High-Resolution Typing of Toxoplasma gondii Using Microsatellite Loci

Blackston, C. R.; Dubey, J. P.; Dotson, Ellen M.; Su, Chunlei; Thulliez, P.; Sibley, David; Lehmann, Tovi

doi:10.1645/0022-3395(2001)087[1472:hrtotg]2.0.co;2

Cited by 48 publications

(23 citation statements)

References 11 publications

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“…The analysis of length polymorphism at seven microsatellite markers was performed using the method of Ajzenberg et al (2005), with minor modifications. We used six microsatellite markers (B18, TUB2, TgM-A, W35, B17, and M33) for genotyping and a seventh marker (M48) for further characterization (Blackston et al, 2001;Ajzenberg et al, 2005). The primers were synthesized by Applied Biosystems (Warrington, UK): the forward primers were 59-end labeled with either 6-carboxyfluorescein (B18, TUB2, and M48), hexachlorofluorescein (TgM-A, W35, and B17), or 2,79,89-benzo-59-fluoro-29,4,7-trichloro-5-carboxyfluorescein (M33).…”

Section: Haresmentioning

confidence: 99%

Natural Toxoplasma Gondii Infections in European Brown Hares and Mountain Hares in Finland: Proportional Mortality Rate, Antibody Prevalence, and Genetic Characterization

Jokelainen

Isomursu

Näreaho

et al. 2011

Journal of Wildlife Diseases

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ABSTRACT:In material examined postmortem in Finland from May 2006 to April 2009, acute generalized toxoplasmosis was the immunohistochemically confirmed cause of death in 14 (8.1%) of 173 European brown hares (Lepus europaeus) and four (2.7%) of 148 mountain hares (Lepus timidus). Sera from 116 of the European brown hares and 99 of the mountain hares were screened with a commercial direct agglutination test for Toxoplasma gondii-specific IgG antibodies at a dilution of 1:40. All sera from cases of fatal toxoplasmosis had high titers of antibodies reactive to T. gondii. In contrast, none of 107 European brown hares and four (4%) of 96 mountain hares that died of other causes were antibody-positive. The proportional mortality rates and the T. gondii antibody prevalences among noncases differed significantly between the two host species (P,0.05). Direct genetic characterization of the causative agent was performed on DNA extracted from formalin-fixed, paraffin-embedded tissue of the hares with fatal toxoplasmosis. Based on the results with six microsatellite markers (B18, TUB2, TgM-A, W35, B17, and M33; all six in 15 cases and four in three cases), all the cases were caused by T. gondii genotype II; the size of the PCR product at the seventh marker (M48) varied (213-229 base pairs). The presence of T. gondii genotype II, which is endemic in Europe, is now confirmed in Finnish wildlife: Natural infections with T. gondii parasites belonging to this widespread genotype caused fatal generalized toxoplasmosis in the two species of wild hares.

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Section: Haresmentioning

confidence: 99%

Natural Toxoplasma Gondii Infections in European Brown Hares and Mountain Hares in Finland: Proportional Mortality Rate, Antibody Prevalence, and Genetic Characterization

Jokelainen

Isomursu

Näreaho

et al. 2011

Journal of Wildlife Diseases

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“…Wing geometric morphometric studies of T. infestans described a spatial structuring pattern (Schachter-Broide et al, 2004), but could not provide definitive explanations for the structuring origin. Analyses of mitochondrial DNA sequences, microsatellites and RAPDs in various species of insect disease vectors and parasites have proven effective (e.g., Monteiro et al, 1999;Borges et al, 2000;Blackston et al, 2001;Lehmann et al, 2003aLehmann et al, , 2003b.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of microsatellite markers in the Chagas disease vector Triatoma infestans (Heteroptera: Reduviidae)

Marcet

Lehmann

Groner

et al. 2006

Infection, Genetics and Evolution

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Triatoma infestans, the main vector of Chagas disease in the southern cone countries, is the principal target of a regional elimination program. A better understanding of its dispersal, sources of reinfestation, and insecticide resistance is key to an effective control program. To address such problems, we identified and characterized 13 microsatellite loci of T. infestans. For each locus, primer sequences and PCR conditions are presented. Allele variability and frequency were analyzed in 59 T. infestans specimens from different rural communities in northwestern Argentina; nine loci were considered suitable for population genetic studies. Departure from Hardy-Weinberg equilibrium was detected in 10/13 loci with F IS values ranging from 0.04 to 0.91, indicating heterozygote deficit and a possible grade of sub-structure in the sample analyzed. Presence of null alleles in some loci cannot be discarded. The present work provides a promising tool to develop a population genetic study of natural populations of T. infestans in tandem with field studies and analyses of bug dispersal and the reinfestation process.

