Am. J. Trop. Med. Hyg., 32(6), 1983, pp.

1251-1259
Copyright @ 1983 by The American Society of Tropical Medicine and Hygiene

VERTEBRATE HOSTS AND VECTORS OF TRYPANOSOMA

RANGELI IN THE AMAZON BASIN OF BRAZIL
M. A. MILES,*]. R. ARlAS,t S. A. S. VALENTE,:!: R. D. NAIFF,t
A. A. de SOUZA,:!:M. M. POVOA,:!:]. A. N. LIMA,:!:AND R. A. CEDILLOS§
*WeUcome Parasitology Unit, Section 01 Parasitology, Instituto Evandro Chagasda Fundação
SESP, Caixa Postal 3, 66.000 Belém, Pará, Brazil, tDivision 01 Medical Sciences,Instituto
Nacional de Pesquisasde Amazônia, 69.000 Manaus, Amazonas, Brazil, :tSeçãode Parasitologia,
Instituto Evandro Chagas da Fundação SESP, 66.000 Belém, Pará, Brazil, and §Pan American
Health Organization, Washington, D.C. 20037

Abstract. A total of 46 Trypanosoma rangeli stocks were isolated from naturally infected
mammals and triatomine vectors. Twenty-two stocks were from the common opossum (Di-
delphis marsupialis), one from the brown "4-eyed" opossum (Metachirus nudicaudatus), one
from the anteater (Tamandua tetradactyla), one from the coati (Nasua nasua), seven from
Rhodnius pictipes and 14 from Rhodnius robustus. Two stocks were also isolated from
recently fed sandflies (Lutzomyia sp., Shannoni group). The stocks were identified as T.
rangeli on the basis of natural or experimental salivary gland infections in Rhodnius, inoc-
ulative (anterior station) transmission to mice, morphological parameters in parasitemic mice
and compari sons of isozyme profiles with a known stock of T. rangeli isolated from mano
Three other trypanosome stocks from D. marsupialis, T. tetradactyla and the three-toed sloth
(Bradypus tridactylus) were morphologically similar to T. rangeli in culture but had quite
different isozyme profiles and were not identified. It is concluded that T. rangeli is widely
distributéd in Amazonas,Pará and Rondonia States of Brazil, and probably extendsinto other
regions where R. pictipes and R. robustus are known to occur. R. pictipes is light-attracted
into houses and occasionally transmits Chagas' diseaseto mano It is likely that T. rangeli is
also occasionally transmitted to man in the Amazon basin.

Trypamosoma rangeli was first described in
1920, by Tejera, on the basis of flagellates
morphologically distinct from those of Trypano-
soma cruzi that were seen in the teces of the tri-
atomine bug Rhodnius prolixus, captured in houses
in Venezuela. In 1936, De Leon found the organ-
ism in man in Guatemala, although it may have
been seen much earlier in Peru under the name
T. escomeli.l T. rangeli infection is pathogenic to
both naturally and experimentally infected vec-
tors, yet human infections are apparently quite
harmless.2,3Transmission is by inoculation of in-
fected saliva during bug-feeding; nevertheless,the
organism (subgenus Herpetosoma) is considered
as belonging to the section Stercoraria in which
transmission is usually contaminative.4 T. rangeli
is widely distributed in Central and northern South
America and is often sympatric with T. cruzi, and
serological cross-reactions between sera from hu-
man T. cruzi and T. rangeli infections are said to
confuse epidemiological studies. The interesting
history and biology of T. rangeli have been re-
viewed in detail by both Hoare4 and D' Alessan-
dro.1
J)' Alessandro acceptsrecords of T. rangeli from
triatomine bugs and mammals in the Americas as
being biologlca1lyproven only when substantiated
by salivary gland infections or by transmissionex-
periments.1Many T. rangeli-like organisms which
may undergo somedevelopmentin triatomine bugs
have been reported, and several are na:rnedas dis-
tinct species,although their relationship to T. ran-
geli is unclear.1,4By D' Alessandro'scriteria p~pv-
en natural vectors of T. rangeli are R. prolixus,
R. paUescens,R. ecuadoriensis, R. pictipes, R.
robustus, Triatoma dimidiata capitata, and pos-
sibly R. brethesi.1.5 Proven naturally infected
mammal hosts are Didelphis marsupialis (com-
mon opossum),Philander opossum(Grey, "4-eyed"
opossum),Tamanduatetradactyla (anteater),Cavia
proceUus(guinea pig), Oryzomysconcolor (rice rat),
Eira barbara (tayra), Procyon lotor (raccoon),and
the primares, Cebusspp. (capuchin), Saimiri spp.

