Toxoplasma Gondii Strains Research Articles

Genotyping of Toxoplasma gondii Strains from Goats in Jahrom District, Southern Iran.

Toxoplasma gondii is transmitted congenitally or acquired by consumption of food and water contaminated with cysts or oocysts. This study aimed at genotyping T. gondii strains from slaughtered goats in Jahrom. A total of 561 specimens (heart, diaphragm, and tongue) from 187 slaughtered goats were collected from Jahrom slaughterhouse. After DNA extraction, the T. gondii strains were genotyped by the nested PCR-RFLP based on GRA6 and 3', and 5' ends of the SAG2 gene. T. gondii infection was present in 18.2% of cases. Among the examined organs, the diaphragm was more disposed to the infection (10.2%). Furthermore, infection rates of the heart and tongue were 8.6% and 3.7%, respectively. Concurrent infection in the heart and diaphragm, tongue and diaphragm, and heart and tongue were 3.2%, 0.5%, and 0.5%, respectively. In genotyping experiments, genotype I was the most frequent genotype of T. gondii (58.8%), followed by type II (23.5%), type III (11.8%), and a combination of type I and II (5.9%). The results of this study showed the presence of different genotypes of T. gondii in goats including three major and mixed genotypes. These results can be useful in toxoplasmosis control and prevention.

Effect of alginate nanoparticles on the immunogenicity of excretory-secretory antigens against acute toxoplasmosis in murine model

Efficacy of alginate nanoparticles (Alg-NPs) as vaccine delivery for the excretory-secretory antigens (ESAs) against the virulent strain of Toxoplasma gondii was evaluated. Swiss albino mice were intraperitoneally immunized with three doses of either in vivo and in vitro-prepared ESA vaccines, 20 µg each, at 2-week intervals, then were challenged with 2500 tachyzoites of the RH HXGPRT (-) Toxoplasma gondii strain, four weeks later. Mice mortality, tachyzoite number in both peritoneal fluid and impression smear, and viability, ultrastructural tachyzoite changes, measuring immunological markers, and histopathological changes of both liver and spleen were studied, in comparison to alum adjuvanted ESAs and infected control subgroups. The in vivo-prepared Alg-NPs loaded ESAs vaccinated subgroups induced significant reduction in mice mortality, tachyzoite count in both peritoneal fluid and impression smears and viability. Scanning electron microscopy revealed tachyzoites deformities with multiple irregularities, while transmission electron microscopy showed tachyzoites distortion, disrupted plasma membranes, loss of nuclear integrities, and absence of dense granules with extensive vacuolations. A statistically significant increase in the level of both IFN-γ and anti-Toxoplasma IgG was noted. Histopathological results recorded amelioration of the pathological changes induced by Toxoplasma infection in both liver and spleen, with scanty parasites. Therefore, Alg-NPs proved its effectiveness in enhancing the ESAs antigencity, and recommended to test its potentiality as drugs carrier for anti-Toxoplasma agents to enhance their therapeutic efficacy.

Global phosphoproteome analysis reveals significant differences between sporulated oocysts of virulent and avirulent strains of Toxoplasma gondii

In this study, the differences in the phosphoproteomic landscape of sporulated oocysts between virulent and avirulent strains of Toxoplasma gondii were examined using a global phosphoproteomics approach. Phosphopeptides from sporulated oocysts of the virulent PYS strain (Chinese ToxoDB#9) and the avirulent PRU strain (type II) were enriched by titanium dioxide (TiO2) affinity chromatography and quantified using IBT approach. A total of 10,645 unique phosphopeptides, 8181 nonredundant phosphorylation sites and 2792 phosphoproteins were identified. We also detected 4129 differentially expressed phosphopeptides (DEPs) between sporulated oocysts of PYS strain and PRU strain (|log1.5 fold change| > 1 and p < 0.05), including 2485 upregulated and 1644 downregulated phosphopeptides. Motif analysis identified 24 motifs from the upregulated phosphorylated peptides including 22 serine motifs and two threonine motifs (TPE and TP), and 15 motifs from the downregulated phosphorylated peptides including 12 serine motifs and three threonine motifs (TP, RxxT and KxxT) in PYS strain when comparing PYS strain to PRU strain. Several kinases were consistent with motifs of overrepresented phosphopeptides, such as PKA, PKG, CKII, IKK, MAPK, EGFR, INSR, Jak, Syk, Src, Ab1. GO enrichment, KEGG pathway analysis and STRING analysis revealed DEPs significantly enriched in many biological processes and pathways. Kinase related network analysis showed that AGC kinase was the most connected kinase peptide. Our findings reveal significant difference in phosphopeptide profiles of sporulated oocysts between virulent and avirulent T. gondii strains, providing new resources for further elucidation of the mechanisms underpinning the virulence of T. gondii.

