Toxoplasmosis In Mice Research Articles

Induction of protective immunity against toxoplasmosis in mice by DNA immunization with a plasmid encoding Toxoplasma gondii GRA4 gene

GRA4 is a dense granule protein of Toxoplasma gondii that is a candidate for vaccination against this parasite. We have inserted the entire coding sequence of GRA4 into an eukaryotic expression vector to determine whether DNA immunization can elicit protective immune response to T. gondii. Susceptible C57BL/6 mice were then vaccinated intramuscularly with GRA4 DNA and orally challenged with a lethal dose of 76 K T. gondii strain cysts. Immunization with pGRA4 resulted in a 62% survival of C57BL/6 infected mice. Mice immunized with GRA4 DNA developed high levels of serum anti-GRA4 immunoglobulin G antibodies as well as a cellular immune response, as assessed by splenocyte proliferation, in response to recombinant GRA4 protein restimulation in vitro. The cellular immune response was associated with IFN-γ and IL-10 synthesis, suggesting a modulated Th1-type response. Splenocyte proliferation was strongly enhanced and protection slightly higher by inoculation with GRA4 DNA combined with a granulocyte-macrophage colony-stimulating factor expressing vector. This is the first report that demonstrates the establishment of a DNA vaccine-induced protective immunity against the acute phase of T. gondii infection.

Protection against Lethal Toxoplasmosis in Mice by an Avirulent Strain of Toxoplasma gondii: Stimulation of IFN-γ and TNF-α Response

Haque, S., Franck, J., Dumon, H., Kasper, L. H., and Haque, A. 1999. Protection against lethal toxoplasmosis in mice by an avirulent strain of Toxoplasma gondii: Stimulation of IFN-γ and TNF-α response. Experimental Parasitology93, 231–240. 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 × 105) of PTN and 5 days later challenged with PLK (1 × 105, LD90) 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-γ and an augmented number of IFN-γ-producing cells in the spleen. Further, PTN + PLK-infected mice showed higher production of TNF-α 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-γ, TNF-α, and NO than PLK-infected mice. Administration of anti-IFN-γ 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-γ 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.

Fas-FasL interaction involved in pathogenesis of ocular toxoplasmosis in mice.

Ocular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation of Toxoplasma gondii. Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8(+) and CD4(+) T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6-lpr) and FasL-deficient (B6-gld) mice. Inoculation of 5,000 T. gondii tachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8(+) than CD4(+) T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6-lpr and B6-gld mice than in wild-type B6 mice (P < 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.

Use of ketolides in combination with other drugs to treat experimental toxoplasmosis.

Because combination therapy is required to treat human toxoplasmosis, we examined combinations of the ketolides HMR 3004 and HMR 3647 with atovaquone, clindamycin or sulphadiazine in a murine model of toxoplasmosis. An oral dose of 50 mg/kg/day of HMR 3004 protected 30% of mice lethally infected with Toxoplasma gondii. The same dose protected 100% of infected mice when administered in combination with non-protective doses of atovaquone, clindamycin or sulphadiazine. Similar results were noted with 25 mg/kg/day of HMR 3647. These results demonstrate that these drug combinations are highly effective for treating toxoplasmosis in mice.

Nifurtimox Plus Pyrimethamine for Treatment of Murine Toxoplasmosis

In the search for novel antitoxoplasmic agents, we evaluated the efficacy of nifurtimox (3-methyl-4[5'-nitrofurfurylidene-amino]-tetrahydroe-4H-1,4- thiazine-1,1-dioxide), an antiprotozoal drug effective against trypanosomiasis, in an experimental model of acute toxoplasmosis in mice. One hundred NIH mice were inoculated intraperitoneally, each with 2,614 RH tachyzoites of Toxoplasma gondii, and randomly allocated into 5 groups (n = 19-21). Animals from each group were orally treated for 10 days either with nifurtimox 25 mg/kg/day (NF1), nifurtimox 50 mg/kg/day (NF2), pyrimethamine 60 mg/kg/day (P), the combination nifurtimox 50 mg/kg/day plus pyrimethamine 60 mg/kg/day (NF2-P), or with corn oil (controls). Survival of mice was recorded daily for 1 mo after the experimental infection. Comparisons of cumulative mortality between groups were made applying the chi2 test. Mean survival time was longer in animals from P and NF2-P groups than those from NF1, NF2, and control groups. Cumulative mortality was less in mice from the NF2-P group (25%), than that in mice from the P (65%), the NF1 (100%), the NF2 (89%), or the control (95%) groups (P < 0.01). The doses of nifurtimox used in the present study were not significantly effective against murine toxoplasmosis. However, when combined with pyrimethamine, a strong anti-toxoplasma effect was obtained in comparison with survival rates associated with pyrimethamine or nifurtimox alone. It seems feasible that nifurtimox inhibits the replication of T. gondii tachyzoites similar to that of other protozoans, e.g., Trypanosoma and Leishmania. It will be important to determine if the reduction of mortality in mice treated with the nifurtimox-pyrimethamine combination results from summation or from synergism. Further studies on the toxic mechanisms exerted by nifurtimox on T. gondii seem warranted.

