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Title: Attachment, persistence and infectivity of Cryptosporidium parvum oocysts in experimentally contaminated fruits and vegetables

item Macarisin, Dumitru
item Santin-Duran, Monica
item Bauchan, Gary
item Fayer, Ronald

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: 3/11/2010
Publication Date: 4/21/2010
Citation: Macarisin, D., Santin, M., Bauchan, G.R., Fayer, R. 2010. Attachment, persistence and infectivity of Cryptosporidium parvum oocysts in experimentally contaminated fruits and vegetables. BARC Poster Day.

Interpretive Summary: Outbreaks of cryptosporidiosis involving hundreds of people have been linked to serving fresh salads and to drinking freshly pressed apple cider. Apples used for cider and leafy vegetables might have been contaminated from irrigation water, from wash water or from contact with the ground or processing equipment. In the present study apples, spinach and green onions were artificially contaminated with Cryptosporidium oocysts suspended in water applied to their surface. Apples were then stored in the cold for six weeks and examined with the aid of a laser scanning confocal microscope. Leafy vegetable were examined by laser scanning confocal microscopy during 5 days after contamination. Oocysts attached to spinach, scallions and the apples showed no morphological changes, suggesting that such oocysts might remain viable and possibly infectious during long periods of storage. Viewed with the aid of a scanning electron microscope, some oocysts were deep in natural crevices in the apple peel, while others were attached to the smooth surface of the peel. Some were seen closely associated with what appeared to be a glue-like substance with which they might have been attached to the apple surface. To test the possibility that oocysts had remained infectious on apples contaminated and stored for 4 weeks, mice were fed the peels. All mice developed cryptosporidiosis. To evaluate the strength of oocyst attachment to apples and leafy vegetables, washing methods were used that were reported to be helpful for recovery of oocysts from various foodstuffs, except that the intensity of washing was greatly increased in the present study. Although the stringency of washing regimens was increased, none of the tested methods succeeded in completely removing oocysts from the apple peel, spinach leaves, and scallions. The most efficient removal of oocysts from apples was achieved by rigorous hand-washing in water with a detergent and agitation in a laboratory buffering solution. Oocysts were still detected in 65% of the apples after washing.

Technical Abstract: Cryptosporidium parvum is an environmentally resistant, abundant, and ubiquitous protozoan parasite that causes severe diarrheal disease in humans and livestock. Consumer dietary preference towards fresh and organically grown produce correlates with a heightened occurrence of foodborne outbreaks of Cryptosporidium. However, drinking or recreational waters are still considered the major source of cryptosporidiosis in humans, thereby prioritizing studies of parasite etiology in aquatic environments while the mechanisms of transmission and parasite persistence in fresh fruits and vegetables remain poorly understood. We hypothesized that water used to irrigate and spray crops might be important source of contamination with Cryptosporidium. Using laser scanning confocal and low temperature scanning electron microscopy it was shown that on experimentally contaminated apples and leafy vegetables waterborne C. parvum oocysts strongly adhered to surfaces and resisted vigorous washing. Furthermore, in spinach plants irrigated with contaminated water oocysts infiltrated through the stomatal openings into the mesophyll layer of the leaf. Neonatal mice become infected after eating apples experimentally contaminated with C. parvum oocysts and stored for 4 weeks under conditions of postharvest storage (6ºC). The ability of the wide spread and chlorine-resistant parasite to adhere and persist on fresh fruits and vegetables raise concerns regarding food safety