ELSEVIER Acta Tropica 68 (1997) 159-173

ACTA TROPICA

Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL

Brener

Bianca Zingales a,,, Maria Elizabeth S. Pereira b Riva P. Oliveira c, Katia A. Almeida d, Eufrosina S. Umezawa d, Ricardo P. Souto a,

Nancy Vargas a, Maria Isabel Cano e, Jos6 Franco da Silveira e, N6dia S. Nehme f, Carlos M. Morel f, Zigman Brener b,

Andrea Macedo c

a Instituto de Quimica, Universidade de Silo Paulo, Caixa Postal 26077, CEP 05599-970 S6o Paulo, Brazil

b Centro de Pesquisas Rene Rachou, FIOCRUZ, Belo Horizonte, Brazil c Instituto de Ci~ncias Biol6gicas, UFMG, Belo Horizonte, Brazil

d Instituto de Medicina Tropical, Universidade de Sgto Paulo, S~o Paulo, Brazil e Escola Paulista de Medicina, UNIFESP, S6o Paulo, Brazil f lnstituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil

Received 17 February 1997; accepted 11 May 1997

Abstract

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28°C in liver infusion-tryptose (LIT) medium is 58 + 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Grace's medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 × 103 parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopuri- nol is inefficient in the treatment of mice experimental infection. The clone infects

* Corresponding author. Tel.: +55 11 8183858; fax: + 55 11 8155579; e-mail: bszodnas@quim.iq.usp.br

0001-706X/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0001-706X(97)00088-0

160 B. Zingales et al./Acta Tropica 68 (1997) 159-173

mammalian cultured cells and performs the complete intracellular cycle at 33 and 37°C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24Sc~ ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed. © 1997 Elsevier Science B.V.

Keywords: Trypanosoma cruzi; Genome project; Reference organism; Molecular typing; Biological characteristics

1. Introduction

In 1994 by initiative of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) the analysis of the Trypanosoma cruzi genome was started. In contrast to other Parasite Genome Projects, the members of the T. cruzi Genome Project decided to select one reference clone (CL Brener) to be used by all laboratories participating in the network. This decision was taken as T. cruzi is an extremely heterogeneous population concerning biolog- ical properties and molecular characteristics (Dvorak, 1984; Zingales and Colli, 1985; Tibayrenc and Ayala, 1988).

Clone CL Brener was isolated by Professor Z. Brener and M.E.S. Pereira (Centro de Pesquisas Ren6 Rachou, FIOCRUZ, Belo Horizonte). This clone derived from the CL strain was chosen since this strain presents important characteristics of T. cruzi: it was isolated from Triatoma infestans, a strictly domiciliary vector (Brener and Chiari, 1963); it is infective to mice (Brener, 1977); it has preferential parasitism of heart and muscle cells (Melo and Brener, 1978); it shows a clear acute phase in accidentally infected humans (Z. Brener, personal communication); and it is highly susceptible to drugs used clinically in Chagas disease (Filardi and Brener, 1987).

Presently approximately 20 laboratories involved in the T. cruzi Genome Project are employing the CL Brener clone. The activities of this network have been recently reported (Zingales et al., 1997) as well as the first analyses of the molecular karyotype of CL Brener (Cano et al., 1995; Henriksson et al., 1995).

In this communication the biological characteristics and the molecular typing of CL Brener clone are presented.

2. Material and methods

2.1. Cultivation of CL Brener and differentiation to trypomastigote metacyclic forms

CL Brener was isolated from the blood of mice infected with the CL strain and cloned on agar plates according to published protocols (Chiari and Camargo,

B. Zingales et al./Acta Tropica 68 (1997) 159 173 161

1984). In some experiments the parental CL strain (Brener and Chiari, 1963) was also employed. Epimastigotes are cultured in LIT (Liver Infusion Tryptose) medium containing 10% of calf serum (Castellani et al., 1967), at 27-28°C, without agitation. Differentiation to metacyclic forms is obtained by incubation of 2 × 10 7

epimastigotes/ml in LIT medium plus 20% (v/v) Grate's Insect medium (Sigma, MO) for 7-10 days. Metacyclics were purified by chromatography on DEAE-cellu- lose (DE-52, Whatman) columns according to published procedures (De Souza, 1983).

