Isolation and Identification of Cyclospora Cayetanensis Oocysts from Fresh Raspberries

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Laboratory Procedure OPFLP-5
September 2008

HEALTH PRODUCTS AND FOOD BRANCH

OTTAWA

ISOLATION AND IDENTIFICATION OF CYCLOSPORA CAYETANENSIS OOCYSTS
FROM FRESH RASPBERRIES

Brent Dixon

Bureau of Microbial Hazards, Food Directorate
Postal Locator: 2204E
Health Canada, Ottawa, ON, K1A 0K9

e-mail: Brent_Dixon@hc-sc.gc.ca

1. Application

These laboratory procedures have been developed in collaboration with Laboratory Services Division, CFIA, and are applicable to the isolation and identification of Cyclospora cayetanensis oocysts from the surface of fresh raspberries.  These procedures are intended for use with small quantities of berries originating from consumer complaints, as well as for larger samples collected in routine inspections.  This revised method replaces ExFLP-3, dated April 1999.

2. Principle

This procedure involves the elution of C. cayetanensis oocysts, along with other debris, from fresh raspberries by means of gentle shaking in a stomacher bag containing a buffer solution.  The material eluted from the berries is concentrated through centrifugation and pooled, and the resuspended pellet is examined microscopically for the presence of C. cayetanensis oocysts. 

3. General characteristics of cyclospora cayetanensis

3.1 Basic Biology and Life Cycle

C. cayetanensis is a newly identified coccidian protozoan parasite (8.1) which appears to infect only humans. The transmission stage, known as an oocyst, is spherical and 8-10 µm in diameter. While unsporulated oocysts may have simply a granular cytoplasm, sporulated (viable) oocysts contain two oval shaped sporocysts each containing two sporozoites. Oocysts autofluoresce blue under UV light.

While a definitive life cycle has not yet been determined for C. cayetanensis , it is thought to be similar to other coccidian protozoan parasites (8.2). Unsporulated (immature) oocysts are shed into the environment with the faeces of infected individuals. Depending upon conditions such as temperature and humidity, the oocysts may sporulate, becoming mature and viable, within a few days or weeks. After being ingested, oocysts pass through the stomach and into the small intestine where they excyst and the individual sporozoites are released and enter cells lining the gut. Sporozoites then undergo asexual multiplication resulting in large numbers of merozoites which leave the cell and invade other host cells. Some merozoites undergo development into male and female gametes which undergo sexual reproduction producing unsporulated oocysts.

3.2 Transmission

C. cayetanensis was initially considered to be primarily a waterborne pathogen, transmitted by means of faecally contaminated water used for drinking. While this continues to be an important mode of transmission in endemic countries such as Nepal, Haiti, Peru, and Guatemala, foodborne outbreaks have been reported annually in North America since 1996. Epidemiological evidence implicated imported fresh raspberries in many of the earlier outbreaks (8.3 to 8.12). Subsequent outbreaks in North America have resulted from the consumption of oocyst contaminated mesclun (baby greens), basil, and possibly other fresh produce items. The means of contamination of foods may involve poor hygiene of workers, contaminated water used in irrigation, mixing pesticides, or washing produce, or some other method not yet identified. Person-to-person transmission (faecal-oral route) is unlikely due to the relatively long sporulation period required. Zoonotic transmission is also unlikely as animal infectivity studies have been largely unsuccessful.

3.3 Symptoms, Diagnosis and Treatment

The disease itself is known as cyclosporiasis and may result in diarrhea, nausea, vomiting, abdominal pain, fatigue, loss of appetite, etc. Symptoms generally appear about a week after ingestion of oocysts and may last for days or weeks if left untreated. Diagnosis usually involves a microcopic examination of stool samples for the presence of oocysts, but may also involve molecular techniques (8.13). Cyclosporiasis is effectively treated with the antibiotic bactrim.

4 Materials, Equipment And Reagents

4.1 Elution

4.1.1 stomacher bags or equivalent (with a capacity of at least 1 L)
4.1.2 TE buffer pH 7.4 (10 mM Tris-HCl, 1 mM EDTA)
4.1.3 orbital shaker with clips or other means to support stomacher bags

4.2 Concentration

4.2.1 gauze sponges (8-ply)
4.2.2 50 mL polypropylene conical centrifuge tubes
4.2.3 centrifuge - swinging bucket rotor with adapters for 50 mL tubes
4.2.4 25 mL pipettes and bulb for removing supernatant
4.2.5 Pasteur pipettes and bulbs
4.2.6 digital micropipettes (100 µL and 1000µL capacity) and appropriate tips for transferring resuspended pellets
4.2.7 1.5 mL polypropylene microcentrifuge tubes
4.2.8 microcentrifuge with adapters for 1.5 mL tubes
4.2.9 vortex mixer

4.3 Microscopy

4.3.1 standard microscope slides and 22x22 mm coverslips
4.3.2 cytoseal 60 (VWR Canlab) or clear nail polish for sealing coverslip
4.3.3 positive control slide of C. cayetanensis oocysts
4.3.4 compound microscope with 40 X and 100 X (oil immersion) objectives; capability of both brightfield and epifluorescence microscopy (330-380 nm ultraviolet excitation filter, 420 nm barrier filter, and mercury lamp).

5. Isolation and identification procedures

5.1 Elution and Concentration of Oocysts

5.1.1 From each sample of fresh raspberries, weigh out approximately 50 g of raspberries into an appropriately labelled stomacher bag. If possible, avoid using raspberries which are either very large or small, or which are obviously damaged or soft.

5.1.2 Carefully pour 120 mL TE buffer (pH 7.4) into each stomacher bag. Avoid pouring buffer directly on the berries.

5.1.3 Place bags on orbital shaker for 2 minutes at 120 rpm. Shaking berries too vigorously, or for too long, will result in excessive debris in the buffer which will in turn produce a pellet that is difficult to handle.

