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Toxoplasma gondii , belonging to the Apicomplexa phylum, is a cosmopolitan protozoan parasite that affects at least 30% of the worldʼs 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 (IC50 = 59.30 µg/mL). The crude bark extract without tannins and pure trachelosperogenin E purified by centrifugal partition chromatography showed the highest activity (IC50 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. [ABSTRACT FROM AUTHOR]

Cheliff River is the most important permanent river of North-west Algeria which plays an essential role for irrigation and drinking water supply, but suffers from high anthropogenic pressure. Multi-dimensional and multi-factorial aspects of water pollution in Cheliff River and its tributary Mina River were evidenced during two contrasting climatic seasons. Urban discharges were identified as a major source of water organic pollution. The inputs of untreated sewages were characterized by maximum concentrations of tryptophan-like (5.7 µmol l−1) and tyrosine-like (9.8 µmol l−1) compounds during the dry period, confirming these amino acids as fingerprints of untreated urban discharges. The synchronous monitoring and assessment of physicochemical parameters, fluorescent organic compounds and metallic elements of Cheliff River waters highlighted its global contamination, mainly in its downstream part. Cheliff River was mainly affected by organic pollution with maximum COD (1536 mg O2l−1) and BOD5(12 mg O2l−1) during the wet period, and by metallic contamination with maximum Fe (287 ± 4.4 µg l−1) and Al (422.4 ± 9.4 µg l−1) during the dry period, exceeding the guideline limits. This chronic contamination was related to untreated domestic sewages, agricultural effluents and technical landfill center discharges in this area. Diffuse pollutions were also evidenced, which made the identification of different contamination sources complicated. Mina River was mostly affected by non-treated domestic sewages with maximum COD (3161 mg O2l−1) during the wet period and maximum BOD5(12 mg O2l−1) during the dry period, exceeding the guideline limits. The correlation Li/Sr showed the contribution of natural influence from saline soils of Mina valley, particularly during the dry period.

The protozoan parasites Cryptosporidium parvum, Cyclospora cayetanensis, and Toxoplasma gondiiare 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. gondiioocysts, 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 Eimeriaparasites, 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. Eimeriaoocysts appear as promising surrogates to investigate in the future the pathogenic coccidian parasites C. cayetanensisand T. gondiithat are the most challenging to work with.

Toxoplasma gondiiis an important zoonotic foodborne parasite. Meat of infected animals appears to be a major source of infection in Europe. Pork is the most consumed meat in France, with dry sausages well represented. The risk of transmission via consumption of processed pork products is largely unknown, mainly since processing will affect viability but may not entirely inactivate all T. gondiiparasites.

•A method based on membrane filtration and molecular detection was used to detect parasites in water.•This is the first study on the presence of protozoan parasites in drinking water in Morocco.•The overall parasitic contamination rate of drinking waters in the region of Marrakech was 67.3%.

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 gondiisubstances. 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 IC50of 2.7 ± 1.2 μM, a CC50greater than 80 μM, and a selectivity index of over 29.6. An additional study of the anti-T. gondiipotential 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 IC50and CC50were similar to that of betulone. Finally, the most active compounds were subjected to the AMIDE reverse docking workflow. A dataset of 87 T. gondiiproteins 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 gondiitissue 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 gondiiin 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< 0.0001) or natural and experimental infection (p< 0.0001). Thoracic and pelvic limbs (3278–1048 parasites/g muscle) were ranked at the top of the list. Toxoplasma gondiiDNA was detected in all the edible parts of lamb studied. These results suggest that lamb meat represents a risk for consumers. Further investigations are needed in order to confirm these differences in larger numbers of animals and in different breeds.

Toxoplasmosis is largely present in rural areas but its spatial distribution in this environment remains poorly known. In particular, it is unclear if areas of high density of cats, the only hosts excreting Toxoplasma gondii, constitute foci of high prevalence. To improve our understanding of the spatial distribution of T. gondii in rural areas, we performed a serological survey in rodents from two villages in France. We trapped 710 rodents including commensal rats and meadow or forest voles and mice. The presence of T. gondii was examined using PCR, mice inoculation and modified agglutination test for antibodies (MAT). We conducted multivariate and discriminant analyses to identify biological, ecological or spatial variables that could explain T. gondii serology in rodents. We then used a logistic regression to assess the relative influence of each explanatory variable. Overall seroprevalence was 4·1%. Commensal-rats were more infected (12·5%) than non-commensal species (3·7%). However, the major determinant of the risk of infection was the distance to the nearest farm (OR = 0·75 for 100 m), which explained the risk in all species or non-commensal species only. We contrast the role of species characteristics and that of the local environment, and discuss the risk of environmental contamination for humans. [ABSTRACT FROM AUTHOR]

