Surrogates of foodborne and waterborne protozoan parasites: A review.

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.

Investigation of the Presence of Toxoplasma gondii, Giardia duodenalis, and Cryptosporidium spp. in Drinking Waters in the Region of Marrakech, Morocco.

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.

Inactivation of Toxoplasma gondii in dry sausage and processed pork, and quantification of the pathogen in pig tissues prior to production.

Toxoplasma gondii is 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. gondii parasites. We investigated the presence and concentration of T. gondii DNA in the shoulder, breast, ham, and heart of pigs orally inoculated with 1000 oocysts (n = 3) or tissue cysts (n = 3) and naturally infected pigs (n = 2), by means of magnetic capture qPCR (MC-qPCR). Muscle tissues of experimentally infected pigs were further used to evaluate the impact of manufacturing processes of dry sausages, including different concentrations of nitrates (0, 60, 120, 200 ppm), nitrites (0, 60, 120 ppm), and NaCl (0, 20, 26 g/kg), ripening (2 days at 16-24 °C) and drying (up to 30 days at 13 °C), by a combination of mouse bioassay, qPCR and MC-qPCR. DNA of T. gondii was detected in all eight pigs, including in 41.7% (10/24) of muscle samples (shoulder, breast and ham) and 87.5% (7/8) of hearts by MC-qPCR. The number of parasites per gram of tissue was estimated to be the lowest in the hams (arithmetic mean (M) = 1, standard deviation (SD) = 2) and the highest in the hearts (M = 147, SD = 233). However, the T. gondii burden estimates varied on the individual animal level, the tissue tested and the parasitic stage used for the experimental infection (oocysts or tissue cysts). Of dry sausages and processed pork, 94.4% (51/54) were positive for T. gondii by MC-qPCR or qPCR, with the mean T. gondii burden estimate equivalent to 31 parasites per gram (SD = 93). Only the untreated processed pork sample collected on the day of production was positive by mouse bioassay. The results suggest an uneven distribution of T. gondii in the tissues examined, and possibly an absence or a concentration below the detection limit in some of them. Moreover, the processing of dry sausages and processed pork with NaCl, nitrates, and nitrites has an impact on the viability of T. gondii from the first day of production. Results are valuable input for future risk assessments aiming to estimate the relative contribution of different sources of T. gondii human infections.

Vaccination of squirrel monkeys (Saimiri spp.) with nanoparticle-based Toxoplasma gondii antigens: new hope for captive susceptible species.

Squirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite.

Comparison of a Commercial Enzyme-Linked Immunosorbent Assay (ELISA) with the Modified Agglutination Test (MAT) for the Detection of Antibodies against Toxoplasma gondii in a Cohort of Hunting Dogs.

Toxoplasmosis is a zoonotic disease, caused by the protozoan Toxoplasma gondii, affecting most warm-blooded animals. Assessing the seroprevalence of T. gondii in different animal species gives a good estimate of the global circulation of the parasite and the risk for human infections. However, the seroprevalence of T. gondii in dogs is not studied as much as other species, despite their close contact with wildlife and humans in rural or urban environments and evidence that dogs can also be a potential source for human contaminations. A commercial enzyme-inked immunosorbent assay (ELISA) kit to detect anti-T. gondii antibodies in sera of hunting dogs potentially naturally infected, was compared to the modified agglutination test (MAT), used as the reference method. The ELISA presented a sensitivity of 76.5% (CI 95%: 60.0-87.6) and a specificity of 87.7% (CI 95%: 76.7-93.9) and a substantial agreement with the MAT for the detection of canine anti-T. gondii antibodies. Both tests can therefore be used widely for epidemiology studies on T. gondii infections in dogs. With a mean seroprevalence of T. gondii infection in hunting dogs from northern Algeria of 36.8% (CI 95%: 34.9-38.7), this study also highlights the importance of T. gondii seroprevalence studies in companion animals to assess infectious risk for human populations.

Electron ionization mass spectrometric fragmentation and multiple reaction monitoring quantification of ferulic and p-coumaric acid trimethylsilyl derivatives in deposited atmospheric particles.

