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Aims: The study was aimed to understand the depuration process of Cryptosporidium parvum and Toxoplasma gondii oocysts by zebra mussel (Dreissena polymorpha), to consider the use of the zebra mussel as a bioremediation tool. Materials and methods: Two experiments were performed: (i) individual exposure of mussel to investigate oocyst transfers between bivalves and water and (ii) in vivo exposure to assess the ability of the zebra mussel to degrade oocysts. Results: (i) Our results highlighted a transfer of oocysts from the mussels to the water after 3 and 7 days of depuration; however, some oocysts were still bioaccumulated in mussel tissue. (ii) Between 7 days of exposure at 1000 or 10 000 oocysts/mussel/day and 7 days of depuration, the number of bioaccumulated oocysts did not vary but the number of infectious oocysts decreased. Conclusion: Results show that D. polymorpha can release oocysts in water via (pseudo)faeces in depuration period. Oocysts remain bioaccumulated and infectious oocyst number decreases during the depuration period in zebra mussel tissues. Results suggest a degradation of bioaccumulated C. parvum and T. gondii oocysts. Significance and Impact of the Study: This study highlighted the potential use of D. polymorpha as a bioremediation tool to mitigate of protozoan contamination in water resources. [ABSTRACT FROM AUTHOR]

Toxoplasmosis is a worldwide parasitosis that is generally benign. The infestation may pose a risk to immunocompromized patients and to fetuses when pregnant women have recently seroconverted. Current treatments have numerous side effects and chemoresistance is emerging, hence the need to find new anti-Toxoplasma gondii substances. This study focuses on the antiparasitic potential of lupane-type pentacyclic triterpenes isolated from the bark of black alder (Alnus glutinosa), as well as the hypothesis of their macromolecular target by an original method of reverse docking. Among the isolated triterpenes, betulone was the most active compound with an IC50 of 2.7 ± 1.2 μM, a CC50 greater than 80 μM, and a selectivity index of over 29.6. An additional study of the anti-T. gondii potential of commercially available compounds (betulonic acid methyl ester and betulonic acid) showed the important role of the C3 ketone function and the C28 oxidation level on the lupane-type triterpene in the antiparasitic activity since their IC50 and CC50 were similar to that of betulone. Finally, the most active compounds were subjected to the AMIDE reverse docking workflow. A dataset of 87 T. gondii proteins from the Protein Data Bank was created. It identified calcium-dependent protein kinase CDPK3 as the most likely target of betulin derivatives.

Consumption of raw or undercooked meat containing Toxoplasma gondii tissue cysts is one of the main sources of infection for humans worldwide. Among the various species intended for human consumption, sheep appear to be a high risk for human infection. The present study focused on the detailed anatomical distribution of Toxoplasma gondii in naturally and experimentally infected lambs using fresh and frozen samples of various pieces of meat, from a public health perspective. The first objective was to rank the edible parts intended for human consumption according to the detectable parasite burden by real-time PCR targeting the 529-bp repeated element. The second objective was to evaluate the impact of freezing by comparing the detection efficiency of the quantitative PCR between fresh and frozen tissues, as imports of lamb carcasses/cuts may arrive frozen or chilled. The highest estimated parasite loads were observed in skeletal muscles, and more particularly in edible portions such as quadriceps femoris muscle, intercostal muscles, deltoid muscle and diaphragm, with a significant difference in detectable parasite burden between fresh and frozen samples (p

Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii are protozoan parasites that have been highlighted as emerging foodborne pathogens by the Food and Agriculture Organization of the United Nations and the World Health Organization. According to the European Food Safety Authority, 4786 foodborne and waterborne outbreaks were reported in Europe in 2016, of which 0.4% were attributed to parasites including Cryptosporidium, Giardia and Trichinella. Until 2016, no standardized methods were available to detect Giardia, Cryptosporidium and Toxoplasma (oo)cysts in food. Therefore, no regulation exists regarding these biohazards. Nevertheless, considering their low infective dose, ingestion of foodstuffs contaminated by low quantities of these three parasites can lead to human infection. To evaluate the risk of protozoan parasites in food, efforts must be made towards exposure assessment to estimate the contamination along the food chain, from raw products to consumers. This requires determining: (i) the occurrence of infective protozoan (oo)cysts in foods, and (ii) the efficacy of control measures to eliminate this contamination. In order to conduct such assessments, methods for identification of viable (i.e. live) and infective parasites are required. This review describes the methods currently available to evaluate infectivity and viability of G. duodenalis cysts, Cryptosporidium spp. and T. gondii oocysts, and their potential for application in exposure assessment to determine the presence of the infective protozoa and/or to characterize the efficacy of control measures. Advantages and limits of each method are highlighted and an analytical strategy is proposed to assess exposure to these protozoa.

Metallopeptidases are a family of proteins with domains that remain highly conserved throughout evolution. These hydrolases require divalent metal cation(s) to activate the water molecule in order to carry out their catalytic action on peptide bonds by nucleophilic attack. Metallopeptidases from parasitic protozoa, including Toxoplasma, are investigated because of their crucial role in parasite biology. In the present study, we screened the T. gondii database using PFAM motifs specific for metallopeptidases in association with the MEROPS peptidase Database (release 10.0). In all, 49 genes encoding proteins with metallopeptidase signatures were identified in the Toxoplasma genome. An Interpro Search enabled us to uncover their domain/motif organization, and orthologs with the highest similarity by BLAST were used for annotation. These 49 Toxoplasma metallopeptidases clustered into 15 families described in the MEROPS database. Experimental expression analysis of their genes in the tachyzoite stage revealed transcription for all genes studied. Further research on the role of these peptidases should increase our knowledge of basic Toxoplasma biology and provide opportunities to identify novel therapeutic targets. This type of study would also open a path towards the comparative biology of apicomplexans.

Oocysts of the protozoan Toxoplasma gondii are found in felid feces and can be washed into coastal waters, where they persist for months, attaching to algae and accumulating in invertebrates. We used wild bivalves to assess contamination of coastal waters of the Kerguelen and Galapagos archipelagos by this zoonotic parasite. Additionally, we leveraged the contrasting situations of these archipelagos to identify some potential drivers of contamination. In the Galapagos, with a cat density reaching 142 per km 2 , 15.38% of the sampled oysters (Saccostrea palmula) tested positive for T. gondii by quantitative real-time PCR (qPCR) (n = 260), and positive samples were found in all eight sampling sites. In Kerguelen, with 1-3 cats per km 2 , 40.83% of 120 tested mussels (Mytilus edulis platensis) were positive, and positive samples were found in four out of the five sampling sites. These findings provide evidence of T. gondii contamination in the coastal waters of these archipelagos. Furthermore, T. gondii-positive bivalves were found on islands located 20 km away (Galapagos) and 5 km away (Kerguelen) from the nearest cat population, indicating that T. gondii oocysts can disperse through waterborne mechanisms over several kilometers from their initial deposition site. In the Galapagos, where runoff is infrequent and all sites are exposed to currents, the prevalence of qPCR-positive bivalves did not show significant variations between sites (p = 0.107). In Kerguelen where runoff is frequent and site exposure variable, the prevalence varied significantly (p < 0.001). The detection of T. gondii in Kerguelen mussels was significantly correlated with the site exposure to currents (odds ratio (OR) 60.2, p < 0.001) and the on-site density of giant kelp forests (OR 2.624, p < 0.001). This suggests that bivalves can be contaminated not only by oocysts transported by currents but also by consuming marine aggregates containing oocysts that tend to form in kelp forests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The protozoan parasites Cryptosporidium parvum , Cyclospora cayetanensis , and Toxoplasma gondii are major causes of waterborne and foodborne diseases worldwide. The assessment of their removal or inactivation during water treatment and food processing remains challenging, partly because research on these parasites is hindered by various economical, ethical, methodological, and biological constraints. To address public health concerns and gain new knowledge, researchers are increasingly seeking alternatives to the use of such pathogenic parasites. Over the past few decades, several non-pathogenic microorganisms and manufactured microparticles have been evaluated as potential surrogates of waterborne and foodborne protozoan parasites. Here, we review the surrogates that have been reported for C. parvum , C. cayetanensis , and T. gondii oocysts, and discuss their use and relevance to assess the transport, removal, and inactivation of these parasites in food and water matrices. Biological surrogates including non-human pathogenic Eimeria parasites, microorganisms found in water sources (anaerobic and aerobic spore-forming bacteria, algae), and non-biological surrogates (i.e. manufactured microparticles) have been identified. We emphasize that such surrogates have to be carefully selected and implemented depending on the parasite and the targeted application. Eimeria oocysts appear as promising surrogates to investigate in the future the pathogenic coccidian parasites C. cayetanensis and T. gondii that are the most challenging to work with., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

