Toxoplasma gondii genotype determines MyD88-dependent signaling in infected macrophages

Infection of mouse macrophages with Toxoplasma gondii elicits MAPK activation and IL-12 production, but host cell signaling pathways have not been clearly delineated. Here, we compared macrophage signaling in response to high virulence type I (RH) vs low virulence type II (ME49) strain infection. Tachyzoites of both strains induced p38 MAPK-dependent macrophage IL-12 release, although ME49 elicited 2- to 3-fold more cytokine than RH. IL-12 production was largely restricted to infected cells in each case. RH-induced IL-12 release did not require MyD88, whereas ME49-triggered IL-12 production was substantially dependent on this TLR/IL-1R adaptor molecule. MyD88 was also not required for RH-stimulated p38 MAPK activation, which occurred in the absence of detectable upstream p38 MAPK kinase activity. In contrast, ME49-driven p38 MAPK activation displayed an MyD88-dependent component. This parasite strain also induced MyD88-dependent activation of MKK4, an upstream activator of p38 MAPK. The results suggest that RH triggers MAPK activation and IL-12 production using MyD88-independent signaling, whereas ME49 uses these pathways as well as MyD88-dependent signaling cascades. Differences in host signaling pathways triggered by RH vs ME49 may contribute to the high and low virulence characteristics displayed by these parasite strains.     

Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines

Virulence in Toxoplasma gondii is strongly influenced by the genotype of the parasite. Type I strains uniformly cause rapid death in mice regardless of the host genotype or the challenge dose. In contrast, the outcome of infections with type II strains is highly dependent on the challenge dose and the genotype of the host. To understand the basis of acute virulence in toxoplasmosis, we compared low and high doses of the RH strain (type I) and the ME49/PTG strain (type II) of T. gondii in outbred mice. Differences in virulence were reflected in only modestly different growth rates in vivo, and both strains disseminated widely to different tissues. The key difference in the virulent RH strain was the ability to reach high tissue burdens rapidly following a low dose challenge. Lethal infections caused by type I (RH) or type II (PTG) strain infections were accompanied by extremely elevated levels of Th1 cytokines in the serum, including IFN-gamma, TNF-alpha, IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by neutralization of IL-18, but not TNF-alpha or IFN-gamma. Nonlethal infections with a low dose of type II PTG strain parasites were characterized by a modest induction of Th1 cytokines that led to control of infection and minimal damage to host tissues. Our findings establish that overstimulation of immune responses that are normally necessary for protection is an important feature of acute toxoplasmosis.     

Dissemination of Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-1 receptor signalling for host resistance assessed by in vivo bioluminescence imaging

Toxoplasma gondii infection can lead to life-threatening systemic disease in the immunocompromised individual and in the developing fetus. Despite intensive investigation in animal models of toxoplasmosis, the processes leading to systemic dissemination remain poorly characterized. In the present study, in vivo bioluminescence imaging (BLI) was applied to the Toxoplasma mouse model to study the dynamics of infection in real time. Photon emission analyses revealed rapid dissemination of parasites in the organism and dissemination to immunoprivileged organs (brain, eyes and testes). Spatio-temporal analysis by BLI in individual mice showed that the virulent RH strain (type I) and the non-virulent ME49/PTG strain (type II) disseminate widely, but the virulent RH strain (type I) exhibits a more dramatic expansion of parasite biomass. Assessment by BLI of the Toll/interleukin-1 receptor (TIR) signalling pathway in host resistance to T. gondii revealed that signal transduction to the adaptor protein MyD88 is probably mediated by Toll-like receptor(s) rather than by IL-1R or IL-18R signalling. However, TLR1(-/-), TLR2(-/-), TLR4(-/-), TLR6(-/-) and TLR9(-/-) animals did not exhibit increased susceptibility to infection. These results suggest that intricate mechanisms regulate TIR-mediated responses during Toxoplasma infection.     

Toxoplasma gondii and Schistosoma mansoni synergize to promote hepatocyte dysfunction associated with high levels of plasma TNF-alpha and early death in C57BL/6 mice.

