Isolation and in vitro cultivation of Trypanosoma evansi Thai strains

Kamyingkird Ketsarin, Chalermwong Piangjai, Inpankaew Tawin, Ngasaman Ruttayaporn, Tattiyapong Muncharee, Tiwananthagorn Saruda, Chimnoi Wissanuwat, Choocherd Suchada, Kengradomkij Chanya, Klinkaew Nutsuda, Desquesnes Marc. 2022. Isolation and in vitro cultivation of Trypanosoma evansi Thai strains. Experimental Parasitology, 239:108289, 10 p.

https://doi.org/10.1016/j.exppara.2022.108289

Article de revue ; Article de recherche ; Article de revue à facteur d'impact

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Résumé : Trypanosoma evansi is a flagellate protozoan parasite responsible for “surra”. To generate T. evansi antigens for serodiagnosis, parasites are generally propagated in laboratory animals before isolation. The alternation of animal models using axenic cultivation systems to produce trypomastigotes of various Trypanosoma species is currently available but has never been applied in Thailand. The isolation protocol for separation of live T. evansi trypomastigotes from animal blood components before in vitro cultivation has not been clearly documented. This study focused on validation of trypomastigote isolation method, in vitro cultivation of T. evansi Thai strains, and its virulence ability in vivo. In this study, two strains of T. evansi collected from Thailand were used. Trypanosoma evansi trypomastigotes were propagated in mice, and three different isolation methods, including: low-speed centrifugation, high-speed centrifugation, and ion exchange chromatography using diethylaminoethyl (DEAE) cellulose (or DE52), were compared. Four solutions of in vitro cultivation media, two different in vitro cultivation containers, and different trypomastigote densities for initiation of in vitro culture were compared. Virulence test using in vitro-adapted parasite for 100 days was conducted in vivo. The results showed that the DE52 isolation method was suitable for separation of live T. evansi trypomastigotes from animal blood components before conducting in vitro cultivation. Trypanosoma evansi Thai strains were successfully cultivated and multiplied in HMI-9 Solution I using 25 cm2 flasks and 12-well plates. The parasite was growing slowly at the initiation of in vitro culture for 15–16 days, and then rapidly increased to 10, 20, 50, 100, and 200 folds, approximately. The doubling times were varied from 11.95 ± 8 h to 41.18 ± 4.29 h in vitro. The maximum densities have reached from 0.14 × 106 to 4.63 × 106 trypomastigotes/ml. Virulence test showed that the in vitro-cultivated T. evansi was virulent in mice. In conclusion, T. evansi Thai strains were successfully isolated and cultivated in vitro for the first time. The isolation and in vitro cultivation protocols were clearly provided. The benefit of using the in vitro cultivation system helps in the production of T. evansi antigen, and replacing the use of experimental animals. It is also useful for the development of diagnostic tests in the future.

Mots-clés Agrovoc : Trypanosoma evansi

Mots-clés libres : Trypanosoma evansi, Isolation, In vitro, Antigen production, Animal replacement

Classification Agris : L73 - Maladies des animaux

Champ stratégique Cirad : CTS 4 (2019-) - Santé des plantes, des animaux et des écosystèmes

Agences de financement hors UE : Kasetsart University

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