RATIONALE: Ferulic and p-coumaric acids are important biological and structural components of plant cell walls and possess antioxidant and antimicrobial properties. These phenolic acids are widespread in environmental samples. However, when they are present at very low concentrations or in very complex lipid extracts, their identification and quantification can be challenging. METHODS: The electron ionization mass spectrometry (EI-MS) fragmentation pathways of ferulic and p-coumaric acid trimethylsilyl (TMS) derivatives were investigated. These pathways were deduced by (i) low-energy collision-induced dissociation (CID) gas chromatography (GC)/EI-MS/MS, (ii) accurate mass measurement, and (iii) 13 C labelling. These compounds were then characterized and quantified in multiple reaction monitoring (MRM) mode in total lipid extracts of deposited atmospheric particles using highly specific transitions based on the main fragmentation pathways elucidated. RESULTS: Low-energy CID-MS/MS analyses, accurate mass measurement and 13 C labelling enabled us to elucidate EI-MS fragmentations of ferulic and p-coumaric acid TMS derivatives. Some specific fragmentations proved useful for subsequent characterization and quantification of these compounds. As an application of some of the described fragmentations, trace amounts of these phenolic acids were characterized and quantified in MRM mode in wet- and dry-deposited atmospheric particles containing low proportions of organic matter. CONCLUSIONS: EI-MS fragmentations of ferulic and p-coumaric acid TMS derivatives exhibit specific fragment ions that can be very useful for the quantification of trace amounts of both phenolic acids in environmental samples.

Toxocara cati and Toxoplasma gondii in French Birds of Prey.

Parasites have developed many strategies to ensure their development, multiplication, and dissemination, including the use of reservoir hosts that are often nondomesticated species. Despite drastic reductions in their populations, wild birds remain widespread worldwide and could constitute some of these reservoirs. We focused on the identification of wild bird species harboring parasite stages in their muscles. Breast muscles of 327 birds of 27 different species were collected at three different sites in France. After artificial digestion, isolated nematode larvae were identified by PCR sequencing or restriction fragment length polymorphism (PCR-RFLP). Toxocara cati was identified mainly in birds of prey. The presence of anti-Toxoplasma antibodies was investigated by modified agglutination test on muscle fluids. Anti-Toxoplasma antibodies were detected in 65 out of 166 samples from various bird species. Avifauna, particularly birds of prey, could help on the surveillance of parasite circulation and play a role as sentinel species.

Anti-Toxoplasma gondii effect of lupane-type triterpenes from the bark of black alder (Alnus glutinosa) and identification of a potential target by reverse docking.

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.

TITLE: Potentiel anti-Toxoplasma gondii de triterpènes de type lupane de l'écorce de l'aulne glutineux, Alnus glutinosa, et identification d'une cible potentielle par docking inverse. ABSTRACT: La toxoplasmose est une parasitose mondiale, généralement bénigne. Les personnes à risque sont les patients immunodéprimés et les fœtus chez les femmes enceintes nouvellement séroconverties. Les traitements actuels ont de nombreux effets secondaires et des phénomènes de chimiorésistance apparaissent, d'où la nécessité de trouver de nouvelles substances actives contre T. gondii. Cette étude porte sur le potentiel antiparasitaire des triterpènes pentacycliques de type lupane isolés de l'écorce de l'aulne glutineux (Alnus glutinosa) et formule une hypothèse quant à leur cible protéique par l'utilisation d'une méthode originale de docking inverse. Parmi les triterpènes isolés, la bétulone s'est révélée être la plus active avec une CI50 de 2,7 µM ± 1,2 µM, une CC50 supérieure à 80 µM et un indice de sélectivité supérieur à 29,6. L'étude complémentaire du potentiel anti-T. gondii de composés disponibles commercialement et analogues à la bétulone (acide bétulonique et methyl ester de l'acide bétulonique) a montré le rôle important de la fonction cétone en C3 et du degré d'oxydation de la position 28 du squelette triterpénique de type lupane dans l'activité antiparasitaire puisque leurs CI50 et CC50 étaient similaires aux valeurs rencontrées pour la bétulone. Enfin, les composés les plus actifs ont été soumis au flux de travail de docking inverse d'AMIDE. Un ensemble de 87 protéines de T. gondii de la Protein Data Bank a été créé. La protéine kinase calcium dépendante CDPK3 a été identifiée comme la cible la plus probable des dérivés de la bétuline.

Anatomical distribution of Toxoplasma gondii in naturally and experimentally infected lambs.

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 < 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 gondii DNA 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.

