A. SOLARI, X. ROZAS, C. CORONADO, S. BOTTO-MAHAN, S. ORTIZ Program of Cellular and Molecular Biology,
ICBM. Faculty of Medicine, University of Chile,
Santiago, Chile.
Abstract
Blood samples from 200 sylvatic and peridomestic animals from an endemic area of Chile were subjected to PCR amplification of Trypanosoma cruzi minicircle sequences. This method enabled to detect parasite DNA in animals of the species. (Thylamis elegans, Octodon degus, Phyllotis darwini, and Abrothrix olivaceuss) as representatives of sylvatic animals, and Capra hircus as representative of the peridomestic one. Altogether, 51% of the sylvatic and 36%
of the peridomestic animals were infected with T.cruzi Amplified DNA products obtained in this study were then studied by Southern analysis with a panel of four radioactive probes prepared from genotyped T.cruzi clones in the endemic areas of Chile and pertaining to T.cruzi lineages I and II. Most of the animal are infected at a rate of 35% with T.cruzi I, however other 85% are infected with T.cruzi II. This method is able to detect mixed infections with two or more different genotypes this figure raise to approximately 40% in this sample.
1. INTRODUCTION
Chagas disease, whose etiological agent is the protozoon parasite Trypanosoma cruzi, is endemic in South America. Parasitemia in chronic cases is low; hence its diagnosis is mainly performed by serologic assays. However, the validation of serological tests with parasitological diagnosis as PCR is necessary since sensitivity of PCR targeted to amplify kinetoplast DNA molecules as minicircles is sensitivity as conventional serology. Moreover, the application of PCR detection of T.cruzi kDNA and amplification of DNA segments are valuable as molecular markers to define parasite genotypes circulating in sylvatic, peridomestic or domestic habitats. Our protocol for kDNA amplification yields products derived from the minicircle variable region. These sequences have proven useful in strain typing of T cruzi by hybridization tests. The present work investigative the presence of T. cruzi, and the characterization of T.cruzi strains circulating in animals of an endemic chagasic area of Chile.
2. MATERIAL AND METHODS.
2.1. Blood samples
These where collected (0.5 to 1.0 mL.) from animals anesthetized with isofurane in the presence of heparin as anticoagulant, and boiled for 15 min before use.
2.2. DNA extraction and PCR amplification.
Half mL of blood was processed for DNA extraction with the E.Z.N.A. kit. The amplification reactions were performed essentially as previously described with primers 121 and 122. PCR products of 330 bp were analysed by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. Amplified DNAs were
then transferred to nylon membranes, denatured, cross-linked with UV irradiation, and hybridized using a total kDNA from T.cruzi as a universal probe, and labelled by the random priming method with [α32P] dCTP [1]
2.3. Generation of T.cruzi specific probes
The four genotype–specific probes corresponding to the T.cruzi clones were sp104cl1 (T.cruzi I), and CBB cl3, v195cl1, and NRcl3 corresponding to T.cruzi IIb, IIc, and IId, respectively. They were prepared with parasite DNA by PCR and the oligonucleotides CV1 and CV2, which are directed from the constant regions of the minicircles as described [2]. Finally DNA products were digested with the restriction endonucleases Sau 96I and Sca I to remove part of the oligonucleotide primer. This procedure generated 250bp probes which are purified by electrophoresis on low melting point agarose.
2.4. Southern blot analysis
Agarose gel containing PCR products are transferred to nylon membranes as described in [1]; and hybridized overnight with the 250 bp genotype-specific radioactive DNA in a hybridisation solution of 10mL (buffer 2X SSE, 5X, Denhardt´s solution) containing 0.5 x 106 cpm of 32P-labelled DNA probe/membrane (specific activity 50-80 x 106 cpm /ug DNA). Washing was carried out in 2 x SSE, 0.1% SDS at 55ºC. The membranes where probed with radioactive DNA autoradiographed and reused with another labelled probe previous to removal and decay of the old probe.
