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Isolation of blood and intracellular forms of Trypanosoma cruzi from rats and other rodents and preliminary studies of their metabolism

Published online by Cambridge University Press:  06 April 2009

W. E. Gutteridge ,
B. Cover  and
Maria Gaborak
W. E. Gutteridge
Affiliation:
Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ
B. Cover
Affiliation:
Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ
Maria Gaborak
Affiliation:
Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ
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Summary

Isolation of blood and intracellular forms of Trypanosoma cruzi was made mainly from rats (90–110 g) which had received 580 rad of whole-body γ-irradiation not more than 24 h before subcutaneous inoculation with 107 trypomastigotes of the Sonya strain of T. cruzi. Unirradiated chinchillas (250–350 g) were, however, used for some experiments. Blood forms were isolated using a technique involving differential centrifugation to remove most of the erythrocytes and DEAE–cellulose chromatography to remove the remaining blood cells. Overall recoveries were usually in the range 30–70%. Parasites were mainly (approximately 98%) broad forms and were motile, metabolically active (as judged by respiratory and radio-tracer incorporation studies) and had lost none of their infectivity for mice. Intracellular forms were isolated from hind-limb muscle tissue. This was disrupted in an MSE tissue homogenizer and the homogenate incubated with DNase, collagenase and trypsin. Parasites, contaminated only by a few blood cells, were then obtained by differential centrifugation. For purer preparations, a terminal sucrose gradient step was used. Recoveries ranged between 40 and 70%. About 1–3% of the parasites isolated were epimastigotes and trypomastigotes; the remainder are probably best collectively termed ‘amastigotes’, though they were pointed and most had a short, free flagellum. They were undamaged as judged by light and electron microscopy and metabolically active as judged by respiratory and radio-tracer incorporation studies. However, the infectivity for mice of both these purified preparations and the initial cell homogenates could be accounted for by the epimastigotes and trypomastigotes present in them. Preliminary biochemical studies with isolated parasites have shown that blood, intracellular and culture forms of T. cruzi have a respiratory system which is in part sensitive to CN- and that all forms synthesize nucleic acids and proteins when incubated in vitro. There appears, however, to be a lack of DNA synthesis in blood stages, and thus it is not surprising that these forms do not divide.

Type
Research Article
Information
Parasitology , Volume 76 , Issue 2 , April 1978 , pp. 159 - 176
Copyright
Copyright © Cambridge University Press 1978