Title: Distribution anatomique de Toxoplasma gondii chez des agneaux infectés naturellement et expérimentalement. Abstract: La consommation de viande crue ou insuffisamment cuite contenant des kystes tissulaires de Toxoplasma gondii est l'une des principales sources d'infection pour l'homme dans le monde. Parmi les différentes espèces destinées à la consommation humaine, le mouton apparaît à haut risque d'infection humaine. La présente étude s'est concentrée sur une distribution anatomique détaillée de Toxoplasma gondii chez des agneaux infectés naturellement et expérimentalement à l'aide d'échantillons frais et congelés de divers morceaux de viande, du point de vue de la santé publique. Classer les parties comestibles destinées à la consommation humaine, selon la charge parasitaire détectable par une PCR en temps réel ciblant l'élément répété de 529 pb était un premier objectif. Un second objectif était d'évaluer l'impact de la congélation en comparant l'efficacité de détection de la PCR quantitative entre les tissus frais et congelés, car les importations de carcasses/coupes d'agneau peuvent arriver congelées ou réfrigérées. Les charges parasitaires estimées les plus élevées ont été observées dans les muscles squelettiques et plus particulièrement dans les parties comestibles telles que le quadriceps fémoral, les muscles intercostaux, le deltoïde et le diaphragme avec une différence significative de charge parasitaire détectable entre les échantillons frais et congelés (p < 0,0001) ou l'infection naturelle et expérimentale (p < 0,0001). Les membres thoraciques et pelviens (3278 à 1048 parasites/g de muscle) ont été classés en tête de liste. L'ADN de T. gondii a été détecté dans toutes les parties comestibles étudiées de l'agneau. Ces résultats suggèrent que l'agneau représente un risque pour les consommateurs. Des investigations supplémentaires doivent être effectuées afin de confirmer les différences mentionnées ci-dessus chez plus d'animaux et dans différentes races.

Investigation of Antiparasitic Activity of 10 European Tree Bark Extracts on Toxoplasma gondii and Bioguided Identification of Triterpenes in Alnus glutinosa Barks.

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/H2O) 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 IC50 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.

Blue mussel (Mytilus edulis)-A bioindicator of marine water contamination by protozoa: Laboratory and in situ approaches.

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.

Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis).

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

Protozoan Parasites and Leafy Greens in Marrakech: Study of Occurrence Using a Molecular Method.

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.

Exposure of pelagic seabirds to Toxoplasma gondii in the Western Indian Ocean points to an open sea dispersal of this terrestrial parasite.

Toxoplasma gondii is a protozoan parasite that uses felids as definitive hosts and warm-blooded animals as intermediate hosts. While the dispersal of T. gondii infectious oocysts from land to coastal waters has been well documented, transmission routes to pelagic species remain puzzling. We used the modified agglutination test (MAT titre ≥ 10) to detect antibodies against T. gondii in sera collected from 1014 pelagic seabirds belonging to 10 species. Sampling was carried out on eight islands of the Western Indian Ocean: Reunion and Juan de Nova (colonized by cats), Cousin, Cousine, Aride, Bird, Europa and Tromelin islands (cat-free). Antibodies against T. gondii were found in all islands and all species but the great frigatebird. The overall seroprevalence was 16.8% [95% CI: 14.5%-19.1%] but significantly varied according to species, islands and age-classes. The low antibody levels (MAT titres = 10 or 25) detected in one shearwater and three red-footed booby chicks most likely resulted from maternal antibody transfer. In adults, exposure to soils contaminated by locally deposited oocysts may explain the detection of antibodies in both wedge-tailed shearwaters on Reunion Island and sooty terns on Juan de Nova. However, 144 adults breeding on cat-free islands also tested positive. In the Seychelles, there was a significant decrease in T. gondii prevalence associated with greater distances to cat populations for species that sometimes rest on the shore, i.e. terns and noddies. This suggests that oocysts carried by marine currents could be deposited on shore tens of kilometres from their initial deposition point and that the number of deposited oocysts decreases with distance from the nearest cat population. The consumption of fishes from the families Mullidae, Carangidae, Clupeidae and Engraulidae, previously described as T. gondii oocyst-carriers (i.e. paratenic hosts), could also explain the exposure of terns, noddies, boobies and tropicbirds to T. gondii. Our detection of antibodies against T. gondii in seabirds that fish in the high sea, have no contact with locally contaminated soils but frequent the shores and/or consume paratenic hosts supports the hypothesis of an open-sea dispersal of T. gondii oocysts by oceanic currents and/or fish.

Validation of the Use of Dried Blood Samples for the Detection of Toxoplasma gondii Antibodies in Stray Cats (Felis s. catus).

If validated beforehand, the analysis of dried blood on blotting paper (BP samples) is very useful for monitoring free-ranging animals. We aimed to validate this method for the detection of antibodies against Toxoplasma gondii in stray cats. We used the modified agglutination test (MAT) in 199 sample pairs of sera and BP samples from 54, 39, 56, and 50 cats trapped during four periods in five dairy farms. Screening was at 1:6, 1:12, and 1:24 dilutions. The cut-off value was at MAT titre ≥ 24, but MAT titre ≥ 12 was also considered for BP samples that often have a higher dilution level. Depending on the period, sample type, and cut-off value, sensitivity of the analysis of the BP sample vs. serum varied from 87.1% to 100% and specificity ranged from 72.22% to 100%. The concordance values and Kappa coefficient showed a substantial to excellent agreement between the results of the two methods, whatever the cut-off value. These findings quantifiably validate the use of MAT on BP samples for the detection of antibodies to T. gondii in stray cats, but we recommend expressing results from BP samples with several cut-off values as the MAT titres tend to be lower than those of sera.