The association between the parasitic illnesses and the consumption of contaminated water has been largely reported. However, there is still a lack of studies investigating the extent of parasitic contamination in water in Morocco. This is the first study in Morocco that aimed at assessing the presence of protozoan parasites, namely Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii, in drinking water consumed in the region of Marrakech. Samples processing was performed by membrane filtration and qPCR detection. A total of 104 drinking water samples (tap water, well, and spring waters) was collected between 2016 and 2020. The analysis revealed an overall protozoa contamination rate of 67.3% (70/104), of which 35 samples were positive for Giardia duodenalis, 18 for Toxoplasma gondii, and 17 for both parasites, whereas no sample was positive for Cryptosporidium spp. This first study showed that drinking water in the region of Marrakech contained parasites which could represent a risk for consumers. For a better understanding and estimation of the risk encountered by local inhabitants, further studies concerned with (oo)cyst viability, infectivity, and genotype identification need to be performed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Purpose: The aim of this study was to assess the presence of T. gondii, Cryptosporidium spp. oocysts, and G. duodenalis cysts, in three leafy greens (coriander, lettuce, and parsley) commonly consumed raw. Despite the recognition of the association between the parasitic illnesses and the consumption of contaminated food, there is still a lack of studies investigating the occurrence of parasitic contamination in food matrices., Methods: A total of 152 leafy green samples were collected in Marrakech from April 2018 to October 2019. Parasites were eluted and concentrated before detection of their DNA by real-time qPCR., Results: The analysis revealed an overall rate of contamination of 32.2% (49/152), with 29.6% (45/152) positive for T. gondii and 2.6% (4/152) for G. duodenalis, while none was positive for Cryptosporidium spp., Conclusion: The results showed that humans can be exposed to protozoan parasites through vegetables consumption. Further investigations can be performed to acquire new epidemiological data to assess the public health impact of these protozoan diseases in Morocco., (© 2021. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Toxoplasmosis is a worldwide parasitosis that affects one-third of the population. People at risk, such as immunocompromised patients (AIDS, chemotherapy treatment) or fetuses (maternal-fetal transmission) can develop severe forms of the disease. The antiparasitic activity of extracts of different polarities ( n -heptane, MeOH, MeOH/H 2 O) of 10 tree species endemic to temperate regions was investigated against Toxoplasma gondii infection in vitro . Our results showed that the n -heptane extract of the black alder ( Alnus glutinosa ) exhibited a significant antiparasitic activity without any cytotoxicity at the tested concentrations, with an IC 50 of up to 25.08 μg/mL and a selectivity index higher than 3.99. The chemical profiling of this extract revealed triterpenes as major constituents. The ability of commercially available triterpene (betulin, betulinic acid, and betulone) to inhibit the growth of T. gondii was evaluated and showed growth inhibition rates of 44%, 49%, and 99% at 10 μM, respectively.

