Acute Toxoplasmosis In Mice Research Articles

The synergetic potential of Lactobacillus delbrueckii and Lactobacillus fermentum probiotics in alleviating the outcome of acute toxoplasmosis in mice

Toxoplasmosis is an immunologically complex disease, particularly in immunocompromised patients. Although there are several therapeutic regimens for such disease, the majority of them have many drawbacks. Therefore, it is of utmost importance to improve the current regimen in an effort to achieve a well-tolerated therapy while also enhancing the host immune response. Famous for their immunomodulatory effect, Lactobacillus delbrueckii and Lactobacillus fermentum probiotics were chosen to be evaluated in this study as an adjuvant therapy against the virulent RH Toxoplasma gondii (T. gondii) strain. Experimental mice were divided into control and treated groups. The control group was further subdivided into two groups: group I: 10 uninfected mice and group II: 20 infected untreated mice. The treated experimental group was subdivided into three groups (20 mice each); group III: sulfamethoxazole-trimethoprim (SMZ-TMP) treated, group IV: probiotics treated, and group V: SMZ-TMP combined with probiotics. The results obtained revealed that combined therapy increased survival rate and time up to 95% and 16 days, respectively, with an 82% reduction of tachyzoites and marked distortion, as detected by the scanning electron microscope (SEM). Additionally, combined therapy alleviated the severity and the extent of the inflammatory cells’ infiltration, thereby reducing hepatocyte degeneration. Intriguingly, serum IF-γ level showed a significant increase to 155.92 ± 10.12 ng/L with combined therapy, reflecting the immunological role of the combined therapy. The current results revealed that probiotics have a high adjuvant potential in alleviating the impact of toxoplasmosis. Using probiotics as a synergistic treatment to modulate conventional therapy in systemic toxoplasmosis may gain popularity due to their low cost and current availability.

Parasitological, Molecular, and Histopathological Investigation of the Potential Activity of Propolis and Wheat Germ Oil against Acute Toxoplasmosis in Mice

Toxoplasmosis is one of the most common parasitic zoonoses that affects all vertebrates. The drugs most commonly used against toxoplasmosis have many side effects, making the development of new antiparasitic drugs a big challenge. The present study evaluated the therapeutic effectiveness of novel herbal treatments, including propolis and wheat germ oil (WGO), against acute toxoplasmosis. A total of 50 albino mice were divided into five groups: group 1 (G1) (non-infected and non-treated); group 2 (G2) (infected without treatment); group 3 (G3) (treated with propolis); group 4 (G4) (treated with WGO); group 5 (G5) (treated with a combination of propolis and WGO). The effects of the herbal substances on different organs, mainly liver, spleen, and lungs, were investigated using parasitological, molecular, and histopathological examinations. The results of parasitological examination demonstrated statistically significant (p < 0.05) differences in the parasitic load between treated groups (G3, G4, and G5) compared to the control positive group (G2). These differences were represented by a significant reduction in the parasite load in stained tissue smears from the liver obtained from the animals treated with propolis (G3) compared to the parasite load in the positive control group. Similarly, animals (G4) treated with WGO exhibited a significant reduction in the parasite load versus the positive control group, while the lowest parasite load was found in G5, treated with propolis and WGO. Quantification of the parasite burden through molecular methods (PCR) revealed similar findings represented by reduction in the parasite burden in all treated groups with WGO and propolis as compared to the control group. Importantly, these previous parasitological and molecular findings were accompanied by a marked improvement in the histopathological picture of the liver, spleen, and lungs. In conclusion, propolis and WGO showed a good combination of therapeutic efficacy against acute toxoplasmosis.