2.2. Curves o f parasitemia in mice

Groups of ten male Balb/c mice were inoculated intraperitoneally (i.p.) with 5 x 105 metacyclic trypomastigotes derived from LIT: Grace's medium or with 5 × 103 bloodstream trypomastigotes. Parasitemia was determined microscopically in 5 #1 blood collected from the tail (Brener, 1962).

2.3. Susceptibility to chemotherapeutic agents

The sensitivity to nifurtimox and benznidazole was assessed by a rapid method (Filardi and Brener, 1984). Twenty male albino mice (18-20 g) were inoculated i.p. with 104 blood trypomastigotes of CL Brener. At the peak of parasitemia, 15 mice presenting a similar number of circulating trypomastigotes were selected. Five mice were submitted to a single oral dose of 500 mg/kg of either nifurtimox (3-methyl-N-((5-nitro-2-furanyl)-methylene)-4-thiomorpholi- namine 1,1-dioxide) or benznidazole (N-benzyl-2-nitroimidazole-l-acetamide). Five non-treated infected mice were used as controls. The number of circulating para- sites was scored microscopically in 5 #1 blood collected from the tail (Brener, 1962) 3 and 6 h after drug administration. The percentage of reduction of parasitemia was calculated comparing the number of parasites obtained at each interval of time before and after drug administration. A traditional protocol (Brener, 1962) was used to assess the therapeutic efficiency of ketoconazole (cis- •-acety•-4-(4-)(2-(2•4-dich••r•pheny•)-2-(•H-imidaz••-•-y•methy•)-••3-di•xa•an-4-y•) methoxy)phenyl)piperazine) and allopurinol (1,5-dihydro-4H-pyrazolo(3,4- D)pyrimidin-4-one). Forty male albino mice (18-20 g) were inoculated i.p. with 10 4 blood forms of CL Brener. On day 6 after infection, the animals were divided into three groups: two groups of 15 mice each for the two drugs trials and one group of ten mice as control. Mice received 20 daily consecutive oral doses of 120 mg/kg of ketoconazole or 64 mg/kg of allopurinol. The therapeutic activity was evaluated by blood examination starting from day 6 after drug administration for a total period of 30 days. Parasitemia and mortality were assessed as described (Brener, 1962). Hemoculture analysis was done 30 days after the end of treatment (Filardi and Brener, 1984).

162 B. Zingales et al./Acta Tropica 68 (1997) 159-173

2.4. Infection of mammalian cultured eells

Metacyclic forms of clone CL Brener obtained in LIT: Grace's medium were inoculated in flasks containing confluent monolayers of monkey kidney epithelial cells (LLCMK2) in RPMI medium containing 2% fetal calf serum (FCS), according to published conditions (Zingales et al., 1982). Tissue culture trypomastigotes (TCT) were recovered in the supernatant 7-10 days after infection. The penetration index of TCT of clone CL Brener employing a parasite/cell ratio of 8:1 was determined as described (Zingales et al., 1982). The number of TCT released in the culture medium of cell monolayers incubated at 37 or 33°C was determined.

2.5. Ribosomal RNA gene typing

PCR amplification of a divergent domain of the 24S~ ribosomal RNA (rRNA) gene was achieved with primers D71 and D72 following a published protocol (Souto and Zingales, 1993). The reaction product was analyzed on 7.5% polyacry- lamide gels. The amplified DNA was cloned into the HincII site of M13mpl9 and sequenced with the Sequenase kit (Amersham).

2.6. Isoenzyme analysis and definition of zymodeme group

Epimastigote extracts were prepared according to Romanha et al. (1979). Isoen- zyme analysis was performed according to Romanha et al. (1979) and Bogliolo et al. (1986) for alanine aminotransferase (ALAT); aspartate aminotransferase (ASAT); glucose phosphate isomerase (GPI); glucose-6-phosphate dehydrogenase (G6PD); malic enzyme (ME) and phosphoglucomutase (PGM). The isoenzyme patterns were analyzed by starch gel electrophoresis. Standard T. eruzi zymodemes Z1, Z2, ZB and ZC were used as reference.

2. 7. Sehizodeme analysis

The kinetoplast DNA was purified as described (Gon~alves et al., 1984) and digested with restriction enzymes. DNA fragments were analyzed in 6-10% poly- acrylamide gels which were silver-stained according to Gonqalves et al. (1990).