Accepted 26 May 1983.

. (squirrel monkey), and Saguinus (=Leontocebus)
geoffroyi (tamarin).l The known distribution of:1
l'1251
,
 1252 MILES ET AL
proven T. rangeli encompassesColombia, Costa
Rica, EI Salvador, Guatemala, Honduras, Pana-
ma, Peru, and Venezuela. The presenceof T. ran-
geli in Brazil, Chile, French Guiana, Paraguay
and Uruguay, which is based only on T. rangeli-
like organisms in triatomine feces or mammal
blood, is controversial.5 In Brazil, flagellates mor-
phologically similar to T. rangeli have been seen
in the intestinal tract of one Panstrongylus megis-
tus used in human xenodiagnosis,one P. megistus
captured in a house,and in the hemolymph of one
fourth instar sylvatic R. domesticus.6-8In exten-
sive studies of the blood protozoa of Brazilian
mammals, Deane and his collaborators have, on
several occasions, noted T. rangeli-like trypano-
somes in the Amazon Basin. Reported hosts are
D. marsuPialis,9 P. (=Metachirops) opossuml0
and Saimiri sciureus;ll in addition, T. diasi
and T. saimiri, considered by both Hoare and
D'Alessandro to be T. rangeli-like, were found
in Cebus and Saimiri, respectively.1.4.12,13None
of these organisms invaded the salivary glands of
experimentally infected R. prolixus. A se~rch by
D'Alessandro and Prado for T. rangeli in Brazil
and Argentina was unsuccessful,5although this
was based only on the examination of domestic
Triatoma infestans and the results of human xe-
nodiagnosis. The same authors concluded that alI
records of T. rangeli in Brazil are equivocal, and
they encouraged further studies.
In this paper we show that T. rangeli is indeed
presentin Brazil, that R. pictipes and R. robustus
are natural vectors widely distributed in the Bra-
zilian Amazon basin, and that D. marsupialis,
Metachirus nudicaudatus (brown, "4-eyed" opos-
sum), T. tetradactyla, and Nasua nasua (coati)are
naturally infected mammal hosts. The methods
used were the discovery of salivary gland infec-
tions in naturally infected triatomine bugs, mor-
phological comparisons, and transmission exper-
iments, supported by comparisons of isozyme
profiles with those of T. rangeli isolated from mano
The epidemiological implications of the presence
of T. rangeli in Brazil are considered.
MATERIALS AND METHODS
Studyareas
Field areas are listed in Table 1 and their geo-
graphicallocations are shown in Figure 1. These
gires were selected initially because they were of
interest for researchon the mammal reservoirsand
F
-;j..
"\
(
" ."
FIGURE1. Collection sites oí mammals, triatomine
bugs, and sandflies that were shown to be naturally in-
íected with Trypanosoma rangeli, 1, Didelphis marsu-
pialisj 2, Metachirus nudicaudatusj3, Tamandua tetra-
dactylaj 4, Nasua nasuaj 5, Rhodnius pictipesj 6,
Rhodnius robustusj 7, Lutzomyia sp. Compare with 10-
calities listed in Table 1.
sandfly vectors of cutaneous leishmaniasis,14,15 in
parallel with which studies of T. cruzi zymodemes16
and the search for Trypanosoma rangeli were
planned. The localities included, in Amazonas
State, the Instituto Nacional de Pesquisas de
Amazonia (INPA) campus and two growing sub-
urbs, Cidade Nova and Parque das Laranjeiras,
of the city of Manaus, the Ducke Forest Reserve
near to Manaus and km 65 on the BR319 highway
(Manaus-Porto Velho). In Rondonia State collec-
tions were made at various points between km 20
and 142 on the BR364 highway (Porto Velho-
Cuiaba). The vegetation, at alI sites, was a com-
bination of disturbed primary forest and second-
~ry growth, cleared of most large trees, as has
been described elsewhere.14, 15,17Palm trees were
abundant in the Manaus suburbs and in R(>ndon-
ia, and this encouraged investigations as palms
are known to be habitats of triatomine bugs of the
genus Rhodnius.17,18
Collection and examination 0/ mammals
Mammals were captured in collapsible,Iive traps
as previously described.19The common opossum,
D. marsupialis, was also caught in oil-drum opos-
sum traps.14A few animaIs were acquired from
hunters or encountered fortuitously whilst work-
ing in the forest. Fresh bIood fiIms and Giemsa-
stained thin fiIms were made from alI mammals
and thoroughly examined microscopically for the
 HOSTS AND VECTORS OF TRYPANOSOMA RANGEU IN BRAZIL 1253

