.I Protozool.. 37(1), 1990, pp. 44-47 ‘C 1990 by the Society of Protozoologists
Developmental Stages of Trypanosoma (Megatrypanum)freitasi Rego, Magalhiies & Siqueira, 1957 in the Opossum Didelphis marsupialis (Marsupialia, Didelphidae) MARIA P. DEANE and ANA MARIA JANSEN Instituto Oswaldo Cruz, Department of Protozoology,Av. Brasil 4365, Manguinhos, 21040, Rio de Janeiro, Brazil
Trypanosoma (Megatrypanurn)freilasi, a parasite of marsupials of the genus Didelphis, has been found to undergo in the lumen of the scent (anal) glands of its vertebrate host, a cycle such as usually occurs in the intestinal tract of the insect vectors of trypanosomatidsand similar to what has been reported for Trypanosoma (Schizotrypanum) cruzi. The invertebratehost of Trypanosoma ,fieitmi is still unknown. Developmental stages of the trypanosome in its mammalian host, especially the dividing epimastigotes, multinucleate plasmodia1 forms and rosettes found in the lumen of the scent glands of a naturally infected Didelphis marsupialis are described and illustrated. Key words. Life cycle, marsupials, scent glands. ABSTRACT.
IDELPHID opossums are certainly the wild mammals most frequently examined in epidemiological investigations of Chagas’ disease in Brazil. Besides the high rates of Trypanosoma cruzi infections throughout the country , these marsupials have been found to harbour Trypanosoma rangeli , Trypanosorna.freitasi [6, 18, 201 and various Leishmania species or subspecies [7, 191. Trypanosoma freitasi was the 2nd species of the genus and the I st Megatrypanurn to be reported as undergoing in the lumen of the scent (anal) glands of its mammalian host, a cycle that usually occurs only in the intestinal tract of insect vectors [9-121. Phases of the development of Trypanosoma ,freitasi in naturally and experimentally infected Didelphis marsupialis are here described. MATERIALS AND METHODS Opossums. Specimens of D. marsupialis were trapped in various localities of the State of Rio de Janeiro, chiefly Jaguanum Island, Miguel Pereira and Itaguai. They were brought to the laboratory and subjected to the routine examinations as given below; when found to be negative they were set free or kept for breeding and for future inoculations; those found positive were maintained for follow-up observations. Examination of wild caught specimens. Soon after capture the animals were examined for ectoparasites and subjected to xenodiagnosis (XENO) with triatomid bugs. They were then bled from the femoral vein for hemoculture and immunofluorescent antibody tests (IFAT) with T. cruzi antigens . After the 1st observation of T. cruzi in the scent (anal) glands (SG), examination of material obtained by manual expression of these glands was included in the routine procedures. The feces of bugs used for XENO were examined within 30 days and hemolymph plus intestinal contents within 45-60 days. Cultures were examined fortnightly up to day 60. Culture media. Triple N with defibrinated rabbit blood was used for isolation. For maintenance, several media were tried, as described elsewhere (Thomaz & Deane, submitted). Inoculations. Cultures of T . freitasi were inoculated subcutaneously (SC) and in the peritoneum (IP) of previously negative adult opossums and also in specimens of various ages (reared
from pouch young and still marsupium-dependent). Some of the young ones were treated with hydrocortisone (Solu-Cortec Upjohn), 0.1 mg/g/day, for 2 days before and 5 days or more after inoculation. Cultures were also inoculated IP in immunodepressed outbred mice and fed through a membrane to three different species of triatomids, Rhodnius neglectus, Triatoma infestans and Dipetalogaster maximus. Follow-up of inoculated opossums was by frequent blood examination, hemoculture and, at necropsy, smears of spleen, liver, lymph nodes, kidneys, bone-marrow, lungs, brain, SG and peritoneal exudate. Material from the SG was also obtained during life, except when the animal was too young. The animals were killed from 3-5 days up to many months after inoculation. Mice blood was examined daily during a month and placed into triple N culture medium after death. Cultures and triatomids were examined as described. Staining. Smears and imprints were stained by Giemsa’s after methanol fixation. Material from the anal glands was fixed in methanol, immersed for 15 min in 5 N HCl at room temperature and washed in tap water, before staining . RESULTS Natural infection of opossums. The 1st hint that we had something different from T . cruzi or T. rangeli in our opossums came when a specimen was found with negative XENO and IFAT, and hemocultures were found showing peculiar epimastigotes, very rich in coarse granular inclusions. Soon after, the 1st infection of the SG was detected with the same type of epimastigote. In Jaguanum Island, where the rate of natural infection was quite high for T. c m z i (87%), T.fieitasi was found, in single or mixed infections, in 12 out of 31 (38.7%) opossums examined. Among 95 opossums caught in all localities 5 were found infected with T . freitasi alone, 26 with T. cruzi alone and 9 had mixed infections, total rates being 36.8% for the latter and 14.7% for the former. All T. freitasi infections were detected through hemocultures and repeated fresh blood examinations made in some cases were always negative; the SG were infected in 2 of the 35 specimens in which they were examined. Only T . freitasi was detected in
+ Fig. 1-9. Trypanosoma (Megatrypanurn)freitasi: multiplicative stages in the scent (anal) glands o f a naturally infected opossum, Didelphis marsupialis. Giemsa stained smears after HC1 treatment. Note the abundance of lipid droplets in the cytoplasma of all forms. Fig. 10. A metacyclic trypomastigote in the same material. Fig. 11. A bloodstream type trypomastigote in the peritoneum of a young opossum inoculated with a culture of T. ,fieitasi. All figures x 1,400.
