./ Prorozool, 37(1), 1990. pp. 40-43 '6 1990 by the Society of Protozoologists

Continuous Growth and Differentiation of Tuypanosoma (Megatuypanum)freitasi Rego, Magalhiies & Siqueira, 1957, In Vitro NEIDE THOMAZ and MARIA P. DEANE' Instituto Oswaldo Cruz, Department of Protozoology, Av. B r a d 4365, 21040, Rio de Janeiro, Brazil

ABSTRACT. Trypanosoma (Megatrypanurn)freitasi, a parasite of didelphid opossum, was known to be very difficult to cultivate in conventional media. Co-cultivation with L929 cell line in Baltz's medium at 27.5" C resulted in luxuriant growth of the trypanosome with the production of epimastigote colonies that adhered to the surface of culture flasks or tubes, and transformation into metacyclics. Further transformation was stimulated by raising the incubation temperature. At 37" C the population was of the bloodstream type and resistant to lysis by complement. Key words. Adhesion, bloodstream trypomastigotes, epimastigote colonies, opossum trypanosome.

I

N a study of the opossum Didelphis marsupialis as reservoir of Trypanosoma cruzi we found specimens naturally infected with a trypanosome later identified as Trypanusoma freifasi. This was the 1st example of a Megatrypanum with a double cycle (vertebrate and invertebrate) in a mammal host [ 1 1 , 121, following reports of a similar cycle for Trypanosoma (Schizotrypanurn) cruzi [13, 141. In both cases the invertebrate cycle occurred in the lumen of the scent (anal) glands of Didelphis tnarsupialis. N o adhesion of the flagellates to the glandular epithelium was seen. References to the finding of Trypanosoma ,freitasi are rare and the species is said to be difficult to maintain in culture [5, 23, 241. Continuous growth of T. freitasi in vitro and transformation into trypomastigotes of the metacyclic and the bloodstream types are here described.

Staining was by Giemsa after methanol fixation, or by immersion in 5N HCI solution at room temperature for 15 min and washing in tap water after methanol fixation [ 3 ] .

MATERIALS AND METHODS Parasites. The parasites were isolated by hemoculture in triple N, from naturally infected opossums captured in localities of the State of Rio de Janeiro. Culture media. Several culture media were used: triple N with defibrinated rabbit blood, with or without an overlay ofYeager's LIT [8] or Eagle's MEM; LIT with 10% (v/v) calf serum; the medium ofCazzulo et al. [4] with 10% (v/v) calf or horse serum; Vero or L929 (mouse fibroblast) cell line cultures with Medium 199, or the "standard" medium of Baltz et al. [ 11. The nutrients of the cell lines were buffered with HEPES and supplemented with 10% (v/v) fetal bovine serum. Media and drugs were Flow, Difco, Merck or Sigma and sera were from Microbiologica (SBo Paulo) or the IOC (Rio). Penicillin G (1 00 U/ml) was incorporated in all media. Cultures in cell lines were made in Leighton tubes with coverslips or in plastic bottles (Descarplast). Incubation temperatures 0.2" C , 34" C or 37" C. were 27.5 Lysis by complement. Resistance of some of the trypanosome phases to the lytic action of complement was tested through microscopical examination of cultures mixed with fresh guinea pig serum (1:7) incubated at 37" C for 30 min compared with controls made with the same serum inactivated at 56 "C for 20 min.

RESULTS Growth and differentiation of the parasite at the lower incubation temperature. The parasite developed slowly and poorly in the axenic biphasic or liquid media, except the one by Cazzulo et al., which promoted good initial growth that was not maintained in subsequent passages, unless horse serum was used instead of calf serum. Very rapid growth was obtained in all cell cultures with any of the nutrients used but by far the best results were seen in the L929 cell line with the medium of Baltz et al. Proliferation of the parasite in the latter cell system at 27.5 _t 0.2" C was luxuriant, with agglomerates of epimastigotes forming colonies that adhered to the plastic surface, or to the glass underneath the coverslip on which the feeder layer of cells grew when Leighton tubes were used. In older cultures the epimastigote colonies tended to fuse. No intracellular parasites were seen and attachment to the living cells themselves did not seem important (Fig. 1-3). Adhesion of the colonies was very strong but, in older cultures, some epimastigotes could be washed out with strong liquid jets after scratching the agglomerates with the point of a pipette. Such a mechanical disorganization of the colonies uncovered much degenerated fibroblast cells or cellular remains that obviously had been serving as support for the epimastigotes (Fig. 6). These were usually small, very pleomorphic and had a short flagellum with an enlarged terminal portion; in some forms this enlargement seemed to occur in the intra-pocket portion of the flagellum with the organism appearing sessile or with a small round dark appendix (Fig. 4, 5, 7). Although most of the epimastigote population remained attached, some were found free, isolated, in division, or loosely arranged in floating rosettes. They were usually elongate, with many coarse inclusions that disappeared with the acid treatment before staining. In the rosettes, the epimastigotes presented a dark strip in their anterior half, along the intracellular portion of the flagellum and extending beyond (Fig. 8). These aspects

