Copyright 1973.

Al rights reserved
BIOLOGY OF 7RYPANOSOMA CRUZ! .:. 1621
Zigman Brener
Departamento de Parasitologia, I.C.B., Universidade Federal de Minas Gerais and
Instituto de Endemias Rurais, Belo Horizonte, Brasil
CONTENTS
INTRODUCTION ............................................................... 347
TRYPANOSOMA CRUZI-LIFE CYCLE IN THE VERTEBRATE .................... 348
Fine Structure of T cruzi Blostream Forms 348
The Signifcance of T cruzi Polymorphism ................................. 350
T cruzi-Intracelular Cycle ............................................. 352
Hosts of T cruzi ....................................................... 354
Factors InRuencing the Cycle in the Vertebrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
TRYPANOSOMA CRUZI-LIFE CYCLE IN THE INVERTEBRATE ................. 356
Factor Whih May InRuence Development in the Vector. . . . . . . . . . . . . . . . . . . . . 357
Some Characteristics of T cruzi Infection in the Invertebrate. 359
Development of T cruzi in Abnormal Invertebrate Hosts ..................... 359
Fine Structure of T cruzi Developmental Stages in the Vector . . . . . . . . . . . . . . . . . 360
DEVELOPMENT IN TISSUE CULTURE ........................................... 360
A vian Embryo Infection ................................................. 360
Tissue Culture Infection ................................................. 360
InRuence of Temperature On T cruzi Morphogenesis 361
InRuence of Interern .................................................. 362
Purne Metabolsm in T cruzi Intracellular Forms . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
Biochemistry of T cruzi-Harborng Cels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
Exprimental Chemotherapy ............................................. 362
Fine Structure of Tissue Forms of T cruzi ................... .............. 363
CULTIVATION OF TRYPANOSOMA CRUZI .................................. 364
Nearly Defned Meia: Growth Factor ................................... . 364
Growth and Diff erentiation in Culture Forms .................. ............. 365
Eff ets of Drugs on Growth, Diff erentiation, and Synthesis of Protein and Nucleic
Acds of Culture Forms ... ....................... ............. 366
DNA-Mediate Hetero-transformation of Culture Forms? ....... . .. . .......... 367
Cultivation of Amastigote Stages in Cel-Free Meium .......... ............. 367
Infetvity of T cruzi Culture ........................................... 368
CYTOCHEMISTRY AND METABOLISM .......................................... 369
Nucleic Acids: Role of Kinetoplast-DNA and Dyskinetoplasty in T cruzi ....... 369
Nuclear and Kinetoplast DNA from T cruzi ............................... 370
Nucleic Acid Metabolsm in T cruzi .. .................................... 371
Amino Acds and Proteins ........ ... . . ........ :......................... 372
Lipids ............................ .... . ................ . .............. 372
Carbohydrate Metabolism and Respiration ................................. 373
OTHER ASPECTS ............................................................... 377
Chemotherapy ................................. .. . ..................... 377
T cruzi Congenital Transmission ......................................... 377
Antigenic Constitution .................................................. 377
Vaccnation Against T cruzi ............................................. 378
Eff ets of T cruzi on Spontaneous and Induce Tumor . . . . . . . . . . . . . . . . . . . . . . 378
CONCLUDING REMARKS ...................................................... 378
347
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ANNUAL
REVIEWS
348 BRENER
INTRODUCTION
Trypanosoma cruzi, the cause of Chagas' disease, is an unbeaten adversary. Im
munological or chemotherapeutic protection is not available and it is becoming, like
other higher Trypanosomatidae, a favorite for studies intended to elucidate how
mitochondrial DNA joins in regulating relations between life in the invertebrate
vector and in the mammalian host. Thanks largely to its easy cultivation and
apparently indefnite retention of infectivity for vector and mammalian hosts: it is
much more amenable to experimental manipulation than the salivarian trypano
somes, especially those of the T brucei group. It shares with Leishmania amenabil
ity to analysis of adapt ion to intracellularity. T cruzi under conditions not well
defned, can form in culture infective metacyclic trypomastigotes, a morphogenetic
process far more difcult to efect with salivarian trypanosomes. This joining of
urgent practical and timely biological motives makes T cruzi despite the hazards
attending its use, an ever more popular object even among workers not specifcally
concerned with prophylaxis and therapy.
TRYPANOSOMA CRUZ/-LIFE CYCLE IN THE VERTEBRATE
T cruzi multiplies discontinuously in the vertebrate host; the amastigote intracellu
lar stages multiply by binary division, then change into nondividing trypomastigotes
which emerge from the parasitized tissues into the bloodstream, where they circulate
for a variable period before penetrating cells and resuming the cycle. In the acute
phase, the host harbors abundant tissue and bloodstream forms; in the chronic phase
parasites are scarce and undetectable in blood. The low parasitemia of the chronic
phase results from buildup of strong immunity and a stable balance between host
and parasite. Spontaneous cure seems rare if it happens at all, since blood parasites
have been detected by indirect laboratory methods in a rather high proportion of
experimental and chronic human infections.
Fine Structure of T crzi Bloodstream Forms
The morphology of T cruzi trypomastigotes by light microscopy and its polymor
phism have been much studied (21, 28, 41, 162, 191). More recently Milder (130)
and Maria et al (121) described the fne structure of T cruzi bloodstream forms and
tried to correlate morphological fndings with organelle function.
T cruzi bloodstream forms have, apposed to the outer surface of the trilaminary
cell membrane, a difuse flamentous coat of variable thickness (25-300 A) (121,
130), which apparently facilitates pinocytosis and has also been related to the
occurrence of the successive surface antigens reported in the relapsing bloodstream
populations of T brucei (216). But for T cruzi no convincing evidence of such
antige