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“…Eight MS markers with a repeat motif consisting of [TG/AC] n or [TC/AG] n were used as typing markers: five markers (TUB2, W35, TgM-A, B18, and B17) were described in our previous report of a multiplex assay (4), M33 was developed by others (6), and two markers (IV.1 and XI.1) were designed for this study. Seven MS markers with a repeat motif consisting of [TA/AT] n were used as fingerprinting markers: five markers (N60, N82, AA, N61, and N83) were described by us in a previous study (2) whereas M48 and M102 were developed by others (6). For some markers, primers sequences were redesigned to allow PCR amplification and allele size discrimination in the multiplex assay.…”

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confidence: 99%

Genotyping of Toxoplasma gondii Isolates with 15 Microsatellite Markers in a Single Multiplex PCR Assay

et al. 2010

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We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers.The commonly used multilocus methods for genotyping Toxoplasma gondii strains employ PCR-restriction fragment length polymorphism (PCR-RFLP) analysis at 10 markers (16), sequencing at 8 introns of 5 loci (14), and length polymorphism analysis of 5 microsatellite (MS) markers (4). Advantages and disadvantages of these different markers have been reviewed elsewhere (16). Based on our experience within the Toxoplasma Biological Resource Center (BRC ToxoBS) and the National Reference Center for Toxoplasmosis, it appears that screening of a large number of clinical isolates for T. gondii strain typing should rely on an easy-to-use method with two different levels of discrimination. The first step of discrimination is the performed at the typing level: that is, employing the ability of markers to distinguish the major clonal lineages from atypical strains. In areas with a marked clonal population structure such as Europe, the genetic screening of clinical isolates should rapidly identify basic type II or III strains, which represent the majority of isolates, and atypical strains, which are the exception to the rule (3, 11). In this context, the identification of atypical strains is of clinical and epidemiological importance because atypical strains are usually associated with severe disease outcomes and contamination of individuals by non-European strains either during residence abroad or after consumption of imported meat (5, 9, 10). The second level of discrimination is the fingerprinting level, representing a high degree of discriminatory power for differentiating closely related strains belonging to the same lineage. This high-resolution analysis is required for identifying laboratory contaminations for diagnosis issues and for establishing a common source of infection among different infected individuals in an outbreak (7,8).Microsatellite (MS) sequences are tandem repeats of short (1 to 6 bp) DNA motifs that are ubiquitous in eukaryotic genomes and undergo length changes due to insertion or deletion of one or multiple repeat units. The most commonly proposed mutation mechanism for MS sequences is strand slippage, occurring predominantly during replication (13). The numbers of repeat motifs differ in a population, thereby creating multiple alleles at an MS locus. MS loci are amplified by PCR using fluorescently labeled forward and unlabeled reverse primers. The dye-labeled products are separated by size using automated electrophoresis and identified by fluorescence detection. We previously designed a multiplex PCR assay with 5 MS markers that were able to reach the typing level but not the fingerprinting level of discrimination between strains (4). Here we developed an easy-to-use and rapid genotyping method which aimed to ensure both levels of genetic discrimina...

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High-Resolution Typing of Toxoplasma gondii Using Microsatellite Loci

Cited by 48 publications

References 11 publications

Natural Toxoplasma Gondii Infections in European Brown Hares and Mountain Hares in Finland: Proportional Mortality Rate, Antibody Prevalence, and Genetic Characterization

Natural Toxoplasma Gondii Infections in European Brown Hares and Mountain Hares in Finland: Proportional Mortality Rate, Antibody Prevalence, and Genetic Characterization

Identification and characterization of microsatellite markers in the Chagas disease vector Triatoma infestans (Heteroptera: Reduviidae)

Genotyping of Toxoplasma gondii Isolates with 15 Microsatellite Markers in a Single Multiplex PCR Assay

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