Detection of Cyclospora, Cryptosporidium, and Giardia in Ready-to-Eat Packaged Leafy Greens in Ontario, Canada

Abstract

Numerous foodborne outbreaks of diarrheal illness associated with the consumption of produce contaminated with protozoan parasites have been reported in North America in recent years. The present study reports on the presence of Cyclospora, Cryptosporidium, and Giardia in precut salads and leafy greens purchased at retail in Ontario, Canada. A total of 544 retail samples were collected between April 2009 and March 2010 and included a variety of salad blends and individual leafy greens. Most of these products were grown in the United States, with some from Canada and Mexico. Parasites were eluted and concentrated before detection by PCR and immunofluorescence microscopy. DNA sequences were aligned with reference sequences in GenBank. Cyclospora spp. were identified by PCR-restriction fragment length polymorphism in nine (1.7 % ) samples and by DNA sequence analysis. Cryptosporidium spp. were identified in 32 (5.9%) samples; 29 were sequenced and aligned with the zoonotic species Cryptosporidium parvum. Giardia duodenalis was identified in 10 (1.8%) samples, and of the 9 samples successfully sequenced, 7 aligned with G. duodenalis assemblage B and 2 with assemblage A, both of which are also zoonotic. The presence of Cryptosporidium oocysts and Giardia cysts was confirmed in some of the PCR-positive samples using microscopy, while Cyclospora -like oocysts were observed in most of the Cyclospora PCR-positive samples. The relatively high prevalence of these parasites in packaged salads and leafy greens establishes a baseline for further studies and suggests a need for additional research with respect to the possible sources of contamination of these foods, the determination of parasite viability and virulence, and means to reduce foodborne transmission to humans.

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation

“…To directly assess the potential of microfluidic inertial separation chips as a concentration and separation device in food microbiological analyses, the Giardia inertial separation chip was incorporated into a method for the recovery and detection of G. duodenalis cysts from lettuce. Lettuce was chosen as an appropriate food matrix for initial testing, due to its relatively high prevalence of contamination (20,21), published methodologies, and relatively low number of background food particles, thus theoretically allowing for greater ease of cyst detection. A direct comparison to an effective, yet more conventional, lettuce sampling method (20) was performed in parallel.…”

“…The limit of detection (LOD) and percent recovery of cysts were assessed on lettuce samples artificially contaminated with G. duodenalis cysts (described above) for both the inertial separation method and a conventional method used by Dixon et al (20), considered the gold standard. Of the 14 total samples in each trial, half of the samples were eluted and concentrated using the inertial separation method, while the second half were eluted and concentrated in parallel using the conventional method of Dixon et al (20). Each sample was screened for the presence of G. duodenalis cysts by epifluorescence microscopy as described above.…”

“…G. duodenalis cysts were eluted from spiked lettuce and initially concentrated according to the method of Dixon et al (20), which is specific for the detection of Cyclospora, Cryptosporidium, and Giardia from ready-to-eat packaged leafy greens. In brief, the method involved weighing 25-g samples into 1-liter stomacher bags and adding 200 ml of elution buffer containing 1% PBS and 0.01% Tween 80, pH 7.4.…”

Emergent and re-emergent parasites in HIV-infected children: immunological and socio-environmental conditions that are involved in the transmission of Giardia spp. and Cryptosporidium spp.

“…For example, researchers have found Giardia spp., Cryptosporidium spp., and Cyclospora spp. in leafy vegetables that were produced in the United States, Canada, and Mexico (11) . Furthermore, these protozoans are remarkably resistant to common disinfectants and may survive in raw foods (2) (11) .…”

Loop-Mediated Isothermal Amplification: An Advanced Method for the Detection of Giardia

“…Waterborne distribution is estimated to be the main source of infection according to various studies of recent years [12,13,23]. The food-borne transmission to humans as well as through consumption of packed salads and/or green leaves by infected food-handlers has been reported [24,25]. Person-to-person contact among schoolchildren atending day care centers and crosscontamination from the staf to their households are also possible and have a signiicant epidemiological impact [26][27][28].…”