Dissemination of extracellular and intracellular Toxoplasma gondii tachyzoites in the blood flow

Toxoplasma gondii is an intracellular parasite. It has been thought that T. gondii can disseminate throughout the body by circulation of tachyzoite-infected leukocytes (intracellular parasite) in the blood flow. However, a small number of parasites exist as free extracellular tachyzoites in the blood flow (extracellular parasite). It is still controversial whether the extracellular parasites in the blood flow disseminate into the peripheral tissues. In this study, we evaluated the dissemination efficiency of the extracellular and intracellular parasites in the blood flow using GFP-expressing transgenic parasite (PLK/GFP) and DsRed Express-expressing transgenic parasite (PLK/RED). When PLK/GFP and PLK/RED tachyzoites were injected, as intracellular and extracellular forms respectively, at the same time into the tail vein of a mouse, many disseminated green fluorescent PLK/GFP tachyzoites were observed in the lung, the spleen, the liver and the brain. However, only a few red fluorescent PLK/RED tachyzoites were detected in these organs. When PLK/GFP and PLK/RED tachyzoites were injected in the opposite manner, that is, as extracellular and intracellular forms respectively, the majority of tachyzoites in these tissues were PLK/RED tachyzoites. Collectively, these results indicate that intracellular tachyzoites mainly disseminate throughout the body and that extracellular tachyzoites hardly contribute to parasite dissemination.     

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.     

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.     

Decreased resistance of B cell-deficient mice to infection with Toxoplasma gondii despite unimpaired expression of IFN-gamma, TNF-alpha, and inducible nitric oxide synthase

The role of B cells in resistance against Toxoplasma gondii was studied using B cell-deficient (muMT) mice. Following peroral infection with 10 cysts of the ME49 strain, all muMT mice survived the acute stage of the infection but died between 3 and 4 wk after infection. In contrast, all control mice were alive at 8 wk after infection. At the stage during which muMT animals succumbed to the infection, parasite replication and pathology were most evident in their brains; small numbers of tachyzoites were also detectable in their lungs. Significantly greater numbers of T. gondii cysts and areas of inflammation associated with tachyzoites were observed in brains of muMT than in control mice. Large areas of necrosis associated with numerous tachyzoites were observed only in brains of muMT mice. Anti-T. gondii IgG Abs were detected only in sera of control mice, whereas similar levels of IFN-gamma were detected in sera of both strains of mice. Amounts of mRNA for IFN-gamma, IL-10, and inducible NO synthase in the brain did not differ between infected muMT and control mice. Expression of mRNA for TNF-alpha was increased in brains of muMT mice. Administration of polyclonal rabbit anti-T. gondii IgG Ab prevented early mortality and pathology associated with tachyzoites in the brain in the infected muMT mice. These results indicate that B cells play an important role, most likely through their production of specific Abs, in resistance to persistent active (tachyzoite) infection with T. gondii in mice, especially in the brain and lung.     

Infection and immunity with the RH strain of Toxoplasma gondii in rats and mice.

Infection and immunity to toxoplasmosis induced by the RH strain of Toxoplasma gondii was compared in Sprague-Dawley (SD) and Wistar rats and in outbred Swiss Webster mice. All rats injected with up to 1,000,000 RH-strain tachyzoites remained clinically normal, whereas mice injected with only 1 live tachyzoite died of acute toxoplasmosis. Rats could be infected with 1 tachyzoite of the RH strain as shown by antibody development and by bioassay in mice. However, after 8 days, RH-strain organisms were recovered only inconsistently from SD and Wistar rat brains. Contrary to a report of sterile immunity to T. gondii infection in rats after immunization with live RH tachyzoites, we found infection immunity after challenge with the VEG strain. Toxoplasma gondii tissue cysts of the VEG strain could be recovered from most SD and Wistar rats, first injected with live RH-strain tachyzoites and then challenged with oocysts of the VEG strain. Our RH strain, and probably many others, passed for 50+ yr as tachyzoites has lost not only the capacity to form oocysts, but also shows a marked reduction or absence of tissue cyst (bradyzoites) formation.     

