Host sensing and signal transduction during Toxoplasma stage conversion.

The intracellular parasite Toxoplasma gondii infects nucleated cells in virtually all warm-blooded vertebrates, including one-third of the human population. While immunocompetent hosts do not typically show symptoms of acute infection, parasites are retained in latent tissue cysts that can be reactivated upon immune suppression, potentially damaging key organ systems. Toxoplasma has a multistage life cycle that is intimately linked to environmental stresses and host signals. As this protozoan pathogen is transmitted between multiple hosts and tissues, it evaluates these external signals to appropriately differentiate into distinct life cycle stages, such as the transition from its replicative stage (tachyzoite) to the latent stage (bradyzoite) that persists as tissue cysts. Additionally, in the gut of its definitive host, felines, Toxoplasma converts into gametocytes that produce infectious oocysts (sporozoites) that are expelled into the environment. In this review, we highlight recent advances that have illuminated the interfaces between Toxoplasma and host and how these interactions control parasite stage conversion. Mechanisms underlying these stage transitions are important targets for therapeutic intervention aimed at thwarting parasite transmission and pathogenesis.

Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

Temporal expression of Toxoplasma stage-specific genes in brain tissue: coincidence with parasitological and histopathological findings in mice models.

In the intermediate hosts, tachyzoites of T. gondii predominate in the acute stage while bradyzoites persist inside tissue cysts with the potential for reactivation. The two stages exhibit different metabolic and antigenic characters. The present study aimed to investigate temporal expression of Toxoplasma SAG1 and BAG1 genes in the brain tissue and the coincident parasitological and histopathological findings in mice models of toxoplasmosis. The study included group A: mice infected with RH strain and sacrificed 7 days post-infection (p.i.); group B: mice infected with RH strain and treated with sulfamethoxazole-trimethoprim (30 mg/kg/day and 150 mg/kg/day respectively) 24 h p.i. until sacrificed at days 5, 10, or 20 post-treatment; group C: mice infected with ME-49 strain and sacrificed at days 7, 27, 47, or 67 p.i; and group D: mice infected with ME-49 strain and received dexamethasone daily starting at day 68 p.i. and scarified at days 6 or 10 post-treatment. All mice were inspected daily for abnormal physical signs. Peritoneal exudate and brain homogenate were examined for detection of Toxoplasma stages. Brain sections were examined histopathologically. SAG1 and BAG1 gene expression was evaluated using reverse transcription real-time polymerase chain reaction and the ΔΔCt method. Results revealed that marked BAG1 upregulation is consistent with detection of Toxoplasma cysts and degenerative changes while predominance of tachyzoites and inflammatory infiltrate is compatible with SAG1 upregulation. The study sheds light on the potential for using stage-specific gene expression pattern as markers for evaluation of toxoplasmosis disease progression in clinical settings.

Development of an in vitro system to study the developmental stages of Toxoplasma gondii using a genetically modified strain expressing markers for tachyzoites and bradyzoites.

Toxoplasma gondii, the agent of toxoplasmosis, is an intracellular parasite that can infect a wide range of vertebrate hosts. Toxoplasmosis causes severe damage to immunocompromised hosts and its treatment is mainly based on the combination of pyrimethamine and sulfadiazine, which causes relevant side effects primarily observed in AIDS patients, including bone marrow suppression and hematological toxicity (pyrimethamine) and/or hypersensitivity and allergic skin reactions (sulfadiazine). Thus, it is important to investigate new compounds against T. gondii, particularly those that may act on bradyzoites, which are present in cysts during the chronic disease phase. We propose an in vitro model to simultaneously study new candidate compounds against the two main causative stages of Toxoplasma infection in humans, using the EGS-DC strain that was modified from a type I/III strain (EGS), isolated from a case of human congenital toxoplasmosis in Brazil and engineered to express markers for both stages of development. One feature of this strain is that it presents tachyzoite and bradyzoite in the same culture system and in the same host cell under normal culture conditions. Additionally, this strain presents stage-specific fluorescent protein expression, allowing for easy identification of both stages, thus making this strain useful in different studies. HFF cells were infected and after 4 and 7 days post infection the cells were treated with 10 µM of pyrimethamine or atovaquone, for 48 or 72 h. We used high-throughput screening to quantify the extent of parasite infection. Despite a reduction in tachyzoite infection caused by both treatments, the atovaquone treatment reduced the bradyzoite infection while the pyrimethamine one increased it. Ultrastructural analysis showed that after treatment with both drugs, parasites displayed altered mitochondria. Fluorescence microscopy of cells labeled with MitoTracker CMXRos showed that the cysts present inside the cells lost their mitochondrial membrane potential. Our results indicate that this experimental model is adequate to simultaneously analyze new active compounds against tachyzoite and bradyzoite forms.

