Molecular detection of Toxoplasma gondii in water samples from Scotland and a comparison between the 529bp real-time PCR and ITS1 nested PCR

Wells, Beth; Shaw, Hannah; Innocent, Giles; Guido, Stefano; Hotchkiss, Emily; Parigi, Maria; Opsteegh, Marieke; Green, James; Gillespie, Simon; Innes, Elisabeth A.; Katzer, Frank

doi:10.1016/j.watres.2015.09.015

Cited by 45 publications

(25 citation statements)

References 26 publications

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“…In fact, the prevalence of T. gondii found in the RTE salads (0.8%) suggests that the dynamics of toxoplasmosis for humans may be different from previous assumptions. T. gondii has recently been listed as the second most harmful foodborne pathogen (Scallan et al, 2015), and is responsible for the highest disease burden of all foodborne pathogens (Wells et al, 2015). Previously, the source of infection for Toxoplasma in humans has always been attributed to consumption of pork and goat meat.…”

Section: Discussionmentioning

confidence: 99%

Detection and prevalence of protozoan parasites in ready-to-eat packaged salads on sale in Italy

et al. 2017

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To investigate the prevalence of protozoan contamination by Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii and Cyclospora cayetanensis, in 'ready to eat' (RTE) salads on sale in Italy, 648 packages were purchased from industrial and local brands. Nine individual packages from each brand were collected per month, pooled and subjected to microscopy and molecular analyses. Microscopic examination of 864 slides detected Cryptosporidium spp. but also Blastocystis hominis and Dientamoeba fragilis. Molecular tools identified G. duodenalis assemblage A, Cryptosporidium parvum and Cryptosporidium ubiquitum, T. gondii Type I and C. cayetanensis. B. hominis and D. fragilis were also molecularly confirmed. The overall prevalence of each protozoan species was 0.6% for G. duodenalis, 0.8% for T. gondii, 0.9% for Cryptosporidium spp., and 1.3% for C. cayetanensis, while prevalence for B. hominis was 0.5% and for D. fragilis 0.2%. Microscopy and/or molecular tools revealed that 4.2% of the samples were contaminated by at least one protozoan species, and 0.6% of samples presented contamination by two protozoan species, with a number of oocysts ranging from 62 to 554 per g of vegetable matter for T. gondii, and 46 to 1.580 for C. cayetanensis. This is Europe's first large-scale study on the presence of protozoans in packaged salads, and shows that RTE sanitation processes do not guarantee a product free from protozoans of fecal origin.

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Section: Discussionmentioning

confidence: 99%

Detection and prevalence of protozoan parasites in ready-to-eat packaged salads on sale in Italy

et al. 2017

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“…Specific primers (FW: 5′-AGC CAC AGA AGG GAC AGA AG-3′ and REV: 5′-TCC AGG AAA AGC AGC CAA G-3′) targeting a 183-bp sequence of the 529-bp repetitive sequence of T. gondii [23,26,[28][29][30] were designed (Prim-er3Plus and BLAST ® ) for DNA detection according to the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines [32,33]. These primers do not bind to any site of the H. hammondi 529-bp repetitive sequence (GenBank: EU493285.1), and no significant similarity was found between the primers' nucleotides and H. hammondi repetitive sequence.…”

Section: Conventional Pcr and Sequencingmentioning

confidence: 99%

“…In this scenario, a laboratory approach was designed based on the experience gained with Method 1623.1/EPA for Cryptosporidium oocyst and Giardia cyst detection [27]. This approach involved: (i) the concentration of oocysts from large volumes of washing water (fruit and vegetables), according to Method 1623.1/EPA; and (ii) subsequent application of PCR for identification and quantification of T. gondii DNA [23,26,[28][29][30]. Herein, we outline findings related with the detection of T. gondii oocysts in vegetables and berry fruits, as a contribution to a better comprehension of oocyst transmission to humans.…”

Section: Introductionmentioning

confidence: 99%

Detection of Toxoplasma gondii oocysts in fresh vegetables and berry fruits

et al. 2020

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Background: Toxoplasma gondii is the third most important contributor to health burden caused by food-borne illness. Ingestion of tissue cysts from undercooked meat is an important source of horizontal transmission to humans. However, there is an increasing awareness of the consumption of fresh fruit and vegetables, as a possible source for oocyst transmission, since this stage of the parasite can persist and remain infective in soil and water for long time. Herein, we outline findings related with detection of T. gondii oocysts in vegetables and berry fruits, which are usually raw consumed. The procedure includes the estimation of the number of oocysts. Methods: Food samples were collected from local producers and supermarket suppliers. Toxoplasma gondii oocysts were concentrated after washing the samples by applying high resolution water filtration and immunomagnetic separation (method 1623.1: EPA 816-R-12-001-Jan 2012), in order to (i) remove potential Cryptosporidium spp. oocysts and Giardia spp. cysts present in the samples; and (ii) select T. gondii oocysts. Toxoplasma gondii oocyst detection and an estimation of their numbers was performed by conventional PCR and real time qPCR, using specific primers for a 183-bp sequence of the T. gondii repetitive DNA region. All PCR-positive DNA samples were purified and sequenced. Restriction enzyme digestion with EcoRV endonuclease confirmed the presence of the T. gondii DNA fragment. In addition, the presence of the parasite was observed by fluorescent microscopy, taking advantage of the oocysts autofluorescence under UV light. Results: Forty percent of the analysed samples (95% CI: 25.5-56.5%) presented the expected PCR and digested DNA fragments. These fragments were confirmed by sequencing. Microscopic autofluorescence supported the presence of T. gondii-like oocysts. The estimated mean (± SE) oocyst concentration was 23.5 ± 12.1 oocysts/g, with a range of 0.6-179.9 oocysts/g. Conclusions: Our findings provide relevant evidence of contamination of fresh vegetables and berry fruits with T. gondii oocysts.

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“…oocysts or Giardia duodenalis cysts, there are no standardized methods available for detection of T. gondii oocysts in water and food samples. Current methods are mainly based on PCR (Villena et al 2004;Karanis et al 2012;Wells et al 2015;Marchioro et al 2016;Cong et al 2017;Lalle et al 2018) and microscopy (Al-Megrin 2010; Harito et al 2017;Caradonna et al 2017); however, they do not provide information regarding the viability of the detected parasites. Interest for molecular-based methods to assess viability of food and waterborne parasites is growing.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of propidium monoazide–based qPCR to detect viable oocysts of Toxoplasma gondii

et al. 2019

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

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Molecular detection of Toxoplasma gondii in water samples from Scotland and a comparison between the 529bp real-time PCR and ITS1 nested PCR

Cited by 45 publications

References 26 publications

Detection and prevalence of protozoan parasites in ready-to-eat packaged salads on sale in Italy

Detection and prevalence of protozoan parasites in ready-to-eat packaged salads on sale in Italy

Detection of Toxoplasma gondii oocysts in fresh vegetables and berry fruits

Evaluation of propidium monoazide–based qPCR to detect viable oocysts of Toxoplasma gondii

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