Toxoplasma gondii: determination of the onset of chronic infection in mice and the in vitro reactivation of brain cysts

Toxoplasma gondii is an intra-cellular parasite that infects humans through vertical and horizontal transmission. The cysts remain dormant in the brain of infected humans and can reactivate in immunocompromised hosts resulting in acute toxoplasmic encephalitis which may be fatal. We determined the onset and progression of brain cysts generation in a mouse model following acute toxoplasmosis as well as the ability of brain cysts to reactivate in vitro. Male Balb/c mice, (uninfected control group, n = 10) were infected orally (study group, n = 50) with 1000 tachyzoites of T. gondii (ME49 strain) and euthanized at 1, 2, 4, 8 and 16 weeks post infection. Brain tissue was harvested, homogenized, stained and the number of brain cysts counted. Aliquots of brain homogenate with cysts were cultured in vitro with confluent Vero cells and the number of cysts and tachyzoites counted after 1 week. Brain cysts but not tachyzoites were detected at week 2 post infection and reached a plateau by week 4. In vitro Vero cells culture showed similar pattern for cysts and tachyzoites and reactivation of cyst in vitro was not influenced by the age of the brain cysts.     

Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR

We have identified a novel 529bp fragment that is repeated 200- to 300-fold in the genome of Toxoplasma gondii. This 529bp fragment was utilised for the development of a very sensitive and specific PCR for diagnostic purposes, and a quantitative competitive-PCR for the evaluation of cyst numbers in the brains of chronically infected mice. The 529bp fragment was found in all 60 strains of T. gondii tested, and it discriminates DNA of T. gondii from that of other parasites. Toxoplasma gondii DNA was detected in amniotic fluid of patients, as well as in various tissues from infected mice. Polymerase chain reaction with the 529bp fragment was more sensitive than with the 35-copy B1 gene. For the quantitative competitive-PCR, a 410-bp competitor molecule was co-amplified with similar efficiency as the 529bp fragment. Quantitative competitive-PCR produced a linear relationship between the relative amounts of PCR product and the number of tachyzoites in the range of 10(2)-10(4) tachyzoites and 100-3000 tissue cysts. A highly significant correlation between visual counting of brain cysts and quantitative competitive-PCR was obtained in mice chronically infected with Toxoplasma. Thus, quantitative competitive-PCR with the 529bp fragment can be used as an alternative for the tedious visual counting of brain cysts in experimental animals. With the quantitative competitive-PCR, furthermore, we could confirm the copy number of the 529bp fragment in tachyzoites and estimate the number of bradyzoites per cyst.     

Toxoplasma gondii: inconsistent dissemination patterns following oral infection in mice

Since Toxoplasma gondii is transmitted in the wild through the ingestion of infective cysts, oral infection is a preferred model for studying the natural mode of parasite dissemination and pathogenesis. Using luciferase-expressing strains of T. gondii and in vivo imaging, we observed different patterns of disease progression in mice depending of the method of oral infection. Oral gavage of infective cysts (e.g., bradyzoites) resulted in an inconsistent pattern of parasite dissemination; in the majority (20/29) of infected mice, luciferase-derived signal (indicating high numbers of Toxoplasma tachyzoites) was first observed in the right chest area. At later time points this signal spread to other parts of the mouse, including the abdominal area. In the remaining mice (9/29), parasites were first observed replicating in the abdominal area, as might be expected. In contrast, when mice were infected naturally (either via ingestion of whole brains from previously infected mice or brain cyst homogenate-soaked bread), parasites were first observed replicating in the abdominal area in all mice examined (10/10). Based on the inconsistency of infections initiated with oral gavage, it is recommended that natural feeding be used to infect mice when a consistent oral infection is desired.     

Immune responses of mice intraduodenally infected with Toxoplasma gondii KI-1 tachyzoites

Toxoplasma gondii Korean isolate (KI-1) tachyzoites were inoculated intraduodenally to BALB/c mice using a silicon tube, and the course of infection and immune responses of mice were studied. Whereas control mice, that were infected intraperitoneally, died within day 7 post-infection (PI), the intraduodenally infected mice survived until day 9 PI (infection with 1 × 10(5) tachyzoites) or day 11 PI (with 1 × 10(6) tachyzoites). Based on histopathologic (Giemsa stain) and PCR (B1 gene) studies, it was suggested that tachyzoites, after entering the small intestine, invaded into endothelial cells, divided there, and propagated to other organs. PCR appeared to be more sensitive than histopathology to detect infected organs and tissues. The organisms spread over multiple organs by day 6 PI. However, proliferative responses of splenocytes and mesenteric lymph node (MLN) cells in response to con A or Toxoplasma lysate antigen decreased significantly, suggesting immunosuppression. Splenic CD4(+) and CD8(+) T-lymphocytes showed decreases in number until day 9 PI, whereas IFN-γ and IL-10 decreased slightly at day 6 PI and returned to normal levels by day 9 PI. No TNF-α was detected throughout the experimental period. The results showed that intraduodenal infection with KI-1 tachyzoites was successful but did not elicit significant mucosal immunity in mice and allowed dissemination of T. gondii organisms to systemic organs. The immunosuppression of mice included reduced lymphoproliferative responses to splenocytes and MLN cells to mitogen and low production of cytokines, such as IFN-γ, TNF-α, and IL-10, in response to T. gondii infection.     

Importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis in mice

The importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis was studied in mice chronically infected with Toxoplasma gondii by using a mAb to this lymphokine. Control mice chronically infected with the ME49 strain that received saline or normal IgG had slight inflammation in their brains whereas those that received the mAb developed severe encephalitis. In contrast to control mice, the mAb-treated mice had many areas of acute focal inflammation and infiltration of large numbers of inflammatory cells in the meninges and parenchyma of their brains. In the areas of acute focal inflammation, tachyzoites and Toxoplasma Ag were demonstrated by immunoperoxidase staining with the use of rabbit anti-Toxoplasma antibody, suggesting that the focal inflammation was induced by Toxoplasma organisms. Acute inflammation was also observed around cysts of Toxoplasma. Immunohistologic staining revealed tachyzoites and Toxoplasma Ag surrounding the periphery of these cysts suggesting cyst disruption had occurred. Mice treated with mAb against IFN-gamma had five times the numbers of cysts in their brains as did control mice. These results clearly indicate that endogenous IFN-gamma plays a significant and important role in prevention of encephalitis in mice chronically infected with Toxoplasma. The mAb-treated mice had the same Toxoplasma antibody titers and the same degree of macrophage killing of Toxoplasma as did untreated controls. These results suggest that IFN-gamma may have a direct role in preventing cyst rupture and toxoplasmic encephalitis.     

Induction of tumor necrosis factor-alpha and inducible nitric oxide synthase fails to prevent toxoplasmic encephalitis in the absence of interferon-gamma in genetically resistant BALB/c mice

Following infection with Toxoplasma gondii, certain strains of mice, such as BALB/c, are genetically resistant to development of toxoplasmic encephalitis (TE) and establish a latent chronic infection as do humans. Thus, these animals appear to be a suitable model to analyze the mechanism of resistance to TE. Since the mechanism for their genetic resistance is unknown, we examined the role of interferon-gamma (IFN-gamma) tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) in the resistance using BALB/c-background IFN-gamma-deficient (IFN-gamma(-/-)) mice. IFN-gamma(-/-) and control mice were infected with the ME49 strain of T. gondii and treated with sulfadiazine to establish chronic infection. After discontinuing sulfadiazine, the IFN-gamma(-/-) mice all died, whereas the control mice all survived. Histological studies revealed remarkable inflammatory changes associated with large numbers of tachyzoites in brains of the IFN-gamma(-/-) mice but not in the control mice after discontinuation of sulfadiazine. Large amounts of mRNA for tachyzoite-specific SAG1 were detected in brains of only the IFN-gamma(-/-) mice. IFN-gamma mRNA was detected in brains of only the control mice, whereas mRNA for TNF-alpha and iNOS were detected in brains of both strains of mice. The amounts of the mRNA for TNF-alpha and iNOS did not differ between these mice. Treatment of IFN-gamma(-/-) mice with recombinant IFN-gamma prevented development of TE. These results demonstrate that IFN-gamma is crucial for genetic resistance of BALB/c mice against TE and that TNF-alpha and iNOS are insufficient to prevent TE in the absence of IFN-gamma.     

Detection of Toxoplasma gondii by polymerase chain reaction in sera of acutely infected mice

The presence of Toxoplasma gondii DNA was detected in sera of acutely infected mice by polymerase chain reaction. Adult mice were inoculated intraperitoneally with 5 x 10(3) T. gondii RH strain tachyzoites. Five mice were killed every 3 hr from 3 to 21 hr post infection (PI) and every day from 1 to 7 days PI. Toxoplasma gondii DNA was first detected in 60% of the infected mice 18 hr PI and in 100% of the animals 21 hr PI and from 1 to 7 days PI. No mice survived longer than 7 days.     

