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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.

This pilot study was aimed at documenting the presence of fecal indicators and enteric pathogens in blue mussels (Mytilus edulis) from 6 communities in Nunavik, Quebec. One to four 2 kg samples of mussels were collected at low tide in each community. Samples were investigated by enumeration methods for the fecal indicators enterococci, Escherichia coli, F-specific coliphages, Clostridium perfringens, and by molecular identification for the pathogens norovirus, Salmonella spp., Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, verocytotoxin-producing E. coli (particularly serovar O157:H7), Shigella spp., and Yersinia enterocolitica. In 5 communities, the presence of Giardia duodenalis and Cryptosporidium spp. was also tested by microscopy and molecular methods and that of Toxoplasma gondii was tested by molecular methods. Apart from small quantities of Clostridium perfringens in 2 samples, no bacterial or viral pathogens were detected in the mussels. Toxoplasma gondii was also not detected. However, G. duodenalis and Cryptosporidium spp. were present in 18% and 73% of the samples investigated for these pathogens, respectively. When considering the indicators and the viral and bacterial pathogens investigated, the mussels examined were of good microbiological quality, but considering the presence of potentially zoonotic protozoa, it should be recommended that consumers cook the molluscs well before eating them.

Giardia duodenalis and Cryptosporidium spp. infections, and the patterns of cyst and oocyst shedding, were observed in a herd of dairy calves in Ontario over a period of 3 mo. Cysts and oocysts were detected and enumerated in fecal samples using immunofluorescence microscopy; Giardia and Cryptosporidium DNA was detected using the polymerase chain reaction. The prevalence of G. duodenalis increased during the course of the study, reaching a peak of 93.1% when calves were 43 to 54 d old, and then decreased. Conversely, Cryptosporidium spp. prevalence was highest (75.9%) when calves were 11 to 22 d old, and subsequently decreased. The numbers of cysts and oocysts shed per gram of feces were positively correlated over time with the respective prevalence rates. Along with genotyping data, temporal changes in prevalence and shedding patterns should be considered when testing dairy calves for the presence and concentrations of cysts and oocysts, and when considering the potential for zoonotic transmission.

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.

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 > or = 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 cut-off 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.

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 analyst's 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.

Three enumeration methods for Escherichia coli in foods, the Health Protection Branch most-probable-number (MPN) method MFHPB-19, a hydrophobic grid membrane filter method MFHPB-26 (HGMF-indole), and a hydrophobic grid membrane filter method utilizing 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide in a (modified) mFC agar (HGMF-FC-BCIG) were compared in 80 food samples that included naturally and artificially contaminated raw vegetables, mung bean and alfalfa sprouts, raw meats, and chicken carcass rinses. The number of samples confirmed as positive for E. coli were 44, 36, and 42 for the MPN, HGMF-indole, and HGMF-BCIG methods, respectively. By the MPN method, E. coli was detected in 3 samples at levels below the limits of detection of the HGMFs; but the MPN method was very time-consuming. With the HGMF-indole procedure E. coli was missed in 4 artificially contaminated samples. With the HGMF-FC-BCIG method E. coli was enumerated in 1 sample of bean sprouts missed by both the MPN and HGMF-indole procedures. High levels of indole-positive Klebsiella spp. in bean sprouts interfered with the HGMF-indole method, but the blue colonies of E. coli were easily observed in the HGMF-FC-BCIG method. Specificity of the HGMF-FC-BCIG method is high enough that routine confirmation should be unnecessary; however, confirmation of presumptive E. coli is easier since no lethal indole-staining step is involved. It appears to be a very simple method for quantifying E. coli in foods or carcass rinses.

Two versions of an electrically powered device (Rotorinser) to sample carcasses or other surfaces in situ for microbiological analysis and several different sampling protocols were evaluated against excision plus stomaching for ability to remove bacteria from pig skin and beef carcass tissue. Both devices sampled circular areas of approximately 14 cm 2 . Ten tissue samples were used for each set of conditions. Rotorinser bacterial removal efficiency was calculated as R/(R + S), where R is the Rotorinser count (CFU cm -2 ) and S is the count on stomached excised tissue after rotorinsing. Stomacher efficiencies were calculated as S1/(S1 + S2), where S1 is the first stomacher count of excised tissue and S2 is the count from a second stomaching. Both Rotorinsers were much better than traditional swabs. Rotorinser 1 gave removal efficiencies of 0.79 to 0.88 for beef, and 0.79 to 0.95 for pork. Prewetting surfaces for 5 min improved removal, but mixtures of enzymes did not. Rotorinser 2 applied with NaCl or NaCl-Tween 80 diluent for either 30 or 60 s was significantly better (0.93 and 0.98) than the stomacher (0.86) at removing aerobic mesophilic bacteria from pork skin. The Rotorinser causes negligible tissue damage and can be used on surfaces at any angle.

The AOAC International official action procedure for performing aerobic colony counts on hydrophobic grid membrane filters (HGMFs) uses Trypticase soy-fast green FCF agar (FGA) incubated for 48 h. Microbial growths are various shades of green on a pale green background, which can cause problems for automated as well as manual counting. HGMFs which had been incubated 24 or 48 h at 35 degrees C on Trypticase soy agar were flooded underneath with 1 to 2 ml of 0.1% triphenyltetrazolium chloride (TTC) solution by simply lifting one corner of the filter while it was still on the agar and adding the reagent. Microbial growths on HGMFs were counted after color had been allowed to develop for 15 min at room temperature. With representative foods, virtually all colonies stained pink to red. Automated electronic counts made by using the MI-100 HGMF Interpreter were easier and more reliable than control HGMF counts made by the AOAC International official action procedure. Manual counting was easier as well because of increased visibility of the microbial growths. Except in the case of dairy products, 24-h TTC counts did not differ significantly from 48-h FGA counts, whereas the FGA counts at 24 h were always significantly lower, indicating that for many food products the HGMF TTC flooding method permits aerobic colony counts to be made after 24 h.