+ Fig. 1. Plastic bottle in which Trypanosomafreitasi was co-cultivated with L929 cells, showing abundant colonies of the trypanosome; darkfield illumination, x 0,8 1. Fig. 2. Coalescent colonies of epimastigotes in an older culture. Fig. 3. The same culture showing that adhesion to living cells does not seem to be essential; phase contrast in an inverted microscope, x 350. Fig. 4-16. Giemsa-stained preparations, x 1,400. 6 . Attachment of epimastigotes to cell remains. 4, 5, 7. Epimastigotes with or without a short free flagellum and with a dark material that is possibly responsible for adhesion and is partially included in the flagellar pocket. 8. A free colony in which epimastigotes show a dark strip along their anterior half, suggesting that the adhesion material is secreted by the flagellate. 9, 10. Metacyclic trypomastigotes and a spheromastigote. 11, 12. Early transformation into the bloodstream type of trypomastigote, some extremely elongated posteriorly. 13,14. Transformationinto bloodstream trypomastigote by unrolling from a spherical body (arrow). 15, 16. Bloodstream type trypomastigotes which predominate at 37" C.

40

THOMAZ & DEANE-CULTIVATION OF TRYPANOSOMA ( M ) FREITASI

41

42

J. PROTOZOOL., VOL. 37, NO. I , JANUARY-FEBRUARY 1990

suggest that some material is secreted by the cell and move along the flagellum up to its tip. The cultures were long-lived and growth of the parasite continued even after most of the cell feeder layer had degenerated and as long as the nutrient was changed periodically. However, the presence of living cells seems to be essential to start epimastigote colonies and produce abundant metacyclogenesis. The 1st metacyclic forms were seen in the nutrient medium of new cell cultures several days after the passage of the parasite and when the epimastigote colonies were already well established. Thereafter they appeared by waves. Close observation of living cultures under an inverted microscope immediately after changing the medium, convinced us that metacyclogenesis takes place at the deeper layers of the colonies, from where the trypomastigotes may be carried by the liquid current and come out already fully transformed or in a spheromastigote stage similar to that described for T. cruzi [2] (Fig. 9, 10). Morphology and transformation at the higher temperatures. Transformation into the bloodstream forms started at 27” C but was accelerated at 34” C; at 37” C most epimastigotes ceased to proliferate and eventually degenerated but bloodstream trypomastigotes were larger and more numerous. The bloodstream forms developed in two ways: (a) by elongation of the small metacyclic especially of its posterior portion, the nucleus becoming rounded and the kinetoplast keeping its postero-nuclear position; or (b) from a long slender epimastigote which, except for the position of the kinetoplast, could be taken as a young bloodstream trypomastigote; indeed these epimastigotes (as well as the metacyclic and the “mature” bloodstream forms) were not lysed by complement. Apparently these epimastigotes can transform directly into trypomastigotes by displacement of the kinetoplast to a post-nuclear position, but they also roll themselves into large spherical forms that, by unrolling and stretching, give rise to trypomastigotes with considerably elongated posterior ends (Fig. 11-14). Presumably, it is by becoming shorter and wider, especially at the level of the undulating membrane, that the trypomastigotes acquire the aspect of the bloodstream “mature” forms (Fig. 15, 16). All trypomastigotes were free from the granular inclusions that fill the epimastigote cytoplasm. These inclusions obstruct the internal structures which are well visualized only when the material passes through an acid solution before staining. Infectivity of the cultures was proven through inoculation of young laboratory-reared opossums. DISCUSSION Except for T. theileri and T. conorhini attempts to cultivate species of the Megatrypanum subgenus have met with limited success [ 15,201. Trypanosoma conorhini was the 1st to be maintained indefinitely in axenic media and was also the 1st mammalian trypanosome for which a pure culture of the bloodstream phases was obtained [6-81. The reported difficulty to grow T.freitasi in an axenic medium [23, 241 was confirmed by us. Some of the media we tested permitted good initial growth but abundant and continuous development of the parasite was possible only by co-cultivating it with some vertebrate cell lines. In this system T.freitasi formed epimastigote colonies of a type not yet described for the Megatrypanum sub-genus. Much attention has been paid in recent years to the phenomenon of attachment of the multiplicative phases of trypanosomatids to the cuticular surface of various segments of the digestive tract of their insect host. For digenetic species it has been suggested that attachment is a pre-requisite for differentiation into the forms that are infective to the vertebrate host. A simili-