Protective effect of vaccination with a combination of recombinant surface antigen 1 and interleukin-12 against toxoplasmosis in mice.

We studied the immune response induced in mice by recombinant Toxoplasma gondii surface antigen 1 (rSAG1) protein, alone or combined with interleukin-12 (IL-12) as an adjuvant, and the protective effect against toxoplasmosis. Immunization with rSAG1 alone induced a specific humoral type 2 immunity and did not protect the animals from infection. In contrast, immunization with rSAG1 plus IL-12 redirected humoral and cellular immunity toward a type 1 pattern and reduced the brain parasite load by 40%.

Lack of Therapeutic Effect of Colchicine on Murine Toxoplasmosis

In a previous report, we showed that addition of colchicine to cultures of glial cells infected with Toxoplasma gondii decreased the number of parasites by up to 80%. To provide support for potential therapeutic use of colchicine in toxoplasmosis, a murine model of T. gondii infection was used. Mice infected with pure RH T. gondii tachyzoites (from 2,233 to 25,000 parasites) were treated daily with either pyrimethamine (80 or 51 mg/kg), colchicine (10 mg/kg), pyrimethamine-colchicine, or vehicle (controls). Survival rates were lower in animals treated with colchicine (from 40% to 27%) and pyrimethamine-colchicine (from 73% to 41%) than in animals treated with pyrimethamine alone (from 100% to 73%). There was no extension of mean survival time in animals treated with colchicine compared to controls. These results demonstrate that colchicine does not improve the course of acute toxoplasmosis in mice, and it is detrimental rather than beneficial at the regimen tested.

Protective Effect of Vaccination with a Combination of Recombinant Surface Antigen 1 and Interleukin-12 against Toxoplasmosis in Mice

Protective Effect of Vaccination with a Combination of Recombinant Surface Antigen 1 and Interleukin-12 against Toxoplasmosis in Mice

The stage-conversion time of Toxoplasma gondii: interpretation of chemical-biologic data out of parasitologic or host context.

Several published biologic times for the stage conversion of Toxoplasma gondii from tachyzoite to bradyzoite in mice are critically examined. There are several reports of 3 days and many of longer times. Possible errors, related to delay from dissemination of the infection, are pointed out. The time to the appearance of the 36-kDa surface antigen, sometimes used for the diagnosis of bradyzoites, should be compared with the biologic attribute of bradyzoites, inducing the short prepatent period in cats. It is recalled that the development of acid pepsin resistance is not exclusively correlated with the biologic definition of bradyzoites. Reactivation of toxoplasmosis in mice does not necessarily follow the administration of a corticosteroid. The daily dose, the type of corticosteroid, its solubility, whether alcohol or ester, the route of administration, the host, and, probably, other factors are important in giving rise to sufficient immunosuppression for reactivation to occur. The plea is made to examine biologic measurements in the context of parasitologic and host factors.

Treatment of Acute Toxoplasma gondii Infections in Mice with Diclazuril or a Combination of Diclazuril and Pyrimethamine

Diclazuril is a benzeneacetonitrile anticoccidial that is also effective in the prevention of toxoplasmosis. The present study was conducted to examine the efficacy of diclazuril alone or in combination with pyrimethamine for the treatment of acute toxoplasmosis in mice. Diclazuril administered at 10 mg/kg beginning 6 days after inoculation and given for 10 days protected 90% of mice over a 56-day observation period. Treatment with diclazuril at 1.0 or 0.5 mg/kg protected 20 and 0% of mice, respectively. Treatment with pyrimethamine at 12.5 or 6.0 mg/kg protected 60 and 0% of mice, respectively. When diclazuril (1.0 or 0.5 mg/kg) was combined with pyrimethamine (12.5 mg/kg) all mice survived the 56-day observation period. When diclazuril (1.0 or 0.5 mg/kg) was combined with pyrimethamine (6.0 mg/kg) 30 and 10% of mice, respectively, survived the 56-day observation period.

