Gondii Genotypes Research Articles

Genetic characterization of Toxoplasma gondii from wild boar ( Sus scrofa) in France

Toxoplasma gondii strains isolated from domestic animals and humans have been classified into three clonal lineages types I–III, with differences in terms of pathogenicity to mice. Much less is known on T. gondii genotypes in wild animals. In this report, genotypes of T. gondii isolated from wild boar ( Sus scrofa) in France are described. During the hunting seasons 2002–2008, sera and tissues of individuals from two French regions, one continental and one insular, were tested for Toxoplasma infection. Antibodies to T. gondii were found in 26 (17.6%) of 148 wild boars using the modified agglutination test (MAT, positivity threshold: 1:24). Seroprevalence was 45.9% when considering a threshold of 1:6. Hearts of individuals with a positive agglutination (starting dilution 1:6) ( n = 60) were bioassayed in mice for isolation of viable T. gondii. In total, 21 isolates of T. gondii were obtained. Genotyping of the isolates using 3 PCR–restriction fragment length polymorphism markers ( SAG1, SAG2 and GRA7) and 6 microsatellite loci analysis ( TUB2, TgM- A, W35, B17, B18 and M33) revealed that all belonged to type II lineage. These results underline that wild boar may serve as an important reservoir for transmission of T. gondii, and that strains present in wildlife may not be different from strains from the domestic environment.

Significant Performance Variation Among PCR Systems in Diagnosing Congenital Toxoplasmosis in São Paulo, Brazil: Analysis of 467 Amniotic Fluid Samples

INTRODUCTIONPerformance variation among PCR systems in detecting Toxoplasma gondii has been extensively reported and associated with target genes, primer composition, amplification parameters, treatment during pregnancy, host genetic susceptibility and genotypes of different parasites according to geographical characteristics.PATIENTSA total of 467 amniotic fluid samples from T. gondii IgM- and IgG-positive Brazilian pregnant women being treated for 1 to 6 weeks at the time of amniocentesis (gestational ages of 14 to 25 weeks).METHODSOne nested-B1-PCR and three one-round amplification systems targeted to rDNA, AF146527 and the B1 gene were employed.RESULTSOf the 467 samples, 189 (40.47%) were positive for one-round amplifications: 120 (63.49%) for the B1 gene, 24 (12.69%) for AF146527, 45 (23.80%) for both AF146527 and the B1 gene, and none for rDNA. Fifty previously negative one-round PCR samples were chosen by computer-assisted randomization analysis and re-tested (nested-B1-PCR), during which nine additional cases were detected (9/50 or 18%).DISCUSSIONThe B1 gene PCR was far more sensitive than the AF146527 PCR, and the rDNA PCR was the least effective even though the rDNA had the most repetitive sequence. Considering that the four amplification systems were equally affected by treatment, that the amplification conditions were optimized for the target genes and that most of the primers have already been reported, it is plausible that the striking differences found among PCR performances could be associated with genetic diversity in patients and/or with different Toxoplasma gondii genotypes occurring in Brazil.CONCLUSIONThe use of PCR for the diagnosis of fetal Toxoplasma infections in Brazil should be targeted to the B1 gene when only one gene can be amplified, preferably by nested amplification with primers B22/B23.

Induction of partial protection against infection with Toxoplasma gondii genotype II by DNA vaccination with recombinant chimeric tachyzoite antigens

Infection with the obligate intracellular parasite Toxoplasma gondii is a significant source of parasitic infections worldwide. In adults, infections may often lead to severe retinochoroiditis. Infection of the foetus causes abortion or congenital pathology that may lead to neurological complications. Although several strategies have been suggested for making a vaccine, none is currently available. Here, we investigate the protection conferred by DNA vaccination with two constructs, pcEC2 (MIC2-MIC3-SAG1) and pcEC3 (GRA3-GRA7-M2AP), encoding chimeric proteins containing multiple antigenic sequences from T. gondii. After challenge with a T. gondii genotype II, but not a genotype III strain, a significant decrease in cerebral cyst load was found compared to the controls. The immune protection involved a cell-mediated immune response with the synthesis of the cytokines IFN-γ and IL-10. In silico structure analysis and the expression profile of EC2, suggest an association between antigen stability, the degree of protein secondary structure and induction of cellular immune responses. Intracellular protein degradation is an important step in the pathway leading to presentation of antigenic peptides on Major Histocompatibility Complex molecules. We suggest that degradation of this chimeric protein may have contributed to the induction of a cellular immune response via enhanced presentation of antigenic peptides on Major Histocompatibility Complex class I molecules.

