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

Summary

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.

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.

Introduction

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 (....

Protocol

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.

1. Animals

1. Keep female Swiss (OF-1) mice aged eight to ten weeks old, 20-25 g, in an animal housing facility fifteen days before each experiment. House them in ventilated boxes that are kept in a protected, temperature (22 °C) and humidity (50%) controlled room, with 12 hours on/off light cycle.
2. Give animals free access to food and water. Minimize pain, suffering and distress and provide enrichment of the environment.

Results

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 pentamidin.......

Discussion

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mL Pipettes	Falcon	357,551
2 mL Pipettes	Falcon	352,507
Centrifugation tube 50 mL	Falcon	352,070
Centrifuge	Sigma Aldrich	4K15
DEAE cellulose	Santa Cruz	s/c- 211213	100 G
filter paper	Whatman	1,001,125
Flat bottom flask narrow neck	Duran	21 711 76	6000 mL
Glucose	VWR	101174Y	500 G
Heparin	Sigma Aldrich	H3149-50KU	5 000 U
KH2PO4	VWR	120 26936.260	500 G
Microscope	Olympus	CH-20
Microscope coverslips	Thermofisher scientific	CB00100RA020MNT0
Microscope slides	Thermofisher scientific	AGAA000001
Na2HPO4	VWR	100 28026;260	500 G
NaCl	VWR	27800.291	1 KG
NaH2PO4	VWR	110 33616;262	500 G
Nalgene Plastic Media Bottles size 125 mL	Thermofisher scientific	342024-0125
Nalgene Plastic Media Bottles size 500 mL	Thermofisher scientific	342024-0500
Pasteur Pipette	VWR	BRND125400
Penicillin 10,000 UI/Streptomycin 10,000 µg	EUROBIO	CABPES01 OU	100 mL
Phenol red	Sigma Aldrich	P0290	100 mL
Syringue	Dutscher	SS+10S21381
Tissue culture hood	Thermoelectro Corporation	MSC-12
Trypanosoma brucei brucei	Institute of Tropical Medicine (Antwerp, Belgium).		ANTAT 1.1
Trypanosoma brucei gambiense	Institute of Tropical Medicine (Antwerp, Belgium).		ITMAP 1893
Trypanosoma musculi	London School of Hygiene and Tropical Medicine (UK)		Partinico II