Publications by authors named "Alvin A Gajadhar"

45 Publications

International Commission on Trichinellosis: Recommendations for quality assurance in digestion testing programs for .

Food Waterborne Parasitol 2019 Sep 5;16:e00059. Epub 2019 Jun 5.

National Academy of Sciences, 500 Fifth Street NW, Washington, DC 20001, USA.

Effective performance of digestion testing methods for , and their use for the detection of infected animals and the prevention of human trichinellosis require system-wide incorporation of appropriate quality assurance (QA) practices. The recommendations of the International Commission on Trichinellosis (ICT) aim to facilitate reliable test results when laboratories operate within a quality management system (QMS) which includes: 1) a quality manual (or similar documentation of the QMS); 2) a validated test method with identified critical control points; 3) a training program; 4) procedures utilizing proficiency testing and other methods to confirm technical capability of analysts; 5) equipment calibration and maintenance; 6) standard operating procedures, related documentation and reporting; 7) procedures to enable continuous monitoring and improvements; and 8) regular internal and third party audits. The quality manual or similar documentation describes the QMS within a testing laboratory, and lists the QA policies and good laboratory practices. Quality assurance goals contained in such documentation are the foundation of an effective QA program and must be explicit, measurable, and expressed in terms of performance criteria for the test method based on purpose for testing. The digestion method is capable of consistently detecting larvae in meat at a level of sensitivity that is recognized to be effective for use in controlling animal infection and preventing human disease. However, consistent performance of the assay is assured only when parameters of the test method have been defined, scientifically validated as fit for purpose, and used within an effective QMS. The essential components of a digestion assay, specifically the critical control points and minimum standards for test performance are described. Reliable proficiency samples and their appropriate use in a quality system are key factors for certifying and maintaining an effective testing laboratory, including qualifying, re-qualifying and disqualifying of analysts as appropriate. Thus recommendations are included for the preparation and use of proficiency samples in a digestion testing laboratory. The minimum training requirements for analysts performing a quality assured digestion assay, as well as suggested requirements for the content of a training manual, are also outlined. Finally, these ICT recommendations include essential components and minimum standards for maintaining and achieving certification and maintenance of a laboratory performing digestion testing for . The certification program for the laboratory, including qualifying analysts, may be administered by a National Reference Laboratory or an authorized third party certifying body, under the auspices of the appropriate competent authority.
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http://dx.doi.org/10.1016/j.fawpar.2019.e00059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033998PMC
September 2019

TRANSMISSION DYNAMICS OF IN ARCTIC FOXES (): A LONG-TERM MARK-RECAPTURE SEROLOGIC STUDY AT KARRAK LAKE, NUNAVUT, CANADA.

J Wildl Dis 2019 07 28;55(3):619-626. Epub 2018 Nov 28.

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

Transmission dynamics of , a parasite of importance for wildlife and human health, are enigmatic in the Arctic tundra, where free-ranging wild and domestic felid definitive hosts are absent and rarely observed, respectively. Through a multiyear mark-recapture study (2011-17), serosurveillance was conducted to investigate transmission of in Arctic foxes () in the Karrak Lake region, Nunavut, Canada. Sera from adult foxes and fox pups were tested for antibodies to by using serologic methods, including the indirect fluorescent antibody test, direct agglutination test, and modified agglutination test. The overall seroprevalence was 39% in adults and 17% in pups. Mature foxes were more likely to be exposed (seroconvert) than young foxes (less than 1 yr old), with the highest level of seroprevalence in midaged foxes (2-4 yr old). Pups in two different litters were seropositive on emergence from the den, around 5 wk old, which could have been due to passive transfer of maternal antibody or vertical transmission of from mother to offspring. The seropositive pups were born of seropositive mothers that were also seropositive the year before they gave birth, suggesting that vertical transmission might not be limited to litters from mothers exposed to for the first time in pregnancy. All recaptured seropositive foxes remained seropositive on subsequent captures, suggesting that antibodies persist or foxes are constantly reexposed or a combination of both. The results of this study provided insights into how foxes were likely exposed to , the dynamics of antibody persistence and immune response, and how the parasite was maintained in a terrestrial Arctic ecosystem in the absence of felid definitive hosts.
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http://dx.doi.org/10.7589/2018-06-144DOI Listing
July 2019

Tongue has higher larval burden of Trichinella spp. than diaphragm in wolverines (Gulo gulo).

