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A simple semi-defined culture medium for the growth of Trypanosoma rangeli
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1Short
I
OF TROPICALMEDICINEAND HYGIENE(1988)82, 241
247
Report 1 1
A simple semi-defined culture medium for the growth of Trypanosoma rangeli G. B. TakIe and A. Young y&t
Grge
s Hospual
Department of ZmmunoloMedacal School, London,
Despite its non-pathogenicity to human and other vertebrates, Ttypanosoma rangeli is considered an important South American parasite since it is frequently found in mixed infections with T. cruzi, the causative agent of Chagasdisease,and often confuses the interpretation of xenodiagnoses (D’ALESSANDRO, 1976; GUHL et al., 1987). There are available a number of methods for differentiating T. cncei from T. rangeli, such as lectin binding (BRETTING & SCHOTTEL~US, 1978), complement sensitivity (SCHOTIELIUS, 1982), assays for sialic acid content (SCHOTTELIUS, 1984) or microscopy. The most sensitive current diagnostic test involves the use of DNA probes in a dot-blot assay (GRBIG & ASHALL, 1987) that can detect as few as 10 parasites and can easily be used to distinguish between T. rangeii and T. cruzi. This assayrelies on the rapid supply of pure parasite DNA free from any contaminating components of the culture medium. In this report a simple monophasic culture medium for the growth of T. rangeli is described that is routinely used in this laboratory for immunochemistry and in the preparation of DNA probes. Eagle’s minimal essential medium (autoclavable, Gibco) was prepared according to the supplier’s instructions. The pH was adjusted to 6.5 with 2M sodium hydroxide and L-glutamine was added to 2Om~. Foetal calf serum (FCS) and antibiotics were added as required. T. rangeli culture-form epimastigotes (San Augustin strain) were seededat 4.5 x 106/ ml, incubated at 27”C, and parasite numbers were counted by haemocytometer. Optimal growth of T. rangeli was obtained in 20% FCS (Figure), parasite numbers reaching approximately 2.5 x lob/ml after one week. After 20 d in 10% FCS cultures were exhausted and rapidly died, whilst cultures in 20% FCS remained stationary. Gentamycin at O-16 mg/ml very rapidly killed all cultures whilst penicillin (100 i$ml) and streptomycin (O-1 mg/ml) allowed parasite growth, albeit to a reduced extent. Young T. rangeii cultures contained predominantly short epimastigote forms, whilst, as the culture aged, long trypomastigotes developed and formed the majority at late stationary phase (data not shown). Previously, T. rangeli has been cultured exclusively
in biphasic media composed of blood-agar slopes overlaid with nutrient medium. This report is the 6rst to describe a monophasic medium for the growth of T. rangeli. It has the advantagesof simplicity and the avoidance of the time-consuming
use of blood-agar.
We recommend the use of 20% FCS and, if antibiotics are required, penicillin and streptomycm. Routinely, T. rangeli is grown in the absence of antibiotics.
Figure. Growth of T. rangeli. Mean (k 1 s.d.) from four 20 ml in culturesin Eagle’sminimumessentialmedium.4 = 20%foetal calf serum (FCS); V = 10% FCS; 0 = Gentamycin (20% FCS); 0 = penicillin/streptomycin (20% FCS).
The development observed from epimastigotes to
trypomastigotes also occurs during haemocoelicinfection of Rhodnius prolixus and Dipetalogaster maximus by T. rangeli (Ar;iu, 1981), and thus the culture
medium may provide conditions similar to those found in the insect haemocoel.It is suggestedthat this medium be used as a convenient substitute to replace the complex media more commonly used for growing T. rangeli.
We acknowledge the advice of Dr J. S. Crowe.