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References

REFERENCES

Bafort, J. M., Kageruka, P. & Timperman, G. T. (1973). A new, highly susceptible laboratory animal for Chagas' disease. Transactions of the Royal Society of Tropical Medicine and Hygiene 67, 434–5.CrossRefGoogle Scholar
Bowman, I. B. R., Srivastava, H. K. & Flynn, I. W. (1972). Adaptations in oxidative metabolism during the transformation of Trypanosoma rhodesiense from bloodstream into culture form. In Comparative Biochemistry of Parasites (ed. Van den Bossche, H.), pp. 329–42. New York: Academic Press.CrossRefGoogle Scholar
Bowman, I. B. R. (1974). Intermediary metabolism of flagellates. In Ciba Foundation Symposium 20 (New Series): Trypanosomiasis and Leishmaniasis with special reference to Chagas' disease, pp. 255–71. Amsterdam: Associated Scientific Publishers.CrossRefGoogle Scholar
Cheto de Queiroz, A. (1973). Tumour-like lesion of the brain caused by Trypanosoma cruzi. American Journal of Tropical Medicine and Hygiene 22, 473–6.CrossRefGoogle Scholar
Childs, G. E., Roberts, M. J. & Foster, K. A. (1976). Partial purification of amastigotes from cutaneous lesions of American leishmaniasis. Journal of Parasitology 62, 676–9.CrossRefGoogle ScholarPubMed
Cook, R. T., Aikawa, M., Rock, R. C., Little, W. & Sprinz, H. (1969). The isolation and fractionation of Plasmodium knowlesi. Military Medicine 134, 866–83.CrossRefGoogle ScholarPubMed
Cross, G. A. M. & Manning, J. C. (1973). Cultivation of Trypanosoma brucei sspp. in semidefined and defined media. Parasitology 67, 315–31.CrossRefGoogle ScholarPubMed
Dixon, M. & Kleppe, K. (1965). D-amino acid oxidase: dissociation and combination. Biochimica et Biophysica Acta 96, 357–67.CrossRefGoogle Scholar
Dvorak, J. A. (1976). New in vitro approach to quantitation of Trypanosoma cruzi-vertebrate cell interactions. In Pan American Health Organization Symposium No. 318: American Trypanosomiasis Research, pp. 109–20. Washington: Pan American Health Organization.Google Scholar
Elsner, Y. Y., Roberts, W. C., Shigematsu, A. & Hammond, D. M. (1974). Isolation of schizonts and merozoites of Eimeria callospermophili from cultured bovine intestinal cells. Journal of Parasitology 60, 531–2.CrossRefGoogle ScholarPubMed
Ferreira, A. L. & Rossi, M. A. (1973). Pathology of the testis and epididymis in the late phase of experimental Chagas' disease. American Journal of Tropical Medicine and Hygiene 22, 699–704.CrossRefGoogle Scholar
Fulton, J. D. & Spooner, D. F. (1957). Comparison of the respiratory activity of an old and of a freshly isolated strain of Trypanosoma rhodesiense. Annals of Tropical Medicine and Parasitolgy 51, 417–21.CrossRefGoogle ScholarPubMed
Garnham, P. C. C. (1956). Isolation of a new strain of Trypanosoma cruzi. Transactions of the Royal Society of Tropical Medicine and Hygiene 50, 613.CrossRefGoogle Scholar
Gutteridge, W. E. (1976 a). Experimental chemotherapy of Chagas' disease. In Pan American Health Organization Symposium No. 318: American Trypanosomiasis Research, pp. 255–62. Washington: Pan American Health Organization.Google Scholar
Gutteridge, W. E. (1976 b). Chemotherapy of Chagas' disease: the present situation. Tropical Diseases Bulletin 73, 699–705.Google Scholar
Gutteridge, W. E. (1976 c). The biochemistry of Trypanosoma cruzi. In Pan American Health Organization Symposium No. 318: American Trypanosomiasis Research, pp. 135–40. Washington: Pan American Health Organization.Google Scholar
Gutteridge, W. E. (1976 d). Isolation of blood and intracellular forms of Trypanosoma cruzi and comparative aspects of nucleic acid metabolism. In Biochemistry of Parasites and Host-Parasite Relationships (ed. Van den Bossche, H.), pp. 245–52. Amsterdam: Elsevier/North Holland Biomedical Press.Google Scholar
Gutteridge, W. E., Cover, B. & Cooke, A. J. D. (1974). Safety precautions for work with Trypanosoma cruzi. Transactions of the Royal Society of Tropical Medicine and Hygiene 68, 161.Google Scholar
Gutteridge, W. E., Knowler, J. & Coombes, J. D. (1969). Growth of Trypanosoma cruzi in human heart tissue cells and effects of aminonucleoside of puromycin, trypacidin and aminopterin. Journal of Protozoology 16, 521–5.CrossRefGoogle ScholarPubMed
Hanson, W. L. & Roberson, E. L. (1974). Density of parasites in various organs and the relation to numbers of trypomastigotes in the blood during acute infections of Trypanosoma cruzi in mice. Journal of Protozoology 21, 512–7.CrossRefGoogle ScholarPubMed