Histopathological and ultrastructural assessment of atovaquone-proguanil hydrochloride combination in chronic murine toxoplasmosis

ABSTRACT Over one billion people worldwide are expected to have Toxoplasma gondii infection with anonymous health problems. Available therapies are ineffective for persistent chronic toxoplasmosis. So, there is an imperative need for effective therapies to eliminate chronic tissue stage. In this study, we aimed to assess the effect of a drug combination of atovaquone and proguanil hydrochloride in the treatment of experimental chronic toxoplasmosis. Fifty Swiss Webster mice were used in the study. Forty mice were infected with Me49 type II cystogenic Toxoplasma gondii strain and allocated into four groups: infected untreated (vehicle-administered), infected and treated with cotrimoxazole (CTX) 370 mg/kg/day, infected and treated with atovaquone (ATV) 100 mg/kg/day, and infected and treated with atovaquone/proguanil (ATV/PROG) 50 mg/kg/day. An additional group of uninfected mice was used as an uninfected control group. Drug treatment was initiated 8 weeks post-infection and continued for two weeks. All mice were sacrificed 12 weeks post-infection. Parasitological and histopathological parameters were assessed. Toxoplasma gondii cysts recovered from brain tissue homogenates of both infected untreated and ATV/PROG-treated groups were examined by scanning electron microscopy. Combined ATV/PROG treatment demonstrated a significant reduction of Toxoplasma gondii cyst count in brain tissue (a reduction rate of 84.87%) compared to untreated group (P < .001). Brain tissues obtained from ATV/PROG treated group showed reduction of inflammatory infiltrate and marked attenuation and deformation of recovered Toxoplasma gondii cysts. We conclude that ATV/PROG drug combination could offer a potential drug therapy for Toxoplasma gondii chronic cystic stage.

Examining the Relationship between Toxoplasma gondii and Seropositivity and Serointensity and Depression in Adults from the United Kingdom and the United States: A Cross-Sectional Study.

Infecting approximately one-third of the world’s population, the neurotropic protozoan Toxoplasma gondii has been associated with cognition and several neuropsychiatric diseases including schizophrenia and bipolar disorder. Findings have been mixed, however, about the relationship between Toxoplasma gondii and depression, with some studies reporting positive associations and others finding no associations. To further investigate the association between Toxoplasma gondii and depression, we used data from the UK Biobank and the National Health and Examination Survey (NHANES). Results from adjusted multiple-regression modeling showed no significant associations between Toxoplasma gondii and depression in either the UK Biobank or NHANES datasets. Further, we found no significant interactions between Toxoplasma gondii and age, sex, educational attainment, and income in either dataset that affected the association between Toxoplasma gondii and depression. These results from two community-based datasets suggest that in these samples, Toxoplasma gondii is not associated with depression. Differences between our findings and other findings showing an association between Toxoplasma gondii and depression could be due to several factors including differences in socioeconomic variables, differences in Toxoplasma gondii strain, and use of different covariates in statistical modeling.