s or variation in the course of infection has appeared. Subpellicular mi

crotubules lying ..10 A beneath the surface membrane are observed, except in some
areas of the reservoir pocket. A group of microtubules seems in close relationship
with the basal body and in apparent continuity with the subpellicular microtubules
(130).
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BIOLOGY OF TRYPANOSOMA CRUZI 349
Two typical basal bodies are found in T cruzi bloodstream forms ( 1 30). Since
trypomastigotes do not multiply in the bloodstream, the occurrence of those two
organelles seems not to indicate self-replication anticipating cell division; rather they
are perhaps vestigial structures. A peculiarity unreported for other trypanosome
species is the presence of central flaments at the intra- and extracytoplasmatic
regions of the basal body, crossing the transitional zone between the two transversal
plates. The flagellum has the 9 + 2 stereotypical axoneme structure. A lattice-like
accessory flamentous complex, within the membrane sheath of the fagellum along
side the axial flaments, lies in the segment of the fagellum attached to the surface
membrane. The fagellum is bound to the pellicle by a complex of two trilaminary
membranes and by desmosome-like fbrous condensations. At the contact surface
the subpelJicular microtubules of the fagellum are thickened markedly ( 1 21 , 1 30).
The reservoir around the base of the fagellum has ofen been considered to be
in trypanosomatids a site of secretion and! or excretion as weJl as a site for ingestion
of macromolecules by pinocytosis (1 98); presumably in T cruzi trypomastigotes the
reservoir acts the same, as suggested by the absence of subpellicular microtubules
underlying parts of the reservoir, the dense debris and double-walled vesicles at
tached to the reservoir wall, and the multi vesicular bodies in the surrounding
cytoplasm ( 1 21 , 1 30). So far a cytostome has not been detected in T cruzi blood
stream trypomastigotes. A new tubulo-membranous structure has been described,
formed by apposition of two cell membranes, which arise from an invagination of
the reservoir wall; it displays some characteristics in common with a cytostome,
such as its emergence from the reservoir wall and its cul-de-sac tubular shape ( 1 21 ).
However, the identity of the two structures cannot be defnitely established owing
to some marked diferences: the tubulo-membranous structure shows microtubules
only at its apex, it is lined by two membrane units instead of a single unit cell
membrane, and vesicles do not surround this structure.
A typical Golgi complex lies between kinetoplast and nucleus. Ramifcations of
the granular endoplasmic reticulum are ofen found close to the Golgi, suggesting
direct communication between both structures and a secretory activity of the Golgi
apparatus in the trypomastigotes (1 30). A' Golgi complex is more developed and
presents more vesicles in stout forms than in slender ones ( 1 21). Vickerman (21 4)
suggested that the increase in Golgi complex in T brucei broad forms results from
a local antigen-antibody reaction elicited by the production of exo-antigens. In T
cruzi such an inference cannot yet be made despite a single, so far unconfrmed,
report on release of soluble antigen by bloodstream forms ( 196).
T cruzi kinetoplast morphology has been detailed ( 1 8, 1 35). Its size and shape
may vary during developmental stages, probably caused by diferences in amounts
of DNA and arrangement of DNA loops. In bloodstream trypomastigotes the loops
are ofen arranged in 3 to 4 layers, imparting a basket-like aspect. Close contact
between nucleus and kinetoplast, disintegration of the membranes, and possible
exchange of genetic material between both organelles have been described in T cruzi
culture epimastigotes but never in bloodstream forms ( 1 35). Slender forms have a
rather developed posterior mitochondrial tubule intimately related to the elongate
shape of the parasite's posterior end, whereas stout forms have a short and less
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350 BRENER
developed posterior mitochondrion. A single tubular anterior mitochondrion ex
tending from the kinetoplast to the parasite's anterior end has been observed. The
occurrence of bifurcations, a more sinuous course, outgrowths, and reticulations
suggests a larger mitochondrial area in stout T cruzi forms (121).
Dense granular bodies similar to the baccilliform or oval bodies in T brucei and
T congolense (215) were also described in both slender and stout T cruzi. Those
bacilliform bodies have been suggested to correspond to extramitochondrial perox
isome-like organelles containing the L-a-glycerophosphate oxidase fguring in the
terminal respiration of slender T brucei forms which lack a functional cytochrome
system. Muitivesicular bodies formed by a variable number of vesicles similar to
those found in the Golgi complex, and enveloped by a dense amorphous substance,
have also been reported (121). Those bodies, originated from condensations of
vesicles found in close association with the tubules of the Golgi complex, may
progress to the reservoir and perhaps figure in secretion transport to the exterior.
Other described vesicles (121, 130) resemble those reported by Herbert (94) in
Trypanosoma theiler and are considered to be lysosomes. Cytochemical techniques
have not, however, been used on T cruzi for detecting specifc lysosomal activity.
The occurrence at the fagellar pocket of vesicles close to dense amorphous material
strongly suggests pinocytosis in this region (121, 130).
Maria et al (121) described round, elongated cytoplasmic protrusions bounded by
a membrane similar to the pellicle overlying the fagellate, clearly connected to the
flagellum and fagellar membrane. They seem transient structures, similar to the
filopodia in T brucei (227). Their function is unclear; they have been suggested to
participate in adhesion of the parasites to the substratum, helping them to pass
through capillary walls and to penetrate host cells (121). Filopodia-like structures
in close contact with Kippfer cells have been reported in an in vivo study of T cruzi
in the liver of experimentally infected mice (187). Comparison of slender and stout
forms of T cruzi showed little detectable variations in the shape and size of the
nucleus and development of the rough endoplasmic reticulum and Golgi complex,
in spite of the physiological diferences between these forms (I2l).
Some problems raised by the few reports on T cruzi fine structure may deserve
further investigation: (8) characterization of the bacilliform bodies which bear some
analogy with the L-a-glycerophosphate-containing organelles of T brucei slender
forms (a functional glycerophosphate system in T cruzi bloodstream forms would
be rather puzzling, since it is generally accepted that cytochrome-mediated respira
tion prevails throughout their life cycle); (b) the function of the filopodia and of
conditions favoring development of these transient structures in the bloodstream
forms; ( c) the nature of the tubulo-membranous structure and its relationship to the
reservoir; (d histochemical characterization of the enzyme activity ofthe lysosome
like bodies and other cytoplasmic inclusions; and (e) studies on the possible localiza
tion of exo-antigens on the surface coat of the bloodstream forms by ferritin
conjugated antibody (216).
The Signifcance of T cruzi Polymorphism
Chagas (41) first reported dimorphism in T cruzi bloodstream trypomastigotes: the
occurrence of both slender and broad forms. Many authors have since studied these
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BIOLOGY OF TRYPANOSOMA CRUZ I 351
interesting morphological variations ( 1 28, 1 62, 1 91). Brener & Chiari (28) studied
seven diferent strains of T cruzi and described, besides slender and broad trypo
mastigotes, a very stout form not yet thoroughly investigated. Brener (21), studying
several T cruzi strains in experimentally infected mice, observed that relative
percentages of the diferent forms in the bloodstream showed at least three distribu
tion patterns: in some strains slender forms predominated from onset of infection
to the animal's death; in other strains slender forms predominated during the frst
days of infection but then gradually diminished while the proportion of stout forms
steadily increased; fnally, in two other strains broad forms predominated during the
entire infection. A similar variation had been reported by Silva ( 1 91 ). Some correla
tion was observed between the predominance of each form in the diferent strains
and some characteristics of the experimental infection, such as duration of prepatent
period, course of parasitemia, and mortality rates (21 ). Animals inoculated with
strains showing predominantly stout forms developed a gradually ascending parasi
temia; the animals died with parasites teeming in the blood. Strains usually showing
predominantly slender forms had an early high parasitemia followed by a sharp
decrease in number of parasites and an irregularly low parasitemia thereafer. Those
diferent patterns prevailed afer eight years of maintenance (32).
The signifcance of these diferent forms is controversial. Chagas (41 ) suggested
that slender and broad forms signify sexual dimorphism. For Brumpt (34), however,
slender trypomastigotes represented young parasites which gradually would develop
into broad mature forms. Meyer & Oliveira ( 1 28), working with tissue cultures
infected with T cruzi, reported that the early intracellular trypomastigotes are
broad and short; these forms may either be liberated by cell rupture or instead
change into slender trypomastigotes when allowed to develop longer in more resis
tant host cells. Slender forms are the frst to appear in the course of experimental
infections but are not usually found in the bloodstream of animals displaying ac
quired immunity. But when immunity is depressed, as in cortisone-treated animals,
an early, marked increase of slim forms is observed (1 62). These facts may be
pertinent to the suggestion that slender forms of African trypanosomes are variants
against which antibodies have not yet been produced, whereas the stumpy forms
originate from slender ones by the action of antibodies (9). T cruzi broad forms,
however, are very unlikely to originate from slender trypomastigotes through host
antibodies' morphogenetic action, since both forms are observed in infected tissue
cultures ( 128, 1 91 , 2 1 1). Antibodies might nevertheless act as selective agents elimi
nating the more sensitive slender forms during chronic infection (25).
It is not yet known whether each form can yield descendents morphologically
similar to the parent. The answer can come only from investigations with clonal
popUlations. Inoculation of only broad forms, isolated by micromanipulation, has
induced infections showing slender and broad blood trypomastigotes ( 1 91 ). Efects
of environment upon phenotypic trypomastigote characteristics have not been thor
oughly investigated. Temperature does influenc the relative number of the diferent
forms in tissue culture: at 26C slender forms greatly increase whereas at 37C stout
forms predominate (21 1).
The diferent morphologies of the blood trypomastigotes apparently denote physi
ological diferences. When T cruzi blood trypomastigotes are intravenously inocu-
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352 BRENER
lated in normal mice the slender forms disappear from the bloodstream within a few
hours, then undergo their normal intracellular cycle. Stout forms, in contrast, can
circulate in the bloodstream for some days without penetrating the host's cells;
slender forms apparently penetrate cells better (25). In immune animals, intrave
nously injected slender forms proved extremely sensitive to the host's immune
mechanism: they disappeared from the blood within 1-2 hr afer inoculation,
whereas stout forms circulated for many days (25). In normal rats, strains showing
large numbers of stout forms displayed high and enduring parasitemia while strains
showing predominantly slender forms preented only a slight, transient parasitemia
(26). These facts strongly suggest that slender and stout blood trypomastigotes may
display diferent behavior in relation to the host's innate and acquired immune
responses.
The number of parasites which develop into epimastigotes in the triatomid vector
is apparentiy proportional to the number of broad forms in the ingested blood. Some
evidence suggests diferences in development of the diferent forms in the inverte
brate host (26, 1 91 ). In hrueei-group trypanosomes the slender trypomastigotes do
not develop in the fy vector, owing apparently to lack of an aerobic ATP-generating
system which seems essential for survival in the new environment. Conversely, the
stumpy forms build up a cytochrome electron-transfer system and so can develop
in the invertebrate host ( 14, 214). However valid the analogy, the mitochondrial
enzyme activity and respiratory chains in the morphologically diferent T eruzi
trypomastigotes remain to be studied.
Prolonged maintenance of brucei-group trypanosomes by syringe passage in
animals renders it monomorphic and unable to develop in the vector, probably
because in the slender forms the mitochondrial apparatus degenerates. Mtilhpfordt
(135) suggested that an exchange of genetic material between nucleus and kineto
plast, most likely occurring in the vector's epimastigote stage owing to the proximity
of both organelles, is required to keep kinetoplast DNA able to code for mitochon
drial enzYmes. Long term maintenance in the vertebrate host would prevent the
cyclical transformation into epimastigotes and hence such interchange. Cyclical
development in both vertebrate and invertebrate hosts seems less critical in T eruzL
which retains polymorphism and the capacity to infect reduviids afer at least eight
years passage in laboratory animals (32).
T cruzi-IntraceJuJar Cyce
T cruzi trypomastigotes can enter a wide range of host cells, but muscle and glia
seem most ofen parasitized. How parasites pass through blood capillaries and
penetrate cells is not yet understood. Kollert (106) followed the in vitro active
penetration of metacyclic culture trypomastigotes in HeLa cells and thought it was
afected at the flagellate's posterior end. This region in bloodstream trypomastigotes
has double-walled vacuoles, multivsicular bodies, and free ribosomes ( 1 21), but
studies are lacking on factors determining cell penetration. The possible role of
transient flopodia-like structures in attachment of blood parasites to cells has been
discussed (121, 1 87).
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BIOLOGY OF TRYPANOSOMA CRUZI 353
Afer penetrating the cell, trypomastigotes soon change into amastigotes which
undergo binary fssion about every 12 hr (128, 163). In tissue culture cells, the
number of parasites in the end of a cycle, which usually lasts 4 to 5 days, may vary
from about 50 to 30, depending on cell size (128). Transformation into trypomas
tigotes seems to begin almost simultaneously just when the cell is flled with amas
tigote forms, the whole cycle being always limited to one cell unit (128). Not all
amastigotes seem competent to develop into trypomastigotes; electron microscopy
of T cruzi in the myocardium of inoculated animals showed that' 25% of the
intracellular amastigotes were clearly degenerated (33). The normal amastigotes
inside the cell-induced cytoplasmic lysis; a clear halo ofen surrounded the parasite
(20). The capsule-like structure that sometimes seems to encircle the intracellular
amastigote stages of Leshmania donovani (179) has not been observed in T cruzL
Nevertheless, in tissue culture, cells seem to withstand parasitism without apparent
damage, at least in the early stages, since they retain motility and ofen show normal
mitosis. Parasitized cells, however, eventually die afer rupture and parasite release
(128). Koberle (105) suggested that ruptured pseudocysts liberate a neurotoxin
which may afect nearby ganglion cells.
Intracellular fusiform and round amastigotes have been described in tissue culture
cells (191). In the host the amastigote show irregularly shaped mitochondria with
few cristae, vacuoles, ribosomes, and dense lipid bodies (186). Phagocytosed para
sites show irregularities in membranes and kinetoplast modifcations. The fne struc
ture of the kinetoplast-chondriome of T cruzi in thin sections of infected tissue
culture has been described (127). A cytostome was detected in intracellular stages
of T cruzi infecting HeLa cells (131), an interesting fnding since this structure has
not been found in bloodstream forms (121, 130). Free amastigotes, probably from
ruptured cells in the bloodstreams of heavily infected animals, have been reported
(167).
Intracellular morphogenesis and transformation of amastigote stages into trypo
mastigotes have been studied in infected tissue culture and in peritoneal liquid
released from cellular elements in infected animals (128, 191,225). A double trans
formation of amastigotes into trypomastigote is believed to occur: in the frst the
fusiform amastigotes tend to elongate along the fagellar line of growth, their kineto
plast migrates to the fagellate'S posterior end, and a slender trypomastigote is then
directly formed. In the second process the round amastigotes develop a vacuole near
the kinetoplast; this grows and eventually ruptures, giving origin through an unfold
ing mechanism to broad trypomastigotes. Such a vacuole has also been described
as a rarefaction of the cytoplasm (137) or a V-shaped splitting (225). The unfolding
process 'and the presence of such vacuole have not, however, been detected by
electron microscopy of T cruzi intracellular stages. The presence of typical epimas
tigote stages in the cells of the vertebrate host still demands investigation. According
to Elkeles (59) epimastigotes are ,very scarce in vertebrate host tissues, which
strongly suggests a direct transformation of round forms into trypomastigotes. An
epimastigote-like stage occurring as a preliminary to the slender trypomastigotes
morphogenesis has, however, been described in tissue culture (191).
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354 BRENER
Hosts of T crzi
T cruzi has a wide host range: besides the various wild mammals belonging to
several orders that are infected with cruzi-like parasites, several domestic animals
have been observed to be naturally or experimentally infected with the organism.
T cruzi can infect mice, rats (26), rabbits (3), hamsters (1 52), and guinea pigs. A
typical acute phase followed by chronic progressive myocarditis (62), dilation of
hollow muscular organs ( 1 50), and neuroganglionic lesions (202) may b produced
in infected mice; in this respect the infection resembles the human disease (62). The
usefulness of T cruzi infection of young and adult dogs for studying pathological
and electrocardiographic changes and for chemotherapeutic studies has been re
ported (8, 78). Cebus monkeys survive the experimental acute disease and afer
wards develop typical myocarditis (209), whereas Rhesus monkeys develop a
nonfatal infection with relatively mild tissue lesions ( 1 23).
Amphibians (52) and birds (57) have proved refractory to T crzi. A substance
which lyses T cruzi culture forms but not bloodstream forms has been described
in normal chicken serum (220, 222) but has not been so far clearly related to the
birds' refractoriness. South American lizards ( Tropidurus) could not be infected
with T cruzi (143) whereas North American Gerrhonotus lizards were easily
infected by feeding them infected Tratoma ( 1 70). Many wild animals widespread
on the American continent are infected with flagellates morphologically identical to
T crzi of human origin. Infection with these so-called cruzi-like trypanosomes has
been detected in over 10 mammalian species from several orders (49), collected in
endemic areas and in areas apparently free from human Chagas' disease, e.g. in some
areas of the United States. Many of those strains have been isolated and apparently
display characteristics identical with those of T cruzi strains from humans in
respect to morphology of bloodstream forms, presence of intracellular stages, devel
opment in culture, life cycle in triatomids, and cross-protection tests. They are,
however, ofen less virulent than human strains: some of them seem not at all
infective to laboratory animals. Most of such strains are very likely to be the same
T CTlzi; their systematic position, however, is still under discussion, since their
infectivity for man would have to be assumed (49).
Factors InRuencin
g
the Cyce in the Vertebrate
Infection in the vertebrate host is clearly infuenced by the particular T cruzi strain.
There is increasing evidence that this parasite comprises an extensive pool of diverse
populations. The T cruzi strains referred to in the literature are usually isolates of
known origin, probably including heterogeneous populations pooled by the ongoing
natural passages of the parasite between diferent hosts. Nevertheless, even under
those conditions, the strains ofen display distinguishing traits, and it is probable
that selective factors in the host from which the parasites are isolated may provide
for a certain degree of homogeneity of the population. Thus, diferent patterns of
blood trypomastigote morphology, mortality rate, and course of parasitemia have
been reported in animals experimentally inoculated with diferent T cruzi strains
( 1 5, 2 1, 1 91 , 21 8). Predominantly myotropic (7) or reticulotropic (203) strains have
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BIOLOGY OF TRYPANOSOMA CRUZI 355
been described. Tafuri & Brener (201 ) observed marked diferences i n degree of
celiac ganglion alterations in mice inoculated with diferent strains. Possible correla
tion of pathogenicity with number of inoculated blood trypomastigotes has been
reviewed by Phillips ( 1 58), who showed that two intrinsically diferent kinds of
infection may ensue according to the strains. In the frst case, infections of low
para