Foodborne infections are of public health importance and deeply impact the global economy. Consumption of bivalve mollusks generates risk for humans because these filtering aquatic invertebrates often concentrate microbial pathogens from their environment. Among them, Giardia, Cryptosporidium, and Toxoplasmaare major parasites of humans and animals that may retain their infectivity in raw or undercooked mollusks. This review aims to detail current and future tools and methods for ascertaining the load and potential infectivity of these parasites in marine bivalve mollusks, including sampling strategies, parasite extraction procedures, and their characterization by using microscopy and/or molecular techniques. Method standardization should lead to better risk assessment of mollusks as a source of these major environmental parasitic pathogens and to the development of safety regulations, similar to those existing for bacterial and viral pathogens encountered in the same mollusk species.

We present a set of cosmological simulations with radiative transfer in order to model the reionization history of the universe from z = 18 down to z = 6. Galaxy formation and the associated star formation are followed self-consistently with gas and dark matter dynamics using the RAMSES code, while radiative transfer is performed as a post-processing step using a moment-based method with the M1 closure relation in the ATON code. The latter has been ported to a multiple Graphics Processing Unit (GPU) architecture using the CUDA language together with the MPI library, resulting in an overall acceleration that allows us to tackle radiative transfer problems at a significantly higher resolution than previously reported: 10243 + 2 levels of refinement for the hydrodynamic adaptive grid and 10243 for the radiative transfer Cartesian grid. We reach a typical acceleration factor close to 100x when compared to the CPU version, allowing us to perform 1/4 million time steps in less than 3000 GPU hr. We observe good convergence properties between our different resolution runs for various volume- and mass-averaged quantities such as neutral fraction, UV background, and Thomson optical depth, as long as the effects of finite resolution on the star formation history are properly taken into account. We also show that the neutral fraction depends on the total mass density, in a way close to the predictions of photoionization equilibrium, as long as the effect of self-shielding are included in the background radiation model. Although our simulation suite has reached unprecedented mass and spatial resolution, we still fail in reproducing the z [?] 6 constraints on the neutral fraction of hydrogen and the intensity of the UV background. In order to account for unresolved density fluctuations, we have modified our chemistry solver with a simple clumping factor model. Using our most spatially resolved simulation (12.5 Mpc h -1 with 10243 particles) to calibrate our subgrid model, we have resimulated our largest box (100 Mpc h -1 with 10243 particles) with the modified chemistry, successfully reproducing the observed level of neutral hydrogen in the spectra of high-redshift quasars. We however did not reproduce the average photoionization rate inferred from the same observations. We argue that this discrepancy could be partly explained by the fact that the average radiation intensity and the average neutral fraction depend on different regions of the gas density distribution, so that one quantity cannot be simply deduced from the other.

ABSTRACTThe evaluation of Toxoplasma gondiiisolates obtained from geographical environments other than Europe and North America revealed the existence of atypical strains that are not included in the three archetypal clonal lineages (lineages I, II, and III). GRA6and GRA7are polymorphic genes that have been used for the genotyping of Toxoplasma. The coding regions of GRA6and GRA7from 49 nonarchetypal strains were sequenced and compared with the sequences of type I, II, and III reference strains. Eighteen and 10 different amino acid sequences were found for GRA6and GRA7, respectively. The polymorphisms found between the different sequences were analyzed, with the objective of defining peptides to be used for the serotyping of Toxoplasmainfections. Two peptides specific for clonal lineages I and III (peptides GRA7I and GRA7III, respectively) were selected from the GRA7locus. Three peptides specific for some atypical strains (peptides Am6, Af6, and Am7) were selected from both the GRA6and the GRA7loci. Serum samples from humans infected with Toxoplasmastrains of known genotypes were serotyped with the selected peptides. Peptide GRA7III seems to be a good candidate for the serotyping of infections caused by type III strains. Peptide GRA7I had a very low sensitivity. Peptides Am6 and Af6 had low specificities, since they reacted with serum samples from patients infected with strains belonging to the three archetypal lineages. Although peptide Am7 was specific, it had low sensitivity.

