Towards improved detection of Cryptosporidium on fresh produce

Authors: Macarisin, Dumitru; Fayer, Ronald; Santin-Duran, Monica; Bauchan, Gary; SHIELDS, JOAN - Food And Drug Administration(FDA)

Submitted to: International Association for Food Protection Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 3/3/2011
Publication Date: 8/2/2011
Citation: Macarisin, D., Fayer, R., Santin, M., Bauchan, G.R., Shields, J. 2011. Towards improved detection of Cryptosporidium on fresh produce. International Association for Food Protection Proceedings.

Interpretive Summary: Foodborne and waterborne outbreaks have resulted from environmental contamination with the environmentally resistant oocyst stage of the parasite Cryptosporidium. Although oocysts have been detected on the surface of various fresh fruits and vegetables worldwide detection has been problematic because they adhere tenaciously to surfaces and cannot be cultured like bacteria that can reproduce into large easily detectable populations. To optimize recovery of oocysts from contaminated fresh foods a commercially available detergent (Alconox) was tested as a washing agent. Spinach leaves were contaminated with known numbers of oocysts and stored under refrigeration overnight. Leaves were then washed with the solution and the number of oocysts in the solution were counted by microscopy to determine the percent recovery. Using molecular methods, the leaves were then tested for the presence of any oocysts that might remain adherent. About 75% of the contaminating oocysts were recovered in the wash solution, indicating its helpful potential in a detection protocol. Because about 25% of the oocysts remained adherent, the present wash method must be enhanced before it can be recommended as a method to remove all oocyst contamination from fresh foods.

Technical Abstract: Introduction: Cryptosporidium has been found contaminating a variety of fresh produce worldwide. Current detection methods lack sensitivity and specificity for this parasite. Furthermore, because this protozoan cannot be cultured like bacteria to produce large numbers that facilitate detection, optimum recovery from vegetal matrices is essential for detection. Based on current recovery and detection methods, data on the extent of parasite foodborne contamination is woefully inadequate. Purpose: Develop improved washing-recovery methods to facilitate detection of contaminated produce. Methods: Spinach leaves were spot-inoculated with 2.7 x 106 C. parvum oocysts /leaf, air dried and incubated overnight at 4°C. Recovery of oocysts from spinach was conducted on a gyratory shaker for 30 min in 50-mL tubes (the seeded side of the leaf facing internally in the tube) in 45 ml of Alconox® 0.1% or water. This washing procedure was repeated twice. Recovered oocysts were immuno-stained with MeriFluor reagent and counted. All inoculation sites were excised from the washed leaves and subjected to nested PCR analysis to determine the presence of C. parvum DNA Results: Most oocysts were released with the first washing. The second wash moderately but significantly (P=0.05) improved recovery rates. The addition of 0.1% Alconox®, greatly increased the percentage of recovery from 53.7 ± 7.2% in water to 73.6 ± 3.2% in Alconox solution. All contaminated sites on the leaves were positive for C. parvum DNA, suggesting that in spite of the improved recovery some oocysts still were not removed from vegetal matrices. This fact should be taken into consideration by fresh produce processors’ washing criteria Significance: Development and application of a superior recovery procedure will enable a much more accurate determination of the extent of contamination of fresh produce and reduce/prevent future foodborne outbreaks of cryptosporidiosis. Potentially these findings can be applicable to other protozoan pathogens that strongly adhere to fresh produce.