ECO 92: Vrotozooíogy InTíkAmazm. Endotrypanum^ a unique intraerythrocytic of New World tree sloths. An evolutionary link or an evolutionary backwater?

JEFFREY J. SHAW

Wellcome Parasítology Unit, Instituto Evandro Chagas, Fundação Nacional de Saúde, Belém, PA 66017-970, Brasil

Endotrypanum is principally a parasite of forest dwelling two-toed sloths, Choloepus species, and apart from being the first trypanosomatid to be named in the neotropics it is also the only haemoflagellate that has an intraerythrocytic phase. In culture it grows as a promastigote and in phlebotomine sandflies it has a peripylarian type development, similar to of the sub- genus Vianna. This review covers the geographical distribution, morphology, ultrastructure, trans- mission, life cycle, vector specificity, antigenic and molecular characterization, taxonomy and phylogeny of Endotrypanum. Attention is drawn to similarities between certain epitopes of Endotrypanum and some rodent Leishmania and it is also suggested that the former may represent a Miocene parasite group. Besides the importance of developing simple methods to distinguish Endotrypanum and Leishmania for epidemiologícal purposes, Endotrypanum^s unique characters also make it a usefui model for both field and laboratory studies on different aspects of trypanosomatid biology especially those related to evolution. Endotrypanum é principalmente um parasita das preguiças reais da flo•taxonomia e filogenia. As similaridades entre alguns epitopos de resta, além de ser o primeiro tripanosomatídeo a ser descrito noEndotrypanum Novo e algumas Leishmania de roedores são salientadas e Mundo e o único hemoflagelado encontrado em células vermelhas. Cres•sugerem também que Endotrypanum representa um grupo de parasitas ce em cultura na forma promastigota e, em flebotomineos, seu desen• da época Mioceno. Além da importância do desenvolvimento de métodos volvimento é do tipo peripilaria como leishmanias do subgênero Vianna. simples para diferenciar entre Endotrypanum e Leishmania em pes• Nesta revisão os seguintes tópicos sobre Endotrypanum são tratados: quisas epidemiológicas, o Endotrypanum é também um bom modelo para distribuição geográfica, moifologia, ultraestrutura, transmissão, cicloestudos de campo e de laboratório sobre vários aspectos da biologia de evolutivo, especificidade vetorial, caracterização antigênica e molecular. tripanosomatídeos, especialmente os relacionados à evolução.

ithout doubt Endotrypanum is one of the most was described a year later by Chagas (2) and Leishmania extraordinary in the world. It belongs braziliensis was named by Vianna in 1911 (3). to the family Trypanosomatidae that includes The literature on Endotrypanum is extremely limited W parasites, such as trypanosomes and leishmania, compared to that of T. cruzi and Leishmania, but this is which produce disease in both man and domestic animais perfectly understandable because of the public health im• in the tropical regions of the New and Old World. Few peo- portance of the latter two parasites. However, the finding ple realize, however, that Endotrypanum was the first that sloths are important reservoirs of Leishmania (4,5,6), member of the family to be described in the neotropics. It and that Endotiypanum also infects sandflies (7), that are was named by Mesnil and Brimont in 1908 (1), the same also the vectors of the former parasite, has led to an in- year that Chagas discovered the first cases of Chagas's dis• creased interest in Endotrypanum during recent years. ease amongst people living along the track of the Central Brazilian railway in Minas Gerais State. Ttypanosoma cruzi Geographical distribution and epízootiology

Correspondence to: Jeffrey J. Shaw, Wellcome Parasítology Unit, Instituto The first records of Endotiypanum were in two-toed sloths Evandro Chagas, Fundação Nacional de Saúde, Caixa Postal 3, Belém, PA (Fig. 1), Choloepus didactylus, from French Guiana (1), 66017-970, Brasil and subsequently it was recorded in the three-toed sloth in

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1953 (8) and the two-toed sloth in 1956 (9) from Costa Rica, and in two-toed sloths from Panamá in 1914 (10) and Brazil in 1925 (11). Sloths of both genera Choloepus and Bradypus, occur in other South American countries, but there is just one record of this parasite from Colômbia (12). This might be considered as surprising, especially as sloths have been examined, and found infected with Leishmania in Colômbia (13) and Ecuador (14). However, in both cases very few animais were examined and Endotrypanum al- most certainly occurs throughout the geographical distribu- tion of two-toed sloths. Habitat preference of any animal plays a key role in determining what infections it is likely to acquire, particu- larly in the case of vector borne parasites. Often important clues in pinpointing the vector of a parasite can be gained by relating incidence of infection to habitats. Both two and three-toed sloths occur in neotropical forests, but they have different habitat preferences, the former being generally commoner in higher forests. Infection rates of Endo• trypanum vary between the two genera of sloths as well as between different geographical áreas, such as Panamá and Costa Rica. This is either a reflection of different habitat preferences of the two genera of sloths or Endotrypanum'^ vector. In Panamá, for example, I found (15) Endo• trypanum in 37/100 two-toed sloths and in 0/39 three-toed sloths, and later workers at the Gorgas Memorial Labora- loepus didactylus. rhe major hosi of tory (4,16) found infections in 49/157 Choloepus and 1/77 in rlie Amazon region. Bradypus. However, if the figures are broken down accord- ing to the ecological background of the sloths, 50% of the two-toed sloths from forested áreas as compared to only Muniz (19) described infections in two-toed sloths from 3% of those from the more open áreas were positive. An Pará State, one of which had been seen in 1936. Infections analysis of the data from Costa Rica (9,10,15,17,18) shows in two-toed sloths have been found in different regions of that 23/63 two-toed sloths were infected with the State, including those to the north of the Amazon river Endotrypanum as were 7/86 three-toed sloths. This infec• and in ali there are records of Endotiypanum in 83/137 two- tion rate in Bradypus is higher than anywhere else in the toed sloths and 1/72 three-toed sloths (5,16,20,21). Américas and may reflect different habitat preferences of The overall picture which emerges throughout most of either the vector or the sloth. Central and South America is that infection rates are signifi- The first record of Endotrypanum (11) in Brazil gave cantly higher in two-toed sloths, reaching almost 50% in ani• no details of the origin of the sloth, but in 1944 Cunha & mais from forested áreas, strongly suggesting that the vector(s) are commoner in this habitat. The size of a piece of forest and the number of sloths that are required to maintain the infection cycle are unknown, but infections have been found in sloths captured in small patches of forest very near . - . . f to the city of Belém located in reserved áreas that belong to the Belém water company. The cutting of the forest for ar- ^ i ^ able and cattle farming has ied to the formation of pockets of d forest over large áreas of Pará State, which may or may not be big enough to support a sufficient number of sloths to maintain Endotrypanum infections. Figure 2. Imraeiylhrocytic epimastigotes of Endotrypanum Morphology, ultrastructure schaudinni (1-13) from Panamanian and virai particles sloths. Choloepus hoffmanni, and The blood forms are morphologically either epimastigotes or trypomastigotes of E. monterogeii trypomastigotes (Figs. 2 and 3) and those seen in sandflies (14-25) from and cultures are typical promastigotes. Early studies on the Costa Rican slotfis morphology of the bloodstream forms of Endotrypanum in 10 u (Ref 15). Panamá (10) and French Guiana (22) showed that they were

108 • Ciência e Cultura (Journal of the Brazilian Association for ttie Advancement of Science) Volume 44(2/3) . Marcti/June 1992 ECO 92: Vrotozootogy InTtieAntazm.