3. RESULTS
3.1. Detection of T.cruzi infected animals
Results for sylvatic animals indicated that 43.3% were positives. Results from Capra hircus indicated that 26.2% were positives. Additional positives were detected by Southern analysis in samples that resulted PCR negative. In sylvatic animals 51% were positives with the radioactive probe, and 35.7% of C. hircus were positives. It is possible to observe based on the reported results that the percentage of positive cases increased when PCR and hybridization results are combined.
3.2. Classification of Trypanosoma cruzi genotypes
DNA amplified from infected animals was subjected to Southern analysis with the four specific probes. Results of positive cases with the T.cruzi I, T.cruzi IIb, IIc, and IId for T. elegans are 33,3% 16.7%, 50% and 16.7%, respectively. Equivalent results for O. degus with T.cruzi I, T.cruzi IIb, IIc, and IId are 47.8%, 43.5%, 21.7%, and 39%, respectively. Meantime results for A. olivaceus with T.cruzi I, T.cruzi IIb, IIc, and IId are 26.9%, 42.3%, 8%, and 15%, respectively. Finally results for the sylvatic animal P.
darwini infected with T.cruzi I, T.cruzi IIb, IIc, and IId are 44%, 28%, 44%, and 40%, respectively. The only one peridomestic animal infected with T.cruzi is C. hircus, and results of reservoirs infected with T.cruzi I, T.cruzi IIb, IIc, and IId are 33.3%, 20%, 46.7%, and 13.3% respectively. It is important to point out that not all infected animals are characterized by this method with the panel of four probes, suggesting that other different T.cruzi genotypes are also circulating in these reservoirs. There are animals as C.hircus where non identified T.cruzi genotypes is close to 0%, meantime in others as A. olivaceus and P. darwini the non identified T.cruzi genotypes range 30-40%. Finally
is worth mentioning that percentages of detected genotypes are over 100%, a clear indication that several animals are infected with more than one T.cruzi genotype, that is, a mixture of parasites.
4. DISCUSSION
This work describes the PCR assay to detect T.cruzi in blood samples of sylvatic and peridomestic animals in a way to improve and confirm serological diagnosis. This method proved useful and more sensitive when a combined hybridization assay is performed with a universal probe. The difference between PCR and Southern analysis results might reflect the very low parasitemia in approximately 8-10% of the animals, which are detected positive by the hybridisation signal, but not by ethidium bromide staining. The other importance to target kinetoplast minicircle DNA by PCR is to determine the infective T.cruzi genotype on each mammalian reservoir with amplified DNA by a panel of well characterized T.cruzi genotype specific probes and hybridization tests. Results of T.cruzi genotypes circulating in sylvatic animals and C.
hircus varied. Both T.cruzi I and T.cruzi II lineages are heterogeneous (IIb, IIc, IId), and frequently animals are infected with mixtures of genotypes which circulate and are transmitted in nature Interestingly sylvatic animals carry T.cruzi I, but also T.cruzi II genotypes which are characteristic of the domestic transmission cycle, a situation completely different as previously thought. This study is relevant to understand the molecular epidemiology of Chagas disease in the wild transmission cycle and to determine reservoirs of parasites with potential risk for humans.
ACKNOWLEDGMENTS
This work was supported by FONDECYT, Chile 104-0762 and IAEA-CHI-11767.
REFERENCES
[1] SOLARI, A., CAMPILLAY, R., ORTIZ, S., WALLACE, A. Identification of Trypanosoma cruzi genotypes circulating in Chilean chagasic patients. Exp.
Parasitol., 97: 226–233 (2001).
[2] VEAS, F., BRENIERE, F., CUNY, G., BRENGUES, C., SOLARI, A., TIBAYRENC, M. General procedure to construct highly specific kDNA probes for clones of Trypanosoma cruzi for sensitive detection by polymerase chain reaction. Cell. Mol. Biology, 37: 73–84 (1991).
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