Cold stress-induced modulation of inflammatory responses and intracerebral cytokine mRNA expression in acute murine toxoplasmosis

The effects of a physical stressor, cold water stress (CWS), within the central nervous system were investigated in the acute phase of infection with Toxoplasma gondii. Female BALB/c mice were subjected to CWS for 5 min each day for 8 days prior to oral infection with 20 cysts of the low virulent ME 49 strain. Animals were killed at 10-day intervals to detect inflammation, gliosis, and expression of intracerebral cytokine mRNAs. Zones of inflammation were detected by Nissl staining and gliosis by immunoreactivity to glial fibrillary acidic protein. Larger zones of inflammation and reactive astrogliosis were consistently observed in mice subjected to CWS and infected (CWS +INF) compared to control infected (INF) mice. Expression of interleukin (IL)-1beta, IL-2, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide synthase (iNOS) were decreased in CWS+INF mice at 10 days postinoculation (PI), followed by a gradual increase after day 20 PI. This was in contrast to increased expression of these cytokines at 10 days PI in INF mice with a gradual decline thereafter. Inflammation and astrogliosis in CWS+INF mice were associated with an increased expression of IL-1beta, IL-6, IL-12, and TNF-alpha between 20 and 30 days PI. These findings correlated with the continuous gene expression of tachyzoite surface antigen (SAG)-1 mRNA in CWS+INF mice compared to its sharp decline in INF mice after 20 days PI. These results suggest that CWS delays regulation and control of intracerebral Toxoplasma gondii during acute infection in BALB/c mice by decreasing the early expression of IFN-gamma, IL-2, TNF-alpha, iNOS, IL-1beta, and IL-12, while increasing the expression of IL-6, a counterregulatory cytokine.     

Toxoplasma gondii: cold stress-induced modulation of antibody responses.

Physical or psychological stressors have been shown to have significant consequences in the immune function and the outcome of disease in human and animal models. Recent work has demonstrated that products released during stress, such as glucocorticoids and catecholamines, can profoundly influence the in vitro growth of pathogens by modulating immune responses. The present study examined the effects of a physical stressor (cold stress) on antigens of Toxoplasma gondii that elicits an antibody-mediated immune response during the acute and chronic phases of infection. Sera obtained from different groups of mice subjected to cold stress during the acute and chronic phases of T. gondii infection were used to measure the levels of antibodies and to localize by Western blot the dominant antigens eliciting IgG and IgM antibody responses. Serum antibodies collected from stressed and infected mice recognized antigens different from those recognized by infected mice without stress. During the acute phase, a stronger IgM antibody response against antigens of 30, 42, 54, and 60 kDa was detected in stressed animals at 3 weeks postinfection. In addition, a 5-kDa antigen was specifically detected in mice subjected to stress during the acute and chronic phases of infection. Levels of specific IgG were increased in infected and in infected and stressed animals that underwent stress in the chronic phase. IgM production did not increase following cold stress in the chronic phase. These results suggest that the strong antibody response in stressed animals is associated with longer parasite persistence in circulation. Stress modulated not only the host immune response but also the ability of parasite antigens to elicit specific antibody responses by the host.     

Cold stress-induced modulation of cell immunity during acute Toxoplasma gondii infection in mice.

Infection with Toxoplasma gondii in the acute phase results in nonspecific suppression of immunologic function in mice and humans. The present study examined the effects of a physical stressor, i.e., cold stress (CS), on macrophage function (nitrite production, parasite survival) and splenic blastogenesis in the acute phase of murine T. gondii infection. In our stress paradigm, female BALB/c mice were placed in cold water (1 +/- 0.5 C), 5 min each day for 8 days. Nitrite production and parasite survival were measured in cultured peritoneal macrophages obtained from mice subjected to CS after in vivo activation with interferon-gamma/lipopolysaccharide (CS + ACT), and in vitro infection with T. gondii tachyzoites. Peritoneal macrophages from CS + ACT mice showed decreased nitrite production compared to control but activated cells (ACT). Spleen cell proliferation to in vitro stimulation with the mitogens concanavalin A (Con A) and anti-CD3, and Toxoplasma lysate antigen (TLA) was measured in splenocytes obtained from BALB/c mice during the acute phase of infection with T. gondii. Mice subjected to CS and infection (CS + INF) had maximum splenocyte proliferation on days 8 and 15 followed by a subsequent decline on day 28 postinoculation (PI). In contrast, infected mice not subjected to stress (INF) showed decreased splenocyte proliferation on days 8 and 15 followed by an increase on day 28 PI. The rate of mortality was decreased in the CS + INF compared to the INF group during acute infection. These results suggest that CS may alter the pathogenesis of T. gondii infection by modulating acute-phase responses, provoking a state of transient disequilibrium between the host and parasite.     

