In vitro Production of Metacyclic Trypomastigotes of Trypanosoma cruzi
1980; American Society of Parasitologists; Volume: 66; Issue: 6 Linguagem: Inglês
10.2307/3280415
ISSN1937-2345
Autores Tópico(s)Research on Leishmaniasis Studies
ResumoBloodstream-forms of Trypanosoma cruzi ingested by reduviid bugs multiply and differentiate through a series of developmental stages into infective, metacyclic, trypomastigote forms. The intestinal tracts of these bugs provide a suitable physical and chemical environment for the complex developmental cycle of this stercorarian parasite. A relatively large number of media are available in which the trypanosomes, when cultivated at room temperature (25-28 C), develop as morphological forms similar to those produced in the vectors (Brener, 1973, Ann. Rev. Microbiol. 27: 347-382). Epimastigote forms predominate during the early phase of their growth and some metacyclics appear later in these media. O'Daly (1975, J. Protozool. 22: 265-270) devised a partially defined medium (SM) containing 5% fetal calf serum (FCS) which promoted the development of up to 90% metacyclic stages during the stationary phase. Maximum growth of the T. cruzi was reported after 13 to 15 days with populations attaining approximately 4 x 106 trypanosomes/ml. Wood and Sousa (1976, Rev. Inst. Med. Trop. 18: 93-96) found excellent growth (8.3 x 107 T. cruzi/ml) and metacyclic differentiation (85%) in a medium composed of Grace's insect cell culture medium, FCS, and an extract of Rhodnius prolixus. Metacyclics did not develop if the Rhodnius extract was excluded from this medium. Pudney and Lanar (1977, Ann. Trop. Med. Parasitol. 71: 109-118) established a cell line from Triatoma infestans in a medium composed of Leibovitz L-15 medium, tryptose phosphate broth, glutamine (ML-15), and FCS. Although reduviid extracts were not included, this medium in combination with FCS, sustained the growth of embryonic Triatoma cells. These T. infestans cell cultures supported the growth of T. cruzi to approximately 1 x 107 trypanosomes/ml (Lanar, 1979, J. Protozool. 26: 457-462). When bloodstream-form trypomastigotes were inoculated into cultures of Triatoma cells, they differentiated into amastigotelike forms and, subsequently, into epimastigotes and metacyclic trypomastigotes. The following study showed that the combined and modified components of the SM and ML-15 in combination with FCS permitted the axenic cultivation of T. cruzi and enhanced differentiation to the metacyclic stage. The Tulahuen strain of T. cruzi was maintained in C3H/Anf mice (Cumberland View Farms, Clinton, Tennessee) by weekly intraperitoneal subinoculations of blood from infected to normal mice. Bloodstream-form trypomastigotes used to inoculate cultures were obtained from mice which received 300 R from a cobalt-60 irradiation source 3 days before injection of the parasites. Six days after inoculation, these animals were bled by cardiac puncture and the blood was collected aseptically from each mouse into 0.1 ml heparinized saline (5 mg heparin/ml 0.85 NaCI). The heparinized blood was pooled and diluted approximately 1:10 with sterile phosphate buffered saline at pH 7.2 (PBS) prepared with 0.025 M NaH2PO4, 0.025 M Na2HPO4, and 0.108 M NaCl. The suspensions were centrifuged at 770 g for 20 min at 4 C and the buffy coats containing the T. cruzi were removed, pooled, and counted in a Neubauer hemacytometer. Primary cultures were inoculated with 1 x 105-1 x 106 bloodstream-forms. Subcultures received inocula of 2 x 104-5 x 105 T. cruzi/ml of medium. During the course of the study, hemacytometer counts were performed on the parasites in each culture, and films were prepared on slides, fixed with methanol, and stained with Giemsa for morphological identifications. The SM (O'Daly, loc. cit.) contained Eagle's Minimum Essential Medium (MEM) with Earle's salts, L-glutamine, nonessential amino acids, MEM vitamins, EPPS (N-2-hydroxyethyl piperazine propane sulfonic acid),
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