Effect of HIV aspartyl protease inhibitors on experimental infection with a cystogenic Me49 strain of Toxoplasma gondii

ABSTRACT Toxoplasmosis is a zoonotic disease of major significant perspectives in public health and veterinary medicine. So far, the available drugs control only the active infection, once the parasite encysts in the tissues, they lose their efficacy. Cytokines; IFN-γ and IL-10, play a critical role in the modulation of toxoplasmic encephalitis and neuro-inflammation in chronic toxoplasmosis. Antiretroviral protease inhibitors applied in the treatment of acquired immunodeficiency syndrome, revealed activity against multiple parasites. Aluvia (lopinavir/ritonavir) (L/R); an aspartyl protease inhibitor, had efficiently treated T. gondii RH strain infection. We investigated the potential activity of L/R against experimental T. gondii infection with a cystogenic Me49 strain in mice, considering the role of IFN-γ and IL-10 in the neuropathology versus pyrimethamine-sulfadiazine combination therapy. Three aluvia regimens were applied; starting on the day of infection (acute phase), 2-week PI (early chronic phase) and eight weeks PI (late chronic phase). L/R reduced the brain-tissue cyst burden significantly in all treatment regimens. It impaired the parasite infectivity markedly in the late chronic phase. Ultrastructural changes were detected in Toxoplasma cyst membrane and wall, bradyzoite membrane and nuclear envelope. The signs of bradyzoite paraptosis and cytoplasmic lipid droplets were observed. L/R had significantly reduced the brain-homogenate levels of IFN-γ and IL-10 in its three regimens however, they could not reach the normal level in chronic phases. Cerebral hypercellularity, perivascular inflammatory response, lymphoplasmacytic infiltrates and glial cellular reaction were ameliorated by L/R treatment. Herein, L/R was proved to possess promising preventive and therapeutic perspectives in chronic cerebral toxoplasmosis.

The delayed death-causing nature of Rosmarinus officinalis leaf extracts and their mixture within experimental chronic toxoplasmosis: Therapeutic and prophylactic implications

BackgroundThe restricted effect, significant toxicity, and emerging resistance of anti-toxoplasmosis synthetic agents impose the search for alternatives. The current research aimed to evaluate the prophylactic and therapeutic efficacy of Rosmarinus officinalis extracts and their mixtures against chronic murine toxoplasmosis and to clarify the phenomenon of delayed death. MethodsThis research included two experimental designs, the first to test the preventive and curative efficacy of the extracts and the second to assess delayed death in mice infected with the ME49 strain of Toxoplasma gondii. The essential oils of the plant were analyzed by gas chromatography/mass spectrometry. Results: Treatment with a mixture of rosemary extracts displayed reduction rates of 81% for T. gondii cyst burden and 23% for cyst viability. The reinfected group with the pretreated cysts reported 93.4% reduction in cyst burden and 95.4% in cyst viability. Moreover, 90% reduction of the infectivity rate was obtained. The therapeutic efficacy of this mixture was superior to its valuable prophylactic effect. Histopathological examination of liver and brain tissue exhibited marked improvement. Both extracts possess free radical scavenging and antioxidant activities evidenced by high expression of iNOS stain. Our results were signified by low BAG-1 gene expression and massive mutilation of T. gondii cyst in the targeted group using scanning electron microscopy. Analysis of R. officinalis revealed the presence of isobornylformate as a novel ingredient. ConclusionsR. officinalis displays a therapeutic rather than prophylactic potential, indicating the emergence of an effective safe alternative therapy.

The High Potency of Green Synthesized Copper Nanoparticles to Prevent the Toxoplasma gondii Infection in Mice.