ABSTRACTSulfadiazine, pyrimethamine, and atovaquone are widely used for the treatment of severe toxoplasmosis. Their in vitro activities have been almost exclusively demonstrated on laboratory strains belonging to genotype I. We determined the in vitro activities of these drugs against 17 strains of Toxoplasma gondiibelonging to various genotypes and examined the correlations among 50% inhibitory concentrations (IC50s), growth kinetics, strain genotypes, and mutations on drug target genes. Growth kinetics were determined in THP-1 cell cultures using real-time PCR. IC50s were determined in MRC-5 cell cultures using a T. gondii-specific enzyme-linked immunosorbent assay performed on cultures. Mutations in dihydrofolate reductase (DHFR), dihydropteroate synthase (DHPS), and cytochrome bgenes were determined by sequencing. Pyrimethamine IC50s ranged between 0.07 and 0.39 mg/liter, with no correlation with the strain genotype but a significant correlation with growth kinetics. Several mutations found on the DHFR gene were not linked to lower susceptibility. Atovaquone IC50s were in a narrow range of concentrations (mean, 0.06 ± 0.02 mg/liter); no mutation was found on the cytochrome bgene. IC50s for sulfadiazine ranged between 3 and 18.9 mg/liter for 13 strains and were >50 mg/liter for three strains. High IC50s were not correlated to strain genotypes or growth kinetics. A new mutation of the DHPS gene was demonstrated in one of these strains. In conclusion, we found variability in the susceptibilities of T. gondiistrains to pyrimethamine and atovaquone, with no evidence of drug resistance. A higher variability was found for sulfadiazine, with a possible resistance of three strains. No relationship was found between drug susceptibility and strain genotype.

Intravenous delivery of nls-lacZ retroviral vectors to the regenerating liver triggers a cytotoxic immune response directed against transduced hepatocytes. We sought to determine whether prior immunization with retroviral vectors impacted on adeno-associated virus (AAV)-mediated muscular expression of the same transgene. The first group of rats first received nls-lacZ retroviral vectors intravenously after a partial hepatectomy. Thirty days later they received AAV vectors intramuscularly in both legs. In the second group, animals received the same vectors in the opposite sequence (i.e., AAV first and retroviruses 20 days later). In the first group, immune response occurred after retrovirus delivery with appearance of anti-β-galactosidase antibodies and elimination of transduced hepatocytes. However, the immune response did not prevent sustained (9-month) β-galactosidase expression in AAV-injected muscles. In the second group, AAV injections did not induce immune response and resulted in β-galactosidase expression in myofibers. In this group, subsequent delivery of retroviral vectors triggered appearance of immune response and elimination of transduced hepatocytes. However, the immune response did not modify β-galactosidase expression in AAV-transduced myofibers for up to 9 months. These results demonstrate a differential susceptibility between retrovirally transduced liver and AAV-transduced muscles to immune response against the transgene product.

Gene transfer of reporter genes may trigger immune responses against the heterologous protein resulting in shortening of gene expression and inflammation. We generated transgenic rats expressing the lacZ gene under the control of the human immunodeficiency virus type 1 (HIV-1) long-terminal repeat (LTR) (HIV-lacZ) to obtain rats with undetectable transgene expression using histologic methods, thus avoiding interference with β-galactosidase (β-gal) expression from gene transfer, and displaying immune tolerance toward β-gal. LacZ transgenic mice with tolerance toward β-gal have already been used for gene transfer but rats constitute unique animal models with several advantages compared to mice. Two transgenic lines displayed low levels of β-gal mRNA in most organs tested, as detected only by reverse transcription-polymerase chain reaction (RT-PCR). The protein was undetectable by immunohistology and was only detected in the thymus and spleen using a sensitive enzyme-linked immunosorbent assay (ELISA). HIV-lacZ transgenic rats displayed immune tolerance to β-gal because immunization with β-gal resulted in markedly lower cellular and antibody responses compared to wild-type controls, whereas immunization with a nonrelated antigen, keyhole limpet hemocyanin (KLH), resulted in comparable immune responses. The usefulness of this model in gene transfer was tested using a retroviral vector, which does not elicit destructive immune responses against transduced cells. Retroviral-mediated nlslacZ gene transfer in the liver resulted in nuclear β-gal expression for longer than 12 months in HIV-lacZ transgenic rats, whereas wild-type controls showed nuclear β-gal expression for less than 1 month. After gene transfer of nlslacZ to the liver, antibodies, cytotoxic T lymphocytes (CTLs), and proliferation against β-gal were detected in wild-type controls but not in HIV-lacZ transgenic rats. In conclusion, HIV-lacZ transgenic rats displaying low β-gal expression and immune tolerance toward β-gal are a useful tool to analyze the spatial and temporal expression of the β-gal protein in gene transfer experiments using lacZ as a reporter gene.