2.8. Randomly amplified polymorphic DNA (RAPD)

Genomic DNA was extracted as described (Medina-Acosta and Cross, 1993). CL Brener DNA (1 ng) was submitted to PCR amplification with 1.0 unit of Taq DNA polymerase (kind gift of Cenbiot, Brazil) using 6.4 /~M of one of the following primers: 3307 (5'-AGTGCTACGT-3'); 3303 (5'-TCACGATGCA-3'); L15996 (5'-CTCCACCATTAGCACCCAAAGC-3'); M13F (5'-GTTTTCCCAGT- CACGAC-3'); M13R (5'-CAGGAAACAGCTATGCA-3'); 2g t l lF (5'-GGTG- GCGACGACTCCTGGAGCCCG-3'); 2gT l lR (5'-TTGACACCAGCCAACTG- GTAATG-3'). The PCR amplification conditions have been described (Steindel et

B. Zingales et a l . /Acta Tropica 68 (1997) 159-173 163

al., 1993). The RAPD products were analyzed on a 4% polyacrylamide gel and visualized by silver staining (Santos et al., 1993).

2.9. DNA fingerprinting

One microgram of DNA was digested with 20 units of BspRI (an isoschizomer of HaeIII). DNA products were electrophoresed in 0.8% agarose gels in TBE (89 mM Tris-borate/2 mM EDTA, pH 8.0) at 1.5 V/cm for 18-20 h until xylene cyanol marker had migrated 6.5 cm. DNA was transferred to a nylon membrane (Biodyne B) and hybridized to the multilocal Nice probe 33.15 (an oligonucleotide linked to alkaline phosphatase, Cellmark diagnostics), according to Pena et al. (1993). Development was done with Lumiphos 480 (a chemioluminescent substrate for alkaline phosphatase, FMC). After 2 h exposure the autoradiogram was developed.

2. I0. Molecular karyotype

The separation of chromosomal bands from 0.45 to 4.0 Mb was achieved by previously established pulsed field gel electrophoresis (PFGE) conditions (Cano et al., 1995). After electrophoresis, chromosomal bands were transferred to nylon membranes (Hybond N, Amersham) and hybridized to radiolabelled probes as described (Cano et al., 1995).

3. Results

3.1. Growth and differentiation

CL Brener epimastigotes have been subcultured weekly for 100 generations in LIT medium, at 28°C. The doubling time was determined as being 58 _+ 13 h. In the recently isolated clone spontaneous differentiation to metacyclic forms was approx- imately 50%. Differentiation can be increased to 70-80% by incubation of epi- mastigotes in LIT medium containing 20% (v/v) Grace's insect medium, under the conditions described in Section 2.

3.2. Infection in mice

Parasitemia curves (Fig. 1) and mortality were determined in Balb/c mice inoculated with 5 × 105 metacyclic trypomastigotes of CL Brener and of the parental CL strain. Pre-patent periods of 6-7 days were observed for both parasite stocks. A very low parasitemia (2-4 × 10 4 trypomastigotes/ml) was observed for CL Brener up to 28 days after inoculation. CL strain parasitemia started to increase from day 16 after infection, reaching 8 × 105 parasites/ml on day 22. After this period, parasitemia decreased. Forty days after inoculation a cumulative mortality of 20 and 40% was observed, respectively, for the CL Brener and CL strain. Blood

164 B. Zingales et al. /Acta Tropica 68 (1997) 159-173

trypomastigotes were maintained by successive passages in mice. Infectivity of bloodstream forms was assessed following the i.p. inoculation of groups of Balb/c mice with 5 × 1 0 3 forms of EL Brener and CL strain. Parasitemia curves were very similar for both parasite stocks, reaching 2 × 1 0 6 trypomastigotes/ml 15 days after inoculation (Fig. 1). The cumulative mortality of the animals was 100% on day 16 after inoculation with both CL Brener and CL strain.

3.3. Susceptibility to chemotherapeutic agents

Male albino mice inoculated with CL Brener blood forms received 500 mg/kg of nifurtimox or benznidazole. The presence of circulating parasites was microscopi- cally scored 3 and 6 h after drug administration. Fig. 2 indicates a 95% parasite clearance after 6 h treatment with either drug.

The sensitivity of CL Brener to allopurinol and ketoconazole was determined. Parasitemia was scored during treatment (Fig. 3A). The cumulative mortality in treated- and control mice is shown in Fig. 3B. Ketoconazole suppressed the presence of circulating parasites in 12/15 animals. The drug was highly curative since 85% of the infected and treated animals had negative hemocultures 30 and 60 days after examination. This is not the case for allopurinol, that, although promoting a reduction in parasitemia (Fig. 3A), gives a cumulative mortality of 100% 30 days after treatment (Fig. 3B).

