Acta Tropiea, 60(1995) 167-177 167

ACTROP 00507

Biological and isoenzymatic characterization of Trypanosoma cruzi strains isolated from sylvatic

reservoirs and vectors from the state of Santa Catarina, Southern Brazil

Mfirio SteindeP; Helena K. Tom#; Maria M. I. Ishid#; Silvane M. F. Murtab; Carlos J. de Carvalho PintoS;

Edmundo C. Grisarda; Bruno R. Schlemper Jr.~; Rodrigo Ribeiro-Rodrigues b and Alvaro J. Romanha b'*

"Departamento de Mierobiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Caixa posta1476, CEP 88040-900, Florian6polis - Santa Catarina, Brazil bLaborat6rio de Parasitologia

Celular e Moleeulat Centro de Pesquisas "Renb Rachou" FIOCR UZ, Av. Augusto de Lima 1715, 30190-002, Belo Horizonte, Minas Gerais, Brazil

'Received 29 March 1995; accepted 4 September 1995)

Sixty eight Trypanosoma cruzi strains were isolated in the state of Santa Catarina, Southern Brazil, from sylvatic reservoirs or naturally infected vectors and characterized by their biological behaviour in mice, morphology of bloodstream forms and isoenzyme profiles. Twenty eight strains were isolated from the triatomine bug (Panstrongylus megistus), 2 from rodents (Echimys dasythrix and Akodon sp) and 38 from opossums (Didelphis marsupialis). The infectivity in mice of 48 T. cruzi strains showed that 2 (4.2%) were of high virulence, 19 (39.6%) of medium virulence, 15 (31.2%) of low virulence and 12 (25.0%) produced subpatent parasitemia in mice. A morphological study of bloodstream trypomastigotes from 8 T. cruzi strains showed a predominance of stout forms. The isoenzyme pattern of 59 T, cruzi strains showed that 54 (91.5%) belonged to zymodeme Z1, 3 (5.1%) to zymodeme Z2 and 2 (3.4%) to mixed zymodemes, Z1 and Z2. All 34 T. cruzi strains analyzed from opossums were Z1. Three out of 5 strains isolated from adults of P. megistus collected inside houses, belonged to zymodeme Z2 and two strains exhibited mixed zymodemes, Z1 and Z2, in 5 out of 6 enzymes studied. Although the State of Santa Catarina is a non endemic region for human Chagas'disease, the presence of zymodeme Z2 parasites in the sylvatic vector, P. megistus, captured in domiciliary environments suggests the possibility of human and/or domestic mammal infection by T. cruzi.

Key words: Trypanosoma cruzi; Strains; Biological behaviour; Zymodeme; Chagas' disease

Introduction

Trypanosoma cruzL the ethiological agent of human Chagas' disease, is a digenetic protozoan that in nature circulates among a large number of triatomine bugs and mammalian species. Intraspecific variations in T. cruzi populations have been shown

*Corresponding author

SSDI 0001-706X(95)00124-7

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at both the biological and molecular level (Brener, 1977; Miles et al., 1977; Morel et al., 1980; Andrade, 1985; Steindel et al., 1993). Isoenzymatic characterization of T. cruzi isolates has shown the existence of distinct parasite populations in both sylvatic and domestic cycles and indicates a possible correlation between zymodeme type and the transmission cycle (Miles et al., 1977).

Although the T. cruzi populations isolated from endemic areas are well charac- terized, there is still little information about the parasite populations in non endemic areas. In the Southern region of Brazil, human Chagas' disease is endemic in the States of Paran~t and Rio Grande do Sul respectively, north and south neighbours of the State of Santa Catarina (Barretto, 1979; Baruffa and Alchntara Filho, 1985). In the State of Santa Catarina, human infection with T. cruzi is rare, due to the absence of domicilar species of triatomines (Ferreira Neto et al., 1971; Schlemper et al., 1985). However, a study of natural infection among sylvatic vectors and reservoirs on the Santa Catarina Island at the city of Florian6polis, situated at 27010 ' and 27050 ' south and 45025 ' and 48035 ' west, showed that 84.5% of Panstrongylus megistus, 66.6% of Rhodnius domesticus and 23.5% of opossums (Didelphis marsupialis) and 4.5% of the rodents (Echimys dasythrix) were positive for T. cruzi (Schlemper et al., 1985). The sylvatic triatomine P. megistus is frequently found associated with the nests of sylvatic mammals. On the other hand, adult triatomines were frequently captured by the local inhabitants inside the houses during the summer months (November to April).