Aims: The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii are identified as public health priorities and are present in a wide variety of environments including the marine ecosystem. The objective of this study was to demonstrate that the marine bivalve blue mussel (Mytilus edulis) can be used as a tool to monitor the contamination of marine waters by the three protozoa over time., Methods and Results: In order to achieve a proof of concept, mussels were exposed to three concentrations of G. duodenalis cysts and Cryptosporidium parvum/T. gondii oocysts for 21 days, followed by 21 days of depuration in clear water. Then, natural contamination by these protozoa was sought for in wild marine blue mussels along the northwest coast of France to validate their relevance as bioindicators in the field. Our results highlighted that: (a) blue mussels bioaccumulated the parasites for 21 days, according to the conditions of exposure, and parasites could still be detected during the depuration period (until 21 days); (b) the percentage of protozoa-positive M. edulis varied under the degree of protozoan contamination in water; (c) mussel samples from eight out of nine in situ sites were positive for at least one of the protozoa., Conclusions: The blue mussel M. edulis can bioaccumulate protozoan parasites over long time periods, according to the degree of contamination of waters they are inhabiting, and can highlight recent but also past contaminations (at least 21 days)., Significance and Impact of the Study: Mytilus edulis is a relevant bioaccumulators of protozoan (oo)cysts in laboratory and field conditions, hence its potential use for monitoring parasite contamination in marine waters., (© 2021 The Society for Applied Microbiology.)

Molecular docking is widely used in computed drug discovery and biological target identification, but getting fast results can be tedious and often requires supercomputing solutions. AMIDE stands for AutoMated Inverse Docking Engine. It was initially developed in 2014 to perform inverse docking on High Performance Computing. AMIDE version 2 brings substantial speed-up improvement by using AutoDock-GPU and by pulling a total revision of programming workflow, leading to better performances, easier use, bug corrections, parallelization improvements and PC/HPC compatibility. In addition to inverse docking, AMIDE is now an optimized tool capable of high throughput inverse screening. For instance, AMIDE version 2 allows acceleration of the docking up to 12.4 times for 100 runs of AutoDock compared to version 1, without significant changes in docking poses. The reverse docking of a ligand on 87 proteins takes only 23 min on 1 GPU (Graphics Processing Unit), while version 1 required 300 cores to reach the same execution time. Moreover, we have shown an exponential acceleration of the computation time as a function of the number of GPUs used, allowing a significant reduction of the duration of the inverse docking process on large datasets.

Background: Protozoan parasites such as Toxoplasma gondii, Giardia duodenalis , and Cryptosporidium spp. , can be transmitted to humans via accidental consumption of contaminated water, fresh produce and foodstuffs. There is a lack of epidemiological data about these pathogens in Morocco. Hence the aim of this study, which is the determination of their prevalence in some leafy greens and root vegetables sold in Marrakech., Methods: A total of 132 vegetable samples including carrot, coriander, lettuce, parsley and radish were purchased monthly from three different markets in Marrakech from March 2017 to January 2018, pre-treated and subjected to microscopic and molecular analyses., Results: Of the 132 samples of vegetables analyzed by qPCR, the overall rate of protozoan was 21.21% (28/132); 22 samples were found to be contaminated with T. gondii , 6 with G. duodenalis , and none was positive for C. parvum / hominis . Whereas, modified Ziehl-Neelsen staining allowed the detection of Cryptosporidium spp . in 3% (4/132) of examined samples., Conclusion: This survey on the presence of protozoan parasites in fresh vegetables revealed that vegetables sold in Marrakech are contaminated by these protozoan parasites, as it showed that leafy green vegetables were more susceptible for parasitic contamination than root ones., (© 2020 Berrouch S et al.)

