Dataset Information

Genetic characteristics of the Korean isolate KI-1 of Toxoplasma gondii.

ABSTRACT: Toxoplasma gondii tachyzoites were isolated from an ocular patient in the Republic of Korea and maintained in the laboratory (designated KI-1). In the present study, its genotype was determined by analyzing dense granule antigen 6 (GRA6) gene and surface antigen 2 (SAG2) gene as typing markers. Digestion of the amplification products of GRA6 and of the 5o and 3o ends of SAG2, respectively, with Mse I, Sau3A I, and Hha I, revealed that KI-1 is included in the genotype I, which includes the worldwide virulent RH strain. In addition, when the whole sequences of the coding regions of SAG1, rhoptry antigen 1 (ROP1), and GRA8 genes of KI-1 were compared with those of RH, minor nucleotide polymorphisms and amino acid substitutions were identified. These results show that KI-1 is a new geographical strain of T. gondii that can be included in the genotype I.

SUBMITTER: Lin A

PROVIDER: S-EPMC2712006 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Genetic characteristics of the Korean isolate KI-1 of Toxoplasma gondii.

Lin Aifen A Shin Eun-Hee EH Kim Tae-Yun TY Park Jae-Hwan JH Guk Sang-Mee SM Chai Jong-Yil JY

The Korean journal of parasitology 20050301 1

Toxoplasma gondii tachyzoites were isolated from an ocular patient in the Republic of Korea and maintained in the laboratory (designated KI-1). In the present study, its genotype was determined by analyzing dense granule antigen 6 (GRA6) gene and surface antigen 2 (SAG2) gene as typing markers. Digestion of the amplification products of GRA6 and of the 5o and 3o ends of SAG2, respectively, with Mse I, Sau3A I, and Hha I, revealed that KI-1 is included in the genotype I, which includes the worldw ...[more]

PMID: 15793356

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Project description:Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite's genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen.Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.

Project description:BackgroundToxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain.MethodsSelected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes.ResultsAs a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms.ConclusionsThe present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity.

Project description:BackgroundThe consumption of uncooked or undercooked food from infected intermediate hosts can result in Toxoplasma gondii infection in humans. However, few studies have investigated the genetic diversity of this protozoan parasite in Iran. The aim of the present study was to genetically characterize isolates of T. gondii from intermediate host animals in Mazandaran Province, Iran.MethodsBlood and heart tissue samples were collected from 204 ruminants, and brain tissue was collected from 335 birds. The prevalence of T. gondii infection in these samples was determined serologically using the modified agglutination test and by conventional PCR assays. Those PCR samples positive for T. gondii DNA and 13 DNA samples extracted from aborted fetuses in a previous study were genotyped with 12 genetic markers using the multilocus-nested PCR-restriction fragment length polymorphism (Mn-PCR-RFLP) technique.ResultsAntibodies for parasites were found in 35.7% of the ruminant (39.1% of sheep and 26.4% of goats) samples and in 51.3% of the bird (100% of geese, 52.3% of free-range chickens and 46% of ducks) samples. Molecular detection by PCR of the repetitive 529-bp DNA fragment revealed contamination of 13.2% of ruminant (14.6% of sheep and 9.4% of goats) samples and of 9.6% of bird (11.1% of chickens, 5.7% of ducks and 0% of geese samples). The results from 30 DNA samples revealed five ToxoDB (genome database for the genus Toxoplasma) PCR-RFLP genotypes, including #1 (Type II), #2 (Type III), #10 (Type I), #27 and #48, with genotype #1 the most predominant.ConclusionsAs evidenced by the results of this study, ruminants and birds are infected with T. gondii in the region, suggesting that they are likely to be involved in the transmission of T. gondii to humans through meat consumption. The identification of different genotypes may suggest a higher genetic diversity of this parasite in Mazandaran, reflecting local environmental contamination. These results have important public health implications for the prevention and control strategies of infection.

Dataset Information

Genetic characteristics of the Korean isolate KI-1 of Toxoplasma gondii.

Publications

Genetic characteristics of the Korean isolate KI-1 of Toxoplasma gondii.

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