Figure 3. Huemoflagellates found in the "hlood of the two-toed stoth. Choloepus hoffmanni, from Central America: Epimasligoies of Endo• Figure 4. Cultural forms of Endotrypanum schaudinni seen in a pri- trypanum schaudinni tl-3i and young forms of the same parasite (6,7;, mary culture in Tohie's hiphasic médium. Forms seen durini; lhe first 4 nypomasiigotes ofE. monterogeii (3-5) from Costa Rica. trypomastigoles days (1-13), after 10 days (14-17) and after 28 days (22-27). Flagel- of Trypanosnma preguici (9-11) and T. leeuwenlioeki 112.131 and lhe lates from colonies on the surface of lhe agar (28-34) with truncated same speçies (14-17) in the blood of a mouse (Ref. 15). ^ anterior ends (Ref. 15). basically the same, having a short free flagellum and some- subsequently in 1941 by Emanuel Dias (19). Cunha and times a poorly developed undulating membrane. In fresh Muniz examined the cultures of these two isolates of Endo• films extracellular forms were noted and in some cases it trypanum and noted that they were predominantly com- was considered that these forms were larger and that the posed of promastigotes, but they also mentioned finding kinetoplasts migrated posteriorly. This is contrary to devel- small numbers of trypomastigotes, Subsequent workers opment seen in primary culturas in which the initial phase (9,15) confirmed the promastigote nature (Fig. 4) of the of multiplication is as amastigotes and subsequently as culture forms of Endotrypanum strains from Central these become more elongated the kinetoplast is anterior in America, but found no trypomastigotes. In cultures of the typical promastigote position. Differences in the mor- Endotrypanum strains that have been shown to be pure in- phology of the actual bloodstream forms are due to varia- fections, by biochemical and immunological techniques, no tions in the position of the nucleus. In E. schaudinni from trypanosomal forms have so far been found. Attempts to both Central and South America the nucleus is in the poste• infect laboratory animais with culture forms have failed rior half of the body while in E. monterogeii it is in the (10,16,19), including hamsters and on one occasion a labo• anterior half of the body and the kinetoplast is posterior to ratory reared sloth (15). it, thus complying with the definition of the trypomastigote The first electron microscopic studies on Endo• form. A similar nuclear position is associated with trypanum were performed on the blood forms (23) to deter• trypanosomes of the subgenus Megatrypanum that occur in mine the exact relationship of the parasite with the sloth's anteaters and /ew/sZ-like trypanosomes of rodents. erythrocyte. Serial transverse and horizontal sections of in- The first in vitro isolation of Endotrypanum was made fected red cells showed conclusively that Endotrypanum in 1938 at the Instituto Evandro Chagas, which was then lies within the sloth red celFs cytoplasm (Fig. 5). Unfortu- called the Instituto de Patologia Experimental do Norte, in nately this material was not well fixed and so details of the Noller's plates by members of Evandro Chagas's team and ultrastructure were not reported. However it was possible to

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see that there was a free flagellum, flagellar canal, blepharoplast, nucleus and a kinetoplast associated with tu• bular structures that ran both posteriorly and anteriorly. Besides this there were also groups of irregularly shaped electron dense and more regularly shaped electron-translu- cent granules in the posterior tip, which in Giemsa stained preparations appear as darkly staining granules. Ultrastructural studies of Endotrypanum culture forms (12,24,25) have revealed a basic promastigote morphology similar to that of Leishmania promastigotes. The kine• toplast DNA forms a thin band that measures from 0.85- 1.00 X 1.5 |.im and the mitochondrial tubule, which arises from it, is distributed evenly throughout the cytoplasm as are lipid inclusions, glycosomes and membrane bound vesi- cles that contain granular, electron dense material. The di- ameter of these different granules varies from 0.2-0.4 |im, but the glycosomes and lipid inclusions of E. monterogeii were significantly bigger and thus represented a greater from Central America have VLP's than those from the Bra- proportion of the total cytoplasm volume (12). zilian Lower Amazon region. The VLP clusters are also The virus-like particles (VLP's) of Endotrypanum were bigger in Central American strains and small numbers of discovered by accident (26). The Liverpool School of VLP's have now been found in strains from Brazil which Tropical Medicine had received a strain of "L. infantum" were previously thought not to have them (12). Genomic from Italy and while examining the ultrastructure of its virai RNA has also been observed in two isolates of L. promastigotes they detected VLP's in the cytoplasm. On (Vianna) guyanensis, whose principal reservoir is the two- checking the enzyme of this strain they realized that it was toed sloth (28). Sloths are thus unusual in having two Endotrypanum and not L. infantum. This observation stimu- trypanosomatid parasites that are associated with virus or lated them to examine other strains of Endotrypanum, and in viral-like particles. 1980, Croft and co-workers (25) reported VLP's in the cul• ture forms of the type strain of E. monterogeii from Costa Rica and 3 strains of E. schaudinni from Panamá, but none Transmission and vector specificity were seen in two strains from Brazil and one from Panamá. The VLP's occur in clusters (Fig. 6) of varying sizes, are ir• The first clues as to the vector of Endotrypanum carne from regular in shape and do not cause cytopathological changes, epizootiological studies that indicated an association of this as do those seen in Leishmania of the hertigi complex (27). parasite with animais that lived in forested áreas. Labora- Lopes and colleagues (12) found reduced numbers of VLP's tory bred sandflies, sanguinária and Lu. in some strains and drew attention to the possibility that be- trapidoi, fed on infected two-toed sloths developed infec- cause of this they could be missed. The relationship of tions in their hind guts (7,15), similar to those seen in wild VLP's to classification and a small 70 kb chromosome is caught flies (29). This development coupled with the com• discussed later. There seems to be some relationship be- plete lack of any development in a series of other haema- tween the geographical origin of a strain and the presence tophagus insects and the survival of Endotrypanum for 6 and quantity of VLP's. A greater percentage of the strains days in Lu. sanguinária led me to suggest (7) that sandflies were most probably the vector of Endotrypanum. Further observations in Panamá by Christensen & Herrer (30,31,32) confirmed development in these same sandfly species, as well as Lu. gomezi, but in addition it was noted that in paired feedings on different sloths a significantly greater number of Lu. gomezi became infected. They also found that there were variations in the distribution of the parasites within the gut of different individuais and that the greatest number of infec- tions were in the pylorus and hindgut. Later, I studied (33) the development of Brazilian strains of E. schaudinni in three other species of sandflies, Lu. flaviscutellata, Lu. furcata and Lu. longipalpis. Significantly more Lu. flaviscutellata became infected than Lu. longipalpis, but besides this there were also significantly fewer pyloric in- fections and significantly more Malpighian tubule infec- Figure 5. A three dimensional diagram of Endotrypanum schaudinni and tions in Lu. furcata. From experimental studies it is clear a íwo-loed sloíh's red cell prepared from ultrathin .serial sections viewed that there are variations in susceptibility of sandflies at both in lhe eleelronmicro.scope. RCC = red cell cylopla.sm: O = hody offlagel- the species and individual leveis. These facts are important late; N = nucleu.i; K = kinetoplast (Ref. 15).