AMIDE v2: High-Throughput Screening Based on AutoDock-GPU and Improved Workflow Leading to Better Performance and Reliability.

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.

Release of particles and metals into seawater following sediment resuspension of a coastal mine tailings disposal off Portmán Bay, Southern Spain.

Portmán Bay in Southern Spain is one of the most extreme cases in Europe of anthropogenic impact on the marine ecosystem by the disposal of mine tailings resulting from the processing of sulphide ores. First, the composition and extent of the surficial deposit were investigated from geochemical and metal analysis on high spatial density of sediment samples. Then, a disturbance experiment was conducted in the coastal area off Portmán Bay in order to investigate the potential impact of mining activities on marine ecosystems. Two research vessels were used for that experiment, one performing as a trawler resuspending bottom sediments while the other monitor the behaviour of turbid plumes thus generated and the evolution of their characteristics through time by using a range of acoustic and optical tools together with water and bottom sediment sampling for biogeochemical and metal analyses. The surficial part of the submarine extension of the mine tailings deposit is highly concentrated in As, Cd, Pb, Fe and Zn with peak concentrations adjacent to the present coastline, from where they decrease seawards before reaching average values for the Mediterranean Sea around 50-m water depth. The artificially triggered resuspension of the surface layer of the deposit led to the formation of resuspension plume about 100 m in width and up to 6 m in height. Resuspended plume was composed of fine particles which rapidly aggregated into flocs of 100 µm. While the biggest particles settled rapidly, the finest fraction remained in suspension during at least 3 h. Resuspended sediment and metal concentrations in particles remained at relatively high levels throughout the experiments following triggering. Fe, Pb, and As concentrations in resuspended particles showed a continuous increase while trawling before decreasing in parallel with the settling down of the resuspended sediments. Those metals have higher affinity with fine particles than with coarse ones, so that while the coarsest fraction from sediment plumes settled first, the finest fraction remained in suspension thus increasing the metals/sediments concentration ratio. On the other hand, Cd and Zn concentrations in suspended particles did not change significantly over time, which is thought to be caused by the fast dissolution of such metals in seawater. Beyond waste dumping itself, the observed increase in some metals in marine particulate material could have a significant impact on the adjacent coastal ecosystems due to their toxicity above certain thresholds. The consequences of the resuspension experiment here presented can be extrapolated to the impact of seafloor mining activities leading to the resuspension of metal-rich particle plumes into the water column. The experimental set-up presented here may be further explored for investigating metal behaviour during seafloor mining activities.

Resilience of Dreissena polymorpha in wastewater effluent: Use as a bioremediation tool?

Nowadays, it is necessary to improve the efficiency of wastewater treatment plant treatments. In this context the use of biofilter species, like Dreissena polymorpha, as a bioremediation tool in wastewater is increasingly highlighted. The innovative aim of this study is to evaluate the zebra mussel survival in the outlet channel of a conventional WWTP to use them as bioremediation tool. For this, mussels were transplanted in the outlet channel for 28 days and different biomarkers were monitored. D. polymorpha is able to maintain itself in good physiological conditions until 21 days, yet at 28 days a high mortality rate (24%), a decrease in filtration efficiency (8/15 mussels filtered and 17.0% of filtration rate) and antioxidant system activation (CAT activity et gpx gene expression increase) suggest an exhaustion. Some biomarkers suggested a hypoxic stress. Despite the unfavourable conditions, bivalves have bioaccumulated pathogenic protozoa (Toxoplasma gondii and Giardia duodenalis) during the exposure. Zebra mussel seems to be a promising tool for bioremediation in wastewater.

Evaluation of a modified method for the detection of Cryptosporidium oocysts on spinach leaves.

Despite the infection risk associated with the consumption of contaminated food, techniques for recovering and detecting Cryptosporidium oocysts 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 Cryptosporidium and Giardia in 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 Cryptosporidium from saponin-rich leaves such as spinach (Spinacia oleraceae), which have previously been implicated in Cryptosporidium parvum outbreaks. 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. parvum from 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% ±â€¯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 Cryptosporidium oocysts from these more challenging leafy greens.