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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 (meningoencephalitic stage) begins when parasites cross the blood brain barrier; neurological signs, including a sleep disorder, which has given its name "sleeping sickness" to this disease, are typical of this second-stage². Related trypanosomes (T. evansi, T. congolense, T. vivax, T. b. brucei) are the causative agents of animal African trypanosomosis (AAT)³.

The World Health Organization (WHO) aims to eliminate HAT as a public health problem by 2020 and to stop transmission by 2030⁴. The recent introduction of rapid diagnosis tests has improved serological diagnosis^1,4,5. Several molecular diagnostic tests have been developed but their role in field diagnostics has not yet been established⁵. They are used to identify the sub-species of the brucei group and atypical trypanosomiasis caused by parasites responsible for animal trypanosomosis⁶.

The detection of the parasite is essential for the diagnosis, treatment and follow-up, as serology can give false positive and unfortunately false negative results¹. The direct microscopical observation of these hemoflagellate protists is difficult in HAT cases that are caused by T. b. gambiense, (more than 95% of cases) as low parasitemias are the rule, whereas for HAT caused by T. b. rhodesiense, a large number of parasites are frequently present in the blood. Various concentration techniques have been used, such as thick drop and capillary tube centrifugation (CTC), but the separation of parasites from blood by a column of anion-exchanger (DEAE cellulose) followed by centrifugation and microscopic observation of the pellet, is the most sensitive method (around 50 parasites/mL of blood can be detected)^1,7. Consequently, the purification of trypanosomes by this anion-exchangers (DEAE cellulose) method is the best and, to date, the reference method for visualizing and isolating parasites from blood for HAT diagnosis. In field conditions, a mini-column of DEAE cellulose has been successfully used and several improvements have facilitated microscopical observation^7,8.

The method of trypanosome separation from blood, described below, depends on parasite surface charge, which is less negative than mammalian blood cells⁹. Interestingly, this method was developed 50 years ago, in 1968 by Dr. Sheila Lanham, and remains the gold standard for detection and preparation of bloodstream trypanosomes. It is fast and reproducible for salivarian trypanosomes from a wide range of mammals, permitting the diagnosis of both animal and human trypanosomiasis¹⁰.

To obtain living, purified parasites, infected blood is added onto an anion-exchanger column. Chromatography conditions (mainly pH, ionic strength of buffers/media) have to be adapted to each trypanosome species, and more generally, to each mix of mammalian blood cells and trypanosomes¹⁰. Elution buffer is precisely adjusted to pH 8 for most African trypanosomes¹⁰. This method favors the concentration of parasites found in the blood of patients, because parasitemias can be too low to be detected by microscopic observation alone, and it also enables laboratory investigations. Working with freshly isolated trypanosomes and on blood from infected animals, using this technique, is more pertinent for various investigations than studies with parasites that have been cultured in axenic conditions in the laboratory for an indefinite period.

Host-parasite relationships are best studied with a parasite infecting its natural host, therefore, T. musculi, a natural murine parasite, which is representative of extracellular trypanosomes, has many advantages as murine infection involves in a small laboratory animal and does not require biohazard safety level (BSL) conditions. T. musculi does not kill immunocompetent mice, unlike many other Trypanosoma species, including human pathogens. T. musculi are not eliminated in T cell-deprived mice and parasitemias can be increased in infected mice by modifying food and nutrient intake¹¹. This parasite modulates the immune response in co-infections with other pathogens¹². T. musculi from infected mice exhibit differences from cultured T. musculi, for example, the expression of membrane Fc receptors is lost in T. musculi axenic cultures, compared to parasites purified from infected mice^13,14. Excreted-secreted factors (ESF) are also qualitatively and quantitatively less expressed in axenic trypanosome cultures and differ between strains isolated in endemic areas¹⁵. ESF are the first antigens to be displayed to the host immune system and so play an important role in the initial host immune response¹⁶.

In experimentally infected animals for laboratory investigations, this protocol facilitates experimentation on a greater number of parasites, minimizing the number of mice required especially when using immunosuppressed animals. The variant surface glycoproteins (VSGs) that are used in the Card Agglutination Test for Trypanosomiasis (CATT) in mass screening are still purified from trypanosomes that are propagated in rats. The two rapid diagnostic tests (individually wrapped cassettes) that are now available for use in the field, are still using an infective model source of native VSGs and not in vitro cultured trypanosomes^1,4,5. The advancement in the study of trypanosome immunology and biology has been facilitated since these DEAE cellulose purified parasites can be easily obtained in large quantities from naturally or experimentally infected hosts, and in particular, rodents.