Enhancement of immune responses by vaccine potential of three antigens, including ROP18, MIC4, and SAG1 against acute toxoplasmosis in mice

Toxoplasma gondii (T. gondii) causes considerable financial losses in the livestock industry and can present serious threats to pregnant women, as well as immunocompromised patients. Therefore, it is required to design and produce an efficient vaccine for controlling toxoplasmosis. The present study aimed to evaluate the protective immunity induced by RMS protein (ROP18, MIC4, and SAG1) with Freund adjuvant, calcium phosphate nanoparticles (CaPNs), and chitosan nanoparticles (CNs) in BALB/c mice. The RMS protein was expressed in Escherichia coli (E. coli) and purified using a HisTrap HP column. Thereafter, cellular and humoral immunity was assessed by injecting RMS protein on days 0, 21, and 35 into four groups [RMS, RMS-chitosan nanoparticles (RMS-CNs), RMS-calcium phosphate nanoparticles (RMS-CaPNs), and RMS-Freund]. Phosphate buffered saline (PBS), CNs, CaPNs, and Freund served as the four control groups. The results displayed that vaccination with RMS protein and adjuvants significantly elicited the levels of specific IgG antibodies and cytokines against toxoplasmosis. There were high levels of total IgG, IgG2a, and IFN-γ in vaccinated mice, compared to those in the control groups, especially in the RMS-Freund, indicating a Th-1 type response. The vaccinated and control mice were challenged intraperitoneally with 1 × 103 tachyzoites of the T. gondii RH strain four weeks after the last injection, and in RMS-Freund and RMS-CaPNs groups, the highest increase in survival time was observed (15 days). The RMS can significantly increase Th1 and Th2 responses; moreover, multi-epitope vaccines with adjuvants can be a promising strategy for the production of a vaccine against toxoplasmosis.

A Novel Calcium-Dependent Protein Kinase 1 Inhibitor Potently Prevents Toxoplasma gondii Transmission to Foetuses in Mouse.

Treatments currently used to prevent congenital toxoplasmosis are non-specific of Toxoplasma gondii and have grievous side effects. To develop a more specific and less toxic drug, we have designed SP230, an imidazo[1,2-b]pyridazine salt targeting the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) and active against acute toxoplasmosis in mice. Efficiency of SP230 to inhibit foetal transmission of the parasite was evaluated in a mouse model of congenital toxoplasmosis. Swiss mice were infected at mid-pregnancy with tachyzoites or cysts of the ME49 strain of T. gondii by intraperitoneal and oral route, respectively, and treated with SP230 at 50 mg/kg for 5 days by the same routes. Parasite burden in organs of dams and in foetuses was measured by quantitative PCR. Intraperitoneal administration of SP230 drastically reduced the number of parasites (more than 97% of reduction) in the brain and lungs of dams, and led to a reduction of 66% of parasite burden in foetuses. Oral administration of SP230 was particularly efficient with 97% of reduction of parasite burdens in foetuses. SP230 did not impact number and weight of offspring in our conditions. This inhibitor of TgCDPK1 is a promising candidate for the development of alternative therapeutics to treat infected pregnant women.

Orally Bioavailable Endochin-Like Quinolone Carbonate Ester Prodrug Reduces Toxoplasma gondii Brain Cysts.

Toxoplasmosis is a potentially fatal infection for immunocompromised people and the developing fetus. Current medicines for toxoplasmosis have high rates of adverse effects that interfere with therapeutic and prophylactic regimens. Endochin-like quinolones (ELQs) are potent inhibitors of Toxoplasma gondii proliferation in vitro and in animal models of acute and latent infection. ELQ-316, in particular, was found to be effective orally against acute toxoplasmosis in mice and highly selective for T. gondii cytochrome b over human cytochrome b Despite its oral efficacy, the high crystallinity of ELQ-316 limits oral absorption, plasma concentrations, and therapeutic potential. A carbonate ester prodrug of ELQ-316, ELQ-334, was created to decrease crystallinity and increase oral bioavailability, which resulted in a 6-fold increase in both the maximum plasma concentration (Cmax) and the area under the curve (AUC) of ELQ-316. The increased bioavailability of ELQ-316, when administered as ELQ-334, resulted in efficacy against acute toxoplasmosis greater than that of an equivalent dose of ELQ-316 and had efficacy against latent toxoplasmosis similar to that of ELQ-316 administered intraperitoneally. Treatment with carbonate ester prodrugs is a successful strategy to overcome the limited oral bioavailability of ELQs for the treatment of toxoplasmosis.