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J. PROTOZOOL., VOL. 31, NO. 1, JANUARY-FEBRUARY 1990
the glands of these two specimens, despite the fact that one of them had a mixed infection revealed by hemoculture. Among the dense, semi-solid granular material, rich in detached epithelial cells and fat globules that make up the contents of the SG, T. freitasi epimastigotes which are, themselves, filled with coarse granules and frequently without a free flagellum, are difficult to detect in fresh preparations, or even in Giemsa stained smears, unless these are treated with HCI before staining. Figures 1-9, illustrate different forms of the parasite found in the SG of one of the naturally infected opossums. Besides epimastigotes of various sizes and shapes, some undergoing binary fission, large multinucleate bodies and rosettes were very frequent. In all these forms the cytoplasma sometimes has the appearance of a lace due to the great amount of droplets, probably lipids. Metacyclic trypomastigotes (Fig. 10) were rare in this material. Aspects of T. freitasi in cultures obtained from the blood of naturally infected opossums are described elsewhere (Thomaz & Deane, submitted). Experimental infections. In young opossums killed 3-5 days following inoculation, bloodstream forms were found in the peritoneum (Fig. 1 1). Trypomastigote invasion however, was exceptional and scanty even when massive inocula were used. Patent parasitemia did occur in two cortisone treated young, but the average was less than one trypomastigote per slide, in thick smears made every other day during a two-month period. U p to this point there was no invasion of the SG in the experimentally infected opossums and, except for rare trypomastigotes of the bloodstream type, no parasites were detected in the imprints and smears made post-mortem. The inoculated mice and all tnatomids fed directly on infected opossums or on cultures of T. freitasi were negative. Other data. Our sample of wild caught opossums has been remarkably free from ectoparasites. In Jaguanum Island, where the rate ofinfection with T.freitasi was 38.7%, no blood-sucking arthropod was found in association with opossums, except rare specimens of Microtriatoma borbai. As mentioned, triatomids have been demonstrated to be refractory to infection with this trypanosome. DISCUSSION The trypomastigotes of T.freitasi, as those of other Megatryp a n u m species, present much variation in size before they reach a final “mature” form in the bloodstream of the host. In our material such forms were rare and found only in thick blood smears, so measurements were not taken. However, in all respects our trypanosome fits with the original description of T . freitasi: by the morphology of the trypomastigotes it is a Megatrypanum; it does not infect the laboratory mouse and, most probably, it is restricted to didelphid opossums; there is no evidence of pathogenicity or of multiplication in the tissues of the vertebrate host; it is not infective to triatomid bugs; and it grows very poorly in triple N medium [ 181. Indeed, in most of these aspects there is a coincidence with what it known and unknown about the trypanosomes included by Hoare in his subgenus Megatrypanum which he regarded as “phylogenetically the most primitive representatives of the genus Trypanosoma in mammals” [ 131. Hoare’s opinion is shared by others [ l , 171 and by ourselves and would justify a greater interest in the study of the biology of this largely neglected group. The finding of T. freitasi multiplying in the lumen of the opossum SG led to the suggestion that other species of the subgenus might be found in similar situations in their vertebrate hosts [lo]. References to dividing stages ofMegatrypanum trypanosomes as amastigotes, multinucleate plasmodia1 forms or epimasti-
