Development of duplex real-time PCR for quick detection of cryptosporidiosis in goats.

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and a leading cause of mortality from foodborne and waterborne gastrointestinal diseases. In neonates of domestic animals, it is associated with consistent diarrhea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy and antigen trap-ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA-based duplex TaqMan® probe PCR was developed to target the Cryptosporidium oocyst wall protein gene and 18SSU rRNA gene in a single tube that can detect metabolically active cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and they will help decipher the active stages of its lifecycle in a host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection (LOD). For COWP and 18SSU rRNA genes, the LOD was 7.08 × 1004 and 5.95 × 1005 , respectively. During active infections, the oocyst wall protein will be active and so its COWP gene transcripts will act as a marker for active infection. While transcripts for 18SSU rRNA are constitutively expressed in cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess the active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among healthy animals.

Molecular and statistical approaches to the delimitation of Eimeriidae species: a case of extreme polymorphism in eimerian oocysts from the plumbeous pigeon Patagioenas plumbea (Vieillot, 1818) (Columbiformes) in South America.

The current work aimed to analyze, morphologically, statistically, and molecularly, oocysts shed from plumbeous pigeons, Patagioenas plumbea (Vieillot, 1818), from a locality at 2197 m of altitude near the Agulhas Negras peak, the highest point of the State of Rio de Janeiro, southeastern Brazil. The oocysts were extremely polymorphic, being subspheroidal, ovoidal, or ellipsoidal, in addition to having the random presence/absence of characteristic features associated with the oocyst wall, such as micropyle, micropyle cap, lateral micropyle, and outer veil/rough wall. Linear regression confirmed the extreme polymorphism of oocysts, showing that if all combinations of taxonomic characters in oocysts (morphotypes) were overestimated, 19 different species could be identified/described. In contrast, the means comparison analysis between oocysts with the presence/absence of characteristic features and the histograms showed equivalences and regularity in the distribution in the classes of measures, which indicate the presence of a single species in the measured oocysts. Molecular analyses were performed from the isolation of individual oocysts of different morphotypes, which had their genetic material extracted, amplified, and sequenced in 4 non-overlapping loci in the cox1 and cox3 genes and fragments of the small and large subunit rDNA of mitochondrial DNA. The sequences were 100% identical between the morphotypes, with the exception of a very small divergence observed at the locus that partially covers the cox3 gene. The phylogenetic analysis was inconclusive for the locus within the cox1 gene traditionally used for eimeriid coccidians; however, the other loci should have a promising future for phylogenetic studies when more sequences for the same genic regions are deposited in GenBank. Finally, the multifactorial analysis of the current work supported that the polymorphic oocysts shed from P. plumbea are a single species, which was named Eimeria patagioenasae, making this the twenty-second eimerian description from Columbiformes.

Finding of an Unusually Colored Sporocyst of the Genus Leucochloridium in a Succinea putris Snail.

A Leucochloridium sp. Carus, 1835 sporocyst with a mature colored broodsac was found in a Succinea putris L., 1758 snail in the Boksitogorsk district of Leningrad Region (Russia). The pigmentation of the sporocyst's broodsac was different from those of other Leucochloridium Carus, 1835 species previously described for Europe. The obtained sporocyst is most similar by the coloration of its broodsac to the trematodes of the same genus from Japan. The genotyping of the investigated sporocyst by the rDNA gene fragments (complete ITS1, ITS2, 5.8S and partial 18S and 28S sequences) was conducted. Genetic distances between the obtained sporocyst and the previously described species of the genus Leucochloridium were higher than intraspecific ones in the most cases. These data and the data of morphological analysis imply that the investigated sporocyst belongs to a separate species of the Leucochloridium genus, previously not described in the European region.

An apicoplast-resident folate transporter is essential for sporogony of malaria parasites.

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.

Development and evaluation of a real-time PCR for genotyping of Cryptosporidium spp. from water monitoring slides.