Extracellular vesicles isolated from Toxoplasma gondii induce host immune response.

This study established a protocol to purify Toxoplasma gondii tachyzoite microvesicles and exosomes, called as extracellular vesicles (EVs). In addition, the investigations were conducted to determine the kinetic of EV release by tachyzoites and whether EV proteins are able to modulate the host immune response. The particle size and concentration released by tachyzoites in culture medium at different incubation-period were characterized by nanoparticle tracking analysis. Tachyzoites (1 × 106 ) released around 4.37 ± 0.81 × 108 EVs/mL/h, with size varying between 138.2 and 171.9 nm. EVs released into the medium were purified by gel-exclusion chromatography and screened by ELISA, using a pool of human positive sera for toxoplasmosis. EV-fractions contained high concentration of proteins, and EVs were analyzed by scanning and transmission electron microscopies. Tachyzoites released EVs into the culture medium throughout all membrane surface, and these vesicles contain small RNAs/miRNA. Pooled sera from chronically infected human or mice (infected with 2 different T. gondii strains) recognized distinct EV electrophoretic patterns in immunoblotting. T. gondii EVs significantly induced IL-10, TNF-α and iNOS in murine macrophages. In conclusion, this study shows that T. gondii secrete/excrete EVs (microvesicles and exosomes) contain miRNA and they were immunologically recognized by host immune response.

Molecular docking to Toxoplasma gondii thymidylate synthase-dihydrofolate reductase and efficacy of raltitrexed in infected mice.

Toxoplasmosis is a zoonosis of worldwide distribution. Currently, two drugs, pyrimethamine and sulfadiazine, are used as a reference in the treatment of toxoplasmosis, but the resistance of Toxoplasma gondii appears as a relevant public health problem. In order to identify new drugs to toxoplasmosis treatment, we performed a molecular docking of raltitrexed to T. gondii thymidylate synthase-dihydrofolate reductase (TS-DHFR) and also evaluated its efficacy in infected mice. Initially, raltitrexed was docked on the crystallographic structures of TS-DHFR from T. gondii and Mus musculus. Then, 48 h after infection with the T. gondii RH strain, different groups of mice received an oral dose of raltitrexed (0.15, 0.75, and 1.5 mg kg-1). Two days after treatments, raltitrexed was able to prevent mortality and reduce the number of tachyzoites in the peritoneal fluid and liver imprints from infected mice. The results showed that raltitrexed has important protective activities against the T. gondii RH strain. Molecular docking still suggests that the effects against the parasite may be dependent on the inhibition of T. gondii thymidylate synthase. This study opens new perspectives for the use of raltitrexed in patients infected with T. gondii, especially when conventional treatments do not exhibit the expected efficacy.

Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle.