Background: Toxoplasma gondii is a parasite of worldwide importance but its burden in indigenous communities remains unclear. In French Guiana, atypical strains of T. gondii originating from a complex rainforest cycle involving wild felids have been linked to severe infections in humans. These cases of Amazonian toxoplasmosis are sporadic and outbreaks are rarely described. We report on the investigation of an outbreak of acute toxoplasmosis in a remote Amerindian village. We discuss the causes and consequences of this emergence. Methods: In May 2017, during the rainy season and following an episode of flooding, four simultaneous cases of acute toxoplasmosis were serologically confirmed in two families living the village. Other non-diagnosed cases were then actively screened by a medical team along with epidemiological investigations. Inhabitants from nine households were tested for T. gondii antibodies and parasite DNA by PCR when appropriate. Samples of water, cat feces and cat rectal swabs, soil, and meat were tested for T. gondii DNA by PCR. Positive PCR samples with sufficient DNA amounts were genotyped using 15 microsatellite markers. Results: Between early May and early July 2017, out of 54 tested inhabitants, 20 cases were serologically confirmed. A fetus infected at gestational week 10 died but other cases were mild. Four patients tested positive for parasite DNA and two identical strains belonging to an atypical genotype could be isolated from unrelated patients. While domestic cats had recently appeared in the vicinity, most families drank water from unsafe sources. Parasite DNA was recovered from one water sample and nine soil samples. Three meat samples tested positive, including wild and industrial meat. Conclusions: The emergence of toxoplasmosis in such a community living in close contact with the Amazon rainforest is probably multifactorial. Sedentary settlements have been built in the last few decades without providing safe water sources, increasing the risk of parasite circulation in cases of dangerous new habits such as cat domestication. Public health actions should be implemented in these communities such as safe water supply, health recommendations, and epidemiological surveillance of acute toxoplasmosis. A "One Health" strategy of research involving medical anthropology, veterinary medicine, and public health needs to be pursued for a better understanding of the transmission routes and the emergence of this zoonosis., (Copyright © 2020 Blaizot, Nabet, Laghoe, Faivre, Escotte-Binet, Djossou, Mosnier, Henaff, Blanchet, Mercier, Dardé, Villena and Demar.)

One of the ways of human parasitic infection is the accidental ingestion of vegetables contaminated with parasites, which represents a major human health hazard. This non-exhaustive review aims to evaluate studies carried out on five types of vegetables (lettuce, parsley, coriander, carrot and radish) since 2000, particularly the methods used for recovery, concentration, detection and identification of protozoan parasites such as Toxoplasma gondii, Giardia duodenalis and Cryptosporidium spp., and the results of each work. Various studies have determined the presence of pathogenic parasites in fresh vegetables with different rates; this variation in rate depends particularly on the detection method used which is related to each parasite and each vegetable type. The variation in parasitic prevalence in food could be due to different factors such as the geographical location, the size of analysed samples and the methods used for parasite detection.