2 5 ~-

r

20

E 15

tn

o - 1 0 ± %

0 0

n

ml ~ •

n'" m " l - - •

_- l : - . ~ . - ~ . - - ~ - - 4 - T - ~ - - 4 =

5 10 15 20 25 30

days

Fig. 1. Parasitemia of CL Brener clone (O) and CL strain (O) in Balb/c mice inoculated with: 5 × 10 5 metacyclic trypomastigotes (. • • ) and 5 × l0 3 blood trypomastigotes ( ).

B, Zingales et al. /Acta Tropica 68 (1997) 159-173 165

o t -

8

"~ ..,,.., 0 0 o._O)

0

0

50

100

0 3 6

hours after drug administration

Fig. 2. Percentage of bloodstream parasite clearance in albino mice inoculated with 104 trypomastigotes of CL Brener and treated with 500 mg/kg of benznidazole (e) or nifurtimox ( I ) . Control non-treated mice (0) .

3.4. Infectivity to cultured-mammalian cells

Previous studies with the CL strain indicated that the intracellular amastigote-to- trypomastigote differentiation took place at 33°C but was inhibited at 37°C (Brener et al., 1976). It was investigated whether clone CL Brener also presented this temperature-dependent characteristic. Monolayers of LLCMK2 cells were exposed to a suspension of TCT for 3 h. The percentage of infected cells was 21 + 3 and 53_ 4 at 33 and 37°C, respectively. The interiorization index expressed as the number of parasites per cell was 1.I and 1.3 at 33 and 37°C, respectively.

The number of TCT liberated in the culture medium was determined. On the fifth day after infection 0.1-0.2 × 106 parasites/ml were observed at either temperature. The yield of TCT increased in the subsequent days reaching maxima values of 2.7 × 106 parasites/ml on day 7 in cultures maintained at 33°C and 3.5 x 106 parasites/ml on day 8 in cultures incubated at 37°C.

3.5. Molecular typing

We have previously shown that two major phylogenetic lineages of T. cruzi can be defined by PCR amplification of a divergent domain of the 24S~ rRNA gene (Souto et al., 1996). Two possible amplification products are obtained: DNA fragments of 125 bp (lineage 1) or 110 bp (lineage 2). Employing the PCR assay previously described (Souto and Zingales, 1993) it was concluded that CL Brener belongs to lineage 1 (data not shown). The 125 bp DNA fragment was sequenced and data have been deposited in GenBank under the accession number U73959.

A

B

6

5

i 4 3

1

0 0 5 10 15 20

days after treatment

100

90 80 70

"~ 60

~ 30

20

10

0 [ ~ ' '

166 B. Zingales et al. /Acta Tropica 68 (1997) 159-173

6 12 13 14 16 18 19 20 24 30

days after treatment

Fig. 3. Susceptibility of CL Brener to ketoconazole and allopurinol. Male albino mice were infected with 10 4 blood forms of CL Brener and treated from day 6 to 25 with daily doses of ketoconazole (120 mg/kg) or allopurinol (64 mg/kg). (A) Parasitemia curves: ketoconazole (O), allopurinol (11) and control (0). (B) Cumulative mortality: control (open bars), allopurinol (solid bars) and ketoconazole (shaded

bars).

B. Zingales et al. / Acta Tropica 68 (1997) 159-173

A B C D E

bp q ~ w - = =

167

.oo-iI . ' 3 5 0 - - "

Fig. 4. Schizodeme analysis of kinetoplast DNA minicircles of CL Brener digested with: (A) EcoRI; (B) Haelll; (C) Hinfl; (D) Taql; (E) Kpnl. Molecular weight markers (in bp) are indicated.

Analysis of six isoenzymes and comparison to standard T. cruzi zymodemes revealed that CL Brener belongs to zymodeme ZB (Romanha et al., 1979). Identical isoenzyme profiles were obtained for the CL Brener and CL strain (data not shown).

The profiles of five restriction endonucleases products of kinetoplast DNA minicircles (schizodeme analysis) of CL Brener are shown in Fig. 4. EcoRI, HaelII, HinfI and TaqI give a high number of fragments smaller than minicircle size being suitable for typing purposes (Morel et al., 1980).

