Infectivity of Cryptosporidium parvum oocysts after storage of experimentally contaminated apples

Authors: Macarisin, Dumitru; Bauchan, Gary; Santin-Duran, Monica; Fayer, Ronald

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/11/2010
Publication Date: 10/1/2010
Citation: Macarisin, D., Bauchan, G.R., Santin, M., Fayer, R. 2010. Infectivity of Cryptosporidium parvum oocysts after storage of experimentally contaminated apples. Journal of Food Protection. 73(10):1824:1829.

Interpretive Summary: Three outbreaks of cryptosporidiosis involving hundreds of people have been linked to drinking freshly pressed apple cider. Some of the apples used for cider might have been contaminated from irrigation water, from wash water, from sick food handlers, or from contact with the ground or processing equipment. In the present study apples 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. Oocysts attached to 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, 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. The most efficient removal of oocysts 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: Irrigation water has been associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to experimentally determine if apples contaminated with waterborne oocysts of Cryptosporidium might represent a food safety concern. By laser scanning confocal microscopy C. parvum oocysts attached to artificially contaminated apples showed no morphological changes after 6 weeks of cold storage, suggesting that such oocysts might remain viable and possibly infectious during long periods of storage. By scanning electron microscopy, some oocysts were seen 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 an amorphous 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, 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. The most efficient removal of oocysts (35%) was achieved by rigorous hand-washing in water with a detergent and agitation in an orbital shaker with Tris SDS buffer. Glycine and PBS buffers had no effect on oocyst removal at all.