Virulence factors from the ROP2-family have been extensively studied in Toxoplasma gondii, but in the closely related Neospora caninum only NcROP2Fam-1 has been partially characterized to date. NcROP40 is a member of this family and was found to be more abundantly expressed in virulent isolates. Both NcROP2Fam-1 and NcROP40 were evaluated as vaccine candidates and exerted a synergistic effect in terms of protection against vertical transmission in mouse models, which suggests that they may be relevant for parasite pathogenicity. NcROP40 is localized in the rhoptry bulbs of tachyzoites and bradyzoites, but in contrast to NcROP2Fam-1, the protein does not associate with the parasitophorous vacuole membrane due to the lack of arginine-rich amphipathic helix in its sequence. Similarly to NcROP2Fam-1, NcROP40 mRNA levels are highly increased during tachyzoite egress and invasion. However, NcROP40 up-regulation does not appear to be linked to the mechanisms triggering egress. In contrast to NcROP2Fam-1, phosphorylation of NcROP40 was not observed during egress. Besides, NcROP40 secretion into the host cell was not successfully detected by immunofluorescence techniques. These findings indicate that NcROP40 and NcROP2Fam-1 carry out different functions, and highlight the need to elucidate the role of NcROP40 within the lytic cycle and to explain its relative abundance in tachyzoites.

Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10(6) tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me2SO without incubation. A cooling rate of ∼1 °C/min to -80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.

Triclosan and triclosan-loaded liposomal nanoparticles in the treatment of acute experimental toxoplasmosis.

Efficacy of triclosan (TS) and TS-loaded liposomes against the virulent strain of Toxoplasma gondii (T. gondii) was evaluated. Swiss albino mice were intraperitoneally infected with 10(4) tachyzoites of RH HXGPRT(-) strain of T. gondii, then were orally treated with 150 mg/kg TS or 100 mg/kg TS liposomes twice daily for 4 days. Mice mortality, peritoneal and liver parasite burdens, viability, infectivity and ultrastructural changes of peritoneal tachyzoites of infected treated mice were studied, in comparison with those of infected non-treated controls. Drug safety was biochemically assessed by measuring liver enzymes and thyroxin. Both TS and TS liposomes induced significant reduction in mice mortality, parasite burden, viability and infectivity of tachyzoites harvested from infected treated mice. Scanning electron microscopy of treated tachyzoites showed distorted shapes, reduced sizes, irregularities, surface protrusions, erosions and peeling besides apical region distortion. Transmission electron microscopy showed that treated tachyzoites were intracellularly distorted, had cytoplasmic vacuolation, discontinuous plasma membranes, nuclear abnormalities and disrupted internal structures. Besides, in TS liposomes-treated subgroup, most tachyzoites were seen intracellularly with complete disintegration of the parasite plasma and nuclear membranes, with complete destruction of the internal structures. Biochemical safety of TS and TS liposomes was proven. Accordingly, TS can be considered as a promising alternative to the standard therapy for treating acute murine toxoplasmosis. Liposomal formulation of TS enhanced its efficacy and allowed its use in a lower dose.

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry.

Toxoplasma gondii is a protozoan parasite infecting almost all warm-blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I-RH strain, Type II-PRU strain, Type II-TgQHO strain, and ToxoDB 9-TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI-TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.

In vitro anti-Toxoplasma effects and apoptotic induction of queen bee acid (10-hydroxy-2-decenoic acid) alone and in combination with atovaquone.

Toxoplasmosis, which is caused by the Toxoplasma gondii parasite, is a parasitic, infectious disease. 10-hydroxy-2-decenoic acid (10-H2DA, queen bee acid (QBA), is one of the most prevalent fatty acids (>40%) present in royal jelly. Studies have pointed to antitumor, anti-inflammatory, antiangiogenic, and antimicrobial effects of 10-H2DA, improving the immune system. This experimental survey aimed to assess the in vitro efficacy of QBA against tachyzoites and intracellular parasites of the T. gondii RH strain. Anti-Toxoplasma effects of QBA against tachyzoites were examined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay for 30, 60, 120, and 180 min. In addition, the effect of QBA on infection rate and intracellular parasites was studied. Real-time polymerase chain reaction (Real-Time PCR) was also applied to assess the expression level of the Caspase-3 gene. The best efficiency of QBA was obtained at 100 and 50 µg/mL, whereas all tachyzoites were diminished, followed by 120- and 180-min treatment, respectively. It was also found that the best repressing efficacy of QBA in the infection rate and the load of parasites into the Vero cells was indicated at 100 µg/mL (P<0.001). Nonetheless, the combination of QBA (12.5 µg/mL) along with atovaquone 30 µg/mL displayed the most marked effect on the infection rate and a load of parasites into the Vero cells in the infected Vero cells. The expression level of the Caspase-3 gene was dose-dependently increased after the exposure of tachyzoites to QBA, mainly at ½ IC50 and IC50 compared to normal saline. The obtained findings exhibited the high in vitro potency of QBA, especially in combination with atovaquone against T. gondii RH strain tachyzoites. Although apoptosis induction can be suggested as one of the principle mechanisms, more studies are required to elucidate its accurate mechanisms, as well as its efficacy and safety in animal models and clinical settings.