Abstract. Toxoplasma gondii is an intracellular, obligate protozoan parasite that actively invades host cells. Phospholipases A2 (PLA2) are widely distributed enzymes that exist in many isoforms [secretory (sPLA2), cytosolic (cPLA2)] in monocytic and other human cells (pancreatic, kidney, etc) and also in parasites. We examined the effects of inhibitors of sPLA2 type II and cPLA2 on the invasion of human monocytic cells (THP1) by T. gondii (RH strain). We also measured sPLA2 type II and cPLA2 enzyme activities and their modulation by interferon γ (IFN γ) in extracts of host cells and parasite. Inhibition of both parasite and THP1 sPLA2 type II, and of parasite cPLA2 reduced the number of infected cells. Enzyme assays and immunoblot analyses demonstrated T. gondii sPLA2 type II and cPLA2 activities and indicated that T. gondii increased the activity of THP1 sPLA2 type II. Incubation with IFN γ (1,000 units/ml) for 20 h reduced the activities of sPLA2 and cPLA2 in infected and non-infected cells. Thus, IFN γ blocks the activities of both THP1 and parasite sPLA2 and cPLA2 in membrane fractions, resulting in protection against active invasion by T. gondii.

Recombinant retroviral vectors are attractive tools for achieving sustained expression of a therapeutic gene in the liver. However, cell division is required for efficient transduction with these vectors. Here we report that two widely used liver mitogens, triiodothyronin (T3) and cyproterone acetate (CPA), enable hepatocyte transduction with recombinant retroviral vectors delivered in vivo into the bloodstream. Treatment with T3 as well as CPA, alone or in combination, resulted in an increase in hepatocyte replication predominantly around the portal tract. The mitogenic activity made it possible to transduce hepatocytes in the same location. Moreover, when administered together, the two drugs synergized and the transduction level reached 5% of hepatocytes. This transduction level is compatible with clinical applications for a number of inherited liver diseases. Since these two compounds have a long history of safe clinical use, we propose that these liver mitogens may have potential for clinical application in liver-directed gene therapy.

Gene transfer in regenerating dog liver using high-titer recombinant retroviral vectors carrying the E. coli beta-galactosidase gene was studied. Supernatants containing amphotropic or gibbon ape pseudotyped recombinant retroviruses were infused into a peripheral vein in beagle dogs after partial hepatectomy. The kinetics of liver regeneration were determined in the animals and daily infusions were carried out for 4 or 5 days during the regeneration period. Up to 2.8% of hepatocytes were beta-galactosidase positive at the end of the procedure. However, the number of positive cells declined rapidly and few positive hepatocytes were detected after 3 weeks. PCR demonstrated the disappearance of the provirus. Histologically, inflammatory lesions were observed in the transduced livers. Finally, we demonstrated the presence of a cytotoxic T lymphocyte immune response directed against beta-galactosidase-expressing cells, which could explain the disappearance of the transgene. This work suggests that the efficiency of in vivo gene delivery using high-titer retroviral vectors directly infused into the circulation may be hampered by a cytotoxic immune response against the infected cells.

Polymerase chain reaction (PCR) technology was used to detect Toxoplasma gondii DNA in 253 immunodeficient subjects, 179 of whom were infected with the human immunodeficiency virus (HIV). The incidence of toxoplasmosis was 12.3% (22/179) in the HIV-infected subjects and 2.7% (2/74) in the remainder. The sensitivity of the PCR during episodes of toxoplasmosis in HIV-infected subjects not on antiparasitic treatment was 86.6% on peripheral blood and 60% on cerebrospinal fluid (CSF), but was only 25% and 16.7%, respectively, in subjects receiving specific treatment or prophylaxis against Pneumocystis carinii. Among the HIV-seronegative population, six patients undergoing anticancer chemotherapy were PCR positive on bronchoalveolar lavage fluid but did not develop pulmonary toxoplasmosis, suggesting transient carriage.