110» Ciência e Cultura (Journal of the Brazilian Association for ttie Advancement of Science) Volume 44(2/3) • Marcti/June 1992 ECO 92: Vrotozooto^y InTík Amazm. in explaining differences seen amongst infections of wild is not clear which group they belonged to. Recent studies caught sandflies with both the same and different trypano- (11,22) indicate that some isolates from Shannoni group somatids. flies previously identified as Endotrypanum are mono- There is also evidence for vector specificity from field xenous insect flagellates. With the above points in mind observations made in Jari, Pará State, Brazil, where sloths there is in my opinion at the moment no strong evidence to are infected with both Leishmania and Endotrypanum incriminate Shannoni group flies as the vectors of Endo- (5,21). Leishmanial infections were found in 27/59 sloths tiypanum. and of these 25 isolates were made from the liver and So far there is not a single record of a mixed haemo- spleen, 1 from liver, spleen and skin and just one from the flagellate infection in a neotropical sandfly, but this could skin alone. Of these same sloths 33/59 had infections of merely be a reflection of the isolation techniques that have Endotrypanum and of these 16 were mixed leishmanial/ been employed. Of ali neotropical sandflies it would seem endotrypanum infections. In Lu. umbratilis 20/20 flagellate that mixed infections, if they occur, should be in those that infections proved to be Leishmania (5,34,35) and none feed on sloths. Endoti-ypanum. As Endotrypanum is a parasite of the blood Mixed infections of Leishmania and Endotiypanum are and marginally more sloths are infected with this parasite common in sloths and there is thus a possibility of mixed than Leishmania in Jari, one would expect that if a sandfly infections of these parasites in wild-caught sandflies. species is equally susceptible to both parasites then there Sandflies take up small quantities of blood and the density of should be a greater number of infections of Endotiypanum. both parasites is relatively low, which suggests that mixed This, however, was not what we found in Lu. umbratilis infections in sandflies are likely to be uncommon. However, which, from the evidence cited above, would seem to be re- as both Leishmania and Endotrypanum develop as pro• fractory to infection with Endotrypanum. In a single Lu. mastigotes it is difficult to differentiate them morphologi- anduzei, which is not one of the commoner species, we did cally. Christensen & Herrer (15,37) drew attention to this find a very heavy natural infection with flagellates in its problem as well as the possibility of mixed infections in Malpighian tubules, which is typical of Endotiypanum in• sandflies and said that the "posterior station" growth pattems fections. This did not infect hamsters and could well have were very similar in both. They are not identical, however, been Endotiypanum, but unfortunately the parasite was not and Endotrypanum infections in sandflies can be distin- isolated in culture. guished from Leishmania of the subgenus Vianna because Results of k-DNA probe identifications of promastigote the latter do not develop in the Malpighian tubules. infections in sandflies captured in Manaus, Amazonas (36) The above experimental studies on the development of at first sight appear to be very different from ours in Jari, Endotiypanum were ali performed by feeding sandflies on Pará. The Endotrypanum k-DNA probe was not tested infected sloths. Attempts to infect sandflies with Endo• against other non-leishmanial flagellates, so the positive re- tiypanum culture forms have generally failed (33) and of 76 actions cannot definitely be identified as Endotrypanum. laboratory bred sandflies fed on promastigotes none be- Bearing this in mind 22/31 Lu. umbratilis with blood re- came infected, including 36 Lu. longipalpis that fed on acted with the Endotrypanum probe while only 2/37 reacted strain MCHO/BR/80/M6159. More recently, however, Bra• with braziliensis k-DNA probe. Flies with flagellates which zil et al (39), infected 18% of a Lapinha Lu. longipalpis did not have blood, however, did not react with either colony with culture forms of this very same strain and flag• probe (3 with the leishmanial probe and 2 with the ellates were present in the mid and hindgut from 5-10 days Endotrypanum probe)! Similarly promastigotes in 1 Lu. after feeding. Because of the above conflicting results we anduzei and 2 Lu. shannoni, ali of which had blood, reacted recently re-fed our Abaetetuba Lu. longipalpis colony on with the Endotiypanum probe. cultures of the same isolate and after 5 days no flagellates The major difference between our data and that from were found in 34 flies. The reasons for these very different Manaus (34) is that the latter dealt predominantly with in• results are unknown, but it is interesting to note that the fections in flies that had old blood meais. This strongly sug- sandfly colonies originated from very different regions of gests that the Manaus Lu. umbratilis had fed on sloths with Brazil. Ward et al (40), found that Amazonian populations of Endotrypanum infections, but that they subsequently died out Lu. longipalpis were sexually isolated from those of the when the blood meai was digested. This reinforces the hy- Lapinha Cave and Phillips et al (41) observed that the com- pothesis that Lu. umbratilis is not a vector of Endotiypanum position of their tergal spots differed. and that care must be taken in interpreting the results of the Finally then, is Endotiypanum transmitted by the bite of presence of flagellates found in blood meais. sandflies? Both myself (15,33) and Christensen and Herrer Blood meai analysis of Shannoni group flies captured (31) postulated that transmission is by bite and it is hard to in Amazonas State indicated that 28% had fed on sloths imagine transmission per os amongst herbivorous animais (37) and later flagellates from flies of this same group were such as sloths. There is a definite forward migration of identified as belonging to two different Endotrypanum Endotiypanum within the sandflies gut and cardial infec• groups (38). The infections were described as "usually in tions have occasionally been observed. Heavy infections association with a recent or old blood-meal" and were were seen (33) in the cardial region of 2 Lu. furcata that present in the Malpighian tubules, which is characteristic of were experimentally infected on a sloth. This animal was Endotiypanum. No mention is made of pyloric infections negative for Leishmania and the flagellates from these flies and although some were said to be in flies with no blood, it failed to infect hamster. The most plausible hypothesis.

