Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups

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Abstract

Previous studies on Toxoplasma gondii population structure, based essentially on multilocus restriction fragment length polymorphism analysis or on multilocus enzyme electrophoresis, indicated that T. gondii comprises three clonal lineages. These studies showed a weak polymorphism of the markers (2–4 alleles by locus). In this study, we used eight microsatellite markers to type 84 independent isolates from humans and animals. Two microsatellite markers were present in the introns of two genes, one coding for beta-tubulin and the other for myosin A, and six were found in expressed sequence tags. With 3–16 alleles detected, these markers can be considered as the most discriminating multilocus single-copy markers available for typing T. gondii isolates. This high discriminatory power of microsatellites made it possible to detect mixed infections and epidemiologically related isolates. Evolutionary genetic analyses of diversity show that the T. gondii population structure consists of only two clonal lineages that can be equated to discrete typing units, but there is some evidence of occasional genetic exchange that could explain why one of these discrete typing units is less clearly individualised than the other.

Introduction

Toxoplasma gondii is an obligate intracellular parasite infecting all warm-blooded animals with a world-wide distribution. It causes a large range of clinical manifestations in humans (Bossi et al., 1998, Dardé et al., 1998). Besides, there are marked biological differences among stocks concerning their pathogenicity to mice: most of the stocks are avirulent in mice producing asymptomatic chronic infections, while few which are highly virulent in mice stocks produce acute toxoplasmosis killing all mice with less than 10 tachyzoites.

Using mAb, it is possible to classify stocks in two or three groups demonstrating antigenic diversity in T. gondii (Bohne et al., 1993, Parmley et al., 1994, Meisel et al., 1996, Jensen et al., 1998). Population genetic analysis of published isoenzyme data led Tibayrenc et al. (1991) to propose that T. gondii exhibits a basically clonal population structure, similar to many other parasitic protozoa. Similar analyses, based on restriction fragment length polymorphism (RFLP) of six independent single-copy loci, amplified by PCR, indicated that T. gondii consists of only three clonal lineages designated types I, II and III, which occur in both animals and humans (Howe and Sibley, 1995). However, the question of the actual nature of these lineages remains entirely open: do they really correspond to clonal groups or, rather, to cryptic biological species within which sexual recombination occurs (Tibayrenc, 1993)?

Isoenzyme analysis using six different enzyme systems allowed the identification of 12 zymodemes among a population of 86 stocks (Dardé et al., 1992, Dardé, 1996; and unpublished data). Eight zymodemes comprise only one stock each, and four zymodemes (Z1, Z2, Z3 and Z4) cluster the majority of the stocks. Twenty-three strains were analysed by both multilocus enzyme electrophoresis (MLEE) and PCR-RFLP analysis of six single-copy genes: Z1 strains were equated to PCR-RFLP type I, Z2 and Z4 to type II and Z3 to type III (Dardé et al., 1996; Howe and Sibley, 1995). With these two kinds of markers (isoenzymes and single-copy genes), the allelic diversity is low (2–3 alleles/locus). These markers lack resolution for phylogenetic and epidemiological studies, and for searching possible associations between genotypes and clinical forms of the disease.

Microsatellites represent another class of genetic markers. They are short tandem repeats of 2–6 nucleotides. Markers generated from these repeats are known to be highly polymorphic because of length variation of these repeats, and consequently, they exhibit multiple alleles, which makes them very informative for genetic studies. Polymorphism can be evaluated by PCR, which requires only a small amount of DNA, and allele sizing can be achieved with fluorescent primers and an automatic sequencer which assures reliability of the results.

We used multilocus genotyping with eight microsatellite markers on 84 stocks of T. gondii to design a molecular tool that has a higher resolution than isoenzymes for molecular epidemiology studies and to reconsider the problem of population structure in T. gondii.

Section snippets

Source of Toxoplasma gondii stocks

We analysed 84 independent Toxoplasma stocks, previously typed by isoenzyme analysis (Table 1). Thirty-five of these stocks were analysed by SAG2 PCR-RFLP by other authors (Howe and Sibley, 1995, Honoré et al., 2000). Most of them originated from Europe (65) and North America (10). Six were from South America (French Guiana, Uruguay and Argentina). The geographic origin was not known for three. Sixty-one stocks were collected from human infections (38 congenital toxoplasmoses, 20 acquired

GeneScan software analysis

PCR products consisted of a single peak of fluorescence after analysis by GeneScan software. Some additional peaks, shorter or longer by 2–4 bp, due to strand slippage of Taq polymerase on microsatellite sequence, were sometimes present, but their fluorescence intensity was always much less important than the main peak. This main peak corresponding to the length of the PCR product was assigned to an allele. The resolution of electrophoresis in the automated sequencer is so high that we can

Discriminatory power of microsatellites

Genetic typing methods of T. gondii strains have been extensively perfected in recent years. From a technical point of view, many tools usable for genetic studies on single-copy loci have been used: RFLP (Sibley and Boothroyd, 1992), PCR-RFLP (Sibley and Boothroyd, 1992, Parmley et al., 1994, Asai et al., 1995, Howe and Sibley, 1995, Howe et al., 1997, Binas and Johnson, 1998, Mondragon et al., 1998, Owen and Trees, 1999), sequencing (Luton et al., 1995, Rinder et al., 1995, Meisel et al., 1996

Acknowledgements

The authors would like to thank their colleagues from Europe, South and North America for providing T. gondii stocks. The authors also thank A. Hehl, I. Manger, M. Marra, L.D. Sibley, J.A. Ajioka, M.A. Aslett, N. Dietrich, T. Dubuque, L. Hillier, T. Kucaba, K.L. Wan, R.H. Waterston, J. Boothroyd, J.C. Fordham, D. Coles, P.M. Taylor-Harris and R.J.M. Wilson for their works in sequencing ESTs or genes which allowed the authors to find their MS markers.

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