Zingiber officinale Ameliorates Acute Toxoplasmosis-Induced Pathology in Mice.

BACKGROUND: Toxoplasma gondii (T. gondii) infects one third of the world's population with significant illness, mainly among immunocompromised individuals and pregnant women. Treatment options for toxoplasmosis are limited which signifies the need for novel, potent, and safe therapeutic options. The goal of this study was to assess the effectiveness of the ethanolic extract of Zingiber officinale (Z. officinale) in treating mice infected with the RH T. gondii strain. MATERIALS AND METHODS: Gas Chromatography/Mass Spectrometry (GC/MS) was used to identify components of ethanolic extract of Z. officinale. A total of 80 mice were randomly allocated into four experimental groups that contained 20 mice each. The first group was left uninfected (uninfected control), while three groups were infected with T. gondii RH virulent strain tachyzoites at 2500 tachyzoites/mouse. One infected group was left untreated (infected, untreated), whereas the other two groups were treated orally with either spiramycin (positive control) or Z. officinale ethanolic extract at doses of 200 mg/kg and 500 mg/kg, respectively for 5 days, starting the day of infection. Ten mice from each group were used to assess mice survival in different groups, whereas the other ten mice in each group were sacrificed on the 5th day post-infectin (dpi) to estimate the treatment efficacy by quantifying liver parasite load, liver function, nitric oxide (NO) production, and levels of antioxidant enzymes. Additionally, histopathological studies were performed to evaluate the therapeutic effect of Z. officinale treatment on toxoplasmosis-induced pathological alterations in liver, brain, and spleen. RESULTS: Treatment with Z. officinale ethanolic extract extended the survival of mice till 9th dpi compared to 7th dpi in infected untreated mice. Higher percentage of mice survived in Z. officinale-treated group compared to spiramycin-treatment group at different time points. Liver parasite loads were significantly lower in Z. officinale extract-treated mice and spiramycin-treated mice compared to infected untreated mice which correlated with significantly lower levels of serum liver enzymes (ALT, AST) and nitric oxide (NO), as well as significantly higher catalase (CAT) antioxidant enzyme activity. Scanning electron microscopy (SEM) examination of tachyzoites from the peritoneal fluid revealed marked damage in tachyzoites from Z. officinale-treated group compared to that from infected untreated mice. Moreover, treatment with Z. officinale ethanolic extract alleviated infection-induced pathological alterations and restored normal tissue morphology of liver, brain, and spleen. CONCLUSION: Our results demonstrated that Z. officinale treatment reduced parasite burden and reversed histopathological and biochemical alterations in acute murine toxoplasmosis. These findings support the potential utility of Z. officinale as a future effective natural therapeutic for toxoplasmosis. Further studies are needed to determine the effective active ingredient in Z. officinale extract that can be further optimized for treatment of toxoplasmosis.

Balamuthia mandrillaris: in vitro interactions with selected protozoa and algae.

