SOP: Isolation and Identification of Cyclospora cayetanensis Oocysts from Fresh Raspberries

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

Fresh raspberries are shaken gently in a stomacher bag with buffer so that Cyclospora cayetanensis oocysts wash off the berry surfaces. The wash liquid is concentrated by repeated centrifugation and pooling, and the final resuspended pellet is examined microscopically for the oocysts.

2. General characteristics of Cyclospora cayetanensis

Background and life cycle

• C. cayetanensis is a coccidian protozoan parasite that appears to infect only humans. The transmission stage, the oocyst, is spherical and 8–10 µm in diameter. Unsporulated oocysts may show only a granular cytoplasm; sporulated (viable) oocysts contain two oval sporocysts, each holding two sporozoites. The oocysts autofluoresce blue under ultraviolet light.
• The full life cycle is not yet established, but it is thought to resemble that of other coccidian protozoa. Unsporulated (immature) oocysts are shed in the faeces of infected people. Depending on temperature, humidity and other conditions, they may sporulate — becoming mature and viable — within days or weeks. After ingestion, oocysts pass through the stomach into the small intestine, where they excyst and release sporozoites that enter the cells lining the gut. The sporozoites multiply asexually, producing many merozoites that leave the cell and invade other host cells; some merozoites develop into male and female gametes whose sexual reproduction produces unsporulated oocysts.

Transmission

• The parasite was first thought of mainly as waterborne, spread through faecally contaminated drinking water — still an important route in endemic countries such as Nepal, Haiti, Peru and Guatemala. Since 1996, foodborne outbreaks have been reported in North America every year. Imported fresh raspberries were implicated in many of the earlier outbreaks; later outbreaks came from oocyst-contaminated mesclun (baby greens), basil and possibly other fresh produce. Contamination may come from poor worker hygiene, contaminated irrigation or wash water, pesticide mixing, or other routes not yet identified. Person-to-person (faecal–oral) transmission is unlikely because of the long sporulation period needed, and zoonotic transmission is also unlikely — animal infectivity studies have been largely unsuccessful.

Symptoms, diagnosis and treatment

• The disease, cyclosporiasis, may cause diarrhea, nausea, vomiting, abdominal pain, fatigue, loss of appetite and similar symptoms. Symptoms generally appear about a week after ingestion of the oocysts and may last days or weeks if left untreated. Diagnosis usually rests on microscopic examination of stool samples for oocysts, though molecular techniques may also be used. Cyclosporiasis is effectively treated with the antibiotic bactrim.

3. Apparatus required

Elution

• Stomacher bags or equivalent (capacity of at least 1 L)
• Orbital shaker with clips or other means to support stomacher bags

Concentration

• Gauze sponges (8-ply)
• 50 mL polypropylene conical centrifuge tubes
• Centrifuge — swinging bucket rotor with adapters for 50 mL tubes
• 25 mL pipettes and bulb, for removing supernatant
• Pasteur pipettes and bulbs
• Digital micropipettes (100 µL and 1000 µL capacity) and appropriate tips, for transferring resuspended pellets
• 1.5 mL polypropylene microcentrifuge tubes
• Microcentrifuge with adapters for 1.5 mL tubes
• Vortex mixer

Microscopy

• Standard microscope slides and 22×22 mm coverslips
• Positive control slide of C. cayetanensis oocysts
• Compound microscope with 40× and 100× (oil immersion) objectives, with both brightfield and epifluorescence capability (330–380 nm ultraviolet excitation filter, 420 nm barrier filter, and mercury lamp)

4. Reagents

• TE buffer, pH 7.4 (10 mM Tris-HCl, 1 mM EDTA)
• Cytoseal 60 (VWR Canlab) or clear nail polish, for sealing coverslips

5. Procedure

Weigh approximately 50 g of fresh raspberries into an appropriately labelled stomacher bag (if possible, avoid raspberries that are very large or small, or obviously damaged or soft)

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Carefully pour 120 mL of TE buffer (pH 7.4) into the bag, avoiding pouring the buffer directly on the berries

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Place the bags on the orbital shaker for 2 minutes at 120 rpm — shaking too vigorously or for too long produces excessive debris and a pellet that is difficult to handle

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Remove the bags from the shaker and carefully decant the liquid from each bag through a gauze sponge into three labelled 50 mL conical centrifuge tubes

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Centrifuge the tubes at 1,500 × g for 10 minutes

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With a 25 mL pipette, remove and discard the supernatant, leaving 1–2 mL in each tube to resuspend the pellet by vortexing

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With a Pasteur pipette, transfer the resuspended pellets from two tubes into the third tube; rinse one of the empty tubes with 1–2 mL of TE buffer using the same pipette, transfer to the second empty tube and rinse again; finally transfer the rinse liquid to the third centrifuge tube containing the pooled pellets

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Centrifuge the tube with the pooled resuspended pellets at 1,500 × g for 10 minutes

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With a Pasteur pipette, remove and discard the supernatant, leaving approximately 0.5 mL to resuspend the pellet; transfer the resuspended pellet into a 1.5 mL microcentrifuge tube

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Centrifuge the tube at approximately 16,000 × g for 10 minutes