Nowadays, due to the lack of an effective vaccine to prevent the toxoplasmosis, chemotherapy with the combination of pyrimethamine and sulfadiazine is considered as the "gold standard" treatment for toxoplasmosis. Recent reports have exhibited that these synthesized chemical drugs are associated with some serious side effects. The present study aims to evaluate the prophylactic effects of copper nanoparticles (CuNPs) green synthesized by Capparis spinosa fruit methanolic extract alone and combined with atovaquone against chronic toxoplasmosis induced by the Tehran strain of Toxoplasma gondii in mice METHODS: Mice were then orally administrated with CuNPs at the doses of 2 and 4mg/kg/day and in combined with atovaquone 50mg/kg for 14days. Male BALB/c mice were divided into two seven groups include C1 (non-treated non-infected); C2 (treated with normal saline); C3 (Infected mice treated with atovaquone 100mg/kg/day); Ex1 (treated with CuNPs 2mg/kg/day); Ex2 (treated with CuNPs 4mg/kg/day); Ex3 (treated with CuNPs 2mg/kg/day + atovaquone 50mg/kg/day); Ex3 (treated with CuNPs 4mg/kg/day + atovaquone 50mg/kg/day). On the 15th day, the mice were infected with the intraperitoneal inoculation of 20-25 tissue cysts from the Tehran strain of T. gondii. The mean numbers of brain tissue cysts and the mRNA levels of IL-12, IFN-γ, and inducible nitric oxide synthase (iNOS) in mice of each tested group were measured. CuNPs were green synthesized by C. spinosa methanolic extract. Scanning electron microscopy showed that the particle size of CuNPs was 17 and 41nm with maximum peak at the wavelength of 414nm. The mean number of T. gondii tissue cysts in mice of tested groups of Ex1, Ex2, Ex3, and Ex4, significantly decreased as a dose-dependent response compared with control group. Moreover, in similar to the control group C3, no T. gondii tissue cysts was observed in mice of experimental group Ex3 and Ex4. The mRNA levels of IFN-γ, IL-12, and iNO was measured in mice of all tested groups. The mRNA levels of IFN-γ, IL-12, and iNO was increased in all mice of experimental groups in comparison with the control group C2; however, a significant enhancement was detected in mRNA level of IFN-γ, IL-12, and iNO in the tested groups of Ex3 and Ex4 when compared with control group C3. The obtained results revealed the high potency of CuNPs alone and combined with atovaquone to prevent toxoplasmosis in mice. Although, the prophylactic effects of CuNPs and other properties, such as improved cellular immunity and low toxicity, are positive topics; however, more studies are required to approve these findings especially in clinical settings.

First isolation and genotyping of Toxoplasma gondii strains from domestic animals in Tunisia

The isolation and molecular typing of Toxoplasma gondii strains provide an essential basis for a better understanding of the parasite’s genetic diversity, determinants of its geographical distribution and associated risks to human health. In this study, we isolated and genetically characterized T. gondii strains from domestic animals in Southern and coastal area of Tunisia. Blood, hearts and/or brains were collected from 766 domestic animals (630 sheep and 136 free-range chickens). Strain isolation from these samples was performed using mouse bioassay and genotyping was carried out with a multiplex PCR technique using 15 microsatellite markers. Thirty viable strains of T. gondii were successfully isolated from tissues of sheep (19/142) and chickens (11/33). In addition, 3 strains could be successfully genotyped from animal tissues for which mouse bioassay was unsuccessful. A large predominance of type II strains (n = 29) was found in the sampled regions, followed by type III (n = 3) and, for the first time in Tunisia, a single isolate of Africa 4 lineage from a sheep. Analyses of population genetics showed the presence of a divergent population of type II lineage in Tunisia, supporting limited recent migrations of strains between Tunisia and other countries of the world.

Effect of spiramycin versus aminoguanidine and their combined use in experimental toxoplasmosis.