TABLE 1
I solation histories of48* Trypanosoma rangeli stocks examined

Host speciesDidelphis Localityt

marsuPialis I AMAZONAS
(common opossum) Manaus Cidade Nova IM449(A)
Manaus Parque das Laranjeiras (*)IM273(A); IM3l0(A); (*)IM5l0(A)
Ducke Forest ReserveAMOlO km 26 IM68(A)
BR3l9 km 65 (*)IM568(B)
RONDONIA
BR364 km 20-65 IM88(A); IM92(A); (*)IMlOl(A);
(*)IM122(A); (*)IM123(A); IMl44(B);
IMl46(B); (*)IMl58(A); (*)IMl70(A);
(*)IM1284(B,C); IM1296(B);
(*)IM1298(B); IMl3l6(B)
km 111 (*)IMl389(B)
Estrada Belnfunte km 11 (*)IMlO6(A)
M etachirus nudicaudatus RONDONIA (*)IMl3l4(B)
(brown, "4-eyed" opossum) BR364 km 54
Tamandua tetradactyla RONDONIA
(anteater) BR364 km 92 IM1269(B)
Nasua nasua RONDONIA
(coati) BR364 km 92 1M 1249(A)
Rhodnius pictipes AMAZONAS
Manaus Parque das Laranjeiras BUG 1333/2 INT(B)j (*)BUG 1333/9
INT(B); BUG 1333/9GL(B); BUG
1814INT(B); BUG 1821 INT(B)j
BUG 1823INT(B)
RONDONIA
BR364 km '23 (*)BUG 861 INT(B)
robustus AMAZONAS
Manaus Parque das Laranjeiras *)BUG 1752 INT(B)j BUG 1752
GL(B)j BUG 1791INT(B); BUG
1798 INT(B)j BUG 1798 GL(B)j
BUG 1801 GL(B); BUG 1946
INT(B); BUG 1946 GL(B)j BUG
1949GL(B); BUG 1958 INT(B)j
BUG 1958 GL(B)j BUG 1959
GL(B)j CX 503(C); CX 507(C)
Lutzomyia sp. RONDONIA
(Shannoni group) BR364 km 20 IMI086 INT(B); IMI087 INT(B)
,..
* Stocksderived by different routes from lhe salDe bost are considered separately"
t Soe Figure 1.
* (*) indicates stocks of T rangeli tbat were mixed with T. c",zi. ,.
§ (A), stocksisolated by culture of cardia.: blood from lhe natural vertebrate host on biphasic blood-agar medium;2' (B), stocksobtained by culture of
naturally infected triatomine bug fetos, salivary glands, or fetos of xenodiagnosisbugs; in addition, two stocks isolated by culture of sandfly intestinal
tra.:tz. INT = culture of intestinal tra.:t or fetos; GL ~ culture of salivary glands; (C), stocksobtained by transmissionto mito during feeding of naturally
infected bugs or by intraperitoneal inoculation of infected xenodiagnosisbug fetos, and subsequentlycultured from infected mouse cardia.: blood.
" Prevalence of T. rangeli infection in D. marsupialis was 10-30% in localities listed.

