Formulation and evaluation of atovaquone-loaded macrophage-derived exosomes against Toxoplasma gondii: in vitro and in vivo assessment.

IMPORTANCE: This study is the first of its kind that suggests exosomes as a nano-carrier loaded with atovaquone (ATQ), which could be considered as a new strategy for improving the effectiveness of ATQ against acute and chronic phases of Toxoplasma gondii.

Green synthesis of zinc nanoparticles using Lavandula angustifolia Vera. Extract by microwave method and its prophylactic effects on Toxoplasma gondii infection.

BACKGROUND: Today, a suitable vaccine has not yet been discovered to prevent Toxoplasma gondii infection. Therefore, prophylaxis can be suggested as the preferred approach to prevent toxoplasmosis. This study aims to evaluate the prophylactic effects of synthesized zinc nanoparticles (ZnNPs) using Lavandula angustifolia Vera., by microwave method on chronic toxoplasmosis in mice. METHODS: BALB/c Mice orally administrated with ZnNPs the doses of 32.5, 75, 150 mg/kg/day for two weeks. On the 15th day, the mice were intraperitoneally infected with the Tehran strain of T. gondii (25 tissue cysts). The mean diameter and the numbers of brain tissue cysts, as well as the mRNA levels of inducible nitric oxide synthesize (iNOs), and interferon-gamma (IFN-γ) in mice of each experimental group were evaluated. RESULTS: The synthesized ZnNPs represent a spherical form with a size ranging from 30 to 80 nm. The results revealed that oral administration of Zn NPs at the doses of 32.5 (p < 0.001) and 75 mg/kg/day (p < 0.001) for 14 days significantly reduced the mean number and diameter of the brain tissue cysts in tested mice. No T. gondii tissue cyst was observed after oral administration of Zn NPs at the doses of 150 mg/kg. Based on the results of Real-time PCR analysis, the expression level of IFN-γ and iNOs was significantly increased (p < 0.001) in mice treated with 32.5, 75, 150 mg/kg/day for two weeks. CONCLUSION: The obtained findings of the current investigation exhibit the significant prophylactic effects of ZnNPs against chronic toxoplasmosis in mice; so that oral administration of ZnNPs the doses 32.5, 75, 150 mg/kg reduced the parasite load and even completely controlled the infection in mice. The results show that the ZnNPs had strengthened the innate immune system which could be the reason for its strong prophylactic effects. However, further in vivo and clinical investigations are required to confirm these results as well as other possible mechanisms that can trigger these pharmacological properties.

Spermatogonia apoptosis induction as a possible mechanism of Toxoplasma gondii-induced male infertility.

OBJECTIVES: The protozoan Toxoplasma gondii as an intracellular protozoan is widely prevalent in humans and animals. Infection generally occurs through consuming food contaminated with oocysts and tissue cysts from undercooked meat. The parasite is carried in sexual fluids like semen but there is little information about the effect of T. gondii on the male reproductive system. In this study, we examined the effect of T. gondii tachyzoites on apoptosis induction in type B spermatogonia (GC-1) cells. MATERIALS AND METHODS: Fresh tachyzoites taken of infected BALB/c mice, GC-1 spg cells were infected with increasing concentrations of tachyzoites of T. gondii, then apoptotic cells were identified and quantified by flow cytometry. The genes associated with apoptosis were evaluated by RT2 Profiler PCR Array. RESULTS: PCR array analysis of 84 apoptosis-related genes demonstrated that 12 genes were up-regulated at least 4-fold and that one gene was down-regulated at least 2-fold in the T. gondii infection group compared with levels in the control group. The number of genes whose expression had increased during the period of infection with T. gondii was significantly higher than those whose expressions had decreased (18 versus 1) and Tnfrsf11b had the highest rate of gene expression. CONCLUSION: T. gondii induce in vitro apoptosis of GC-1 spg cells. This effect shows a trend of concentration-dependent increase so that with an increase in the ratio of parasite burden to spermatogonial cells, in addition to an increase in the number of genes whose expression has changed, the fold of these changes has increased as well.

Prophylactic Activity of Biogenic Selenium Nanoparticles Against Chronic Toxoplasma gondii Infection.