itemia and morbidity are obtained merely by decreasing the inoculum; in the
second, the host's death may be delayed but not prevented by progressive reduction
in the number of inoculated parasites. Apparently, some inherent strain characteris
tics such as tropism for specific tissues, localized reproduction, wide dispersal, etc,
may account for this diferent behavior. In spite of strain diferences, strong cross
immunity is the rule among the various strains inoculated in laboratory animals
(23), even when distinct immunological types are used (1 01).
The host's sex (92), age (48), and genetic constitution (163) may infuence the
course of T cruzi infection in the vertebrate. Some authors have described the
infuence of environmental temperature (108, 1 22, 2 1 2) and seasonal fuctuations
( 107) on the course of T crzi infection. The number of blood and tissue parasites
definitely decreased in animals kept at relatively high temperature (35-37C); lower
temperatures ( 1 01 8C) resulted in higher parasitemia and mortality. The increased
mortality of mice infected with T cruzi and treated with chlorpromazine has also
been attributed to the hypothermic action of the drug (71 ). Nutritional deficiencies
(thiamine, pyridoxine, vitamin A, and lysine) may produce a higher parasitemia in
the rat and more severe lesions, whereas riboflavin and pantothenate defciencies had
no infuence (228).
Interferon, induced by double-stranded polyinosinic-polycytidyJic acid, appar
ently exacerbated T cruzi infection in' experimentally infected mice (124). On the
other hand, persistent amounts of interferon, probably released by the increasing
cellular involvement, have been detected in mice (1 83).
Adrenal steroid hormones, especially cortisone, have long been known to enhance
T cruzi acute infections ( 1 78), although apparently unable to afect chronic infec
tions (29). Some chemical and physical agents have recently been used to study the
efects of immunosuppression in experimental Chagas' disease. Cyclophosphamide
regularly increased parasitemia, mortality. and severity of myocarditis in mice in the
acute phase (110). No efect of azathioprine in mice in the chronic phase was
detected (149). A study of the efects of gamma radiation, cyclophosphamide.
azathioprine. and 6-mercaptopurine 0) the course of chronic Chagas' disease was
carried out in mice infected with four diferent strains (3 1). Gamma radiation and
cyclophosphamide induced, only in animals inoculated with the CL strain, an acute
phase characterized by outbreaks of intense parasitemia accompanied by high mor
tality. This peculiar response has been suggested as being caused by the predomi
nance in this strain of stout trypomastigotes, which are far more resistant to the host
immune mechanisms (25). As antibody molecules are radioresistant and unafected
by the chemical immunosupressive agents. the residual antibody concentration
present afer immunosuppression would probably be sufcient to maintain, at sub
patent levels. the parasitemia of the slender predominant trypomastigote strains. but
not that produced by the stout forms of CL strain
.
Azathioprine and 6-mercaptopu-
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356 BRENER
rine did not afect the smoldering chronic disease in all animals. Neonotal thymec
tomy led to delayed appearance of antibodies, higher parasitemia, and shorter
survival of mice inoculated with T. cTuzi, strongly suggesting that thymus-depend
ent immunity plays an important role in the infection (189).
TRYPANOSOMA CRUZ/-LIFE CYCLE IN THE INVERTEBRATE
Basic features of T cruzi development in the invertebrate host (Hemipter, Reduvi
dae) have long been known (34, 41 ). Dias (53) was frst to ofer a comprehensive
account of the development of T cruzi in hematophagous triatomids as a rather
schematic cycle, the ingested bloodstream forms passing along the bug's digestive
tract through irreversible morphological transformations in fxed sequence, each
stage developing in a particular portion of the gut. Thus, in the stomach most blood
trypomastigotes soon change into epimastigotes and a few into round forms; in the
intestine the bulk of the fagellate population consists of actively dividing epimas
tigotes which support the apparently perennial insect infection; fnally, in the rectum
a certain proportion of the epimastigotes diferentiate into infective metacyclic
trypomastigotes which are eliminated in the feces along with epimastigotes.
This description stayed nearly unchanged until
<
uite recently. Then Brack ( 1 8)
reported that in experimentally infected Rhodnius prolxus, apart from epimas
tigote and trypomastigote stages, rounded forms possessing a fagellum (sphaer
omastigotes) very soon appeared in the stomach. A certain proportion of those
round forms may change either into short epimastigotes, which start multiplying in
the intestine, or into long epimastigotes, which are unable to multiply but actively
reach the rectum 3- days afer the infective'meal. A few days later the sphaeromas
tigotes suddenly appear in the rectum where they are detectable before the metacy
clie forms are formed. The long epimastigotes apparently cannot diferentiate, the
sphaeromastigotes being the only stages able to change into metacyclic trypomas
tigotes. Brack assumes that the early presence of long epimastigotes in the rectum
probably led to the conclusion that they are precursors of the infective metacyclic
stages. Opposed to Dias's interpretation, T cruzi would .then, undergo two parallel
cycles in the invertebrate host, the sphaeromastigotes being able to change either
into epimastigotes or into infective trypomastigotes. Short epimastigotes might
develop into round forms which would resume the described cycle and probably
constitute the stock which sustains the long term infection in the vector.
In exami)ing stages of T cruzi in the blood-harboring stomach of T infestans
fed on experimental mice, Brener (27) observed that most bloodstream trypomas
tigotes very soon changed into round or pear-shaped forms ofen seemingly arranged
in closely attached pairs, sometimes connected by cytoplasmic bridges and mem
brane leaks. Besides the apposed pairs, large masses of round, aggregated parasites
were seen 1 20-144 hr afer the infective meal. The next developmental stage con
sisted of a fusion of the parasites, an apparent disorgariization of the DNA-contain
ing organelles, thickening of the borders of the fused parasite masses and apparent
detachment of newly formed parasites. This sequential development in the vector
has been suggested to represent a method of reproduction involving genetic ex-
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BIOLOY OF TRYPANOSOMA CRUZI 357
change, thus reopening the problem of syngamy in T cruzi discussed in some of
Chagas' early papers. A previous attempt to demonstrate syngamy in T cruzi was
reported by Amrein (6). who fed triatomids on cultures of two diferent drug
resistant strains, recovered the fagellates from the feces. and cultivated them. But
further experiments failed to demonstrate resistance of the culture forms to both
drugs. and therefore genetic exchange was not demonstrated. As culture forms may
not be suitable stages for this kind of experiment. syngamy could not be defnitely
excluded. So far drug resistance or other genetic markers have not yet been reported
in T cruzi bloodstream forms.
The fndings reported by Brener (27). on the other hand. somewhat resemble the
phenomenon reported by Adler (2) who found that under the action of specifc
antiserum, T cruzi epimastigote stages change into leishmanoids which agglutinate
and form large multinucleated syncytial masses. The fusion of biological membranes
as described may have more general implications than to a T cruzi cycle. The
mechanism of cell adhesion. cell fusion, and further formation of polykaryocytes
and heterokaryons by diferent agents such as lysolecithin or inactivated Sendai
virus has been reviewed by Poole et al (165). who emphasized the importance of the
study of experimentally induced alterations in the integrity of lipoprotein membrane
which precede cell fusion.
The features in the stomach of triatomids, along with the already known extensive
multiplication of epimastigotes which occurs simultaneously in the intestine, led to
the suggestion that both series of events are either independent phenomena. possibly
occurring as concomitant cycles. or phase of the same cycle. These recent observa
tions underscore the need for critical reexamination of T cruzi development in the
invertebrate host, notably morphogenesis of the early stages in the blood-containing
stomach, the relationship between these forms and the multiplying epimastigotes
found in the midgut, and the origin and morphogenesis of the infective stages.
Factor Which May Infuence Development in the Vector
The host-parasite relationship in T crzi vectors has not yet been satisfactorily
clarified; discussions about conditions which afect ability to develop in the inverte
brate host are necesarily rather speculative. Some factors infuencing the cycle in
the vector are already known and will be detailed here.
Fluctuations in infection rate of diferent triatomid species known to have in
gested large numbers of T cruzi bloodstream forms have been reported quite ofen.
e.g. the same T cTuzi strain infected 90.4% of PanstrngyJus megistus and only
54.7% of Rhodnius prlxus fed on experimentally infected animals (56). A study
of the sensitivity of diferent species of vectors in the xenodiagnosis of human
chronic Chagas' disease showed that nymphs of T infestans became 83.3% positive
as compared with only 18.2% of R. prolxus (4). Apart from this varying capacity
of the diferent vectors in allowing full development of the parasite, some bugs from
susceptible laboratory-bred colonies may be highly refractory to T crzi infection
even after repeated feeding on blood of animals with high parasitemias. Phillips &
Bertram (159) reported that in a group of bugs which did not contract the infection
after the frst intake of bloodstream parasite, only 13% of the specimens were
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358 BRENER
infected after a second infective meal; some of the remaining insects continued
negative even afer three or fve attempts with diferent T cruzi strains. Several
numbers of T infes/ans, a highly susceptible Brazilian vector, which had fed on
experimentally inoculated mice in the acute phase and definitely ingested 1 to 2
million bloodstream trypomastigotes also failed, nevertheless, to become infected
(26).
The geographical origin of the vector and parasite strains has been suggested as
infuencing the cycle. Dias (55) demonstrated that a Venezuelan strain of trypano
somes infected a signifcantly higher percentage of R. prolxus from Venezuela
(56. 8%) than of T infetans and P megistus of Brazilian origin (38. 1 %), whereas
a Brazilian strain was signifcantly more infective for the local bugs. T crzi readily
develops in both adult and larval stages, but with a decrease in susceptibility of R.
prfxus larvae at each successive stage despite the increasing intake of blood and
trypomastigotes (159). As blood digestion in the ffh larval stage i 20 times faster
than in the first instar, Dias (54) suggested that rapid blood digestion may harm the
parasite and so account for the negative results. In insects showing a rapid hemolysis
of the ingested blood, the rate of degeneration and lysis of blood trypomastigotes
was correspondingly higher (54).
Symbiotic microorganisms occur in the gut of T cruzi vectors (38, 77, 1 33).
Mihlpfordt (I 33) studied the influence of one of those symbionts, Nocardia rhodni;
on the development and diferentiation of T cruzi in R. prolxus, showing that the
parasite multiplied more intensely in the Norcard-free ones. However, N rhodni
apparently provides no essenti
a
l factor for diferentiation of epimastigotes into
trypomastigotes, since both T cruzi forms were regularly found in sterile and
Nordia-bearing insects. The role played by Nocardia and many other Gram
positive bacteria in the gut of triatomids (38) remains unelucidated.
The possibility that genetic factors of the vector are involved in the cycle of T
cruzi in the invertebrate host has been invoked by Phillips & Bertram (159). They
reported that the experimental infection rate of the progeny of a group of vectors
which failed to get infected afer ingesting T cruzi was signifcantly lower (57%)
than that obtained for the whole vector population (80. 5%).
Although some authors have detected no signifcant diference in T cruzi strains
in respect to capacity to infect vectors (159), there is some evidence that slender and
broad trypomastigotes behave diferently in the invertebrate host, and that some
diference are likely to occur when popUlations of bloodstream trypomastigotes,
with predominance of either form, are fed to the triatomids. Silva (191) reported that
in the vector's digestive tract most broad forms soon develop into epimastigotes or
directly change into round forms, wheres most slender forms usually keep their
morphology and movements for 4 or 5 days and degenerate only thereafer. A
signifcantly higher percentage (18. 6%) of bugs failed to acquire infection when fed
on mice inoculated with strains presenting predominantly slender forms than when
they had ingested a larger number of stout forms (5.2% of negative insects) (26).
However, the role played by the diferent forms as well as their fate during blood
digestion could not be thoroughly investigated, since unmixed populations could not
b provided to the vectors.
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BIOLOGY OF TRYPANOSOMA CRUZI 359
Some Characterstics of T cruzi Infction in the In vertebrate
The period intervening between infective meal and elimination of the frst infective
metacyc1ic trypomastigotes has usuaJly been considered as strongly infuenced by
the instars undergone by the insect and by temperature. Dias (29) reported that in
P megistus, metacyclic trypomastigotes are detected on the sixth to seventh days