presence of trypanosomes. Trypanosomes were usewas restricted as susceptibility to salivary gland

isolated and grown from mammal hosts using the
three p~oceduresof blood culture, xenodiagnosis,
and mouse inoculation. Fifth instar R. prolixus
or, initially, Dipetalogaster maximus were em-
ployed for xenodiagnoses. Although D. maximus
readily acquired intestinal T. rangeli infections its
infection was uncertain.20The methods used for
individual T. rangeli stocks are indicated in Table
1 by A, B, and C, A refers to stocks isolated by
the direct culture of cardiac blood from the nat-
ural vertebrate host on biphasic, blood-agar me-
dium;21 B refers to stocks isolated by xenodiag-

Rhodnius
 MILES ET AL.
nosis and grown in vitro by culture of
xenodiagnosis bug teces or salivary glands using
gentamycin and 5'-fluorocytosine to prevent con-
tamination;22C refers to stocks isolated by xeno-
diagnosis, passagedinto mice by intraperitoneal
inoculation of xenodiagnosisbug teces,and grown
in vitro by the culture of mouse cardiac blood.
Collection and examination of triatomine bugs
The majority of triatomine bugs were collected
by the systematic dissection of palm trees18-the
"inajá" (Maximiliana regia) palm in the vicinity
of Manaus and the "babaçu" palm (Orbignya spe-
ciosa) in Rondonia.17A smalI number of adultR.
pictipes were captured in light-traps like those de-
scribed by Whitlaw and Chaniotis.23The intesti-
nal tracts and salivary glands of samples of R.
pictiPes and R. roliustus from eachfield colIection
were dissectedand examined by phase microscopy
for T. rangeli and/or T. cruzi. Hemolymph was
not routinely examined. Almost alI isolares of T.
rangeli from naturalIy infected R. pictipes and R.
robustus were made by the direct culture of in-
fected bug feces or salivary glands (B in Table 1).
Occasionally, T. rangeli stocks were obtained by
feeding bugs on mice or by intraperitoneal inoc- Experimentaltransmission
ulation of infected fecesinto mice and subsequent
culture of mouse cardiac blood (C in Table 1).
Two T. rangeli stocks were also isolated by direct perimentally, through the entire life cycle, by
culture of the intestinal tracts of recently fed Lut- membrane feeding laboratory-reared Rhodnius
zomyia sp. (Shannoni group) sandflies.
M ouse infections and morphological comparisons described in detail in a later reporto
Mouse infections were established either by al-
lowing naturally infected triatomines to feed on
mice, by intraperitoneal inoculation of organisms
from cultures past peak growth into mice, or by
intraperitoneal inoculation of naturally infected or
xenodiagnosisbug teces. Laboratory-bred 4- to 5-
week-old mice were used throughout. Fresh blood
films and Giemsa-stained thin films were exam-
ined periodically from days 4-30. Trypanosomes
were photographed, measured and comparative
índices were calculated according to Hoare.4
Enzymeelectrophoresis
Bulk growth of T. rangeli stocks was in large
tubes of blood-agar medium.21 Harvesting of or-
ganisms, preparation of enzyme extracts and en-
zyme electrophoresis were as used for the char-
251
ESULTS
acterization of T. cruzi stocks,21and included the
routine preparation and exarnination of Giemsa-
stained films of each population of organisms that
was harvested. The 11 enzymesused in this study
were: malate dehydrogenase (E.C. 1.1.1.37,
MDH); malate dehydrogenase (oxaloacetate de-
carboxylating) (NADP+) (E.C. 1.1.1.40, ME); iso-
citrate dehydrogenase (NADP+) (E.C. 1.1.1.42,
ICD); phosphogluconate dehydrogenase (decar-
boxylating) (E.C. 1.1.1.44, 6PGDH); glucose-6-
phosphatedehydrogenase(E.C. 1.1.1.49, G6PD);
aspartateaminotransferase(E.C. 2.6.1.1, ASAT);
alanine arninotransferase (E.C. 2.6.1.2, ALAT);
phosphoglucomutase(E.C. 2.7.5.1, PGM); ami-
nopeptidase (cytosol)(E.C. 3.4.11.1; PEP); acon-
itate hydratase (E.C. 4.2.1.3, ACON), and glu-
cosephosphateisomerase(E.C. 5.3.1.9, GPI). The
isozyme profiles of T. rangeli stocks were com-
pared visually with those of T. rangeli stock R1625
isolated from a patient in EI Salvador by culture
of venous blood on Seneckjie'smedium, and kind-
ly provided by Dr M. Sauerbrey(CARS/CDC). T.
cruzi stocksrepresentingT. cruzi zymodemeswere
algoincluded in electrophoresisto detect mixed T.
rangeli/T. cruzi infections.
Trypanosomarangeli stocks were passagedex-
spp. on cultures and subsequently refeeding on
uninfected, juvenile mice. These studies will be
A total of 46 trypanosome stocks identified as
T. rangeli were isolated from naturally rnfected
mammals or triatomine bugs (Table 1). Twenty-
two stocks were from D. marsupialis, one from
M. nudicaudatus, one from T. tetradactyla, one
from N. nasua, seven from R. pictipes and 14
from R. robustus. In addition, two stocks were
isolated from sandflies, which contained blood
meals of an unknown mammal host. The stocks
from triatomine bugs included 10isolated directly
or indirectly from salivary glands (Table 1).
Trypanosomes were very rarely seen in fresh
films of naturally infected mammals and Deverin
sufficient numbers to allow satisfactory morpho-
logical studies in Giemsa-stainedthin films of par-
asitemic blood. AlI stocks grew rapidly in the bi-
 HOSTS AND VECTORS OF TRYPANOSOMA RANGEU IN BRAZn. 1255