Vet Parasitol 2018 Apr 21;253:94-97. Epub 2018 Feb 21.

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

Trichinella is an important zoonotic parasite found in a range of wildlife species harvested for food and fur in Canada. We compared larval intensity from tongue and diaphragm, the best predilection sites in other animal species, from naturally infected, wild wolverines (Gulo gulo) (n = 95). Muscle larvae of Trichinella spp. were recovered by the pepsin/HCl artificial digestion method (gold standard) using double separatory funnels, and species were identified using multiplex PCR. Prevalence was 83% (79/95). Of those positive for Trichinella spp. (n = 79), 76 (96.2%) were detected in both tissues, 2 (2.5%) were positive only on diaphragm, and 1 (1.3%) only on tongue. A total of 62 of 79 wolverines (78.5%) had higher larval burden in tongue than in diaphragm, whereas 17 wolverines (21.5%) had higher larval burden in diaphragm. The predilection site (higher larval burden) of Trichinella spp. larvae did not vary significantly between juvenile and adult wolverines (P = 0.2), between male and female wolverines (P = 0.9), and among wolverines classified as having low and high larval intensities overall (P = 0.2). Trichinella T6 was the predominant genotype (63 of 79; 80%), followed by T. nativa (T2) (6 of 79; 8%). Mixed infections of T2 and T6 were observed in 9 of 79 (12%) wolverines. Larval intensity of Trichinella T6 was higher in tongues than diaphragms. No statement can be made for T2 due to insufficient T2 positive samples. In conclusion, tongues are a better site for sampling than diaphragms in future surveys of Trichinella larval intensity in wolverines; however, either tissue is suitable for prevalence studies.
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http://dx.doi.org/10.1016/j.vetpar.2018.02.032DOI Listing
April 2018

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis.

Parasit Vectors 2018 03 2;11(1):125. Epub 2018 Mar 2.

Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada.

Background: Equine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay's utility for regulatory testing.

Results: We developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay's performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5-90.7%), 90.5% (95% CI: 87.3-92.3%) and 87.4% (95% CI: 84.0-90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9-12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7-10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9-63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR.

Conclusions: The duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.
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http://dx.doi.org/10.1186/s13071-018-2751-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834856PMC
March 2018

Pathology, clinical signs, and tissue distribution of in experimentally infected reindeer ().

Int J Parasitol Parasites Wildl 2017 Dec 15;6(3):234-240. Epub 2017 Aug 15.

University of Saskatchewan, Department of Veterinary Microbiology, 52 Campus Drive, Saskatoon, Saskatchewan S7N5B4, Canada.

is a zoonotic parasite found in vertebrates worldwide for which felids serve as definitive hosts. Despite low densities of felids in northern Canada, Inuit people in some regions show unexpectedly high levels of exposure, possibly through handling and consumption of Arctic wildlife. Free-ranging caribou () are widely harvested for food across the Canadian North, show evidence of seroexposure to , and are currently declining in numbers throughout the Arctic. We experimentally infected three captive reindeer (conspecific with caribou) with 1000, 5000 or 10,000 oocysts of via stomach intubation to assess clinical signs of infection, pathology, and tissue distribution. An unexposed reindeer served as a negative control. Signs of stress, aggression, and depression were noted for the first two weeks following infection. By 4 weeks post infection, all infected reindeer were positive on a modified agglutination test at the highest titer tested (1:200) for antibodies to . At 20 weeks post infection, no gross abnormalities were observed on necropsy. Following histopathology and immunohistochemistry, tissue cysts were visualized in the reindeer given the highest and lowest dose of oocysts. Focal pleuritis and alveolitis were associated with respiratory problems in reindeer given the middle dose. DNA of was detected following traditional DNA extraction and conventional PCR on 25 mg samples from 17/33 muscles and organs, and by magnetic capture DNA extraction from 100 g samples from all 26 tissues examined. This research demonstrated that reindeer/caribou can serve as intermediate hosts for , and that the parasite may be associated with health effects in wildlife. The presence of in all tissues tested, many of which are commonly consumed raw, smoked, or dried in northern communities, suggests that caribou may serve as a source of human exposure to .
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http://dx.doi.org/10.1016/j.ijppaw.2017.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573777PMC
December 2017

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

ESTIMATING TOXOPLASMA GONDII EXPOSURE IN ARCTIC FOXES (VULPES LAGOPUS) WHILE NAVIGATING THE IMPERFECT WORLD OF WILDLIFE SEROLOGY.

