Gondii Strains Research Articles

Quantitative and morphometric changes of subpopulations of myenteric neurons in swines with toxoplasmosis

The consequences of the infection caused by Toxoplasma gondii in myenteric neurons of the jejunum of swines reactive to NADH-diaphorase and NADPH-diaphorase were evaluated in this study. Ten 88-day-old mixed-breed swines (Pietrain and Wessex) were assigned into two groups: Control ( n = 5) and Experimental ( n = 5), which orally received 5000 sporulated oocysts from a genotype III T. gondii strain. After 30 days, the animals were anesthetized, having part of their jejunum removed and stained with NADPH-diaphorase and NADH-diaphorase. NADPHd-p neurons (nitrergic) presented increase of the number of cells per ganglion and hypertrophy. The number of NADHd-p neurons (metabolic more active) and their nuclear area decreased.

Apoptosis and S Phase of the Cell Cycle in BeWo Trophoblastic and HeLa Cells are Differentially Modulated by Toxoplasma gondii Strain Types

Transplacental transmission of Toxoplasma gondii causes congenital toxoplasmosis, one of the most severe forms of infection. The ability of the parasite to survive intracellularly largely depends on the blocking of different proapoptotic signaling cascades of the host cells. During pregnancy, however, alterations in the incidence of apoptosis are associated with abnormal placental morphology and function. The aim of this study was to evaluate the incidence of apoptosis and cell proliferation in trophoblastic (BeWo cell line) and uterine cervical (HeLa cell line) cells infected with a highly virulent RH strain or a moderately virulent ME49 strain of T. gondii. BeWo and HeLa cells were infected with RH or ME49 tachyzoites (2:1 and 5:1; parasite:cell) or medium alone (control). After 2 h, 6 h and 12 h of incubation, cells were fixed in 10% formalin and analyzed by immunohistochemistry to determine the apoptosis (expression of cytokeratin 18 neo-epitope – clone M30) and cell in S phase (expression of proliferating cell nuclear antigen – PCNA) indices. RH strain-infected BeWo and HeLa cells showed a lower apoptosis index than non-infected controls, whereas a higher apoptosis index was found in ME49 strain-infected cells compared to controls. In addition, RH-infected cells displayed lower apoptosis index than ME49-infected cells, even though active caspase-3 was detected in both cell types infected with either RH or ME49 strains as well in non-infected cells in all analyzed times of infection. Also, the cell S phase indices were higher in ME49 strain-infected BeWo and HeLa cells as compared to non-infected controls and RH strain-infected cells. These results indicate that RH and ME49 strains of T. gondii possess opposing mechanism of interference in apoptosis and cell cycle S phase of both BeWo and HeLa cells and these differences can be associated to evasion strategies of the parasite to survive inside the host cells.

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.

Hypertrophy of the neurons in the ileum of rats infected with cysts of <em>Toxoplasma gondii</em> (genotype II)

This paper verified possible alterations caused by a genotype II Toxoplasma gondii strain with respect to the total number and morphometry of the myenteric neurons in the terminal ileum and descending colon of rats. Eight rats were divided into two groups: control (n = 4) and experimental (n = 4). This group was inoculated orally with 20 tissue cysts of T. gondii from a genotype II strain (ME-49). Whole mounted from the terminal ileum and the descending colon were stained with Giemsa. There was not any neuronal loss on both organs. The neurons became hypertrophied in the terminal ileum, whereas morphometric alterations were not observed for the neurons in the descending colon.

Toxoplasmose disséminée sévère avec choriorétinite atypique : à propos d’un cas de primo-infection

The clinical diagnosis of ocular toxoplasmosis is based on clinical features and biological tests: polymerase chain reaction (PCR) and the determination of intraocular specific antibody secretion (Goldmann-Witmer coefficient) on aqueous humor. Older patients may have a higher prevalence of ocular involvement and more severe ocular disease during the acute phase of recently acquired systemic infection because of altered cell-mediated immunity. Moreover, the genotype of the infecting parasite (particularly involving neotropical Type I Toxoplasma gondii strain), appears to be an important determinant of disease severity.

