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Isolation and in vitro cultivation of Trypanosoma evansi Thai strains. | LitMetric

Isolation and in vitro cultivation of Trypanosoma evansi Thai strains.

Exp Parasitol

Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Ladyao, Chatuchak, Bangkok, 10900, Thailand; CIRAD, UMR InterTryp, Bangkok, Thailand; InterTryp, Univ Montpellier, CIRAD, IRD, Montpellier, France.

Published: August 2022

AI Article Synopsis

  • Trypanosoma evansi is a protozoan parasite that causes "surra," and its antigens are typically generated by propagating the parasite in laboratory animals before isolation for serodiagnosis.
  • This study aimed to validate an isolation method for T. evansi trypomastigotes from animal blood, compare various isolation techniques, and explore in vitro cultivation of Thai strains of the parasite.
  • Results indicated that the ion exchange chromatography method using DEAE cellulose was effective for isolating live parasites, and the cultivated Thai strains grew significantly in specific media and containers, exhibiting varied growth rates and densities over time.

Article Abstract

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 cm 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 × 10 to 4.63 × 10 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.

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Source
http://dx.doi.org/10.1016/j.exppara.2022.108289DOI Listing

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