A search for T. cruzi infection in 138 adult triatomines captured inside houses in Florian6polis, State of Santa Catarina, in the last 10 years, showed that 56.5% were infected with the parasite and that human blood was detected in the stomach content of 25 out of 31 insects assayed by the precipitin test (Steindel et al., 1994).

A study of T. cruzi infection rate among opossums (D. marsupialis) on the Arvoredo island, situated at 27°17'10 " south and 48o21'05 " west, 11 km from Santa Catarina Island, showed that 59.1% of the opossums were naturally infected by the parasite and one of them, also presented 72. cruzi in its anal glands (Steindel et al., 1988). In contrast with Santa Catarina island, in the Arvoredo island the high infection rate in opossums cannot be explained by the presence of triatomines once they are scarce there.

The State of Santa Catarina is non endemic for human Chagas' disease and located between two endemic states for this disease. In order to understand the role of the parasite in the epidemiology of human Chagas' disease in a non endemic area, 68 T. cruzi strains isolated from sylvatic vectors an reservoirs from the State of Santa Catarina were biologically and isoenzymatically characterized.

Material and methods

Trypanosoma cruzi isolation Parasites were isolated from nymphs and adults of the triatomine (P. megistus),

from rodents (Echimys dasythrix and Akodon sp) and opossums (D. marsupialis) captured from sylvatic, peridomiciliar and domiciliar environments by different methods: a) mice inoculation with faeces of naturally infected triatomines; b) hemo- culture in LIT (Liver Infusion Tryptose) medium; c) mice inoculation with infected blood from opossums; d) xenodiagnosis and e) LIT medium culture of anal gland

169

contents from naturally and experimentally infected opossums (Table 1). Isolated T. cruzi strains were maintained in the laboratory by monthly blood passages in outbread male albino mice, cyclical passages (mice-triatomine-mice), and mainte- nance in culture with weekly passages.

Parasitemia Each strain was subjected to an adaptation period of 3 passages in mice. Groups

of 10 male albino mice weighting 15-18 g were inoculated i.p. with 5 × 103 blood trypomastigotes. The parasitemia was followed every two days from the 6 th to the 50 th day of infection by fresh blood smears and the number of parasites was estimated as described by Brener (1962). Curves were plotted using the mean parasitemia of

TABLE 1

Host, isolation method, origin and zymodeme of Trypanosoma cruzi strains from the State of Santa Catarina

Host Strain Isolation Origin Zymodeme method

Z1 Z2 Z I + Z 2 ND

P. megistus SC-1 a (02) S~o Jo~o do Sul 01 SC-3 01 SC-49 a (01) Chapec6 01 SC-51 a (01) Arvoredo Island 01

SC-5, SC-7, SC-8, SC-13, a (24) Santa Catarina SC-14, SC-16, SC-17, Island 15 SC-18, SC-19, SC-20, SC-21, SC-41, SC-43, SC-50, SC-57

SC-4, SC-6 02 SC-56, SC-60

SC-2, SC-9, SC-10, SC-11, SC-22

E. dasythrix SC-24 d (01) 0l Akodon sp SC-55 d (01) 01 D. rnarsupialis SC-15, SC-23, SC-25, 09

SC-35, SC-36, SC-38, SC-39, SC-40, SC-44

SC-26 SC-28, SC-32, SC-33, c (04) 04 SC-34 A-88, A-89, A-99, A-100, b (10) 10 A-102, A-103, A-104, A-118, A-121, A-129

D. marsupialis SC-42, SC-59 d (05) Arvoredo Island 02 SC-52, SC-53, SC-54

A-112sg*, A-136, A-139, b (06) 06 A-140sg, A-141, A-202sg

A-112gl*, A-140gl, A-202gl e (03) 03

Total (68) 54 03

02 05

01

03

02 09

ND = not determined; a = mice inoculation with infected faeces of triatomine bugs; b = hemoculture; c = mice inoculation with opossum infected blood; d = xenodiagnosis and e = culture of infected anal glands of opossums. ( ) = number of strains. * = opossum experimentally infected with strain SC-42.