in larval stages and on the tenth to ffeenth days in adult specimens. Similar results
were obtained with Tratoma protracta (226). Phillips (157), however, demon
strated the development of T cruzi in the vector to be directly related to environ
mental temperature whatever the instars. Infectivity determined by inoculation of
voluntarily released feces started for all stages of R. prlixus on the seventh day at
20C, the third day at 25C, and the second day at 3035C. Most data are from
experiments with insects fed on heavily infected hosts and may not be relevant for
vectors with low parasite intakes.
Elimination of metacyclic trypomastigotes is generally assumed to persist during
the vector's entire life. Luz & Borba (119), for instance, demonstrated that T
infestans kept eliminating infective fagellates even 812 days afer infection despite
long starvation periods. On the other hand, however, only 4. 8% of R. prolxus
infected larvae retained their infection afer being reared to the adult stage or
submitted to a seven-week starvation period (159). Infected nymphs of T infetans,
examined on the thirtieth and ninetieth days afer a meal of heavily infected mice,
showed a marked decrease of positivity (92.6% and 25.0% for ffh instar and 64.0%
and 20.0% for third instar, respectively) (199). Notwithstanding these data, the
importance of the vector species in respect to persistence of T cruzi infection has
not been thoroughly investigated. Likewise, the fate of metacyclic trypomastigote
elimination has not yet been investigated nor has their number been estimated,
owing to lack of a reliable method for counting those fagellates, despite a few
attempts to score infective form densities in the feces (159).
T cruzi developmental stages do not reach the salivary glands nor the hemocoele
but may be detected in the Malpighian tubes (53). Evidence of T cruzi transmission
by the bite of reduviids, reported by early authors, has been reviewed by Pipkin (161)
who practically ruled out this possibility. Some attempts to inject blood or culture
forms into the hemocoele resulted in the flagellate'S survival for only a limited period
(51, 206). In spite of some controversial records, intracellular stages are now consid
ered not to occur in T cruzi vectors (53). No harmful efects on the invertebrate
hosts have been recorded from naturally or experimentally infected bugs.
Flagellates may survive in the vector's intestine for as long as nine days after death
(118). Nevertheless, cyst or resistant forms are not observed; the long-resistant T
cruzi cysts described (190) are very likely stages of a Blastocrthidia which infects
triatomids (39).
Development of T cruzi in Abnormal Invertebrate Hosts
T cruzi' normal invertebrate hosts are Triatominae (Reduvidae), all hematopha
gous. However, other Arthropoda have been experimentally infected with the or
ganism [reviewed by Pipkin (161)], including other Hemiptera (Cimicidae: Cimex
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360 BRENER
lectularus and other bedbugs; Lygaeidae: Clerada apiicomis); Diptera (Hippobos
cidae: Melophagus ovinus); Lepidoptera (Pyralidae); Acarina (Ixodidae, including
ticks belonging to Rhipiephalus! Omithodorus, and Amblyiomma). Most of those
arthropods are obviously abnormal hosts unable to support the full parasite cycle.
However, feces of Cimex hemipterus and C letularius fed on experi1entally
infected animals had many metacyclic trypomastigotes which proved infective to
mice (231). Transmission of T cruzi to normal dogs by naturally infected ticks
(Rhipicephalus sanguineus) has also been reported (141). A possible role for nonbit
ing flies Juch as Musca domestica as well as experimentally infected Culex ttigans
has been discussed by Pipkin (161) in his comprehensive review on transmission of
T crzi.
Goldman (81) inoculated pupae of a plant-feeding Lepidoptera (Philosamia
cynthia) with T cruzi culture forms and reported the presence of free parasites in
the body fluid and midgut as well as intracellular stages.
Fine Structure of T cruzi Developmental Stages in the Vector
Sanabria (185), besides studying the ultrastructure of epimastigotes and trypomas
tigotes in R. prolxus, also pointed out the abundance and size of their mitochondria
contrasted with their scarcity in the vertebrate tissue forms. Brack (18) described
the fne structure of sphaeromastigotes, eJimastigotes, and trypomastigotes in R.
prolxus, and presented a detailed study of kinetoplast morphology during the
developmental stages, including the arrangement of the DNA loops in all stages,
types of kinetoplast division, and relationships between this DNA-containing struc
ture and fJrmation of mitochondria.
DEVELOPMENT IN TISSUE CULTURE
The intracellular development of T cruzi in avian embryos and in tissue culture has
provided insight into intracellular morphogenesis, influence of diferent factors on
intracellular growth, metabolism of intracellular stages, biochemistry of infected
cells, and drug activity against extra- and intracellular parasites.
A vian Embryo Infection
Chick embryos have been used by several authors to propagate T cruzi [reviewed
by Pipkin (160)]. Despite a few unsuccessful attempts, avian embryos have proved
susceptible to infection, showing fagellates in the blood (74) and dividing forms in
various organs as well as in the chorioallantoic membrane (46, 74). Blood invasion
seems transitory and is characterized by very low parasitemia (160). Chickens
hatched from inoculated eggs had a transient parasitemia (46).
Tissue Culture Infection
Intracellular T cruzi development was obtained early in plasma clot tissue. cultures
and hanging drop cultures (128, 176) of embryonic and adult cells [reviewed by
Pipkin (160)]. Later, monolayer tissue culture infections were obtained in primary
cultures from trypsinized cell suspensions (22, 142) or cell lines (106, 125, 191).
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BIOLOGY OF TRYPANOSOMA CRUZI 361
Diferent tissues from various animals and from man have proved susceptible to
intracellular infection with T cruzi ( 1 25, 1 91). Neva et al (142) readily infected
several embryonic tissues of chick and rat, human embryonic skin muscle, human
kidney, and human amnion. Among primary cultures of chick embryo cells and
various tissue cell lines, human heart cells were considered the best (86).
Inocula were generally suspensions of metacyclic trypomastigote forms harvested
from blood-agar culture. Strain diferences in T cruzi cultures in respect to infec
tivity for tissue culture cells have been reported (142). Culture forms maintained in
the laboratory for long periods tend to lose their infectivity for tissue culture cells.
To obtain a great yield of cell-harboring parasites, recent isolates from the vertebrate
host were recommended as inocula (1 93). Bloodstream forms did not, apparently,
produce sustained or progressive intracellular infection, and very mild parasitism
resulted from inoculation of blood containing trypomastigotes (1 42). Other experi
ments, however, showed a cell line derived from a murine chondrosarcoma to be
highly susceptible to infection with bloodstream forms, single parasites readily
initiating intracellular growth in such cells ( 1 25). Since inocula consisting of para
sites newly released from infected cells do not regularly produce high infection rates,
the infectivity of these forms is unclear. Metacyclic trypomastigotes from LIT and
HIL media were signifcantly more infective for monkey's heart cells than forms
obtained from fve-day infected tissue culture fluid medium (1 69).
Infuence of Temperature on T crzi Morphogenesis
Most papers dealing with diferentiation of amastigotes into trypomastigotes and
with morphogenesis of the diferent trypomastigotes in tissue culture have been
mentioned previously ( 128, 1 75, 1 76, 1 91 , 227). The infuence of d
.
iferent incubation
temperature upon intracellular growth of the T crzi Brazil strain was investigated
by Neva et al (142). At 33C amastigote forms steadily multiplied and changed into
trypomastigotes, cellular infection progressed, and the proportion of infected cells
reached 20 to 50% afer 20 days. As a result released fagellates- increasingly accu
mulated in the fuid from infected cultures; 104 to lOG trypomastigotes per milliliter
could be detected afer 2 to 3 weeks. At 38C, however, the percentage of infected
cells was usually < 10%; amastigotes could divide but remained in this stage with
neither diferentiation into trypomastigote forms nor progressive cell invasion. An
influence by temperature, however, was not observed in similar experiments with
the Y strain (192). This discrepancy suggests a parallel with thermosensitivity
mechanisms in polio viruses and emphasizes the need for further experiments with
several strains to ascertain whether thermosensitivity in the amastigote-trypomas
tigote transformation involves mutational adaptation. The infuence of diferent
temperatures on the percentage of intracellular infection in monkey heart cells has
been studied (1 69). The morphogenetic action of temperature on tissue culture
forms was also described by Trejos et al (211), who reported that at 26C infected
cells released slender trypomastigote forms identical to those in the bloodstream of
the vertebrate host, whereas at 37C rounded leishmanoids and stout trypomas
tigotes predominated among the parasites liberated from ruptured cells.
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362 BRENER
InDuence of Interferon
Intracellular growth of T cruzi in a murine chondrosarcoma cell line was un
afected by the high levels of interferon induced by a synthetic polynucleotide,
double-stranded polyinosinic-polycytidylic acid ( 1 24). The lack of protection
against intracellular protozoa, including T cruzi, aforded by this antiviral agent
might be explained by the fact that interferon supposedly hinders synthesis of viral
proteins by disturbing the translation of mRNA on the host cell ribosomes. In the
case of the parasites, protein synthesis is coded by their nucleic acids and syntheized
by their own ribosomes.
Punne Metabolism in T crzi Intracellular Fors
As noted, the biosynthesis of nucleotides and polynucleotides by T cruzi culture
forms is performed through the uptake of nitrogenous bases in the medium (63, 64),
de novo pathways being at best negligibly low. Yoneda (229) investigated the
biosynthesis of purines and pyrimidines by intracellular forms of T cruzi in infected
monkey heart cells to determine the metabolic pathways which satisfy the high
demand for nucleic acids by dividing parasites. By the use of labeled glycine and
adenine as well as metabolic inhibitors, e.g. azaserine and the aminonucIeoside of
stylomicin, it was demonstrated autoradiographically that the preferential biosyn
thetic pathway of purine compounds in T cruzi intracellular stages is apparently
a de novo synthesis, whereas extracellular forms keep using the salvage pathway.
A gradual replacement of the de novo biosynthetic pathway by salvage seems to
occur as long as amastigotes transform into trypomastigotes.
Biochemistry of T cruzi-Harborng Cels
Parasitized chick macrophages have more glycolytic activity than do noninfected
cells (220). This elevation in fermentation is not caused by glucose comsumption of
host cell + parasites but rather by the higher glycolysis of the infected cells them
selves and does not appear related to the intensity of intracellular parasitism. T
cruzi can accomplish the whole intracellular cycle even with small amounts of
exogenous glucose available. The same amino acids accumulate in infected cultures
and incubated controls (220).
Expermental Chemotherapy
T cruzi-infected monolayer tissue cultures have often been used as an experimental
model for chemotherapy. Despite obvious inconveniences such as difculty in de
tecting activity of drug metabolites, the impossibility of using highly insoluble
compounds, and the absence of drug excretion and of an immune response, this
method presents some advantages for studying drug activity. Therapeutic activity
may be detected against both extra- and intracellular forms. Stained preparations
give an accurate picture of drug action against the intracellular forms and quantita
tive data may be obtained by using graded drug concentrations. Silva & Kirchner
(1 93) studied the action of primaquine, carbidium sulfate, and stylomycin aminonu
cIeoside on T cruz i-infected monolayers of monkey heart cells. Activity against
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BIOLOGY OF TRYPANOSOMA CRUZI 363
extra- or intracellular forms was determined by counting trypomastigotes in the
culture fuid and noting some characteristics of intracellular infection. such as
disturbances of the dividing forms. Carbidium and stylomycin aminonucleoside
were clearly active against intracellular forms whereas primaquine seemed active
only against extracellular forms. Nitrofurans tested in infected primary tissue cul
tures from trypsinized chicken cells and in HeLa cells proved highly active against
tissue forms (22, 1 29). This in vitro action of nitrofurans has been confrmed in vivo;
electron microscopy of the myocardium of mice inoculated with T cruzi and treated
with nitrofurazone showed the whole sequence of degenerative changes and destruc
tion of the intracellular stages by this active compound (33).
The usefulness of tissue culture in chemotherapy studies of T cruzi was also
evidenced by an investigation of the mode of action of stylomycin aminonucleoside,
which is practically inactive against extracellular parasites but remarkably damages
intracellular forms (1 93, 1 94). Gutteridge et al (87) studied this diferential suscepti
bility and suggested that it might be explained either by fundamental diferences
between purine metabolism in both developmental stages or by metabolitic transfor
mation of the drg to a more active compound. When human heart tissue cells were
incubated with tritiated drug, two compounds were obtained; some evidence
