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Efficacy of Wash Solutions in Recovering <I>Cyclospora</I>

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Efficacy of Wash Solutions in Recovering <I>Cyclospora</I> 1348 Journal of Food Protection, Vol. 77, No. 8, 2014, Pages 1348–1354 doi:10.4315/0362-028X.JFP-13-381 Copyright G, International Association for Food Protection Efficacy of Wash Solutions in Recovering Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from Basil VENESSA CHANDRA, MARIA TORRES, AND YNE´ S R. ORTEGA* Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/8/1348/1686703/0362-028x_jfp-13-381.pdf by guest on 30 September 2021 Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, Georgia 30223-1797, USA MS 13-381: Received 13 September 2013/Accepted 23 March 2014 ABSTRACT Parasitic diseases can be acquired by ingestion of contaminated raw or minimally processed fresh produce (herbs and fruits). The sensitivity of methods used to detect parasites on fresh produce depends in part on the efficacy of wash solutions in removing them from suspect samples. In this study, six wash solutions (sterile E-Pure water, 3% levulinic acid–3% sodium dodecyl sulfate, 1 M glycine, 0.1 M phosphate-buffered saline, 0.1% Alconox, and 1% HCl–pepsin) were evaluated for their effectiveness in removing Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from basil. One hundred or 1,000 oocysts of these parasites were inoculated onto the adaxial surfaces of 25 g of basil leaves, placed in stomacher bags, and stored for 1 h at 21uC or 24 h at 4uC. Leaves were hand washed in each wash solution for 1 min. DNA was extracted from the wash solutions and amplified using PCR for the detection of all parasites. Oocysts inoculated at a concentration of 1,000 oocysts per 25 g of basil were detected in all wash solutions. At an inoculum concentration of 100 oocysts per 25 g, oocysts were detected in 18.5 to 92.6% of the wash solutions. The lowest variability in recovering oocysts from basil inoculated with 100 oocysts was observed in 1% HCl–pepsin wash solution. Oocyst recovery rates were higher at 1 h than at 24 h postinoculation. Unlike most bacteria, parasites cannot be enriched; therefore, an optimal recovery process for oocysts from suspected foods is critical. The observations in this study provide guidance concerning the selection of wash solutions giving the highest retrieval of parasite oocysts. Foodborne diseases are a major cause of illness and cayetanensis require time to sporulate and are not infectious death in the United States. Over the decades, there has been when excreted, Cryptosporidium oocysts undergo sporula- an increase in the number of reported cases of foodborne tion inside the host and are infectious when shed into the illness linked to fresh produce. An epidemiologic investi- environment. Toxoplasma gondii oocysts in cat feces gation revealed that there were 190 produce-associated sporulate 24 to 48 h after being excreted. Toxoplasmosis outbreaks from 1973 through 1997 and listed Cyclospora is acquired by ingestion of contaminated drinking water, and Escherichia coli O157:H7 as previously unrecognized fresh produce containing T. gondii oocysts, or meat causes of foodborne illness (34). Outbreaks of parasitic containing Toxoplasma tissue cysts. foodborne diseases are on the increase in parallel with the Numerous outbreaks of parasitic infections associated globalization of the food supply, increased consumption of with the consumption of contaminated fruits (primarily fresh produce, and increased international travel. Scallan raspberries) (14, 15, 19), fresh produce (basil, mesclun, et al. (32) estimated 9.4 million episodes of foodborne green onions, and lettuce) (2, 3, 13, 21, 24), and water (1, illnesses annually in the United States, in which 0.2 million 12, 22, 26) have been reported since 1990, with recurring illnesses are caused by parasites. Toxoplasma is one of cyclosporiasis outbreaks from contaminated Thai basil in the leading causes of foodborne illness requiring hospital- 2001 (16) and snow peas in 2005 (4) and cryptosporidiosis ization (8%) and one of the leading causes of death (24%) outbreaks in 2003 and 2009 implicating not only unpas- (32). teurized milk and apple cider but also salads from salad Cyclospora cayetanensis and Cryptosporidium parvum bars, salad mixtures, and green onions (5, 6). A recent are emerging human pathogens that have been associated outbreak of cyclosporiasis involving more than 600 cases with numerous outbreaks of diarrheal illness. Both are has been linked to consumption of a salad mix (7). coccidian parasites that cause intracellular infections, According to a 2011 U.S. Food and Drug Administration primarily in the epithelial cells of the intestinal tract when (FDA) import alert, fresh produce, including basil, from infectious oocysts are ingested. While the oocysts of C. Mexico has been refused for admission to the United States as it appeared to contain C. cayetanensis (36). Although