Toxoplasma gondii , belonging to the Apicomplexa phylum, is a cosmopolitan protozoan parasite that affects at least 30% of the world's population. In West Africa, the leaves and bark of the tree species Anogeissus leiocarpa (DC.) Guill. & Perr. are used against zoonosis in traditional medicine and play a key role in controlling diseases induced by Apicomplexans such as malaria. In this study, extracts, fractions, and pure compounds obtained from an ethanol extract of the bark of A. leiocarpa were evaluated against T. gondii infection in vitro and in vivo . The crude bark extract showed significant activity on tachyzoites from the T. gondii RH strain (IC 50  = 59.30 µg/mL). The crude bark extract without tannins and pure trachelosperogenin E purified by centrifugal partition chromatography showed the highest activity (IC 50 s = 12.83 and 26.63 µg/mL, respectively) with satisfying selectivity indexes of 9.61 and 9.75, respectively. The crude bark extract without tannins and pure trachelosperogenin E were able to significantly inhibit host cell invasion by the parasite in vitro , while the crude bark extract without tannins was able to increase mice survival in our murine model of chronic toxoplasmosis. These results provide new biological data for natural compounds that could enhance the current panoply of treatments against toxoplasmosis., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Documenting the extent of soil contamination by Toxoplasma gondii oocysts is a key issue to prevent the worldwide infection caused by this protozoan. Our aim was to improve the practicability and sensitivity of a low-cost method to detect T. gondii DNA in soil samples developed a few years ago. Various parameters of the reference protocol were modified to determine their effect on the detection of T. gondii DNA in soil samples ("natural soil" and "sand") spiked with oocysts. We tested i) filtration using stomacher bags, ii) Tween 80, Tween 20, SDS and Triton X100 as dispersion solutions, iii) sucrose solution, zinc chloride solution, Optiprep and Percoll as density gradients, iv) freeze/thaw versus mechanical grinding as lysis methods, and v) Qiagen versus Fastprep as extraction kits The optimized protocol is quicker and easier to use than the previous one, and includes the following items: 0.1% Tween80/PBS for dispersion, sucrose solution for flotation, mechanical grinding, and FastDNA spin kit for extraction. It accurately detects T. gondii DNA in both fresh and frozen soil samples and displays a detection limit below 1 oocyst/g of fresh soil., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Toxoplasma gondii is a ubiquitous foodborne protozoan that can infect humans at low dose and displays different prevalences among countries in the world. Ingestion of food or water contaminated with small amounts of T. gondii oocysts may result in human infection. However, there are no regulations for monitoring oocysts in food, mainly because of a lack of standardized methods to detect them. The objectives of this study were (i) to develop a reliable method, applicable in biomonitoring, for the rapid detection of infectious oocysts by cell culture of their sporocysts combined with quantitative PCR (sporocyst-CC-qPCR) and (ii) to adapt this method to blue and zebra mussels experimentally contaminated by oocysts with the objective to use these organisms as sentinels of aquatic environments. Combining mechanical treatment and bead beating leads to the release of 84% ± 14% of free sporocysts. The sporocyst-CC-qPCR detected fewer than ten infectious oocysts in water within 4 days (1 day of contact and 3 days of cell culture) compared to detection after 4 weeks by mouse bioassay. For both mussel matrices, oocysts were prepurified using a 30% Percoll gradient and treated with sodium hypochlorite before cell culture of their sporocysts. This assay was able to detect as few as ten infective oocysts. This sporocyst-based CC-qPCR appears to be a good alternative to mouse bioassay for monitoring infectious T. gondii oocysts directly in water and also using biological sentinel mussel species. This method offers a new perspective to assess the environmental risk for human health associated with this parasite. IMPORTANCE The ubiquitous protozoan Toxoplasma gondii is the subject of renewed interest due to the spread of oocysts in water and food causing endemic and epidemic outbreaks of toxoplasmosis in humans and animals worldwide. Displaying a sensitivity close to animal models, cell culture represents a real alternative to assess the infectivity of oocysts in water and in biological sentinel mussels. This method opens interesting perspectives for evaluating human exposure to infectious T. gondii oocysts in the environment, where oocyst amounts are considered to be very small., (Copyright © 2019 American Society for Microbiology.)

Information on the viability of Toxoplasma gondii oocysts is crucial to establish the public health significance of this environmental transmission stage that can contaminate water and foods. Interest for molecular-based methods to assess viability is growing and the aim of our study was to assess, for the first time, a propidium monoazide (PMA)-qPCR approach to determine the viability of T. gondii oocysts. Untreated and heat-killed (99 °C, 5 min) oocysts were incubated with PMA, a photoreactive DNA binding dye, and analyzed by confocal microscopy and flow cytometry to characterize oocysts' dye permeability. Different PMA concentrations (50 to 150 μM), incubation temperatures (22, 37, and 45 °C), amplicon length, selected targeted gene, and dyes (PMA, PMAxx™) were evaluated to define optimal conditions to discriminate specifically viable oocysts by PMA-qPCR. In theory, PMA binding to DNA would inhibit PCR amplification in dead but not in viable oocysts. Incubation at 22 °C with 100 μM PMA coupled to qPCR targeting a 123-bp sequence of the 529-bp repeat element allowed the distinction between viable and heated oocysts. However, the reduction of viability following heating of oocysts at high temperature was slight and, contrarily to reverse transcriptase-qPCR, the qPCR signal was not totally suppressed in heated suspensions. Therefore, PMA-qPCR is able to assess the impact of heating on T. gondii oocysts' viability but underestimates the efficacy of this treatment. The relevance of this technique to evaluate the efficacy of other inactivation processes and assess exposure of humans to this pathogen requires further investigations.