tude between this “in vivo” situation and the “in vitro” adhesion of the parasites to glass or plastic surfaces has been proposed [221. In cultures the reproducing phases of mono and digenetic trypanosomatids tend to form colonies that may adhere to substrata, as in the case of T. freitasi, or float in the liquid media as globular aggregates which are flattened in “rosettes” when fixed on microscopical slides. Strikingly regular floating colonies were described for T. conorhini [8]. In the globular colonies the flagellates are radially disposed with their flagella directed inwards toward the center of the aggregate where there is an accumulation of debris [lo]. According to Jardin & Creemers [ 171, such an arrangement of promastigotes of Leishmania tropica represents a temporary gathering that facilitates endocytosis, through the flagellar pocket, of food material supplied by the remains of lysed organisms. On the other hand, the necessity of a support was illustrated by Maraghi et al. [18] who demonstrated that several trypanosomatids that did not attach to plastic surfaces promptly formed linear colonies along scratches made on these surfaces. As for T.freitasi, we can speculate that degenerated cells, or cell fragments may offer support for attachment of epimastigotes, or a supply of food on which to scavenge, or both. Anyway, the fact that the colonies, adhered or floating, are difficult to dissociate [8,22] does not seem to qualify them as a transitory getting together only for feeding. For many trypanosomes aggregation of reproductive forms stimulates multiplication and/or differentiation into metatrypanosomes [16, 18, 221. Differentiation seems to be programmed to occur after a certain number of cell division cycles and we suggest that aggregation might favour intercell communication and the passage of “signals” that trigger differentiation. The closeness of cells in epimastigote aggregations could also supply the opportunity for exchange of genetic material as it has been suggested for T. conorhini [9, 101. Various factors, such as electrostatic charge and the presence of specific lectins in insect guts, have been invoked as possibly involved in these parasite/substrata interactions [22]. Some of the illustrations presented in this paper suggest that the material responsible for adhesion may be secreted by T. freitasi epimastigotes. This material moves along the flagellum up to its tip and, possibly discharge in the medium when the organism transforms or dies. Specific and strain differences in responsiveness to the stimuli present in the various media and situations are to be expected, especially if one considers separately each phase of these phenomena-aggregation, adhesion, multiplication and transformation. Temperature is a known morphogenetic factor in the life cycle of mammalian trypanosomes and this was confirmed for T. freitasi. At 37” C the population of T. freitasi consisted of large epimastigotes and trypomastigotes of the bloodstream type, both in frequent division and similar to those described for T. conorhini and T. theileri [7, 8, 19, 251. The possibility ofmaintaining the mammalian cycle in vitro, for long periods, has been demonstrated for T. theileri and for species of the subgenera Herpetosoma and Trypanozoon; in all cases a feeder layer of vertebrate cells has been used, but some success has been obtained with cell-free media [ 1, 19, 2 I]. We are presently trying to understand the role of living cells in promoting growth of T. freitasi and other species that do not have an intracellular cycle in the vertebrate host. ACKNOWLEDGMENTS This work was supported by grants of the CNPq and FINEP. We thank Drs. Radovan Borojevic and Raul Machado of the

THOMAZ & DEANE-CULTIVATION OF 7'R YPANOSOMA ( M ) FREITASI

Federal University o f R i o de J a n e ir o ( U F R J ) for supplying t h e L929 cell line and for facilities to photograph our material i n an inverted phase-contrast microscope. L I T E R A T U R E CITED 1. Baltz, T., Baltz, D., Giroud, Ch. &Crockett, J. 1985. Cultivation