To address the question of how the murine host responds to a prototypic type 1 cytokine inducer while concurrently undergoing a helminth-induced type 2 cytokine response, C57BL/6 strain animals with patent schistosomiasis mansoni were orally infected with the cystogenic Toxoplasma gondii strain ME49. Schistosoma mansoni infection resulted in a significantly higher mortality rate when mice were subsequently orally infected with ME49, and these animals displayed a defective IFN-gamma and NO response relative to animals infected with T. gondii alone. Plasma levels of TNF-alpha and aspartate transaminase in double-infected mice were greatly elevated relative to mice infected with either parasite alone. Consistent with the latter observation, these animals exhibited severe liver pathology, with regions of coagulative necrosis and hepatocyte vacuolization unapparent in mice carrying either infection alone. Interestingly, mean egg granuloma size was approximately 50% of that in mice with S. mansoni infection alone. The exacerbated liver pathology in coinfected mice did not appear to be a result of uncontrolled tachyzoite replication, because both parasite-specific RT-PCR analysis and immunohistochemical staining demonstrated a low number of tachyzoites in the liver. We hypothesize that mortality in these animals results from the high level of systemic TNF-alpha, which mediates a severe liver pathology culminating in death of the animal.     

Characterization of a spontaneous cyst-forming strain of Toxoplasma gondii isolated from Tokachi subprefecture in Japan

Apicomplexan parasite Toxoplasma gondii has three distinct clonal lineages: high, medium and low virulent strains, type I, II and III, respectively. T. gondii avoids the immune response by transforming from fast multiplying tachyzoite to slow multiplying bradyzoite, and establishing a chronic infection. In the present study, we isolated a new strain of T. gondii from cat feces in the Tokachi subprefecture, Hokkaido, Japan and named it as TgCatJpObi1 (Obi1) strain. Genotyping analysis of 12 loci revealed atypical characters close to type II, genotype 4 according to ToxoDB classification. Phenotypically, Obi1 strain shows slow growth rate and the ability of spontaneous cyst formation in both human foreskin fibroblast (HFFs) and mouse peritoneal macrophages in vitro without bradyzoite induction. Parasite virulence was assessed by means of mouse survival upon infection with either Obi1 or ME49 strains. Obi1 strain displayed no mortalities in comparison to type II clonal lineage, ME49 at LD₅₀ to LD₁₀₀ range (1 × 10³–10⁶ tachyzoites). Although virulence of Obi1 strain is significantly lower than that of ME49, nucleotide sequences analyses revealed that genes of virulence factors such as Gra15, Rop5, 16, 17, and 18 in Obi1 strain were 100% identical to those in the type II strain. Thus, characterization of a newly isolated strain, Obi1, is crucial to clarify the development of toxoplasmosis in both humans and animals.     

Kinetics of parasite burdens in blood and tissues during murine toxoplasmosis

A sensitive real-time PCR technique was used to examine the distribution of Toxoplasma gondii in the blood and tissues of mice during acute and chronic infection. Groups of Swiss Albino mice, inoculated i.p. with 10(2) or 10(6) tachyzoites of the RH strain as a typical type-1 strain, or fed 10 cysts of the Me49 strain as a typical type-2 strain, were killed at different time points post-infection (p.i.), and blood and organs including the lungs, brain and liver were harvested for DNA extraction. Toxoplasma DNA was quantified by a real-time PCR targeted at the 529bp gene fragment, with a detection limit of a single parasite per g/ml of tissue. The results showed a strain- and dose-dependent spread of Toxoplasma. In infection with type-1 parasites, in case of a high infective dose, Toxoplasma DNA was detected within 24h p.i. in all analyzed tissues including the brain. Conversely, in case of a low infective dose, parasitaemia was undetectable early p.i., at a time when Toxoplasma DNA was detected in the tissues, but reached very high levels as infection progressed. With both infective doses, pre-death parasite burdens were higher in the blood than in the tissues, whereas the same loads in the lungs suggest that reaching these Toxoplasma burdens may be critical for survival. In infection with Me49 parasites, steady high parasite burdens were noted up to the end of the experiment at d42 only in the brain, parasitaemia was low but detectable throughout, and Toxoplasma DNA was completely cleared only from the liver. These data are important to better understand the pathogenesis of toxoplasmosis, and also as baseline data for the experimental evaluation of novel chemotherapeutics.     

