Publications by authors named "Brent R Dixon"

25 Publications

  • Page 1 of 1

A cloth-based hybridization array system for rapid detection of the food- and waterborne protozoan parasites , spp. and .

Food Waterborne Parasitol 2021 Sep 11;24:e00130. Epub 2021 Aug 11.

Bureau of Microbial Hazards, Food Directorate, Health Canada, Ottawa, ON K1A 0K9, Canada.

Protozoan parasites in food or water samples are generally detected using microscopy or PCR followed by Sanger sequencing. However, microscopy is subjective, requires a high degree of expertise and has limited sensitivity, while DNA sequencing requires expensive and specialized equipment and facilities. This study describes a cloth-based hybridization array system (CHAS) that is an alternative to Sanger sequencing to confirm PCR-positive samples. CHAS is an inexpensive, rapid and reliable method for the simultaneous detection of multiple protozoan parasite species based on the colorimetric detection of PCR amplicons on a polyester cloth. PCR primers and CHAS hybridization probes were developed to detect the protozoan parasites , spp. and . In addition, CHAS probes were designed for the differentiation of Assemblages A and B. In artificially contaminated fresh produce (lettuce, parsley) and water samples (river water, wastewater), this CHAS assay allowed for the successful detection of , spp., and . The present study demonstrates that the CHAS detection method is a simple and inexpensive alternative to DNA sequencing for the confirmation of PCR-positive results in laboratories testing for parasites in food or water samples. This assay may also be beneficial in developing countries, where DNA sequencing facilities may not be readily available.
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http://dx.doi.org/10.1016/j.fawpar.2021.e00130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8379661PMC
September 2021

A review of Cryptosporidium spp. and their detection in water.

Water Sci Technol 2021 Jan;83(1):1-25

C.R.E.M. Co Labs, Units 1-2, 3403 American Drive, Mississauga, ON, Canada, L4V 1T4.

Cryptosporidium spp. are one of the most important waterborne pathogens worldwide and a leading cause of mortality from waterborne gastrointestinal diseases. Detection of Cryptosporidium spp. in water can be very challenging due to their low numbers and the complexity of the water matrix. This review describes the biology of Cryptosporidium spp. and current methods used in their detection with a focus on C. parvum and C. hominis. Among the methods discussed and compared are microscopy, immunology-based methods using monoclonal antibodies, molecular methods including PCR (polymerase chain reaction)-based assays, and emerging aptamer-based methods. These methods have different capabilities and limitations, but one common challenge is the need for better sensitivity and specificity, particularly in the presence of contaminants. The application of DNA aptamers in the detection of Cryptosporidium spp. oocysts shows promise in overcoming these challenges, and there will likely be significant developments in aptamer-based sensors in the near future.
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http://dx.doi.org/10.2166/wst.2020.515DOI Listing
January 2021

Highly sensitive magnetic-microparticle-based aptasensor for Cryptosporidium parvum oocyst detection in river water and wastewater: Effect of truncation on aptamer affinity.

Talanta 2021 Jan 5;222:121618. Epub 2020 Sep 5.

Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, K1S 5B6, Canada. Electronic address:

Many methods have been reported to detect Cryptosporidium parvum (C. parvum) oocysts in the water environment using monoclonal antibodies. Herein, we report the use of DNA aptamers as an alternative ligand. We present the highly sensitive detection of C. parvum oocysts in wastewater samples based on aptamer-conjugated magnetic beads. A previously selected DNA aptamer (R4-6) that binds to C. parvum oocysts with high affinity and selectivity was rationally truncated into two minimer aptamers (Min_Crypto1 and Min_Crypto2), and conjugated to micro-magnetic beads. In flow cytometry tests with phosphate buffer, river water, and wastewater samples, both the minimers showed improved affinity and specificity toward C. parvum oocysts than the parent R4-6. Moreover, Min_Crypto2 showed higher affinity to its target than the parent aptamer when testing in wastewater, indicating superior binding properties in a complex matrix. Using a fluorescence microplate-based assay, and when incubated with different numbers of oocysts, Min_Crypto2 showed a limit of detection as low as 5 C. parvum oocysts in 300 μL of wastewater. Results described here indicate that Min_Crypto2 has superior specificity and sensitivity for the detection of C. parvum oocysts, and has a strong potential to be used successfully in a sensor.
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http://dx.doi.org/10.1016/j.talanta.2020.121618DOI Listing
January 2021

Giardia duodenalis in humans and animals - Transmission and disease.