The RAPD profiles of CL Brener using seven different primers are shown in Fig. 5. A complex multiband pattern was observed for each primer. On average 18 ___ 7 bands were detected, varying in intensity and size. With 3303 primer the lowest number of bands (12 bands) was observed, while 2gtl 1R primer gave the highest score (25 bands).

The human multilocus probe 33.15 (Jeffreys et al., 1985) recognizes multiple minisatellites regions in nuclear DNA of T. cruzi generating strain-specific banding patterns (Macedo et al., 1992). The DNA fingerprinting of CL Brener obtained after hybridization to the 33.15 probe showed a complex multiband profile with approximately 20 bands from 2 to 23 kb. The banding pattern remains stable during prolonged parasite cultivation (from 10 to 70 generations) (Fig. 6). Stability of isoenzymatic profiles, schizodeme and RAPD patterns was also observed up to 100 generations (data not shown).

168 B. Zingales et a l . /Acta Tropica 68 (1997) 159-173

3.6. Stability o f molecular karyotype

The molecular karyotype of CL Brener has been established using more than 20 T. cruzi homologous probes (Cano et al., 1995). To verify the stability of the chromosomal location and of physical linkage of some genes after 100 generations of subcultivation, Southern blots of the chromosomal bands separated by PFGE were hybridized to radiolabelled probes. Hybridization to a T. brucei telomere sequence (Van der Ploeg et al., 1984) and to a T. cruzi interspersed repetitive

eq 0

b p

1 , 7 2 2 -

1,107 -

9 8 4 -

8 6 1 -

7 3 8 -

6 1 5 -

4 9 2 -

3 6 9 -

2 3 6 -

1 2 3 - i

Fig. 5. RAPD profiles of CL Brener obtained with seven arbitrary primers (specified on the top of the figure). Molecular weight markers (in bp) are indicated.

B. Zmga&setal./ActaTropica68(199D 159-I~

generations

kb

23.1 -

10 20 30 40 50 60 70

169

9 . 4 -

6 . 6 -

4 . 4 -

2 . 3 -

Fig. 6. D N A fingerprinting of CL Brener cultivated for several generations. Total D N A was digested with BspRI. The Southern blot was hybridized to the 33.15 multilocal probe linked to alkaline phosphatase. Molecular weight markers (in bp) are indicated.

element (C6) reproduced the chromosomal banding pattern obtained before (Cano et al., 1995). It was also confirmed the chromosomal assignment of several genes encoding antigen proteins (B12, B13, H49, JL8), of the fl-tubulin locus and the ribosomal RNA genes. Previously established gene linkage groups (B12/B13; H49/ JL8; 24S~/24Sfl ribosomal RNA) were also maintained after 100 generations. Comparison to published data (Cano et al., 1995) indicate that the molecular karyotype of CL Brener is stable.

4. Discussion

To our knowledge this is the first extensive characterization of a clone of T. cruzi concerning biological and molecular parameters. CL Brener is the reference organ- ism used in the T. cruzi Genome Project and was chosen a priori in a Planning Meeting held in 1994 (cf Zingales et al., 1997). Following the biological character-

170 B. Zingales et al./Acta Tropica 68 (1997) 159-173

ization here presented, we are confident that CL Brener was a right choice since this organism shows important characteristics of T. cruzi concerning infectivity to the mammalian host, ability to differentiate in vitro and susceptibility to chemothera- peutic agents used in Chagas disease. Furthermore it presents stable genetic markers that allow molecular typing of the parasite.

Doubling times ranging from 22 to 200 h have been reported for different T. cruzi clones (Dvorak, 1984). For CL Brener the doubling time of epimastigotes calculated from measures taken along 100 generations of continuous subculturing is 58 + 13 h. The high standard deviation is due to the different batches of the complex non-defined LIT medium used. Differentiation to metacyclic trypomastig- otes can be induced by incubation of epimastigotes in LIT-20% Grace's medium. Differentiation indices up to 70-80% are obtained with the recently isolated CL Brener clone. However, after prolonged subcultivation of epimastigotes, this value is drastically reduced to 10-30%.

Metacyclic forms of CL-Brener give very low parasitemia in Balb/c mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10 3.