J Wildl Dis 2016 Jan;52(1):47-56

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

Although the protozoan parasite Toxoplasma gondii is ubiquitous in birds and mammals worldwide, the full suite of hosts and transmission routes is not completely understood, especially in the Arctic. Toxoplasma gondii occurrence in humans and wildlife can be high in Arctic regions, despite apparently limited opportunities for transmission of oocysts shed by felid definitive hosts. Arctic foxes (Vulpes lagopus) are under increasing anthropogenic and ecologic pressure, leading to population declines in parts of their range. Our understanding of T. gondii occurrence in arctic foxes is limited to only a few regions, but mortality events caused by this parasite have been reported. We investigated the exposure of arctic foxes to T. gondii in the Karrak Lake goose colony, Queen Maud Gulf Migratory Bird Sanctuary, Nunavut, Canada. Following an occupancy-modeling framework, we performed replicated antibody testing on serum samples by direct agglutination test (DAT), indirect fluorescent antibody test (IFAT), and an indirect enzyme-linked immunosorbent assay (ELISA) that can be used in multiple mammalian host species. As a metric of test performance, we then estimated the probability of detecting T. gondii antibodies for each of the tests. Occupancy estimates for T. gondii antibodies in arctic foxes under this framework were between 0.430 and 0.758. Detection probability was highest for IFAT (0.716) and lower for DAT (0.611) and ELISA (0.464), indicating that the test of choice for antibody detection in arctic foxes might be the IFAT. We document a new geographic record of T. gondii exposure in arctic foxes and demonstrate an emerging application of ecologic modeling techniques to account for imperfect performance of diagnostic tests in wildlife species.
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http://dx.doi.org/10.7589/2015-03-075DOI Listing
January 2016

Toxoplasma gondii exposure in arctic-nesting geese: A multi-state occupancy framework and comparison of serological assays.

Int J Parasitol Parasites Wildl 2014 Aug 30;3(2):147-53. Epub 2014 Jun 30.

Department of Veterinary Microbiology, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan S7N5B4, Canada.

The zoonotic parasite, Toxoplasma gondii, has a worldwide distribution and a cosmopolitan suite of hosts. In arctic tundra regions, the definitive felid hosts are rare to absent and, while the complete transmission routes in such regions have yet to be fully elucidated, trophic and vertical routes are likely to be important. Wild birds are common intermediate hosts of T. gondii, and in the central Canadian arctic, geese are probable vectors of the parasite from temperate latitudes to the arctic regions. Our objective was to estimate seroprevalence of T. gondii in Ross's and Lesser Snow Geese from the Karrak Lake ecosystem in Nunavut, Canada. After harvesting geese by shotgun, we collected blood on filter paper strips and tested the eluate for T. gondii antibodies by indirect fluorescent antibody test (IFAT) and direct agglutination test (DAT). We estimated seroprevalence using a multi-state occupancy model, which reduced bias by accounting for imperfect detection, and compared these estimates to a naïve estimator. Ross's Geese had a 0.39 probability of seropositivity, while for Lesser Snow Geese the probability of positive for T. gondii antibodies was 0.36. IFAT had a higher antibody detection probability than DAT, but IFAT also had a higher probability of yielding ambiguous or unclassifiable results. The results of this study indicate that Ross's Geese and Lesser Snow Geese migrating to the Karrak Lake region of Nunavut are routinely exposed to T. gondii at some point in their lives and that they are likely intermediate hosts of the parasite. Also, we were able to enhance our estimation of T. gondii seroprevalence by using an occupancy approach that accounted for both false-negative and false-positive detections and by using multiple diagnostic tests in the absence of a gold standard serological assay for wild geese.
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http://dx.doi.org/10.1016/j.ijppaw.2014.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4142267PMC
August 2014

Endoparasites in the feces of arctic foxes in a terrestrial ecosystem in Canada.