gotes, in extra or intravascular localizations are found for T. theileri, T . hoarei, T. conorhini and T. talpae [3, 8, 13, 15, 161. However, the data available indicate that a multiplicative phase of Megatrypanum trypanosomes in their vertebrate hosts, if present, is very limited and this could explain why parasitemia is low or subpatent in most infections and of short duration in experimental primo-infections [ 131. The possibility of reinfection within one year after the primo-infection, has been demonstrated at least for T. melophagium [I31 and T. conorhini (Deane & Yoshida, unpubl.). Under natural conditions, the frequent contact with a parasite transmitted by a vector which is in permanent association with the vertebrate host, probably keeps immunity at a level sufficient to maintain in check the multiplication of the parasite. Such would be the case, for instance, with T . melophagium and T . theodori and their Hippoboscidae vectors. The opossum SG can be thought of as a “vector” in permanent association with the vertebrate host, since in this habitat T..freitasi undergoes a cycle that corresponds to the one that probably occurs in its as yet unknown insect vector. The presence of T . freitasi in the opossum SG opens fascinating new perspectives for the study of the immunological aspects of parasite/host relationship. ACKNOWLEDGMENTS This work had financial support from CNPq and FINEP. The authors are thankful to Maria de Fitima Bernard0 for her technical assistance and to Marlene Lopes de Lucena for her secretarial help. LITERATURE CITED 1. Baker, J. R. 1963. Speculations on the evolution of the family Trypanosomatidae Doflein, 1901. Expl. Parasitol.. 13:219-233. 2. Barretto, M. P. & Ribeiro, R. D. 1979. Reservatorios silvestres do Trypanosoma (Schizotrypanum) cruzi. Rev. Inst. Adolfo Lutz, 39: 25-36. 3. Carpano, M. 1932. Localization du Trypanosoma theileri dans les organes internes des bovines. Son cycle Cvolutif. Ann. Parasit. Hum. Comp., 10:305-322. 4. Carvalho, A. L. M. & Deane, M. P. 1974. Trypanosomatidae isolated from Zelus leucogramrnus (Perty, 1834) (Hemiptera, Reduviidae) with a discussion on flagellates of insectivorous bugs. J. Protozool., 21~5-8. 5. Deane, L. M. 1958. Encontro de tripanosomos do tipo rangeli em gambds da espkie Didelphis marsupialis no Estado d o Para. Rev. Bras. Malariol. DoenGus Trop.. 10:45 1-458. 6. Deane, L. M. 1964. Tripanosomideos de mamiferos da RegiZo AmazBnica. 111. Hemoscopia e xenodiagnostico de mamiferos silvestres dos arredores de Belim, Para. Rev. Inst. Med. Trop. Sa^oPaulo, 6:225232. 7. Deane, L. M. & Grirnaldi, G., Jr. 1985. Leishmaniasis in Brazil. In: Chang, K.-P. & Bray, R. S. (ed.), Leishmaniasis, Human Parasitic Diseases. I. Elsevier Science Publishers, Amsterdam, pp. 247-28 1. 8. Deane, M. P. 1969. On the life cycle of Trypanosomes of the lewisi group and their relationships to other mammalian trypanosomes. Rev. Inst. Med. Trop. Srio Paulo, 11:34-43. 9. 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, 81:131-1 32. 10. Deane, M. P. & Jansen, A. M. 1986b. Besides Trypanosoma (Schizotrypanum)cruzi, T. (Megatrypanum)freitasi is found in the anal glands of opossums. Speculations on the significance of these findings. Mem. Inst. Oswaldo Cruz, 81(Suppl.):53. 1 1. 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, 79513-5 15. 12. Deane, M. P., Lenzi, H. L. & Jansen, A. M. 1986. Double development cycle of Trypanosoma cruzi in the opossum. Parasitology Today, 2:144-147.