Cryptosporidium is an important cause of gastroenteritis globally and the main agent of waterborne outbreaks caused by protozoan parasites. Water monitoring for Cryptosporidium oocysts is by detection and enumeration using stained slide microscopy. Species identification (known as genotyping) may be undertaken post hoc and remains a specialist test, only undertaken in some laboratories. The benchmark method is nested PCR-sequencing of part of the SSU rRNA gene, but not all slides are typable and the workflow is cumbersome. We report the development, in-house validation and application of a real-time PCR-sequencing assay based on that gene, using a hydrolysis probe, for the detection and genotyping of all Cryptosporidium spp. The assay was investigated in two formats; a high volume DNA template for analysing all the DNA extracted from Cryptosporidium-positive water monitoring slides with <5 oocysts seen, and a lower volume DNA template permitting several technical replicates from slides with ≥5 oocysts seen where multiple species are more likely to be present. Each format conformed to the MIQE guidelines for amplification dynamics and was specific for Cryptosporidium spp. With high sensitivity, being capable of detecting and genotyping single oocysts by sequencing of a 435 bp amplicon. When 65 water monitoring slides with <5 oocysts seen were tested, slide typeability varied by sending laboratory (n = 9), and ranged from 22 to 60%. Typeability was 75% for slides with ≥5 oocysts seen that were submitted by a single laboratory. The laboratory workflow was improved by using real-time PCR, and decreased the time to result compared with nested PCR-sequencing. In practical application, there was no loss of typeability when the ≥5 oocysts assay was applied to all slides, irrespective of the number of oocysts present.

Anopheles dirus yellow-g mediates Plasmodium vivax infection.

OBJECTIVE: Our previous transcriptome analysis of Anopheles dirus revealed upregulation of the An. dirus yellow-g gene upon ingestion of Plasmodium vivax-infected blood. This gene belongs to the yellow gene family, but its role regarding P. vivax infection is not known and remains to be validated. The aim of this study was to investigate the role of the An. dirus yellow-g gene in P. vivax infection. METHODS: The qRT-PCR was used to detect the expression of the yellow-g gene in many organs of both male and female mosquitos. The yellow-g gene silencing was performed by dsRNA membrane feeding to An. dirus. These mosquitoes were later challenged by P. vivax-infected blood. The oocyst numbers were determined. RESULTS: The yellow-g transcript was detected in several organs of both male and female An. dirus mosquitoes. Successful knockdown of yellow-g was achieved and resulted in reduced P. vivax infection in the mosquitoes. The decrease in yellow-g expression had no effect on the life span of the mosquitoes. CONCLUSIONS: These results support the yellow-g gene as having an important function in Plasmodium development in Anopheles mosquitoes.

Eimeria ninakohlyakimovae casts NOX-independent NETosis and induces enhanced IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in caprine PMN.

Eimeria ninakohlyakimovae represents a highly pathogenic coccidian parasite causing severe haemorrhagic typhlocolitis in goat kids worldwide. NETosis was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites in vitro and in vivo. In vitro interactions of caprine PMN with parasitic stages of E. ninakohlyakimovae (i. e. oocysts and sporozoites) as well as soluble oocyst antigens (SOA) were analyzed at different ratios, concentrations and time spans. Extracellular DNA staining was used to illustrate classical molecules induced during caprine NETosis [i. e. histones (H3) and neutrophil elastase (NE)] via antibody-based immunofluorescence analyses. Functional inhibitor treatments with DPI and DNase I were applied to unveil role of NADPH oxidase (NOX) and characterize DNA-backbone composition of E. ninakohlyakimovae-triggered caprine NETosis. Scanning electron microscopy (SEM)- and immunofluorescence-analyses demonstrated that caprine PMN underwent NETosis upon contact with sporozoites and oocysts of E. ninakohlyakimovae, ensnaring filaments which firmly entrapped parasites. Detailed co-localization studies of E. ninakohlyakimovae-induced caprine NETosis revealed presence of PMN-derived DNA being adorned with nuclear H3 and NE corroborating molecular characteristics of NETosis. E. ninakohlyakoimovae-induced caprine NETosis was found to be NOX-independent since DPI inhibition led to a slight decrease of NETosis. Exposure of caprine PMN to vital E. ninakohlyakimovae sporozoites as well as SOA resulted in up-regulation of IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in stimulated PMN. Since vital E. ninakohlyakimovae-sporozoites induced caprine NETosis, this effective entrapment mechanism might reduce initial sporozoite epithelial host cell invasion during goat coccidiosis ultimately resulting in less macromeront formation and reduced merozoites I production.