The GASA gene family previously identified in Arabidopsis belongs to a wide-spread class of genes found in mono- and dicotyledonous plants, all structurally related to the original GA-regulated GAST1 gene from tomato. They encode small peptides (97 to 112 residues) of unknown function sharing a 60 amino acid conserved C-terminal domain comprising twelve conserved cysteine residues which define a pattern not related to other known cysteine-rich motifs. Northern blot hybridization analysis revealed sequential expression of three genes during flowering, silique development and seed germination. GASA4 transcripts were detected in flower buds. GASA1 transcripts markedly accumulated in siliques, about five days after pollination, and correlated with the peak of GA biosynthesis at this stage of silique development. GASA3 transcripts accumulated at the end of the maturation stage of the silique, and transcripts were still present in dry seeds but degraded rapidly during imbibition. In addition, the GASA4 gene was again actively transcribed after germination and this expression was shown to be dependent on the presence of GAs in GA-deficient mutants. Immunoblot analysis confirmed the presence of the GASA4 gene product in flower buds, seedlings and roots. We focused on the GASA4 gene and characterized its expression. The upstream region (-890 to +128) was fused to the GUS reporter gene. GASA4/GUS expression was detected in transgenic Arabidopsis primarily in all meristematic regions, including vegetative, inflorescence and floral meristems, as well as primary and lateral root tips. In a GA-deficient background (ga1–3), GUS activity in the vegetative meristem was detected only in the presence of supplied GA. In root and flower meristems, basal GUS activity was slightly enhanced by exogenous GA. Interestingly, GA strongly inhibit GUS activity in expanding cotyledons and leaves in ga1–3 mutants supplied with exogenous GAs, as well as in the wild type. The GA-dependent meristem-specific expression pattern suggests that the GASA4 protein plays a role in dividing cells rather than in elongating cells.

In France, the consumption of cattle and sheep meat appears to be a risk factor for infection of pregnant women with Toxoplasma gondii. Several nation-wide surveys in France have investigated the prevalence of T. gondiiin sheep and pig meat, but little is known at present about the prevalence of the parasite in beef. The main objective of the present cross-sectional survey was to estimate the seroprevalence of T. gondiiinfection in beef consumed in France. A secondary objective was to attempt to isolate T. gondiifrom cattle tissues and to study the geographical and age variations of this seroprevalence. The overall estimate of seroprevalence of T. gondiiin bovine carcasses (n= 2912), for a threshold of 1:6 was 17.38%. A strong age effect was observed (p< 0.0001) with a seroprevalence of 5.34% for calves (<8 months) and 23.12% for adults (>8 months). Seroprevalence estimates given by area of birth and area of slaughtering for adults showed that the areas with the highest seroprevalence were not the same between these two variables. Only two strains, corresponding to genotype II, were isolated from heart samples, indicating that there is a limited risk of human infection with T. gondii, which needs to be correlated with the food habit of consuming raw or undercook (bleuor saignant) beef. However, new questions have emerged, especially concerning the isolation of parasites from beef and the precise role of bovines, generally described as poor hosts for T. gondii, in human infection.

Despite the infection risk associated with the consumption of contaminated food, techniques for recovering and detecting Cryptosporidiumoocysts from fruit and vegetables are generally inadequate due to the variable recovery efficiencies and high reagent costs, such as those presented by ISO 18744:2016 “Microbiology of the food chain -Detection and enumeration of Cryptosporidiumand Giardiain fresh leafy green vegetables and berry fruits”. Although an improved method for recovering these parasites from Iceberg lettuce, which reported increased recovery efficiency as well as lower costs, has been published, it appears to have limitations for the recovery of Cryptosporidiumfrom saponin-rich leaves such as spinach (Spinacia oleraceae), which have previously been implicated in Cryptosporidium parvumoutbreaks. In this study, we refined the method to improve its use with these more challenging samples. The use of alkaline elution buffer (1 M glycine) of different pH values was evaluated for their effectiveness in removing C. parvumfrom spinach leaves. The refinement of Utaaker's method showed, from spinach leaves inoculated with 100 oocysts, an increased oocyst recovery rate with an overall mean recovery rate of 33.79% ± 2.82%. The emergence of parasitic foodborne illnesses and outbreaks associated with the consumption of fresh produce demonstrates the need for the development of an optimal recovery process for parasites from suspected foods. Results showed that refinement of existing protocols could improve the retrieval of Cryptosporidiumoocysts from these more challenging leafy greens.