emerged that one of these metabolic products might act against intracellular stages.
Attempts to use infected tissues as an in vitro screening test for useful drugs have
been reported (14, 86). A test has been devised which permits assessment of drug
action on T cruzi in respect to intracellular growth, cell invasion, and intracellular
development, as well as the evaluation of drug toxicity upon un infected cells (14).
The use of tissue cultures for screening was not, however, endorsed by Mieth &
Seiden hat (1 29) who considered this test oflimited value, having tested in HeLa cells
some compounds active in vivo, with negative results.
Table I shows the results obtained with diferent compounds active in vivo in the
study of their activity against intra- and extracellular T cruzi forms in tissue
culture.
Fne Structure of Tissue Culture Forms of T cruzi
The structure of the fagellum and kinetoplast-mitochondrion of T cruzi in tissue
culture forms has been described by Meyer (1 27). Besides the usual fbrous kineto
plast, some extracellular trypomastigotes showed in their kinetoplasts electron
lucent formations arranged like mitochondrial cristae, imparting to the structure a
basket-like aspect. These formations were not observed in the numerous amastigote
forms contained in the parasitized cells nor in intracellular trypomastigotes. It has
been suggested that the extracellular parasites exhaust all their kinetoplast DNA
during their life span outside the cells and are unable, at this stage, to synthesize
new material; the parasites must therefore fnd a new host to resume their life cycle.
A cytostome in intracellular T cruzi forms in HeLa cells has been described
( 1 3 1). The rough endoplasmic reticulum and the Golgi complex of trypomastigotes
from tissue culture are less developed than in bloodstream forms, presumably be
cause those exo-antigen producing structures are not sufciently stimulated by the
in vitro conditions ( l 30).
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364 BRENER
Table 1 Activity of different compounds against T. cri intra- and extacllular tissue
culture forms
Compounds
Nitrofuran
Primaquine
(8-ainouinoline)
Carbidium
(phenanthridinium sulfate)
Aminonucleoside
of Stylomycin
"Cruzon"
(Bisquinaldine)
"Spirotrypan "
(Trivalent arsnical)
Tris (aminophenyl carbonium)
Trypacidin
Aminopterin
Activity
Aginst Against
intracllular extracellular
fors forms
+ +
t
+
+
t
+
+
+ +
CULTIVATION OF TRYPANOSOMA CRUZl
Referencs
14, 22, 129
193
22, 193
87, 193, 1 94
129
1 29
14
87
87
Undefned media for cultivating T cruzi mainly blood agars, have been reviewed
(20, 205). A few authors used autoclaved diphasic agar-blood medium ( 1 40) or
liquid medium ( 1 39); T cruzi seems not to require heat-labile materials. The efec
tiveness of dialysate media (207) is evidence that T cruzi may grow without mac
romolecules or nondialyzable lipids. Two complex undefined liquid media, the LIT
medium (liver infusion-Tryptose) (35) and brain-heart infusion + blood (221), have
ben used for mass cultivation and morphogenetic studies.
Nerly Defne Media: Growth Factor
T cruzi although less so than other mammalian trypanosomes, is still rather
exacting and has not been cultivated in defined media. Little & Subbarow ( 1 1 6) and
Sampath & Little ( 1 84) cultivated it in a fltrate of a mixture of peptone, Casamino
acids, the usual growth factors, and rabbit blood coagulum. This mixture was
improved ( 1 1 7) by replacing peptone and Casamino acids with amino acids, vita
mins, glucose, and miscellaneous compounds (adenine, uracil, urea, purines and
pyrimidines, etc); the parasite was subcultured for long periods in such media. A
partly defned medium was devised, containing hemin, purifed fraction of serum
albumin, and tomato juice (44); the latter was replaced by known growth factors
including vitamins, RNA, cytidylic acid, and creatinine. This partly defined me-
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BIOLOY OF TRYPANOSOMA CRUZI 365
dium, in which casein hydrolysate was the only undefned component, supported
growth comparable to that in blood media for 1. 5 years (45), but Bone & Parent
(1 7) did not obtain satisfactory growth with it.
Bone & Parent ( 1 7) reported that the Tryptose-liver infusion medium described
for T mega did not suit T cruzi unless fresh calf serum was added. Egg yolk
lecithin had growth activity when added to the Tryptose-liver infusion. From the
composition of egg yolk lecithin, which suggested trial of fatty acids, only stearate
and to some extent behenate promoted growth. These results were confirmed neither
by Lambrecht ( 1 1 2), who attempted to grow T cruzi in Bone & Parent's stearate
free medium, nor by Fernandes & Castellani (65), who reported that stearate could
not replace serum in LIT medium. CO2 increased growth in a stearate-free medium,
probably by enhancing the biosynthesis oHatty acids (1 7). With Tryptose as the only
undefned component, liver extract was replaced by thiamine + folic acid without
decreasing the growth rate ( 1 7); again Fernandes & Castellani (65) could not con
frm these fndings.
Montes ( 1 32) studied the growth of four T cruzi strains in the semisynthetic
media of Citri & Grossowicz (45), Bone & Parent ( 1 7), Little & Subbarow ( 1 1 6),
and Little & Oleson (1 1 7). Bone & Parent's medium supported growth of all strains,
making it the most promising for development of a defned medium. Diferences
among strains in Citri & Grossowicz and Bone & Parent's media point to diferences
in nutritional requirements.
Growth and Diferentiation in Culture Forms
Soon afer cultivation of T cruzi it was recognized that the origin in cultures of the
infective metacyc1ic trypomastigotes is from epimastigotes (forms having an anterior
undulating membrane) and that metacyclics are usually unable to multiply. An
increase in metacyclics in old cultures has been commonly observed, but the factors
cOtrolling the morphogenesis of metacyclic trypomastigotes could not be properly
studied in the usual diphasic culture media. In a series of papers based on use of
the LIT liquid medium, which supports fast growth and allows use of electronic
counters (35, 42, 65), cultures with original densities ranging from 3 to 20 x 1 0
6
/ml
show exponential growth for four days, then a stationary phase (35). The number
of metacyclic trypomastigotes decreased during the exponential phase as a result of
extensive multiplication of epimastigotes. At the end of exponential growth, how
ever, the number of metacyclic forms steadily increased as a result of the epimas
tigote --- trypomastigote diferentiation which proceeded as cultures aged. In
cultures kept in an exponential phase by daily subculture, the proportion ofmetacy
clie forms was extremely low. Subcultures into fresh LIT medium of cultures
already displaying ongoing diferentiation checked further morphogenesis of meta
cycJics whereas transfer to a poorer, incomplete medium (lactalbumin-serum) did
not slow the diferentiation (35). These observations support the idea that the
diferentiation favored by the stationary phase may depend on exhaustion of nutri
ents. Nevertheless, ac
c
umulation of unknown factors in the medium cannot be
ignored, since the formation of organic acids may stimulate transformation of
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366 BRENER
epimastigotes into trypomastigotes (67). Acetic and pyruvic acids, added to a glu
cose-free medium having a slightly acid pH, speed up diferentiation, suggesting
participation of the tricarboxylic acid cycle metabolism in diferentiation (67). Re
placement of ox liver infusion, usually employed in LIT medium, by dog heart
infusion, and concomitant lowering of pH from 7. 2 to 6.7, strongly stimulated
metacycIogenesis. In this medium (HIL), cultures preincubated at 21 C for 48 hr,
then incubated at 28C, had about 75% of metacyclic trypomastigotes within fve
days (37). The change in pH seems essential for the intensifed diferentiation since
replacement of the ox liver infusion by dog heart infusion did not increase metacy
clogenesis.
Ferandes & Castellani (65), using LIT medium, showed that log phase organ
isms were larger and have a higher RNA and protein content, whereas those in
stationary phase synthesized protein and nucleic acids much more slowly while
using less O2,
Synthesis of protein and nucleic acids has been studied by incorporating labeled
1
4
C-leucine into protein, 3H-uridine in RNA, and 3H-thymidine in DNA (67). In
HIL medium at pH 7.2, which supports growth but not good diferentiation, the
highest incorporation rates were at the end of exponential growth; they then de
creased during the stationary phase. When, however, T cruzi was grown in HIL
medium at pH 6. 7, which favored diferentiation, in addition to the peak of inc or po
ration at the end of the growth phase there was a second peak of protein, RNA,
and DNA synthesis which "follows and runs parallel to rate of diferentiation." This
burst of synthesis seems, at least for DNA, to be carried out predominantly by the
metacyclic trypomastigotes, not the epimastigotes.
In the above-mentioned studies only the Y strain seems to have been used. Chiari
(42) compared growth and diferentiation of Y and MR strains maintained in vitro
for diferent periods afer isolation from experimentally infected animals. The gen
eral growth characteristics were confrmed: diferent isolates had similar growth
curves; nevertheless, variations in respect to diferentiation were apparent. The Y
strain maintained in LIT for about 3 years had 50-60% of metacyclic trypomas
tigotes in the stationary phase, whereas other Y cultures maintained in LIT for 1 . 5
and 7 years, respectively. had at most 1 5-25% metacyclics. On the other hand, the
PF culture of the Y strain (1 26) kept in blood-agar medium for "17 years, then 1
year in LIT. consistently had an extremely low proportion of metacyclics 05%).
MR cultures maintained 2 to 6.5 years in LIT generally showed higher diferentia
tion rates than did the Y strain.
These latter experiments apparently show that besides subtle repressing or stimu
lating infuences prevailing in the medium, marked diferences are inherent in the
diferentiation potentialities of several cultures, revealed by prolonged laboratory
maintenance in vitro.
Efects of Drugs on Growth, Diferentiation, and Synthesis of Protein and
Nucei Acds of Culture Forms
Attempts to suppress development of T cruzi culture forms with inhibitors of
protein and nucleic acid synthesis have also usually been performed with the aim
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BIOLOGY OF TRYPANOSOMA CRUZI 367
of preventing normal morphogenesis, thereby obtaining populations of fagellates
unable to diferentiate but still retaining characteristic antigenicity (66, 69).
The inability of T cruzi culture forms to synthesize purine and pyrimidine
nucleotides, with consequent dependence on exogenous bases (63, 1 68), led some
authors to try to inhibit incorporation of nitrogenous bases into nucleotides and
nucleic acids with purine and pyrimidine analogs (36, 66, 69). The aminonucleoside
of stylomycin blocked incorporation of adenine into purine nucleotide and nucleic
acids (64, 66); 5-fluorouracil (FU) and 5-fluorouracil deoxyriboside (FUDR) inhib
ited incorporation of uracil or uridine into pyrimidine nucleotides and nucleic acids
(66, 69, 1 68). The rapid inhibition by drugs of DNA synthesis distorted growth: cell
size increased and population growth slowed (36, 66). Certain antibiotics, mitomy
cin C, actinomycin D, and puromycin, inhibit growth by combining with nucleic
acids. Mitomycin inhibits synthesis of DNA and protein and later of RNA a well;
it unbalances growth as evidenced by cytokinesis without duplication of DNA
containing organelles (66, 69). Puromycin also strongly inhibited protein synthesis
by T cruzi probably by its well-known activity as an analog of aminoacyl tRNA.
Although rather active against tissue culture and vertebrate host forms, the
aminonucleoside moiety of puromycin did not inhibit culture forms (66, 69).
Actinomycin, extensively applied to T cruzi cultures, strongly inhibited RNA
synthesis and, at higher concentrations, protein and DNA synthesis. Multiplication
was blocked as demonstrated by nonincorporation of thymidine into DNA but,
although it prevented infectivity to tissue culture and animals, it apparently did not
lessen antigenicity. These efects were irreversible: normal synthesis was not
resumed when the drug was removed and the parasites put in fresh media (66, 69).
DNA-Mediated Heterotransforation of Culture Fors?
In localizing dehydrogenases in culture forms, Lehmann ( 1 1 5) found that two
cultures contaminated with a Candida had an enzyme patter diferent from that
of noncontaminated cultures of the same strains. Before exposure to CandIda both
strains showed only one dehydrogenase each, either isocitric dehydrogenase or lactic
dehydrogenase; afer 1 01 4 days of contact with Candida, however, both strains
contained the two enzymes. As Candida itself possessed the respective primitive
enzymes lacking in T cruzi he suggested that genetic transfer had taken place
between Candida and T cruzi.
Cultivaton of Amasti
g
ote Sta
g
es in Cel-Free Medium
Trager (21 0), in discussing difculties in obtaining developing stages of
Trypanosomatidae in cell-free medium as are found in the vertebrate host, reviewed
some of the environmental conditions which might trigger diferentiation towards
the peculiar forms of Leishmania and Trypanosoma usually present in mammalian
hosts. In T cruzi cultures, amastigote forms isolated or agglutinated into large
masses have been reported sporadically (41 , 145). When T cruzi was grown in a
medium containing specifc antiserum prepared in rabbits, large colonies of amas
tigotes (leishmanoids) produced by binary fission appeared (2). Amastigotes also
occur in infected blood kept at room temperature (191) and in the fluid of infected
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368 BRENER
