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)

Authors: CAZEAUX, CATHERINE - Actalia Securite Des Aliment; LALLE, MARCO - Universite De Reims Champagne-Ardenne; DURAND, LOIC - Universite De Reims Champagne-Ardenne; AUBERT, DOMINIQUE - Universite De Reims Champagne-Ardenne; FAVENNEC, LOIC - University Of Rouen; Dubey, Jitender; GEFFARD, ALAIN - Universite De Reims Champagne-Ardenne; VILLENA, ISABELLE - Universite De Reims Champagne-Ardenne; LA CARBONA, STEPHANIE - Actalia Securite Des Aliment

Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2021
Publication Date: 7/17/2021
Citation: Cazeaux, C., Lalle, M., Durand, L., Aubert, D., Favennec, L., Dubey, J.P., Geffard, A., Villena, I., La Carbona, S. 2021. 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). Food Microbiology. 103870. https://doi.org/10.1016/j.fm.2021.103870.
DOI: https://doi.org/10.1016/j.fm.2021.103870

Interpretive Summary: Food safety due to microbial contamination of foods remains a public health concern worldwide. Among the parasitic contaminants, fecal contamination of foods due to protozoans such as Cyclospora, Cryptosporidium, Giardia, and Toxoplasma are of prime concern. Detection of these contaminants in foods is technically difficult because of low numbers present. These protozoa are concentrated by filter feeders such as mussels and humans can become infected if these are eaten uncooked. Here, authors report on a method optimized to detect parasite DNA in mussel tissues. These results will be of interest to diagnosticians, parasitologists, and public health workers. Work related to toxoplasmosis was completed prior to the redirection of USDA’s research program towards other food safety parasites.

Technical Abstract: The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfishes’ consumption. No standardized methods are available for their detection in these foods, and the performances of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performances criteria (including limit of detection (LD95METH) and 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, using 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 showed poor performances (LD95METH > 300 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performances (LD95METH: 4-40 parasites/g, DNA-RR: 19 to 80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (LD95METH: 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.