Toxoplasmosis is one of the widest spread parasitic infections which is caused by Toxoplasma gondii protozoon. Many experimental studies have evaluated the effect of aminoguanidine upon parasitic load and inflammatory process. However, few reports have illustrated the impact of combining aminoguanidine with spiramycin in the treatment of toxoplasmosis. Therefore, our study aimed to explore the possible effects of spiramycin used alone and combined with aminoguanidine against the avirulent (ME49) Toxoplasma gondii strain in experimental toxoplasmosis. Fifty-five Swiss albino mice were included in the study and were divided into five groups: (GI): non-infected control group; (GII): infected untreated control group; (GIII): infected- spiramycin treated group; (GIV): infected-aminoguanidine treated group; (GV): infected and received combination of spiramycin and aminoguanidine. Obtained results exhibited a significant increase in brain cysts numbers in aminoguanidine treated groups compared to infected untreated control groups. Histopathological studies denoted that combination between spiramycin and aminoguanidine improved the pathological features only in liver and heart tissues of the studied groups. Moreover, it was noticed that spiramycin administered alone had no effect on nitric oxide expression, whereas its combination with aminoguanidine had an inhibitory effect on inducible nitric oxide synthase enzyme in brain, liver and heart tissues of different study groups. In conclusion, the combination of spiramycin and aminoguanidine significantly reduced the parasitic burden, yet, it failed to resolve the pathological sequels in brain tissues of Toxoplasma gondii infected mice.

Characterization of extracellular vesicles isolated from types I, II and III strains of Toxoplasma gondii

This study investigated the participation extracellular vesicles (EVs) in Toxoplasma gondii-host interaction. EVs of three T. gondii strains (RH, ME-49 and VEG) were purified by chromatography and ELISA. Results of “nanoparticle tracking analysis” and scanning electron microscopy showed that RH strain released more EVs than other strains. Images of transmission electron microscopy showed that in beginning of incubation (culture medium), EVs were inside of tachyzoites preparing to be released. After 24 hours, they were largely produced inside tachyzoites and were released through plasma membrane. The parasite burden of mice infected with RH strain plus EVs was increased and with early death of 1-2 days compared of those that received only parasites. EV proteins of ME-49 and VEG strains were poorly reactive to sera of infected patients in imunoblot. However, those from RH strain were reactive against sera of patients with cerebral toxoplasmosis. EVs stimulated murine splenocytes caused similar production of IFN-γ and IL-10 levels. RH strain derived EVs stimulated more TNF-α than those stimulated by other strains. T. gondii and infected hosts can express the same miRNAs (miR-155-5p, miR-125b-5p, miR-423-3p). In conclusion, T. gondii derived EVs promote host-parasite interactions, modulate host immune responses, carry virulent factors and cause an imbalance in cellular immune response.

Rosuvastatin revert memory impairment and anxiogenic-like effect in mice infected with the chronic ME-49 strain of Toxoplasma gondii.

The aim of this study was to investigate the effect of rosuvastatin treatment on memory impairment, and anxiogenic-like effects in mice chronically infected with Toxoplasma gondii. For this, Balb/c mice were infected orally with chronic ME-49 strain of Toxoplasma gondii. Oral treatment with rosuvastatin (40mg/kg/day) started on the 51st day post-infection and was performed daily for 21 days. After completion of treatment, anxiety-like effects and locomotion were investigated in the open field (OF) test, whereas novel object recognition (NOR) test was used for evaluation of short- and long-term memory. At the end of the experiments, the brain was collected for Toxoplasma gondii DNA quantification and histopathological analysis. Infection with ME-49 strain decreased the time spent in the center of OF, indicating an anxiogenic effect, without affecting total and peripheral locomotion. Rosuvastatin treatment inhibited the change in the center time. Besides, pharmacological treatment increased total and central locomotion in both non-infected and infected animals. Infection also impaired both short- and long-term memory in the NOR test, and these effects were reverted by rosuvastatin treatment. In addition to effects in behavioral changes, rosuvastatin also reduced parasite load in the brain and attenuated signs of brain inflammation such as perivascular cuffs, inflammatory cell infiltration and tissue damage. These findings indicate for the first time the efficacy of rosuvastatin in treatment of memory impairment and anxiogenic effect evoked by infection with Toxoplasma gondii. These effects might be mediated by reduced cyst load, which in turn decrease inflammation and damage in the brain.