Although Balamuthia mandrillaris was identified more than two decades ago as an agent of fatal granulomatous encephalitis in humans and other animals, little is known about its ecological niche, biological behavior in the environment, food preferences and predators, if any. When infecting humans or other animals, Balamuthia feeds on tissues; and in vitro culture, it feeds on mammalian cells (monkey kidney cells, human lung fibroblasts, and human microvascular endothelial cells). According to recent reports, it is believed that Balamuthia feeds on small amebae, for example, Acanthamoeba that are present in its ecological niche. To test this hypothesis, we associated Balamuthia on a one-on-one basis with selected protozoa and algae. We videotaped the behavior of Balamuthia in the presence of a potential prey, its ability to hunt and attack its food, and the time required to eat and cause damage to the target cell by direct contact. We found that B. mandrillaris ingested trophozoites of Naegleria fowleri, Naegleria gruberi, Acanthamoeba spp., Trypanosoma cruzi epimastigotes, Toxoplasma gondii tachyzoites, and Giardia. However, it did not feed on Acanthamoeba cysts or algae. Balamuthia caused cytolysis of T. cruzi epimastigotes and T. gondii tachyzoites by direct contact. Balamuthia trophozoites and cysts were, however, eaten by Paramecium sp.

Cryo-Electron Tomography of Toxoplasma gondii Indicates That the Conoid Fiber May Be Derived from Microtubules.

Toxoplasma gondii (T. gondii) is the causative agent of toxoplasmosis and can infect numerous warm-blooded animals. An improved understanding of the fine structure of this parasite can help elucidate its replication mechanism. Previous studies have resolved the ultrastructure of the cytoskeleton using purified samples, which eliminates their cellular context. Here the application of cryo-electron tomography to visualize T. gondii tachyzoites in their native state is reported. The fine structure and cellular distribution of the cytoskeleton are resolved and analyzed at nanometer resolution. Additionally, the tachyzoite structural characteristics are annotated during its endodyogeny for the first time. By comparing the structural features in mature tachyzoites and their daughter buds, it is proposed that the conoid fiber of the Apicomplexa originates from microtubules. This work represents the detailed molecular anatomy of T. gondii, particularly during the budding replication stage of tachyzoite, and provides a reference for further studies of this fascinating organism.

The role of Curcuma longa essential oil in controlling acute toxoplasmosis by improving the immune system and reducing inflammation and oxidative stress.

Background: Chemotherapy with synthetic drugs is the principal approach for toxoplasmosis treatment; however, recent studies reported the limitations and adverse side effects of these chemical drugs. Objective: This study aimed to examine the in vitro and in vivo effects of Curcuma longa essential oil (CLE) against the Toxoplasma gondii RH strain. Methods: The in vitro effect of different concentrations of CLE on T. gondii tachyzoites was assessed by cell viability assay. Flow cytometry and apoptosis analysis were performed, and nitric oxide production by CLE was also evaluated in tachyzoites. BALB/c mice were orally treated with various doses (1.25, 2.5, and 5 mg·kg-1·day-1) of CLE for 2 weeks. After the induction of acute toxoplasmosis in the mice, their survival rate and the mean number of peritoneal parasites were checked. The hepatic level of antioxidant enzymes and oxidative stress markers was evaluated by commercial kits. The mRNA expression level of proinflammatory cytokines such as interleukin 1-beta (IL-1ß) and interferon-gamma (IFN-γ) was evaluated by quantitative real-time PCR. Results: CLE, especially at 50 µg/ml, showed potent inhibitory effects on T. gondii tachyzoites. It increased the survival rate (ninth day) and reduced the mean number of peritoneal tachyzoites in the infected mice. CLE dependently increased (p < 0.01) the number of necrotic and apoptotic cells as well as NO production. CLE significantly (p < 0.05) reduced the hepatic level of oxidative stress markers but increased (p < 0.001) the antioxidant enzymes and proinflammatory cytokines in the infected mice, with no important toxicity for vital organs. Conclusion: The findings of this survey revealed the significant in vitro inhibitory effects of CLE on T. gondii tachyzoites. The results also exhibited promising in vivo effects of CLE. CLE improved the survival rate of infected mice and reduced the parasite number in them. Although the mechanisms of action of CLE are not clear, our study demonstrated its beneficial effects on acute toxoplasmosis by strengthening the immune system and reducing inflammation and oxidative stress. Still, more studies are required to confirm these results.

Antibacterial and anti-Toxoplasma activities of Aspergillus niger endophytic fungus isolated from Ficus retusa: in vitro and in vivo approach.