Protection against lethal toxoplasmosis in mice by an avirulent strain of Toxoplasma gondii: stimulation of IFN-gamma and TNF-alpha response

In this study, we examined whether the PTN strain (isolated from an AIDS patient) of Toxoplasma gondii could induce cross-protection in mice against infection with a lethal dose of the PLK strain. Mice were first infected with tachyzoites (5 x 10(5)) of PTN and 5 days later challenged with PLK (1 x 10(5), LD(90)) parasites. None of these mice succumbed to infection until day 21 after infection, whereas 100% of the mice given the same dose of PLK infection alone died between 5 and 11 days after infection. The protection was accompanied by an increased expansion of NK cells and CD4 + T cells. This condition was associated by increased production of IFN-gamma and an augmented number of IFN-gamma-producing cells in the spleen. Further, PTN + PLK-infected mice showed higher production of TNF-alpha and nitrite compared to PLK-infected mice. Mice infected with the PTN strain had an enhanced capacity to activate the immune system early in infection since they produced higher levels of IFN-gamma, TNF-alpha, and NO than PLK-infected mice. Administration of anti-IFN-gamma mAb or anti-asialo GM1 antibody resulted in 100 and 20% mortality, respectively, in PTN-infected mice but no death in PTN + PLK-infected mice. Together, these results suggest that early production of IFN-gamma and NK-cell activity is important in protection against PTN infection, whereas in PTN + PLK infection components of adaptive immunity rapidly developed following elaboration of an effective early innate immune response.     

Subcutaneous and intestinal vaccination with tachyzoites of Toxoplasma gondii and acquisition of immunity to peroral and congenital toxoplasma challenge

Mice were immunized s.c. or intraintestinally with two injections of a temperature-sensitive mutant of Toxoplasma gondii (ts4). Nonpersistence of the vaccine strain was documented by subinoculation of tissues of a subgroup of mice 3 mo or more after the second immunization. Mice were immune to other-wise lethal parenteral challenges with tachyzoites of the M7741 strain or to peroral challenge with bradyzoites of the Me49 strain of T. gondii. Although two s.c. or intraintestinal immunizations did not completely protect against development of T. gondii in the brains of mice, fewer cysts developed in the s.c. immunized mice than in control mice (2 +/- 3 cysts/0.01 ml in immunized mice compared with 75 +/- 48 cysts/0.01 ml in controls (p less than 0.002)). Reduction in cyst number after intraintestinal immunization was more variable, but also statistically significant (p less than 0.02). Female mice were first immunized, then mated, and then challenged perorally. Neonates of the s.c. immunized mice were not protected. Neonates of intraintestinally immunized mice were protected in part (36% of 115) against congenital infection compared with controls (7% of 107).     

Evaluation of the mood-stabilizing agent valproic acid as a preventative for toxoplasmosis in mice and activity against tissue cysts in mice

Toxoplasma gondii is a common intracellular protozoan infection of humans worldwide. Severe disease can occur in immunocompromised individuals and the in the fetuses of nonimmune pregnant women. Chronic infection is associated with vision and hearing problems, and functional mental alterations, including schizophrenia. The mood-stabilizing agent valproic acid has been shown to inhibit the development of T. gondii in vitro at dosages that are normally achieved in the serum and cerebral spinal fluid of human patients and to have positive effects on the behavior of rats chronically infected with T. gondii. The present study was done to examine the in vivo activity of valproic acid against acute toxoplasmosis in mice. Two studies were done with valproic acid given in the drinking water at concentrations of 1.5 mg/ml (Experiment 1) or 3.0 mg/ml (Experiment 2). In a third experiment (Experiment 3), valproic acid was injected intraperitoneally (i.p.) at doses of 200 or 300 mg/kg every 12 hr. Valproic acid was not effective in preventing acute toxoplasmosis. All mice treated with valproic acid died or were killed and did not (P > 0.05) live significantly longer than the controls. Tachyzoites were demonstrated in the tissues of infected valproic-acid-treated mice. A fourth study was done to determine if valproic acid has activity against T. gondii tissue cysts in chronically infected mice. Mice were chronically infected with the ME-49 strain of T. gondii for 8 wk and then treated orally with valproic acid at approximately 6.6 mg/ml (800 mg/kg/day) in the drinking water for 10 wk (amount was varied due to increasing mouse weights). No significant differences (P > 0.05) were present in tissue cyst numbers in valproic-acid-treated T. gondii chronically infected mice and in mice chronically infected with T. gondii but not given valproic acid. Our results indicate that valproic acid, although effective in vitro against T. gondii tachyzoites, is not effective as a preventative in mice inoculated with T. gondii tachyzoites. Additionally, no activity against tissue cysts was observed in chronically T. gondii-infected valproic-acid-treated mice.     