Detection of Toxoplasma gondii Infections using Virus-Like Particles Displaying T. gondii ROP4 Antigen

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.     

Evaluation of Toxoplasma gondii placental transmission in BALB/c mice model

Toxoplasma gondii infection is common worldwide and highly important to pregnant women as it can be transmitted to the fetus via the placenta. This study aimed at evaluating the prevention of placental transmission in two different strains after chronic infection with each one of the strains. A BALB/c mice model was inoculated 30 days before breeding (immunization) and re-infected 12 and 15 days after pregnancy (challenge). Seven experimental groups were assayed: G1: ME49-immunization (type II), M7741-challenge (type III); G2: M7741-immunization, ME49-challenge; G3, ME49-immunization; G4: M7741-immunization; G5: ME49-challenge; G6: M7741-challenge; G7: saline solution inoculation. Serology, mouse bioassay, PCR and RLFP of the uterus, placenta and fetus were performed to determine the congenital transmission of the strains challenged after chronic infection. IgG T. gondii antibodies were detected in G1, G2, G3 and G4, but not in G5, G6 and G7. All animals of G5 and G6 were IgM-positive. Congenital infection was not detected by bioassay and PCR. Nonetheless, placentas from G3 and G4 resulted positive but no corresponding fetal infection was detected. G1 and G2 did not show the genotype of the strain challenged during pregnancy, only those of chronic infection. Thus, the chronically infected BALB/c mice showed no re-infection after inoculation with another strain during pregnancy. Further studies with different parasite loads and different mice lineages are needed.     

Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt-2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt-2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt-2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite-immune mice with anti-Lyt-2 antibody, but not anti-L3T4 antibody, before challenge significantly increased mortality.     

Role of CD28 in the generation of effector and memory responses required for resistance to Toxoplasma gondii.

CD28 deficient (CD28-/-) mice were used to study the role of costimulation in the T cell-mediated, IFN-gamma-dependent mechanism of resistance to Toxoplasma gondii. These mice were resistant to infection with the ME49 strain of T. gondii. Analysis of the immune response of acutely infected CD28-/- mice revealed that IL-12 was required for T cell production of IFN-gamma and this was independent of the CD40/CD40 ligand interaction. A similar mechanism of IL-12-dependent, CD28/B7 independent production of IFN-gamma by T cells was also observed in wild-type mice. Interestingly, although chronically infected wild-type mice were resistant to rechallenge with the virulent RH strain of T. gondii, chronically infected CD28-/- mice were susceptible to rechallenge with the RH strain. This deficiency in the protective memory response by CD28-/- mice correlated with a lack of IL-2 and IFN-gamma in recall responses and reduced numbers of CD4+ T cells expressing a memory phenotype. Together, our findings demonstrate that CD28 is not required for the development of a protective T cell response to T. gondii, but CD28 is required for an optimal secondary immune response.     

Mic1-3KO tachyzoite a live attenuated vaccine candidate against toxoplasmosis derived from a type I strain shows features of type II strain

Vaccination with live attenuated parasites has been shown to induce high level of protection against Toxoplasma gondii. In this study we compared the Mic1-3KO tachyzoite (a live attenuated strain) with the parental wild type (WT) tachyzoite in terms of virulence in mice in vivo, dissemination in mouse tissues and persistence in mouse brain. Survival of mice infected with the Mic1-3KO parasites correlated with reduced parasite burden in mouse tissues compared to the parental strain. Like the WT parasite, Mic1-3KO is able to form tissue cysts in vivo which are not, in our experimental conditions, infectious when given by oral route. Infection with the attenuated tachyzoite induced lower levels of cytokine and chemokine than with the parental strain. These data demonstrate that the deleted strain derived from a type I strain behaves like type II strain in outbred mice in terms of virulence, dissemination in mouse tissue and persistence in brain.     