Whole-genome sequencing of a Toxoplasma gondii strain from a Turkish isolate using next-generation sequencing technology

PurposeToxoplasma gondii is an intracellular parasite that can affect all vertebrae and is the causative agent of toxoplasmosis. At present, the United States CDC (Centers for Disease Control and Prevention) recognizes this infection as a neglected disease. Toxoplasma gondii infection profiles exhibit differences because of the different regional and climatic responses to these parasites in Turkey, and these protozoan infections are notably common in this country. In this study, we attempted to obtain the whole-genome sequence of T. gondii using next-generation sequencing technology. MethodsToxoplasma gondii isolates were isolated from an infant with congenital toxoplasmosis by Ekmen et al. (1974) in Ankara, Turkey. Whole-genome sequencing (WGS) was performed using the Illumina HiSeq 2500 and HiSeq SBS Kit v2. A T. gondii library was created on this device in the initial stage. After the completion of the library phase, sequence analysis was begun with a next-generation sequencing device. The resulting fragments were combined using paired-end (PE) reading and converted into a single DNA fragment. Bioinformatic analysis was performed using the Geneious 2.1. program. ResultsIn our study, WGS was successfully performed on T. gondii. The T. gondii whole-genome sequence has a coverage value of 50x, a size of 61,5763 Mb and a GC ratio of 52.6%. Data from this sequence were submitted to the National Center for Biotechnology Information (NCBI) GenBank (www.ncbi.nlm.nih.gov) database under the name Toxoplasma gondii TR01 (TG_TR01). The accession number of the genome obtained in this study is WOEV00000000.1. The biological sample access number is SAMN13338796. The genome of the T. gondii strain obtained in this study was compared with the reference genome, and 8312 CDSs (coding sequences), 183 tRNAs, 294 rRNAs and 8789 genes were identified. Among the 8312 CDSs, 4284 encoded hypothetical proteins (hypothetical protein CDSs/proteins of unknown function). The entire genome sequence of T gondii TR01 was compared with that of Toxoplasma gondii ME49. The results of this comparison demonstrate that the analyzed genome was 99,98% similar to the reference genome. The accession numbers of 14 chromosomes belonging to the genome sequences of T. gondii TR01 (TG_TR01) are CM019722.1, CM019723.1, CM019724.1, CM019725.1, CM019726.1, CM019727.1, CM019728.1, CM019729.1, CM019730.1, CM019731.1, CM019732.1, CM019733.1, CM019734.1, and CM019735.1. ConclusionIn this study, a whole-genome sequences of T. gondii was conducted for the first time in Turkey. The analyzed strain was named T. gondii TR01. The data obtained from this study may contribute to a better understanding of T. gondii. T. gondii is an important pathogen with an unusual population structure. Although T. gondii is highly zoonotic and has a complicated life cycle, some strains of this parasite have exhibited high genetic sequence similarity, and our study supports this knowlegde. The characterization of this strain may be very useful for the scientific community of our country and may help to establish a foundation for further research investigating the genome of T. gondii.

Myrtus communis Essential Oil; Anti-Parasitic Effects and Induction of the Innate Immune System in Mice with Toxoplasma gondii Infection.

Background: Myrtus communis (M. communis) is a wild aromatic plant used for traditional herbal medicine that can be demonstrated in insecticidal, antioxidant, anti-inflammatory, and antimicrobial activity of its essential oils (MCEO). Aim: The present study aimed to evaluate the prophylactic effects of M. communis essential oil (MCEO) against chronic toxoplasmosis induced by the Tehran strain of Toxoplasma gondii in mice. Methods: Gas chromatography/mass spectrometry (GC/MS) analysis was performed to determine the chemical composition of MCEO. Mice were then orally administrated with MCEO at the doses of 100, 200, and 300 mg/kg/day and also atovaquone 100 mg/kg for 21 days. On the 15th day, the mice were infected with the intraperitoneal inoculation of 20–25 tissue cysts from the Tehran strain of T. gondii. The mean numbers of brain tissue cysts and the mRNA levels of IL-12 and IFN-γ in mice of each tested group were measured. Results: By GC/MS, the major constituents were α-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%), respectively. The results demonstrated that the mean number of T. gondii tissue cysts in experimental groups Ex1 (p < 0.05), Ex2 (p < 0.001) and Ex3 (p < 0.001) was meaningfully reduced in a dose-dependent manner compared with the control group (C2). The mean diameter of tissue cyst was significantly reduced in mice of the experimental groups Ex2 (p < 0.01) and Ex3 (p < 0.001). The results demonstrated that although the mRNA levels of IFN-γ and IL-12 were elevated in all mice of experimental groups, a significant increase (p < 0.001) was observed in tested groups of Ex2 and Ex3 when compared with control groups. Conclusion: The findings of the present study demonstrated the potent prophylactic effects of MCEO especially in the doses 200 and 300 mg/kg in mice infected with T. gondii. Although the exceptional anti-Toxoplasma effects of MCEO and other possessions, such as improved innate immunity and low toxicity are positive topics, there is, however, a need for more proof from investigations in this field.