Volume 44(2/3) • March/June 1992 Ciência e Cultura (Journal of the Brazillan Association for tfie Advancement of Science) «111 ECO 92: Frotozooíbgy InTfieAmazm. based on experimental evidence, is that Endotrypanum is specific while others were genus specific. The strain spe- transmitted by the bite of certain species of phlebotomine cific epitopes were located on 4 molecules whose molecu• sandflies. However, since Endotrypanum develops more lar masses were 24, 54, 110 and 160 kDa. In Western blots successfully in some sandfly species than in others, it is the reaction with the 54 kDa band was very strong and likely that in nature only certain species act as vectors al- weak with the other three. Immunofluorescent studies sug- though many may be found to be infected. gest that the 54 kDa epitope is located in surface structures of both the body and flagellum. The genus specific monoclonals reacted only with the body surface and in Developmental cycle in sloths Western blots these antigens were located in a discrete 48 kDa band and a diffuse band that extended from 64 to 200 No dividing forms have ever been detected in sloth tissues, kDa. Attention was drawn to the similarity of the latter however, in a heavy infection of a Panamanian sloth I saw bands with those produced by macroglycolipids. (15) small rounded intracellular forms that resembled We have screened a number of Endotiypanum strains amastigotes (Fig. 3). This led me to postulate that against a panei of leishmanial monoclonal antibodies in a Endotrypanum divides as an amastigote in haemopoetic tis- biotin/avidin immunofluorescent test (45,46). The only sue, such as bone marrow, where it invades the red cell. ones which were positive were LI, a group specific The method of invasion must be very different from that of monoclonal that is known to recognize Leishmania (47), coccidial sporozoites and merozoites which is considered to Endotrypanum (36), monoxenous insect flagellates (48) but be mediated by the secreted products of the apical or- so far not trypanosomes, an L. major/L. trópica mono• ganelles, such as rhoptries (42). Trypanosomes, such as T. clonal, T3 (49) and WIC 79.3 which recognizes L. major (Schizotiypanum) cruzi, have an intracellular phase in non- lipophosphoglycan (LPG) (50). The latter two epitopes phagocytic cells, and although invasion is considered to be have so far not been found in any Leishmania of the by endocytosis the method is unknown. subgenus Vianna nor those of the Mexicana complex, but Recently workers at the Universidade Federal do Rio they do occur in Leishmania of the hertigi complex (4-6). de Janeiro successfully infected mouse peritoneal macro- The surface saccharides of 3 Endotrypanum isolates phages in vitro with promastigotes of three strains of E. were examined (51) with 18 lectins and both quantitative schaudinni from Pará State (43). They noted an increase in and qualitative differences were noted between the strains. the number of amastigotes over 96 hours which adds These preliminary results suggest that lectins can be used to weight to my hypothesis that Endotiypanum multiplies in identify strains with the genus, but it is questionable the form of an amastigote. whether they would be useful at the generic levei. Lastly, could the endoerythrocytic parasites known as Immunological studies of the culture forms of Endo• Endotrypanum be a phase in the life cycle of a trypanosome? tiypanum show that it is an antigenically complex parasite This is a possibility that cannot be completely excluded, es- that has some epitopes that have so far only been found in pecially as trypanosomes are commonly found in the sloths certain Leishmania species. For instance the finding of the that harbor Endotiypanum, but there is presently no evidence T3 and WIC 79.3 epitopes in Endotiypanum is intriguing as at ali to support this hypothesis. it suggests antigenic links between some rodent Leishmania of both the Old and New World. The possible functional importance of strain specific epitopes in the life cycle is Antígenic relationships discussed later in this paper. There is no Information on the sloth's immune reaction Cunha and Muniz (19) compared the agglutination pattems to Endotiypanum infections. Such studies are presently dif- of their two Endotrypanum strains from Pará State using ficult because of mixed leishmanial infections, the lack of antisera prepared against the first isolate, a strain of T. cruzi specific immunoglobulin conjugates and an absence of In• and strains of Leishmania isolated from patients with cuta- formation on the cellular responses of sloths. neous and visceral leishmaniasis and a dog with visceral leishmaniasis. There were cross reactions between the T. cruzi antisera and Endotrypanum promastigotes, but not Biochemistry and taxonomy with the leishmanial antisera. The Endotrypanum antisera reacted strongly with the homologous strain but only weakly In his 1964 revision of the classification of the genus with the other strain, however, after 3 years in culture this Tiypanosoma, Cecil Hoare (52) followed Noller who had same strain reacted strongly. considered Endotiypanum as a subgenus of the genus Using the Noguchi-Adler technique I examined strains Tiypanosoma. However, I felt that Endotiypanum had so of Leishmania from Panamá and Costa Rica and noted that many characters that differed from those of the try• there was a weak cross reaction with the strain of L. (V.) panosomes that in the same year I supported (53) the reten- panamensis, but none with the strain of E. monterogeii tion of Endotiypanum as a valid genus, which was the posi- from Costa Rica. tion of Latin American workers at the time. Hoare later Lopes and McMahon-Pratt (44) produced a series of 26 agreed with this opinion and in this monograph on mamma- monoclonal antibodies against a strain of E. schaudinni lian trypanosomes, that was published in 1972 (54), he also from Pará State. Of these, they found that some were strain considered Endotiypanum as a genus. Up to the time of