Authors:
Brent R Dixon

Res Vet Sci 2021 Mar 30;135:283-289. Epub 2020 Sep 30.

Bureau of Microbial Hazards, Food Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, Ontario K1A 0K9, Canada. Electronic address:

Giardia duodenalis is a protozoan parasite infecting the upper intestinal tract of humans, as well as domestic and wild animals worldwide. Transmission of giardiasis occurs through the faecal-oral route, and may be either direct (i.e., person-to-person, animal-to-animal or zoonotic) or indirect (i.e., waterborne or foodborne). While asymptomatic infections are common in both humans and animals, a wide range of enteric symptoms have been reported, along with extra-intestinal and post-infectious complications. A definitive diagnosis of giardiasis is generally made by detection of cysts in stool specimens through microscopical examination of wet mounts, or through the use of permanent or fluorescent antibody stains. More recently, molecular methods have become popular for diagnosis and for testing environmental samples. Symptomatic giardiasis is often treated to reduce the duration of symptoms, to prevent complications, and to minimize transmission of the parasite to other hosts. Direct faecal-oral transmission of giardiasis can be largely controlled thorough improved hygiene and sanitation. In the case of waterborne transmission, a multiple barrier approach, including limiting access of people and animals to watersheds and reservoirs, and treatment using flocculation, filtration and disinfection, is necessary to minimize the risk. Since foodborne transmission is often associated with the consumption of fresh produce, a number of control measures can be taken during pre- and post-harvest, as well as at the food handler/consumer level to minimize the risk of contamination, or for removing or inactivating parasites. Good husbandry and farm management practices are important in controlling the spread of giardiasis in livestock and companion animals.
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http://dx.doi.org/10.1016/j.rvsc.2020.09.034DOI Listing
March 2021

Benchmarking hybrid assemblies of Giardia and prediction of widespread intra-isolate structural variation.

Parasit Vectors 2020 Feb 28;13(1):108. Epub 2020 Feb 28.

Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.

Background: Currently available short read genome assemblies of the tetraploid protozoan parasite Giardia intestinalis are highly fragmented, highlighting the need for improved genome assemblies at a reasonable cost. Long nanopore reads are well suited to resolve repetitive genomic regions resulting in better quality assemblies of eukaryotic genomes. Subsequent addition of highly accurate short reads to long-read assemblies further improves assembly quality. Using this hybrid approach, we assembled genomes for three Giardia isolates, two with published assemblies and one novel, to evaluate the improvement in genome quality gained from long reads. We then used the long reads to predict structural variants to examine this previously unexplored source of genetic variation in Giardia.

Methods: With MinION reads for each isolate, we assembled genomes using several assemblers specializing in long reads. Assembly metrics, gene finding, and whole genome alignments to the reference genomes enabled direct comparison to evaluate the performance of the nanopore reads. Further improvements from adding Illumina reads to the long-read assemblies were evaluated using gene finding. Structural variants were predicted from alignments of the long reads to the best hybrid genome for each isolate and enrichment of key genes was analyzed using random genome sampling and calculation of percentiles to find thresholds of significance.

Results: Our hybrid assembly method generated reference quality genomes for each isolate. Consistent with previous findings based on SNPs, examination of heterozygosity using the structural variants found that Giardia BGS was considerably more heterozygous than the other isolates that are from Assemblage A. Further, each isolate was shown to contain structural variant regions enriched for variant-specific surface proteins, a key class of virulence factor in Giardia.

Conclusions: The ability to generate reference quality genomes from a single MinION run and a multiplexed MiSeq run enables future large-scale comparative genomic studies within the genus Giardia. Further, prediction of structural variants from long reads allows for more in-depth analyses of major sources of genetic variation within and between Giardia isolates that could have effects on both pathogenicity and host range.
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http://dx.doi.org/10.1186/s13071-020-3968-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048089PMC
February 2020

, sp. and like parasites in seals from northern and eastern Canada: potential risk to consumers.