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10 mice. Mortality rate was expressed as a percentage of accumulated deaths within the 60 days period after the inoculation. The virulence of the strains was determined according to prepatent period, parasitemia level and mortality rate. For each strain, 3 parasitemia curves were generated at 3 monthly intervals in order to evaluate parasitemia stability.

Morphology of bloodstream trypomastigotes The morphology of bloodstream forms was studied microscopically on Giemsa

stained blood smears obtained from infected mice at 2 day intervals during a period of 14-18 days of infection and in one naturally infected opossum. The results are expressed as percentage of slender, broad or stout forms among 300 randomly observed parasites (Brener and Chiari, 1963).

Isoenzyme patterns Before the isoenzymes were studied, the strains were maintained in mice for a

period which varied from 1 to 36 months. Three strains (SC-1, SC-3 and SC-6), were isolated every 3 months during one year for isoenzyme determination. After the isolation of the T. cruzi strains from either experimentally infected mice or naturally infected hosts or vectors, the parasites were maintained and amplified in LIT medium at 28 ° C. The parasite culture forms were harvested and washed 3 times by centrifugation, 2000 x g, 4 ° C, 10 min. in KRT (Krebs Ringer Tris, pH 7.4) buffer. The parasite pellet was stored at - 7 0 ° C. The pellet was thawed at room temperature and submitted to osmotic lysis in an enzyme stabilizer (2.0 mM dithiothreitol, 2.0 mM a-amino caproic acid, 2.0 mM Na2 EDTA, pH 7.4) at 4 ° C at a volume ratio 1:1. The lysate was centrifuged at 15 000 x g for 1 h. at 4 ° C. The supernatant, called enzymatic extract, was collected and stored as 15 I~1 beads in liquid nitrogen. The following six enzymes were analysed: alanine aminotrans- ferase (ALAT) [E.C.2.6.1.2]; aspartate aminotransferase (ASAT) [E.C.2.6.1.1]; glucose phosphate isomerase (GPI) [E.C.5.3.1.9]; phosphoglucomutase (PGM) [E.C.2.7.5.1 ]; glucose-6-phosphate dehydrogenase (G6PD) [E.C. 1.1.1.49] and malic enzyme (ME) [E.C.I.I.I.40]. Electrophoretic separation and enzyme staining were as described by Carneiro et al. (1990). Standard T. cruzi zymodemes Z1, Z2 and ZB were used as references.

Results

Trypanosoma cruzi isolation Sixty eight T. cruzi strains were isolated from sylvatic reservoirs and vectors from

the State of Santa Catarina (Fig. 1). Fifty one of them from Santa Catarina Island, 14 from Arvoredo Island and 3 from other counties. Fourteen strains were obtained from nymphs and 14 from adult P. megistus, 2 strains were obtained from rodents (E. dasythrix and Akodon sp) and 38 from opossum (D. marsupialis). Three of the T. cruzi strains from opossum were obtained from the anal glands of natural (A-140 gl and A-202 gl) and experimentally infected (A-112 gl) animals (Table 1).

Parasitemia Thirty six of 48 T. cruzi strains studied produced patent parasitemia in mice with

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PARAN/~ STATE

eARVOREDO ISLAND

Fig. 1. Map of the State of Santa Catarina showing the origin of the Trypanosoma cruzi strains.

