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Home , Cryptosporidium, Cryptosporidium parvum, Cyclospora cayetanensis

1957

Journal of Food Protection, Vol. 69, No. 8, 2006, Pages 1957–1960 Copyright ᮊ, International Association for Food Protection

Microwave Inactivation of cayetanensis Sporulation and Viability of parvum Oocysts

YNES R. ORTEGA* AND JYEYIN LIAO

Center for and Department of Food Science and Technology, University of , Griffin, Georgia 30223, USA

MS 06-056: Received 31 January 2006/Accepted 29 March 2006

ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/8/1957/1681726/0362-028x-69_8_1957.pdf by guest on 01 October 2021

The efficacy of microwave heating on the viability of oocysts and on the sporulation of Cy- clospora cayetanensis oocysts for various periods of cooking times (0, 10, 15, 20, 30, and 45 s) at 100% power was determined. Cyclospora oocysts were stored in 2.5% dichromate at 23ЊC for 2 weeks, and sporulation rates were then determined. The 4Ј,6-diamidino-2-phenylindole and propidium iodide vital stain and the neonate animal infectivity assay determined Crypto- sporidium oocyst viability. Cryptosporidium oocysts could be completely inactivated with as little as 20 s of cooking time, whereas Cyclospora sporulation was observed up to 45 s. Two of the examined microwave ovens were more effective at reducing sporulation and viability than the third one. Because of the variability of temperature achieved by the various ovens, cooking time was not an accurate parameter for parasite inactivation. Cryptosporidium oocysts could be inactivated only when temperatures of 80ЊC or higher were reached in the microwave ovens.

Cryptosporidium spp. and are MATERIALS AND METHODS parasites that can cause diarrheal illness in humans (6, 9). Microwave ovens. Three microwave ovens were used for Cryptosporidium parvum and this experiment: Kenmore model 721.61102100 (700 W, 0.7 cu ft have been frequently isolated from individuals in endemic [19,821.8 cm3]), Samsung model MW640WA (650 W, 0.6 cu ft and epidemic areas and are associated with food- or water- [16,990.1 cm3]), and General Electric model JE1340 (1,100 W, borne outbreaks. Additionally, other Cryptosporidium spe- 1.3 cu ft [36,811.9 cm3]). cies have been reported less frequently in humans. Cryp- tosporidium outbreaks have been reported in association Oocysts. Unsporulated C. cayetanensis oocysts were ob- with consumption of prepared foods, cilantro, and apple tained from naturally infected individuals from Lima, Peru. Feces cider (12). Cyclospora has been mostly associated with containing C. cayetanensis oocysts were sieved and stored in 2.5% foodborne outbreaks, particularly with imported raspber- potassium dichromate (Sigma, St. Louis, Mo.). Only samples con- taining unsporulated oocysts were used in this study. An initial ries, basil, or lettuce (8, 11). In 2004, the first association concentration of oocysts was performed by a modified ethyl ac- of a Cyclospora outbreak with snow peas was reported (1). etate method (14). Briefly, fecal samples were resuspended in dis- Microwave ovens are found in most U.S. households. tilled water. Then, 20 ml of this suspension was mixed thoroughly The effects of microwave cooking and the inactivation of with 5 ml of ethyl acetate (Fisher Scientific, Pittsburgh, Pa.) and pathogens have been researched by various investigators. centrifuged at 800 ϫ g for 5 min. The supernatant and organic The microbicidal effect of microwave irradiation has been layers were discarded, and the pellet was resuspended in saline studied in a variety of microorganisms, including Bacillus solution and centrifuged at 1,500 ϫ g for 10 min. This process subtilis, Clostridium perfringens, Enterococcus faecalis, was repeated three times. Pellets were resuspended in distilled , Listeria monocytogenes, Pseudomonas water, layered over a primary discontinuous sucrose gradient, and aeruginosa, Salmonella Typhimurium, and Staphylococcus centrifuged at 1,160 ϫ g for 20 min. The discontinuous sucrose aureus. A range of 7 to 25.5 s was needed for bacterial gradient was prepared with a Sheather’s solution (500 g of sucrose inactivation (5). The type of food matrix and salt concen- in 320 ml of distilled water) to make a 1:2 solution with a specific gravity of 1.103 (300 ml of Sheather’s, 600 ml of 0.025 M phos- tration affect the bacterial inactivation process in Bacillus phate-buffered saline [PBS], and 9 ml of Tween 80) and a 1:4 sp. and E. coli (4, 16). Other investigators have described solution with a specific gravity of 1.064 (200 ml of Sheather’s, the limitations of microwave heating on the -forming 800 ml of 0.025 M PBS, and 9 ml of Tween 80). With 50-ml bacteria present in spices and herbs (10). Conventional polypropylene tubes, 10 ml of the 1:4 solution was layered over cooking and temperature inactivation of parasites have been 10 ml of the 1:2 solution. Five milliliters of three-layer gauze- described by various investigators. The objective of this filtered fecal samples was carefully layered over the sugar layers study was to determine the effect of timed microwave cook- and centrifuged at 1,160 ϫ g for 25 min. The sugar layers and ing on the sporulation of C. cayetanensis and the infectivity interphase were then collected from each sample and washed by of C. parvum. centrifugation with a 0.85% saline solution (0.85 g of NaCl in 100 ml of distilled water). Pellets containing oocysts were stored * Author for correspondence. Tel/Fax: 770-233-5587; E-mail: in 2.5% potassium dichromate solution at 4ЊC until used (within [email protected]. 2 months) (13). 1958 ORTEGA AND LIAO J. Food Prot., Vol. 69, No. 8