Food Waterborne Parasitol 2019 Dec 2;17:e00067. Epub 2019 Nov 2.

Bureau of Microbial Hazards, Food Directorate, Health Canada, Ottawa, ON, K1A 0K9, Canada.

Zoonotic parasites of seals that are harvested for food may pose a health risk when seal meat or organ tissues of infected animals are eaten raw or undercooked. In this study, 124 tissue samples from 81 seals, comprising four species, were collected from northern and eastern Canada. Tissues from 23 ringed seals (), 8 hooded seals (), 21 harp seals (), and 29 grey seals () were tested for parasites of the Sarcocystidae family including , spp., and spp. using nested PCR followed by Sanger sequencing. DNA was present in 26% of ringed seals, 63% of hooded seals, 57% of harp seals, and 31% of grey seals. sp. DNA was found in 9% of ringed seals, 13% of hooded seals, 14% of harp seals, and 4% of grey seals, while like DNA was present in 26% of ringed seals. While it is unclear how pinnipeds may become infected with these protozoans, horizontal transmission is most likely. However, one harp seal pup (4 days old) was PCR-positive for , suggesting vertical transmission may also occur. Phylogenetic analysis of the 18S gene region indicates that sp. in these seals belongs to a unique genotype. Furthermore, this study represents a new host report for in harp seals, a new host and geographic report for like parasites in ringed seals, and four new hosts and geographic reports for sp. These results demonstrate that parasites of the Sarcocystidae family are prevalent in northern and eastern Canadian seals. While the zoonotic potential of sp. and the -like parasite are unclear, consumption of raw or undercooked seal meat or organ tissues pose a risk of infection to consumers.
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http://dx.doi.org/10.1016/j.fawpar.2019.e00067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033983PMC
December 2019

Multi-scale occupancy approach to estimate Toxoplasma gondii prevalence and detection probability in tissues: an application and guide for field sampling.

Int J Parasitol 2016 08 4;46(9):563-70. Epub 2016 May 4.

Department of Veterinary Microbiology, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan S7N 5B4, Canada.

Increasingly, birds are recognised as important hosts for the ubiquitous parasite Toxoplasma gondii, although little experimental evidence exists to determine which tissues should be tested to maximise the detection probability of T. gondii. Also, Arctic-nesting geese are suspected to be important sources of T. gondii in terrestrial Arctic ecosystems, but the parasite has not previously been reported in the tissues of these geese. Using a domestic goose model, we applied a multi-scale occupancy framework to demonstrate that the probability of detection of T. gondii was highest in the brain (0.689, 95% confidence interval=0.486, 0.839) and the heart (0.809, 95% confidence interval=0.693, 0.888). Inoculated geese had an estimated T. gondii infection probability of 0.849, (95% confidence interval=0.643, 0.946), highlighting uncertainty in the system, even under experimental conditions. Guided by these results, we tested the brains and hearts of wild Ross's Geese (Chen rossii, n=50) and Lesser Snow Geese (Chen caerulescens, n=50) from Karrak Lake, Nunavut, Canada. We detected 51 suspected positive tissue samples from 33 wild geese using real-time PCR with melt-curve analysis. The wild goose prevalence estimates generated by our multi-scale occupancy analysis were higher than the naïve estimates of prevalence, indicating that multiple PCR repetitions on the same organs and testing more than one organ could improve T. gondii detection. Genetic characterisation revealed Type III T. gondii alleles in six wild geese and Sarcocystis spp. in 25 samples. Our study demonstrates that Arctic nesting geese are capable of harbouring T. gondii in their tissues and could transport the parasite from their southern overwintering grounds into the Arctic region. We demonstrate how a multi-scale occupancy framework can be used in a domestic animal model to guide resource-limited sample collection and tissue analysis in wildlife. Secondly, we confirm the value of traditional occupancy in optimising T. gondii detection probability in tissue samples.
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http://dx.doi.org/10.1016/j.ijpara.2016.04.003DOI Listing
August 2016

Detection of Cryptosporidium parvum Oocysts on Fresh Produce Using DNA Aptamers.

