Assessing Toxoplasma gondii oocyst infectivity using a sporocyst-based cell-culture assay combined with qPCR for environmental application

Authors: ROUSSEAU, ANGÉLIQUE - Universite De Reims Champagne-Ardenne; ESCOTTE-BINET, SANDIE - Universite De Reims Champagne-Ardenne; LA CARBONA, STEPHANIE - Actalia Securite Des Aliment; DUMÈTRE, AURÉLIEN - Aix-Marseille University; CHAGNEAU, SOPHIE - Laboratoire Parasitologie-Mycologie; FAVENNEC, LOÏC - University Of Rouen; KUBINA, SOPHIE - Universite De Reims Champagne-Ardenne; Dubey, Jitender; MAJOU, DIDIER - Actia; BIGOT-CLIVOT, BIGOT-CLIVOT - Universite De Reims Champagne-Ardenne; VILLENA, ISABELLE - Universite De Reims Champagne-Ardenne; AUBERT, DOMINIQUE - Universite De Reims Champagne-Ardenne

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/9/2019
Publication Date: 10/1/2019
Citation: Rousseau, A., Escotte-Binet, S., La Carbona, S., Dumètre, A., Chagneau, S., Favennec, L., Kubina, S., Dubey, J.P., Majou, D., Bigot-Clivot, B., Villena, I., Aubert, D. 2019. Assessing Toxoplasma gondii oocyst infectivity using a sporocyst-based cell-culture assay combined with qPCR for environmental application. Applied and Environmental Microbiology. 85(20):e01189-19. https://doi.org/10.1128/AEM.01189-19.
DOI: https://doi.org/10.1128/AEM.01189-19

Interpretive Summary: Toxoplasmosis, caused by the single celled parasite, Toxoplasma gondii, continues to be a public health problem worldwide. This parasite infects all warm-blooded hosts, including humans. It causes mental retardation and loss of vision in children, and abortion in livestock. The ingestion of food and water contaminated with resistant stage of the parasite, the oocyst, is a major mode of transmission of this parasite. Of all the hosts infected, only cats are known to excrete oocysts in feces. Cats can excrete millions of oocysts after eating an infected prey, such as a mouse or a bird. Oocysts can survive outdoors for months and they are highly infectious to humans. Detection of oocysts in the environment is a technically difficult. In the present paper, the authors describe a protocol for efficient detection of Toxoplasma oocysts by bioassay in cell culture. The paper will be of interest to biologists, epidemiologists and parasitologists.

Technical Abstract: Toxoplasma gondii is a ubiquitous foodborne protozoan that infects several levels of the world's population. However, no standardized method is currently available to detect cysts in foods and therefore there are no regulations. Ingestion of food or water contaminated with small amounts of T. gondii oocysts may result in human infection. However, there is no regulations for monitoring oocysts in food mainly because of a lack of standardized methods to detect them. The objectives of this study are (i) to develop a method, applicable in biomonitoring, for the fast detection of infectious T. gondii by cell culture combined with qPCR (sporocyst-CC-qPCR), (ii) to compare CC-qPCR and mouse bioassay for assessing oocysts infectivity, and (iii) to apply this method in blue and zebra mussels experimentally contaminated by oocysts. Combining mechanical treatment and bead beating leads to the release of 84 ' 14% of free sporocysts. The sporocyst-CC-qPCR can detect fewer than ten infectious oocysts in water after three days of cell culture, compared to four weeks by mouse bioassay. For both mussel matrices, oocysts were pre-purified by using a 30%-Percoll gradient and sodium hypochlorite treatment before cell culture of their sporocysts. The limit of detection (LOD100) was less than 10 oocysts. However, the in vitro model was sensitive but not quantitative like in vivo. This sporocyst-CC-qPCR appears as a good alternative to mouse bioassay for monitoring infectious T. gondii oocysts in water and biosentinel mussel species. Moreover, this method could be useful to evaluate inactivation treatments on oocysts and reconsider the environmental risk for human health associated to this parasite.