FIGURE 1. Temperatures reached by mi- crowaves at various periods of time. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/8/1957/1681726/0362-028x-69_8_1957.pdf by guest on 01 October 2021

Oocysts were washed three times with distilled water by cen- examined by light at ϫ400 magnification. Evidence trifugation at 2,000 ϫ g for 5 min each to remove all the potas- of infection was defined as the observation of C. parvum parasite sium dichromate. Oocysts were then resuspended in distilled water developmental stages in the microvilli of prepared ileal tissue sec- and enumerated with a Neubauer hemocytometer (Reichert, Buf- tions. Tissues from each mouse were scored plus (infected) or falo, N.Y.). minus (not infected) by microscopic observation, and the propor- C. parvum oocysts, isolate, were purified from experi- tion of animals infected at each dose was calculated and recorded. mentally infected calves and were obtained from the Parasitology Because there is currently no in vivo viability or infectivity Laboratory at the University of Arizona, Tucson. C. parvum oo- assay available for C. cayetanensis, treated Cyclospora oocysts cysts were purified with a discontinuous sucrose gradient and then were incubated at 23ЊC in potassium dichromate for 2 weeks. The a cesium chloride discontinuous gradient (2). Oocysts were stored sporulation percentage of the treated samples was determined and at 4ЊC in an antibiotic solution (100 U of penicillin, 0.1 mg of compared with untreated oocysts. streptomycin, and 0.1 mg of gentamycin per 10 ml) until used (within 2 months). Statistical analysis. Statistical Analysis Software (version 9.0, SAS Institute, Cary, N.C.) was used to analyze results. Anal- Microwave cooking process. One thousand unsporulated C. ysis of variance, with the general linear model (PROC GLM), was cayetanensis oocysts were placed in 5 ml of deionized water in a used to determine the means and standard deviations among sam- 30-ml Kimax beaker. Each beaker was covered with Reynolds ples for all treatments, the three microwave machines, and the six plastic wrap. Each sample was microwaved according to specific cooking times. Fisher’s least significant difference test was per- cooking times (0, 10, 15, 20, 30, and 45 s) at 100% power. The formed to determine which sample means were significantly dif- cooking temperature for each sample was recorded immediately ferent at P Ͻ 0.05. after each treatment with a digital thermocouple. The samples were quickly cooled until the water temperature reached 23ЊC. Suspensions were transferred to 15-ml centrifuge tubes and cen- RESULTS AND DISCUSSION trifuged at 3,220 ϫ g for 10 min. The pellets were transferred to Heating temperatures reached at various time periods a 1.5-ml microcentrifuge tube and resuspended with 2.5% potas- were reproducible in all microwaves (Fig. 1). The Samsung Њ sium dichromate. Cyclospora oocysts were incubated at 23 C for and General Electric microwaves reached temperatures 2 weeks. higher than 80ЊC after 20 s of heating. The Kenmore mi- Inactivation of Cryptosporidium oocysts was also examined by the same experimental design and with the same microwaving crowave reached the same temperature after 45 s. conditions. Cyclospora sporulation was reduced in all experiments; however, sporulation was not completely inhibited under Viability and infectivity. Viability was determined by the the conditions examined in the present study (Table 1). Ј 4 ,6-diamidino-2-phenylindole and propidium iodide (DAPI-PI) Cryptosporidium oocyst viability also diminished with time vital staining method (3) and the neonate mouse infectivity assay. All experiments were performed in triplicate. Viable oocysts did of microwaving. Inactivation was achieved if oocytes were not incorporate the stain, whereas those that incorporated the stain heated with the General Electric microwave for 20 s, fol- were considered nonviable. lowed by the Samsung for 30 s and the Kenmore for 45 s, Eighteen term-pregnant CD1 mice were obtained from Har- when examined by DAPI-PI vital staining. Oocysts were lan Sprague Dawley Inc. (Madison, Wis.). Upon delivery, each rendered noninfectious to neonate mice if they were heated mother was housed separately with ten 5-day-old neonate mice. with the General Electric microwave for 20 s, the Samsung Each mouse pup was orally inoculated with a 50-␮l aqueous sus- for 30 s, and the Kenmore for 45 s. All positive control 5 pension containing 10 microwave-treated C. parvum oocysts. mice (n ϭ 20) were infected, but none of the negative con- Each treatment was evaluated in 9 to 10 mice. Ten neonate mice trol mice (n ϭ 10) were infected (Table 2). The results from were used as negative controls (inoculated only with water). the DAPI stain used in the current study coincided with the Five days postinoculation, all pups were euthanized by CO2 inhalation. A 1-cm segment of terminal was removed, fixed animal infectivity assay; however, caution should be taken in 10% formalin, embedded in paraffin, sectioned, and stained when this vital stain protocol is used, as it may not correlate with hematoxylin and eosin. The 4-␮m histological sections were adequately when and sanitizers are used. This J. Food Prot., Vol. 69, No. 8 MICROWAVE INACTIVATION OF CYCLOSPORA AND CRYPTOSPORIDIUM 1959