TABLE 2
Morphological comparisonsbetweenBrazilian T. rangeli stocks,T. rangeli isolatedfrom man, and published records

No.
mea- . Parameterst
Stock code sured* L PK KN F NI KI

IM144 (D. marsupialis) 20 28.3-36.7 2.7-4.3 7.0-11.7 7.0-12.0 7.0-11.0 1.0-1.8 1.3-1.5
(32.7) (3.4) (9.4) (10.4) (9.6) (1.3) (1.4)
BUG 1768GL:I: (R. robustus) 27 27.7-35.3 2.7-4.3 8.0-10.7 6.7-12.3 7.3-13.0 1.0-2.0 1.3-1.5
(31.7) (3.5) (9.6) (9.2) (9.3) (1.5) (1.4)
BUG 1798GL (R. Tobustus) 25 27.0-37.0 1.7-5.7 7.0-12.7 6.6-11.7 5.0-10.3 0.9-2.2 1.2-1.5
(29.1) (3.3) (9.2) (9.1) (7.5) (1.4) (1.4)
BUG 1801 GL (R. robustus) 10 25.7-33.3 2.7-4.0 7.0-12.0 5.0-10.6 7.3-12.7 1.à-2.7 1.2-1.5
(30.8) (3.2) (9.9) (7.9) (9.7) (1.8) (1.3)
R1625 (man) 23 28.0-36.0 2.7-4.0 9.3-12.7 6.3-11.7 7.0-10.7 1.1-2.2 1.2-1.4
(32.1) (3.3) (10.7) (8.8) (9.4) (1.6) (1.3)

Various§ (27.0-32.2)
25.0-37.0 1.8-7.0
---(7.9-9.5)8.2-10.0 (1.2-1.7) (1.6-2.0)