Detection of Toxoplasma gondii oocysts on organic and conventionally grown produce.

Toxoplasma gondii infection can result in toxoplasmosis and potential psychological effects. Research commonly focuses on infection through contact with cat fecal matter or consumption of contaminated meat. However, T. gondii oocysts can persist in the environment for years and may be present in soils and on soil-grown produce. Rates of oocyst DNA recovery from produce were high, with 18% of vegetable samples testing positive for T. gondii via PCR test and melt curve analysis. Radishes had significantly higher oocyst counts than arugula, collard greens, kale, lettuce, and spinach. There were no significant differences in oocyst detection rates between samples taken from organic farmer's markets and conventional grocery stores. This study demonstrates that these oocysts can transfer to produce grown both conventionally and using organic techniques.

Modifications of the U.S. food and drug administration validated method for detection of Cyclospora cayetanensis oocysts in prepared dishes: Mexican-style salsas and guacamole.

Although multiple outbreak clusters of Cyclospora cayetanensis have been traced back to consumption of dishes in Mexican-style restaurants, the FDA Bacteriological Analytical Manual (BAM) does not currently provide methods to detect C. cayetanensis in dishes that contain multiple produce ingredients, such as salsas and guacamole. These complex food matrices also may contain high levels of fats, which can interfere with the detection. Several modifications to the BAM Chapter 19b method (washing produce, DNA extraction, and a TaqMan real-time PCR assay targeting the 18S rRNA gene of C. cayetanensis) were assessed with the goal to detect as few as 5 oocysts of C. cayetanensis in 25 g samples of commercial salsa/pico de gallo, guacamole, and salsa verde. Both freshly prepared and frozen versions of these foods were seeded with 5, 10 and 200 oocysts. For salsa samples, using a gentler washing step than recommended by BAM, we achieved detection of 5 oocysts in the samples (81.8%, n = 11). Increasing the amount of Alconox® in the wash solution to 1%, rather than the 0.1% used in BAM, and adjusting the DNA extraction protocol to process large wash pellets, enabled detection of 5 oocysts in guacamole. To reach the desired level of detection in salsa verde, two types of modifications were necessary: gentler washing and DNA extraction modifications. The use of these same method modifications on previously frozen food samples, provided levels of detection similar to those achieved with fresh dishes. Our modifications enabled robust and reproducible detection of C. cayetanensis in multi-ingredient Mexican dishes, detecting as few as 5 oocysts in 25 g samples. Validating and deploying effective methods to detect C. cayetanensis in high risk fresh produce and prepared dishes are critically important for prevalence studies and outbreak investigations of this parasite.

Detection of Cyclospora cayetanensis on bagged pre-cut salad mixes within their shelf-life and after sell by date by the U.S. food and drug administration validated method.

