Adhesive-tape recovery combined with molecular and microscopic testing for the detection of Cryptosporidium oocysts on experimentally contaminated fresh produce

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

Submitted to: Parasitology Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/17/2012
Publication Date: 4/2/2013
Citation: Fayer, R., Santin, M., Macarisin, D., Bauchan, G.R. 2013. Adhesive-tape recovery combined with molecular and microscopic testing for the detection of Cryptosporidium oocysts on experimentally contaminated fresh produce. Parasitology Research. 112(4):1567-1574.

Interpretive Summary: Human parasitic protists such as Cryptosporidium, contaminate a variety of fresh produce worldwide. Existing detection methods often lack sensitivity and specificity, require many hours or days for confirmation of detection, and are costly. Furthermore, detection has been problematic because these parasites adhere tenaciously to plant surfaces and cannot be enriched in culture medium like bacteria to produce large numbers that facilitate detection. To determine the sensitivity and specificity of a rapid and inexpensive detection method, Cryptosporidium oocysts (the infectious particles) were suspended in water and then applied to the surface of apples, cucumbers, peaches, and tomatoes at low concentrations. After the surface of the produce was dry, short strips of double sided clear adhesive tape were pressed against the surface of the produce and then removed after several seconds. Tape strips were either examined microscopically after staining with a fluorescent dye, or analyzed using molecular methods. The microscopic method was found to be rapid, sensitive, and relatively inexpensive. This method could be used for the detection of pathogenic protists on fresh produce or surfaces where food is processed, enabling preventive action to be taken in the event that contamination is detected. This information will be useful for other researchers, regulatory agencies or the produce industry.

Technical Abstract: Cryptosporidium parvum suspended in water were applied to the surface of apples, cucumbers, peaches, and tomatoes at concentrations of 100, 50 or 10 oocysts within circles drawn with a permanent marker. Approximately 18 hr later, skin containing uncontaminated and contaminated circles from each produce was excised and placed in microcentrifuge tubes to extract DNA. Pieces of transparent double sided adhesive tape were lightly pressed onto the surface of other contaminated circles, then placed in microcentifuge tubes before those circles were excised and placed in additional microcentifuge tubes. Pieces of adhesive tape were pressed against the surfaces of remaining circles, applied to a glass microscope slides, stained with fluorescein labeled anti-Cryptosporidium antibody, and examined by fluorescence microscopy. At concentrations of 100 and 50 oocysts per spot, the sensitivity of detection using adhesive tape examined either by PCR (polymerase chain reaction) or IFA (immuno-fluorescence microscopy). These methods were indistinguishable from detection levels achieved by PCR of contaminated skin without prior tape application for all four types of produce, with the exception of one tomato which was contaminated with 50 oocysts. At 10 oocysts per circle the most sensitive methods of detection was PCR of skin without prior tape application followed by examination of adhesive tape by IFA. Detection of oocysts by IFA examination of adhesive tape from peaches was problematic because of the complex surface structure, as demonstrated by scanning electron microscopy. For the other produce, the findings suggest that the adhesive tape IFA method is a rapid, sensitive, and relatively inexpensive method for detection of pathogenic protists on fresh produce.