Identification of Trypanosoma cruzi isolates

Isolates were analysed from all the geographical regions in Chile where Chagas disease occurs (Table 1). Isolates from the northern political regions of Chile, I and II (18°S–23°S); were from T. infestans and chronic chagasic patients. Isolates from regions III and IV (25°S–31°S), where the disease is most endemic in Chile, also included stocks from the vector Mepraia spinolai. Isolates from the Southern region V and the Metropolitan region (31°S–34°S) were from chronic patients with T. infestans and M. spinolai. The experimental results obtained after hybridization of the digested kDNA from each isolate with DNA probes are shown in Figs 1 and 2, and summarized in Table 1.

Fig. 1. Detection of Trypanosoma cruzi isolates. kDNA from each isolate was digested with HaeIII and run on 2% agarose gels (A). The blotted gels were hybridized with NR probe (B). The results are from different gels, and hence RFLPs are not identical. a, b, c, and d represent the full size, ¾, ½, and ¼ minicircle, respectively. The isolate SPA9 gave a RFLP of partially degraded minicircles.

Fig. 2. Detection of Trypanosoma cruzi isolates. kDNA from each isolate was digested and electrophoresed as in Fig. 1. Ethidium bromide staining is shown (A, B, and C). The blotted gels were hybridized with v195, CBB, and sp104 probes, respectively (D, E, and F). The molecular weight markers are the same as in Fig. 1. *Represent isolates which cross-reacted with two different probes. The isolates CHI22, and spTi9 gave RFLP with partially degraded minicircles.

Parasites were obtained from chagasic patients by xenodiagnosis with Triatoma infestans followed by cultivation of the parasites in liquid medium l (Diamond, 1968), from the whole digestive tract or faeces of the insect vector.

Schizodeme analysis

Kinetoplast DNA of parasites were obtained as previously described (Sanchez et al. 1993). kDNA samples were digested to completion with an excess of restriction endonuclease according to the manufacturer's buffer conditions. The digestion products were electrophoresed in a 4·5–10% polyacrylamide gel gradient and stained with silver nitrate, as previously described (Goncalves, Nehme & Morel, 1990).

Mouse infection

Metacyclic trypomastigote forms were obtained by metacyclogenesis from epimastigotes grown in Diamond's medium supplemented with calf serum as described (Wallace et al. 2001). These trypomastigote forms were inoculated into irradiated (450 Rad.) Balb/c mice weighing 20–22 g. From these mice normal male mice were infected with each isolate by intraperitoneal inoculation of blood trypomastigote forms collected at the peak of parasitaemia. Several passages (4–6) were used to adapt each isolate to mice, producing a patent infection. The parasites were re-isolated by haemocultivation in Diamond's medium supplemented with 10% foetal calf serum, and then grown to a final yield of approximately 10⁹ cells in exponential growth for schizodeme re-analysis.

kDNA purification, DNA probes and hybridization conditions

HVRm from 4 different T. cruzi clones were used as probes to determine by Southern analysis with total kDNA the parasite populations infecting insect vectors and chagasic patients in Chile. Construction of subspecific HVRm probes for T. cruzi clones was performed as previously described (Veas et al. 1991). These fragments, close to 250 bp in size, do not contain the constant region that can cross-hybridize with minicircles of other T. cruzi genotypes (Solari et al. 2001). Each probe presents a great variety of different molecules, even though they are very similar in sequence since minicircle heterogeneity exists within a single parasite clone (Macina et al. 1986). This is an advantage over using cloned fragments as probes because in the latter cases only molecules of the same minicircle sequence class are detected (Macina et al. 1985). Thus it is possible, using HVRm probes, to detect similarities among genetically related groups of parasites as previously demonstrated (Breniere et al. 1998). A very good correspondence between hybridization patterns and lineages determined by isoenzyme characterization has been found (Veas et al. 1991; Solari et al. 2001). Therefore the method is validated for epidemiological purposes, using total kDNA as probes. This association also has been found previously (Macina et al. 1987). However, we cannot completely rule out the possibility that some parasites would be recognized by several probes, due for exchange to evolutionary convergence of sequences or to horizontal transfer of minicircles among lineages.

kDNA purification was performed as described before (Goncalves et al. 1984). kDNA was digested with HaeIII, electrophoresed on 2% agarose gels, further blotted onto nylon membranes, and hybridized with the random-primed labelled DNA probe. DNA probes NR, CBB and sp 104 corresponding to HVRm of the parasite were prepared and used as already described (Veas et al. 1991; Solari et al. 2001). T. cruzi clone v195 was used here for the first time. The polymerase chain reaction was carried out in order to generate selectively important amounts of the HVRm from the kDNA minicircles. Oligonucleotides were selected from constant regions of the minicircle in order to anneal sites flanking the variable region. An artificial restriction site was introduced close to the 3′ end of each oligo primer, allowing a fast and easy purification of HVRm ready to use as a probe. High stringency hybridization was used: 55 °C in 2×SSC, washed at 55 °C in 0·1×SSC. To achieve good signals in an overnight exposure, preparations were hybridized in a solution (10 ml) containing 2×10⁶ cpm of ³²P-labelled DNA probe (specific activity 50–80×10⁶ cpm/μg DNA), or using the alkaline phosphatase chemiluminescent detection method (Amersham, England).