Recently, outbreaks of Cyclospora cayetanensis in the U.S. were linked to the consumption of a variety of salads containing romaine and/or iceberg lettuce, carrots and/or red cabbage. The Bacteriological Analytical Manual (BAM) Chapter 19b method was validated for the detection of C. cayetanensis in carrots, cabbage and romaine lettuce, but has not been previously evaluated in ready-to-eat (RTE) salad mixes. In addition, the only samples available for traceback investigations are sometimes leftovers in bad conditions. This study evaluated the validated BAM method for detection of C. cayetanensis in two different RTE mixed salads (mix 1: romaine and iceberg lettuces, carrots, and red cabbage and mix 2: romaine and iceberg lettuces, carrots, red cabbage, radish, and pea pods) in good condition and after their sell by date. Individual samples (25 g) were seeded with five and 200 C. cayetanensis oocysts. Unseeded produce was used as negative control. The method included washing of the produce, concentration and extraction of C. cayetanensis DNA and molecular detection of C. cayetanensis 18 S rRNA gene. As few as five oocysts were detected in both fresh and after sell by date mix salads. All unseeded samples were negative, and all samples of both salad types seeded with 200 oocysts were positive. In samples seeded with 200 oocysts, average 18 S rRNA C. cayetanensis CT values were significantly higher in fresh salad mix 1 compared to fresh salad mix 2; CT values were significantly higher in the after sell by date salads compared to their respective fresh mixes (p < 0.05). In conclusion, the BAM method was able to detect as few as five oocysts even in after sell by date RTE mix salads. However, the differences in detection observed, highlight the importance of evaluating the performance of the validated C. cayetanensis detection method in different food matrices and conditions, in advance for future outbreak investigations.

Contamination of fresh produce sold on the Italian market with Cyclospora cayetanensis and Echinococcus multilocularis.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.

Morphological and molecular identification of Eimeria tetartooimia oocysts from a Japanese green pheasant (Galliformes; Phasianidae; Phasianus versicolor) at a zoo in Japan.

We detected Eimeria oocysts from Japanese green pheasants (Phasianus versicolor) at a zoo in Osaka, Japan. The oocyst isolates were subspherical or ovoidal shaped and measured 17.2 (range 14.7-20.0) µm in length and 14.8 (13.3-16.7) µm in width with a length/width (L/W) ratio of 1.2 (1.0-1.4) and each had one polar granule. The oocysts lacked a residuum and micropyle. Sporocysts measured 9.8 (6.7-13.3) µm in length and 5.9 (4.7-7.3) µm in width, with a L/W ratio of 1.2 (1.1-1.4). Compared to previously published values, this strain shows morphological similarities with an isolate of E. teetartooimia from ring-necked pheasants from other countries. Phylogenetic analysis of the 18S rRNA and mitochondrial cytochrome c oxidase subunit I genes places the isolate in a clade related to chicken Eimeria spp., such as E. acervulina or E. brunetti. Although further analysis is needed, this information can be helpful for the diagnosis and determination of virulence of Eimeria spp. in pheasants.

Phosphoproteomic Comparison of Four Eimeria tenella Life Cycle Stages.

Protein phosphorylation is an important post-translational modification (PTM) involved in diverse cellular functions. It is the most prevalent PTM in both Toxoplasma gondii and Plasmodium falciparum, but its status in Eimeria tenella has not been reported. Herein, we performed a comprehensive, quantitative phosphoproteomic profile analysis of four stages of the E. tenella life cycle: unsporulated oocysts (USO), partially sporulated (7 h) oocysts (SO7h), sporulated oocysts (SO), and sporozoites (S). A total of 15,247 phosphorylation sites on 9514 phosphopeptides corresponding to 2897 phosphoproteins were identified across the four stages. In addition, 456, 479, and 198 differentially expressed phosphoproteins (DEPPs) were identified in the comparisons SO7h vs. USO, SO vs. SO7h, and S vs. SO, respectively. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEPPs suggested that they were involved in diverse functions. For SO7h vs. USO, DEPPs were mainly involved in cell division, actin cytoskeleton organization, positive regulation of transport, and pyruvate metabolism. For SO vs. SO7h, they were related to the peptide metabolic process, translation, and RNA transport. DEPPs in the S vs. SO comparison were associated with the tricarboxylic acid metabolic process, positive regulation of ATPase activity, and calcium ion binding. Time course sequencing data analysis (TCseq) identified six clusters with similar expression change characteristics related to carbohydrate metabolism, cytoskeleton organization, and calcium ion transport, demonstrating different regulatory profiles across the life cycle of E. tenella. The results revealed significant changes in the abundance of phosphoproteins during E. tenella development. The findings shed light on the key roles of protein phosphorylation and dephosphorylation in the E. tenella life cycle.