CL Brener is highly susceptible to nifurtimox and benznidazole, two drugs used clinically in the acute phase of Chagas disease (Brener et al., 1993). Ketoconazole, an antifungic drug whose activity against T. cruzi has been previously described (Raether and Seidenath, 1984; McCabe et al., 1987), is also efficient in the elimination of CL Brener in experimental infection. However, previous studies indicate that administration of ketoconazole to chronic chagasic patients at doses used in the treatment of deep mycosis failed to induce cure as demonstrated by parasitological and serological tests (Brener et al., 1993). Allopurinol, an analogous of hypoxantin, was inefficient in the treatment of the experimental infection by CL Brener since a cumulative mortality of 100% of the animals was observed 10 days after the end of treatment. These data should be taken into consideration in accidental contamination with CL Brener.

Recently, the efficacy of a new drug D0870, a bis-triazole derivative which inhibits sterol biosynthesis, has been reported in experimental models of Chagas disease (Urbina et al., 1996). In the future, this drug should be tested for CL Brener.

An inhibitory effect of temperature on the intracellular differentiation of the CL strain was described (Brener et al., 1976). The transformation amastigote to trypomastigote readily occurred at 33°C but was strongly inhibited at 37°C. This phenomenon was not observed with other strains (Y, Gilmar, G) where differentia- tion to intracellular trypomastigotes occurred at both temperatures. Data presented herein indicate that clone CL Brener is able to penetrate and differentiate in cultured-mammalian cells at 33 and 37°C. The thermosensitivity observed for the CL strain suggests that the original population isolated from the insect vector was heterogeneous, with predominance of temperature-sensitive clones.

The characterization of CL Brener at the genotype level has been done using several independent markers. The typing approach based on PCR amplification of a 24Se rRNA sequence indicates that CL Brener belongs to T. cruzi lineage 1

B. Zingales et al. /Acta Tropica 68 (1997) 159-173 171

(Souto et al., 1996). Zymodeme ZB (Romanha et al., 1979) was assigned to CL Brener following the analysis of six isoenzymes. The molecular typing of CL Brener was also accomplished by schizodeme analysis, RAPD analysis using arbitrary- primed PCR and by DNA fingerprinting. For each typing approach, the patterns obtained do not change after prolonged parasite subcultivation. Therefore, either typing technique could be used for periodical verification of the authenticity of CL Brener culture and the chosen approach will depend on the laboratory facilities. For zymodeme determination, a considerable mass of parasites is required. This is not the case for RAPD analysis where a small amount of DNA (1 ng) is employed. However, it has been shown that the band pattern obtained by RAPD varies greatly with the source of Taq DNA polymerase and the thermocycler apparatus used (Penner et al., 1993). As a consequence, each laboratory has to generate its own RAPD profile of CL Brener using one or more arbitrary primers that can be easily obtained from different manufacturers or be synthesized. The typing sensitiv- ity and stability of DNA fingerprinting and schizodeme is very good. However, since nuclear DNA constitutes the major interest in the T. cruzi Genome Project and the alkaline phosphatase-linked 33.15 probe is now commercially available, we propose that the DNA fingerprinting typing approach here described should be used to verify the authenticity of CL Brener clone.

Previous characterization of the molecular karyotype of CL Brener identified some genes that have unique chromosomal locations and that could be used as specific mapping tools for the identification of single chromosomes (Cano et al., 1995; Henriksson et al., 1995). The physical linkage of some genes was also established. In the present study the karyotype stability of CL Brener was confi- rmed using some T. cruzi cloned sequences.

The Trypanosoma cruzi Genome Network has already constructed large fragment genome libraries of CL Brener in yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid vectors. Furthermore, many expressed sequence tags (ESTs) and chromosome markers are available (Zingales et al., 1997). Data originated in the T. cruzi Genome Project are available from the WWW server at the address http://www.dbbm.fiocruz.br/genoma/tcruzi/tcruzi.html. For those researchers interested in joining the T. eruzi Genome Project or to perform studies with CL Brener, the clone can be obtained from a Reference Laboratory at the University of Silo Paulo, Brazil (B.Z. E-mail: bszodnas@quim.iq.usp.br).

Acknowledgements

This work received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases; the Fundagfio de Amparo ~t Pesquisa do Estado de S~o Paulo (FAPESP); the Funda~ao de Amparo

Pesquisa do Estado de Minas Gerais (FAPEMIG); the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) and the Financiadora de Estu- dos e Projetos (FINEP).

172 B. Zingales et a l . /Acta Tropica 68 (1997) 159-173

References

Bogliolo, A.R., Chiari, E., Silva-Pereira, R.O., Silva-Pereira, A.A., 1986. A comparative study of Trypanosoma cruzi enzyme polymorphism in South America. Brazilian J. Med. Biol. Res. 19, 673-683.