Variousll 25.0-37.0 1.8-7.0 8.2-10.0 5.0-12.0 5.0-11.0 -1.1-2.8

(26.4-33.8) (3.4-4.4) (9.5-9.7) (6.9-8.9) (8.1-9.5) -(1.6-2.0)
.Trypomastigotes in Giemsa-stained tmn tilms of parasitemic mouseblood.
t Mler Hoare.' L, totallength; PK, distance from posterior to kinetoplast; KN, distance from kinetoplast to nucleus; NA, distance from nucleus to
anterior; F, length of free flaKOllum;NI, nuclear index (PN/NA); KI, kinetoplast index (PN/KN). Rangesgiven with means in parentheses.
* But 1768 GL, isolated from R. robustus salivary glands (Manaus, Parque das Laranjeiras) is not listed in Table 1 as it was not characterized
biochemically.
§ From Hoare.'
II From O'Alessandro. '

phasic blood agar culture medium and their
morphology, as assessedby phase microscopy and
examination of Giemsa-stained thin films, was
eitherthat of T. rangeli or T. rangeli/T. cruzi mix-
tures. Similar morphological forms were seen in
naturally infected triatomine bugs or xenodiag-
nosis bugs. A total of 16 stocks were shown to be
T. rangeli/T. cruzi mixtures on the basis of mor-
pholQgy and/or the presenceof superimposed iso-
zyme profiles (Table 1). Infections of 14 stocks
were established in mice by inoculation of cultures
past peak growth; they were: three from D. mar-
supialis, two from R. pictipes, eight from R. ro-
bustus and the T. rangeli standard stock R1625.
The morphology of trypomastigotes in infected
mice was characteristic of T. rangelij occasionally
T. cruzi was algOnoted in mice inoculated with
mixed T. rangeli/T. cruzi stocks. Trypomastigotes
of four stocks were measured in Giemsa-stained
thin films of parasitemic mouse blood and their
dimensions were compared with those of the stan-
dard R1625 as shown in Table 2.
The isozymeprofiles of the newly isolated stocks
corresponded with that of the T. rangeli standard
R1625 (Fig. 2) except for slight variation in the
position of the principal, fast, ALA T band or mi-
nor mobility differences in the main PEP com-
ponents, similar to those reported for T. cruzi.21
Mixed T. rangelilT. cruzi infections were easily
recognized by the presence of two superimposed
characteristic patterns within a single enzyme ex-
tract. The intensity of either the T. rangeli or T.
cruzi pattern in thesemixed stocks was frequently
weak, and somemixtures in which one population
ovêrgrew the other may have been missed.
Several of the Brazilian T. rangeli stocks were
transmitted experimentally through the entire life
cycle using laboratory-bred triatomine bugs and
mice. In alI aspectsbehavior and morphology were
consistent with those of T. rangeli. These results
will be described in detail elsewhere. '
The geographical distribution of the original
hosts of the T. rangeli isolaresis shown in Figúre
1. D. marsuPialis was commonly infected in the
vicinity of Manaus, and both R. pictipes and R.
robustus were vectors. In Rondonia StateD. mar-
supialis, M. nudicaudatus, T. tetradactyla andN.
nasua were infected, as was R. pictipes. One of
38 R. pictipes and 12 of 61 R. robustus examined
from the Parque das Laranjeiras had natural sal-
ivary gland infections. Morphological forms in the
glands were as described for T. rangelij1.4 some
infections were massive, with glands packed with
metatrypanosomes. No salivary gland infections
1256 MILES ET AL.

FIGURE2. Examples of isozyrneprofiles. The enzyrnesshown are: (a) ME; (b) ASATj (c) PGM; .and (d) GPI.
In each casethe trypanosomestocks are (from left to right): I, R 1625-Trypanosoma rangeli from man, EI Salvador;
2, IM92-T. rangeli from Didelphis marsupialis, Rondonia State, Brazilj 3, Bug 333/9 GL-T. rangeli from Rhod-
nius PictiPes, Manaus, Amazonas State, Brazil; 4, Bug 1752 GL-T. rangeli from Rhodnius robustus, Manaus,
Amazonas State, Brazil; 5, IMI087-T. rangeli from Lutzomyia sp. (Shannoni group), Rondonia State, Brazilj 6,
IM418-unidentified, from Bradypus tridactylus, Manaus, Amazonas State, Brazilj 7, 1M 1269-T. rangeli from
Tamandua tetradactyla, Rondonia State, Brazil. Scale: the application threads at the origin measureapproximately
1 cm.