InfecÇão crônica por Toxoplasma gondii induzindo hipertrofia de neurônios do plexo mientérico do cólon descendente de Rattus norvegicus

The effects of the chronic infection due to Toxoplasma gondii tachyzoites on the myenteric neurons of the adult rat descending colon were assessed in this study. Ten male, 60-day-old, Wistar rats, divided into control and experimental group were orally inoculated with 105 tachyzoites from Toxoplasma gondii genotype I strain. After 30 days, the animals were anesthetized and submitted to laparotomy. The descending colon was removed, dissected, and the whole-mounts were staining by Giemsa, in order to observe neurons of the myenteric plexus, followed by quantitative and morphometric analysis. It was verified that the infection caused alterations neither with respect to the dimensions of the organ nor the neuronal population; however, there was a significant increase of the perikarion area and the cytoplasm.

Molecular characterization and expression analysis of a P-glycoprotein homologue in Toxoplasma gondii

ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present study reports on the characterization of a P-glycoprotein (Pgp), TgABC.B1, in the protozoan parasite Toxoplasma gondii. The protein encoded by the TgABC.B1 gene displays the typical (TMD-NBD)2 structural organization of the “full” ABC transporter and shows significant identity and similarity with two apicomplexan Pgps; Pgh1 in Plasmodium falciparum and CpABC3 in Cryptosporidium parvum. The TgABC.B1 gene is a single copy gene transcribed into a full-length mRNA of 4.3kb and expressed as a protein of approximately 150kDa, which cellular localization revealed a membrane-associated labelling in tachyzoites. The TgABC.B1 gene is constitutively expressed in the three major T. gondii genotypes but demonstrated a higher expression in virulent type I, at both transcriptional and translational levels. Further characterization of this Pgp-like protein will increase our knowledge of the membrane transport system in this parasite and could result in the identification of a new therapeutic target in Toxoplasma.

Direct high-resolution genotyping of Toxoplasma gondii in arctic foxes ( Vulpes lagopus) in the remote arctic Svalbard archipelago reveals widespread clonal Type II lineage

Characterization of Toxoplasma gondii genotypes in hosts living in remote, isolated regions is important for elucidating the population structure and transmission mode of this parasite. Herein, we report the results of direct genotyping of T. gondii in brain tissue of arctic foxes ( Vulpes lagopus) from the remote, virtually cat-free, high arctic islands of Svalbard. DNA extracts from brains of 167 seropositive arctic foxes (including four cases of fatal toxoplasmosis) and 11 seronegative arctic foxes were genotyped at 10 loci (SAG1, SAG2, SAG3, BTUB, GRA6, L358, c22-8, c29-2, PK1, and Apico) using the polymerase chain reaction-restriction fragment length polymorphism method. Of the 167 samples from seropositive foxes (including toxoplasmosis cases), 31 were genotyped at all 10 loci and 24 were genotyped at four to nine loci. To ensure confidence in T. gondii strain genotyping, samples for which less than four loci were genotyped were not considered positive. None of the 11 samples from seronegative foxes was positive for the 10 markers. Of the 55 samples that genotyped positively, 46 were of the Type II strain, 7 were of the Type III strain, and 2 were of atypical T. gondii strains. Five representative samples of the three genotypes were sequenced at loci SAG2, SAG3, GRA6, PK1, and UPRT-1. The DNA sequences confirmed the genotyping results. This study shows that the archetype Type II T. gondii strain, which is most widely distributed in North America and Europe, also predominates in arctic foxes on the Svalbard archipelago. This suggests that the T. gondii at this location originate from continental Europe and that transmission may be mediated by migrating birds. This study highlights the significance of long-distance transport of T. gondii and demonstrates that high-resolution genotyping protocols are useful for direct genetic studies of T. gondii when isolation of live parasites is infeasible.

Toxoplasma gondii genotyping in a dog co-infected with distemper virus and ehrlichiosis rickettsia

This paper reports a toxoplasmosis, ehrlichiosis and distemper co-infection in a dog with an exuberant neuropathological clinical picture. Primary involvement was discussed based on information collected in the analysis of the clinical case, such as neurological impairment, epidemiological data, poor immunoprophylactic scheme of the dog affected and the role of these diseases on immunosuppression. Canine distemper and ehrlichiosis were diagnosed based on epidemiologic data, clinical signs, hematological and cytological evaluation. Toxoplasma gondii was isolated and genetically characterized as Type I using restriction analysis (RFLP) with SAG-2 genes. Immunosuppression features of both dogs and human beings are discussed, as well as implications on animal and public health. This is the first report on toxoplasmosis, ehrlichiosis and distemper co-infection in a dog in Brazil, associated with genotyping determination of the T. gondii strain involved.