Efficacy and Safety of Zataria multiflora Boiss Essential Oil against Acute Toxoplasmosis in Mice

Broad spectrums of pharmacological properties, including antimicrobial activity have been attributed to Zataria multiflora Boiss (Laminaceae). The in vivo efficacy and safety of Z. multiflora essential oil (ZM-EO) were evaluated against acute toxoplasmosis caused by Toxoplasma gondii (Sarcocystidae) in mice. Z. multiflora (aerial parts) was obtained from the rural districts of Kerman city (Kerman Province) Southwestern Iran, in May of 2016. Male NMRI mice were orally treated with normal saline (control group) and ZM-EO at the doses of 0.2 and 0.4 mL/kg once a day for 14 d (8 mice in each group). On the 15th day, the mice were infected with 104 tachyzoites of T. gondii RH strain by intraperitoneal route. The mortality rate and parasite load were determined in the infected mice. Additionally, 24 mice were applied to examine the sub-acute toxicity of ZM-EO at the above doses after treatment during 14 d. GC/MS analysis displayed that the key constituents were thymol (45.4%), carvacrol (23%) and p-cymene (10.6%), respectively. Overall, 100% mortality was observed on the 8th and 9th days in treated mice with the concentrations of 0.2 and 0.4 mL/kg, respectively. The mean number of tachyzoites in the mice treated with 0.2 and 0.4 mL/kg of ZM-EO were 189×104 and 76×104 cell/mL, respectively, meaningfully (P<0.05) reduced compared with the control mice. Results also demonstrated that ZM-EO had no important toxicity on mice. The results demonstrated the efficacy of ZM-EO against acute toxoplasmosis. Nevertheless, supplementary surveys are mandatory to examine its precise effects, mainly immunomodulatory effect on toxoplasmosis.

Indoleamine 2,3-Dioxygenase Activity During Acute Toxoplasmosis and the Suppressed T Cell Proliferation in Mice.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite and belongs to the phylum Apicomplexa. T. gondii is of medical and veterinary importance, because T. gondii causes the parasitic disease toxoplasmosis. In human cells, the interferon-gamma inducible indoleamine 2,3-dioxygenase 1 (IDO1) is an antimicrobial effector mechanism that degrades tryptophan to kynurenine and thus limits pathogen proliferation in vitro. Furthermore, IDO is described to have immunosuppressive properties, e.g., regulatory T cell differentiation and T cell suppression in humans and mice. However, there is only little known about the role of IDO1 in mice during acute toxoplasmosis. To shed further light on the role of mIDO1 in vivo, we have used a specifically adjusted experimental model. Therein, we infected mIDO1-deficient (IDO−/−) C57BL/6 mice and appropriate wild-type (WT) control mice with a high dose of T. gondii ME49 tachyozoites (type II strain) via the intraperitoneal route and compared the phenotype of IDO−/− and WT mice during acute toxoplasmosis. During murine T. gondii infection, we found mIDO1 mRNA and mIDO1 protein, as well as mIDO1-mediated tryptophan degradation in lungs of WT mice. IDO−/− mice show no tryptophan degradation in the lung during infection. Even though T. gondii is tryptophan auxotroph and rapidly replicates during acute infection, the parasite load was similar in IDO−/− mice compared to WT mice 7 days post-infection. IDO1 is described to have immunosuppressive properties, and since T cell suppression is observed during acute toxoplasmosis, we analyzed the possible involvement of mIDO1. Here, we did not find differences in the intensity of ex vivo mitogen stimulated T cell proliferation between WT and IDO−/− mice. Concomitant nitric oxide synthase inhibition and interleukin-2 supplementation increased the T cell proliferation from both genotypes drastically, but not completely. In sum, we analyzed the involvement of mIDO1 during acute murine toxoplasmosis in our specifically adjusted experimental model and found a definite mIDO1 induction. Nevertheless, mIDO1 seems to be functional redundant as an antiparasitic defense mechanism during acute toxoplasmosis in mice. Furthermore, we suggest that the systemic T cell suppression observed during acute toxoplasmosis is influenced by nitric oxide activity and IL-2 deprivation.