Evaluation of the U.S. Food and Drug Administration validated molecular method for detection of Cyclospora cayetanensis oocysts on fresh and frozen berries.

Outbreaks and sporadic cases of Cyclospora cayetanensis have been linked to consumption of berries. The efficacy of the U.S. Food and Drug Administration (FDA) method for detection of C. cayetanensis was evaluated in fresh berries (blackberries, strawberries, blueberries and mixed berries) and in frozen mixed berries. The protocol included seeding with C. cayetanensis oocysts, produce washing, DNA extraction and a dual TaqMan assay. As few as five oocysts were detected in every type of fresh berry analyzed. All berry samples seeded with 200 oocysts were positive and all unseeded berry samples were negative. No significant differences were observed among any of the berry types analyzed in detection rates, CT values and estimated oocyst recovery percentages. Mixed berries were seeded and frozen for up to seven weeks. As few as five oocysts were also detected. No significant differences were observed in C. cayetanensis CT values between fresh and frozen mixed berries at any seeding level. In conclusion, the FDA BAM Chapter 19B method for the detection of Cyclospora was robust, consistent, and showed high sensitivity in all types of berries analyzed. Evaluation of the FDA detection method in berries will provide reliable laboratory support for surveillance programs and for outbreak investigations.

Morphological and molecular characterization of Cystoisospora laidlawi oocysts (Apicomplexa: Eimeriidae) in farmed American mink (Neovison vison) in Denmark.

From a longitudinal survey conducted on 30 Danish mink farms in 2016, 11.0% of faecal samples (456/4140) were positive for Cystoisospora laidlawi oocysts by microscopy, with 60% (189/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts identified Cystoisospora oocysts measuring 34.3 × 29.5 µm with an oocyst length/width (L/W) ratio of 1.2. The morphological features of the oocysts were identical to Isospora laidlawi previously morphological identified in farmed mink from Denmark and elsewhere. Phylogenetic analysis of 18S rDNA sequences (1221 bp) from three positive mink indicated that Cystoisospora from mink shared the highest genetic similarity to C. canis from a Canadian dog (99.6%). The phylogenetic analysis placed Cystoisospora from mink in a clade with other Cystoisospora isolates.

Molecular and morphological description of Sarcocystis kutkienae sp. nov. from the common raven (Corvus corax).

Until now, two Sarcocystis species, S. cornixi and S. corvusi, were known to employ members of the family Corvidae as intermediate hosts. Between 2013 and 2019, having examined leg muscles of 23 common ravens in Lithuania, sarcocysts were detected in 18 birds (78.3%). Using light microscopy, transmission electron microscopy (TEM), and molecular analysis (three genetic loci, 18S rDNA, 28S rDNA, and ITS1), sarcocysts found in the common raven were described as a new species S. kutkienae. Under a light microscope, the observed sarcocysts were ribbon-shaped (1500-8147 × 53-79 µm) and had a wavy striated cyst wall that reached up to 1.5 µm. Lancet-shaped bradyzoites were 7.7 × 2.2 µm (6.1-9.0 × 1.2-3.0 µm) in size. Ultrastructurally, the sarcocyst wall was 1.5-1.8 µm in thickness and had conical-like protrusions with minute invaginations of a parasitophorous vacuolar membrane. The cyst wall was type 1e-like. Limited genetic variability was observed between the 18S rDNA and 28S rDNA sequences of S. kutkienae and other Sarcocystis spp. using birds as intermediate hosts. In contrast, S. kutkienae could be clearly identified by comparing sequences. At this locus, sequences of S. kutkienae shared the highest similarity (89.5-89.7%) with those of S. cornixi. Phylogenetic analysis showed that S. kutkienae was most closely related to Sarcocystis spp. that employs birds as intermediate and definitive hosts. The issue relating to which species might serve as definitive hosts of S. kutkienae in Lithuania is addressed.