tissue cultures (21 1). Fresh cultures of newly isolated blood forms of three T crlzi
strains revealed trypomastigotes steadily developing into amastigotes which gradu
ally changed into epimastigotes; there was some evidence that broad and stout
bloodstream trypomastigotes are more prone to change into amastigotes than are
the slender ones (30).
Cultivatin of T crlzi "serially and predominantly as leishmaniforms" in cell
free media was reported (155). By using the TPH medium (Trypticase, salts, horse
serum, rabbit blood, plus Ender's tissue culture fuid) and raising the temperature
to 35. 5C Pan (1 55), starting with a culture containing 98% epimastigotes, ob
tained -90% of amastigote forms after 1 5 passages. At 24.SoC most culture forms
continued to grow in TPH medium as epimastigotes, showing that temperature was
the prime factor in morphogenesis of the predominant amastigotes. However, with
a simpler medium (modified tissue culture medium 199, hemolyzed rabbit blood,
chicken plasma, and chicken embryo extract), > 90% amastigotes were obtained,
whatever the temperature. A new medium without chicken embryo extract but still
containing chicken plasma permitted serial cultivation of leishmaniforms, likewise
irrespective of temperature (2436C) (1 56). Besides playing an important role in
eliciting development of amastigotes in cell-free culture, chicken serum probably
helps eliminate the remaining epimastigotes apparently because it contains a lytic
antiepimastigote factor (220, 222). As noted earlier. amastigotes are apparently
better fitted to survive at higher temperatures than epimastigotes. which resemble
amastigotes of Lishmania that are likewise better equipped to survive at 37C than
are the promastigotes. Perhaps motile stages have a greater energy demand but a
decreased biosynthetic capability at higher temperature (98).
Infetivity of T crzi Culture
Among the T cTuzi flagellate forms usual in cultures, only the metacyclic trypomas
tigotes are believed to be infective to the vertebrate. This infectivity is retained in
cultures maintained for long periods without animal passage. Thus. mice developed
full infections with culture forms kept 1 3 years in blood agar (154), and lethal
infections have been observed in mice inoculated with ten or even one metacyclic
trypomastigote from cultures maintained for about 5 years (1 77). Some cultures.
however. steadily diminish in virulence (42, 1 26); mice given even 8 x 107 flagellates
did not develop a patent parasitemia (1 64). This apparent variability in retention of
infectivity makes one wonder whether trypomastigote metacyclics continually de
velop in long-term culture. Diferent cultures from diferent strains kept for 1 .5 to
9 years (l S) and from l . S to 1 7 years (42) contained a variable but almost always
signifcant number of metacyclic trypomastigotes. How infectious are those trypo
mastigotes? By counting the metacyclics and inoculating mice with a similar number
of infective forms. Bice & Zeledon (15) and Chiari (42) discovered fluctuations in
virulence of various cultures maintained for diferent periods in the laboratory. The
decreased virulence of some cultures, therefore, seemed to be caused by loss of
infectivity of the metacyclics rather than by decrease in the number of metacyclics.
Length of time in artifcial media may go along with decrease in virulence (IS, 42).
It has been suggested that prolonged maintenance in artifcial medium selects for
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BIOLOGY OF TRYPANOSOMA CRUZI 369
variants better ftted to develop in vitro than in the vertebrate (I l l ). Nevertheless,
a single passage through the vertebrate host of attenuated cultures has not signif
cantly increased virulence in experiments performed afer reisolation and cultivation
in LIT medium (42).
Actinomycin-treated T cruzi culture forms apparently lost infectivity for mice
while retaining antigenic competence, and inoculation of treated flagellates aforded
a certain protection against challenge with virulent blood forms (68).
Clear-cut evidence remains to be obtained as to whether T cruzi needs still
unidentifed nutrient. Once this is settled, factors for morphogenesis can be profta
bly analyzed. This in turn imparts more realism to the use of cultures for drug
testing; cultures might then more closely approximate in vitro conditio
n
s including
sustained growth at blood and tissue pH and temperatures.
CYTOCHEMISTRY AND METABOLISM
Nuceic Acids: Role of Kinetoplast DNA and Dyskinetoplasty in T cruii
As frst demonstrated by Schildkraut (1 88), DNA from intact trypanosomatids
could be dissociated by CsCI density-gradient centrifugation into a major band and
a satellite band. The satellite component, whose base composition difers from
nuclear DNA, corresponds to kinetoplast DNA and represents, at least in T cruzi
-20% of the total extracted DNA (174). This remarkably large amount of extranu
clear DNA is unusual among plant or animal cells; their mitochondrial DNA is only
0. 5-1 .0% of total cells DNA. Kinetoplast DNA has been suggested to participate
in coding of protein moieties of oxidative enzymes and cytochromes in
Trypanosomatidae [reviews: Newton (1 4); Hill & Anderson (95)
]
.
The kinetoplast as determinant of enzyme balance in Trypanosomatidae has been
discussed in the aforementioned reviews. There is no present reason to think that
T crzi diverges fundamentally from these patterns, therefore this subject will be
mentioned only in outline.
Permanent loss of Peulgen-positive material from the kinetoplast (dyskineto
plasty) is common among trypanosomes which are mechanically transmitted. Acri
dine dyes and ethidium bind to kinetoplast DNA and may induce dyskinetoplastic
forms in cyclically transmitted trypanosomes. This specifc binding has been sug
gested to be caused by the absence of histone in the kinetoplast, by the peculiar
nonlinear structure of the kinetoplast DNA, or by diferences between nuclear and
kinetoplast membranes (14). As demonstrated by Miihlpfordt (1341 36), dys
kinetoplastic forms lose the Feulgen-positive fibers but retain the double-membrane
envelope. As now demonstrated by thymidine incorporation and electron micro
scopic radioautography in T evansi dyskinetoplastic bloodstream forms, DNA is
still present in the two-layered kinetoplast membranes ( 1 5 I); this condition is
thought to be characteristic of the T evansi strain. As for T cruzi incorporation
studies with thymidine showed silver grains in the kinetoplast but not in the kineto
plast double membranes (1 53). In brucei-group trypanosomes, bloodstream dys
kinetoplastic forms may multiply indefnitely in the vertebrate host but cannot
develop in vector or culture, which accords with the concept that brucei-group
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370 BRENER
trypomastigotes must elaborate a cytochrome-mediated respiratory system to sur
vive and multiply in the vector. The stercoraria trypanosomes, including T cruzi,
probably depend on a functional cytochrome system in all stages in both hosts and
are therefore unable to multiply in the vertebrate host if dyskinetoplastic. Miihlp
fordt (1 34) surveyed various genera of Trypanosomatidae and saw dyskinetoplastic
forms in aU species, with the exception of T cruzi bloodstream trypomastigotes. In
three strains of T cruzi a low proportion of culture forms (0.20 to < 1 . 20%) were
spontaneously dyskinetoplastic. Similar results were reported by Ono et al ( 1 5 1)
who observed < I % of such forms in cultures. Culture forms in LIT medium
treated with acriflavine had a high proportion of dyskinetoplastic epimastigotes (50,
1 80); those forms, which could not, however, be subcultured, nevertheless could
di
f
erentiate and in some cultures accounted for the existence of ..50% of dyskineto
plastic metacyclic trypomastigotes (50). These abnormal metacyclics were infective
to HeLa cells and gave rise within 24-48 hr to dyskinetoplastic amastigotes; but very
soon most intracellular stages had normal kinetoplast and there was evidence that
more tha
n
one generation of dyskinetoplastic parasites had been produced.
The first recorded spontaneous dyskinetoplastic T cruzi bloodstream parasites
were reported by Deane & Kloetzel (50) who observed a few forms in a slide from
the blood of an acute h\man case of Chagas' disease. A few dyskinetoplastic trypo
mastigote and amastigote forms have also been observed in and outside cells of the
peritoneal exudate of mice inoculated with acriflavine-treated cultures (50). Al
though dyskinetoplastic forms have not been regularly obtained in the mammalian
host and in tissue culture, these fndings suggested that an impaired kinetoplast may
not abort development of T cruzi in the vertebrate host and hence that dependence
of T cruzi on mitochondrial respiratory enzymes might not be quite as essential as
supposed for its development. More studies on the occurrence of natural dyskineto
plastic parasites and long term observations of the behavior of spontaneous and
induced dyskinetoplastic forms in culture and hosts may be necessary to decide
whether kinetoplast DNA is critical for the parasite's heteroxenous development as
mediated by mitochondrial enzymes .
. Nucear and Kinetoplast DNA /om T cruzi
Total DNA from T cruzi culture forms showed, besides a nuclear band with
buoyant density 1 .7 10, two satellite bands with buoyant densities of 1 . 699 and 1 . 686,
respectively (1 73). The light satellite DNA was not, however, detected in further
experiments; its appearance has been related to some unknown condition of the
growth medium (1 74). These results have been confrmed by Ono et al (1 5 1), who
found densities of 1 . 709 and 1 . 699, respectively, for the nuclear and satellite bands.
The base composition of nuclear and heavy satellite DNA has been determined; the
GC content was 5 1 .0% and 39. 8% (via density), respectively ( 1 73, 1 74). These
reports confict with those of Fernandes & Castellani (65) who found in DNA of
whole organisms a high proportion of adenine and thymine and GC of only 27%.
T cruzi kinetoplast DNA isolated by buoyant-density centrifugation in ethidium
CsCI density gradients comprises open and closed circles of 0.45 ,.m monomer size
as well as long linear flaments 2 to 1 2 ,.m long ( 172). DNAp 1 . 699 was formed of
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BIOLOGY OF TRYPANOSOMA CRUZI 371
minicirc1es 0.45 /m long whereas the DNA 1 .688 was built of linear strands ( 1 71 ).
Circular DNA molecules have been detected in nuclear and kinetoplast nucleic
acids; a study of the frequency distribution of the circular molecules showed a larger
range than reported in the previous papers: most of the molecules were 0.20-0.29
/m length and molecules between 0. 1 1 /m and 12 /m have been found. Most of
the kinetoplast DNA, however, consisted of long linear strands ( 1 5 1).
When two trypanocids (Berenil and hydroxystilbamidine) known to bind selec
tively to kinetoplast DNA of trypanosomes were used in T cruzi cultures, DNAp
1 . 688 increased from 1 to -10% and DNAp 1 .699 decreased from 20 to 4%. Both
drugs apparently enhance replication of one DNA and inhibit the other (1 71 ). From
the DNA content of each parasite, the number of circles in the T cruzi kinetoplast
has been estimated as 24,00 (1 74). The existence of replicating kinetoplast DNA
circles was described for the frst time in trypanosomes in Berenil-treated T cruzi
culture forms ( 1 9). Berenil increased the proportion of replicating molecules by 103 J
probably not by a general increase of DNA synthesis but rather by blocking its
replication at AT-rich specifc points. The lengths of the replicating branches corre
sponded to multiples of 1 5% of the total contour length of the circular DNA
molecules, which suggests a discontinuous replication process.
Some of the reported fndings warrant additional investigation owing to discrep
ancies: (a) The existence, in diferent experiments, ofa single or two distinct satellite
bands detected by density-gradient centrifugation of whole-cell DNA. Those two
satellite bands, displaying diferent buoyant densities and molecular components,
have been regularly reported by some authors ( 1 71 , 1 73) but are not mentioned, for
instance, by Ono et al ( 1 5 1), who detected only one band. ( b) A certain
heterogeneity of data for occurrence and size of kinetoplast circular molecules.
Although Riou & DeJain ( 1 72) reported that most of kinetoplast DNA was formed
by circular molecules, Ono et al ( 1 5 1) clearly stated that kinetoplast DNA consisted
mainly of long linear DNA molecules and that minicircles were not the major
component of the kinetoplast. On the other hand, most authors reported the pres
ence of free or complex associations of minicircles of 0.45-.50 /m contour length.
Ono et all ( 1 5 1), however, although not separating minicircles from nuclear and
kinetoplast sources, described circular DNA molecules ranging from 0. 1 0 to 1 5
.
0
/m. (c) The coding properties of the minicircles have not yet been disclosed,
although they probably can code for a few polypeptides (95). Those peculiar DNA
arrangements have been interpreted either as evolutionary artifacts without genetic
function or as an amplifcation of genes involved in mitochondrial biogenesis (1 95).
Their independent replication apparently confers upon them with genetic signif
cance ( 1 9). The presence of linear DNA flaments mixed with minicirc1es has been
explained either as contamination of nuclear origin or by the supposition that this
makeup of DNA plays a role in replication of kinetoplast DNA (1 72).
Nuceic Acd Metabolsm in T cruzi
Purine nuc1eotides may be synthesized either by conversion of precursors, i.e. gly
cine and CO2 (de novo synthesis), or by utilization of exogenous preformed purines
(salvage pathway). Use of 14C-IabeJed glycine revealed that T cruzi culture forms
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372 BRENER
may synthesize purines but at a very low rate; uptake of radioactive adenine,
however, has shown that preformed purine utilization is preferred for synthesis of