Int J Parasitol Parasites Wildl 2013 Dec 14;2:90-6. Epub 2013 Mar 14.

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

The parasites of arctic foxes in the central Canadian Arctic have not been well described. Canada's central Arctic is undergoing dramatic environmental change, which is predicted to cause shifts in parasite and wildlife species distributions, and trophic interactions, requiring that baselines be established to monitor future alterations. This study used conventional, immunological, and molecular fecal analysis techniques to survey the current gastrointestinal endoparasite fauna currently present in arctic foxes in central Nunavut, Canada. Ninety-five arctic fox fecal samples were collected from the terrestrial Karrak Lake ecosystem within the Queen Maud Gulf Migratory Bird Sanctuary. Samples were examined by fecal flotation to detect helminths and protozoa, immunofluorescent assay (IFA) to detect Cryptosporidium and Giardia, and quantitative PCR with melt-curve analysis (qPCR-MCA) to detect coccidia. Positive qPCR-MCA products were sequenced and analyzed phylogenetically. Arctic foxes from Karrak Lake were routinely shedding eggs from Toxascaris leonina (63%). Taeniid (15%), Capillarid (1%), and hookworm eggs (2%), Sarcocystis sp. sporocysts 3%), and Eimeria sp. (6%), and Cystoisospora sp. (5%) oocysts were present at a lower prevalence on fecal flotation. Cryptosporidium sp. (9%) and Giardia sp. (16%) were detected by IFA. PCR analysis detected Sarcocystis (15%), Cystoisospora (5%), Eimeria sp., and either Neospora sp. or Hammondia sp. (1%). Through molecular techniques and phylogenetic analysis, we identified two distinct lineages of Sarcocystis sp. present in arctic foxes, which probably derived from cervid and avian intermediate hosts. Additionally, we detected previously undescribed genotypes of Cystoisospora. Our survey of gastrointestinal endoparasites in arctic foxes from the central Canadian Arctic provides a unique record against which future comparisons can be made.
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http://dx.doi.org/10.1016/j.ijppaw.2013.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3862500PMC
December 2013

A new multi-host species indirect ELISA using protein A/G conjugate for detection of anti-Toxoplasma gondii IgG antibodies with comparison to ELISA-IgG, agglutination assay and Western blot.

Vet Parasitol 2014 Feb 1;200(1-2):66-73. Epub 2013 Dec 1.

Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada. Electronic address:

Toxoplasma gondii is a zoonotic protozoan parasite which can cause significant disease and losses in livestock and wild animals. It is increasingly recognized as an important foodborne pathogen in a broad range of food animals and products. Effective control strategies require rapid, reliable and cost-effective detection methods for large scale surveys and diagnostic applications in a broad range of warm-blooded animals. To overcome one or more of these shortcomings in the currently available detection methods for T. gondii infection a non-species-specific protein A/G conjugate was used in the development of an indirect ELISA (ELISA-A/G) for the detection of IgG antibodies in serum samples obtained from experimentally infected pigs. The performance of the assay was evaluated using serum samples from pigs, cats, mice and seals with known positive or negative status for T. gondii infection. Results of the ELISA-A/G obtained with pig serum samples were compared with those generated by traditional ELISA using host specific IgG conjugate (ELISA-IgG), modified agglutination test (MAT) and Western blot analysis (WB). Using protein A/G conjugate, comparative analysis of results from 77 samples obtained from T. gondii infected pigs showed excellent agreement between the ELISA-A/G and in-house ELISA-IgG (0.917 κ). Similar agreements were also observed when these samples were tested by a commercial ELISA kit (0.816 κ), MAT (0.816 κ) and WB (0.79 κ). A total of 86 serum samples obtained from cats, mice and seals experimentally infected with T. gondii and tested by the ELISA-A/G as well as MAT for the presence of anti-Toxoplasma IgG antibodies yielded Kappa value of 1.0 for cats and mice and 0.79 for seals. These results show that the ELISA-A/G is a suitable method for serological detection of T. gondii infection in multiple host species and has the potential for testing samples from a broad range of domestic, wild, and aquatic mammalian host species. Simultaneous testing of samples from multiple host species on the same ELISA plate, and the use of multiple plates in a single run for large scale screening will enhance the cost effectiveness and speed of the test in the control and management of toxoplasmosis. This study also shows the effectiveness of the protein A/G conjugate in a modified WB assay for confirmation of T. gondii infection in mammalian hosts. Appropriate validation studies using field samples from various host species to validate the performance of ELISA-A/G is recommended prior to its application for diagnostic and surveillance programs.
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http://dx.doi.org/10.1016/j.vetpar.2013.11.004DOI Listing
February 2014