TABLE 1. Cyclospora cayetanensis sporulation when exposed to microwave heating with three different ovens Samsung General Electric Kenmore

Heating Temp (ЊC) % sporulation Temp (ЊC) % sporulation Temp (ЊC) % sporulation time (s) (SD) (SD) (SD) (SD) (SD) (SD)

0 22.6 (0.78) 41.88 (0.98) 22.5 (0.46) 46.37 (2.33) 22.6 (0.21) 43.41 (3.81) 10 55.7 (0.85) 14.33 (3.29) 65.3 (4.71) 15.94 (0.25) 49.3 (2.75) 16.71 (3.19) 15 68.8 (2.62) 6.16 (3.29) 76.0 (1.45) 17.84 (7.12) 53.4 (1.74) 12.51 (3.33) 20 80.4 (3.99) 7.59 (2.57) 84.8 (6.74) 9.92 (2.29) 62.4 (2.71) 7.81 (0.47) 30 97.0 (0) 1.0 (0) 95.0 (2.75) 7.37 (2.06) 73.7 (1.64) 7.39 (1.78) 45 96.0 (0.27) 1.9 (1.92) 96.8 (0.93) 4.26 (2.05) 80.4 (3.0) 5.47 (0.98)

vital stain did not work for Cyclospora viability, probably tributes to the control of parasite transmission. This coin- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/8/1957/1681726/0362-028x-69_8_1957.pdf by guest on 01 October 2021 because of the composition of the Cyclospora oocyst wall. cides with the conventional cooking inactivation of Cryp- The heating temperature achieved by each microwave tosporidium. Whether different parasite species and geno- oven used in this study was very reproducible between ex- types behave similarly during the process of temperature periments; however, variability was found among the inactivation needs to be further studied. brands of microwaves. The General Electric microwave reached higher temperatures at shorter periods of time than ACKNOWLEDGMENTS the other microwaves. When heating times were the same, This study was funded by a U.S. Food and Drug Administration the Kenmore microwave used in this study was always 10 Center for Food Safety and Applied Nutrition grant (FD-U-001948-01). to 20Њ cooler than the temperature of the other microwaves. The authors are grateful to Patricia Torres for providing insight on this C. parvum oocysts are more sensitive to microwave project and to Alison Payton for technical assistance with the animal mod- el used in this study. heating than Cyclospora oocysts. Cryptosporidium oocysts were completely inactivated with as little as 20 s of cooking REFERENCES time, whereas C. cayetanensis sporulation was observed up 1. Anonymous. 2004. Outbreak of associated with snow to 45 s. The Samsung and General Electric microwaves peas—Pennsylvania, 2004. Morb. Mortal. Wkly. Rep. 53:876–878. were more effective at reducing sporulation and viability 2. Arrowood, M. J., and K. Donaldson. 1996. Improved purification than the Kenmore microwave. Because of the variability of methods for calf-derived Cryptosporidium parvum oocysts using dis- temperature achieved by the various ovens, cooking time continuous sucrose and cesium chloride gradients. J. Eukaryot. Mi- was not an accurate parameter for parasite inactivation. crobiol. 