purine nucIeotides (63). Using 14C-Iabeled uracil and orotic acid as well as metabolic
inhibitors, Rey & Fernandes (168) suggested that T cruzi culture forms cannot
synthesize pyrimidines de novo and would therefore depend on preformed pyrimi
dines for nucleotide biosynthesis. This dependence on preformed pyrimidines seems
unusual since monoxenic Trypanosomatidae cultivated in defned media require
exogenous purine but not pyrimidine (98). Dependence of T cruzi on exogenous
purine and pyrimidine bases has suggested that the uptake of these nucleotides for
the biosynthesis of nucleic acids may also supply additional energy . for anabolic
reactions (96).
The chemotherapeutic action of some selective inhibitors of trypanosome nucleic
acid metabolism has been studied in mice (99). Mitomycin C (an inhibitor of DNA
synthesis), 6-azauracil (inhibitor of de novo synthesis of pyrimidine nucleotides),
and actinomycin D (inhibitor of RNA synthesis) did not prolong the life of the
experimentally infected animals. An in vitro inhibitor of T cruzi purine nucleotide
synthesis, the aminonucleoside of stylomycin, exerted some activity in experimen
tally infected mice. changing the acute disease into a chronic one but without
afecting a cure (70).
Amino Acids and Proteins
T cruzi culture forms contain 43-53% of protein by weight (219). The protein
contents of culture forms in exponential and stationary phases of T cruzi in LIT
medium showed, respectively, 67.0 and 42.0 Ig X 17 per fagelJate (65). Culture
forms examined by paper chromatography contained, among others, alanine and
aspartic acid, presumably formed by transamination (224) since transaminase activ
ity had been observed in T cruzi culture forms (1 3).
Uptake ofJysine and arginine is mediated by active transport and seems somewhat
diferent from their uptake by mammalian cells (90, 91). Proteins and some amino
acids may be utilized by T cruzi culture forms as energy sources in sugar-free media
(208, 230). In a medium devoid of glucose or in which sugar depletion was demon
strated, the pH rose, caused by production of ammonia from nitrogenous substrates
(208). According to Zeled6n (230) oxidation of amino acids in organisms having a
tricarboxylic acid cycle is probably mediated by transaminase systems. Honigberg
(96) speculates that this oxidation is likely to be performed only in those blood
stream forms of trypanosomes having a functional tricarboxylic acid cycle.
Lipids
Lipids in T cruzi culture forms comprise up to 20. 1 % of their dry weight (219).
Cholesterol has been identifed in culture forms in media containing this substance
(219). The presence of an ergosterol-like sterol (89) was not confrmed by William
son (223). More recently, however, six sterols have been detected in T cruzi culture
forms grown on serum-free medium. Of these, four were ergosterol compounds and
the two others were probably 24-methylene-7-dehydrocholesterol and 24-ethyli
dene-7-dehydrocholesterol (109); cholesterol was detected only in flagellates grow-
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BILOY-O TRYPANOOA CRUZI 373
-jng in serum-containing medium. ThIn-layer chromat{graphy of lipid extracts fmm
T cruzi culture forms demonstrated several phospholipids: phosphatidylethanola
mine, phosphatidylcholine, and inositol (88).
Gas-liquid chromatography of T cruzi blood and culture forms (grown in a
medium where cholesterol was freely available) showed three only partially identifi
able sterols (A, C, and D); evidence pointed to the A fraction being cholesterol (58).
Culture forms had A and D sterols, bloodstream forms had A and C. Phytosterols
are apparently the only important sterols in pathogenic trypanosomes since zoos
terols, chiefly cholesterol, are of exogenous origin and not apparently further meta
bolized. Sterols with structural function are probably obtained exogenously whereas
the metabolically active sterols are likely to arise endogenously (58). It seems to be
worth investigating the characteristics of flagellates grown in cholesterol-free media
as opposed to those cultivated in media with serum, since cholesterol may help
determine cell permeability and development of other lipid-containing structures.
Activity of the antifungal polyene (Amphotericin B) against T crzi (1) sug
gested the presence of sterols in the parasite's delimiting membrane since polyene
antibiotics are known to act by attachment to sterols, especially ergosterol, in
sterol-phospholipid complexes, thereby incr.easing--the permeability of the cell mem
hrane and permitting escape of low-molecular components (58, 99).
Carbohydrate Metabolsm and Respiration
1 cruzi -does not store polysaccharide; endogenous me-tabolism is apparently sup
ported by utilization of fat and protein rather than by carbohydrate ( 1 82). Small
quantities of a immunogenic polysaccharide-containing galactose, mannose,
xylose, glucse, and glucosamine have been detected in T cruzi cultl1re forms (82).
Another polysaccharide yielding galactose but contaminated with nucleic acid frag
ments was reported in culture forms (21 9). Small amounts of glucose (0.06% of dry
material) and trehalose (0.05%) have also been detected (61}.
Utilization of glucose by culture and bloodstream forms of T cruzi has been
reported (180). The culture f{fms failed to utilize maltose, mannitol, lactose, su
crose, trehalose, arabinose, and galactose; glucose, fructose, and xylose were utilized
( 1 14). Several endproducts of anaerobic fermentation-acetic acid and succinic acid
in culture forms; acetic acid, succinic acid, lactic acid, and small amounts of pyruvic
acid in bloodstream forms-have been detected [review von Brand (21 7)]. More
over, the following glycolytic -enzymes were detected in T cruzi culture forms:
hexokinase, phosphohexose isomerase, phosphohexokinase, aldolase, phosphotriose
isomerase, and phosphotriose dehydrogenase [review von Brand (21 7)]. Glucose-6-
phosphate and 6-phosphogluconic dehydrogenases, enzymes of the pentose-phos
phate pathway, have also been reporte ( 1 63). A 6-phosphogluconic-splitting
enzyme has been described (1 63) suggesting that pyruvic acid and glyceraldehyde-3-
phosphate are formed by this reaction. Utilization of 6-phosphogluconic acid has
not, however, been confrmed by Ryley ( 1 8 1). This disagreement might be due to
diferent strain behavior as suggested by data reported by Mancilla & Naquira (120),
who compared the ability of culture forms from two T cruzi strains to metabolize
labele 14C-glucose. They showed that the Tulahuen strain consumed glucose and
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374 BRENER
incorporated labeled glucose-1
4
C into CO2 at a higher rate than the Peruvian strain;
moreover, the pentose-phosphate pathway in the Tulahuen strain was responsible
for 41 .4% of glucose catabolism as compared with 27.9% in the Peruvian strain.
As the Tulahuen strain is clearly the more pathogenic, the authors suggested that,
although the pentose phosphate pathway is operative in both strains, the quantita
tive diferences detected in this glucose catabolic pathway may have to do with
pathogenicity. As in other cells, carbohydrate metabolism in trypanosomes is car
ried out through anaerobic glycolysis with conversion of glucose to pyruvate or
through the pentose-phosphate pathway, followed by an aerobic degradation of
pyruvate via a functional tricarboxylic acid cycle, an active cytochrome system, and
production of CO2 However, some diferences between the terminal respiration of
trypanosomes from the brucei group and those of the lewisi group ( T lewisi, T
cruzl) have been reported (73, 1 80). Thus, African trypanosomes showed cyclical
changes from a cytochrome to a noncytochrome metabolic pathway in the diferent
developmental stages in vertebrate hosts, whereas T cruzi forms have an active
cytochrome system during the whole life cycle in both hosts. Those diferences have
been detected (a) through study of the sensitivity of the various species and stages
to cyanide (an inhibitor of a + a
3
cytochromes), which may presumably indicate
whether a functional cytochrome system is present or not; (b) by direct spectro
scopic characterization of diferent cytochromes; and (c) by cytochemical character
ization of respiratory enzymes such as cytochrome oxidase NADH and NADPH
diaphorase as well as L-a-glycerophosphate oxidase (95, 14). Such suggested meta
bolic diferences demand a brief review of the respiratory chains characterizing both
group of pathogens.
Slender bloodstream forms of brucei-group trypanosomes are cyanide insensitive;
their electron transfer system is probably not mediated by cytochromes; these forms
use instead a particulate, cyanide-insensitive L-a-glycerophosphate oxidase system,
localized in extramitochondrial granules [(85), reviewed by Honigberg (96)]. In
stumpy forms originating from the slender tripomastigotes, however, the site of
oxidation shifs to the chondriome where NADH diaphorase was demonstrable by
tetrazolium reduction (21 3), suggesting the presence of a functional tricarboxylic
acid cycle. This respiratory switch apparently occurs in the bloodstream, probably
representing a preadaptation of the stumpy forms to the conditions to be faced by
the parasite in the invertebrate host; it seems essential for heteroxenous develop
ment. In bloodstream forms glucose is not degraded beyond pyruvic acid + glycerol,
and the respiratory quotient is <0. 1 0, with negligible production of CO2 (1 80).
Midgut and culture forms, on the other hand, are cyanide sensitive, have a func
tional cytochrome system, (1 1 , 73), and have a signifcantly higher respiratory
quotient of -1. 0 ( I 80). More recently, however, it has been demonstrated that
oxidation of succinate, NADH, and L-a-glycerophosphate by whole homogenates
of T brucei culture forms, obtained during the transformation of bloodstream forms
into established culture, were completely insensitive to cyanide (60). Afer passing
through this phase, the early culture forms developed cyanide sensitivity and grew
in a medium with a low blood concentration. Those data do not apparently agree
with the concept that the respiration switch occurs only in the bloodstream, since
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BIOLOY OF TRYPANOSOMA CRUZI 375
it is very unlikely that. the newly cyanide-insensitive culture forms were using an
electron-transfer system mediated by cytochro
m
e.
The respiration of bloodstream and culture forms of T crzi is cyanide sensitive
(73, 1 80) and the respiratory quotient is high, usually -l .O.Glucose is degraded by
bloodstream and culture forms beyond pyruvate to a mixture of lactic, acetic, and
succinic acids ( 1 80). A cytochrome system has been detected in T crzi culture
forms but, quite unusually, cytochrome c seems absent ( 1 2, 1 80). A NADH-cyto
chrome b5 system was detected in the microsomal hemoprotein of T cruzi culture
forms ( 1 6). The same authors, however, could not detect mammalian-type a, b, and
c cytochromes in the particulate and soluble fractions. Nevertheless, evidence that
a tricarboxylic acid cycle is active in T cruzi culture forms has been provided by
identifcation of various Krebs cycle enzymes, e.g. malic dehydrogenase and fuma
rase (1 0), succinic dehydrogenase (4, 1 66), and isocitric dehydrogenase (5, 1 66). The .
endproducts and enzymes involved in. T cruzi carbohydrate metabolism are shown
in Figure 1 . Cytochrome oxidase activity was very low in homogenates of T cruzi
culture forms and was not increased by exogenous cytochrome c, ( 1 80). Spectro
scopic investigations of cytochromes in T cruzi bloodstream forms, as well as a .
selective study of the respiratory enzymes in slender and broad forms, have appar
ently not yet been. made. However, cytochemical methods have shown the presence
in the kinetoplast of T crzi culture forms, as well as in trypomastigotes and
amastigotes of the vertebrate host of such enzymes as cytochrome oxidase, NADH,
and NADPH diaphorases (1 03). Although these studies indicate the presence of a
functional cytochrome system, cytochemical investigations must be confrmed by
more direct experiments (95). In sum, the existence of a cytochrome system in all
T cruzi developmental stages seems uncertain.
The possibility of an analogy between the difering biochemical behaviors of
slender and broad trypomastigotes of the. brucei group and the diferent T cruzi
bloodstream forms seems to be ignored. Thus, new studies on cyanide sensitivity and
further characterization of respiratory enzymes in T crzi should be made, with
some attention devoted to comparing slender and broad forms. Such studies would
probably be hampered by the absence of pure monomorphic T. cruzi strains and
by difculties in separating the parasites from the blood of infected animals. Motile
trypomastigotes, well separated from blood cells and platelets, have been obtained
by passing blood of animals infected with diferent trypanosomes ( T brucei T
gambiense, T rhodesiense, T evansi T congoinse, T vivax, T lewisl) through an
anion-exchange column. Owing to the small diference in surface charge between
trypanosomes and blood cells, T cruzi was the one species defying separation (1 1 3).
Carbohydrate metabolism of the intracellular stages of T cruzi freed of host or
tissue culture cells has apparently not yet been investigated. That such a study is
feasible is supported by the investigation of glucose uptake and respiratory quotient
performed with Leishmania donovani free from tissue (72). T. cruzi amastigote
forms may be readily obtained in vitro (30, 1 56) but decisive proof of their identity
with intracellular stages has not been demonstrated so far. Parasites from tissue
cultures are more likely to provide practicable material for this study.
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376 BRENER
GLUCOSE
8 7
RI 8ULOS E 5 _ P ..6 _ PHOSPHOGLUCONIC ACID +- GLUCOSE 6. P
( PY RUVI C ACID)
9
F RUCT OSE 6 _ P
1 3
5
DI PHOSHOGL YCERI C AC 1 0
i
I
+
PHOSPHOENOLPYRUVI C ACI D
CO.
I
( ACETI C ACI D)