112. Ciência e Cultura {Journal of the Brazilian Association for the Advancement of Science) Volume 44(2/3) • March/June 1992 ECO 92: VrcftozooCogy InTík Amazon. writing the only other major taxonomic decision since the similar within the groups, except for the E. monterogeii original description was the naming of Endotrypanum strain that had a small 70-kb chromosome. The intriguing monterogeii in 1969 (15). question now is whether or not the 70-kb chromosome is The first isoenzyme studies of Endotrypanum were contained in the viral-like particles. Group A contained those of Croft and co-workers (25) who noted that the strains in which Croft's team had failed to detect VLP's electrophoretic mobilities of the enzymes malate de- (25), Group B coincided with their group in which they had hydrogenase (MDH), isocitrate dehydrogenase (ICDH) and found VLP's and Group C was a group apart whose strains malic enzyme (ME) were different for the strains that had had not been previously examined. Isolates from Brazil oc- VLP's from those that did not. Using 14 enzymes we found curred in groups A and C while those from Central (55) olear differences between the strains from Central and America werew present in Groups A and B. South America. Regional variations were less marked and Cloned sequences of the small 70-kb chromosome hy- amongst 16 stocks from Pará we detected 8 different bridized strongly with medium-sized chromosomes of the zymodemes that were defined on variations of a smaller group B strains, to which E. monterogeii belongs, but number of enZymes. The position of the mannosephosphate which do not have the small chromosome. Weaker hybridi• isomerase (MPI) bands were reminiscent of variants of a zation was noted with Group A's médium sized chromo• monomeric enzyme with 3 alleles. Attention has also been re- some and none occurred with the Group C isolates. cently drawn (56) to the fact that Endotiypanum aconitate The degree of sequence divergence between the hydratase (ACON) migrates anodically whereas that of n-DNA of the 3 Endotiypanum groups is on the same levei Leishmania of the subgenus Vianna, that also have a as seen between Leishmania which are considered to be peripylarian development in sandflies, does not. The migratory separate species, such as L. (L.) trópica and L. (L.) major nature of Endotrypanum ACON is therefore like that of (63) . Based on this, Lopes et al (11) felt that isolates desig- Leishmania of the subgenus Leishmania, monoxenous nated as Endotiypanum schaudinni might be a species com• trypanosomatids and trypanosomes which are also anodic. plex. They felt, however, that before any taxonomic revi- The buoyant density of Endotrypanum nuclear DNA sion was undertaken the biological characters of the groups (n-DNA) is 1.712 g/mL and that of the kinetoplast DNA (k- should be investigated in greater depth, as molecular diver• DNA) is either 1.693 or 1.697 g/mL (25). The n-DNA gence is not always coupled with organismal divergence. value is close to those of T. dionisii and Crithidia oncopelti More detailed examinations are needed of isolates from and the 1.693 g/mL k-DNA buoyant density is similar to intermediary sloth populations and new isolates are needed, leishmania of the braziliensis complex, while the other is especially from Central America. For instance, the type closer to those of T. congolense, Herpetomonas and strain of E. monterogeii, MCHO/CR/62/A9, was isolated in Blastocrithidia (57). The greater k-DNA buoyant density 1962 and has been passaged so many times that one won- was associated with strains that had VLP's. A reflexion of ders if it bears any similarity to the original isolate. Such similarity between n-DNA sequences of Endotiypanum and doubts can only be investigated and controUed by examin- Leishmania is indicated by the fact that a simply prepared ing new isolates that are cryopreserved immediately after probe from total nuclear L. donovani DNA did not un- isolation. Some authorities consider that there are three spe• equivocally differentiate between parasites of the two gen- cies of three-toed sloths and two species of two-toed sloths era (58). Reactions were, however, weaker with Endo• (64) , but chromosomal data suggests that there are races of tiypanum and this same probe did not react with trypano- Choloepus which could be grouped into four species (65). It somal culture forms. is against the background of host and vector variation that The Liverpool workers (25) concluded that the seven we will now have to consider Endotrypanum speciation. strains of Endotiypanum they had examined fell into two taxonomic units based on their k-DNA buoyant densities, enzyme electrophoretic mobilities and the presence or ab- Phylogeny and evolutionary sence of VLP's. The most extensive and sophisticated stud• importance of Endotrypanum ies of the n-DNA of Endotrypanum are those of Angela Lopes and her colleagues (59) who examined the karyotype Sloths are considered to have evolved from the basic by pulsed field gel electrophoresis (PFGE) and Southern Xenathran armadillo-like stock some 60-70 million years blots of the products of 6 different endorestriction enzymes. ago during the Palaeocene period. The separation between The results of the hybridization of the restriction frag- the two and three-toed sloths occurred later during the ments with 3 gene loci probes showed that 17 isolates pre- Miocene period and they are considered to be living relics of viously identified as Endotiypanum fell into 4 groupswhich the two Miocene sloth groups (66,67). Does Endotrypanum they called A, B, C and D. Group D was so different that it represent a Miocene haemoflagellate group? It is debatable was considered that strains belonging to this group were as to which sloth line Endotiypanum evolved in, but the most probably in fact not Endotiypanum. A view that was higher incidence in two-toed sloths suggests to me that they later substantiated in ultrastructural studies (12) which are the primary vertebrate host and that it probably evolved showed that the k-DNA was structurally more similar to in them, spilling over into three-toed sloths in certain habi• that of monoxenous insect flagellates. As with Leishmania tats where both commonly occur. The geographical distri- (60,61,62) they noted considerable chromosomal size varia- bution of three-toed sloths is greater than that of two-toed tion between strains, however, the overall karyotypes were sloths, but so far Endotrypanum has not been recorded in