Toxoplasma gondii is a cosmopolitan protozoan parasite which affects approximately 30% of the population worldwide. The drugs currently used against toxoplasmosis are few in number and show several limitations, such as drug intolerance, poor bioavailability, or drug resistance mechanism developed by the parasite. Thus, it is important to find new compounds able to inhibit parasite invasion or proliferation. In this study, the 400 compounds of the open-access Pathogen Box, provided by the Medicines for Malaria Venture (MMV) foundation, were screened for their anti- Toxoplasma gondii activity. A preliminary in vitro screening performed over 72 h by an enzyme-linked immunosorbent assay (ELISA) revealed 15 interesting compounds that were effective against T. gondii at 1 μM. Their cytotoxicity was estimated on Vero cells, and their 50% inhibitory concentrations (IC 50 ) were further calculated. As a result, eight anti- Toxoplasma gondii compounds with an IC 50 of less than 2 μM and a selectivity index (SI) value of greater than 4 were identified. The most active was MMV675968, showing an IC 50 of 0.02 μM and a selectivity index value equal to 275. Two other compounds, MMV689480 and MMV687807, also showed a good activity against T. gondii , with IC 50 s of 0.10 μM (SI of 86.6) and 0.15 μM (SI of 11.3), respectively. Structure-activity relationships for the eight selected compounds also were discussed on the basis of fingerprinting similarity measurements using the Tanimoto method. The anti- Toxoplasma gondii compounds highlighted here represent potential candidates for the development of new drugs that could be used against toxoplasmosis., (Copyright © 2018 Spalenka et al.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Treatment options for toxoplasmosis in humans are generally limited to the use of sulfonamide and/or pyrimethamine-based compounds. However, there is increasing evidence for clinical therapy failures in patients suggesting the existence of drug resistance in these classes of drug. In vitro resistance to sulfadiazine has been detected in three strains of Toxoplasma gondii isolated from clinical cases. In order to begin to understand the mechanisms of resistance, we undertook a difference-gel electrophoresis (DIGE) approach combined with mass spectrometry to identify proteins that are differentially expressed in sulfadiazine-resistance strains of the parasite. Naturally resistant strains TgA 103001 (Type I), TgH 32006 (Type II) and TgH 32045 (Type II variant) were compared to sensitive strains RH (Type I) and ME-49 (Type II) using DIGE and the modulated proteins analyzed using LC-MS/MS. In total, 68 differentially expressed protein spots were analyzed by mass spectrometer and 31 unique proteins, including four hypothetical proteins, were identified. Among the differentially expressed proteins, 44% were over-expressed in resistant strains and 56% were over-expressed in sensitive strains. The virulence-associated rhoptry protein, ROP2A, was found in greater abundance in both naturally resistant Type II strains TgH 32006 and TgH 32045 compared to the sensitive strain ME-49. Enolase 2 and IMC1 were found to be in greater abundance in sensitive strains RH and ME-49, and MIC2 was found to be more abundant in the sensitive strain ME-49. Proteins regulation of ROP2, MIC2, ENO2, IMC1 and GRA7 were confirmed by Western blot analysis. In addition, gene expression patterns of ROP2, MIC2, ENO2 and IMC1 were analyzed with qRT-PCR. This study provides the first proteomics insights into sulfadiazine resistance in T. gondii resistant strains isolated from clinical cases.

Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-R(SDZ) and ME-49-R(SDZ), by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied., (© C. Doliwa et al., published by EDP Sciences, 2013.)