1 4
"",
ACETYL CoA
.
OXALACETATE
10
/
CI TRATE
MALATE \ 1
5
I I /
F U MA RATE
.
12 \
( SU CCI NAT f )

S UCC I NIL C o A
ACONI T A TE
t
1 5
I SOCI T R A T E
/
1
3
o KE T OGLUT ARA T E
/
(GLyCEROL)
Fgre 1 Terms i parentheses are endproducts of T. crzi carbohydrate metabolism.
Enzymes involved in carbohydrate metabolism desribed in T. crzi: 1 . hexokinase;
2. phosphohexose isomerase; 3. phosphohexolinase; 4. aldolase; 5. phosphotriose
isomerase; 6. phosphotriose dehydrogenase; 7. glucose--phosphate dehydrogenas;
8. 6-phosphogluconic dehydrogenase; 9. Wood & Schwerdt enzyme; 10. malic dehy
drogenase; 1 1 . fumarase; 12. succinic dehydrogenas; 1 3. isocitric dehydrogenas;
14. malc enzyme; 15. aconitase.
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OTHER ASPECTS
Chemotherapy
BIOLOY OF TRYPANOSOMA CRUZI 377
Drugs that will cure Chagas disease are not known with certainty. Present screening
methods appear efcient for detecting suppressive activity on the experimental
disease (20, 24). However, no other parasitic disease has so badly misled chemother
apists: standard methods ofen fail to assess drug activity realistically because the
spontaneous low parasitemia in the chronic phase arouses hopes that are eventually
dashed. Lack of defnite cures means a corresponding uncertainty as to the reliability
of serological methods for monitoring infections. The dismal state of afairs can be
gauged from the fact that xenodiagnosis is widely used; i.e. allowing uninfected
triatomids to feed on the patient and so to develop, if positive, flagellate populations
in the gut. Since negative results are inconclusive, the patient may have to be bitten
repeatedly. Better parasitological and more sensitive immunological tests are
needed. Immunological tests demand a far more detailed knowledge of the specifc
antigens of T cruzi than is yet available.
A more attainable goal is elimination of T cruzi from banked blood; many
infections have been traced to survival of T cruzi in blood (47). Gentian violet is
routinely added to blood in endemic areas. Prospects for finding a less objectionable,
preferably colorless agent, appear good, since one is not coping with nearly occult
intracellular forms. It seems strange that the screening of drugs for this purpose has
not been instituted.
T cruzi Congenital Transmission
T cruzi as other trypanosomes had done, is learning to dispense with the insect
vector, as witnessed by a disturbing increase in the reported number of congenital
cases (97). As a highly successful parasite, T cruzi has much scope for evolution.
Antigenic Constitution
Study of the antigenic constitution of several T cruzi strains (isolated from man,
triatomids, and wild animals) has identifed three immunological types (A, B, and
C) (83, 146, 147). These types could not be correlated with host species or with
geographical distribution of strains. Cross-protection tests in mice could not distin
guish the in vitro typed strains (148).
An immunological relationship between T cruzi and T lewisi has been suggested
(75, 76): mice inoculated with T /wisi culture forms were highly resistant to a T
cruzi challenge infection; double-difusion tests and immunoelectrophoresis showed
common antigens between both species. Those results were not confrmed by Kloet
zel & Deane (104) who readily infected rats previously inoculated with T lewisiwith
T cruzi. The authors were also unable to build up resistance against T crzi in
mice repeatedly inoculated with T lewisi bloodstream forms. Some degree of cross
protection against T cruzi challenge was observed afer inoculation of killed Cr
thidia fsciculata and Leptomonas collosoma (10), as well as of a living suspension
of "Leptomonas" pessoai a monoxenic trypanosomatid isolated from a reduviid
(197).
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378 BRENER
Vaccination against T cruzi
Vaccination has been tried with diferent preparations: bloodstream forms killed by
chemical agents (93) or by freezing-thawing (1 0); living culture forms attenuated
by actinomycin (68), gamma radiation (43), or repeated in vitro passages (1 26);
culture forms killed by Merthiolate (1 38), sonic oscillation (79), or pressure (84).
Living attenuated vaccines are likely to cause cryptic infections which may simulate
an induced immune response. Physical agents used for: killing and disrupting flagel
,lates apparently enhance the efcacy , of the 'antigens; nevertheless, only partial
protection against T cruzi challenge has been obtained with nonliving antigens
[review Goble (80)].
Efcts of T cruzi on Spontaneus and Induced Tumors
The. contradictory literature on the therapeutic efects of T cruzi endotoxines and
Iysates on the development of malignant tumors was reviewed by Kagan et al (1 02).
The authors reported that the proportion of spontaneous mammary tumors in mice
inoculated with American T crzi strains was signifcantly less than in no
.
ninfected
controls. This decrease in the number of spontaneous tumors is probably caused by
nonspecifc stimulation of the reticuloendothelial system.
CONCLUDING REMARKS
The foregoing complexities, the ever more clearly understood near intractability of
the T cruzi therapeutic problem, should not lead one to succumb to "Abandon
hope all ye who enter a Chagas' disease laboratory. " Afer all, T cruzi cannot have
exactly the same physiology as man; somewhere there will be found a chink in its
metabolic armor.
ACKNOWLEDGMENTS
Dr. S. H. Hutner assisted in preparation of the English manuscript.
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i
o
l
.

1
9
7
3
.
2
7
:
3
4
7
-
3
8
2
.

D
o
w
n
l
o
a
d
e
d

f
r
o
m

a
r
j
o
u
r
n
a
l
s
.
a
n
n
u
a
l
r
e
v
i
e
w
s
.
o
r
g
b
y

U
n
i
v
e
r
s
i
d
a
d

N
a
c
i
o
n
a
l

d
e

C
o
l
o
m
b
i
a

o
n

0
7
/
1
0
/
0
9
.

F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.