Effects of Goussia infecting the blue whiting and phylogenetic placement of Goussia infecting marine fish off Northern Portugal.

Coccidian parasites of fish have received considerably less attention than their terrestrial counterparts, and within piscine hosts, most studies have focused on freshwater fish. The present study aimed to describe oocyst morphology, phylogenetic affinities, and the impacts of coccidian parasites infecting the internal organs of a commercially valuable marine fish, the blue whiting (Micromesistius poutassou), captured off the Portuguese coast. As part of the phylogenetic analysis, sequences from coccidians infecting the pout (Trisopterus luscus) and the Atlantic chub mackerel (Scomber colias) were included, and the oocyst morphology of the coccidians infecting the former was also reported. Results showed that the prevalence of coccidiosis in the blue whiting was very high (> 82%), occurring in all analyzed organs, despite being more abundant in the liver. A significant negative correlation was found between the abundance of the parasites in the liver and host condition index (p < 0.05), which indicates a negative effect on the fitness of this host. Phylogenetic analyses of the parasites found in all three species examined identified three different species of Goussia, closely related to Goussia clupearum. Adding to previous research, we propose the existence of a fourth group of Goussia, the clupearum type, able to infect multiple organs and phylogenetic related with G. clupearum.

Polymorphism and genetic diversity of Isospora parnaitatiaiensis Silva, Rodrigues, Lopes, Berto, Luz, Ferreira & Lopes, 2015 (Eimeriidae) from antbirds (Thamnophilidae) in Brazil.

Isospora parnaitatiaiensis Silva, Rodrigues, Lopes, Berto, Luz, Ferreira & Lopes, 2015 was identified from a new host, the plain antvireo Dysithamnus mentalis (Temminck), and also from the white-shouldered fire-eye Pyriglena leucoptera Vieillot, in its type-locality, the Itatiaia National Park in the southeastern Brazil, and a preliminary genotypic characterisation by sequencing the mitochondrial cytochrome c oxidase subunit 1 gene is provided. The oöcysts recovered from P. leucoptera and D. mentalis were polymorphic and have genotypic differences that were not considered sufficient for the description of new species, but only different genotypes and morphotypes of I. parnaitatiaiensis related to each host. These morphological and molecular variations were associated with a process of ongoing speciation and in adaptive development to their respective host species.

Toxoplasma gondii Oocyst Infectivity Assessed Using a Sporocyst-Based Cell Culture Assay Combined with Quantitative PCR for Environmental Applications.

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.

Development and application of DNA-aptamer-coupled magnetic beads and aptasensors for the detection of Cryptosporidium parvum oocysts in drinking and recreational water resources.

Environmentally stable and disinfectant-resistant oocysts of Cryptosporidium spp. shed in the feces of infected humans and animals frequently contaminate water resources and are subsequently spread via potable and recreational waters. The current monoclonal-antibody-based methods for detecting them in water are slow, labor-intensive, and demand skills to interpret the results. We have developed DNA-aptamer-based aptasensors, coupled with magnetic beads, to detect and identify the oocysts of C. parvum for monitoring recreational and drinking water sources. A sensitive and specific electrochemical aptasensor (3'-biotinylated R4-6 aptamer) was used as a secondary ligand to bind the streptavidin-coated magnetic beads. This was incorporated into a probe using gold nanoparticle modified screen-printed carbon electrodes. Square wave voltammetry allowed for specific recognition of C. parvum oocysts. The aptamer-coated probes had an oocyst detection limit of 50. It did not bind to the cysts of Giardia duodenalis, another common waterborne pathogen, thus indicating its high specificity for the target pathogen. The system could successfully detect C. parvum oocysts in spiked samples of the raw lake and river waters. Therefore, the combined use of the aptasensor and magnetic beads has the potential to monitor water quality for C. parvum oocysts in field samples without relying on monoclonal antibodies and skill-demanding microscopy.