BACKGROUND: Studies showed that biogenic selenium nanoparticles (SeNPs) have a number of pharmacological properties, such as antimicrobial ones. OBJECTIVE: The present investigation assesses the efficacy of biogenic selenium nanoparticles (SeNPs) as a new patent against latent toxoplasmosis in a mice model. METHODS: Male BALB/c mice were orally treated with SeNPs at the doses of 2.5, 5, 10 mg/kg once a day for 14 days. On the 15th day, the mice were infected with the intraperitoneal inoculation of 20-25 tissue cysts from the Tehran strain of Toxoplasma gondii. The mean numbers of brain tissue cysts and the mRNA levels of TNF-α, IL-12, IL-10, IFN-γ, and inducible nitric oxide synthase (iNOS) in mice of each tested group were measured. Moreover, serum clinical chemistry factors in treated mice were examined to determine the safety of SeNPs. RESULTS: The mean number of the brain tissue cysts was significantly (P<0.001) decreased in mice treated with SeNPs at doses 2.5 (n=37), 5 (n=11), and 10 mg/kg (n=3) based on a dose dependent manner compared with the control group (n=587). The mRNA levels of IFN-γ, TNF-α, IL-12, and iNO were significantly increased in mice treated with SeNPs at the doses 10 mg/kg compared with control subgroups (p<0.05). No significant variation (p>0.05) was observed in the clinical chemistry parameters among the mice in the control subgroups compared with groups treated with SeNPs. CONCLUSION: The results of the present study showed a new patent in the treatment of toxoplasmosis; so that taking the biogenic selenium nanoparticles in concentrations of 2.5-10 mg/kg for 2 weeks was able to prevent severe symptoms of the toxoplasmosis in a mice model. This indicated the prophylactic effects of SeNPs with no considerable toxicity against latent toxoplasmosis. However, more studies are required to elucidate the correct anti-Toxoplasma mechanisms of SeNPs.

Biogenic selenium nanoparticles target chronic toxoplasmosis with minimal cytotoxicity in a mouse model.

Introduction. Nanoparticles (NPs) have numerous biological benefits due to their large surface-volume ratio and convenient entry into cells compared to other particles. Previous research has shown the antimicrobial properties of biogenic selenium NPs (SeNps) and their effects on cellular immunomodulatory cytokines that play a key role in controlling infections.Aim. This study aimed to evaluate the therapeutic effects of SeNPs against chronic toxoplasmosis in mice.Methodology. Infected mice with Toxoplasma gondii (Tehran strain) were orally treated with SeNPs at doses of 2.5, 5 and 10 mg kg-1 once a day for 14 days. On the fifthteenth day, the mean number of brain-tissue cysts and the mRNA levels of TNF-α, IL-12, IL-10, IFN-γ and inducible nitric oxide synthase (iNOS) in the mice of each group were recorded. Moreover, serum clinical chemistry factors in the treated mice were examined to determine the safety of SeNPs.Results. The mean number of tissue cysts was significantly (P<0.001) decreased in mice treated with SeNPs in a dose-dependent manner compared with the control group. The mRNA levels of inflammatory cytokines were significantly increased in mice treated with SeNPs at a dose of 10 mg kg-1 compared with the control subgroup (P<0.05). No significant variation (P>0.05) observed in clinical chemistry parameters among the mice in the control subgroup compared with those treated with SeNPs.Conclusion. The findings demonstrated the therapeutic effects of SeNPs with no considerable toxicity against latent toxoplasmosis in the mouse model. Nevertheless, further studies are obligatory to reveal the exact anti-Toxoplasma mechanisms of SeNPs.

Nanosilver Colloid Inhibits Toxoplasma gondii Tachyzoites and Bradyzoites in Vitro.

BACKGROUND: Toxoplasma gondii, the coccidian protozoan parasite with worldwide distribution, is the agent of toxoplasmosis. The disease is life threatening in congenital form and in immunocompromised patients. The present study was carried out in 2016 to evaluate the in vitro effects of nanosilver colloid on tachyzoites and bradyzoites of T. gondii, RH and Tehran strains. METHODS: Different concentrations (5, 10, 20 ppm) of nanosilver colloid were added to tachyzoites of T. gondii, RH strain (type I) and bradyzoites and tissue cysts of T. gondii, Tehran strain (type II) and incubated for 30, 60, 90 and 120 minutes. The mortality rates of tachyzoites and bradyzoites were evaluated by trypan blue dye and MTT assay. Then SEM carried out to show the changes between control and exposed parasites. RESULTS: The greatest mortality rate was seen in 20 ppm concentration and after 120 minutes of exposure. By electron microscopy, the structural changes were seen in tachyzoites of RH and tissue cyst of Tehran strain in comparison with control groups. CONCLUSION: Nanosilver colloid was effective on both tachyzoites and bradyzoites of T. gondii, RH and Tehran strains.