PLoS One 2015 3;10(9):e0137455. Epub 2015 Sep 3.

Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, Canada.

There are currently no standard methods for the detection of Cryptosporidium spp., or other protozoan parasites, in foods, and existing methods are often inadequate, with low and variable recovery efficiencies. Food testing is difficult due to the low concentrations of parasites, the difficulty in eluting parasites from some foods, the lack of enrichment methods, and the presence of PCR inhibitors. The main objectives of the present study were to obtain DNA aptamers binding to the oocyst wall of C. parvum, and to use the aptamers to detect the presence of this parasite in foods. DNA aptamers were selected against C. parvum oocysts using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Ten rounds of selection led to the discovery of 14 aptamer clones with high affinities for C. parvum oocysts. For detecting parasite-bound aptamers, a simple electrochemical sensor was employed, which used a gold nanoparticle-modified screen-printed carbon electrode. This aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and the anti- C. parvum aptamer. Square wave voltammetry was employed to quantitate C. parvum in the range of 150 to 800 oocysts, with a detection limit of approximately 100 oocysts. The high sensitivity and specificity of the developed aptasensor suggests that this novel method is very promising for the detection and identification of C. parvum oocysts on spiked fresh fruits, as compared to conventional methods such as microscopy and PCR.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137455PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559477PMC
May 2016

Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

PLoS One 2015 3;10(9):e0136102. Epub 2015 Sep 3.

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada; Inflammation Research Network, University of Calgary, Calgary, Alberta, Canada; Host-Parasite Interactions, University of Calgary, Calgary, Alberta, Canada.

Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136102PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559405PMC
May 2016

Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in diarrhoeic patients in the Qikiqtani Region, Nunavut, Canada.

Int J Circumpolar Health 2015 19;74:27713. Epub 2015 Jun 19.

Bureau of Microbial Hazards, Food Directorate, Health Canada, Ottawa, ON, Canada;

Background: Although the prevalences of infection with the protozoan parasites Cryptosporidium spp. and Giardia duodenalis in humans appear to be relatively high in the Canadian North, their transmission patterns are poorly understood.

Objective: To determine the detection rate and the molecular characteristics of Cryptosporidium spp. and Giardia duodenalis in diarrhoeic patients in the Qikiqtani (Baffin Island) Region of Nunavut, Canada, in order to better understand the burden of illness and the potential mechanisms of transmission.

Study Design/methods: Diarrhoeal stool specimens (n=108) submitted to the Qikiqtani General Hospital for clinical testing were also tested for the presence of Cryptosporidium spp. and Giardia duodenalis using epifluorescence microscopy and polymerase chain reaction (PCR). DNA sequencing and restriction fragment length polymorphism (RFLP) analyses were performed on PCR-positive specimens to determine the species, genotypes and sub-genotypes of the parasites.

Results: Cryptosporidium was detected in 15.7% of the diarrhoeic patients, while Giardia was detected in 4.6%. DNA sequencing of a fragment of the small subunit rRNA gene indicated that all of the Cryptosporidium amplicons had a 100% homology to C. parvum, and a gp60 assay showed that all aligned with C. parvum sub-genotype IIa. Microsatellite analysis revealed 3 cases of sub-genotype IIaA15G2R1, 2 of IIaA15G1R and 1 case each of sub-genotypes IIaA16G1R1 and IIaA15R1. For Giardia, results based on the amplification of both the 16S rRNA gene and the gdh gene were generally in agreement, and both DNA sequencing and RFLP demonstrated the presence of the G. duodenalis Assemblage B genotype.

Conclusions: Both C. parvum and G. duodenalis Assemblage B were present in human diarrhoeal stool specimens from Nunavut, which was suggestive of zoonotic transmission, although human-to-human transmission cannot be ruled out. To fully understand the public health significance of the different Cryptosporidium and Giardia species and genotypes in diarrhoeic patients, it will be imperative to establish the extent of genetic diversity within these parasites through comprehensive studies of the molecular epidemiology of cryptosporidiosis and giardiasis in the Nunavut region.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475686PMC
http://dx.doi.org/10.3402/ijch.v74.27713DOI Listing
November 2016

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

Appl Environ Microbiol 2015 Jun 3;81(12):3925-33. Epub 2015 Apr 3.

Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-μm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy.
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http://dx.doi.org/10.1128/AEM.03868-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524145PMC
June 2015

Tradition and transition: parasitic zoonoses of people and animals in Alaska, northern Canada, and Greenland.

Adv Parasitol 2013 ;82:33-204

Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.

Zoonotic parasites are important causes of endemic and emerging human disease in northern North America and Greenland (the North), where prevalence of some parasites is higher than in the general North American population. The North today is in transition, facing increased resource extraction, globalisation of trade and travel, and rapid and accelerating environmental change. This comprehensive review addresses the diversity, distribution, ecology, epidemiology, and significance of nine zoonotic parasites in animal and human populations in the North. Based on a qualitative risk assessment with criteria heavily weighted for human health, these zoonotic parasites are ranked, in the order of decreasing importance, as follows: Echinococcus multilocularis, Toxoplasma gondii, Trichinella and Giardia, Echinococcus granulosus/canadensis and Cryptosporidium, Toxocara, anisakid nematodes, and diphyllobothriid cestodes. Recent and future trends in the importance of these parasites for human health in the North are explored. For example, the incidence of human exposure to endemic helminth zoonoses (e.g. Diphyllobothrium, Trichinella, and Echinococcus) appears to be declining, while water-borne protozoans such as Giardia, Cryptosporidium, and Toxoplasma may be emerging causes of human disease in a warming North. Parasites that undergo temperature-dependent development in the environment (such as Toxoplasma, ascarid and anisakid nematodes, and diphyllobothriid cestodes) will likely undergo accelerated development in endemic areas and temperate-adapted strains/species will move north, resulting in faunal shifts. Food-borne pathogens (e.g. Trichinella, Toxoplasma, anisakid nematodes, and diphyllobothriid cestodes) may be increasingly important as animal products are exported from the North and tourists, workers, and domestic animals enter the North. Finally, key needs are identified to better assess and mitigate risks associated with zoonotic parasites, including enhanced surveillance in animals and people, detection methods, and delivery and evaluation of veterinary and public health services.
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http://dx.doi.org/10.1016/B978-0-12-407706-5.00002-2DOI Listing
September 2013

Prevalence and genotypes of Giardia duodenalis in dairy and beef cattle in farms around Charlottetown, Prince Edward Island, Canada.

Can Vet J 2011 Sep;52(9):967-72

Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada.

Prevalence of Giardia duodenalis in dairy and beef cattle on farms around Charlottetown, Prince Edward Island (Canada) was determined by analyzing feces using direct immunofluorescence antibody microscopy. Genotypes were determined by 16S-rRNA sequencing. Fecal samples (n = 892) were collected from adult cattle in dairy tie-stall, dairy free-stall, and beef herds (10 herds each), and from calves (n = 183) from 11 dairy farms. Prevalence rates were 38% and 51% in cows and calves, respectively. Giardia duodenalis was present in all dairy herds, in 9/10 beef herds and in calves from 10/11 herds examined. Prevalence rates were 40% and 41% for cows in tie- and free-stall herds, respectively, and 27% for beef cows. Zoonotic Assemblage A was found in 12.2% of calves concomitantly infected with Assemblage E. All successfully sequenced samples (114/128) from cows corresponded to Assemblage E. Giardia duodenalis is highly prevalent in cattle herds in Prince Edward Island and Assemblage A in calves is a potential public health concern.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157069PMC
September 2011

Occurrence of Cryptosporidium and Giardia on beef farms and water sources within the vicinity of the farms on Prince Edward Island, Canada.

Vet Parasitol 2012 Feb 25;184(1):1-9. Epub 2011 Oct 25.

Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, PE, C1A 4P3, Canada.

