Lorna J. Parrington

The potential for zoonotic transmission of Giardia duodenalis and Cryptosporidium spp. from beef and dairy cattle in Ontario, Canada.

The objective of this study was to compare the occurrence and the genotypes and species of Giardia duodenalis and Cryptosporidium spp. in beef and dairy cattle from farms in the Regional Municipality of Waterloo, Ontario, in an effort to determine the potential for zoonotic transmission from these animals. Pooled manure samples were collected from 45 dairy cattle farms and 30 beef cattle farms. The presence of Giardia cysts and Cryptosporidium oocysts was determined by immunofluorescence microscopy, while nested-PCR and DNA sequencing were used to determine genotypes and species. The overall farm prevalence was very high for both Giardia and Cryptosporidium, and was similar for dairy cattle farms (96 and 64%, respectively) and beef cattle farms (97 and 63%, respectively). However, on dairy cattle farms, G. duodenalis and Cryptosporidium spp. were detected in 44% and 6% of total pooled pen manure samples, respectively, with the occurrence of both parasites being generally higher in calves than in older animals. Most Giardia isolates were identified as either the host-adapted genotype G. duodenalis Assemblage E or the zoonotic Assemblage B. Cryptosporidium parvum and Cryptosporidium andersoni were the most frequently identified species in dairy cattle, while the non-zoonotic species Cryptosporidium ryanae and Cryptosporidium bovis were also found. On beef cattle farms, 72% and 27% of the total pooled pen manure samples were positive for Giardia and Cryptosporidium, respectively, with no obvious correlation with age. All Giardia isolates in beef cattle were identified as G. duodenalis Assemblage E, while all Cryptosporidium isolates were identified by sequence analysis as C. andersoni, although microscopic analyses, and subsequent restriction fragment length polymorphism analyses, indicated that other Cryptosporidium species were also present. The results of this study indicate that although Giardia and Cryptosporidium were identified in a higher overall percentage of the pooled beef cattle manure samples than in dairy cattle, firmly established zoonotic genotypes and species were much more common in dairy cattle than in beef cattle in this region. Dairy cattle, and especially dairy calves, may, therefore, pose a greater risk of infection to humans than beef cattle. However, these results may also provide evidence of potential zooanthroponotic transmission (human to animal).

Giardia duodenalis and Cryptosporidium Spp. in the Intestinal Contents of Ringed Seals (Phoca hispida) and Bearded Seals (Erignathus barbatus) in Nunavik, Quebec, Canada

The prevalence of Giardia duodenalis and Cryptosporidium spp. was determined for ringed and bearded seals harvested for food in the Nunavik region in northern Quebec, Canada. Flow cytometric results demonstrated that G. duodenalis was present in the intestinal contents of 80% of the ringed seals and 75% of the bearded seals tested, while Cryptosporidium spp. were present in 9% of the ringed seals and none of the bearded seals. Prevalence of both parasites was highest in animals less than 1 yr of age. Giardia sp. isolates from ringed seals were identified as G. duodenalis Assemblage B, which is commonly identified in human infections. The high prevalence of G. duodenalis in ringed seals, and the presence of Assemblage B in these animals, highlights the potential for zoonotic transmission to the Inuit people, who consume dried seal intestines and uncooked seal meat.

Detection of Cyclospora, Cryptosporidium, and Giardia in ready-to-eat packaged leafy greens in Ontario, Canada.

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.

Detection of Cyclospora cayetanensis Oocysts in Human Fecal Specimens by Flow Cytometry

ABSTRACT A diagnosis of cyclosporiasis typically involves stool examinations for the presence of Cyclospora oocysts by means of microscopy. In recent years, flow cytometry has been gaining in popularity as a novel method of detecting pathogens in environmental and clinical samples. The present study is an evaluation of a flow cytometric method for the detection and enumeration of Cyclospora oocysts in human fecal specimens associated with food-borne outbreaks of cyclosporiasis in Ontario, Canada. Flow cytometry results were generally very comparable to the original microscopy results for these specimens, in terms of both presence or absence of oocysts and relative oocyst concentrations. Of the 34 fecal specimens confirmed positive for Cyclospora by microscopy, 32 were also found positive by flow cytometry, and 2 others were considered equivocal. Of the eight fecal specimens reported to be negative by microscopy, two were found positive by flow cytometry and five others were considered equivocal. These two flow cytometry-positive samples and one of the equivocal samples were confirmed by microscopic reexamination, suggesting that flow cytometry may be more sensitive than microscopy. While the sample preparation time for flow cytometry is similar to or slightly longer than that for microscopy, the actual analysis time is much shorter. Further, because flow cytometry is largely automated, an analysts levels of fatigue and expertise will not influence results. Flow cytometry appears to be a useful alternative to microscopy for the screening of large numbers of stool specimens for Cyclospora oocysts, such as in an outbreak situation.

Detection and characterization of Giardia duodenalis and Cryptosporidium spp. on swine farms in Ontario, Canada.