Brener, Z., 1962. Therapeutic activity and criterion of cure in mice experimentally infected with Trypanosoma cruzi. Rev. Inst. Med. Trop. $5o Paulo 4, 389-396.

Brener, Z., Chiari, E., 1963. Variag6es morfol6gicas observadas em diferentes amostras de Trypanosoma cruzi. Rev. Inst. Med. Trop. Sho Paulo 5, 220-224.

Brener, Z., Golgher, R., Bertelli, M.S., Teixeira, J.A., 1976. Strain-dependent thermosensitivity influenc- ing intracellular differentiation of Trypanosorna cruzi in cell culture. J. Protozool. 23, 147-150.

Brener, Z., (Ed.), 1977. Intraspecific variation in Trypanosoma cruzi: two types of parasite populations presenting distinct characteristics. In: Chagas' disease. Proceeedings of the International Symposium of the Fifth International Congress Protozoology. Pan American Health Organization, Scientific Publication, Washington, 347, pp. 11-21.

Brener, R., Cangado, J.R., Galv~o, L.M.C., da Luz, Z.M.P., Filardi, L.S., Pereira, M.E.S., Santos, L.M.T., Canqado, S.B., 1993. An experimental and clinical assay with kecotonazole in the treatment of Chagas disease. Mem. Inst. Oswaldo Cruz 88, 149 153.

Cano, M.I., Gruber, A., Vazquez, M., Cort6s, A., Levin, M.J., Gonz~ilez, A., Degrave, W., Rondinelli, E., Zingales, B., Ramirez, J.L., Alonso, C., Requena, J.M., Silveira, J.F., 1995. Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi Genome Project. Mol. Biochem. Parasitol. 71, 273-278.

Castellani, O., Ribeiro, L.V., Ferreira, J.F., 1967. Differentiation of Trypanosoma cruzi in culture. J. Protozool. 14, 447-451.

Chiari, E., Camargo, E.P., 1984. Culturing and cloning of Trypanosoma cruzi. In: Morel, C.M. (Ed.), Genes and Antigens of Parasites--A laboratory Manual. Funda~o Oswaldo Cruz, Rio de Janeiro, pp. 23-26.

De Souza, M.A., 1983. A simple method to purify biologically and antigenically preserved bloodstream trypomastigotes of Trypanosoma cruzi using DEAE cellulose columns. Mem. Inst. Oswaldo Cruz 78, 317-333.

Dvorak, J.A., 1984. The natural heterogeneity of Trypanosoma cruzi: biological and medical implica- tions. J. Cel. Biochem. 24, 357-371.

Filardi, L.S., Brener, Z., 1984. A rapid method for testing in vivo the susceptibility of different strains of Trypanosoma cruzi to active chemotherapeutic agents. Mem. Inst. Oswaldo Cruz 79, 221-225.

Filardi, L.S., Brener, Z., 1987. Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease. Trans. R. Soc. Trop. Med. Hyg. 81, 755-759.

Gonqalves, A.M., Nehme, N.S., Morel, C.M., 1984. Trypanosomatid characterization by schizodeme analysis. In: Morel, C.M. (Ed.) Genes and Antigens of Parasites--A laboratory Manual. Fundaq~o Oswaldo Cruz, Rio de Janeiro, pp. 95-109.

Gon~alves, A.M., Nehme, N.S., Morel, C.M., 1990. An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels. Mem. Inst. Oswaldo Cruz 85, 101-106.

Henriksson, J., Porcel, B., Rydaker, M., Ruiz, A., Sabaj, V., Galanti, N., Cazzulo, J.J., Frasch, A.C.C., Pettersson, U., 1995. Chromosome specific markers reveal conserved linkage groups in spite of extensive chromosomal size variation in Trypanosorna cruzi. Mol. Biochem. Parasitol. 73, 63-74.

Jeffreys, A.J., Wilson, V., Thein, S.L., 1985. Hypervariable 'minisatellite' regions in Human DNA. Nature 314, 67-73.

Macedo, A.M., Martins, S.M., Chiari, E., Pena, S.D.J., 1992. DNA fingerprinting of Trypanosoma cruzi: a new tool for characterization of strains and clones. Mol. Biochem. Parasitol. 55, 147 154.

Medina-Acosta, E., Cross, G.A.M., 1993. Rapid isolation of DNA from trypanosomatid protozoa using a simple 'mini-prep' procedure. Mol. Biochem. Parasitol. 59, 327-330.