distinct parasitemia profiles (Fig. 2 ). Two strains, SC-1 and SC-28, presented a high virulence characterized by a short prepatent period ranging from 6 to 12 days, displaying a rapid raising parasitemia reaching 2.6 × 103 parasites/ml of blood and causing a 100%o mortality within 20-24 days after infection. Nineteen of the strains (SC-5, SC-7, SC-8, SC-13, SC-15, SC-16, SC-17, SC-18, SC-19, SC-20, SC-21, SC-25, SC-33, SC-36, SC-37, SC-38, SC-41, SC-50 and SC-56) displayed a medium virulence with prepatent period ranging from 8 to 16 days. This group presented long term parasitemia with parasite peaks around the 24 th t o 28 th day of infection reaching 0.58 × 103 parasites/ml and a mortality rate varying from 10 to 70%. Fifteen of the strains (SC-3, SC-4, SC-6, SC-14, SC-24, SC-32, SC-34, SC-35, SC-40, SC-43, SC-44, SC-49, SC-55, SC-57 and SC-60) were characterized by a low virulence with prepat- ent period of 12 to 22 days. In this group a peak of parasitemia of 0.23 x 103 parasites/ml occurred around the 24 th to 30 th day of infection and mortality ranged from 0 to 40%. Twelve strains (SC-2, SC-9, SC-10, SC-11, SC-22, SC-23, SC-42, SC-51, SC-52, SC-53, SC-54 and SC-59) displayed a subpatent parasitemia in mice. Maintenance of these strains by 8 to 14 serial mice-triatomine-mice passages for a period of 18-36 months altered neither the virulence nor the parasitemia profile of any of the strains studied.

172

15 - -

A

O o

O

E E

N

O , u

m I I E O a .

z

10

5 -

0

0

r - ]

I ---D--- A

B t C

I I I I I I 1 ' ' ~ 5 10 15 2 0 2 5 3 0 3 5 4 0 4 5 5 0

D a y s a f t e r i n f e c t i o n

Fig. 2. Representative parasitemia curves from sylvatic Trypanosoma cruzi strains from the State of Santa Catarina in albino mice inoculated with 5 x 103 blood trypomastigotes. Each profile represents the mean and the standard deviation of the mean of 3 distinct experiments (n= 10 mice/experiment). The strains chosen as representative for the different parasitemia curves were: SC-1 =high, SC-20=medium and

SC-3 = low parasitemia (y-axis lettering should be "5 mm 3 of blood (10-3)-).

Morphology of bloodstream trypomastigotes Morphological studies of blood trypomastigotes from 7 strains (SC-28, SC-41,

SC-43, SC-44, SC-50, SC-56, SC-60) showed the presence of slender and broad forms varying from 6-40% during the beginning of infection, then replaced by stout forms comprising more than 90% of the parasites. The predominance of stout forms was also observed in one naturally infected opossum.

Isoenzyme pattern Isoenzyme analysis for 6 enzymes was undertaken with 59 strains of T. cruzi,

together with three T. cruzi strains representative of zymodeme Z1, Z2 and ZB. Figure 3a shows the results of one of the enzymes (GPI) and Fig. 3b shows a diagrammatic representation of the results of the six enzymes. Fifty four of 59 strains studied (91.5%) presented the zymodeme Z1 pattern (Table 1). From these strains, 11 were isolated from nymphs captured in the sylvatic environment and 6 from adults of P. megistus collected inside houses, 2 from rodents E. dasythrix and Akodon

GPI

173

(a)

+

t 0

-

ALAT ASAT

m m m m H H H i m m

i N N m m a n

GPI i i i i

[ ] I i D im

PGN G6PD ME

m i i + i

T mmmm imam m n mm m

m m m m m m

(b)

n m m m i i

| , i m i

Fig. 3. Isoenzyme patterns of the Trypanosoma cruzi strains, a representative zymogram for the enzyme glucose phosphate isomerase (GPI) and 5 a diagrammatic representation of the six enzymes studied. For

enzyme and strain abbreviations refer to Material and Methods and Table 1.

sp. and 35 from opossums (D. marsupialis). Of the other 5 strains isolated from adults of P. megistus captured in the intradomicilary environment, 3 strains (SC-3, SC-4 and SC-6) were of zymodeme Z2, two strains (SC-56 and SC-60) were a mixture of zymodeme Z1 and Z2 according to all enzymes studied except malic enzyme. Isoenzyme patterns were not determined for 9 strains.

Discussion

T. cruzi populations have been characterized on the basis of biological behaviour in mice (Brener, 1977; Andrade, 1985). The present study of sylvatic T. cruzi strains from the State of Santa Catarina, an area non endemic for Chagas' disease, showed that 36 out of 48 of the strains produced patent and 12 subpatent parasitemia in mice. Of the 36 strains that produced patent parasitemia, two were of high, 19 of medium and 15 of low virulence in mice. These results confirm previous data derived from T. cruzi strains isolated from humans in endemic areas of Chagas' disease

174

(Andrade, 1974; Schlemper et al., 1983; Lana and Chiari, 1986; Carneiro et al., 1991). In contrast to other regions of the State of Santa Catarina, all 6 strains from Arvoredo Island produced subpatent parasitemia in mice.