Emergent records propose that Aspergillus niger endophytic fungus is a vital source for various bioactive molecules possessing many biological properties. The current study was designed to inspect the antibacterial and anti-Toxoplasma potentials of Ficus retusa-derived endophytic fungi. After isolation and identification (using 18S rRNA gene sequencing) of A. niger endophytic fungus, LC/MS was utilized for identification and authentication of the chemical profile of the A. niger endophyte extract. Then, the fungal extract was assessed for its antibacterial and antibiofilm activities against Klebsiella pneumoniae clinical isolates. Additionally, its efficacy against Toxoplasma gondii was elucidated in vivo. The fungal extract displayed antibacterial activity against K. pneumoniae isolates with minimum inhibitory concentration values of 64-512 µg/mL. It also possessed a membrane potential dissipating effect using flow cytometry. Moreover, it formed distorted cells with rough surfaces and deformed shapes using a scanning electron microscope (SEM). Regarding its antibiofilm activity, it resulted in a dysregulation of the genes encoding biofilm formation (fimH, mrkA and mrkD) using qRT-PCR in nine K. pneumoniae isolates. The in vivo anti-Toxoplasma potential was demonstrated by decreasing the mortality rate of mice and reducing the tachyzoites' count in the peritoneal fluids and liver impression smears of mice. In addition, the deformities of the parasite decreased, as revealed by SEM and the inflammation in tissues diminished. Thus, A. niger endophytic fungi could be a valuable source of antibacterial and anti-Toxoplasma compounds.

Inhibitory Effect of Hemiscorpius lepturus Scorpion Venom Fractions on Tachyzoites of Toxoplasma gondii.

Background: The present study determined the effect of the fractions obtained from Hemiscorpius lepturus scorpion venom on the tachyzoite of Toxoplasma gondii. Methods: The fractions of dried venom of He. lepturus scorpion of Khuzestan Province, southern Iran in 2019 were isolated through gel filtration chromatography, and then tachyzoites were exposed to fractions of venom at different concentrations. Trypan blue counting and MTT were applied to assay tachyzoite viability, and the inhibition of the cellular growth of fractions in Vero cells was evaluated. Results: The maximum effect on tachyzoite was observed in fraction 5 of venom. To further separate the protein, fraction 5 was used in high-performance liquid chromatography assay to purify its proteins. Based on the results of HPLC of fraction 5, among which the second peak, a peptide with <10 KDa representing a more potent effect in eliminating the tachyzoite of T. gondii. Conclusion: The scorpion venom-purified fractions possess anti-parasitic activity against the tachyzoite of T. gondii and can be used in parasite-controlling studies.

Potential effects of alpha-pinene, a monoterpene commonly found in essential oils against Toxoplasma gondii infection; an in vitro and in vivo study.

This survey designed to assess the in vitro and in vivo activity of α-pinene, a monoterpene commonly originated in essential oils on Toxoplasma gondii. The in vitro effect of various concentration of α-pinene against tachyzoites of T. gondii Rh strain was assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The activity of α-pinene on the stimulation of apoptosis in tachyzoites of T. gondii was also examined using the caspase 3 colorimetric activity assay. In vivo assay, mice were orally received α-pinene at 2 and 4 mg/kg/day for 14 days, then, pre-treated mice were daily tested and the rate of death was recorded. α-pinene meaningfully declined (p < 0.001) the tachyzoites viability with the IC50 value of 23.3 µg/mL. α-pinene induced the apoptosis through increasing the caspase-3 activity by 35.6%. Oral treatment with α-pinene significantly (p < 0.01) improved the survival rate infected mice with by 9th day. α-pinene + atovauone (50 mg/kg) significantly (p < 0.01) improved the survival rate infected mice up to 11 days compared with the control groups. α-pinene especially in combined atovaquone at 50 mg/kg for 2 weeks meaningfully (p < 0.05) declined oxidative stress. We found the promising in vitro anti-Toxoplasma effects of α-pinene on T. gondii RH strain. In addition, we found that α-pinene therapy particularly along with the reference drug declined the mortality rate of infected mice. Although, we just confirmed the stimulation of apoptosis and anti-inflammatory effects as the main anti-Toxoplasma mechanisms of α-pinene; however, more surveys concerning the accurate mechanisms, toxicity, and efficacy on other T. gondii strains are required to confirm these results.