The objectives of this study were to determine the prevalence and assemblages of Giardia and species of Cryptosporidium on beef farms in Prince Edward Island (PEI), Canada, including the water sources associated with the farms, and to determine risk factors for infection of cattle with these parasites. Twenty beef farms were selected based on the presence of surface water< 500 m from the barn. Prevalence was determined by direct immunofluorescence microscopy, while genotyping and species determination were performed by nested-PCR and DNA sequencing. Giardia was detected in 42% (95% CI: 38-46%) of fecal samples from 100% farms while Cryptosporidium was detected in 17% (95% CI: 14-19%) of fecal samples from 80% of farms. The most predominant Giardia assemblage isolated was the livestock specific assemblage E (89%). The zoonotic assemblages A and B were found in 4 and 7% of the Giardia isolates that were genotyped, respectively. The Giardia assemblages were detected equally between the cows and calves examined. Overall, the most common Cryptosporidium species detected in this study was Cryptosporidium andersoni (49%), predominantly found in cattle > 6 mo of age, while most Cryptosporidium bovis and Cryptosporidium pestis (previously Cryptosporidium parvum 'bovine genotype') isolates were detected in calves ≤ 6 mo of age. All Cryptosporidium ryanae isolates (four) were found in calves. Giardia cysts and Cryptosporidium oocysts were detected in 14 and 93% of surface water samples of 14 farms, respectively. Cryptosporidium oocysts were detected in three (15%) ground water samples of 20 farms. One Cryptosporidium-positive water sample, which was the only surface water sample amenable to genotyping, contained C. parvum. The farm-level risk factors investigated in this study, age of animals and location of the farm, were not associated with the risk of infection in cattle with either Cryptosporidium spp. or Giardia duodenalis. We conclude that beef cattle are a potential reservoir of Cryptosporidium spp. and G. duodenalis that could contaminate source water. There is the possibility of further transmission to humans on PEI if the source water is not properly treated prior to consumption.
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http://dx.doi.org/10.1016/j.vetpar.2011.10.027DOI Listing
February 2012

Foodborne illness associated with Cryptosporidium and Giardia from livestock.

J Food Prot 2011 Nov;74(11):1944-55

Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island, Canada C1A 4P3.

Waterborne outbreaks caused by Cryptosporidium and Giardia are well documented, while the public health implications for foodborne illness from these parasites have not been adequately considered. Cryptosporidium and Giardia are common in domestic livestock, where young animals can have a high prevalence of infection, shedding large numbers of oocysts and cysts. Molecular epidemiological studies have advanced our knowledge on the distribution of Cryptosporidium and Giardia species and genotypes in specific livestock. This has enabled better source tracking of contaminated foods. Livestock generate large volumes of fecal waste, which can contaminate the environment with (oo)cysts. Evidence suggests that livestock, particularly cattle, play a significant role in food contamination, leading to outbreaks of cryptosporidiosis. However, foodborne giardiasis seems to originate primarily from anthroponotic sources. Foodborne cryptosporidiosis and giardiasis are underreported because of the limited knowledge of the zoonotic potential and public health implications. Methods more sensitive and cheaper are needed to detect the often-low numbers of (oo)cysts in contaminated food and water. As the environmental burden of Cryptosporidium oocysts and Giardia cysts from livestock waste increases with the projected increase in animal agriculture, public health is further compromised. Contamination of food by livestock feces containing Cryptosporidium oocysts and Giardia cysts could occur via routes that span the entire food production continuum. Intervention strategies aimed at preventing food contamination with Cryptosporidium and Giardia will require an integrated approach based on knowledge of the potential points of entry for these parasites into the food chain. This review examines the potential for foodborne illness from Cryptosporidium and Giardia from livestock sources and discusses possible mechanisms for prevention and control.
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http://dx.doi.org/10.4315/0362-028X.JFP-11-107DOI Listing
November 2011

Occurrence of Giardia and Cryptosporidium in pigs on Prince Edward Island, Canada.

Vet Parasitol 2012 Feb 4;184(1):18-24. Epub 2011 Aug 4.

Department of Health Management Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, PE, C1A 4P3, Canada.

