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Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)
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Summary 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.
Transcript
African trypanosomes are responsible for human African trypanosomiasis, or sleeping sickness and animal African trypanosomiasis. The trypanosomes that cause sleeping sickness are tsetse fly transmitted protist parasites present in many geographical foci in sub Saharan Africa. The detection of the parasite is essential for the diagnosis, treatment, and follow-up.
A serology can give false positive and unfortunately, false negative results. To aid detection in the blood, trypanosomes have to be concentrated. Various parasite concentration techniques have been used.
The most sensitive method is the separation of parasites from blood by a column of anion exchanger, DEAE cellulose followed by centrifugation and microscopic observation. Consequently, the DEAE cellulose method is the best and remains to date, the reference method for visualizing and isolating parasites from blood for human African trypanosomiasis diagnosis, especially in field conditions. This method, the most fitting diagnostic for African trypanosomiasis also provides purified parasites for immunological, biological, biochemical, pharmaceutical, and molecular biological investigations.
All investigations conformed to the guide for the care and use of laboratory animals and agreement has been obtained from the French authorities and all the protocols used have been approved by our local ethics committee. Weigh out each substance according to the written protocol. Add distilled water and adjust precisely to pH 8 with potassium dihydrogen phosphate to make the following buffered solutions, concentrated phosphate buffer saline 2X.
Add distilled water and glucose for phosphate buffered saline glucose. For 100 milliliters of elution buffer, add 100 units per milliliter of penicillin, 100 micrograms per milliliter of streptomycin, and five micrograms milliliter of phenol red to phosphate buffered saline glucose. 100 grams of DEAE cellulose is first washed with three liters of distilled water and then left to settle.
Fine particles are discarded. The washes are repeated until the supernatant is clear. Concentrated phosphate buffered saline 2X is added and the DEAE cellulose is stirred.
The pH is adjusted to eight with one molar potassium phosphate. The cellulose is then washed twice with distilled water and twice with phosphate buffered saline glucose. The DEAE cellulose and phosphate buffered saline glucose are mixed in equal volumes.
Alloy quartered and stored at room temperature or four degrees celsius or at minus 20 degrees celsius for prolonged storage. Trypanosome infected blood is stored in liquid nitrogen. An alloy quart of one milliliter is thawed at room temperature.
Parasites are carefully collected from the cryotube using a needle and a one milliliter syringe. The viability of thawed parasites is assessed by the motility as visualized by light microscopy observation before infection. Parasites are injected into the peritoneal cavities of two mice, 500 microliters per mouse.
Each day post infection, parasitemia is assessed by collecting a drop of blood from the tail with a needle and checked by microscopy. When the parasitemia is at a suitable level, infected blood is collected. A 10 milliliter syringe is supported in a clamp or holder and a precut piece of filter paper is added.
Prepared DEAE cellulose is poured into the syringe up to the eight or nine milliliter level. The column is washed with the elution buffer. Two milliliters of infected blood is carefully placed on top of the column and trypanosomes are collected in the column eluate in a 50 milliliter centrifugation tube.
Elution buffer is regularly added according to the transit of trypanosomes. The transit of trypanosomes through the column is checked by taking a drop of column eluate at regular intervals and observing for trypanosomes using light microscopy. When trypanosomes are no longer observed in the eluate, the conical tube is centrifuged at 1, 800 times G for 10 minutes at four degrees celsius.
The supernatant is discarded and trypanosomes in the pallet are counted with a hemocytometer. Collected trypanosomes are subsequently diluted in the medium required for the planned investigation. This method, the most fitting diagnostic for African trypanosomiasis provides purified parasites for immunological, biological, biochemical, pharmaceutical, and molecular biological investigations.
These studies have been developed because DEAE cellulose purified parasites can be easily obtained in large quantities from naturally or experimentally infected hosts and in particular, rodents. Trypanosome viability in the presence of pharmaceutical agents is assessed by microscopic observation. Parasite viability is associated with motility and therefore, can be checked by eye under a light microscope at 40 times or higher magnification.
Parasite viability is assessed by measuring the percentage of motile forms. Dilutions of test compounds are added into each well of a 96 well tissue culture plate while control wells are mock treated. Each concentration is assessed in triplicate.
The number of viable parasites for each dilution is compared to the number of parasites in control wells. The effects of Pentamidine, a reference drug, used in human African trypanosomiasis therapy is displayed in this figure. Trypanosome macrophage co-cultures allow the investigation of host parasite interaction at the cellular level.
In macrophage parasite co-cultures, extra cellular trypanosomes induce production of macrophage arginase that hydrolyzes arginine to ornithine. Ornithine is the precursor of polyamines and trypanothione, essential for parasite survival and growth. Furthermore, arginine depletion decreases the production of trypanocidal nitric oxide.
Subsequently, addition of an arginase inhibitor, S-L-cysteine to co-cultures dramatically reduces macrophage induced parasite growth. Arginase induction is mediated by trypanosome excreted and or secreted factors. This arginase inducing factor has been identified as an orthine kinesin.
The kinesin binds to C-type lectin mannose binding receptors. Arginase is induced and produces ornithine which favors parasite growth. Arginase is not induced by kinesin in macrophages cultured with 12.5 millimolar mannose or in macrophages from mice where the macrophage mannose receptors have been deleted.
Further examples of cell biology and biochemical experimentation using DAEA cellulose purified trypanosomes are demonstrated in studies where novel cytoskeletal proteins such as BILBO1 has been identified. BILBO1 is required for the biogenesis of important cytoskeletal structures and parasite survival. Thus, BILBO1 represents an important target for intervention in pathogenic trypanosomes.
An image showing immunoflourescence labeling of a bloodstream culture form, trypanosoma brucei cell probed with an anti-BILBO1 antibody is shown in this figure. Additionally, glucose and threonine metabolism have been described using DEAE cellulose purified trypanosomes and the enzymes involved in these processes have been experimentally validated. A schematic representation of glucose and threonine metabolism in the bloodstream-formed trypanosomes is shown in this figure.
Purification of African trypanosomes from blood by anion exchanges DEAE cellulose columns with recent improvements remains the gold standard for trypanosome detection, not only in natural hosts with low parasitemias in endemic areas, but also for the requirement of parasites in large numbers for experimental investigations. Investigations have allowed the identification of trypanosome molecules playing essential roles in parasite survival and growth. Identified targets might represent the basis for a future vaccine with potential antigens for both humans and animals in a one health approach.
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