The genotype of Toxoplasma gondii strains causing ocular toxoplasmosis in humans in Brazil

To determine Toxoplasma gondii genotype in scars of retinochoroiditis in humans. Immunopathology analysis. DNA was isolated from retinochoroidal specimens taken from the borders of the scars from eye bank eyes as well as enucleated eyes from uveitis patients with retinochoroidal toxoplasmic scars. The SAG2 region was amplified by polymerase chain reaction and analyzed by restriction fragment length polymorphism. Of the 92 eyes studied, 9 had toxoplasmic scars. All of them, as well as the 2 enucleated eyes, disclosed the presence of type I parasites by the gel electrophoresis analysis of the restriction fragment length polymorphism from SAG2. These results suggest that type I strain seems to be responsible for the ocular infections in the population studied.

Transmission dynamics of Toxoplasma gondii on a pig farm

Transmission of Toxoplasma gondii infection on a pig farm in New England was investigated using genetic and ecological methods to (i) determine if infection of pigs was a result of a single source, such as in an epizootic situation (e.g. outbreak) or of multiple sources, such as in an enzootic situation, (ii) identify the main source species of infection to pigs and (iii) evaluate the role of the environment surrounding the farm as the source of infection on the farm. Genetic characterization of 25 T. gondii isolates from market pigs revealed three distinct genotypes with no evidence of recombinants. These data imply that at least three distinct exposure events occurred during the 7-month lifespan of these pigs. This genotype diversity is consistent with enzootic transmission of T. gondii on the farm. Cats were suspected as the main source of pig infection based on the high seroprevalence (>95%) in pigs. The presence of the two most common T. gondii genotypes in eight isolates from free ranging chickens on this farm corroborated the role of cats because chickens were probably infected through ingestion of oocysts in the soil. The seroprevalence of toxoplasmosis in 163 wild mammals and birds captured around the pig sties (overall 13.1%) increased with proximity to the pig sties. Thus, transmission of T. gondii was higher near the pig sties than in the surrounding environment probably because of increased density of oocysts there. We propose that the farm does not simply reflect its surroundings in terms of strain composition and risk of infection, but that it acts as a reservoir of strains from which the outflow of new infections into its surrounding environment is higher than the inflow.

Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings.

To study the influence of Toxoplasma gondii genotypes on the severity of human congenital toxoplasmosis (asymptomatic, benign, or severe infection or newborn or fetal death), 8 microsatellite markers were used to analyze 86 T. gondii isolates collected from patients with congenital toxoplasmosis. Seventy-four different genotypes were detected, some identical genotypes originating probably from the same source of contamination. The 3 less polymorphic microsatellite markers associated with 6 isoenzymatic markers allowed a classification of isolates into the 3 classical types and detected atypical genotypes. Whatever the clinical findings, type II isolates were largely predominant (84.88% in the whole collection and 96.49% in 57 consecutive cases). Type I and atypical isolates were not found in asymptomatic or benign congenital toxoplasmosis. However, in 4 cases in which children were not infected despite isolation of T. gondii from placenta, only type I isolates were found.

Successful reinfection of chronically infected mice by a different Toxoplasma gondii genotype

It is generally assumed that primary infection by Toxoplasma gondii protects from reinfection. A recent study using a murine model has questionned this dogma using indirect procedures to detect the reinfecting strain. We have reinvestigated this issue using a transfected strain of T. gondii (Prugniaud β galactosidase: Pru β gal) which expresses Escherichia coli β-galactosidase. Detection of enzyme activity on fixed parasites allows a direct distinction between transfected and untransfected strains. We have found that in OF1 mice primary infection with the 76 K strain of T. gondii fully protects mice against tissue cyst production upon reinfection with the Pru β gal T. gondii strain whereas primary infection with the Pru β gal T. gondii strain does not impair tissue cyst formation upon reinfection with the Ned strain of T. gondii, which belongs to another T. gondii genotype. These results suggest that the immune protection conferred by one strain of T. gondii can be breached by reinfection with a strain belonging to another genotype; which can have significant consequences in human or veterinary medicine.