Hoare, C. A. (1972). The trypanosomes of mammals. A Zoological Monograph. Oxford: Blackwell Scientific Publications.Google Scholar
Jaffe, J. J., McCormack, J. J. Jr & Gutteridge, W. E. (1969). Dihydrofolate reductases within the genus Trypanosoma. Experimental Parasitology 25, 311–18.CrossRefGoogle ScholarPubMed
Köberle, F. (1968). Chagas' disease and Chagas' syndromes: the pathology of American trypanosomiasis. Advances in Parasitology 6, 63–116.CrossRefGoogle ScholarPubMed
Köberle, F. (1974). Pathogenesis of Chagas' disease. In Ciba Foundation Symposium No. 20 (New Series): Trypanosomia8is and Lei8hmaniasis with special reference to Chagas' disease, pp. 137–58. Amsterdam: Associated Scientific Publishers.Google Scholar
Krebs, H. A. & Eggleston, L. V. (1940). The oxidation of pyruvate in pigeon breast muscle. Biochemical Journal 34, 442–59.CrossRefGoogle ScholarPubMed
Lanham, S. M. (1968). Separation of trypanosomes from the blood of infected rats and mice by anion exchangers. Nature, London 218, 1273–4.CrossRefGoogle ScholarPubMed
Lanham, S. M. (1971). The separation of stercorarian trypanosomes from infected blood using DEAE cellulose. Transactions of the Royal Society of Tropical Medicine and Hygiene 65, 248–9.CrossRefGoogle Scholar
Lanham, S. M. & Godfrey, D. G. (1970). Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Experimental Parasitology 28, 521–34.CrossRefGoogle ScholarPubMed
Lumsden, W. H. R. (1974). Leishmaniasis and trypanosorniasis: the causative agents compared and contrasted. In Ciba Foundation Symposium No.20 (New Series): Trypanosomiasis and Leishmaniasis with special reference to Chagas' disease, pp. 3–27. Amsterdam: Associated Scientific Publishers.CrossRefGoogle Scholar
Meyer, H. & De souza, W. (1976). Electron microscopic study of Trypanosoma cruzi periplast in tissue cultures. I. Number and arrangement of the peripheral microtubules in the various forms of the parasite's life-cycle. Journal of Protozoology 23, 385–90.CrossRefGoogle ScholarPubMed
Newton, B. A., cross, G. A. M. & Baker, J. (1973). Differentiation in the Trypanosomatidae. In Microbial Differentiation (ed. Ashworth, J. M. and Smith, J. E.), pp. 339–74. Cambridge:Cambridge University Press.Google Scholar
Pan, S. C. (1976). In vitro cultivation of amastigotes of Trypanosoma cruzi in cell-free media. In Pan American Health Organization Symposium No. 318: American Trypanosomiasis Research, pp. 121–6. Washington: Pan American Health Organization.Google Scholar
Remington, J. S., Bloomfield, M. M., Russell, JR., E.& Robinson, W. S.(1970). The RNA of Toxoplasma gondii. Proceedings of the Society for Experimental Biology and Medicine 133, 623–6.CrossRefGoogle ScholarPubMed
Riou, G., & Gutteridge, W. E. (1973). Comparative study of kinetoplast DNA from intracellular, blood and culture forms of Trypanosoma cruzi. In Progress in Protozoology, Proceedings of the 4th International Congress of Protozoology (ed. de Puytorac, P. and Grain, J.), p. 470. Clermont-Ferrand: University de Clermont.Google Scholar
Scorza, C.(1972). Early cytochemical changes in the hearts of rats infected with Trypanosoma cruzi. Transactions of the Royal Society of Tropical Medicine and Hygiene 66, 889–96.CrossRefGoogle ScholarPubMed
Shoemaker, J. P. & HoffmanJR., R. V. JR., R. V. (1974). Trypanosoma cruzi: possible stimulatory factor(s) in brown adipose tissue of mice. Experimental Parasitology 35, 272–4.CrossRefGoogle ScholarPubMed
Simpson, L.(1968). The leishmania-leptomonad transformation of Leishmania donovani: nutritional requirements, respiration changes and antigenic changes. Journal of Protozoology 13, 201–7.CrossRefGoogle Scholar
Spurr, A. R.(1969). The low-viscosity epoxy resin embedding media for electron microscopy. Journal of Ultrastructure Research 26, 31–43.CrossRefGoogle Scholar
stohlman, S. A., Kttwahara, S. S.&Kazan, B. H.(1973). Enzyme, protein and nucleic acid content of two morphological forms of Trypanosoma (Schizotrypanum) cruzi. Archives of Microbiology 92, 301–11.Google ScholarPubMed
Trigg, P. I. & Gtjtteridge, W. E. (1977). Morphological, biochemical and physiological changes occurring during the life cycles of parasitic protozoa. In Parasite Invasion (ed. Taylor, A. E. R. and MÜller, R.), pp.57–81.Oxford: Blackwell Scientific Publications.Google Scholar
Williamson, J.&Cover, B. (1966). Separation of blood cell-free trypanosomes and malaria parasites on a sucrose gradient. Transactions of the Royal Society of Tropical Medicine and Hygiene 60, 426–7.CrossRefGoogle Scholar