Curcumin nanoemulsion as a novel chemical for the treatment of acute and chronic toxoplasmosis in mice.

BACKGROUND: The aim of this study was to prepare curcumin nanoemulsion (CR-NE) to solve the problems associated with poor water solubility and low bioavailability of CR and to test its efficiency in the treatment of acute and chronic toxoplasmosis in mouse models. MATERIALS AND METHODS: CR-NE 1% was prepared using spontaneous emulsification by soybean as oil phase; a mixture of Tween 80 and Tween 85 as surfactant; ethanol as cosurfactant and distilled water. Particle size and zeta potential of NE were assessed using Nano-ZS90 dynamic light scattering. Stability testing of NE was assessed after storage for 2 months at room temperature. In vivo experiments were carried out using 50 BALB/c mice inoculated with virulent RH strain (type I) and 50 BALB/c mice inoculated with avirulent Tehran strain (type II) of Toxoplasma gondii and treated with CR-NE (1% w/v), CR suspension (CR-S, 1% w/v), and NE without CR (NE-no CR). RESULTS: The mean particle size and zeta potential of CR-NE included 215.66±16.8 nm and -29.46±2.65 mV, respectively, and were stable in particle size after a three freeze-thaw cycle. In acute phase experiment, the survival time of mice infected with RH strain of T. gondii and treated with CR-NE extended from 8 to 10 days postinoculation. The differences were statistically significant between the survival time of mice in CR-NE-treated group compared with negative control group (P<0.001). Furthermore, CR-NE significantly decreased the mean counts of peritoneum tachyzoites from 5,962.5±666 in negative control group to 627.5±73 in CR-NE-treated mice (P<0.001). Growth inhibition rates of tachyzoites in peritoneum of mice receiving CR-NE, CR-S, and NE-no CR included 90%, 21%, and 11%, respectively, compared with negative control group. In chronic phase experiment, the average number and size of tissue cysts significantly decreased to 17.2±15.6 and 31.5±6.26 µm, respectively, in mice inoculated with bradyzoites of T. gondii Tehran strain and treated with CR-NE compared with that in negative control group (P<0.001). Decrease of cyst numbers was verified by downregulation of BAG1 in treatment groups compared with negative control group with a minimum relative expression in CR-NE (1.12±0.28), CR-S (11.76±0.87), and NE-no CR (14.67±0.77), respectively, (P<0.001). CONCLUSION: Results from the current study showed the potential of CR-S and CR-NE in treatment of acute and chronic toxoplasmosis in mouse models for the first time. However, CR-NE was more efficient than CR-S, and it seems that CR-NE has a potential formula for the treatment of acute and chronic toxoplasmosis, especially in those with latent bradyzoites in brain.

Nanoemulsion of atovaquone as a promising approach for treatment of acute and chronic toxoplasmosis.

Treatment of toxoplasmosis is necessary in congenital form and immunocompromised patients. Atovaquone is a powerful suppressor of protozoan parasites with a broad-spectrum activity, but an extremely low water solubility and bioavailability. In this study, nanoemulsion of this drug was prepared with grape seed oil using spontaneous emulsification method to increase bioavailability and efficacy of atovaquone for treatment of toxoplasmosis. In vitro activity of atovaquone nanoemulsion against T. gondii, RH and Tehran strains, was assessed in HeLa cell culture. For in vivo assessment, BALB/c mice were infected with RH and Tehran strains and then treated with nanoemulsion of atovaquone, compared to that treated with free atovaquone. Concentration of atovaquone nanoemulsion showed in vitro anti-parasitic effects in both strains of T. gondii. Furthermore, oral administration of atovaquone nanoemulsion increased oral bioavailability, tissue distribution and mice survival time and reduced parasitemia and number and size of the brain cysts. Decrease of cyst numbers was verified by down regulation of BAG1 using real-time polymerase chain reaction (real-time PCR) assay. Effective therapeutic activity of atovaquone at a reduced dose is the major achievement of this study.