Evaluation of protective immunity induced by recombinant calcium-dependent protein kinase 1 (TgCDPK1) protein against acute toxoplasmosis in mice

Toxoplasma gondii is an intracellular zoonotic parasite that causes toxoplasmosis, which can cause economic losses and serious public health problems worldwide. A member of the T. gondii calcium-dependent protein kinases family, TgCDPK1 was recently identified as an essential regulator of exocytosis in T. gondii, and participated in direct parasite motility, host-cell invasion and egress. In the present study, the protective immunity of recombinant TgCDPK1 protein (rTgCDPK1) was evaluated against acute toxoplasmosis in mice. rTgCDPK1 were expressed and purified, BABL/c mice were intraperitoneally immunized with rTgCDPK1 and challenged with the highly virulent RH strain of T. gondii. The specific immune responses were analyzed by measuring the cytokine and serum antibody, and lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival curve were employed to evaluate the protective efficacy. From the results we found that special humoral and cellular responses could be elicited in vaccine mice, and higher level of IgG antibody, and the significant increased levels of Th1-type cytokines IFN-γ, IL-12 (p70), IL10 and CD3+CD4+CD8− and CD3+CD8+CD4− T cells could also be detected comparing to control mice (P < 0.05). All vaccinated mice prolonged survival time (14.90 ± 2.89 days) challenge with 1000 tachyzoites of RH, while the control mice died within 8 days. These results indicated that TgCDPK1 protein was a potential vaccine candidate against acute toxoplasmosis.

Vaccination with recombinant adenoviruses expressing Toxoplasma gondii MIC3, ROP9, and SAG2 provide protective immunity against acute toxoplasmosis in mice

Toxoplasmosis is a worldwide zoonosis caused by the protozoan parasite Toxoplasma gondii, an obligate intracellular parasite. Currently, no viable vaccine or effective drug strategies exist to prevent and control toxoplasmosis. T. gondii microneme protein 3 (MIC3), rhoptry protein 9 (ROP9), and surface antigen 2 (SAG2) are related to active invasion of the parasite. Hence, we constructed recombinant adenoviruses expressing TgMIC3, TgSAG2, or TgROP9 and evaluated the recombinant adenoviruses as potential vaccines against acute T. gondii infection in BALB/c mice. Mice immunized with the recombinant adenoviruses were measured the antibody levels, percentages of lymphocytes and actived T lymphocytes, cytokine productions, and the survival rates and time to evaluate the protective efficacy. Results showed that immunization with the bivalent or trivalent recombiant adenoviruses could strongly stimulate humoral and cellular immune responses in mice, resulting in effective immune protections against lethal challenge with the tachyzoites of T. gondii RH. These results indicated that the divalent and trivalent adenoviruses, especially Ad-ROP9-MIC3-EGFP, may be promising vaccine candidates against acute T. gondii infection.

Evaluation of Protective Immune Response Induced by a DNA Vaccine Encoding GRA8 against Acute Toxoplasmosis in a Murine Model.

Toxoplasma gondii is an apicomplexan zoonotic protozoan parasite that infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicate a need for the development of an effective vaccine. T. gondii GRA8 is a member of the dense granules protein family and is used as a marker of acute infection. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pDsRed2-GRA8, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with the pDsRed2-GRA8 plasmid and then challenged by infection with the highly virulent GFP-RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii of this vaccine were analyzed by measuring cytokine and serum antibody titers, splenocyte proliferation assays, and the survival times of mice after challenge. Our results showed that mice immunized with pDsRed2-GRA8 demonstrated specific humoral and cellular responses, induced higher IgG antibody titers with predominant IgG2a production; increased levels of IL-10, IL-12 (p70), IFN-γ, TNF-α, and splenocyte proliferation; and prolonged survival times compared to those of control mice. The present study showed that DNA immunization with pDsRed2-GRA8 induced humoral and cellular immune responses, and all immunized mice showed greater Th1-type immune responses and longer survival times than those of control mice. These results indicated that T. gondii GRA8 DNA immunization induces a partial protective effect against acute toxoplasmosis.