5.1.4 Remove stomacher bags from shaker and carefully decant the liquid from each bag into three labelled 50 mL conical centrifuge tubes. The liquid should be decanted through a gauze sponge to exclude berries and other large debris.

5.1.5 Centrifuge the tubes at 1,500 x g for 10 minutes.

5.1.6 With a 25 mL pipette, remove and discard supernatant leaving 1-2 mL in each tube to resuspend pellet (by vortexing).

5.1.7 Using a Pasteur pipette, transfer resuspended pellets from two tubes into the third tube and then, using the same pipette, rinse one of the empty tubes with 1-2 mL of TE buffer, transfer to the second empty tube and rinse again. Finally, transfer the rinse liquid to the third centrifuge tube containing the pooled pellets.

5.1.8 Centrifuge the tube containing the pooled resuspended pellets at 1,500 x g for 10 minutes.

5.1.9 Using a Pasteur pipette, remove and discard supernatant leaving approximately 0.5 mL to resuspend the pellet. Transfer the resuspended pellet into a 1.5 mL microcentrifuge tube.

5.1.10 Centrifuge the tube at approximately 16,000 x g for 10 minutes.

5.1.11 Using a Pasteur pipette, remove and discard supernatant. With a digital micropipette, add 500 µL TE buffer to the tube and resuspend pellet by vortexing.

5.2 Identification of Oocysts

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

5.2.2 Using a digital micropipette, transfer 30 µL of the resuspended pellet to a standard microscope slide. Smaller volumes may be required in more dilute samples. Place a coverslip over the drop and seal it with cytoseal 60. Using a 40 X objective, carefully examine the entire slide (left to right and top to bottom) under UV light. C. cayetanensis oocysts will autofluoresce and appear as bright, pale blue circles, with a darker blue inside. They should measure 8-10 µm in diameter. Other debris may be observed with a similar colour but can usually be quickly rejected based on size and shape. If a filter system suitable for the fluorochrome FITC (450-490 nm excitation filter) is available, a confirmation of identification can be made as oocysts will fluoresce a green colour at this wavelength.

5.2.3 Confirm the identity of oocysts under brightfield using either 40 X or, if necessary, 100 X (oil immersion) objective. The degree of sporulation may also be determined at this point. An unsporulated oocyst will have a granular cytoplasm, while a fully sporulated oocyst will contain two oval shaped sporocysts each containing two sporozoites (which may or may not be visible).

5.2.4 If possible, a colour photograph should be taken, or an image captured by computer image analysis, of each positive or questionable microscope slide. The corresponding resuspended pellet should also be retained for further examination, if necessary.

6. Recording results

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

7. Interpretation

While there are currently no HPFB guidelines for parasites on raspberries, the consumption of fresh raspberries containing any number of C. cayetanenis oocysts 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 yet available concerning the heat and freezing resistance of C. cayetanensis oocysts. However, preliminary studies indicate that contaminated berries which have been heated (eg. bakery products) may be safe to consume. Similarly, contaminated berries which have been commercially frozen, or subsequently frozen by the consumer (eg. freezer jams), may be safe to consume. While good practice, washing of raspberries is difficult, given their delicate nature, and is not fully effective in removing C. cayetanensis oocysts.

8. References

8.1 Ortega, Y. R., et al. 1993. Cyclospora species - a new protozoan pathogen of humans. New Engl. J. Med. 328: 1308-1312.

8.2 Eberhard, M. L. and M. J. Arrowood. 2002. Cyclospora spp. Curr. Opin. Infect. Dis. 15: 519-522.

8.3 DeGraw, E., et al. 1997. Update: outbreaks of cyclosporiasis - United States and Canada, 1997. Morb. Mort. Weekly Rep. 46: 521-523.

8.4 Herwaldt, B. L., et al. 1997. An outbreak in 1996 of cyclosporiasis associated with imported raspberries. New Engl. J. Med. 336:1548-1556.

8.5 Toronto Public Health et al., 1998. Outbreak of cyclosporiasis - Ontario, Canada, May 1998. Morb. Mort. Weekly Rep. 47:806-809.

8.6 Cáceres, V. M., et al., 1998. A foodborne outbreak of cyclosporiasis caused by imported raspberries. J. Family Pract. 47: 231-234.

8.7 Fleming, C. A., et al. 1998. A foodborne outbreak of Cyclospora cayetanensis at a wedding. Arch. Intern. Med. 158: 1121-1125.

8.8 Manuel, D. G., et al. 1999. The first reported cluster of food-borne cyclosporiasis in Canada. Can. J. Publ. Health 90: 399-402.

8.9 Herwaldt, B. L., et al. 1999. The return of Cyclospora in 1997: another outbreak of cyclosporiasis in North America associated with imported raspberries. Ann. Int. Med. 130: 210-220.

8.10 Manuel, D. G., et al. 2000. An outbreak of cyclosporiasis in 1996 associated with consumption of fresh berries - Ontario. Can. J. Infect. Dis. 11: 86-92.

8.11 Herwaldt, B. L., et al. 2000. Cyclospora cayetanensis: a review, focusing on the outbreaks of cyclosporiasis in the 1990s. Clin. Infect. Dis. 31: 1040-1057.

8.12 Ho, A. Y., et al. 2002. Outbreak of cyclosporiasis associated with imported raspberries, Philadelphia, Pennsylvania, 2000. Emerg. Infect. Dis. 8: 783-788.

8.13 Mansfield, L. S. and A. A. Gajadhar. 2004. Cyclospora cayetanensis, a food- and waterborne coccidian parasite. Vet. Parasitol. 126: 73-90.