43:89S. 3. Campbell, A. T., L. J. Robertson, and H. V. Smith. 1992. Viability When cooking with conventional heating, sporulation of Cryptosporidium parvum oocysts: correlation of in vitro excys- of Cyclospora is completely inactivated when oocysts are tation with inclusion or exclusion of fluorogenic vital dyes. Appl. exposed at 70ЊC for 15 min. Few oocysts sporulate at 50ЊC Environ. Microbiol. 58:3488–3493. when cooked from 15 to 60 min (15). Cryptosporidium 4. Canumir, J. A., J. E. Celis, J. de Bruijn, and L. V. Vidal. 2002. oocysts are rendered noninfectious when heated at 72.4ЊC Pasteurisation of apple juice by using microwaves. Lebensm.-Wiss. Technol.-Food Sci. Technol. 35:389–392. for 1 min; however, oocysts are infectious when heated at 5. Firouzi, R., S. S. Shekarforoush, and F. Hajikhani. 2003. Effect of 67.5ЊC for 1 min (7). microwave radiation on survival of some bacterial species. J. Appl. Cryptosporidium oocysts were inactivated only when Anim. Res. 24:207–211. temperatures of 80ЊC or higher were reached in the micro- 6. Harp, J. A. 2003. Parasitic infections of the . wave ovens. Because C. cayetanensis and Cryptosporidium Curr. Opin. Gastroenterol. 19:31–36. 7. Harp, J. A., R. Fayer, B. A. Pesch, and G. J. Jackson. 1996. Effect spp. can be acquired by ingestion of contaminated water or of pasteurization on infectivity of Cryptosporidium parvum oocysts foods, temperature inactivation, particularly through micro- in water and milk. Appl. Environ. Microbiol. 62:2866–2868. wave cooking, is important in the food industry and con- 8. Herwaldt, B. L. 2000. Cyclospora cayetanensis: a review, focusing

TABLE 2. Cryptosporidium parvum viability when exposed to microwave heating with three different ovens Samsung General Electric Kenmore

Heating Temp (ЊC) Viable Temp (ЊC) Viable Temp (ЊC) Viable time (s) (SD) (SD)a Miceb (SD) (SD) Mice (SD) (SD) Mice

0 21.4 (0.25) 96.67 (1.5) 20/20 21.5 (0.1) 86.95 (5.17) 20/20 21.4 (0.38) 97.17 (1.84) 20/20 10 56.8 (2.37) 71.16 (11) 10/10 61.1 (2.35) 21.48 (21.37) 10/10 46.9 (1.04) 70.34 (25.83) 10/10 15 67.8 (1.91) 8.57 (6.1) 10/10 72.2 (2.66) 8.66 (13.5) 5/10 53.7 (1.6) 86.84 (4.18) 10/10 20 74.0 (5.49) 3.44 (2.9) 2/10 79.0 (2.27) 0 (0) 0/10 61.5 (1.04) 55.94 (18.67) 9/9 30 96.9 (0.55) 0 (0) 0/10 96.0 (1.05) 0 (0) 0/10 71.8 (1.72) 32.55 (18.69) 3/10 45 95.7 (0.96) 0 (0) 0/10 97.4 (1.23) 0 (0) 0/10 80.1 (1.54) 0 (0) 0/10 a Viability of oocysts determined by DAPI-PI. b Infectivity in mice values are presented as number of infected mice/total number of mice per treatment. 1960 ORTEGA AND LIAO J. Food Prot., Vol. 69, No. 8

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