OPFLP-05 Isolation and Indentification of Cyclospora cayetanensis oocysts from fresh raspberries
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BCC9658493E04557B3FEF82CBD65E604 |
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0.03 |
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5 |
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日期： |
2024-7-30 |
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Published on the Food Directorate (Health Canada's) website at,http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index_e.html.,Government of Canada Gouvernement du Canada,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 :m 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,OPFLP-5,September 2008,-2-,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 ……
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