Neospora caninum: identification of 19-, 38-, and 40-kDa surface antigens and a 33-kDa dense granule antigen using monoclonal antibodies.

Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, can infect a broad host range and is regarded as an important cause of bovine abortion worldwide. In the present study, four antigens of N. caninum were partially characterized using monoclonal antibodies. Immunofluorescence of viable tachyzoites as well as the immunoprecipitation of antigens extracted from tachyzoites previously labeled by surface biotinylation revealed that three of these antigens with apparent molecular weights of 40, 38, and 19 kDa are located in the outer surface membrane of this parasite stage. Further evidence for the surface localization of the 38-kDa antigen was obtained by immunoelectron microscopy. In addition to the surface molecules, an antigen located in dense granules and in the tubular network of the parasitophorous vacuole was detected by another monoclonal antibody. When tachyzoite antigens separated under nonreducing conditions were probed on Western blots, this antibody reacted mainly with a 33-kDa antigen. Immunohistochemical analysis of infected tissue sections indicated that the 33-kDa dense granule antigen is present in both tachyzoites and bradyzoites, while the 38-kDa surface antigen from tachyzoites seems to be absent in bradyzoites.     

Simultaneous depletion of CD4+ and CD8+ T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii.

C57BL/6 mice chronically infected with an avirulent strain (ME-49) of Toxoplasma gondii were used to study the mechanisms by which T lymphocytes and IFN-gamma prevent reactivation of latent infection. Infected animals were treated with mAb, either anti-CD8, anti-CD4, anti-CD4 plus anti-CD8, anti-IFN-gamma, or anti-CD4 plus anti-IFN-gamma and the mice followed for survival, histopathology, cyst numbers, and spleen cell cytokine responses. In agreement with previously published findings, treatment with anti-IFN-gamma antibodies fully reactivated the asymptomatic infection, inducing massive necrotic areas in the brain with the appearance of free tachyzoites and death of all animals within 2 wk. Mice treated with the combination of anti-CD4 plus anti-CD8 antibodies showed augmented pathology and mortality nearly identical to the anti-IFN-gamma- treated animals. In contrast, treatment with anti-CD4 or anti-CD8 mAb alone failed to result in significantly enhanced brain pathology or mortality. In additional experiments, full reactivation of infection was observed in mice treated with anti-CD4 plus anti-IFN-gamma indicating that CD4+ lymphocytes are not required for the pathology resulting from IFN-gamma neutralization. Cytokine measurements on parasite Ag-stimulated spleen cells from mAb-treated mice indicated that both CD4+ and CD8+ cells produce IFN-gamma whereas only CD4+ cells contribute to parasite Ag-induced IL-2 synthesis. Together, these results suggest that CD4+ and CD8+ lymphocytes act additively or synergistically to prevent reactivation of chronic T. gondii infection probably through the production of IFN-gamma.     

Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with 125I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi . All cell surface antigens of T. gondii that were labeled with 125I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi . The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.     

Monoclonal antibody directed against Neospora caninum tachyzoite carbohydrate epitope reacts specifically with apical complex-associated sialylated beta tubulin

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.     

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue is difficult because the number of latent cysts is small and reactivation is a transient event. To detect the early stage of reactivation in mouse brain tissue, we constructed a cyst-forming strain of T. gondii in the tachyzoite stage, specifically expressing red fluorescence. The PLK strain of T. gondii was stably transfected with a red fluorescent protein gene, DsRed Express, under the control of a tachyzoite-specific SAG-1 promoter and the resulting parasite was designated as PLK/RED. Tachyzoites of PLK/RED growing in Vero cells showed red fluorescence. When C57BL/6J mice were i.p. infected with tachyzoites of PLK/RED, red fluorescent tachyzoites were detected in their brains at the fourth day p.i. However, red fluorescent tachyzoites were not detected in BALB/c mice latently infected with PLK/RED, although non-fluorescent cysts were detected in their brains. After treatment of latently infected mice with dexamethasone for 1 month, the mice showed neurological symptoms. In mice with symptoms, red fluorescent tachyzoites were again detected in their brains and in other organs. To detect the initial site of reactivation, BALB/c mice latently infected with the strain were treated with dexamethasone for 3 weeks, and brains were excised before any symptoms appeared. Excised brains were examined for red fluorescence-positive sites. By a histological study of red fluorescent-positive sites, we detected a cyst containing red fluorescent zoites, which still had a PAS stain-positive cyst wall. A few red fluorescent zoites breaking away from the cyst were also observed. The stage-specific expression of fluorescent protein facilitates detection of a rare transient event and makes it possible to detect the initial site of reactivation.     

Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.     

Toxoplasma gondii: detection of MIC10 antigen in sera of experimentally infected mice

We developed a sandwich ELISA for the detection of circulating Toxoplasma gondii MIC10 antigens. In T. gondii culture supernatant, MIC10 was detected in a growth dependent manner. Mice were infected with a lethal dose of either a virulent RH strain, an avirulent Beverley strain or a sub-lethal dose of a PLK strain of T. gondii. MIC10 appeared 2 days after infection and increased gradually in the sera of RH-infected mice. A detectable but significantly lower amount of MIC10 was observed in the sera of mice infected intraperitoneally with Beverley tachyzoites. In contrast, the MIC10 antigen in mice sera following oral infection with Beverley cysts was below detectable levels during the course of the experiment. In sera of PLK-infected mice, MIC10 was predominantly observed between late acute and early chronic phase. Our data show that the kinetics of circulating MIC10 differs depending on the strain and route of infection.     

Is stage conversion the initiating event for reactivation of Toxoplasma gondii in brain tissue of AIDS patients?

Reactivation of chronic toxoplasmosis resulting in Toxoplasma encephalitis (TE) is a common event in acquired immune deficiency syndrome (AIDS) patients. Conversion from Toxoplasma gondii bradyzoites to tachyzoites is a prerequisite for reactivation. Until recently, the study of stage conversion in human tissue was not possible due to the lack of antibodies that recognize stage-specific epitopes after long-term formaldehyde fixation. Using the combination of a polyclonal anti-T. gondii antibody, the cyst-stage-specific monoclonal antibody CC2, and a tachyzoite-specific polyclonal antibody (anti-SAG1, recombinant), we tried to demonstrate parasite differentiation in the brain tissue of 10 AIDS patients with clinically suspected TE. Double labeling of the stage-specific antibodies enabled us to demonstrate interconversion between tachyzoites and bradyzoites for the first time in human tissue. The study confirmed that the transformation process is nonsynchronous and that the manifestation of TE depends on the degree and site of tissue destruction caused by invading tachyzoites. The original source of tachyzoites could never be located, but a few samples suggested that tachyzoites may invade by dissemination across the blood-brain barrier. Cyst rupture as the first event in the process of reactivation was not seen. We conclude that the initial site(s) of reactivation will be destroyed by tissue-destructive tachyzoites long before clinical symptoms occur.     

Ultrastructural and biochemical studies on the immunohistochemistry of Toxoplasma gondii antigens using monoclonal antibodies

To determine the cellular distribution of Toxoplasma antigens, RH strain tachyzoites were incubated with either one of three monoclonal antibodies (FMC 19, FMC 20, FMC 22) to T. gondii, or one of two controls (the murine myeloma protein MOPC 21, or phosphate buffered saline), and then incubated with peroxidase-labelled goat-antimouse IgG. Diaminobenzidine was added as substrate and electron microscopy was used to localize the reaction. All three antibodies bound to the entire periphery of the tachyzoite surface membrane. To ascertain the chemical composition of the antigens against which seven monoclonal antibodies (FMC 18, FMC 19, FMC 20, FMC 22, FMC 23, 2G11, 3E6) to T. gondii reacted, untreated, pronase-treated, or periodate-treated tachyzoites were incubated with the antibodies or MOPC 21, and then with [125I]-Protein A. The pronase-treated tachyzoites showed reduced binding for six of the antibodies, compared with the reduction in binding of MOPC 21 with the pronase-treated parasites. The periodate-treated tachyzoites had reduced binding for FMC 18 only. The results of these experiments confirm that most Toxoplasma surface antigens are protein in nature, and are consistent with the hypothesis that at least one cytoplasmic antigen is secreted onto the parasite cell surface.     

Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites

Since we had previously demonstrated the protective role played by Toxoplasma excreted-secreted antigens, the aim of the present work was to produce monoclonal antibodies directed against these antigens in order to determine if their localization in the parasite is compatible with a mechanism of excretion or secretion. Western immunoblotting analysis revealed three monoclonal antibodies (TG17-179, TG17-43, and TG17-113) raised against excreted-secreted antigens of 28.5, 27, and 21 kDa, respectively. The TG17-179 which reacts with antigens isolated by Concanavalin A affinity chromatography is directed against a glycosylated 28.5-kDa component. Colloidal immunogold labeling showed the ultrastructural localization of the 21-, 27-, and 28.5-kDa antigens in the matrix of the dense granules of tachyzoites and associated with the microvilli network of the parasitophorous vacuole, after host cell invasion. These observations suggest the following mechanism of Toxoplasma secretion: secreted antigens are first stored in tachyzoite-dense granules and are then released inside the parasitophorous vacuole. Among the secretory molecules characterized here, the native 27-kDa antigen recognized by TG17-43 is a calcium-binding protein found to be intermixed with the 21- and 28.5-kDa antigens inside the dense granules and hence could play a role in the packaging of secretory products. In addition, the 21- and 28.5-kDa antigens were also located beneath the parasite plasma-lemma. This particular location could reflect a transient step characteristic of T. gondii secretion.     

Analysis of antibody cross-reactivity in experimental American trypanosomiasis.

Susceptible C3H/He mice were immunized with the avirulent Corpus Christi strain of Trypanosoma cruzi and subsequently infected with virulent Brazil stain organisms. Seventy days after infection sera were isolated and analyzed on western blots of electrophoretically separated T. cruzi antigens prepared from culture-form parasites (primarily epimastigotes). More than 25 bands were identified. The antibodies were fractionated by elution from various regions of western blots corresponding to average molecular weights of approximately greater than 130, 77, 70, 60, 48, or 38 kDa. Each of these antibody preparations was then incubated with strips of nitrocellulose containing all of the electrophoretically separated T. cruzi, and cross-reactivity was determined. Antibodies isolated from the 130-, 77-, and 70-kDa regions all cross-reacted with each other. Antibodies eluted from the 60-kDa region bound antigens in the 60-, 70-, and the 77-kDa regions. More importantly, antibodies eluted from every region bound antigens in the 70-kDa region. Conversely, antibodies eluted from this region bound to antigens in all of the other regions. These data indicate the presence of (a) common antigenic epitope(s) in T. cruzi infections in these mice that is predominantly found in the 70-kDa antigen-antibody complex on western blots.     

Bradyzoite-Induced Murine Toxoplasmosis: Stage Conversion, Pathogenesis, and Tissue Cyst Formation in Mice Fed Bradvzoites of - Different Strains of Toxoplasma gondii

ABSTRACT. The development of Toxoplasma gondii was studied in mice fed bradyzoites. At one hour after oral inoculation (HAI), bradyzoites were found in cells of the surface epithelium and the lamina propria of the small intestine, primarily the ileum. Division into two tachyzoites was first observed at 18 HA1 in the intestine. At 24 HAI, organisms were also seen in mesenteric lymph nodes. Organisms were first detected in the brain at six days after oral inoculation with bradyzoites (DAI) but not consistently until 10 DAI. Immunohistochemical staining with bradyzoite specific (BAG-5 antigen) anti-serum showed that bradyzoites retained their BAG-5 reactivity even after the first division into two tachyzoites in the intestine at 18 HAL BAG-5 positive organisms were not seen 2–5 DAI. BAG-5 antigens reappeared in T. gondii at 6 DAI. Whole mice and individual tissues of mice fed bradyzoites were bioassayed in cats and mice for the presence of bradyzoites. Feces of cats fed murine tissues were examined for oocyst shedding for short prepatent periods. Bradyzoites were present in the intestines of mice up to 12 HA1 but not at 18 HAI, and tachyzoites and not bradyzoites disseminated to other tissues from the intestine. Bradyzoites were again detected 6 DAI. Using the mouse bioassay, T. gondii was first detected in peripheral blood at 24 HA1 and more consistently at 48 HAL Using a pepsin-digestion procedure and mouse bioassay, organisms were demonstrated in many tissues of mice 15 and 49 DAI.     

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.     

Effects of specific monoclonal antibodies to dense granular proteins on the invasion of Toxoplasma gondii in vitro and in vivo

Although some reports have been published on the protective effect of antibodies to Toxoplasma gondii surface membrane proteins, few address the inhibitory activity of antibodies to dense granular proteins (GRA proteins). Therefore, we performed a series of experiments to evaluate the inhibitory effects of monoclonal antibodies (mAbs) to GRA proteins (GRA2, 28 kDa; GRA6, 32 kDa) and surface membrane protein (SAG1, 30 kDa) on the invasion of T. gondii tachyzoites. Passive immunization of mice with one of three mAbs following challenge with a lethal dose of tachyzoites significantly increased survival compared with results for mice treated with control ascites. The survival times of mice challenged with tachyzoites pretreated with anti-GRA6 or anti-SAG1 mAb were significantly increased. Mice that received tachyzoites pretreated with both mAb and complement had longer survival times than those that received tachyzoites pretreated with mAb alone. Invasion of tachyzoites into fibroblasts and macrophages was significantly inhibited in the anti-GRA2, anti-GRA6 or anti-SAG1 mAb pretreated group. Pretreatment with mAb and complement inhibited invasion of tachyzoites in both fibroblasts and macrophages. These results suggest that specific antibodies to dense-granule molecules may be useful for controlling infection with T. gondii.     