Evaluation of protective immunity induced by DNA vaccination with genes encoding Toxoplasma gondii GRA17 and GRA23 against acute toxoplasmosis in mice

Toxoplasma gondii, an obligatory intracellular protozoan, can cause serious public health problems and economic losses worldwide. Two novel dense granule proteins (GRA17 and GRA23) were recently identified as T. gondii-secreted proteins which are localized to the parasitophorous vacuole membrane (PVM) and can mediate the movement of small molecules between the host cell and parasitophorous vacuole (PV). In the present study, we evaluated the protective immunity induced by DNA vaccination with genes encoding GRA17 and GRA23 against acute toxoplasmosis in mice. Eukaryotic expressing plasmids pVAX-TgGRA17 and pVAX-TgGRA23 were constructed. Then, BALB/c mice were intramuscularly immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 followed by challenge infection with the highly virulent RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii were examined by cytokine and serum antibody measurements, lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival time after challenge. Our results showed that mice immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 induced specific humoral and cellular responses, with higher level of IgG antibody, increased levels of Th1-type cytokines IFN-γ and IL-12 (p70), and CD3+CD4+CD8− and CD3+CD8+CD4− T cells, as well as prolonged survival time (9.1 ± 0.32 days for pVAX-TgGRA17, 10.8 ± 0.79 days for pVAX-TgGRA23, and 12.6 ± 2.55 days for pVAX-TgGRA17 + pVAX-TgGRA23) compared to the blank control (7.11 ± 0.33 days), PBS control (7.22 ± 0.44 days), and pVAX I control (7.11 ± 0.33 days). These results demonstrated that both TgGRA17 and TgGRA23 are potential vaccine candidates, TgGRA23 has a better immunogenicity, and co-immunization of pVAX-TgGRA17 and pVAX-TgGRA23 induces better protective efficacy.

Effects of azithromycin and Chlorella vulgaris treatment on certain cytokine values and NK cell activity in an acute murine toxoplasmosis model

Toxoplasmosis is a common infection with a complicated treatment process. Azithromycin (AZT) is a macrolide antibiotic that can be effectively used in patients with cerebral and ocular toxoplasmosis and has fewer side effects. Chlorella vulgaris (CV), a single-cell green algae that contains nutrients and has various biological effects. CV extract (CVE) has been shown to have protective effects against infections via immune enhancement by increasing the cytotoxicity of NK cells, IL-12 and IFN-γ levels. The aim of this study was to investigate the protective effects of AZT and CV, individually and in combination, against acute toxoplasmosis in mice, and their effects on NK cell cytotoxixity, IL-12, IFN-γ, and IL-2 levels. Six groups of mice (Balb/c) were formed. With the exception of the healthy control (HC) group, all other groups were infected with 1 ml (11 x 104 trofozoit/ml) Toxoplasma gondii RH strain trophozoites. No further action was performed for infected control (IC) group. After 24 hours from trophozoite infection, CVE was given to CV group, AZT to azithromycin group and CVE + AZT combination to CV + AZT group by oral gavage for 6 days. All of the mice from IC, CV, AZT and CV + AZT groups were sacrified on the 8th day of the infection and serum, peritoneal fluid and spleen samples were collected. Trophozoite count of the groups were determined in all groups except HC group and the average growth inhibition activity was calculated by using the growth inhibition formula. In all groups IL-12, IFN-γ, IL-2 levels were measured with ELISA method and cytotoxicity of the NK cells were measured using Cytotox 96 Non-Radioactive Cytotoxicity Assay. The number of trophozoites were significantly lower in the CV group than the IC group (p< 0.001), and also significantly lower in CV + AZT combination group than the AZT group. According to the growth inhibition calculations CV treatment showed 88.6%, AZT treatment 98.46%, AZT + CV combination treatment 99.4% antiprotozoal activity against T.gondii compared with the IC group. NK cell cytotoxicity in the CV and the combination group were significantly higher than all the other groups (p< 0.001). IL-12 and IFN-γ levels were highest in IC group and the lowest in AZT + CV group. This situation has been linked to the fact that the severity of the infection has fallen considerably. IL-2 levels were significantly higher in CV, CV + AZT groups than in the other groups (p< 0.001). In our study, even CV administration alone caused a significant decline in infection.This may be related to the increased NK cytotoxicity, IL-2, IL-12 and IFN-γ levels. CV + AZT combination seems to be an effective treatment option than AZT alone, particularly in patients who are difficult to treat with common methods or in patients with immunosuppression.