4-Arylthiosemicarbazide derivatives as a new class of tyrosinase inhibitors and anti-Toxoplasma gondii agents

We report herein anti-proliferation effects of 4-arylthiosemicarbazides, with a cyclopentane substitution at N1 position, on highly virulent RH strain of Toxoplasma gondii. Among them, the highest in vitro anti-Toxoplasma activity was found with the meta-iodo derivative. Further experiments demonstrated inhibitory effects of thiosemicarbazides on tyrosinase (Tyr) activity, and good correlation was found between percentage of Tyr inhibition and IC50 Tg . To confirm the concept that thiosemicarbazides are able to disrupt tyrosine metabolism in Toxoplasma tachyzoites, the most potent Tyr inhibitors were tested for their efficacy of T. gondii growth inhibition. All of them significantly reduced the number of tachyzoites in the parasitophorous vacuoles (PVs) compared to untreated cells, as well as inhibited tachyzoites growth by impeding cell division. Collectively, these results indicate that compounds with the thiosemicarbazide scaffold are able to disrupt tyrosine metabolism in Toxoplasma tachyzoites by deregulation of their crucial enzyme tyrosine hydroxylase (TyrH).

Investigating Immunological Parameters and Quantification of Rhoptry Pseudokinases Gene Expression in Toxoplasmosis

Toxoplasma affects about one-third of the world’s population and is a common disease between humans and animals. The most common cause of toxoplasmosis is infection with Toxoplasma gondii. The main virulence factors of Toxoplasma gondii in toxoplasmosis are rhoptry pseudokinases. The aim of this study was to evaluate the seroprevalence and immunological parameters of toxoplasmosis and investigate the expression of rhoptry protein 5, rhoptry protein 16 and rhoptry protein 18 genes in women in Erbil. Also, evaluation of the serum levels of interleukin-12 and interleukin-6 in women with recurrent miscarriage were performed. In this study, 300 women at their reproductive age who referred to Maternity Teaching Hospital of Erbil were registered. Enzyme linked immunosorbent assay technique has been applied to detect anti toxoplasma immunoglobulin G and immunoglobulin M. Statistical analysis was done via statistical package for the social sciences version 21.0 and the p value of ≤0.05 was considered as statistically significant. A total of 50 seropositive samples with and without history of miscarriage were tested by polymerase chain reaction for detection of rhoptry protein 5, rhoptry protein 16 and rhoptry protein 18 genes using home designed primers. A significant association was found between toxoplasmosis and history of abortion, but no association was found between toxoplasmosis and education level, socioeconomic status, age and residence. According to the results, the interleukin-6 level was non-significantly (p>0.05) changed in infected women. Nevertheless, serum interleukin-12 level was remarkably (p<0.05) elevated in infected women compared to control group. Polymerase chain reaction targeting both toxoplasma genes detected more abortive cases than non-abortive one among anti-toxoplasma seropositive women. The results of quantification and detection of rhoptry protein 5, rhoptry protein 16 and rhoptry protein 18 genes of Toxoplasma gondii among 50 samples by polymerase chain reaction, confirmed the expression of these genes in toxoplasmosis condition. Finally, it was concluded that the prevalence of toxoplasmosis has increased in Erbil women and the level of interleukin-12 in the serum of women infected with Toxoplasma gondii has increased significantly. Also, rhoptry protein 5, rhoptry protein 16 and rhoptry protein 18 genes can be used as markers in the first level of toxoplasmosis. Therefore, quantification of these genes can help in determining Toxoplasma gondii strain virulence.

Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain.

BACKGROUND Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-β signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains.OBJECTIVES To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole.METHODS Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence.FINDINGS Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole.MAIN CONCLUSIONS These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Immunological, histopathological, and ultrastructural evidence of steroid-induced reactivation of chronic murine toxoplasmosis

ABSTRACT We developed a model of steroid-induced reactivation of chronic murine toxoplasmosis to mirror similar effects of steroids or other immunosuppressants in infected humans. Immunological, histopathological, and ultrastructural parameters were reported. Prior to steroid administration, mice were infected with 10 cysts of the Me49 strain of Toxoplasma gondii. Mice were treated with dexamethasone (DXM, 2.5 mg/kg/day in drinking water), alone or combined with Solu-Cortef (SOLU, 50 mg/kg by subcutaneous injection 3 times a week) for 7 weeks or left untreated as control. Histopathological changes and ultrastructural effects of steroids on the course of chronic toxoplasmosis were recorded. By electron microscopy, the brains of infected combined treated mice showed an increase in number of tachyzoites and bradyzoites, degeneration, and necrosis of neural cells and hydropic degeneration besides the observed rupture of toxoplasma cysts releasing free tachyzoites in brain tissue. DXM+SOLU-combined treatment also significantly increased mortality, mean brain cyst count as compared to infected untreated mice (P = .01 and). Moreover, 3/12 (25%) treated animals developed clinical signs of toxoplasmic encephalitis. This simple model of drug-induced reactivation of chronic toxoplasmosis permits investigation of host-parasite interaction and may be used for the evaluation of chemotherapeutics in immunocompromised infected patients.

HDAC inhibitors Tubastatin A and SAHA affect parasite cell division and are potential anti-Toxoplasma gondii chemotherapeutics

The redirectioning of drugs in the pharmaceutical market is a well-known practice to identify new therapies for parasitic diseases. The histone deacetylase inhibitors Tubastatin A (TST) and Suberoylanilide Hydroxamic Acid (SAHA), firstly developed for cancer treatment, are effective against protozoa parasites. In this work, we aimed to demonstrate the activity of these drugs as potential agents against Toxoplasma gondii, the causative agent of toxoplasmosis. TST and SAHA were active against different genotypes of Toxoplasma gondii, such as, RH (type I), EGS (I/III) and ME49 (type II) strains. The IC₅₀ values for the RH strain were 19 ± 1 nM and 520 ± 386 nM for TST and 41 ± 3 nM and 67 ± 36 nM for SAHA, for 24 and 48 h, respectively. Both compounds were highly selective for T. gondii and their anti-proliferative effect was irreversible for 8 days. The calculated selectivity indexes (39 for TST and 30 for SAHA) make them lead compounds for the future development of anti-Toxoplasma molecules. Western blotting showed TST led to a significant increase of the nuclear histone H4 and a decrease of H3 acetylation levels. Treatment with 1 μM TST and 0.1 μM SAHA for 48 h decreased the amount of global α-tubulin. Fluorescence and electron microscopy showed that both drugs affected the endodyogeny process impairing the budding of daughter cells. The drugs led to the formation of large, rounded masses of damaged parasites with several centrosomes randomly dispersed and incorrect apicoplast division and positioning. TST-treated parasites showed a rupture of the mitochondrial membrane potential and led to a failure of the IMC assembling of new daughter cells. SAHA and TST possibly inhibit HDAC3 and other cytoplasmic or organelle targeted HDACs involved in the modification of proteins other than histones.

Isolation and genotyping of local Toxoplasma gondii strains in infected chicken

Isolation and genotyping of local Toxoplasma gondii strains in infected chicken