Monitoring of Toxoplasma infection in animals destined for human consumption is a great challenge for human toxoplasmosis prevention. This study aimed to compare results obtained from a naturally infected population of sheep using different tests and targeting an original matrix: meat samples and muscle fluids collected at the slaughterhouse. A commercial ELISA test was performed on diaphragm fluids from 419 ovine carcasses collected at the slaughterhouse. A MAT (modified agglutination test) was performed on heart fluids obtained from the same animals. In addition, all hearts were bioassayed in mice. Serological test agreement, the relative sensitivity of ELISA MAT and mouse bioassay as well as a correlation between titres and parasite isolation probability were statistically evaluated. The overall agreement (kappa coefficient=0.64) of ELISA on diaphragm fluids and MAT on heart fluids is substantial and subsequently both tests can be used for epidemiological studies. Relative sensitivity was higher for MAT performed on cardiac fluids (90%) than ELISA on diaphragm fluid (61%). For both serological tests, relative sensitivity is lower in lambs younger than 12 months. Relative sensitivity of mouse inoculation was 42%. A significant correlation was obtained between increasing MAT titres and probability to isolate live parasite from the heart. When the fluid titre was higher than 1:16, parasites were isolated in 65% of cases. When it was lower, isolation failed in 95% of the cases. According to our results, cardiac fluids appear to be a relevant matrix for toxoplasmosis survey in meat., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Consumption of sheep meat presents a risk of human contamination by Toxoplasma gondii. A nationwide study was conducted in France to evaluate the prevalence of Toxoplasma in fresh ovine meat. A sampling procedure was established to guarantee the representativity of consumption. As is the case for meat consumed, half of the samples were from France and half were imported from other countries. Animals were selected according to their age, as lamb (<12months) represents 90% of the meat consumed. Available data for French samples allowed the selection of 16 districts distributed in seven areas according to their density of production. Diaphragms and hearts from 433 sheep were collected. Diaphragms were collected from 398 imported carcasses. Fluids from hearts and diaphragms were tested serologically. All hearts were bioassayed in mice and parasite isolates were genotyped using PCR-restriction fragment length polymorphism and microsatellite markers. Prevalence estimates were calculated, taking into account uneven distribution of production and age. For French meat, the effect of area, age and their interactions was evaluated. The overall estimate of Toxoplasma seroprevalence was 17.7% (11.6-31.5%) for lambs and 89% (73.5-100%) for adults (P<0.0001). No significant difference was observed between imported and French meat. In France, seroprevalence in lambs showed an increasing North-western to Southern gradient. The proportion of French carcasses carrying live parasites according to bioassay results was estimated at 5.4% (3-7.5%) (45 genotype II; one genotype III). This study offers an accurate drawing of the toxoplasmosis pattern amongst sheep consumed in France and a model for a zoonosis hazard control survey., (2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The ATP-binding cassette (ABC) superfamily is one of the largest protein families with representatives in all kingdoms of life. Members of this superfamily are involved in a wide variety of transport processes with substrates ranging from small ions to relatively large polypeptides and polysaccharides, but also in cellular processes such as DNA repair, translation or regulation of gene expression. For many years, the role of ABC proteins was mainly investigated for their implication in drug resistance. However, recent studies focused rather on their physiological functions for the parasite. In this review, we present an overview of ABC proteins in major protozoan parasites including Leishmania, Trypanosoma, Plasmodium, Toxoplasma, Cryptosporidium and Entamoeba species. We will also discuss the role of characterized ABC transporters in the biology of the parasite and in drug resistance.

ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present study reports on the characterization of a P-glycoprotein (Pgp), TgABC.B1, in the protozoan parasite Toxoplasma gondii. The protein encoded by the TgABC.B1 gene displays the typical (TMD-NBD)2 structural organization of the "full" ABC transporter and shows significant identity and similarity with two apicomplexan Pgps; Pgh1 in Plasmodium falciparum and CpABC3 in Cryptosporidium parvum. The TgABC.B1 gene is a single copy gene transcribed into a full-length mRNA of 4.3kb and expressed as a protein of approximately 150kDa, which cellular localization revealed a membrane-associated labelling in tachyzoites. The TgABC.B1 gene is constitutively expressed in the three major T. gondii genotypes but demonstrated a higher expression in virulent type I, at both transcriptional and translational levels. Further characterization of this Pgp-like protein will increase our knowledge of the membrane transport system in this parasite and could result in the identification of a new therapeutic target in Toxoplasma.