Volume 44(2/3) • March/June 1992 Ciência e Cultura (Journal of the Brazilian Association for tfie Advancement of Science) «113 ECO 92: Vrotozooíogy InTfkAmazan. the former outside the ranges of the latter. the immune response so that they are not eliminated too Based on the morphology of the different stages of quickly. This can be accomplished in various ways such as Endotiypanum I suggested (53) that this genus may have becoming unavailable to white cells or by producing evolved from a leishmanial-hke ancestral stock and at the antigenic variants. Living in an erythrocyte is a good way same time said that T. cruzi may have had similar origins. of evading the hosts immune system and at the same time Molyneux (68) misinterpreted my hypothesis by saying that being available to haematophagous insects. A situation that I had said that "Endotiypanum was an ancestral form and malarial parasites have so successfully explored. the link between Leishmania and Schizotrypanum". He Endotiypanum may well have adopted this same mecha- complicated the issue even further by saying that this hy• nism, but what happens when the erythrocyte dies? Either pothesis was untenable as Schizotiypanum occurred in bats the endotrypanums invade another red cell, are eliminated around the world. Godfrey (63) pointed out, however, that or start another internai multiplication cycle. However, the zymodemes of Schizotiypanum species of bats bear no whatever their fate is they make themselves available to the resemblance to those of typical South American T. (S.) vector presumably for the lifetime of the red cell, which for cruzi. He concluded that we must contemplate the idea that sloths is unknown, but in man is on the average about 115 they are not closely related and that their morphology is a days! result of convergent evolution. However, Molyneux is cor- There are fascinating similarities between sloth specific rect in pointing out that the presence of an amastigote viruses and Endotiypanum that may be related to the para• phase does not necessarily imply ancestral affinity with the sites survival. The strain specific epitopes revealed by genus Leishmania. The rounded amastigote of phylo- monoclonal antibodies of Endotiypanum are dominant on genetically different trypanosomatids is most likely a con• the surface of the body and flagellum (44) and thus prob- vergent evolution due to the physiological pressure of the ably provoke initial immune responses. Subsequent infec• intracellular habitat. My argument was not, however, based tions with strains that have antigenically different surface on the presence of amastigotes, but on the culture forms epitopes would therefore stand a better chance of evading being promastigotes and the development in insects. I still any immune reaction and strain specific antigens could feel that Endotiypanum may well have evolved from a therefore be an important method of survival. Another ad- primitive leishmanial stock which may or may not have vantage of the presence of different parasite populations in been a parasite of macrophages. the same host is that there would be a greater opportunity Lopes et al (59), prepared molecular evolutionary trees for genetic exchange which, by some workers, has been from their endorestriction fragment hybridization results considered as a form of sexual reproduction. In respect to and concluded that Endotiypanum originated in South the latter, enzymic evidence for genetic exchange has been America. The spread to Central America depended on the observed for MPI (55). This method of reproduction would formation of the land bridge which occurred about 3 mil- favour karyotypic conservation that could be an advantage lion years ago (69). Using this as a yardstick they calcu- for a specialized parasite, such as Endotiypanum, whose lated that the sequence divergence between their groups A host is a slowly evolving mammal with a relatively long life and B was in the order of 0.056-0.56% per million years span and a restricted range. It is interesting that the which is comparable to estimates of rates in other taxa (70). karyotype of L. (V.) panamensis, whose major host is also They also felt that the disjunctive distribution of the 70-kb sloths, shows only a small degree karyotypic variation be• DNA reflects an event that occurred after the group C line tween strains of the same consensus karyotype, but more and the line that gave rise to groups A and B separated than one consensus karyotype occurs within a restricted from the ancestral stock, and that it might represent hori• area (72). zontal gene transfer. Since the group C parasites are the oldest, I feel that they could well represent a Miocene para• site line. Besides being hosts of different haemoflagellates, Conclusions sloths are also hosts of 8 viruses that are not specific for The above review shows that Endotiypanum shares many sloths and others that are specific for sloths. In discussing characters with other trypanosomatids, particularly those of the latter, Charles Seymour (71) drew attention to the fact the genus Leishmania. Its restricted geographical distribution that sloths are long lived and have a slow population tumo- is related to its major host, the two-toed sloth, which is a liv• ver. This situation favours virus populations that are capa- ing member of a group of mammals that have always been ble of phenotypic variations which will aid their survival. restricted to the American continent, where they were part of Genetic reassortment which would promote this is favoured the dominant mammal group of the Miocene period. Such in mixed infections which are likely to occur in sloths. The facts suggest that Endotiypanum may be a useful model for sloth specific viruses are antigenically complex and their studies on the evolution of trypanosomatids. Similarly, complement fixation antigens are identical, but their neu- studies on the host/vector specificity of Endotiypanum may tralization-test antigens, associated with surface antigens also help us to understand this presently little understood and functional immunity, are variable. aspect of trypanosomatid biology. Similarly the survival of haemoflagellates, especially Biogeographical data suggests that Endotiypanum is an highly specific ones, such as Endotiypanum, in long lived evolutionary cu! de sac, but since its vector is in close con- populations with a slow tumover is dependant on evading tact with a whole range of more recent mammals, one won-