A pilot study on the efficacy of epiroprim against developmental stages ofToxoplasma gondii andPneumocystis carinii in animal models

The new bacteriocidal drug epiroprim (Ro-11-8958) was tested (alone or in combination with dapsone) on its efficacy against Toxoplasma gondii and Pneumocystis carinii in their hosts: laboratory mice and/or nude mice/rats, and was compared to the curative effects of the recent drugs of choice. The experiments clearly pointed out that epiroprim has significant effects on the reduction of both parasites when given alone. In combination with dapsone epiroprim led to a complete cure of toxoplasmosis in mice. This finding is of some importance for AIDS patients mostly suffering from bacteriosis and parasitosis at the same time.

Enhancement by recombinant human interleukin 2 of host resistance to Toxoplasma gondii infection in pregnant mice.

The lymphokine production by pregnant mice infected with a lethal dose of Toxoplasma gondii was evaluated in comparison with that by virgin mice infected with a sublethal dose of this protozoan parasite. Splenocytes taken from mice before and on the day after infection produced considerable amounts of IL-2 in response to concanavalin A (Con A) stimulation, but the titers rapidly declined in both pregnant and virgin mice as infection progressed. A trace amount or undetectable level of IL-2 was produced by splenocytes from acutely infected mice when stimulated with Toxoplasma lysate antigen (TLA). In contrast to the kinetics of IL-2 production, the levels of IFN-gamma produced by splenocytes cultured with Con A or TLA increased steadily in the later stage of infection in both pregnant and virgin mice. Thus, the response to Con A or TLA of splenocytes to produce IL-2 and IFN-gamma differed strikingly in acute toxoplasmosis in mice. The administration of rHuIL-2 resulted in a significant decrease in the mortality of pregnant mice infected with a lethal dose of Toxoplasma. The combination of rHuIL-2 and rMuIFN-gamma increased the survival rate slightly but not significantly compared with pregnant mice receiving either rHuIL-2 or rMuIFN-gamma. Moreover, exogenously administered rHuIL-2 enhanced the production of both IFN-alpha and IFN-gamma in the bloodstreams of pregnant mice, in accordance with the decreased mortality. These results indicate that IL-2 may play a significant role in modulating the host defense against Toxoplasma infection in pregnant mice.

Synergistic combination of azithromycin and sulfadiazine for treatment of toxoplasmosis in mice

Experiments were performed in vivo in a mouse model of acute toxoplasmosis to evaluate the effectiveness of the combination azithromycin/sulfadiazine. Azithromycin alone or sulfadiazine alone, at doses that did not provide any protection against death due to toxoplasmosis, were remarkably and significantly synergistic against murine toxoplasmosis when administered in combination.

The effect of anti-IFN-gamma antibody on the protective effect of Lyt-2+ immune T cells against toxoplasmosis in mice.

The effect of an IFN-gamma mAb on the protective activity of immune T cells against Toxoplasma infection was examined in a murine model of toxoplasmosis. Mice that received anti-IFN-gamma antibody and immune spleen cells all died of toxoplasmosis after challenge with Toxoplasma tachyzoites. In contrast, mice that received normal IgG and immune spleen cells all survived the infection. The protective activity of Lyt-2+ immune T cells, previously shown to be the principal mediators of resistance against Toxoplasma in mice was completely ablated by the anti-IFN-gamma mAb. These results suggest that IFN-gamma is the major mediator of the resistance against Toxoplasma infection in mice which is conferred by immune T cells.

Immunological response in experimentally reactivated toxoplasmosis in mice.

Both humoral and cell-mediated immune responses were studied in reactivated toxoplasma infection in mice. The infection was established by immunosuppressing chronically infected animals by thymectomy followed by irradiation. The reactivated infection was confirmed by reappearance of toxoplasma tachyzoites in peritoneal exudate. The animals survived until the 8th day post thymectomy. The percentage of splenic T lymphocytes was depressed after 4th day post thymectomy in both the uninfected thymectomized and infected immunosuppressed animals as compared to the intact uninfected controls and intact chronically infected animals. However, there was no significant change (P greater than 0.05) between the uninfected immunosuppressed control and infected immunosuppressed mice. When the infection progressed to more acute phase there was significant depression (P less than 0.01) in the percentage of T lymphocytes in comparison to the uninfected thymectomized mice. However, there was no change in the percentage of B lymphocytes (P greater than 0.05). The function of lymphocytes as assessed by blast transformation was depressed in the presence of nonspecific mitogens, i.e. phytohemagglutinin and concanavalin A until the end of the study. The response to toxoplasma antigen was increased during early stages in the infected immunosuppressed mice (stimulation index = 4.15 +/- 0.1 and 4.5 +/- 0.15 on 4th and 6th day post thymectomy). On the 8th day post thymectomy, there was a sudden fall in the stimulation index (3.5 +/- 0.11). The antitoxoplasma antibodies as detected by indirect hemagglutinin and fluorescence analysis and enzyme-linked immunosorbent assay (ELISA) tests and toxoplasma-specific IgM antibodies as detected by ELISA test were present throughout the study period though at low levels. The significance of these findings is discussed.