McCabe, R.E., Remington, J.S., Aratijo, F.G., 1987. Ketoconazole promotes parasitological cure of mice infected with Trypanosoma cruzi. Trans. R. Soc. Trop. Med. Hyg. 81, 613-615.

B. Zingales et al. /Acta Tropica 68 (1997) 159-173 173

Melo, R.C., Brener, Z., 1978. Tissue tropism of different Trypanosoma cruzi strains. J. Parasitol. 64, 475-482.

Morel, C., Chiari, E., Camargo, E.P., Mattei, D.M., Romanha, A.J., Simpson, L., 1980. Strains and clones of Trypanosoma cruzi can be characterized by restriction endonucleases fingerprint of kinetoplast DNA molecules. Proc. Natl. Acad. Sci. USA 77, 6810-6814.

Pena, S.D.J., Santos, P.C., Campos, M.C.B.N., Macedo, A.M., 1993. Paternity testing in Brazil by DNA fingerprinting with multilocus probes F10. Cirncia e Cultura 45, 236 240.

Penner, G.A., Bush, A., Wise, R., Kim, W., Domier, L., Kasha, K., Laroche, A., Scoles, G., Molnar, S.J., Fedak, G., 1993. Reproducibility of Random Amplified Polymorphic DNA (RAPD) Analysis among Laboratories. PCR Methods and Applications. Cold Spring Harbor Laboratory Press, New York, pp. 341-345.

Raether, W., Seidenath, H., 1984. Ketoconazole and other potent antimycotic azoles exhibit pronounced activity against Trypanosoma cruzi, Plasmodium berghei and Entamoeba histolytica in vivo. Z. Parasitenjd. 70, 135-138.

Romanha, A.J., Pereira, A.A.S., Chiari, E., Kilgour, V., 1979. Isoenzyme patterns of cultured Try- panosoma cruzi: changes after prolonged subculture. Comp. Biochem. Physiol. 62B, 139 142.

Santos, F.R., Pena, S.D.J., Epplen, J.T., 1993. Genetic population study of Y-linked tetranucleotide repeat DNA polymorphism with a simple non-isotopic technique. Hum. Genet. 90, 655 656.

Souto, R.P., Zingales, B., 1993. Sensitive detection and strain classification of Trypanosoma cruzi by amplification of a ribosomal RNA sequence. Mol. Biochem Parasitol. 62, 45-52.

Souto, R.P., Fernandes, O., Macedo, A.M., Campbell, D.A., Zingales, B., 1996. DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol. Biochem. Parasitol. 83, 141-152.

Steindel, M., Dias Neto, E., Menezes, C.L.P., Romanha, A.J., Simpson, A.J.G., 1993. Random amplified polymorphic DNA analysis of Trypanosoma cruzi strains. Mol. Biochem. Parasitol. 60, 71 80.

Tibayrenc, M., Ayala, F., 1988. Isoenzyme variability in Trypanosoma cruzi, the agent of Chagas' disease: genetical, taxonomic and epidemiological significance. Evolution 42, 277-292.

Urbina, J.A., Payares, G., Molina, J., Sanoja, C., Liendo, A., Lazardi, K., Piras, M.M., Piras, R., Perez, N., Wincker, P., Ryley, J.F., 1996. Cure of short- and long-term experimental Chagas' disease using D0870. Science 273, 969 971.

Van der Ploeg, L.H.T., Liu, A.Y.C., Borst, P., 1984. Structure of the growing telomeres of try- panosomes. Cell 35, 459 468.

Zingales, B., Andrews, N.W., Kuwajima, V.Y., Colli, W., 1982. Cell surface antigens of Trypanosoma cruzi: possible correlation with the interiorization process in mammalian cells. Mol. Biochem. Parasitol. 6, 111 - 124.

Zingales, B., Colli, W., 1985. Trypanosoma cruzi: Interaction with host cells. Curr. Topics Microbiol. Immunol. 117, 129-152.

Zingales, B., Rondinelli, E., Degrave, W., Franco da Silveira, J., Levin, M., Le Paslier, D., Modabber, F., Dobrokhotov, B., Swindle, J., Kelly, J.M., Aslung, L., Hoheisel, J.D., Ruiz, A.M., Cazzulo, J.J., Pettersson, U., Frasch, C.A., 1997. The Trypanosoma cruzi initiative. Parasitol. Today 13, 16-22.