Protokół

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.
3. For housing, use clear-walled cages, enrichment with wooden sticks and cardboard tunnels. Gently draw an animal into a tunnel to transfer it from the cage to the palm of the hand.
4. Perform daily monitoring to assess signs of prostration, social isolation, body injury, ruffled hair, or lack of grooming.
5. Weigh each animal once per week. Perform regular inspections by a veterinarian.
6. For natural parasites, collect blood at the peak of parasitemia and for parasites causing animal death, collect blood the day before presumed death.
   NOTE: All experiments with infectious agents are performed in dedicated rooms, according to university approved guidelines.

2. Buffers, media preparations

1. Weigh out each substance and add distilled water for the following buffers:
   1. Prepare Concentrated Phosphate-Buffered Saline (2x):
      Na₂HPO₄ (anhydrous) (MW 141.96 g) 10.14 g
      NaH₂PO₄∙2H₂O (MW 156.01 g) 0.62 g
      NaCl (MW 58.44 g) 2.55 g
      Distilled H₂O to 1 L
   2. Prepare Phosphate-Buffered Saline-Glucose:
      Na₂HPO₄ (anhydrous) (MW 141.96 g) 5.39 g
      NaH₂PO₄∙2H₂O (MW 156.01 g) 0.31 g
      NaCl (MW 58.44 g) 1.70 g
      Glucose (MW 180 g) 10 g
      Distilled H₂O to 1 L
   3. Prepare 1 M KH₂PO₄.
   4. Prepare Elution Buffer: Supplement Phosphate-Buffered Saline-Glucose with
      Penicillin (100 U/mL), Streptomycin (100 µg/mL), and Phenol red (5 µg/mL).

3. Preparation of DEAE-cellulose

1. Plan around 5 hours for the DEAE-cellulose preparation.
2. Wash 100 g of DEAE-cellulose with distilled water in a flask with a narrow neck and allow to settle, then discard fine particles. Repeat washes until the supernatant is clear.
3. Add 3 L of concentrated 2x Phosphate-Buffered Saline and stir.
4. Adjust pH to 8.0 with 1 M KH₂PO₄ and discard supernatant.
5. Wash twice with distilled water and leave to settle.
6. Wash and allow to settle twice with 3 liters Phosphate-Buffered Saline-Glucose and discard the supernatants.
7. Measure the volume of cellulose and add an equal volume of Phosphate-Buffered Saline-Glucose, distribute into plastic bottles and store at -20 °C.

4. Parasites

1. Collect trypanosome strains in areas endemic for human and animal trypanosomiasis. Keep parasites frozen in liquid nitrogen.
   NOTE: Trypanosoma musculi is non-pathogenic to humans and is an extracellular trypanosome used to safely replace pathogenic trypanosomes in various laboratory experiments.
2. In the case of laboratory investigations requiring human pathogens, handle experiments with care in dedicated appropriate biohazard safety level (BSL) conditions and precautions; BSL2 for T. b. gambiense and BSL3 for T. b. rhodesiense. In field conditions, standard microbiological practice is established: secure sampling, mechanical pipetting, frequent decontamination of surfaces, and sterilization of waste.

5. Mouse infection

1. Rapidly thaw parasites in a water bath at 37 °C.
2. Observe a drop of thawed infected blood with a microscope. Assess parasite viability by measuring the percentage of motile forms¹⁷.
3. Inject parasites intraperitoneally into mice (0.5 mL per mouse). Each day, post-infection, collect 20 µL of tail blood by needle puncture and observe under a microscope. Evaluate parasitemia according to Herbert and Lumsden¹⁸ by counting parasites in several microscope fields, or by using a haemocytometer.
4. When the parasitemia reaches a threshold that is defined for each strain, collect the blood (1 mL/mouse) in the elution buffer (5 mL/mouse) containing heparin (10 U/mL).
   NOTE: The original and novel work of Lanham and Godfrey reported the optimal ionic strength of phosphate buffered saline glucose for several host/parasite species from a pH 8.0 stock solution.

6. Parasite separation

NOTE: All experiments from this point onwards must be done in a tissue culture hood wearing gloves. The room temperature and humidity in the laboratories used were 22 °C and 45% respectively. In field conditions, parasite separation has been successfully performed at 34 °C.

1. Place a 10 mL syringe in a vertical support and add a previously cut circular, piece of filter paper, glass wool or cellulose sponge.
2. Pour the DEAE cellulose into the syringe until the 8 mL level is reached, and then wash with 25 mL of elution buffer.
3. Carefully add 2 mL of diluted blood at the top of the column and then add elution medium. Regularly add elution buffer according to the transit of the trypanosomes.
4. Collect effluent drops from the columns in a centrifuge tube and check regularly for the presence of parasites with a microscope.
5. When parasites are no longer detected in the column effluent, centrifuge the tube (1,800 x g, 10 minutes, at 4 °C in the laboratory and at ambient temperature in field conditions).
6. Remove the supernatant and suspend the parasites in 1 mL of the relevant medium required for the next investigative step.
7. Count parasites with a hemocytometer and dilute them in appropriate medium if necessary.

Wyniki

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...

Dyskusje

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

Materiały

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