were seenin bugs from Rondonia State but glands DISCUSSION

of only 18 R. robustus have been dissected.*
In addition to the T. rangeU stocks given in
Table 1, three other stocks, morphologically sim-
ilar to T. rangeli in culture, were isolated from D.
marsupialis (IM1013, t BR364, km 49), T. tetra-
dactyla (1M311, BR364, km 92)and Bradypus tri-
dactylus (IM41.8, 3-toed sloth; Manaus, Parque
das Laranjeiras). The isozyme profiles of these
stocks were quite different from those of the 48
T. rangeli stocks listed in Table 1 (Fig. 2): they
were separated from the T. rangeli stocks by the
mobilities of alI but two (MDH, ME) of the en-
zymes examined. We have yet to perform mor-
phological studies in mice and transmission ex-
periments to determine whether this organism
conforms to the criteria adopted for identifying T.
rangeli or, as we suspect, represents another try-
panosomespecies.
* Massive salivary gland infections were algOseenin
one of 14R. pictiPes from Mosqueiro Island, Pará State,
and one R. PictiPes collected from the city of Belém..
Pará State (the latter infection found fortuitously during
electron rnicroscopy24),but neither of these stocks was
isolated for morphological or biochemical characteriza-
tion.
t INPA Manaus stock code numbers.
The presence of T. rangeli in Brazil has long
been suspected from records of morphologically
similar trypanosomes in the intestinal tract of
triatomine bugs and in the blood of Didelphis
marsupialis and the grey "4-eyed" opossum (Phi-
lander opossum).1.4Conclusive evidencehas been
lacking becausesalivary gland infections have not
been seen in naturally or experimentally infected
bugs,I. 4Dor have intrinsic characters, such as iso-
zyme profiles, beenused to compare Brazilian iso-
lates with biologically-proven T. rangeli from
elsewhere.We have found natural salivary gland
infections in both R. pictipes and R. robustus,
and demonstrated inoculative (anterior station)
transmission to mice. The morphological param-
eters of trypanosomesfrom parasitemic mice were
very similar to those reported for T. rangeli (Table
2). The isozyme profiles of stocks isolated from
the mammal and vector specieslisted in Table 1
were, with the exception ofminor ASAT and PEP
differences,indistinguishable from the profile of a
standard T. rangeli stock isolated from man in El
Salvador. We consider that these results, which
fulfill the criteria of D' Alessandro, are irrefutable
evidence that T. rangeli is indeed present in Bra-
zil.
 HOSTS AND VECTORS OF TRYPANOSOMA RANGELI IN BRAZIL
Trypanosoma rangeli infections of R. pictipes
and R. robustus were recently discovered in Ven-
ezuela.19Although this was not the impetus for
the present study, in retrospectthese observations
in Venezuelasuggestthat T. rangeli was likely to
be found in the same triatomine species in the
Brazilian Amazon. The known geographical dis-
tribution of R. pictipes and R. robustus is, for R.
pictipes: Bolivia, Brazil (Amazonas, Goiás, MatO
Grosso, Pará and Rondonia States), Colombia,
Ecuador, French Guiana, Guyana, Peru, Suri-
nam, Trinidad and Venezuela, and for R. robus-
tus: Bolivia, Brazil (Amazonas, Pará and Ron-
donia States),Colombia, Ecuador, French Guiana,
Peru and Venezuela.17,26,27It seems predictable,
therefore, that T. rangeli will be found in other
arfas such as Goiás State, Brazil, and Trinidad,
in association with the same vector species, es-
pecially as the most common mammal host of T.
rangeli in our localities (Table 1), the opossumD.
marsupialis, is ubiquitous throughout the ranges
of R. pictipes and R. robustus. Both R. pictipes
and R. robustus are associated with palm tree
habitats.17Collection of bugs from palms, and ex-
amination by either the cultivation of teces fol-
lowed by isozyme characterization of trypanosome
