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J Vis Exp. 2019 Apr 6;(146). doi: 10.3791/58415.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns).

Author information

1
Univ. Bordeaux UMR INTERTRYP 177 IRD CIRAD; IRD UMR 177 INTERTRYP CIRAD.
2
CNRS, UMR 5234, Microbiologie Fondamentale et Pathogénicité, Univ. Bordeaux.
3
Univ. Bordeaux UMR INTERTRYP 177 IRD CIRAD; IRD UMR 177 INTERTRYP CIRAD; Centre Hospitalier Université Bordeaux.
4
Univ. Bordeaux UMR INTERTRYP 177 IRD CIRAD; IRD UMR 177 INTERTRYP CIRAD; Centre Hospitalier Université Bordeaux; philippe.vincendeau@u-bordeaux.fr.

Abstract

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations. HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity. Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal. These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

PMID:
31009012
DOI:
10.3791/58415

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