Viability of Toxoplasma gondii tachyzoites in different conditions for parasite transportation.

Background and Aim: Toxoplasma gondii tachyzoite is the infective stage that causes acute infection, leading to severe toxoplasmosis. The tachyzoite stage has been extensively used for several inoculation purposes, including antigen production, immunological studies, nutrition mechanisms, and in vitro drug trials. The use of fresh tachyzoites is required for inoculation in either in vitro or in vivo studies. However, there is a lack of information on preserving live tachyzoites during transportation from laboratories to inoculation sites. Therefore, this study aimed to validate suitable preservative conditions for maintaining live parasites by determining the survival and viability of T. gondii tachyzoites on the basis of different media, temperatures, and incubation times. Materials and Methods: The free live T. gondii tachyzoites were evaluated on their viability when maintained in different media without 5% Carbon dioxide (CO2). The purified tachyzoites of the RH and PLK strains were individually suspended in normal saline (NS), phosphate-buffered saline (PBS), minimum essential medium (MEM), and MEM with 10% fetal bovine serum (MEM-FBS) and incubated for 6 h at ice-cold (IC; 3-9°C) and room temperature (RT; 25°C). Parasite survival was measured at the 0, 1st, 2nd, 3rd, 4th, 5th, and 6th h post-incubation using the trypan blue exclusion test. Results: The viability was in the range of 85.0%-91.0% for IC using NS and 81.0%-85.1% (IC) and 75.3%-77.5% (RT) using PBS. The viability was approximately 75.0%-83.0% (IC) and 70.0%-79.0% (RT) using MEM and MEM-FBS. There was a significant difference in the viability between the seven periods on the basis of one-way repeated Analysis of variance and Friedman analyses. Parasite survival slightly reduced (20.0%-30.0%) in NS and MEM-FBS at both temperatures during incubation. Notably, PBS could not support tachyzoite viability after 3 h post-incubation. Conclusion: NS was a suitable preservative for maintaining purified T. gondii tachyzoites during transportation at IC and RT without 5% CO2 supplementation. This could be a valuable medium for parasite transportation, especially when there is a large distance between the laboratory and inoculation site.

Synthesis and anti-Toxoplasma activity of indole-triazole compounds on tachyzoites of RH strain.

BACKGROUND: Conventional treatment for toxoplasmosis have severe side effects and the inability to completely eradicate the disease. Therefore, the acquisition of new anti-Toxoplasma drugs has always been of interest among researchers. In the present study, we prepare a new indole-triazole derivatives and evaluated their potential anti-parasitic activity against tachyzoites of Toxoplasma RH strain. MATERIALS AND METHODS: In this study, after synthesis of the two new compounds of indole-triazole, the effect of their different concentrations (2-1024 µg/ml) were determined on Toxoplasma tachyzoites using flow cytometry. Furthermore, tachyzoites were exposed to different concentrations of compounds (4, 16, 64, 265, 1024 µg/ml) for 1.5 h and their infectivity were evaluated in BALB/c mice. RESULTS: The flow cytometry results indicated the benzyl derivative of indole-triazole in various concentrations had a lethal effect on tachyzoites between 11.93% and 89.66%, while the naphthalene derivative had a lethality of 26.63%-66.82%. The infectivity analysis showed that the survival time of mice at concentrations of 1024 µg/ml and 512 µg/ml of benzyl derivatives was significantly increased (P = 0.008 and P = 0.016, respectively), compared to that in the negative control group. Furthermore, survival time of mice was statistically significant at the concentration of 1024 µg/ml for naphthyl derivative (P = 0.012). CONCLUSION: Findings of the current study suggested indole triazole compounds, based on their structure and enzymes targeting, have a considerable effect on tachyzoites of T. gondii RH strain and can be considered as a new anti-Toxoplasma agent.