Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 96, Num. 2, 2001, pp. 251-256

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 96(2) Feb. 2001, pp. 251-256

Relationship between BiologicaI Behaviour and Randomly Amplified Polymorphic DNA Profiles of Trypanosoma cruzi Strains

Rafael A Martínez-Díaz/⁺, José A Escario*, Juan J Nogal-Ruiz*, Alicia Gómez-Barrio*

Departamento de Medicina Preventiva (Parasitología), Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo s/n, 28029 Madrid, España *Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, España

⁺Corresponding author. Fax: +34-1-397.5353. E-mail: rafael.martinez@uam.es

Received 17 February 2000
Accepted 4 October 2000

Code Number: oc01037

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.

Key words: Trypanosoma cruzi - strains - characterization - randomly amplified polymorphic DNA (RAPD)

Attempts to correlate biological, biochemical and genetic characteristics of Trypanosoma cruzi isolates with the clinical variability of Chagas disease have been reported (Gonzalez et al. 1995, Montamat et al. 1996). Studies of natural populations of the parasite have revealed interesting data. Tibayrenc et al. (1986), Tibayrenc and Ayala (1987) and Laurent et al. (1996) suggest a clonal structure in T. cruzi by isoenzymes analysis and kDNA sequences. Miles et al. (1980), Bogliolo et al. (1986) and Carneiro et al. (1991) define three zymodemes in several surveys performed in Brazil by using immunoenzymatic markers. Steindel et al. (1993) suggest the existence of a great number of individual genotypes into the species, based on the diversity of randomly amplified polymorphic DNA (RAPD) profiles. Bogliolo et al. (1996) even report evidences of genetic recombination from polymorphisms in T. cruzi. The presence of principal clones in some geographic areas, showing a high percentual of similarity by restriction fragment length polymorphism (RFLP) analysis (Campos et al. 1999) could also contribute to the pattern of clinico-pathological manifestations of Chagas disease.

The present work attempts to correlate the heterogeneity of the biological characteristics shown by several strains of the parasite with their RAPD profiles. For this purpose, unweighted pair groups method analysis (UPGMA) phenograms are built both with biological and genetic data.

MATERIALS AND METHODS

Parasites and DNA preparation - Four strains of T. cruzi were used to DNA extraction: Bolivia strain (Funayama & Prado Junior 1974), RAL strain (Ribeiro et al. 1993), GM strain (Prado Junior et al. 1992a) and MC strain (Prado Junior et al. 1992b). After infecting groups of NMRI mice by the intraperitoneal route, samples of blood obtained by cardiac puncture were cultured in glass tubes with 5 ml LIT medium (10% heat-inactivated fetal calf serum) and two subcultures were made before beginning the assays.

Biological characteristics of these four strains, as well as Y strain have been previously determined and a summary is indicated in Table I. As can be seen, some parameters for Tulahuen strain (Segura et al. 1980), as growth of epimastigotes in LIT and susceptibility to drugs (data not published), have been included.

To DNA preparation, parasites were cultured for 7-9 days in LIT medium at 28ºC using 80 ml glass flasks. Cultures were centrifuged at 900 g 10 min at 4°C and washed three times in PBS (phosphate buffered saline), pH 7.4. Pellet of parasites was resuspended in 50 mM Tris-HCl/ 50 mM EDTA/100 mM NaCl/0.5% SDS, pH 7.6 and incubated together with 20 mg/ml proteinase K for 2 h at 55°C. Following two phenol extractions and 3M ethanol-acetic acid precipitation, DNA was resuspended in 10 mM Tris-HCl/1 mM EDTA pH 8.0 (TE). Valuation was made in spectrophotometer at 260-280 nm to determine the purity rate.

Promastigotes of cultures of Leishmania infantum and larvae of Trichinella spiralis isolated by digestion were processed as above to extraction of DNA.

PCR amplification and polyacrilamide gel electrophoresis - Each amplification reaction was done in a final volume of 10 µl containing 0.5 units of Replitherm DNA polymerase (Epicentre Technologies)/ 2.5 mM of each dNTP/ 200 mM Tris-ClH, pH 8.3/ 1M ClK/ 0.1% Tween 20/ 0.1% NP-40/ 25 mM Cl₂Mg together with 0.01 µg of template DNA. The primers B1 (5´-CTTTCGCTCC-3´), B2 (5´-CTGCTGGGAC-3´) and Z3 (5´-CAGCACC GCA-3´), from Operon Technologies, were arbitrarily selected. Amplification was made in Genetic Thermal Cycler GTC-2 (Precision Scientific) with an initial denaturation at 95°C for 5 min, following two cycles at 30°C for 2 min for annealing, 72°C for 1 min for extension and 30 sec at 95°C for denaturation; next, 33 cycles at 40°C for 2 min, 72°C for 1 min and 30 sec at 95°C. In the final cycle the extension step was for 5 min (Steindel et al. 1993). Amplified DNA samples were subjected to electrophoresis through a 4% polyacrilamide gel. Gels were fixed with 10% ethanol/ 0.5% acetic acid for 5 min, stained with 0.2% silver nitrate for 10-20 min and reduced with NaOH and formaldehyde until appearance of bands.