In vivo evaluation of Berberis vulgaris root methanolic extract against acute toxoplasmosis in mice

Objective : Since last centuries, herbal medicines have been considered as the main source for prevention and treatment a broad spectrum of illnesses as well as infectious diseases. Here we evaluated the in vivo activity of Berberis vulgaris methanolic root extract against acute toxoplasmosis induced by Toxoplasma gondii , RH strain. Materials and Methods: Male NMRI mice orally treated with B. vulgaris methanolic extract at the dose of 1 and 2 g/kg once a day for 14 days . After 24 h mice were infected intraperitonealy with 10 4 tachyzoites of T. gondii, RH strain. The mortality rate in all infected mice and also parasite load were determined to assess the efficacy B. vulgaris methanolic extract against acute toxoplasmosis in mice model. Results: The obtained results demonstrated that the mortality rate of infected mice was 100%, in 9 th day after orally administration of B. vulgaris at the concentrations of 1 and 2 g/kg; while all mice in the control group were death in 5th day. Moreover obtained findings revealed that the mean number of tachyzoites was 131 × 10 4 and 79 × 10 4 for infected mice treated with B. vulgaris at the concentrations of 1 and 2 g/kg, respectively, whereas in control group the mean number of tachyzoites was 288×10 4 parasite. Conclusion : The obtained findings demonstrated the potential of B. vulgaris as a natural source for the production of new prophylactic agent for use in toxoplasmosis.

Immune responses and protection after DNA vaccination against Toxoplasma gondii calcium-dependent protein kinase 2 (TgCDPK2).

Toxoplasma gondii, an intracellular zoonotic protozoan parasite, is possibly the most widespread parasite of warm-blooded animals and can cause serious public health problems and economic losses worldwide. TgCDPK2, a member of the T. gondii calcium-dependent protein kinase family, was recently identified as an essential regulator for viable cyst development in T. gondii. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pVAX-TgCDPK2, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with pVAX-TgCDPK2 plasmid and then challenged by infection with the highly virulent RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii were analyzed by cytokine and serum antibody measurements, lymphocyte proliferation assays, flow cytometric on lymphocytes and the survival time of mice after challenge. Our results showed that mice immunized with pVAX-TgCDPK2 could elicit special humoral and cellular responses, with higher levels of IgG antibody, and increased levels of Th1-type cytokines IFN-γ, IL-12(p70), and CD3 + CD4 + CD8 − and CD3 + CD8 + CD4 − T cells, and had a prolonged survival time (14.0 ± 2.32 days) compared to control mice. These results demonstrate that pVAX-TgCDPK2 is a potential vaccine candidate against acute toxoplasmosis.

Effect of honey on mRNA expression of TNF-α, IL-1β and IL-6 following acute toxoplasmosis in mice

This study analyzed the mRNA expression of tumor necrosis factor (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6) in mice experimentally infected with T. gondii undergoing honey treatment. Thirty male mice were divided in groups: pre-treatment/infected (1), infected/non-treated (2), infected/treated (3), non-infected/treated (4) and control (5). Honey was applied for groups 1, 3, 4 by gavage and the mice in group 1–3 were infected by T. gondii tissue cysts. The parasite load and the level of mRNA expression of the aforementioned cytokines in the brains of mice were assessed by qPCR. The mean number of T. gondii tachyzoite in 1mg brain tissue was 32, 73 and 59 in groups one, two and three, respectively. The mRNA expression of TNF-α increased in group 1, 2 and 3, about 49.1%, 307.3% and 63.2%, respectively but it was down-regulated by 53% in group 4. The mRNA expression of IL-1β and IL-6 was also up-regulated in all groups except group 2. The mRNA level of TNF-α was reduced by 2.7-fold and 1.18-fold in pre-treated/infected (group 1) and infected/treated (group 3) compared with infected/non-treated (group 2). The mRNA level of IL-1β and IL-6 were increased in these groups. The current study demonstrated that honey can stimulate or suppress the mRNA expression of some pro-inflammatory cytokines in mice brains. Furthermore, honey suppresses the TNF-α mRNA expression in the presence of T. gondii infection but it stimulates the IL-1β and IL-6 mRNA expression. Treatment of the mice with honey reduces parasite multiplication in the brain.