Experimental infection of Peromyscus californicus with Toxoplasma gondii

Eight female Peromyscus californicus were infected with 10(2) or 10(4) Toxoplasma gondii culture-derived tachyzoites (Type II or X) isolated from southern sea otters. All but 2 mice survived infection and developed antibodies to T. gondii. The 2 fatally infected mice were inoculated with 10(4) tachyzoites of the Type X strain. Parasite detection by immunohistochemistry (IHC) and DNA amplification with 2 polymerase chain reaction (PCR) methods was compared for brain, heart, lung, liver, spleen, biceps muscle, and tongue, at a mean of 41 days postinfection. Parasites were detected most commonly by IHC in spleen (8/8) and brain (6/8). DNA amplification by PCR was most successful from brain, heart, and spleen.     

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.     

Efficacy of ponazuril in vitro and in preventing and treating Toxoplasma gondii infections in mice

Toxoplasma gondii is an important apicomplexan parasite of humans and other warm-blooded animals. Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States. We determined that ponazuril significantly inhibited T. gondii tachyzoite production (P < 0.05) at 5.0, 1.0, or 0.1 microg/ml in African green monkey kidney cells. We used outbred female CD-1 mice to determine the efficacy of ponazuril in preventing and treating acute toxoplasmosis. Each mouse was subcutaneously infected with 1,000 tachyzoites of the RH strain of T. gondii. Mice were weighed daily, and ponazuril was administered orally in a suspension. Mice given 10 or 20 mg/kg body weight ponazuril 1 day before infection and then daily for 10 days were completely protected against acute toxoplasmosis. Relapse did not occur after prophylactic treatments were stopped. Toxoplasma gondii DNA could not be detected in the brains of these mice using polymerase chain reaction (PCR). One hundred percent of mice treated with 10 or 20 mg/kg ponazuril at 3 days after infection and then daily for 10 days were protected from fatal toxoplasmosis. Sixty percent of mice treated with 10 mg/kg ponazuril at 6 days after infection and 100% of mice treated with 20 mg/kg or 50 mg ponazuril 6 days after infection and then daily for 10 days were protected from fatal toxoplasmosis. Relapse did not occur after treatments were stopped. Toxoplasma gondii DNA was detected in the brains of some, but not all, of these mice using PCR. The results demonstrate that ponazuril is effective in preventing and treating toxoplasmosis in mice. It should be further investigated as a safe and effective treatment for this disease in animals.     

Toxoplasma gondii: in vivo expression of BAG-5 and cyst formation is independent of TNF p55 receptor and inducible nitric oxide synthase functions

Wild type, TNFRp55(-/-), iNOS(-/-) and IFN-gamma(-/-) mice were infected with Toxoplasma gondii strain ME-49, and the central nervous system (CNS), lungs, liver, spleen, heart and kidneys were examined for the presence of parasites expressing tachyzoite-specific (SAG-1) and bradyzoite-specific (BAG-5) antigens. During the acute phase of infection, the peripheral organs, but not the CNS, of the IFN-gamma(-/-) mice are heavily parasitized by tachyzoites and there are no signs of parasites expressing BAG-5. In contrast, the tissues from TNFRp55(-/-) and inducible nitric oxide synthase (iNOS)(-/-) mice, mainly the CNS, presented high numbers of parasites expressing SAG-1 and/or BAG-5. Tachyzoite transformation into bradyzoite and cyst development was shown to be normal in the tissues from TNFRp55(-/-) and iNOS(-/-) mice, as indicated by the high numbers of BAG-5/PAS positive cysts. Consistently, reactivation of infection in IFN-gamma(-/-) mice was rapid and characterized by a dramatic increase in SAG-1, contrasting with slow course in the TNFRp55(-/-) or iNOS(-/-) mice associated with a relatively small increase in SAG-1- and/or BAG-5-positive parasites. In conclusion, our results suggest that the control of multiplication of tachyzoites is largely dependent on endogenous IFN-gamma with partial involvement of TNFRp55 and iNOS. In contrast, induction of BAG-5 expression and cyst formation during toxoplasmosis seems to be dependent on IFN-gamma, but independent of TNFRp55 and iNOS functions.     

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.