Application of a qPCR assay with melting curve analysis for detection and differentiation of protozoan oocysts in human fecal samples from Dominican Republic.

Am J Trop Med Hyg 2013 Nov 9;89(5):892-8. Epub 2013 Sep 9.

Centre for Food-Borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada; Facultad de Ciencias de la Salud, Pontificia Universidad Católica Madre y Maestra, Santiago, Dominican Republic.

A quantitative polymerase chain reaction assay with melt curve analysis (qPCR-MCA) was applied for the detection of protozoan oocysts in 501 human fecal samples collected in Dominican Republic. Samples were subjected to qPCR using universal coccidia primers targeting 18S rDNA to detect oocysts followed by MCA to identify oocyst species based on amplicon melting temperature. Putative positive samples were also tested by conventional PCR and microscopy. Cystoisospora belli (×3), Cryptosporidium parvum (×3), Cryptosporidium hominis (×5), Cryptosporidium meleagridis (×1), Cryptosporidium canis (×1), and Cyclospora cayetanensis (×9) were detected by qPCR-MCA and confirmed by sequencing. This assay consistently detected 10 copies of the cloned target fragment and can be considered more efficient and sensitive than microscopy flotation methods for detecting multiple species of oocysts in human feces. The qPCR-MCA is a reliable protozoan oocyst screening assay for use on clinical and environmental samples in public health, food safety and veterinary programs.
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http://dx.doi.org/10.4269/ajtmh.13-0106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820332PMC
November 2013

Performance of commercial ELISA and agglutination test kits for the detection of anti-Toxoplasma gondii antibodies in serum and muscle fluid of swine infected with 100, 300, 500 or 1000 oocysts.

Vet Parasitol 2012 Dec 17;190(3-4):362-7. Epub 2012 Jul 17.

Centre for Food Borne and Animal Parasitology, Saskatoon Laboratory, Canadian Food Inspection Agency, 116 Veterinary Road, Saskatoon, SK S7N 2R3, Canada.

Serum and tissue fluid samples from experimentally infected swine were tested for antibodies to Toxoplasma gondii using both an indirect ELISA and a modified agglutination test (MAT) available commercially in kit form. Ten 8-9 week-old swine were fed meatballs containing 100, 300, 500 or 1000 T. gondii oocysts and three control animals were fed meatballs with no oocysts. Post-inoculation blood samples were collected weekly until euthanasia at 35-63 days post inoculation (DPI). Tissue fluid was obtained from diaphragm, heart and sternomastoideus muscles post-mortem. By 16 DPI, nine of 10 inoculated pigs were detected serologically using ELISA at a pre-test serum dilution of 1:50 and all ten pigs were detected by the MAT at a serum dilution of 1:25. The last pig became positive on ELISA by 21 DPI and the 10 pigs maintained their serological status for the duration of the experiment. Heart muscle was the best overall source of tissue fluid for ELISA and all six pigs inoculated with either 500 or 1000 oocysts were positive using either diaphragm or heart tissue fluid samples. However, 10 of 18 fluid samples from pigs receiving ≤ 300 oocysts were not detected using ELISA, including 5 of 6 from sternomastoideus muscle. The MAT used at a 1:10 pre-test dilution of tissue fluid correctly identified all 10 inoculated pigs regardless of the source muscle. Based on these data, we conclude that either assay would be useful for herd evaluation or surveillance testing using sera, and the MAT would be a good candidate assay for testing tissue fluid for the same purposes.
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http://dx.doi.org/10.1016/j.vetpar.2012.06.040DOI Listing
December 2012

Serological evidence of Besnoitia spp. infection in Canadian wild ruminants and strong cross-reaction between Besnoitia besnoiti and Besnoitia tarandi.