Data analysis - Data obtained both from biological parameters and RAPD profiles were subjected to the same statistical procedure. NTSYS (Applied Biostatistics Inc., Setauket, NY) informatic program was used. This program allows the establishment of possible strain groups based on the similarities and differences observed. Briefly, we elaborated matrices of similarity assigning 0 or 1 values to the results of the assays. These matrices were processed using the Simple Matching coefficient (SM; Sneath & Sokal 1973) and phenetic trees were plotted using the UPGMA. Although artificial, UPGMA dendogram constructed from biological parameters, makes comparison of results easier.

RESULTS

Criteria from biological parameters used for performing the matrix of similarity are shown in Table II. By the Simple Matching coefficient, the matrix of similarity (Table III) was used to cluster analysis, resulting the UPGMA phenogram indicated in Fig. 1.

Regarding to RAPD profiles, 0 or 1 values of the matrix are based in the presence or absence of bands in the electrophoretic patterns (Figs 2, 3, 4, respectively). As above, the resulting matrix of similarity coefficients is shown in Table IV and the corresponding UPGMA phenogram is shown in Fig. 5.

DISCUSSION

The extent of heterogeneity of T. cruzi isolates has been widely demonstrated from biological and genetic studies. Early isoenzymatic analysis or studies with restriction endonucleases have allowed the establishment of groups of T. cruzi strains according to different characteristics as geographical source or pathogenicity (Miles et al. 1978, 1980, Tibayrenc & Desjeux 1984). T. cruzi taxon is presently subdivided into two major phylogenetic lineages (Tibayrenc 1995, Souto et al. 1996), each retaining considerable heterogeneity. Zymodeme Z1 stocks belong to lineage 2, which cycles in a sylvatic environment but has the capacity to colonize human settlements, whereas zymodeme Z3, which also cycles in a sylvatic environment, is clustered into lineage 1 (Tibayrenc 1995, Souto et al. 1996). Zingales et al. (1998) found strong association of lineage 1 with the domestic cycle, though in the sylvatic cycle both lineages circulate equally.

In this paper, diversity of individual genotypes by RAPD analysis and its relation to the previously determined biological behaviour is approached. For this purpose, cluster analysis using the UPGMA was performed. A phenogram based on band sharing was constructed to show genetic relationships among the strains studied. By RAPD analysis and isoenzyme studies, Steindel et al. (1993) and Lopez-Olmos et al. (1998) have found similarities among isolates from the same geographical source. In our case, the variability found is not related to factors previously known as geographical or biological source or pathogenicity.

Because of the number of strains characterized, results obtained must be considered as preliminary data; however, an interesting and unexpected finding is the same association among strains, both by their biological characteristics and their RAPD profiles. So, the phenogram constructed from biological data revealed that there are two principal lines; the first one clusters Bolivia and GM strains, and the second one shows two groups of pairs: RAL/MC and Y/Tulahuen.

When RAPD fingerprintings were used in a cluster analysis of combined electrophoretic patterns, the phenogram allowed the separation of two groups based on their divergence: Trichinella and the protozoa. The latter shows clear differences among T. cruzi strains and the reference sample from Leishmania. These results emphasize the usefulness of RAPD to the study of parasite populations. According to Steindel et al. (1993), the technique is ideal for searching genetic markers for the various biological characteristics of the parasite and the clinical variations in the disease it causes.

As above, phenon line of T. cruzi strains shows a new division in two pairs: Bolivia/GM and RAL/MC strains.

After all, the two methods of analysis led us to draw the same conclusion. It seems obvious that the differences in the biological behaviour are related to variations in the genomic constitution expressed by the amplification of DNA fragments. However, having in mind the low number of strains analyzed, it would be necessary to perform new assays with more sensitive techniques, or increase the number of compared strains in a similar study. Furthermore, the establishment of clones from the strains must be the next step in the continuation of this study. After the enlargement of these studies we will be able to check if the two major branches found in this work, both for biological parameters and RAPD profiles, correspond to the two major phylogenetic lineages TC 1 and TC 2.

Determination of the number of amplified DNA fragments in common is a technique extremely simple to study phylogeneytic relationships among organisms. However, the characterization of T. cruzi strains by measuring of biological parameters, such as growth kinetics in axenic liquid medium, in vitro metacyclogenesis, parasitemias and mortality rates in mice, infection of cultured cells (Gonzalez et al. 1995, Lauria-Pires et al. 1997, Lopez-Olmos et al. 1998, Solari et al. 1998, Almeida-de-Faria et al. 1999) follow nowadays applying to knowledge of diversity Chagas disease. The correlation found by us between RAPD profiles and biological behaviour of T. cruzi strains confirms the importance of the biological analysis, and this is the main aim of this paper.

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