Maintenance of the 12 T. cruzi strains that displayed subpatent parasitemia in mice by 8 to 14 cyclic passages (mice-triatomine-mice), did not induce any change in parasitemia or virulence. These results suggest that the subptatent parasitemia in mice may be a characteristic of some strains and express the homogeneity of these parasite populations. However, studies with two clones obtained from the Sylvio X-10 T. cruzi strain showed distinct parasitemia levels and virulence for mice (Postan et al., 1983). In addition, changes in the parasitemia, isoenzyme and k-DNA profiles were observed with one subpatent strain after cyclical (mice-triatomine-mice) pas- sages (Carneiro et al., 1991). Therefore it is possible that the number of cyclic passages that our subpatent strains were submitted were not enough to select more virulent clones. Difficulty in the adaptation to mice of T. cruzi strains isolated from sylvatic reservoirs or from chronic chagasic patients has been described (Deane et al., 1963; Schlemper et al., 1983; Carneiro et al., 1991). Therefore these strains are frequently excluded from studies and little is known about their biological and biochemical characteristics.

In recent years, a new multiplication cycle of T. cruzi in the anal glands of experimentally infected opossums has been demonstrated (Deane et al., 1984). This cycle was also confirmed in naturally infected opossums D. marsupialis from the Amazon region (Naiff et al., 1987), from Arvoredo Island in the State of Santa Catarina (Steindel et al., 1988) and in Didelphis albiventris from Bambui county in the State of Minas Gerais (Fernandes et al., 1989). The ability to infect the anal glands of opossums occurs possibly only with some T. cruzi strains. This is confirmed in the present study by the absence of the parasite in the anal glands of naturally or experimentally infected opossums from Santa Catarina Island. In contrast, para- sites from Arvoredo Island were detected in the anal glands of both naturally and experimentally infected opossums. The distinct behaviour in mice between T. cruzi strains from Santa Catarina Island and from Arvoredo Island probably reflects a heterogeneity between these parasite populations.

A study of sylvatic reservoirs and vectors shows that 23.5% of the opossums (D. marsupialis), 4.5% of the rodents (E. dasythrix), 84.5% of the triatomine (P. megistus) and 66.6% of the/L domesticus were naturally infected by T. cruzi on Santa Catarina Island (Schlemper et al., 1985). A survey for T. cruzi infection in opossums from Arvoredo Island showed that 55.0% presented parasites in the blood and 5.0% also harboured parasites in their anal glands (Steindel et al., 1988). This high infection rate among opossums contrast with the very low number of triatomines found there and suggest that a homogeneous T. cruzi population highly adapted to opossums exists on Arvoredo Island. The absence of T. cruzi infection in all 12 rodents (Oryzomys sp) captured on the same island supported this hypothesis (unpublished data). Differences between T. cruzi strains from the islands of Santa Catarina and Arvoredo were also recently demonstrated by randomly amplified polymorphic DNA (RAPD) profiles (Steindel et al., 1993).

In the present work a morphological study of blood trypomastigotes from 8 sylvatic T. cruzi strains showed a predominance of stout forms. This characteristic was also observed in 3 T. cruzi strains from the neighbouring State of Rio Grande do Sul (Brener and Chiari, 1963) and in 5 sylvatic strains from the State of Santa

175

Catarina (Schlemper et al., 1986). Stout forms of the parasite seems to be more adapted to develop in the insect vector. Therefore this particular character from southern Brazil, might explain the high natural infection rate of sylvatic P. megistus (84.5%) and ~ domesticus (66.6%) from Santa Catarina (Schlemper et al., 1985) and T. infestans (56.6%) from Rio Grande do Sul (Coutinho et al., 1952).