References Aftcz, N. (1981). Studies on the Trypanosomatidaeof Veneau& with specialreference to Trypanosoma rangeli. Ph.D Thesis, University of London. Bretting, H. & Schottelius, J. (1978). Differentiation by microimmunofluorescence of T. cnui and T. cruzi-like strains from T. conorhini and T. rungek using the protectin from the spongeAapws papillata. Zeitschriftfir Parasitenkunde, 57, 213-219. D’Alessandro, A. (1976). Biology of Ttypanosoma(Herpetosoma)rangeli Tejera, 1920. In: Biology of theKinetoplastida. Lumsden, W. H. R. and Evans, D. A. (editors), London: Academic Press, Vol. 1, pp. 327403. Greig, S. & Ashall, F. (1987). Detection of South American, trypanosomes in insects using total parasite DNA probes. Parasiwhgy Today 3, 375-376. Guhl, F., Hudson, L., Marinkelle, C. J., Jaramillo, C. A. & Bridge, D. (1987). Clinical Typanumna rangeli infection as a complication of Chagas’ disease. Parasitology, 94, 475-484. Schottelius, J. (1982). Differentiation between Typanomna crui and Ttypanosomarangeli by their different compley4y;5enskivi,. Tropenmedizin und Parasitologic, 33, Schottelius; J. (1984). Differentiation between Typanosoma cruzi and Trypanosomarangeli on the basis of their sialic acid content. Tropenmedizinund Parasiwlogie, 35, 160162. Received 17 September 1987; acceptedfor publication 28 October I987
1Short
I
OF TROPICALMEDICINEAND HYGIENE(1988)82, 241
247
Report 1 1
A simple semi-defined culture medium for the growth of Trypanosoma rangeli G. B. TakIe and A. Young y&t
Grge
s Hospual
Department of ZmmunoloMedacal School, London,
Despite its non-pathogenicity to human and other vertebrates, Ttypanosoma rangeli is considered an important South American parasite since it is frequently found in mixed infections with T. cruzi, the causative agent of Chagasdisease,and often confuses the interpretation of xenodiagnoses (D’ALESSANDRO, 1976; GUHL et al., 1987). There are available a number of methods for differentiating T. cncei from T. rangeli, such as lectin binding (BRETTING & SCHOTTEL~US, 1978), complement sensitivity (SCHOTIELIUS, 1982), assays for sialic acid content (SCHOTTELIUS, 1984) or microscopy. The most sensitive current diagnostic test involves the use of DNA probes in a dot-blot assay (GRBIG & ASHALL, 1987) that can detect as few as 10 parasites and can easily be used to distinguish between T. rangeii and T. cruzi. This assayrelies on the rapid supply of pure parasite DNA free from any contaminating components of the culture medium. In this report a simple monophasic culture medium for the growth of T. rangeli is described that is routinely used in this laboratory for immunochemistry and in the preparation of DNA probes. Eagle’s minimal essential medium (autoclavable, Gibco) was prepared according to the supplier’s instructions. The pH was adjusted to 6.5 with 2M sodium hydroxide and L-glutamine was added to 2Om~. Foetal calf serum (FCS) and antibiotics were added as required. T. rangeli culture-form epimastigotes (San Augustin strain) were seededat 4.5 x 106/ ml, incubated at 27”C, and parasite numbers were counted by haemocytometer. Optimal growth of T. rangeli was obtained in 20% FCS (Figure), parasite numbers reaching approximately 2.5 x lob/ml after one week. After 20 d in 10% FCS cultures were exhausted and rapidly died, whilst cultures in 20% FCS remained stationary. Gentamycin at O-16 mg/ml very rapidly killed all cultures whilst penicillin (100 i$ml) and streptomycin (O-1 mg/ml) allowed parasite growth, albeit to a reduced extent. Young T. rangeii cultures contained predominantly short epimastigote forms, whilst, as the culture aged, long trypomastigotes developed and formed the majority at late stationary phase (data not shown). Previously, T. rangeli has been cultured exclusively
in biphasic media composed of blood-agar slopes overlaid with nutrient medium. This report is the 6rst to describe a monophasic medium for the growth of T. rangeli. It has the advantagesof simplicity and the avoidance of the time-consuming
use of blood-agar.
We recommend the use of 20% FCS and, if antibiotics are required, penicillin and streptomycm. Routinely, T. rangeli is grown in the absence of antibiotics.
Figure. Growth of T. rangeli. Mean (k 1 s.d.) from four 20 ml in culturesin Eagle’sminimumessentialmedium.4 = 20%foetal calf serum (FCS); V = 10% FCS; 0 = Gentamycin (20% FCS); 0 = penicillin/streptomycin (20% FCS).
The development observed from epimastigotes to
trypomastigotes also occurs during haemocoelicinfection of Rhodnius prolixus and Dipetalogaster maximus by T. rangeli (Ar;iu, 1981), and thus the culture
medium may provide conditions similar to those found in the insect haemocoel.It is suggestedthat this medium be used as a convenient substitute to replace the complex media more commonly used for growing T. rangeli.
We acknowledge the advice of Dr J. S. Crowe.
References Aftcz, N. (1981). Studies on the Trypanosomatidaeof Veneau& with specialreference to Trypanosoma rangeli. Ph.D Thesis, University of London. Bretting, H. & Schottelius, J. (1978). Differentiation by microimmunofluorescence of T. cnui and T. cruzi-like strains from T. conorhini and T. rungek using the protectin from the spongeAapws papillata. Zeitschriftfir Parasitenkunde, 57, 213-219. D’Alessandro, A. (1976). Biology of Ttypanosoma(Herpetosoma)rangeli Tejera, 1920. In: Biology of theKinetoplastida. Lumsden, W. H. R. and Evans, D. A. (editors), London: Academic Press, Vol. 1, pp. 327403. Greig, S. & Ashall, F. (1987). Detection of South American, trypanosomes in insects using total parasite DNA probes. Parasiwhgy Today 3, 375-376. Guhl, F., Hudson, L., Marinkelle, C. J., Jaramillo, C. A. & Bridge, D. (1987). Clinical Typanumna rangeli infection as a complication of Chagas’ disease. Parasitology, 94, 475-484. Schottelius, J. (1982). Differentiation between Typanomna crui and Ttypanosomarangeli by their different compley4y;5enskivi,. Tropenmedizin und Parasitologic, 33, Schottelius; J. (1984). Differentiation between Typanosoma cruzi and Trypanosomarangeli on the basis of their sialic acid content. Tropenmedizinund Parasiwlogie, 35, 160162. Received 17 September 1987; acceptedfor publication 28 October I987