Induction of a protective antibody-dependent response against toxoplasmosis by in vitro excreted/secreted antigens from tachyzoites of Toxoplasma gondii.

Toxoplasma gondii is a worldwide protozoan parasite which causes severe disease in congenitally infected children and in immunocompromised patients. Besides the well-defined cytoplasmic and membrane antigens of tachyzoites, we felt that excreted/secreted antigens could play a major role in the immune response. We first report the development of a well-controlled procedure for obtaining tachyzoite excreted/secreted antigens (E/SA) in cell-free incubation media. The E/SA immunogenic in human, rat and mouse toxoplasmosis were then characterized. The major E/SA recognized by human sera from the chronic phase of toxoplasmosis had molecular weights of 108, 97, 86, 69, 60, 57, 42, 39, 28.5, 27 and 26 kD. When injected into +/+ Fischer rats, E/SA elicited high antibody titres. In addition, passive transfer of these sera to highly susceptible nu/nu littermates induced a significant degree of protection towards the virulent RH strain of T. gondii. This work, which demonstrates the key role played by E/SA in the protective immune response, suggests that these antigens should be of value both for diagnostic purposes and for the development of new strategies for immunization against toxoplasmosis.

Effect of roxithromycin on acute toxoplasmosis in mice.

Roxithromycin effectively treated acute peritoneal murine toxoplasmosis. After five doses, starting 24 h after challenge, the 100 and 50% survival doses were 540 and 336 mg/kg per day, respectively. After 14 doses, starting 3 h after challenge, the 50% survival dose was 360 mg/kg per day. Toxoplasma gondii was recovered from the brain in 59 and 28% of surviving mice treated with 5 and 14 doses, respectively.

Live and Killed Vaccines against Toxoplasmosis in Mice

Mice were immunized with live organisms of the different stages (i.e., tachyzoites, bradyzoites, or sporozoites) of Toxoplasma gondii, or with killed tachyzoites with or without adjuvants. The adjuvants used were liposomes, anhydrides of myristic or lauric acid, levamisole and Freund's complete or incomplete adjuvant. The following strains of T. gondii were used: RH, M-7741, the nonpersisting, temperature-sensitive mutants ts-1, ts-4, or ts-5, and the "back mutant" of ts-1 (Pfefferkorn and Pfefferkorn, 1976). The protection afforded was measured by challenge with the pathogenic M-7741 strain. Killed tachyzoites alone, or with adjuvants, offered only slight protection against challenge with M-7741 and no protection against challenge doses that were lethal to all control mice. Chronic infection and live nonpersisting vaccines conveyed a strong immunity to challenge, except strain ts-1. Because it was less pathogenic and did not require chemoprophylaxis, strain ts-4 best fulfilled the requirements for a good vaccine; its effect in hosts other than the mouse remains to be determined. The immunity induced by tachyzoites, bradyzoites, or sporozoites appeared equally strong when challenged with sporozoites.

Effect of three coccidiostatics against toxoplasmosis in mice.

Duocoxin, nicrazine and amprol plus showed no therapeutic effect in 50 mice each of which was infected with 25,000 organisms of a virulent Toxoplasma strain.

Effect of Clindamycin on Acute and Chronic Toxoplasmosis in Mice

The effect of clindamycin on survival of mice during acute infection with the RH and C56 strains of Toxoplasma and the ability of this drug to prevent congenital transmission during the acute stage of the infection in the mother and to eradicate the parasite from tissues of mice chronically infected with the C56 strain were evaluated. The drug effectively prevented death due to the acute infection and, in the experimental model employed, eradicated the organism at least from the liver, spleen, and brain of approximately 30 to 50% of the acutely infected animals which survived. Clindamycin also effectively prevented congenital transmission during the acute infection in the mother. During short-term treatment (7 days), persistent parasitemia in the chronic infection was effectively diminished or eradicated. More prolonged treatment resulted in a significant clearing of the organisms from the spleens and livers, but not from the brains, of chronically infected mice.