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.     

Role of Toxoplasma gondii HSP70 and Toxoplasma gondii HSP30/bag1 in antibody formation and prophylactic immunity in mice experimentally infected with Toxoplasma gondii.

Production of antibodies against Toxoplasma gondii (T. gondii)-derived stress proteins, T. gondii HSP70 (T.g.HSP70) and T.g.HSP30/bagl, in C57BL/6 and BALB/c mice perorally infected with cysts of the avirulent Fukaya strain of T. gondii was analyzed. Production of anti-T.g.HSP70 IgG antibodies was transient, whereas production of anti-T.g.HSP30/bag1 IgG antibodies persisted after infection in both C57BL/6 and BALB/c mice. C57BL/6 mice, a susceptible strain, predominantly produced IgG antibodies specific for T.g.HSP70, whereas BALB/c mice, a resistant strain, predominantly produced IgG antibodies specific for T.g.HSP30/bag1, after T. gondii infection. Immunization with rT.g.HSP30/bag1 enhanced, whereas immunization with rT.g.HSP70 reduced host protective immunity against T. gondii infection with a cyst-forming avirulent strain, Fukaya, and a virulent strain, RH.     

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.     

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.     

Toxoplasma gondii: protective immunity against toxoplasmosis with recombinant actin depolymerizing factor protein in BALB/c mice

Toxoplasmosis is one of the most world-wide spread zoonosis representing a very serious clinical and veterinary problem. There is still need for vaccines for toxoplasmosis. In the present study, we evaluated the protective efficacy of a recombinant actin depolymerizing factor (ADF) subunit vaccine against Toxoplasma gondii infection in BALB/c mice. The recombinant T. gondii ADF protein (rADF) was expressed in Escherichia coli and used as antigens for BALB/c mice immunization. The results indicated that specific antibody and the increased percentage of CD4(+) T lymphocyte were found in vaccinated BALB/c mice with rADF, when compared with adjuvant or PBS groups. After challenged with T. gondii (RH strain) tachyzoites, the survival time of the mice in rADF group was longer than those in the control group. The numbers of brain cysts of the mice in rADF group reduced significantly when compared with those in control groups (P<0.05), and the rate of reduction could reach to around 30%. These results suggest that rADF can generate protective immunity against T. gondii infection in BALB/c mice.     

Effect of rIFN-gamma and IL-2 treatments in mouse and nude rat infections with Toxoplasma gondii.

Mice and nude rats lethally infected with T. gondii and treated with recombinant rat interferon-gamma (rIFN-gamma) or recombinant human interleukin-2 (rIL-2) were protected against death, when compared with untreated infected controls. In mice rIFN-gamma and rIL-2 played an important role in "prophylactic treatment", but not in "curative therapy". The survival rate was 42% in mice treated with 3 doses of 20,000 U of rIFN-gamma at days -2, -1, 0 before challenge and up to 66% in mice treated with 3 doses of 10,000 U of rIFN-gamma at days -2, 0, +2 before and after infection. Whereas the survival rate was 33% in mice that received 3 doses of 500 U rIL-2 at days -2, -1, 0 before infection, or -2, 0, +2 before and after infection respectively, up to 50% of the mice treated with 3 doses of 1,000 U rIL-2 at days -2, -1, 0 survived. In nude rats rIFN-gamma had a slight effect in "prophylactic treatment", whereas rIL-2 was active only in "curative treatment". The survival rate was 25% both in nude rats treated with doses of 400,000 U of rIFN-gamma at days -3, 0 before challenge, or with doses of 5,000 U of rIL-2 at days +2, +6, +9 after infection. These results lead us to hypothesise that the mechanism by which the lymphokine treatment exerts a protective effect on Toxoplasma infected mice is different from that on nu/nu rats. We conclude that these cytokines may play a notable role in modulating the host's immune defence against T. gondii infection.     

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.     

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.     

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).     

Suppressed cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with Toxoplasma gondii tachyzoites

Mechanisms of host immunosuppression after infection with Toxoplasma gondii are unclear. This study was performed to observe cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with live, nonreplicating (irradiated) RH tachyzoites on stimulation with concanavalin A (Con A) or lipopolysaccharide (LPS). For lymphocyte cultivation, 3 groups were prepared: coculture with live nonirradiated tachyzoites separated by a transwell (group T), live irradiated tachyzoites without a transwell (group R), and no tachyzoites (group C). Compared with group T, groups R and C, on stimulation with Con A, revealed significantly (P < 0.05) lower levels of interleukin-2 (IL-2) and IFN-gamma, but not IL-10. The levels of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were also significantly (P < 0.05) lower in groups R and C than in group T after stimulation with LPS. The results suggest that intracellular infection of murine splenic lymphocytes with T. gondii tachyzoites could impair their capacity to produce cytokine and immunoglobulin secretions.     

