of a specific fragment of the Trypanosoma sp. GAPDH gene. L – ladder marker. In the columns numbered from 1 to 3, the DNA of the reference strains Trypanosoma cruzi strain INPA 3663, Trypanosoma mega and Trypanosoma rangeli strain Peita Gonzales was tested, respectively. In columns 4 to 8, the results of the GAPDH specific fragment PCR made with DNA samples purified from the Trypanosoma sp. hemoculture isolates obtained in this study (C125, C3, C4, C538, C678) were tested. It is possible to observe bands with sizes of about 600bp in all reference and test isolates. These bands were purified from the gel and submitted to sequencing, and then the nucleotide sequences were used for the identity analysis (BLASTn) and the phylogenetic study. Band formation was not observed in the well destined for the negative control (CN) (PNG 831 kb)
Supplementary Figure 3
PCR results for amplification of a specific fragment of the Trypanosoma sp. SSU rRNA gene. L – ladder marker. Columns 1 and 2 contain the duplicate DNA test of the reference strains T. mega and T. rangeli strain Peita Gonzales. In columns 3 to 7, also in duplicate, are the results of the 18S gene specific fragment PCR made with the DNA samples purified from the hemoculture isolates of Trypanosoma sp. obtained in this study (C125, C3, C4, C538, C678). It is possible to observe the formation of the main band between the 3,000 pb and 1,500 pb lines. These bands were purified from the gel and submitted to sequencing, and then the nucleotide sequences were used for the identity analysis (BLASTn) (PNG 991 kb)
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Fonseca, M.S., Santos, A.J., Mendonça, M.A. et al. Trypanosoma sp. infection in Boa constrictor snakes: morphological, hematological, clinical biochemistry, molecular, and phylogenetic characteristics. Parasitol Res 123, 21 (2024). https://doi.org/10.1007/s00436-023-08023-x
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DOI: https://doi.org/10.1007/s00436-023-08023-x