As part of the C-EnterNet surveillance program of the Public Health Agency of Canada, 122 pooled swine manure samples from 10 farms in Ontario, Canada were collected and tested for Giardia and Cryptosporidium. Giardia duodenalis cysts and Cryptosporidium spp. oocysts were detected using immunofluorescence microscopy. Nested-polymerase chain reaction protocols were performed to amplify the small subunit rRNA gene and the β-giardin gene for G. duodenalis, and the small subunit rRNA gene and the heat shock protein-70 gene for Cryptosporidium spp. The DNA amplicons were sequenced to determine genotypes and species. A mixed multivariable method was used to compare the presence of Giardia and Cryptosporidium in different stages of production. Both Giardia cysts and Cryptosporidium oocysts were present on all tested farms, with 50.8% of the samples positive for G. duodenalis and 44.3% positive for Cryptosporidium spp. by microscopy, and 66.4% and 55.7%, respectively, positive by polymerase chain reaction (PCR). No significant agreement was observed between microscopy and PCR method to detect Giardia and Cryptosporidium (p<0.05). The prevalence of Giardia in manure pits and finisher pigs did not differ (p>0.05), however, it was less frequent (odds ratio, OR=0.21 [0.07, 0.63]) among sows. Cryptosporidium was more likely (OR=3.6 [1.3, 9.9]) to be detected in manure pits and weaners (OR=3.3 [1.1, 10.0]) compared to finisher pigs, and it was less frequent (OR=0.06 [0.007, 0.55]) in sows than in finishers (p<0.05). DNA sequencing demonstrated that 92.1% of the Giardia isolates were Assemblage B and 7.9% were Assemblage E. The most prevalent Cryptosporidium were Cryptosporidium parvum (55.4%), and Cryptosporidium sp. pig genotype II (37.5%). These findings indicate that the occurrence of zoonotic isolates of G. duodenalis and Cryptosporidium is very high on swine farms in southern Ontario, and that there is a potential for transmission between swine and humans by means of cyst and oocyst contaminated water or foods.

Immunomagnetic Separation Significantly Improves the Sensitivity of Polymerase Chain Reaction in Detecting Giavdia Duodenalis and Cryptosporidium spp. in Dairy Cattle

The effectiveness of molecular methods for the detection of species of Giardia and Cryptosporidium in fecal samples is often reduced by low or intermittent cyst and oocyst shedding, and/or the presence of polymerase chain reaction (PCR) inhibitors. The present study investigates the use of immunomagnetic separation (IMS) as an additional concentration step before PCR in the detection of these common protozoan parasites in dairy cattle. The IMS-PCR assays were optimized for amplifying fragments of the 16S ribosomal RNA (rRNA), β-giardin, and glutamate dehydrogenase (GDH) genes of Giardia duodenalis, as well as fragments of the 18S rRNA, heat shock protein (HSP)-70, and Cryptosporidium oocyst wall protein (COWP) genes of Cryptosporidium spp. In all cases, IMS-PCR was more sensitive than PCR alone. A significantly greater number of Giardia–positive samples were identified using IMS-PCR of the 16S rRNA gene (P < 0.01) and of the GDH gene (P < 0.01), as compared with PCR without any additional concentration step. In the case of Cryptosporidium, IMS-PCR of the COWP gene (P = 0.02) resulted in a significantly greater number of positives than did PCR without the IMS concentration step. The greatest number of positives, however, was obtained using IMS-PCR to amplify a portion of the 16S rRNA gene of Giardia and a portion of the HSP-70 gene of Cryptosporidium. A further comparison of the optimized IMS-PCR assays to immunofluorescence microscopy suggested that the IMS-PCR assays were considerably more sensitive than microscopy was in the detection of Giardia cysts and Cryptosporidium oocysts in fecal samples.

Comparison of flow cytometry and immunofluorescence microscopy for the detection of Giardia duodenalis in bovine fecal samples

The performance of flow cytometry (FC) was compared with immunofluorescence microscopy (IM) for detection of Giardia duodenalis in bovine feces. Samples from 36 adult dairy cows and 208 dairy calves were collected. Flow cytometry test characteristics were calculated using continuous, ordinal, and dichotomized results. Spearman correlation coefficients comparing the results of the 2 tests were 0.47 and 0.68 for cows and calves, respectively. Using IM as indicative of presence or absence of G. duodenalis cysts in each sample, likelihood ratios of FC results with 0, 1, and ≥2 gated events indicated that samples with 1 gated event were likely to be positive in the cows but not in the calves. Immunofluorescence microscopy detected G. duodenalis in 69.7% and 48.1% of cows and calves, respectively. When dichotomizing the FC results at a cutoff point of 1 or 2 gated events, 46.3% and 19.9% of the cow and 51.9% and 35.1% of the calf samples, respectively, were classified as G. duodenalis-positive. Relative to IM, the sensitivity in the cows was 0.59 and 0.28, respectively, and 0.76 and 0.64, respectively, in the calves. At a cut-off point of 1, 65.7% and 73.1% of the cow and calf samples, respectively, were correctly classified in FC, and at a cut-off point of 2, 49.3% and 78.4% were correctly classified in the cows and calves, respectively. Flow cytometry was less sensitive than IM. Possible reasons and research needed to improve FC for G. duodenalis detection are discussed.