Toxoplasma gondii: Effects of Artemisia annua L. on susceptibility to infection in experimental models in vitro and in vivo

Considering that the treatment for toxoplasmosis is based on drugs that show limited efficacy due to their substantial side effects, the purpose of the present study was to evaluate the effects of Artemisia annua on in vitro and in vivo Toxoplasma gondii infection. A. annua infusion was prepared from dried herb and tested in human foreskin fibroblasts (HFF) or mice that were infected with the parasite and compared with sulfadiazine treatment. For in vitro experiments, treatment was done on parasite before HFF infection or on cells previously infected with T. gondii and the inhibitory concentration (IC50) values for each treatment condition were determined. Viability of HFF cells in the presence of different concentrations of A. annua infusion and sulfadiazine was above 72%, even when the highest concentrations from both treatments were tested. Also, the treatment of T. gondii tachyzoites with A. annua infusion before infection in HFF cells showed a dose–response inhibitory curve that reached up to 75% of inhibition, similarly to the results observed when parasites were treated with sulfadiazine. In vivo experiments with a cystogenic T. gondii strain demonstrated an effective control of infection using A. annua infusion. In conclusion, our results indicate that A. annua infusion is useful to control T. gondii infection, due to its low toxicity and its inhibitory action directly against the parasite, resulting in a well tolerated therapeutic tool.

Experimental reinfection of BALB/c mice with different recombinant type I/III strains of Toxoplasma gondii: involvement of IFN-³ and IL-10

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-gamma and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-gamma were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

Vaccines against Toxoplasma gondii: challenges and opportunities

Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge.

Toxoplasma gondii and the Immunity-Related GTPase (IRG) resistance system in mice: a review

The Immunity Related GTPases (IRG proteins) constitute a large family of interferon-inducible proteins that mediate early resistance to Toxoplasma gondii infection in mice. At least six members of this family are required for resistance of mice to virulent T. gondii strains. Recent results have shown that the complexity of the resistance arises from complex regulatory interactions between different family members. The mode of action against T. gondii depends on the ability of IRG proteins to accumulate on the parasitophorous vacuole of invading tachyzoites and to induce local damage to the vacuole resulting in disruption of the vacuolar membrane. Virulent strains of T. gondii overcome the IRG resistance system, probably by interfering with the loading of IRG proteins onto the parasitophorous vacuole membrane. It may be assumed that T. gondii strains highly virulent for mice will be disadvantaged in the wild due to the rapid extinction of the infected host, while it is self-evident that susceptibility to virulent strains is disadvantageous to the mouse host. We consider the possibility that this double disadvantage is compensated in wild populations by segregating alleles with different resistance and susceptibility properties in the IRG system.

The temperature-sensitive mutants of Toxoplasma gondii and ocular toxoplasmosis

The risk of blindness caused by ocular toxoplasmosis supports efforts to improve our understanding for control of this disease. In this study, the involvement of CD8 +, CD4 +, B cell, and IL-10 gene in the immune response of primary ocular infection with the temperature-sensitive mutant (ts-4) of the RH Toxoplasma gondii strain, and in the protective immunity of ocular ts-4 vaccination and challenge with RH strain was investigated in murine models utilizing inbred C57BL/6 mice-deficient in CD4 +, CD8 +, B cells (μMT), or IL-10 gene. Compared to naive mice, all WT and mutant mice had different degree of ocular pathological changes after ts-4 ocular infection, in which both CD8 KO and IL-10 KO mice showed the most severe ocular lesions. Immunized by ts-4 intracameral (i.c.) inoculation, all mutant mice had partially decreased vaccine-induced resistance associated with increased ocular parasite burdens after RH strain challenge. A significant increase of the percentages of B cells and CD8 + T cells in the draining lymph nodes were observed in WT and IL-10 KO mice after either infection or challenge. The levels of specific anti-toxoplasma IgG in both eye fluid and serum from all the mice were significantly increased after ts-4 i.c. immunization, except μMT mice. These results suggest that the avirulent ts-4 of T. gondii inoculated intracamerally can induce both ocular pathology and ocular protective immunity; CD4 +, CD8 +, B cell, and IL-10 gene are all necessary to the vaccine-induced resistance to ocular challenge by virulent RH strain, in which CD8 + T cells are the most important component.

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.

Prevalent Genotypes of Toxoplasma gondii in Pregnant Women and Patients from Crete and Cyprus

Molecular genotyping has been used to characterize Toxoplasma gondii strains into the three clonal lineages known as types I, II, and III. To characterize T. gondii strains from Greece and Cyprus, polymerase chain reaction-restriction fragment length polymorphism analysis on the GRA6 gene was performed directly on 20 clinical samples from 18 humans (11 pregnant women, six patients with lymphadenopathy, and one patient positive for human immunodeficiency virus) and two rats. Characterization of T. gondii types was performed after digestion of amplified products with Mse I. The 20 strains were characterized as type II (20%) and type III (80%). Of these strains, 19 originated from the island of Crete (4 strains type II and 15 strains type III), and 1 from the island of Cyprus (type III). Although both type II and type III strains were found, type III was the most prevalent in Crete.

