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

Abstract

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.

Survival and infectivity of Toxoplasma gondii and Cryptosporidium parvum oocysts bioaccumulated by Dreissena polymorpha

“…Toxoplasma gondii infectivity assessment by CC‐qPCR was highly sensitive (LOD < 10 oocysts per mussel; Rousseau et al . 2019) and consistent with the results observed in bioassay although not quantitative. Previously, CC‐qPCR had only been tested on bivalve tissues spiked with oocysts ex vivo (Rousseau et al .…”

“…For T. gondii oocysts, Vero cells (ATCC, CCL‐81) were used, as previously described by Rousseau et al . (2019). For C. parvum , HCT‐8 cells (CCL‐244) were used and obtained from the American Type Culture Collection (Rockville, MD).…”

“…2000) and 72 h for Vero cells with T. gondii (CC3; Rousseau et al . 2019). Three cell monolayer wells were analysed at CC0 and CC2/CC3.…”

“…Parasite DNA detection was detected by real‐time qPCR using a SimpliAmp™ thermal cycler (Thermo Fisher Scientific) according to Rousseau et al . (2019). Infectivity was highlighted by parasite multiplication and therefore by the reduction of quantification cycle (Cq) value obtained at cell culture stop (CC2 for C. parvum or CC3 for T. gondii ) compared to Cq value obtained at CC0.…”

“…Infectivity of bioaccumulated oocysts was evaluated with two methods: by cell culture associated with a real‐time PCR assay (Rousseau et al . 2019), a non‐quantitative in vitro approach in mussels which is an alternative to animal bioassays, and by in vivo assay (Villena et al . 2004), a quantitative approach which is the ‘gold standard’.…”