stocks, or by the simple technique described by
Anez,28would rapidly enhance understanding of
the geographical distribution of T. rangeli.
We have found slight isozyme heterogeneity
amongst the T. rangeli stocks examined. Other
authors have reported heterogeneity on the basis
of enzyme electrophoresis29or the variable sus-
ceptibility of triatomine bug speciesto T. rangeli
isolated in different regions.1A complex T. rangeli
epidemiology is implied from the number of T.
rangeli-like species,with controversial taxonomic
status, that have been described.L 4 The ecotopes
of R. pictipes and R. robustus are not fuIly under-
stood,17and there may be differencesbetweentheir
mammal hosts and roles as vectors of T. rangeli.
As far as we are aware, little work has beendone
to assessthe capacity of other Amazonian triato-
mine species,18including species of the genus
Rhodnius such as R. paraensis, to transmit T.
rangeli. The role of primate speciesas potential
hosts of T. rangeli in the Amazon Basin clearly
merits further investigation. T. rangeli-like organ-
isms have been seenin S. sciureus; arboreal tria-
tomine bugs such as Panstrongylus lignarius and
R. pictipes may live in close association with pri-
mates and even form part of their diet.11
The value of isozyme profiles in epidemiological
1257
studies of this kind is emphasized by the clear
distinction (Fig. 2) of three unidentified"stocksfrom
single specimens of D. marsupialis, T. tetradac-
tyla and B. tridactylus, from the T. rangeli stocks
listed in Table 1. The importance of biochemical
characterization in elucidating complex epide-
miologies has been clearly demonstrated previ-
ously with Leishmania and T. cruzi. 30Monoclonal
antibodies31and genetic probes32provide alter-
native techniques directly applicable to such epi-
demiological investigations.
Trypanosomarangeli and T. cruzi are sympatric
in many regions; in some localities T. rangeli in-
fections in man may be much more common than
T. cruzi infections. I. 33In natu,rally infected pri-
mate speciesT. rangeli has beenfound to be more
prevalent than T. cruzi. 34.35This difference prob-
ably reflects the ease of transmission by the in-
oculative rather than the contaminative route. Al-
though T. rangeli is not pathogenic to man,
serological cross reaction may hinder epidemio-
logical surveys and individual diagnosis of T. cru-
zi infection.1 A differential diagnostic test is not
available but has become more feasible with the
advent of species-specific monoclonal antibod-
ies.36Chagas'diseaseis rare in the Amazon basin
and only sporadic autochthonous caseshave been
reported.37R. pictiPes, light-attracted into houses
from infested palm trees, has been implicated as
a source of such infections.17The fact that R. pic-
tipes and R. robustus are vectors of T. rangeli in
the Brazilian Amazon basin implies, especially in
view of the inoculative transmission route, that
occasional human T. rangeli infections in the re-
gion are to be expected.
ACKNOWLEDGMENTS
We thank F. S. Gomes,R. M. Almeida, R. N.
L. Santos,J. Vidal and R. A. Freitas for assis-
tance in the field; C. R. Sena and D. S. Lima of
the Instituto Nacional de Pesquisasda Amazpnia
(CNPq) for technial assistance, and Colo Jorge
Teixeira, Govemor of the State of Rondonia, for
accessto study areasand logistical support. MAM
is a Wellcome Trust Senior Lecturer and we are
grateful to the following for financial support:
the Wellcome Trust (London); the UNDP/World
Bank/WHO Special programme for researchand
training in tropical diseases;CNPq Grandes En-
demias Grant No. 222.8.087/80 (Brazil)j Funda-
ção SESP(Brazil); Instituto Nacional de Pesquisas
da Amazônia CNPq (Brazil); and the Federal
Govemment of the State of Rondonia (Brazil).
1258 MILES ET AL.
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