in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum. T. evansi, T. rhodesiense and 7'. gambiense. EMBO J., 4:1273-1277. 2. Brack, C. 1968. Elektronmikroskopische Untersuchungen zum Lebeenszyklus von Trypanosoma cruzi. Acta Trop.. 25:289-356. 3. Carvalho, A. L. M. & Deane, M. P. 1974. Trypanosomatidae isolated from Zelus leucogramus (Perty, 1834) (Hemiptera, Reduviidae), with a discussion on flagellates ofinsectivorous bugs. J. Protozool., 21~5-8. 4. Cazzulo, J. J., Cazzulo, B. M. F., Engel, J. C. & Cannata, J. J. B. 1985. End products and enzyme levels of aerobic glucose fermentation in Trypanosomatids. Mol. Biochem. Parasitol., 16:329-343. 5. Deane, L. M. 1964. Trypanosomideos de mamiferos da RegiHo Amazhica. 111. Hemoscopia e xenodiagnostico de animais silvestres dos arredores de Beltm, Para. Rev. Inst. Med. Trop. Sa'o Paulo, 6:225232. 6. Deane, M. P. 1948. OcorrEncia d o Trypanosoma conorhint em barbeiros e em ratos na cidade de Belim, Para e seu cultivo em meio NNN. Rev. ServiCo Especial Salide Ptiblica, 1:433-488. 7. Deane, M. P. & Deane, L. M. 1961. Studies on the life cycle of Trypanosoma conorhini. In vitro development and multiplication of the bloodstream trypanosomes. Rev. Inst. Med. Trop. Sa'o Paulo, 3: 149160. 8. Deane, M. P. & Kirchner, E. 1963. Life cycle of Trypanosoma conorhini. Influence of temperature and other factors on growth and morphogenesis. J. Protozool., 10:39 1400. 9. Deane, M. P. & Milder, R. 1966. A process of reproduction of Trypanosoma conorhini different from binary or multiple fission. J. Protozool., 13:553-559. 10. Deane, M. P. & Milder, R. 1972. Ultrastructure of the "cystlike bodies" of Trypanosoma conorhini. J. Protozool., 19:2842. I I . Deane, M. P. & Jansen, A. M. 1986a. Another Trypanosoma, distinct from T. cruzi, multiplies in the anal glands of the opossum Didelphis marsupialis. Mem. Inst. Oswaldo Cruz. 81: 13 1-132. 12. Deane, M. P. & Jansen, A. M. 1986b. Besides Trypanosoma (Schizotrypanurn), another species, T. (Megatrypanum)freitnsi is found in the anal glands of opossums. Speculations on the significance of these findings. Mem. Inst. Oswaldo Cruz, 81(Suppl.):53.

43

13. Deane, M. P., Lenzi, H. L. & Jansen, A. M. 1984. Trypanosoma cruzi: vertebrate and invertebrate cycles in the same mammal host, the opossum Didelphis marsupialis. Mem. Inst. Oswaldo Cruz, 19:5 13-5 15. 14. Deane, M. P., Lenzi, H. L. & Jansen, A. M. 1986. Double development cycle of Trypanosoma cruzi in the opossum. Parasitology Today, 2:146-147. 15. Gardner, R. A. & Molyneux, D. H. 1988. Trypanosoma (Megatrypanum) incertum from Pipistrellus pipistrellus: development and transmission by cimicid bugs. Parasitology, 96:433447. 16. Hendry, K. A. K. & Vickerman, K. 1988. The requirement for epimastigote attachment during division and metacyclogenesis in Trypanosoma congolense. Parasitol. Rex, 74:403408. 17. Jadin, J. M. & Creemers, J. 1966. L'ultrastructure des formes en rosace de Leishmania tropica Wright, 1903. Ann. SOC.Belge Med. Trop., 46:349-354. 18. Maraghi, S., Mohamed, H. A., Wallbanks, K. R. & Molyneux, D. H. 1987. Scratched plastic as a substrate for trypanosomatid attachment. Ann. Trop. Med. Parasitol., 81:457-458. 19. McHolland-Raymond, L. E., Kingston, N. & Trueblood, M. S. 1978. Continuous cultivation of Trypanosoma theileri at 37" C in bovine cell culture. J. Protozool., 29388-394. 20. Mohamed, H. A., Molyneux, D. H. 8~Wallbanks, K. R. 1987. On Trypanosoma (Megatrypanum) talpe from Talpa europaea: method of division and evidence of Haemogamasinae as vectors. J. Parasitol., 73:1050-1052. 21. Mohamed, H. A,, Maraghi, S., Wallbanks, K. R. & Molyneux, D. H. 1988. In vitro cultivation of Herpetosorna trypanosomes on embryonic fibroblasts and in semidefined cell-free media. J. Parasitol., 74:421426. 22. Molyneux, D. H., Wallbanks, K. R. & Ingram, G. A. 1987. Trypanosomatid-vector interfaces-in vitro studies on parasite substrate interactions. In: Chang, K. P. & Snary, D. (ed.), Host-Parasite Cellular and Molecular Interactions in Protozoal Infections. SpringerVerlag, Berlin, Heidelberg, NATO AS1 Series Vol. Hl1:387-396. 23. Rego, S. F. M., Magalhies, A. E. A. & Siqueira, A. F. 1957. Um novo trypanosomo d o gamba, Trypanosoma freitasi N. Sp. Rev. Bras. Malariol. D. Trop., 9:277-284. 24. Silva, E. 0. R., Pattoli, D. B. G. & Camargo, J. C. 1976. Novo encontro d o Trypanosoma (Megatrypanurn)freitasi,parasita d o gamba. Rev. Satide Publ. S. Paulo, 10:12 1-1 24. 25. Wink, M. 1979. Trypanosoma theileri in vitro cultivation in tsetse fly and vertebrate cell culture systems. Int. J. Parasitol., 9585589.

Received 4-28-89; accepted 9-5-89