In a cross-sectional study of 633 pigs from 21 herds on Prince Edward Island, Canada (PEI), the prevalence of infection with Cryptosporidium and Giardia, and the genotypes and species of isolates were determined in order to establish the zoonotic potential of pigs in this region. As determined by direct immunofluorescence microscopy (DFA), 18 herds (86%) and 163 animals (26%; 95% CI: 22-29%) tested positive for Cryptosporidium, while just 3 herds (14%) and 6 animals (1%; 95% CI: 0.4-2%) tested positive for Giardia. Cryptosporidium spp. isolates were detected in 39% (95% CI: 34-44%) of weanlings (1-3 months of age) and 9% (95% CI: 6-13) of sows (>8months of age). Molecular characterization using the 18S rDNA and HSP70 gene fragments revealed the presence of Cryptosporidium sp. pig genotype II, C. suis, C. parvum, and Cryptosporidium sp. mouse genotype. Among the 113 isolates of Cryptosporidium spp. successfully genotyped, pig genotype II (61%) predominated, with C. suis (36%) being the next most prominant isolate. C. parvum (2%; two isolates) and Cryptosporidium sp. mouse genotype (0.9%; one isolate) were only occasionally isolated. The only two Cryptosporidium-positive genotyped isolates from sows included one each of C. suis and Cryptosporidium sp. pig genotype II. All but one of the six Giardia positive isolates were detected in weanling pigs. None of the Giardia-positive isolates was amenable to PCR. This study demonstrates that Cryptosporidium spp. are highly prevalent in pigs on PEI, Canada, are found mostly in weanlings (1-3 months of age). Furthermore, the pigs are primarily infected by the host-specific genotypes and species, Cryptosporidium sp. pig genotype II and C. suis, whereas the zoonotic C. parvum is rare. Giardia duodenalis is only occasionally found in pigs. These findings suggest that domestic pigs on PEI, Canada, likely do not pose a significant health risk to humans from these parasites.
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http://dx.doi.org/10.1016/j.vetpar.2011.07.047DOI Listing
February 2012

Immunomagnetic separation significantly improves the sensitivity of polymerase chain reaction in detecting Giardia duodenalis and Cryptosporidium spp. in dairy cattle.

J Vet Diagn Invest 2011 Mar;23(2):260-7

Canadian Food Inspection Agency, 1400 Merivale Road, T1-4-307, Ottawa, ON, Canada K1A 0Y9.

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.
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http://dx.doi.org/10.1177/104063871102300210DOI Listing
March 2011

Microbiological quality of blue mussels (Mytilus edulis) in Nunavik, Quebec: a pilot study.

Can J Microbiol 2010 Nov;56(11):968-77

Département de médecine sociale et préventive, Faculté de médecine, Université Laval, 945 avenue Wolfe, QC G1V 5B3, Canada.

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.
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http://dx.doi.org/10.1139/w10-078DOI Listing
November 2010

Temporal changes in the prevalence and shedding patterns of Giardia duodenalis cysts and Cryptosporidium spp. oocysts in a herd of dairy calves in Ontario.

Can Vet J 2010 Aug;51(8):841-6

Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada, Postal Locator 2204E, Ottawa, Ontario.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905001PMC
August 2010

Prevalence and molecular characterization of Cryptosporidium spp. in dairy calves from 11 farms in Prince Edward Island, Canada.

Vet Parasitol 2009 Mar 5;160(3-4):323-6. Epub 2008 Nov 5.

Microbiology Research Division, Bureau of Microbial Hazards, Health Canada, 2204E, Ottawa, Ont., Canada.

Cryptosporidium spp. are common intestinal protozoan parasites that infect a wide range of hosts, including humans and livestock, worldwide. The objective of this study was to determine the prevalence of Cryptosporidium spp. in dairy calves in Prince Edward Island, Canada, and the potential for transmission of this parasite between dairy calves and humans. Fecal samples were collected from 183 dairy calves from 11 farms in Prince Edward Island. The prevalence of Cryptosporidium spp. infections in these animals was determined by examining for the presence of oocysts in the fecal samples, using immunofluorescence microscopy. Molecular characterization was done using a nested-PCR protocol to amplify fragments of the Cryptosporidium heat-shock protein 70 gene, followed by DNA sequencing. Ten calves (6.2%), representing 4 out of 11 farms tested, were positive for Cryptosporidium spp. DNA sequence analysis on five PCR positive samples demonstrated that Cryptosporidium parvum was the only species present in the calves tested, suggesting that there is a potential risk of zoonotic transmission between dairy calves and humans in this region.
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http://dx.doi.org/10.1016/j.vetpar.2008.10.096DOI Listing
March 2009

Giardia duodenalis and Cryptosporidium spp. in the intestinal contents of ringed seals (Phoca hispida) and bearded seals (Erignathus barbatus) in Nunavik, Quebec, Canada.