Giardia duodenalis, Cryptosporidiumspp. and Toxoplasma gondiiare 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, Giardiaand Trichinella. Until 2016, no standardized methods were available to detect Giardia, Cryptosporidiumand 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. duodenaliscysts, Cryptosporidiumspp. and T. gondiioocysts, 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. gondiidatabase 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 Toxoplasmagenome. 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 Toxoplasmametallopeptidases 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 Toxoplasmabiology and provide opportunities to identify novel therapeutic targets. This type of study would also open a path towards the comparative biology of apicomplexans.

ABSTRACTToxoplasma gondiiis 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 gondiiactivity. A preliminary in vitroscreening performed over 72 h by an enzyme-linked immunosorbent assay (ELISA) revealed 15 interesting compounds that were effective against T. gondiiat 1 μM. Their cytotoxicity was estimated on Vero cells, and their 50% inhibitory concentrations (IC50) were further calculated. As a result, eight anti-Toxoplasma gondiicompounds with an IC50of less than 2 μM and a selectivity index (SI) value of greater than 4 were identified. The most active was MMV675968, showing an IC50of 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 IC50s 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 gondiicompounds highlighted here represent potential candidates for the development of new drugs that could be used against toxoplasmosis.

Echinococcus multilocularis, Toxoplasma gondiiand Toxocaraspp. are foodborne parasites whose eggs or oocysts are spread in the environment via canid or felid faeces. They can cause infections in humans following the raw consumption of contaminated fruit or vegetables. In this study, their occurrence was investigated by quantitative polymerase chain reaction (qPCR) in 254 carnivore faeces deposited in 94 kitchen gardens of northeastern France that were sampled between two and six times from October 2011 to April 2013. Less than 25% of the sampled kitchen gardens contained more than 75% of the collected faeces. Of the 219 faeces that could be attributed to an emitter, cat accounted for 58%, fox for 32% and dog for 10%. Echinococcus multiloculariswas detected in 35%, 11% and 7% of fox, dog and cat faeces, respectively, and Toxocaraspp. in 33%, 12% and 5.5% of cat, fox and dog faeces, respectively. Toxoplasma gondiiwas detected in 2/125 cat faeces and 2/21 dog faeces. The 34 faeces that tested positive for E. multiloculariswere found in only 19 out of the 94 sampled kitchen gardens, and the 40 faeces that tested positive for Toxocaraspp. were found in 28 of them. Consequently, some kitchen gardens appeared particularly at risk of human exposure to foodborne parasites, including E. multilocularisresponsible for alveolar echinococcosis (AE), which is a serious zoonosis. In endemic areas, kitchen garden owners should be informed about the zoonotic risk linked to carnivore faeces deposits and encouraged to set up preventive measures.

Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondiistrains naturally resistant to sulfadiazine and we developed in vitrotwo sulfadiazine resistant strains, RH-RSDZand ME-49-RSDZ, by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhpsand dhfrand/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 dhpsand dhfr, three ABC genes, two Pgp, TgABC.B1and 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. gondiisulfadiazine resistance is not related to these toxoplasmic genes studied.

Detection of Toxoplasma gondiiDNA in cat feces is considered indicative of the presence of T. gondiioocysts. This study aims to demonstrate that the high sensitivity of qPCR can lead to T. gondiiDNA detection in cat feces in the absence of oocysts. A cat immune to toxoplasmosis was fed with a mouse experimentally infected with T. gondii.Detection of DNA of this parasite was performed by qPCR on feces passed: (i) on the day the cat ingested the infected prey; (ii) during the three previous days; and (iii) during the three following days. The kinetics of qPCR results are clearly not linked to oocyst shedding and this result demonstrates that qPCR can detect T. gondiiDNA related to bradyzoites from an infected prey, in the absence of oocysts. Caution is thus recommended when interpreting T. gondiiqPCR results for samples of cat feces.