Isoenzymatic characterization of T. cruzi strains has shown that distinct popula- tions are associated with either a sylvatic or a domiciliary transmission cycle of the parasite (Miles et al., 1977; De Luca D'Oro et al., 1993). T. cruzi zymodemes Z1 and Z3 have been found associated with sylvatic vectors and hosts and with some acute human cases of Chagas' disease in Brazil (Miles et al., 1978; Barrett et al., 1980). Isoenzyme profiles of T. cruzi strains isolated from patients, sylvatic and domestic mammalian hosts and insect vectors from Argentina indicated the presence of 12 zymodemes. None of them were identical to the Brazilian zymodemes (De Luca D'Oro et al., 1993). We show here that the analysis of 59 T. cruzi strains from Santa Catarina demonstrated the presence of zymodemes Z1 and Z2. Zymodeme 1 was found in all strains isolated from opossums, rodents, nymphs and from 7 adults of P. megistus, six of them captured in the domiciliar environment. Although reported previously for some T. cruzi Y strain clones (Alves et al., 1993), the zymodeme profiles of strains from Santa Catarina were neither affected by the isolation procedure nor the duration of strain maintenance in mice. Comparison of the electrophoretic profile of T. cruzi strains isolated from blood and anal glands of naturally or experimentally infected opossums showed that these two parasite populations belonged to exactly the same zymodeme. Identical results were also reported with T. cruzi stocks isolated from the blood and anal glands of one naturally infected opossum (D. albiventris) from Bambui (Fernandes et al., 1989).

Only zymodeme Z1 was found among 145 parasite stocks isolated by different methods from 44 naturally infected opossums, D. albiventris, from Bambui city, State of Minas Gerais (Fernandes et al., 1991). On the other hand, nymphs and adults of P. megistus captured in the domiciliar and peridomiciliar environments in the same county were found harbouring both Z 1 and Z2 T. cruzi parasites (Fernandes et al., 1992). In the present study 3 out of 5 T. cruzi strains isolated from sylvatic adults of P. megistus captured inside houses presented the zymodeme 2 and the remaining 2 strains displayed a mixture of Z1 +Z2 in five out of the 6 enzymes analysed. Due to the absence of domiciliar triatomines in the State of Santa Catarina, adults of P. megistus captured inside houses were considered of sylvatic origin. In Brazil, only zymodeme 2 parasites have been isolated from chronic chagasic patients while zymodeme 1 has been found only in some acute infections (Luquetti et al., 1986).

A comparative study of T. cruzi from Brazil and Venezuela showed that in Brazil Z1 and Z2 strains were strongly associated with the sylvatic and the domiciliary cycle respectively while, in Venezuela, Z1 was found in both transmission cycles (Miles et al., 1981). Conversely, a recent study of T. cruzi isolated from wild mammals from the Chaco forest in Argentina, showed that the parasites belonged to a zymodeme closely related to Z2 (Wisnisvesky-Colli et al., 1992).

The State of Santa Catarina is not an endemic area of human Chagas' disease. A recent serological survey among 7374 inhabitants from Santa Catarina Island showed that only 27 (0.36%) presented anti-T, cruzi antibodies in an indirect immunofloure- scence test (Carobrez, personal communication). Due to the absence of domiciliary triatomines, the 3 adults of P. megistus presenting Z2 strains were of sylvatic origin

176

and probably acquired the parasites in the domestic environment from man or domestic mammals. Likewise, the other 2 adults presenting mixed zymodeme infec- tion may indicate that they got possibly infected by Z1 parasites in the sylvatic environment and by Z2 parasites within the domicile or peridomicile. T. cruzi Z2 has been isolated from man, dog and cat in endemic regions of Chagas' disease in Brazil (Miles et al., 1977; Barrett et al., 1980; Fernandes et al., 1992). A serological survey in dogs on Santa Catarina Island showed that 11/300 (3.6%) presented anti- T. cruzi antibodies in an immunoflurescence test (Ishida, personal communication). The infection of dogs may be caused by adults of P. megistus from sylvatic origin that visit the domestic environment as well as by contact with infected opossums. Therefore, the sole presence of Z2 parasites in adults of P. megistus captured inside houses contrasts with the low prevalence of human infection and suggests that dogs may act as a reservoir of T. cruzi Z2 in the peridomestic environment on Santa Catarina Island.

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