Cyst Formation from Virulent RH Strain of Toxoplasma gondii Tachyzoite: In Vitro Cultivation.

BACKGROUND: This study was performed to induce conversion of RH strain tachyzoites of Toxoplasma gondii to bradyzoites by pH changing of the culture medium. METHODS: HeLa cell monolayers were infected at a 1:1 tachyzoite to cell ratio. Four hours after infection, the culture medium was removed and replaced with culture medium and 5% FCS, adjusted to pH 8 with NaOH. The culture was maintained at 37 °C without CO2 until the end of the experiment. Cyst-like structures were collected and stained with periodic acid schiff (PAS) staining method. The soluble antigens of cyst-like structures of RH strain, in addition to RH tachyzoite, bradyzoites of avirulent Tehran strain and uninfected HeLa cells were electrophoresed on 12.5% polyacrylamide gel. The gel was stained by coomassie brilliant blue R-250. RESULTS: Four days after infection of HeLa cells with tachyzoites of T. gondii, RH strain, cyst- like structures were noticed and stained with PAS. In the SDS-PAGE, protein bands of these structures had some differences with tachyzoites of RH strain, but there was quite similarity between protein bands of these structures and tissue cysts (bradyzoites) of Tehran strains. P34 and P36 (bradyzoite-specific proteins) were observed only in T. gondii bradyzoites of RH (cyst like structures) and bradyzoites of Tehran strains. CONCLUSION: Alkalization of culture medium to pH 8 induced expression of bradyzoite- specific proteins and production of RH cysts in cell culture.

Acetylcholinesterase Inhibitor Improves Learning and Memory Impairment Induced by Toxoplasma gondii Infection.

BACKGROUND: Here, we established the mouse models of chronic toxoplasmosis by T. gondii Tehran strain to provide a good understanding about defining the possible association between T. gondii exposure and learning and memory impairments. Moreover, as secondary objective of the present study, we hypothesized whether administration of an acetylcholinesterase (AChE) inhibitor could reduce learning and memory impairments induced by T. gondii infection. METHODS: Twenty-four male BALB/c mice were used to establishment of latent toxoplasmosis. The animal model of Toxoplasma infection was established by the intraperitoneal inoculation of 20-25 tissue cysts from Tehran strain of T. gondii. Donepezil (2 mg/kg) an AChE inhibitor to treat Alzheimer disease was injected intraperitoneally once a day for two weeks starting from post-infection day 90. Morris water maze (MWM) task was used to assay spatial learning and short term spatial memory in all groups. One-way ANOVA with Tukey's post-hoc test was used to assess differences between experimental groups. P<0.05 was considered statistically significant. RESULTS: Toxoplasma infection impaired spatial leaning and short term spatial memory of the infected BALB/c mice, whereas donepezil, an AChE inhibitor, improved impairments induced by Toxoplasma infection. CONCLUSION: T. gondii infection through increasing AChE reduces the level of Acetylcholine (Ach) and consequently affects learning and memory activity in infected hosts, whereas, donepezil as an AChE inhibitor improves these impairments by restoring ACh levels at synapses of neurons in brain.

Conjugated Linoleic Acid Stimulates Apoptosis in RH and Tehran Strains of Toxoplasma gondii, in Vitro.

BACKGROUND: The aim of this study was to evaluate the effects of conjugated linoleic acid (CLA) on apoptosis of tachyzoites of T. gondii, RH strain (type I) and the cyst-forming Tehran strain (type II) in vitro. METHODS: Toxoplasma strains were injected into the peritoneal cavity of BALB/c mice. The Tehran strain forms cysts in the brain of mice. Bradyzoites within the cysts are reactivated to proliferative tachyzoites, by dexamethasone. Tachyzoites were aspirated from the peritoneum of infected mice, and the percentage of viable parasites was estimated with trypan blue staining. Tachyzoites were inoculated into HeLa cells cultivated in DMEM medium. Different concentrations of CLA were evaluated on T. gondii in HeLa cells by the tetrazolium (MTT) colorimetric assay. Differentiation between apoptosis and cell death was determined by flow cytometry using Annexin V and propidium iodide (PI) double staining. The statistical analysis performed by GraphPad Prism version 6.00. RESULTS: CLA induces apoptosis in virulent (RH) and avirulent (Tehran) strains of T. gondii. The results of MTT indicated that CLA could decrease the proliferation of tachyzoites of both strains in HeLa cells. CONCLUSION: Conjugated linoleic acid has anti-toxoplasmacidal activity on tachyzoites of T. gondii. Therefore, we recommended further studies on this component in order to achieve a new drug against the parasite.