Strain and stage specific variation in Toxoplasma gondii antigens

The antigenic profile of virulent (RH, ENT, Martin) and avirulent (RRA, DEG, ME49) Toxoplasma strains was compared directly by western blotting using a panel of immune mouse sera. Dominant antigens of approximate MR 30-33, 21 and 25 x 10(3) were common to tachyzoites of all strains, however, there were significant quantitative and qualitative differences in the antigen profiles, indicating a moderate degree of strain specific polymorphism in tachyzoite antigens. We found no specific association between antigenic variation and strain virulence. Comparison of tachyzoite and bradyzoite antigens from homologous strains (RRA, DEG, ME49) confirmed the existence of stage specific antigens and demonstrated a conserved antigen profile among bradyzoites.     

Virulence for BALB/c mice and antigenic diversity of eight Toxoplasma gondii strains isolated from animals and humans in Brazil

With the purpose of establishing alternative parameters to determine the virulence of Toxoplasma gondii strains, the antigenic diversity of eight strains of the parasite isolated in Brazil was evaluated. BALB/c mice were inoculated i.p. with 10(0), 10(1), 10(2) and 10(3) tachyzoites from each strain. The mortality and time to death of the animals showed that T. gondii strains may be divided in three groups: three strains resulted in 100% of mortality, 5-10 days post inoculation (DPI); three strains resulted in 100% of mortality, 7-19 DPI and brain cysts were observed in the mice which were inoculated; two strains resulted in 0% of mortality, 30 DPI. The analysis of the antigenic profile of different T. gondii strains through Western blotting, using rabbit antiserum to T. gondii, revealed that most antigens are similar to all strains. The mAb 4C3H4 recognized antigens only in the RH, N, AS28 and ME49 strains.     

A human origin type II strain of Toxoplasma gondii causing severe encephalitis in mice

Despite its capacity for sexual reproduction and global distribution, Toxoplasma gondii has a highly clonal structure, with the majority of isolates belonging to three distinct clonal types. Congenital toxoplasmosis has been associated with type I and type II strains. We here present the first characterization of a T. gondii strain (BGD1) from South-East Europe, isolated from the umbilical blood of a 24-week-old fetus in Serbia. Genotyping, performed by PCR-RFLP using a set of nested PCR markers (5'SAG2, 3'SAG2, BTUB, SAG3, and GRA6), showed that the BGD1 strain possessed a type II genotype. The cytokine patterns in Swiss-Webster mice inoculated with brain cysts of BGD1 and the prototype type II ME49 strain were similar until 180 days post-infection, with highly elevated IFN-gamma, IL-12 and IL-10 by d7 and decreasing thereafter. While both strains induced pneumonia and hepatitis in acute infection (d14), chronic infection (d56) was characterized, in addition to hepatitis, by severe meningoencephalitis, associated with numerous brain cysts. Thus, the BGD1 strain of T. gondii has type II genotypic and immunologic characteristics, but unlike other type II strains of human origin, induces severe encephalitis, making it an alternative to the sheep-derived ME49 strain for experimental models of infection.     

Population biology of Toxoplasma gondii and its relevance to human infection: do different strains cause different disease?

Toxoplasma gondii is a protozoan parasite that is globally widespread and causes a common infection of many warm-blooded animals. It has an unusual population structure with a few clonally reproducing strains apparently dominating in many of its hosts, which include humans. In mice, the various strains of the parasite differ enormously in their virulence and disease presentation. In humans, disease manifestations are highly variable, ranging from asymptomatic to severe, especially in cases of brain and eye infection. Recent data suggest that, as with mice, at least part of this variability in human infection may be tied to the type of strain that causes the infection. Improvements in our knowledge of this parasite's population biology and ways to determine the genotype of an infecting strain should make it possible to test this relationship in various disease scenarios. Clear correlations will substantially affect the management of human disease, matching an aggressive infection with an equally aggressive treatment.     

Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation

European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-κB, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are?protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate?macrophage activation influence the ability of Toxoplasma to establish a chronic infection.     

Toxoplasma gondii maintenance in tissue culture: a new efficient method for culturing RH tachyzoites

We describe here a new tissue culture method for prolonged laboratory maintenance of tachyzoites of the highly virulent RH strain of Toxoplasma gondii. Using a rapidly proliferating murine tumor cell line (YAC-1), the method described is easy to perform and is as or more efficient (both in terms of yield and cost) than other traditional methods for maintenance of the parasite. Furthermore, upon prolonged maintenance (greater than 160 days) in YAC-1 tissue culture, the pathogenicity of the parasite, as well as its capacity to elicit an immune response, are comparable to that of organisms maintained in mice. We conclude therefore, that the method described herein is a suitable alternative to the traditional method of maintenance of virulent RH strain T. gondii tachyzoites.     

Attenuation of the virulent RH strain of Toxoplasma gondii by passages in mice immunized with Toxoplasma lysate antigens

The virulent RH strain of Toxoplasma gondii was attenuated after a few passages or just one long passage in mice immunized twice with a four-week interval between immunizations with an emulsion of Toxoplasma lysate antigens and complete Freund's adjuvant. Three avirulent strains, RH-cyst III, IV and VIII were established from the RH strain. The RH-cyst III strain was effective for vaccination against challenge with the original, virulent RH strain. The attenuation of T. gondii is an expression of the innate attributes of this parasite necessary to maintain its parasitic life cycle in nature.     

An investigation of sterile immunity against toxoplasmosis in rats

The non-persistent BK strain was examined for its ability to induce sterile immunity in Wistar rats. Groups of 2-9 Wistar rats were inoculated subcutaneously with 5 x 10(4) BK strain tachyzoites per rat. Two months later, 46 rats were dosed by gavage with 2 x 10(1) cysts of the C, ME-49, Prugniaud, C-56, Elg, M-7741 or M3 strains. Another 26 rats were inoculated with 10(3) oocysts of the ME49, M7741, Bear or Hopa-Hopa strains of Toxoplasma gondii. After 2 months, the rats were euthanized and their brains screened microscopically for toxoplasma tissue cysts and bioassayed in mice if negative. As judged by bioassay, the BK strain of Toxoplasma induced statistically significant protection against reinfection only when rats were challenged with cysts of the C and Prugniaud strains or with oocysts of the ME49 strain. Nonetheless, cysts were detected microscopically only in 23% of brains of immunized rats challenged with oocysts of the Bear and Hopa-Hopa strains of Toxoplasma and none of those challenged with tissue cysts of any strain. Tissue cysts were detected in 43 and 48% of non-immunized control rats infected with tissue cysts and oocysts, respectively. The overall infection in control rats (microscopy and bioassay) was 70 and 72% for rats inoculated with cysts and oocysts, respectively. These results are consistent with the divergent results obtained by other investigators with regard to protection after challenge with different complete strains (cyst and oocysts forming) of the parasite, of rats immunized with incomplete strains.     

Toxoplasma gondii: Impaired maturation and pro-inflammatory response of dendritic cells in MIF-deficient mice favors susceptibility to infection

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Toxoplasma gondii, we investigated host resistance in MIF−/− mice (BALB/c background) during natural oral infection. We focused on the potential involvement of MIF in Dendritic Cell (DC) maturation and IL-12 production. Following oral T. gondii infection, wild type mice developed a strong IL-12 response with an adequate maturation of their draining mesenteric lymph node DC (MLNDC) population and were resistant to challenge with either 40 or 100 cysts (ME49 strain). In contrast, similarly infected MIF−/− mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to both type of challenges. Lack of maturation and IL-12 production of DCs in response to T. gondii antigens was confirmed by in vitro studies, and these effects were reversed following treatment with recombinant MIF. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. gondii infection.