Epidemiological and genetic characterization of Toxoplasma gondii in urban pigeons from the area of Lisbon (Portugal)

Circumstantial evidence suggests that birds may be a good indicator of soil contamination, since they feed from the ground. Therefore a study was conducted to assess the prevalence of Toxoplasma gondii in urban pigeons ( Columba livia) from different areas of the city of Lisbon and to genetically characterize T. gondii strains isolated from infected animals, in order to assess circulating genotypes. Sera from 695 pigeons captured at 33 different localities of the city of Lisbon were assayed by the direct agglutination test (DAT) showing a 4.6% prevalence. One third of flocks sampled revealed to be infected with T. gondii, with prevalence ranging between 5% and 62.5%. T. gondii DNA could be characterized directly from brain tissue from 12 of 23 seropositive pigeons. Microsatellite typing revealed that 9 strains belonged to type II, 2 were type III and one was type I. No recombinant or atypical genotypes were found. Attempts to recover viable T. gondii strains through bioassay in mice resulted in the isolation of 9 strains.

Patterns and role of diversifying selection in the evolution of Toxoplasma gondii SAG5 locus

The higher intergenotypic polymorphism of the surface antigen genes 5 (SAG5)A, SAG5C, and SAG5E in Toxoplasma gondii was proposed to be the outcome of positive selection pressure favoring variation within these loci. However, the exact nature and magnitude of this selection is not completely known. To address this issue, the amino acids on which natural selection may operate were identified by comparing the ratios of nonsynonymous and synonymous substitutions (pN/pS) of homologous DNA sequences in strains belonging to the three major genotypes of T. gondii. Both positive and negative selections were detected and are likely to have contributed to shaping the patterns of nucleotide substitution and polymorphism in SAG5 genes. Several sites identified in SAG5 loci as likely to be under positive selection suggesting that diversifying selection may have promoted divergence in these genes. Also, it was noted that the SAG5 genetic loci contain many areas that exhibit signs of purifying selection; some of these areas might be the attractive candidates for drug targets. Phylogenetic analysis using the neighbor-joining and maximum parsimony methods grouped the SAG5 sequences of T. gondii strains into three distinct statistically well-supported evolutionary lineages. These findings carry important implications for human and veterinary toxoplasmosis epidemiology and may provide important insights into the pathways through which virulence has evolved in T. gondii.

Control of Toxoplasma gondii infection by athymic LEW‐Whnrnu rats

In immunocompetent rats and humans infection with Toxoplasma gondii remains mostly without overt clinical symptoms, but can be fatal, if the T-cell response is impaired. For a better understanding of the lack of control of T. gondii infection under immunosuppressed conditions, congenitally athymic rats were used as the experimental model. Whereas athymic F344-Whn(rnu) (F344 nude) rats die from a generalized infection during the first 3 weeks after peritoneal inoculation with 10(6) tachyzoites of T. gondii strain NTE, LEW-Whn(rnu) (LEW nude) rats and euthymic LEW rats infected with a 10-fold higher number of parasites developed chronic infection. To identify underlying mechanisms of LEW rats resistance to T. gondii infection and to investigate a possible contribution of residual T-cells to LEW-Whn(rnu) rat resistance, we characterized the immune response of LEW rats by determination of cellularity and composition of lymphocyte population, antigen-specific IgG2b response as well as assays of antigen-specific proliferation and production of IL-2, IFN-gamma and TNF-alpha. As only euthymic LEW rats developed production of antigen-specific IgG and cellular in vitro responses, these results strongly suggest that the genetic background of LEW rats permits a control of the infection independent of an adaptive immune response.