T. * Author for correspondence. Tel: 770-233-5587; Fax: 770-233-5587; gondii has not been implicated in foodborne outbreaks E-mail: [email protected]. linked to fresh produce, oocysts in feces of felines could J. Food Prot., Vol. 77, No. 8 RECOVERY OF PARASITES FROM BASIL 1349 contaminate water or soil, thereby possibly leading to HCl (Fischer, Fairlawn, NJ), pH 2.0, plus 6.4% pepsin (Fischer, contamination of produce. Nazareth, PA). The PBS solution contained 0.14 M sodium Unlike bacteria, parasites do not multiply in the chloride, 0.002 M potassium phosphate monobasic, 0.003 M environment. Because contaminated foods may contain potassium chloride, and 0.01 M sodium phosphate dibasic in very low numbers of oocysts, very sensitive isolation and E-Pure water. detection methods are essential. Detection of C. cayetanen- Recovery of oocysts. Bags containing inoculated samples and sis, Cryptosporidium spp., and T. gondii oocysts in fresh wash solutions (200 ml of E-Pure water, 3% levulinic acid–3% produce can be done using microscopy techniques or PCR. SDS, 1 M glycine, 0.1 M PBS, or 0.1% Alconox or 100 ml of 1% However, these methods are highly dependent on the HCl–pepsin) were vigorously shaken by hand for 15 s, hand efficient recovery of oocysts from produce tissues. Washing rubbed for 30 s, and shaken vigorously for another 15 s. Wash of fresh produce with elution buffers has been used to solutions were decanted into 50-ml conical tubes. Stomacher bags remove oocysts. Typically, the fresh produce is washed with containing basil leaves were squeezed by hand to remove as much a solution of choice by manually agitating the sample for a of the wash solution as possible. Oocysts in the wash solution were Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/8/1348/1686703/0362-028x_jfp-13-381.pdf by guest on 30 September 2021 period of time. The resulting wash solutions are concen- concentrated by centrifugation (Allegra 6 Centrifuge, Beckman trated by centrifugation and further analyzed by microscopy Coulter, Palo Alto, CA) for 15 min at 2,060 | g. Most of the or PCR. supernatant wash solution was decanted, leaving 3 to 4 ml in each The purpose of this study was to determine the tube. The resultant pellets from each tube were pooled and transferred to a sterile 15-ml conical tube, followed by centrifu- effectiveness of several wash solutions in recovering C. gation as described above. The supernatant wash solution was cayetanensis, C. parvum, and T. gondii from basil. aspirated, and the pellet was brought to 1 ml with sterile E-Pure Optimization of elution methods has implications for water in a 1.5-ml microcentrifuge tube and stored at 4uC. For each outbreak investigations, as well as for surveillance studies. wash solution, one uninoculated control sample was treated as Basil was selected because it has been implicated as a described above. vehicle in numerous outbreaks of cyclosporiasis. DNA extraction. DNA was extracted from wash solution MATERIALS AND METHODS samples using a FastDNA Spin for Soil Kit (MP Biomedicals, Irvine, CA) with slight modifications to the manufacturer’s Parasites. C. cayetanensis oocysts were obtained from a protocol as follows: 200 ml of each sample was added to a Lysing naturally infected individual from Peru courtesy of Dr. Robert Matrix E Tube (MP Biomedicals). Samples were processed in a Gilman, A.B. Prisma, and C. parvum oocysts were obtained from infected calves and kindly provided by the Centers for Disease FastPrep FP120 (Thermo Savant, Holbrook, NY) at a speed setting Control and Prevention (CDC). The oocysts of both parasites were of 5.5 for 30 s. All samples were centrifuged (Eppendorf stored in 2.5% potassium dichromate. T. gondii oocysts were Centrifuge 5415 D, Eppendorf AG, Hamburg, Germany) at | | obtained courtesy of Dr. J. P. Dubey, U.S. Department of 15,700 g instead of 18,200 g as stated in the manufacturer’s Agriculture, and were stored in 2% sulfuric acid. The oocysts of instructions. The final sample volume for the DNA extract (80 ml) each parasite were counted (10 repetitions each) using a Bright- was stored at 220uC. Positive controls were extracted simulta- Line hemacytometer (Hausser Scientific, Horsham, PA), further neously. diluted to give 100 and 1,000 oocysts per 100 ml, and used to inoculate the basil leaves. PCR. A nested PCR assay was used to amplify fragments of the 18S rRNA gene of Cyclospora (20). For the primary PCR, 1 ml Inoculation of basil. Sweet basil stems (Tom Pontano & of DNA extract from each sample was used. One microliter of this Sons Farms, Vineland, NJ, Pontano Farms, Inc., Boynton Beach, primary reaction mixture was used for the secondary nested PCR. FL, and Rock Garden, Miami, FL) were purchased from a farmers’ The amplified product was about 500 bp. A nested PCR protocol market in Atlanta, GA, and stored at 4uC for no longer than 3 days for Cryptosporidium was used to amplify fragments of the 18S prior to inoculation.
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