Rapid detection of human and canine visceral leishmaniasis: assessment of a latex agglutination test based on the A2 antigen from amastigote forms of Leishmania infantum.

The diagnosis of visceral leishmaniasis (VL) in humans and animal reservoir hosts is difficult, particularly in rural areas where the disease is endemic and laboratory facilities are limited. This study aimed to develop a latex agglutination test (LAT) for the rapid detection of anti-Leishmania antibodies against the A2 antigen derived from the amastigote form as well as those against crude antigens derived from the promastigote form of an Iranian strain of Leishmania (Leishmania) infantum. The A2 antigen (42-100 kDa) was prepared from the amastigote form of L. infantum, purified with electroelution and compared with the crude antigen from the promastigote form of L. infantum. Both antigens showed appropriate intensity reactions, were selected using dot blotting of positive and negative pooled sera and used to sensitize 0.9-µm latex beads. The tests were carried out on sera from 43 symptomatic, human patients with VL confirmed by parasitological examination and direct agglutination test (DAT), 30 healthy controls and 32 patients with other infections but without VL. Canine sera were collected from 63 domestic dogs with VL confirmed using parasitological examinations and DAT and 31 healthy dogs from areas non-endemic for VL. Compared with the controls, human sera from DAT-confirmed patients yielded a sensitivity of 88.4% (95% CI, 82.1-94.5%) and specificity of 93.5% (95% CI, 87.0-99.7%) on A2-LAT (amastigote) when 1:3200 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.914). LAT required 3-5 min to complete, versus the 12-18 h needed for DAT. Compared with the controls, A2-LAT of canine sera from DAT-confirmed cases yielded a sensitivity of 95.2% (95% CI, 95.0-95.4%) and specificity of 100% (95% CI 100%) when 1:320 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.968). Similarly, the sensitivity and specificity of Pro.-LAT (promastigote) was calculated to be 88.4% and 91.9%, respectively for human sera and 96.8% and 90.3%, respectively for canine sera. No statistically significant differences were observed between A2-LAT and Pro.-LAT for the detection of human and canine L. infantum infections. In conclusion, A2-LAT and Pro.-LAT could be used in parallel to screen for L. infantum infections in humans and dogs in areas endemic for VL in Iran.

In Vitro and In Vivo Potential of RH Strain of Toxoplasma gondii (Type I) in Tissue Cyst Forming.

BACKGROUND: Based on recent studies, there are controversial reports on the capacity of tissue cyst forming of Toxoplasma gondii RH strain. In this study, the capacity was evaluated by in vivo and in vitro experiments. METHODS: RH strain was subcutaneously inoculated to ten Wistar rats. After one month, their blood, brain, tongue and diaphragm were collected and evaluated by MAT, PCR, pathological and bioassay methods. The parasite was cultivated in the cell monolayer. To change to bradyzoite, the media pH was altered to 6.8. Biological aspect of the bradyzoites was evaluated by incubation in acidic pepsin and it's inoculation in ten BALB/c mice. RESULTS: All rats showed antibodies to Toxoplasma at titers ≥1:320 but no DNA and tissue cyst were detected in the tissues. Following intraperitoneal inoculation of rats' brain homogenate into BALB/c mice, no infection was established in none of the animals. During presence of cell culture, in acid media for a 3-5 days period, cyst-like structures were noticed when they were stained with PAS. The visible bradyzoites in the cysts that were incubated in acid pepsin medium were not able to kill any mice. CONCLUSION: This study confirmed that Iranian RH strain has lost the potential of tissue cyst forming in rats and bradyzoites cultivated in cell culture lost their resistance to acidic condition, so this strain can be a candidate for future vaccine researches.

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.