An unexpected response to vaccination with a purified major membrane tachyzoite antigen (P30) of Toxoplasma gondii

A purified Toxoplasma gondii tachyzoite membrane protein (P30) and a monoclonal antibody directed against this antigen were used to immunize mice. The P30 protein has an apparent m.w. of 30,000 and is the major radioiodinated tachyzoite membrane antigen identified by human and mouse antitoxoplasma antisera. Polyclonal and monoclonal antibody to the P30 antigen are parasiticidal in the presence of human serum. A series of mice were immunized with affinity column-purified P30 protein. This produced a dose-dependent, antigen-specific IgG and IgM response. The mice were challenged with the less virulent C strain tachyzoite. Immunized mice showed a statistically significant increase in mortality over nonimmunized control mice. In addition, vaccinated mice had an increased number of intracerebral tissue cysts when compared with the control group. Similar results were obtained with passive transfer immunization by using monoclonal antibody directed against the P30 antigen. Immunofluorescence assay of brain tissue cyst bradyzoites revealed a total absence of P30 antigen. Bradyzoites were also deficient in another major tachyzoite antigen of approximate m.w. 22,000 (P22). Mouse antibradyzoite serum absorbed with tachyzoites recognized bradyzoites but failed to identify tachyzoites. This suggests that there are stage-specific bradyzoite antigens of Toxoplasma gondii.     

Inhibition of inducible nitric oxide synthase exacerbates chronic cerebral toxoplasmosis in Toxoplasma gondii-susceptible C57BL/6 mice but does not reactivate the latent disease in T. gondii-resistant BALB/c mice

Infection of C57BL/6 mice with Toxoplasma gondii leads to progressive and ultimately fatal chronic Toxoplasma encephalitis (TE). Genetic deletion or inhibition of inducible nitric oxide synthase (iNOS) from the beginning of infection increased the number of T. gondii cysts in the brain and markedly reduced the time-to-death in this mouse strain. In the present study, we addressed whether iNOS also contributes to the control of intracerebral parasites in a clinically stable latent infection that develops in T. gondii-resistant BALB/c mice after resolution of the acute phase of TE. iNOS was expressed in the inflammatory cerebral infiltrates of latently infected BALB/c mice, but the number of iNOS+ cells was significantly lower than in the brains of chronically infected T. gondii-susceptible C57BL/6 mice. In BALB/c mice with latent TE (> 30 days of infection), treatment with the iNOS inhibitors L-N6-iminoethyl-lysine or L-nitroarginine-methylester for < or = 40 days did not result in an increase of the intracerebral parasitic load and a reactivation of the disease, despite the presence of iNOS-suppressive inhibitor levels in the brain. However, L-nitroarginine-methylester treatment had remarkably toxic effects and induced a severe wasting syndrome with high mortality. In contrast to BALB/c mice, L-N6-iminoethyl-lysine treatment rapidly exacerbated the already established chronic TE of C57BL/6 mice. Thus, the containment of latent toxoplasms in T. gondii-resistant BALB/c mice is independent of iNOS, whereas the temporary control of intracerebral parasites in T. gondii-susceptible C57BL/6 mice with chronic TE requires iNOS activity.     

Molecular cloning of the 82-kDa heat shock protein (HSP90) of Toxoplasma gondii associated with the entry into and growth in host cells

Among the monoclonal antibodies (mAb) against Toxoplasma gondii, mAb Tg485 specifically reacted with an 82-kDa cytoplasmic protein of tachyzoites. The protein was secreted from extracellular tachyzoites, but was not released into the parasitophorous vacuole after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg485. The full-length cDNA was amplified by the 5(')-RACE method and sequenced. The deduced amino acid sequence of the 82 kDa protein reacting with Tg485 revealed a polypeptide of 708 amino acids showing significant homology to the heat shock protein 90 (HSP90) family of other organisms, especially to those of apicomplexan species. Treatment with geldanamycin, a drug known to interfere with HSP90 function, did not affect the secretion of TgHSP90 from extracellular tachyzoites, but the entry of the tachyzoites into host cells and the intracellular growth of the parasite were significantly disturbed.