The role of MHC haplotypes H2d/H2b in mouse resistance/susceptibility to cyst formation is influenced by the lineage of infective Toxoplasma gondii strain

Toxoplasma gondii strains displaying the Type I/III genotype are associated with acquired ocular toxoplasmosis in humans. Here, we used a mice model to characterize some immunological mechanisms involved in host resistance to infection with such strains. We have chosen the Type I/III strains D8, G2 and P-Br, which cause a chronic infection in mice that resembles human toxoplamosis. Mice deficient of molecules MyD88, IFN-gamma, and IL-12 were susceptible to all three parasite strains. This finding indicates the importance of innate mechanisms in controlling infection. On the other hand, MHC haplotype did not influenced resistance/susceptibility; since mice lineages displaying a same genetic background but different MHC haplotypes (H2b or H2d) developed similar mortality and cyst numbers after infection with those strains. In contrast, the C57BL/6 genetic background, and not MHC haplotype, was critical for development of intestinal inflammation caused by any of the studied strains. Finally, regarding effector mechanisms, we observed that B and CD8+ T lymphocytes controlled survival,whereas the inducible nitric oxide synthase influenced cyst numbers in brains of mice infected with Type I/III strains. These findings are relevant to further understanding of the immunologic mechanisms involved in host protection and pathogenesis during infection with T. gondii.

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: New linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.

Atypical Toxoplasma gondii Strain from a Free-living Jaguar (Panthera onca) in French Guiana

Like domestic cats, wild felids are involved in the complete infective cycle of Toxoplasma gondii because they can host in their gastrointestinal tract sexually mature parasites and shed infective oocysts in their feces. We report, to our knowledge, the first isolation and molecular characterization of a T. gondii strain from the heart tissue of a free-living jaguar (Panthera onca) in French Guiana. Sequencing at six polymorphic markers indicated that the jaguar isolate had an atypical genotype, including an allele at TgM-A previously found only in isolates from South America, and an allele at GRA6, which was previously reported only in Californian sea otter isolates. These findings are consistent with the recent description of atypical T. gondii strains involved in severe toxoplasmoses in immunocompetent patients in French Guiana that seemed to be linked to a neotropical forest-based cycle involving wild cats and their prey.

Congenital toxoplasmosis: Priorities for further health promotion action

Toxoplasmosis is a disease of considerable public health impact. As the transmission, occurrence and phenotype of this disease are influenced in a complex way by host genetics, immunity, behaviour and by the agent characteristics, prevention will not be simple. This article aimed to review studies defining seroprevalence of and characteristic sociodemographic, biological and lifestyle risk factors for Toxoplasma gondii infection in pregnant women, to evaluate screening and educational programmes, and to assemble recommendations for combating toxoplasmosis in populations at risk. Electronic databases were searched, using a specific search strategy, from 1975 to 2007. There is a high prevalence of T. gondii antibodies in pregnant women worldwide, with some geographic discrepancies attributed to climatic conditions, local food customs, hygiene, lifestyle and cultural differences. The main risk factors for toxoplasmosis in pregnant women are unsanitary feeding habits, poor immune system, contact with cats, contact with soil, pregnancy, number of births, older age, race, travelling outside the country, drinking beverages prepared with unboiled water, consumption of municipal or uncontrolled (well/spring) water and T. gondii strain virulence. Knowledge of these risk factors helps to identify priorities for further epidemiological work and defines effective preventive measures along five main themes of action: information and health education; screening of pregnant women and infants; limiting harm from risk behaviour; treatment of cases found to be at risk; and vaccination.

Toxoplasma gondii: Genotyping of strains from Brazilian AIDS patients with cerebral toxoplasmosis by multilocus PCR–RFLP markers

This study investigated the genetic characteristics of the Toxoplasma gondii strains isolated from 87 patients with cerebral toxoplasmosis and AIDS, treated in Sao Paulo State, Brazil. The laboratorial diagnosis of cerebral toxoplasmosis was based on positive serological exams and PCR of blood and/or cerebrospinal fluid. Four markers (5′-SAG2, 3′-SAG2, SAG3 and GRA6) were chosen to analyze the samples. Each having clear resolution to distinguish the three clonal lineages after PCR amplified targets were treated with restriction enzyme digestion (PCR–RFLP). The genotyping provided the following results: 40 patients (46%) were infected with strains classified as type I; 4 (4%), as type III; 13 (15%) were infected with polymorphic strains (unusual genotype); 6 patients with type I or II alleles; and 15 (17%) patients had strains not classified for any marker. PCR–RFLP, also classified 9 (11%) clinical isolates as type II, which is uncommon in South America. However, the sequencing of the nested-PCR products (of SAG3 marker) of type II and polymorphic isolates (of 5′-SAG2, SAG3 and GRA6 markers) showed a nucleotide polymorphism compared with the archetypal clonal genotypes (types I, II and III) and these isolates were considered as polymorphic strains. The markers used here were inappropriate to distinguish the most isolates considered as polymorphic strains. These data confirm other studies showing the high rate of genetic polymorphism in T. gondii strains isolated in Brazil.