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13. Hoare, C. A. 1972. The trypanosomes of mammals. A zoological monograph. Blackwell Scient. Publ., pp. 740. 14. Jansen, A. M., Moriearty, P. L., Galvlo-Castro, B. & Deane, M. P. 1985. Trypanosoma cruzi in the opossum Didelphis marsupialis: an indirect fluorescent antibody test for the diagnosis and follow-up of natural and experiemntal infections. Trans. R. SOC.of Trop. Med. and Hyg., 79:47447 I . 15. Matthews, D. M., Kingston, N., Maki, L. R. & Nelms, G. 1979. Trypanosoma theileri, 1902, in Wyoming Cattle. Am. J. Vet. Res., 40: 623-629. 16. Mohamed, H. A,, Molyneux, D. H. & Wallbanks, K. R. 1987. On Trypanosoma (Megatrypanum)talpae from Talpa europaea; method ofdivision and evidence of Haemogamasinae as vectors. J. Parasitol., 73: 1050-1052. 17. Molyneux, D. H. 1986. Evolution of Trypanosomatidae. Con-
siderations on polyphyletic origins of mammalian parasites. IMEEE (MontpeNier):231-240. 18. Rego, S. F. M., Magalhles, A. E. A. & Siqueira, A. F. 1975. Um novo trypanosomo d o gamba, Trypanosoma freitasi n. sp. Rev. Bras. Malariol. DoenGus Trop., 9:277-284. 19. Sherlock, I. A., Miranda, J. C., Sadigurski, M. & Grimaldi, G., Jr. 1984. Natural infection of the opossum Didelphis albiventris (Marsupialia, Didelphidae) with Leishmania donovani in Brazil. Mem. Inst. Oswaldo Cruz, 79:5 1 1. 20. Silva, E. 0. R., Pattoli, D. B. G. & Camargo, J. C. 1976. Novo encontro d o Trypanosoma (Megatrypanum)freitasi, parasita do gamba. Rev. Saride Publ. S. Paulo, 10:121-124.
Received 4-28-89; accepted 9-6-89
J . Protozool., 37(1), 1990, pp. 47-49 0 1990 by the Society of Protozoologists
PZasmodium fakiparum: Increased and Multiple Invasion During Short Periods of Time I
JEAN PAUL VERNOT' and MOISES WASSERMAN'.L Grupo de Bioquhica, Instituto Nacional de Salud, Bogotri. Colombia and Facultad de Ciencias. Universidad Nacional, Bogotb. Colombia
ABSTRACT. We describe how to obtain an increased merozoite invasion of Plasmodium fakiparum into human erythrocytes during short periods of time. Using this procedure, infected erythrocytes show multiple invasions ( 2 4 merozoites per erythrocyte), amplifying,
several times, the effects of parasite entry into host cells. The procedure yields synchronous cultures (2-h age range) with parasitemia as high as 15%. It is possible to reach parasitemia of 50% or higher allowing for a 6-h invasion period. Key words. Plasmodium fakiparum invasion. s nmiocnii
HE establishment of a system for long term cultivation of Plasmodium,falciparum erythrocytic stages ,is a major advance towards the development ofantimalarial drugs, malaria vaccines and the elucidation of molecular mechanisms involved in the growth and the multiplication of the parasite. However, in this culture system, growth is always asynchronous and parasitemia rarely reaches values higher than 15%. This fact limits the usefulness of the culture; modifications of the erythrocyte due to invasion are barely detectable because of the large number of uninfected cells masking any variation. One of us reported  40-50% parasitemias after a 5-h invasion period. In those experiments, mature parasites were concentrated using Plasmagel  and left to culture with fresh erythrocytes. The experiments could be reproduced neither with a knobless variant of the FCR3-Gambia strain nor with the FCB 1 -Colombia strain, both concentrated by isopynic Percoll centrifugation. In these cases, during the time of invasion, parasitemia was two to three times lower than that reached with the knobby variant (this report and Vernot unpubl. data). Here we present an easy procedure to attain high invasion rates in short periods of time. The procedure is used by us to study the modifications induced by merozoite entry, in the erythrocyte membrane proteins. MATERIALS AND METHODS Cultures. The FCBI strain from Colombia was maintained in continuous culture according to the method of Trager & Jensen ,in human O(+) erythrocytes at a hematocrit of 5%. The culture medium was RPMl 1640 supplemented with 25 m M Hepes, 32 m M NaHCO3, 50 mg/L gentamycin, 0.2 mM
lb P A R A S I T A E W I I O F
Fig. 1. Mature parasites were concentrated by 65% Percoll centrifugation; A parasitemia of 90% was obtained. Schizonts were diluted with fresh erythrocytes in culture medium and invasion was stopped after 3 h by sorbitol treatment. (-----) Parasitemia of schizonts was changed while hematocrit was maintained constant at I%. (-) Parasitemia was set up at 10% and hematocrit was vaned between 0.5 and 2.5%.