Tumor necrosis factor-independent protective effect of recombinant IFN-gamma against acute toxoplasmosis in T cell-deficient mice

rIFN-gamma conferred remarkable resistance against acute infection with Toxoplasma gondii in T cell-deficient (athymic nude) mice. Mice that received an i.p. injection of rIFN-gamma every other day beginning 24 h before infection for a total of eight doses survived significantly longer than untreated control mice although all of the treated mice died after the lymphokine was discontinued. Mice that received 14 doses of rIFN-gamma survived significantly longer than those that received eight doses of the lymphokine although mice started dying soon after the final (14th) injection of rIFN-gamma and eventually all of the treated mice died. Histologic study revealed that the IFN-gamma treatment prevented proliferation of the organisms in all organs examined, including brain, lung, heart, liver, and spleen. The treatment was effective even when started 1 day after infection. Peritoneal macrophages obtained from mice injected with rIFN-gamma were activated and effectively killed tachyzoites of T. gondii in vitro. TNF activity could not be detected in sera of the infected mice during treatment with rIFN-gamma. Administration of anti-TNF antibody did not affect the protective effect of rIFN-gamma against T. gondii infection. These facts indicate that rIFN-gamma can confer resistance to acute infection with T. gondii without collaboration of lymphokines derived from T cells and TNF. This suggests that rIFN-gamma may be effective for therapy of toxoplasmosis in immunosuppressed patients who have impaired activity of T cell function, especially those with AIDS.     

Buprenorphine does not affect acute murine toxoplasmosis and is recommended as an analgesic in Toxoplasma gondii studies in mice

Groups of mice were infected with tachyzoites of the RH strain of Toxoplasma gondii, treated with the opioid analgesic buprenorphine, sodium sulfadiazine, a combination of buprenorphine and sodium sulfadiazine, or nothing in the drinking water, on days -1 to 12 postinfection. Mice in the T. gondii-infected buprenorphine-treated group did not live significantly longer (P > 0.05) than mice given T. gondii and not treated with buprenorphine. Clinical observations of mice indicated that buprenorphine treatment reduced distress and pain in mice with acute toxoplasmosis. Mice treated with sodium sulfadiazine alone or sodium sulfadiazine combined with buprenorphine survived the 28-day study. Mice treated with buprenorphine and not infected with T. gondii also survived the 28 days. This study demonstrates that buprenorphine does not adversely interfere with acute T. gondii infection and indicates that buprenorphine can be given to mice to alleviate pain and distress associated with a T. gondii infection, and not adversely influence the results of toxoplasmosis studies. Analgesic (buprenorphine) treatment should now be the standard of care for mice in acute toxoplasmosis studies.     

Use of mouse macrophage cell lines for in vitro propagation of Toxoplasma gondii RH tachyzoites

In most laboratories, Toxoplasma gondii is maintained in mice and is studied in vitro using nonlymphoid cell lines or primary mouse macrophages. In this study, three rapidly dividing mouse macrophage cell lines (J774 A.1, P388D1, RAW264.7) were evaluated for their suitability for studying the RH strain of T. gondii. For comparison, tachyzoites were also grown in two slowly dividing epithelial cell types: a rat lung cell line (L2) and a bovine turbinate cell line (BT). Various inocula of T. gondii were added to the above cells and tachyzoites were harvested from the culture supernatants after 2-8 days of infection. The mouse macrophage cell lines supported rapid growth of T. gondii RH allowing up to a 300-fold increase of the inoculum in 2-4 days. L2 and BT supported slower growth of T. gondii (10- to 90-fold increase of inoculum in 5 to 8 days) and, thus, may be more suitable for assessment of host cell-parasite interactions and drug activity. Toxoplasma gondii RH isolated from each of the cell cultures described were able to multiply in all cell types used. Protein profiles of whole tachyzoite isolated from mice or cell cultures and protein profiles of the corresponding soluble and membrane fractions of the intraphagosomal membrane network were similar as seen after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In mice, intraperitoneal injection of 10(6), 10(5), and 10(3) tachyzoites isolated from the cell cultures or from infected mice caused death after 4, 5, and 8 days, respectively, indicating that parasites grown in vitro retained virulence.     

Stage conversion of Toxoplasma gondii RH parasites in mice by treatment with atovaquone and pyrrolidine dithiocarbamate

The mouse-virulent RH strain of Toxoplasma gondii is generally considered to have lost its cyst-forming capacity, and conversion of RH tachyzoites into cysts in non-immune mice has previously been shown exclusively following early treatment with sulfadiazine (SDZ). We here describe the development of tissue cysts in mice infected with RH strain parasites and treated with atovaquone (ATO) combined with pyrrolidine dithiocarbamate (PDTC). Groups of Swiss-Webster mice infected intraperitoneally (i.p.) with 10(2) RH tachyzoites were treated with 5, 25 and 100 mg of ATO/kg per day alone or combined with PDTC at 250 mg/kg per day from day 1 postinfection (p.i.) for 14 days. A total of 19 mice survived the 6-week observation period. Of these, brain cysts were recovered in nine (47%), with burdens ranging from 50 to 3120 (mean +/- S.D. = 622 +/- 963). All cyst-harboring mice had high specific IgG antibody levels (1:10,240-1:40,960, corresponding to 500-2000 IU/ml), as did one mouse in which cysts were not demonstrated, which was therefore included in the group of mice with residual infection. Bioassay performed to test the infectivity of these cysts produced acute lethal toxoplasmosis following i.p. inoculation in all instances (100%), and importantly, following peroral inoculation in four (29%). The recovered tachyzoites were highly infectious. In addition, significantly elevated interferon gamma (IFN-gamma) in the treated mice which developed residual infection compared with any group of infection-free (treated or subinoculated) mice, indicates immunological control of the parasite in the latent form. In conclusion, early treatment of mice infected with T. gondii RH tachyzoites with ATO and PDTC induces conversion into tissue cysts, thus providing a new model for studying the mechanism(s) of T. gondii stage conversion.     