J Parasitol 2008 Oct;94(5):1161-3

Microbiology Research Division, Food Directorate, Health Canada, Banting Research Centre 2204E, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, 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.
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http://dx.doi.org/10.1645/GE-1485.1DOI Listing
October 2008

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

J Vet Diagn Invest 2008 Mar;20(2):178-85

Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3, Canada.

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.
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http://dx.doi.org/10.1177/104063870802000206DOI Listing
March 2008

Giardia duodenalis and Cryptosporidium spp. in a veterinary college bovine teaching herd.

Vet Parasitol 2006 Dec 14;142(3-4):231-7. Epub 2006 Aug 14.

Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PEI, Canada C1A 4P3.

In a preliminary study, we commonly identified Giardia duodenalis in adult dairy cattle from a veterinary college teaching herd. Therefore, the present study was carried out in order to better understand the potential of adult cattle to act as a source for G. duodenalis infections for students and staff at the veterinary college. Fecal samples were collected bi-weekly from this herd of adult cattle (n=30) over an 8-month period to determine the prevalence of G. duodenalis and Cryptosporidium spp. within the herd. Nested PCR followed by DNA sequencing was then performed on a subset of positive samples in order to better understand the zoonotic potential of these infections. Every cow was sampled between 11 and 18 times, depending on the date the animal joined the teaching herd. In total, 507 fecal samples were collected from 30 different cows and examined for cysts and oocysts using epifluorescence microscopy. G. duodenalis prevalence during the course of the study ranged from 37% (11/30) to 64% (18/28), with a mean of 49%. Cumulative G. duodenalis prevalence was 73% (22/30). Zoonotic G. duodenalis assemblage A genotype was identified in 43% (6/14) of the G. duodenalis-positive samples on which PCR and genetic sequencing were successfully performed. G. duodenalis assemblage E was identified in 57% (8/14) of these samples. Cryptosporidium spp. oocysts were not detected in the feces of any cows during the study period. The presence of the zoonotic G. duodenalis assemblage A in 43% of the sequenced samples indicates that there is a potential risk of infection for students and staff at this research and teaching facility, although the roles of cows as sources of giardiasis in humans remain uncertain. Furthermore, due to the large amount of feces they produce, adult cattle may serve as important sources for G. duodenalis infections in young cattle, or other animals in the facility, despite relatively low numbers of cysts excreted per gram of feces. In contrast, the results of this study indicate that this herd posed a negligible risk of transmitting Cryptosporidium parvum infections to humans.
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http://dx.doi.org/10.1016/j.vetpar.2006.07.007DOI Listing
December 2006

Genetic characterization of Cryptosporidium isolates from ringed seals (Phoca hispida) in Northern Quebec, Canada.

J Parasitol 2005 Jun;91(3):712-6

Environmental Microbial Safety Laboratory, Animal and Natural Resources Institute Agricultural Research Service, United States Department of Agriculture, Building 173, BARC-East, 10300 Baltimore Avenue, Beltsville, Maryland 20705, USA.

This study reports the molecular characterization of Cryptosporidium spp. isolates identified from intestinal contents of ringed seals (Phoca hispida) from Nunavik (Quebec, Canada). Cryptosporidium spp. fragments of 18S rRNA, HSP-70, and actin loci were amplified by PCR from seal intestinal contents. PCR-positive specimens were sequenced and compared with other Cryptosporidium species and genotypes reported previously. Sequence analysis showed the presence of C. muris and 2 novel genotypes in ringed seals.
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http://dx.doi.org/10.1645/GE-3438RNDOI Listing
June 2005

Detection of Cyclospora cayetanensis oocysts in human fecal specimens by flow cytometry.

J Clin Microbiol 2005 May;43(5):2375-9

Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada.

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.
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http://dx.doi.org/10.1128/JCM.43.5.2375-2379.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1153738PMC
May 2005
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