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With a Pasteur pipette, remove and discard the supernatant; with a digital micropipette, add 500 µL of TE buffer and resuspend the pellet by vortexing

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Briefly examine the C. cayetanensis positive control slide before and occasionally during examination of the samples

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With a digital micropipette, transfer 30 µL of the resuspended pellet to a standard microscope slide (smaller volumes may be needed for more dilute samples); place a coverslip over the drop and seal it with cytoseal 60; using the 40× objective, carefully examine the entire slide (left to right and top to bottom) under UV light — C. cayetanensis oocysts autofluoresce and appear as bright, pale blue circles with a darker blue inside, measuring 8–10 µm in diameter; other debris of a similar colour is usually quickly rejected by size and shape; if a filter system suitable for the fluorochrome FITC (450–490 nm excitation filter) is available, confirm the identification — the oocysts fluoresce green at that wavelength

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Confirm the identity of oocysts under brightfield using the 40× objective or, if necessary, the 100× oil-immersion objective; the degree of sporulation may be determined at the same time — an unsporulated oocyst has a granular cytoplasm, while a fully sporulated oocyst contains two oval sporocysts each with two sporozoites (which may or may not be visible)

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If possible, take a colour photograph, or capture an image by computer image analysis, of each positive or questionable microscope slide; retain the corresponding resuspended pellet for further examination if necessary

6. Recording of results

• Record in a log book all pertinent information for each berry sample received (berries received from, date received, origin, lot numbers, etc.)
• If the raspberries were in poor condition or any other problems were encountered during the examination, note these in the log book
• Record the total weight and number of berries examined for each sample
• Label each tube and microscope slide carefully to correspond with the berry sample being tested
• Along with the examiner’s name and the date of examination, record the results for each slide as negative or positive (confirmed or unconfirmed)
• If positive, indicate the total number (or an approximation) of oocysts observed on the slide
• If morphological confirmation was possible, record whether the oocysts were sporulated, unsporulated, or both

7. Interpretation

• There are currently no HPFB guidelines for parasites on raspberries; the consumption of fresh raspberries containing any number of C. cayetanenis oocysts (as the source prints it) may be considered a health hazard
• The minimum infectious dose for C. cayetanensis is unknown but is probably relatively low, as it is for the related parasite Cryptosporidium spp.
• Little data are available on the heat and freezing resistance of C. cayetanensis oocysts; preliminary studies indicate that contaminated berries which have been heated (e.g. bakery products) may be safe to consume, and likewise berries that have been commercially frozen or subsequently frozen by the consumer (e.g. freezer jams)
• While good practice, washing raspberries is difficult given their delicate nature and is not fully effective in removing C. cayetanensis oocysts

References

• Health Canada, Compendium of Analytical Methods, Volume 5 — Laboratory Procedure OPFLP-05, September 2008: “Isolation and Identification of Cyclospora cayetanensis Oocysts from Fresh Raspberries” (replaces ExFLP-3, April 1999).
• Ortega, Y. R., et al. 1993. Cyclospora species — a new protozoan pathogen of humans. New England Journal of Medicine, 328: 1308–1312.
• Eberhard, M. L. and M. J. Arrowood. 2002. Cyclospora spp. Current Opinion in Infectious Diseases, 15: 519–522.
• DeGraw, E., et al. 1997. Update: outbreaks of cyclosporiasis — United States and Canada, 1997. Morbidity and Mortality Weekly Report, 46: 521–523.
• Herwaldt, B. L., et al. 1997. An outbreak in 1996 of cyclosporiasis associated with imported raspberries. New England Journal of Medicine, 336: 1548–1556.
• Toronto Public Health et al. 1998. Outbreak of cyclosporiasis — Ontario, Canada, May 1998. Morbidity and Mortality Weekly Report, 47: 806–809.
• Cáceres, V. M., et al. 1998. A foodborne outbreak of cyclosporiasis caused by imported raspberries. Journal of Family Practice, 47: 231–234.
• Fleming, C. A., et al. 1998. A foodborne outbreak of Cyclospora cayetanensis at a wedding. Archives of Internal Medicine, 158: 1121–1125.
• Manuel, D. G., et al. 1999. The first reported cluster of food-borne cyclosporiasis in Canada. Canadian Journal of Public Health, 90: 399–402.
• Herwaldt, B. L., et al. 1999. The return of Cyclospora in 1997: another outbreak of cyclosporiasis in North America associated with imported raspberries. Annals of Internal Medicine, 130: 210–220.
• Manuel, D. G., et al. 2000. An outbreak of cyclosporiasis in 1996 associated with consumption of fresh berries — Ontario. Canadian Journal of Infectious Diseases, 11: 86–92.
• Herwaldt, B. L., et al. 2000. Cyclospora cayetanensis: a review, focusing on the outbreaks of cyclosporiasis in the 1990s. Clinical Infectious Diseases, 31: 1040–1057.
• Ho, A. Y., et al. 2002. Outbreak of cyclosporiasis associated with imported raspberries, Philadelphia, Pennsylvania, 2000. Emerging Infectious Diseases, 8: 783–788.
• Mansfield, L. S. and A. A. Gajadhar. 2004. Cyclospora cayetanensis, a food- and waterborne coccidian parasite. Veterinary Parasitology, 126: 73–90.