Toxoplasma gondii Hsp70 as a danger signal in toxoplasma gondii-infected mice

Toxoplasma gondii Hsp70, T gondii Hsp30/bag1, and surface antigen 1 messenger RNAs were shown to be useful in analyzing stage conversion of T gondii between bradyzoites and tachyzoites. The high-level expression of T gondii Hsp70 was correlated with mortality in interferon-gamma knockout mice infected with T gondii. Tgondii Hsp70 inhibited the induction of nitric oxide release by peritoneal macrophages of T gondii-infected mice. These findings identify T gondii Hsp70 as a danger signal during lethal, acute T gondii infection.     

Experimental congenital toxoplasmosis in Wistar and Holtzman rats

Congenital toxoplasmosis was evaluated in Wistar and Holtzman rats using two strains of Toxoplasma gondii isolated in Brazil. Pregnant rats were inoculated by subcutaneous or intraperitoneal routes with 10(6) or 8 x 10(6) tachyzoites of N strain (virulent for mice) and by subcutaneous or oral routes with 10(2) or 1.2 x 10(3) cysts of P strain (avirulent for mice). The tissues of rat pups born from these rats were bioassayed for T. gondii infection. T. gondii was not observed in the pups born from rats inoculated with N strain. In the animals inoculated with P strain, congenital toxoplasmosis occurred in 22.8% (Wistar rats inoculated with 10(2) cysts by the subcutaneous route), 11.4% (Wistar rats inoculated with 10(2) cysts by the oral route), 21.2% (Wistar rats inoculated with 1.2 x 10(3) cysts by the oral route) and 2.9% of fetal infection (Holtzman rats inoculated with 10(2) cysts by the oral route). None of the pups born from chronically infected mother were infected with T. gondii.     

Serologic response of the opossum Didelphis virginiana to a temperature-sensitive mutant (ts-4) of Toxoplasma gondii.

A study was designed to measure the Toxoplasma gondii-specific IgM and IgG antibody responses of opossums inoculated with tachyzoites of the temperature-sensitive mutant of T. gondii, ts-4, and to examine its persistence in the tissues. Four young opossums seronegative for anti-Toxoplasma gondii IgM and IgG antibodies immediately after capture and 4 wk later were injected subcutaneously with 1.8 x 10(6) ts-4 tachyzoites; a fifth opossum (also seronegative) received an injection of saline only. Serum was collected weekly and titered by modified direct agglutination for anti-Toxoplasma gondii IgM and IgG. IgM titers were detectable from week 1 to week 6 postinoculation (PI). IgG was measurable by week 3 and remained high for 30 wk PI when the opossums were killed and examined. The control opossum did not develop a specific antibody response. At necropsy major lesions were not found. No anti-Toxoplasma gondii IgG was detected in serum collected from mice injected with tissues prepared from the opossums at necropsy, and no T. gondii was found on impression smears made at necropsy from these mice. Modified direct agglutination performed with or without 0.2 M 2-mercaptoethanol worked well for measuring specific IgM and IgG antibodies in experimentally infected opossums.     

Parasiticidal activity of Haemaphysalis longicornis longicin P4 peptide against Toxoplasma gondii

The Haemaphysalis longicornis longicin P4 peptide is an active part peptide produced by longicin which displays bactericidal activity against both Gram-negative and Gram-positive bacteria and other microorganisms. In the present study, the effect of the longicin P4 peptide on the infectivity of Toxoplasma gondii parasites was examined in vitro. Tachyzoites of T. gondii incubated with longicin P4 had induced aggregation and lost the trypan blue dye exclusion activity and the invasion ability into the mouse embryonal cell line (NIH/3T3). Longicin P4 bound to T. gondii tachyzoites, as demonstrated by fluoresce microscopic analysis. An electron microscopic analysis and a fluorescence propidium iodide exclusion assay of tachyzoites exposed to longicin P4 revealed pore formation in the cellular membrane, membrane disorganization, and hollowing as well as cytoplasmic vacuolization. The number of tachyzoites proliferated in mouse macrophage cell line (J774A.1) was significantly decreased by incubation with longicin P4. These findings suggested that longicin P4 conceivably impaired parasite membranes, leading to the destruction of Toxoplasma parasites in J774A.1 cells. Thus, longicin P4 is an interesting candidate for antitoxoplasmosis drug design that causes severe toxicity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that longicin P4 causes aggregation and membrane injury of parasites, leading to Toxoplasma tachyzoite destruction.     

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.