Immunology and Infection
Published: April 6th, 2019
This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.
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.
The method presented described here allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits robust serological and research investigation.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense1. These protozoan parasites multiply extracellularly in the bloodstream, lymph, and interstitial fluids during the first stage of the disease (hemolymphatic stage). The second stage (....
Investigations conformed to the Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85±23, revised 1996). Protocols were approved by our local ethics committee.
Purified trypanosomes have been used in pharmaceutical tests. Parasites are transferred into culture wells containing serial dilutions of specific drugs, either alone or mixed19. Microscopic observations, evaluating motility is a marker of viability, can be performed when only a few dugs are being tested, whereas AlamarBlue cell viability assay is an excellent method for large motility assays during drug screening20. The effect of penta.......
Purified trypanosomes represent a powerful means to study immunology, biochemistry, cellular and molecular biology. Large expanses of data and results have been obtained from trypanosomes, which has then helped to obtain information from other eukaryotic cells30. Trypanosomes are also the subject of important and interesting research because they have devised numerous mechanisms that permit them to survive and grow in two very different environments: the tsetse fly vector and the mammalian host
We thank all members of UMR 177 INTERTRYP IRD CIRAD Université de Bordeaux. This research was supported by internal funding from University of Bordeaux and support from the ANR, LABEX ParaFrap ANR-11-LABX-0024, and from the Association pour le développement de la recherche en parasitologie et médecine tropicale and the Service de coopération et d'action culturelle de l'Ambassade de France à Bangui (Centrafrique).....
|10 mL Pipettes
|2 mL Pipettes
|Centrifugation tube 50 mL
|Flat bottom flask narrow neck
|21 711 76
|5 000 U
|Nalgene Plastic Media Bottles size 125 mL
|Nalgene Plastic Media Bottles size 500 mL
|Penicillin 10,000 UI/Streptomycin 10,000 µg
|Tissue culture hood
|Trypanosoma brucei brucei
|Institute of Tropical Medicine (Antwerp, Belgium).
|Trypanosoma brucei gambiense
|Institute of Tropical Medicine (Antwerp, Belgium).
|London School of Hygiene and Tropical Medicine (UK)
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