114» Ciência e Cultura (Journal of the Brazilian Association for tfie Advancement of Science) Volume 44(2/3) • Maroh/June 1992 ECO 92: Vratozoo[ogy InTfieAmazon. ders if some mutant form might not, at some point in time, 15. Shaw JJ 1969 The haemoflagellates of sloths. Lon Seh Hyg Trop become adapted to the intraerythrocytic habitat of some Med, Memoir 13, HK Lewis & Co Ltd, London other mammal group. The fact that both neotropical mon- 16. Deane LM 1961 Tripanosómideos de mamíferos da região Amazônica. I. Alguns flagelados encontrados no sangue de keys and sloths have been together in the same environ- mamíferos silvestres do Estado do Pará. Rev Inst Med Trop S Paulo ment since the Miocene period (64), however, suggests that 3: 15-28 Endotijpanum may be too far down the evolutionary path- 17. Zeledon R, C Ponce, J Murillo 1979 Leishmania herreri sp.n. íiom way of specialization to become adapted to another type of sloths and sandflies of Costa Rica. / Parasit 65: 273-279 18. Zeledon R, C Ponce, E de Ponce 1975 The isolation of Lrá/íman/a host. The leishmanial parasites of sloths, on the other hand, hraziliensis from sloths in Costa Rica. Amer J Trop Med Hyg 24: infect a wide range of South American mammals, and also 706-707 man. Is this because there is a greater similarity between 19. Cunha AM, J Muniz 1944 Pesquisas sobre o Endotrypanum the white cells than the red cells of the different mamma- sehaudinni Mesnil e Brimont, 1908, parasita do Choloepus didaetylus (L.). Mem Inst Oswaldo Cruz 41: 179-193 lian groups or are we dealing with less specialized parasites 20. Deane LM, RG Damasceno 1961 Tripanosómideos de mamíferos de that can adapt more easily to different hosts? In considering região Amazônica. II. Tripanosomas de macacos da Zona do the intraerythrocytic habit one also asks why are there no Salgado, Estado do Pará. Rev Inst Med Trop S Paulo 3: 61-70 malarial parasites in sloths, especially as two species occur 21. Shaw JJ, R Lainson 1985 (Unpublished observations quoted in ref. 73) in monkeys living in the same habitat? Such tantalizing 22. Labemadie VGP, A Hubac 1923 Sw VEndotrypanum sehaudinni de questions cannot presently be answered, but the reasons are rUnau edenté de la Guyane (Choloepus didactxlus). C R Séane Soe most probably linked to chance mutations and vector/host Biol 88: 664-666 contacts. Undoubtedly, future studies on Endotrypanum 23. Shaw JJ, RG Bird 1969 The endoerythrocytic habitat of a member of the Trypanosomatidae, Endotrypanum sehaudinni, Mesnil and with the ever increasing array of molecular biological tools Brimont, 190%. Ztschr Tropenmed Parasit 20: 144-150 will begin to answer some of these questions and may even 24. Jadin JM, J Creemers 1969 L'ultrastructure des formes de culture help US understand the nature of the evolution of neotropical á'Endotiypanum sehaudinni, Mesnil and Brimont, 1908. Protis- trypanosomatid. Where possible such studies should, how• tologiea 5: 383-386 25. Croft SL, ML Chance, P Gardener 1980 Ultrastructure and biochemi- ever, be performed on both the standards and new isolates cal characterization of stocks of Endotrypanum. Ann Trop Med from the very same geographical áreas as the standards. • Parasit IA: 585-589 26. Croft SL 1983 (Personal Communication) 27. Molyneux DH 1974 Virus-like particles in Leishmania parasites. Nature 249: 588-589 References and notes 28. Widmer G, AM Comeau, DB Furlong, DF Wirth, JL Patterson 1989 Characterization of a RNA virus from the parasite Leishmania. Proc 1. Mesnil F, E Brimont 1908 Sur un hématozaire nouveau (Endotry• Nat Acad Sei USA 86: 5979-5982 panum n.gen.) d'un édenté de Guyane. C R Hebd Séanc Acad Sei 29. Johnson PT, E McConnell, M Hertig 1963 Natural infections of Paris 65: 581-583 leptomonad flagellates in Panamanian Phlehotomus sandflies. Expl 2. Chagas C 1909 Ueber eine neue Trypanosomiasis des Menschen. Parasit 14: 107-122 Studien tiber die morphologie und Entwicklungszyklus des 30. Christensen H, A Herrer 1976 Neotropical sandflies (Diptera: Schizotrypanum cruzi n.gen., n.sp., Erreger einer neuen Krankheit Psychodidae), invertebrate hosts of Endotrypanum sehaudinni des Menschen. Mem Inst Oswaldo Cru: 1: 159-218 (Kinetoplastidae)./Med£ní 13: 299-303 3. Vianna G 1911 Sobre uma nova espécie de Leishmania (Nota 31. Christensen HA, A Herrer 1977 The use of phlebotomine sandflies in Preliminar). Bras Méd 25: 411 xenodiagnosis. Coll Inter Centre Nat Rec Sei 239: 129-130 4. Herrer A, HA Christensen 1969 Leishmania hraziliensis isolated 32. Christensen HA, A Herrer 1979 Susceptability of sandflies (Diptera: from sloths in Panamá. Science 164: 1419-1420 Psychodidae) to trypanosomatidae from two-toed sloths (Edentata: 5. Lainson R, JJ Shaw, M Póvoa 1981 The importance of edentates Bradypodidae). J Med Ent 16: 424-427 (sloths and anteaters) as primary reservoirs of Leishmania 33. Shaw JJ 1981 The behavior of Endotrypanum sehaudinni (Ki- hraziliensis guyanensis, causative agent of "pian bois" in North Bra- netoplastidae: Trypanosomatidae) in three species of laboratory- zil. Trans Roy Soe Trop Med Hyg 75: 611-612 bred neotropical sandflies (Diptera: Psychodidae) and its influence 6. Gentile B, F Le Pont, FX Pajot, T Besnard 1981 Dermal leish- on the classification of the genus Leishmania. In Parasitologieal maniasis in French Guiana: the sloth (Choloepus didaciylus) as a topies, a presentation volume to PCC Garnham, FRS, on the occa- reservoir host. Trans Roy Soe Trop Med Hyg 75: 612-613 sion ofhis 80th hirthday 1981. EU Canning Ed., Society of Protozo- 7. Shaw JJ 1963 A possible vector of Endotrypanum sehaudinni of the ologists, Special Publication no. 1, p 232-241, AUen Press Inc., sloth, Choloepus hojfmanni, in Panamá. Nature 201: 417-418 Lawrence, Kansas 8. Trejos A, F Montero-Gei 1953 Estúdios sobre tripanosómideos de 34. Lainson R, JJ Shaw, PD Ready, MA Miles, M Póvoa 1981 edentata en Costa Rica. Rer Biol Trop 1: 21-27 Leishmaniasis in Brazil: XVI. Isolation and Identification of 9. Montero-Gei F 1956 Contribución al estúdio de Endotrypanum Leishmania species from sandflies, wild mammals and man in North sehaudinni (Trypanosomatidae). Rer Biol Trop 4: 41-68 Pará State, with particular reference to Leishmania hraziliensis 10. Darling ST 1914 The Endotrypanum of Hoffman's sloth. / Merf guyanensis causative agent of "pian bois". Trans Rox Soe Trop Med 31: 195-204 Hyg 75: 530-536 11. Wenyon CM, HH Scott 1925 Endotrypanum sehaudinni in the two- 35. Lainson R, RD Ward, JJ Shaw 1976 Cutaneous leishmaniasis in toed sloth. Trans Roy Soe Trop Med Hyg 19: 280-281 North Brazil: Lutzomyia anduzei as a major vector. Trans Roy Soe 12. Soares MM, AHCS Lopes, W de Souza 1990 Ultrastructural and Trop Med Hyg 10: 171-172 steriological analysis of trypanosomatids of the genus Endo• 36. Rogers WO, PF Bumheim, DF Wirth 1988 Otltcúon of Leishmania trypanum. Mem Inst Oswaldo Cruz 86: 175-180 within sandflies by kinetoplast DNA hybridization. Amer J Trop Med 13. Loyola EG, A Alzate, A Sanchez, A Gonzales 1988 Epidemiology Hyg 39: 434-439 of a natural focus of Leishmania hraziliensis in the Pacific lowlands 37. Christensen HA, JR Arias, AM Vasquez, RA Freitas 1982 Host of of Colômbia. III. Natural infections in wild mammals. Trans Rov sandfly vectors of Leishmania hraziliensis guyanensis in the Central Soe Trop Med Hyg 82: 406-407 Amazon of Brazil. Amer J Trop Med Hyg 31: 239-242 14. Hashiguchi Y, EA Gomez, VV de Coronel, T Mimori, M Kawabata 38. Arias JR, MA Miles, RD Naiff, MM Póvoa. RA de Freitas, CB 1985 Leishmania isolated from wild mammals caught in endemic Biancardi, EG Castellon 1985 Flagellate infections of Brazilian áreas of leishmaniasis in Ecuador. Trans Roy Soe Trop Med Hyg 79: sandflies (Diptera: Psychodidae): isolation in vitro and biochemical 120-121 identification of Endotrypanum and Leishmania. Amer J Trop Med