Toxoplasma gondii: Vaccination with a DNA vaccine encoding T- and B-cell epitopes of SAG1, GRA2, GRA7 and ROP16 elicits protection against acute toxoplasmosis in mice

Toxoplasma gondii (T. gondii) is an obligate, intracellular, protozoan parasite that infects large variety of warm-blooded animals including humans, livestock, and marine mammals, and causes the disease toxoplasmosis. Although T. gondii infection rates differ significantly from country to country, it still has a high morbidity and mortality. In these circumstances, developing an effective vaccine against T. gondii is urgently needed for preventing and treating toxoplasmosis. The aim of this study was to construct a multi-epitopes DNA vaccine and evaluate the immune protective efficacy against acute toxoplasmosis in mice. Therefore, twelve T- and B-cell epitopes from SAG1, GRA2, GRA7 and ROP16 of T. gondii were predicted by bioinformatics analysis, and then a multi-epitopes DNA vaccine was constructed. Mice immunized with the multi-epitopes DNA vaccine gained higher levels of IgG titers and IgG2a subclass titers, significant production of gamma interferon (IFN-γ), percentage of T lymphocyte subsets, and longer survival times against the acute infection of T. gondii compared with those of mice administered with empty plasmid and those in control groups. Furthermore, a genetic adjuvant pEGFP-RANTES (pRANTES) could enhance the efficacy of the multi-epitopes DNA vaccine associating with humoral and cellular (Th1, CD8+ T cell) immune responses. Above all, the DNA vaccine and the genetic adjuvant revealed in this study might be new candidates for further vaccine development against T. gondii infection.

Dual Role for Inflammasome Sensors NLRP1 and NLRP3 in Murine Resistance to Toxoplasma gondii

ABSTRACTInduction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1β (IL-1β), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1β in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis.

Protective immunity induced by a DNA vaccine expressing eIF4A of Toxoplasma gondii against acute toxoplasmosis in mice

Toxoplasma gondii is an obligate intracellular protozoan parasite infecting humans, mammals and birds. Eukaryotic translation initiation factor (eIF4A) is a newly identified protein associated with tachyzoite virulence. To evaluate the protective efficacy of T. gondii eIF4A, a DNA vaccine (pVAX-eIF4A) encoding T. gondii eIF4A (Tg-eIF4A) gene was constructed. The expression ability of this recombinant DNA plasmid was examined in Marc145 cells by IFA. Then, Kunming mice were intramuscularly immunized with pVAX-eIF4A and followed by challenge infection with the highly virulent T. gondii RH strain. The results showed that vaccination with pVAX-eIF4A elicited specific humoral responses, with high IgG antibody titers and specific lymphocyte proliferative responses. The cellular immune response was associated with significant production of IFN-γ, IL-2 in Kunming mice, and a mixed IgG1/IgG2a response with predominance of IgG2a production, indicating that a Th1 type response was elicited after immunization with pVAX-eIF4A. In addition, the increase of the percentage of CD8+ T cells in lymphoid in mice suggested the activation of MHC class I restricted antigen presentation pathways. After lethal challenge, the mice vaccinated with the pVAX-eIF4A showed a significantly prolonged survival time (23.0±5.5 days) compared with control mice which died within 7 days of challenge (P<0.05). These results demonstrate that pVAX-eIF4A could elicit strong humoral, Th1-type cellular immune responses and increase survival time of immunized mice, suggesting that eIF4A is a promising vaccine candidate against acute T. gondii infection in mice.

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.

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.