Volume 44(2/3) • March/June 1992 Ciência e Cultura (Journal of the Brazilian Assocíation for tfie Advancement of Science) «US .ECO 92: Frotozooíqgtf InTfie Amazm.

Hyg 34: 1096-1108 59. Lopes AHCS, D lovannisci, MP Peixoto, D McMahon-Pratt, SM 39. Brazil RP, AHCS Lopes, BG Brazil. AL Falcão 1991 Experimental Beverley 1990 Evolution of nuclear DNA and the occurrence of infection with the culture forms of Endotrypanum schaudinni sequence related to new small chromosomal DNA's in the (: Trypanosomatidae) in Lurromyia longipalpis trypanosomatid genus Endotrypanum. Mol Biochem Parasit 40: (Diptera: Psychodidae). Mem Inst Oswaldo Cruz 86: 275 151-161 40. Ward RD, AL Ribeiro, PD Ready, A Murtagh 1983 Reproductive 60. Giannini SH, M Schittini, JS Keithly, PW Warburton, CR Cantor, isolation between different forms of Lutzomyia longipalpis (Lutz LHT Van der Ploeg 1986 Karyotype analysis of Leishmania spe- and Neiva), (Diptera: Psychodidae). the vector of Leishmania cies and its use in classification and clinicai diagnosis. Science 232: donovani chagasi Cunha and Chagas and its significance to kala- 762-765 azar distribution in South America. Mem Insi Oswaldo Cruz 78: 61. Samaras N, TW Spithill 1987 Molecular karyotype of five species of 269-280 Leishmania and analysis of gene locations and chromosomal 41. Phillips A, R Ward, L Ryan, DH Molyneux, R Lainson, JJ Shaw rearrangements. Mol Biochem Parasit 25: 279-291 1986 Chemical analysis of compounds extracted from the tergal 62. Bishop RP, F Akinsehinwa 1989 Characterization of Leishmania "spots" oíLutzomyia longipalpis from Brazil. Acta Trópica 43: 271- donovani stocks by genomic DNA heterogeneity and molecular 276 karyotype. Trans Roy Soe Trop Med Hyg 83: 629-634 42. Perkins ME 1992 Rhoptry organelies of apicomplexan parasites. 63. Beverley SM, RB Ismach, D McMahon-Pratt 1987 Evolution of the Parasil Today 8: 28-32 genus Leishmania as revealed by comparisons of nuclear DNA re- 43. Saraiva E, E Lopes, M Vemier, W de Souza 1992 (Personal Com- striction fragment pattems. Proc Nat Acad Sei USA 84: 484-488 munication) 64. Wetzel RM 1985 The Identification and distribution of recent 44. Lopes AHCS, D McMahon-Pratt 1989 Monoclonal antibodies spe- Xenathra (Edentata). In The evolution and ecology of armadillos, cific for members of the genus Endotrypanum. J Protozool 36: 354- sloths and vermilinguas. p 5-21, GG Montgomery Ed., Smithsonian 361 Institution Press, Washington, London 45. Shaw JJ, EAY Ishikawa, R Lainson 1989 A rapid and sensitive 65. Jorge W, AT Orsi-Souza, R Best 1985 The somatic chromosomes of method for the Identification of Leishmania with monoclonal anti• Xenathra. In The evolution and ecology of armadillos, sloths and bodies using fluorscein-labelled avidin. Trans Roy Soe Trop Med vermilinguas, p 121-129, GG Montgomery Ed., Smithsonian Institu• Hyg 83: 783-784 tion Press, Washington, London 46. Shaw JJ, EAY Ishikawa, R Lainson 1992 (Unpublished observations) 66. Webb SD 1985 The interrelationships of the tree sloths and ground 47. McMahon Pratt D, E Bennett, G Grimaldi, CL Jaffe 1985 Subspe- sloths. In The evolution and ecology of armadillos, sloths and cies and species-specific antigens of Leishmania mexicana charac- vermilinguas, p 105-112, GG Montgomery Ed., Smithsonian Institu• terized by monoclonal antibodies. / Immunol 134: 1935-1940 tion Press, Washington, London 48. Dedet JP, F Gay, G Chatenay 1989 Isolation of LWs/7mamo species 67. Sarich VM 1985 Xenathran systematics: albumin immunological from wild mammals in French Guiana. Trans Roy Soe Trop Med evidence. In The evolution and ecology of armadillos, sloths and Hyg 83: 613-615 vermilinguas, p 77-81, GG Montgomery Ed., Smithsonian Institu• 49. Jaffe CL, D McMahon-Pratt 1983 Monoclonal antibodies specific tion Press, Washington, London for Leishmania trópica. I. Characterization of antigens associated 68. Molyneux DH 1986 Evolution of Trypanosomatidae. Considera- with stage- and species-specific determinants. J Immunol 131: tions of polyphyletic origins of mammalian parasites. In Leishmania 1987-1993 taxonomie et phylogenése applications éco-épidémiologiques 50. Handman E, CL Greenblatt, JW Goding 1984 An ampiphatic colloque International, p 231-240, J-A Rioux Ed., (2-6 July 1984), sulphated glyconjugate of Leishmania: Characterization with IMEEE, Montpellier monoclonal antibodies. EMBO J 3: 2301-2306 69. Marshall LG, SD Webb, JJ Sepkoski, DM Raup 1982 Mammalian 51. Lopes AHCS, MJG Esteves, J Augluster, AFB Andrade 1987 Lectin evolution and the great American interchange. Science 215: 1351- binding to surface saccharides on Endotrypanum strains. Mems Inst 1357 Oswaldo Cruz 82: 42 70. Britten RJ 1986 Rates of DNA sequence evolution differ between 52. Hoare CA 1964 Morphological and taxonomic studies on mamma- taxonomic groups. Science 231: 1393-1398 lian Trypanosomes. X. Revision of the systematics. J Protozool 11: 71. Seymour C 1985 Sloths as hosts of arboviruses. In The evolution 200-207 and ecology of armadillos, sloths and vermilinguas, p 269-278, GG 53. Shaw JJ 1966 The relationship of Endotrypanum to other members Montgomery Ed., Smithsonian Institution Press, Washington, Lon• of the Trypanosomatidae and its possible bearing upon the evolu- don tion of certain haemoflagellates of the New World. Proc Ist Int 72. Giannini SH, NG Saravia 1990 Molecular karyotype analysis of Cong Parasit Rome 1: 332-333 Leishmania braziliensis panamensis from the Pacific coast of Co• 54. Hoare CA 1972 The Trypanosomes of mammals. A zoological lômbia. In Parasites: molecular biology, drug and vaccine design, monograph p 60. Blackwell Scientific Publications, Oxford and Ed- p 465-478, N Agabian, A Cerami Eds., Wiley-Liss, New York, USA inburgh 73. Shaw JJ 1985 The hemoflagellates of sloths, vermilinguas (anteat- 55. Shaw JJ, R Lainson, MM Póvoa, MA Miles 1985 (Unpublished ob• ers), and armadillos. In The evolution and ecology of armadillos, servations quoted in ref. 73) sloths and vermilinguas, p 279-292, GG Montgomery Ed., Smith• 56. Shaw JJ, RR Braga, R Lainson, EAY Ishikawa 1991 Aconitate sonian Institution Press, Washington, London hydratase (ACON), an enzyme that distinguishes Leishmania of the 74. Acknowledgments: The author is deeply grateful to the Wellcome subgenus Vianna from other trypanosomatids. Trans Roy Soe Trop Trust, London, and the Instituto Evandro Chagas of the Fundação Med Hyg 85: 597-598 Nacional de Saúde, Brazil, for financial support which not only ena- 57. Newton BA, JK Bumett 1972 DNA of kinetoplastidae: a comparative bled him to study Endotrypanum in the Amazonian region of Brazil, study. In Comparative hiochemistry of parasites, p 185-198, H Van but also during the 60's in Central America. The author would also den Bossche Ed., Academic Press, New York and London like to thank Prof. Ralph Lainson for his unstinting encouragement 58. Greig SR, FA Akinsehinwa, F Ashall, R Lainson, JJ Shaw, MA Miles, and Dra. Angela Lopes for helpful discussions. He is also indebted DC Barker 1989 The feasibility of discrimination between to the London School of Hygiene and Tropical Medicine and to the Leishmania and Endotiypanum using total DNA probes. Trans Roy editor of the Memórias do Instituto Oswaldo Cruz for their permis- Soe Trop Med Hyg 83: 196-197 sion to use previously published illustrations.

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For microfilms, the top should be guese and other subjects of regional in• indicated. If possible, the Figures sub• terest. Contributions in Portuguese will mitted should be the size they will ap• Submission of manuscripts only be published on the last pages of pear when published, so that no reduc- the issue at the discretion of the Editorial tion is necessary. If they must be re- Submit ali manuscripts to: Board. duced, make sure that ali elements, in• Luiz R. Travassos cluding labeling, can withstand reduc- Sénior Editor References and notes — The references tion and remain legible. Electron and Ciência e Cultura, SBPC and notes that appear at the end of con• light micrographs must be direct copies Rua Costa Carvalho, 222 tributions in ali categories should be of the original negative. Indicate magni- São Paulo, SP 05429-000 Brasil numbered according lo their introduction fication with a scale marker on each mi- in the lext; the numbering of the refer• crograph. 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Investigations on..., Contribution Unpublished work should be referred Typing the manuscript — Manuscripts to..., Preliminary results on..., Survey to in the next with a number and listed in (and copies) must be typed on one side of... " and so on. h) Names of authors. References and notes. Acknowledge- of the paper only. Double-space every- In the case of numerous family names, ments should be in incorporated in a thing, including references, Tables and use initials and the first and last names brief text and have a number, usually to legends. Leave side margins of 3 cm. in full. í) Institution address (Labora- be listed at the end of References and Indent the first line of the paragraph 6 tory, Center, Division, Department, Uni- notes. The same number can refer to spaces from the left-hand margin. versity, etc). Indicate also locality (city, more than one citation and more than Number ali pages consecutively and in- State, country) and postal code, ali in one note, technical detail, etc. Refer• clude the author"s name in the right-hand italic or with underlined words. Do not ences and notes can be quite versatile as comer of each page. use abbreviations. Use superscripts it can accomodate several details and re- Altematively, manuscripts can also be etc, to indicate the author's affiliation to marks that would otherwise disturb the submitted in a fioppy disk compatible to an institution. d) Corresponding au- main fiow of the scientific thought of the IBM-PC system. thor. Indicate the complete name and text. address of the corresponding author. Also, a telephone or FAX number for a Table — Tables should be understand- prompt contact. able without references to the text. Over-