Vet Parasitol 2012 Nov 23;190(1-2):19-28. Epub 2012 Jun 23.

SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain.

Bovine besnoitiosis, caused by Besnoitia besnoiti, is considered to be emergent in Europe and responsible for severe economic losses due to the chronic and debilitating course of the disease but has not been reported in North America. Besnoitia tarandi is a related species and it has been reported in reindeer and caribou from different locations of the Arctic Pole, including North America. Diagnosis of clinical besnoitiosis is largely based on the recognition of dermal grossly visible tissue cysts of Besnoitia. Nothing is known of cross reactivity between B. besnoiti and B. tarandi species. Here, we evaluated the use of serological tests employed in the diagnosis of bovine besnoitiosis for the detection of Besnoitia spp. infections in different wild ruminant species (caribou, elk, mule-deer, white-tailed deer, moose, muskox and bison) from Canada and investigated cross-reactivity between B. besnoiti and B. tarandi species by indirect immunofluorescence antibody test and Western blot. For this, species-specific antibodies were obtained in rabbits experimentally infected with B. besnoiti and B. tarandi. Marked cross reactivity was found between B. besnoiti and B. tarandi. For the first time, antibodies to Besnoitia spp. infection were found in 16 of 20 caribou (Ranginfer tarandus), seven of 18 muskox (Ovibos moschatus), one of three bison (Bison bison), but not in 20 elk (Cervus canadensis), 20 white tailed deer (Odocoileus virginianus), and 20 moose (Alces alces) in Canada; results were similar using B. besnoiti and B. tarandi as antigen. There was no cross reactivity between the two Besnoitia species, Neospora caninum and Toxoplasma gondii with the cut-offs applied that prevented to observe it. The present study provides evidence that the serological assays can be useful to accomplish large scale prevalence studies in caribou and other wildlife species. Further studies are needed to study sylvatic and domestic cycle of B tarandi and B. besnoiti.
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http://dx.doi.org/10.1016/j.vetpar.2012.06.017DOI Listing
November 2012

Serological cross-reactivity between Anaplasma marginale and an Ehrlichia species in naturally and experimentally infected cattle.

J Vet Diagn Invest 2011 Nov;23(6):1181-8

Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada.

Seroconversion and cross-reactivity in cattle infected with Anaplasma marginale or a recently described Ehrlichia species (BOV2010 from British Columbia, Canada) were investigated. The study used 76 samples from 20 animals, a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for bovine anaplasmosis, and an indirect fluorescent antibody test (IFAT). Blood smear examination and/or polymerase chain reaction assay were performed to confirm or rule out the presence of Anaplasma or Ehrlichia. Samples comprised 3 groups. Group 1 consisted of 24 samples from 9 cattle naturally infected with Ehrlichia sp. BOV2010. Group 2 had 13 samples from 3 A. marginale-infected cattle from Manitoba, Canada. Group 3 had 39 samples, consisting of 26 from 5 calves experimentally infected with Ehrlichia sp. BOV2010, 10 from 2 calves experimentally infected with A. marginale from cattle (Manitoba) or bison (Saskatchewan), and 3 from an uninfected calf. All samples from cattle naturally or experimentally infected with Ehrlichia sp. BOV2010 or A. marginale were seropositive for A. marginale by both cELISA and IFAT, except 3 calves euthanized at 28 and 33 days post-inoculation (DPI) that did not seroconvert. Antibodies were detected in 2 experimental animals inoculated with Ehrlichia sp. BOV2010, as early as 28 and 33 DPI by the cELISA and IFAT, respectively, and by 42 DPI for both tests. The current study demonstrates that the specificity of the recombinant major surface protein 5 (MSP5) antigen is not restricted to Anaplasma spp., which reduces the utility of the test for serological diagnosis of bovine anaplasmosis in regions where Ehrlichia sp. BOV2010-infected cattle might exist.
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http://dx.doi.org/10.1177/1040638711425593DOI Listing
November 2011

Validation of an immunohistochemical assay for bovine cysticercosis, with comparison to a standard histological method.

Vet Parasitol 2012 May 12;186(3-4):301-11. Epub 2011 Nov 12.

Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon Laboratory, 116 Veterinary Road, Saskatoon, Saskatchewan S7N 2R3, Canada.

The larval stage (syn Cysticercus bovis) of the human tapeworm Taenia saginata causes cysticercosis in cattle, which has both aesthetic and food safety implications to consumers of beef. A monoclonal antibody-based immunohistochemical (IHC) assay developed to improve postmortem diagnosis of this parasite and a standard histological method were assessed to determine their fitness for intended use. Sections from 169 known-positive specimens of T. saginata from experimentally or naturally infected cattle, and from 30 known-negative specimens and lesions of various etiologies from non-infected cattle, were tested. The IHC assay identified significantly more known positive bovine cysticerci than the histological method (91.7% and 38.5%, respectively). Positive IHC staining occurred on sections from other cestode species, but should not affect the diagnostic specificity of this assay for bovine cysticercosis, due to the different host and/or tissue preferences amongst these parasites. Use of the IHC assay should improve the reliability of diagnosing lesions caused by degenerated cysticerci, facilitating more effective and efficient control of bovine cysticercosis.
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http://dx.doi.org/10.1016/j.vetpar.2011.11.014DOI Listing
May 2012

Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis.

J Parasitol 2011 Aug 11;97(4):725-30. Epub 2011 Feb 11.

Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, 116 Veterinary Road, Saskatoon, S7N 2R3, Canada.

Rapid and reliable detection and identification of coccidian oocysts are essential for animal health and foodborne disease outbreak investigations. Traditional microscopy and morphological techniques can identify large and unique oocysts, but they are often subjective and require parasitological expertise. The objective of this study was to develop a real-time quantitative PCR (qPCR) assay using melting curve analysis (MCA) to detect, differentiate, and identify DNA from coccidian species of animal health, zoonotic, and food safety concern. A universal coccidia primer cocktail was designed and employed to amplify DNA from Cryptosporidium parvum, Toxoplasma gondii, Cyclospora cayetanensis, and several species of Eimeria, Sarcocystis, and Isospora using qPCR with SYBR Green detection. MCA was performed following amplification, and melting temperatures (T(m)) were determined for each species based on multiple replicates. A standard curve was constructed from DNA of serial dilutions of T. gondii oocysts to estimate assay sensitivity. The qPCR assay consistently detected DNA from as few as 10 T. gondii oocysts. T(m) data analysis showed that C. cayetanensis, C. parvum, Cryptosporidium muris, T. gondii, Eimeria bovis, Eimeria acervulina, Isospora suis, and Sarcocystis cruzi could each be identified by unique melting curves and could be differentiated based on T(m). DNA of coccidian oocysts in fecal, food, or clinical diagnostic samples could be sensitively detected, reliably differentiated, and identified using qPCR with MCA. This assay may also be used to detect other life-cycle stages of coccidia in tissues, fluids, and other matrices. MCA studies on multiple isolates of each species will further validate the assay and support its application as a routine parasitology screening tool.
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http://dx.doi.org/10.1645/GE-2706.1DOI Listing
August 2011

An update on bovine anaplasmosis (Anaplasma marginale) in Canada.

Can Vet J 2010 Aug;51(8):837-40

Terrestrial Animal Health Division, Canadian Food Inspection Agency, 1400 Merivale Road, Ottawa, Ontario K1A 0Y9.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905000PMC
August 2010

A novel Ehrlichia genotype detected in naturally infected cattle in North America.

Vet Parasitol 2010 Oct 25;173(3-4):324-9. Epub 2010 Jun 25.

Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan S7N 2R3, Canada.

During a research investigation to determine if cattle from British Columbia (BC), Canada were infected with Anaplasma marginale or other related rickettsial blood parasites, a novel Ehrlichia genotype was revealed. Blood from seven BC source cattle was bioassayed by intravenous inoculation into naïve splenectomised calves. Additional splenectomised calves were used as uninoculated negative control or A. marginale-inoculated positive control. Newly designed sets of primers specific for the msp5 gene of A. marginale or for the 16S rRNA gene were used to test blood samples collected from all source cattle and from all recipient calves prior to inoculation and up to 72 days post-inoculation. Results of the PCR assays as well as microscopic examination of stained blood smears failed to demonstrate A. marginale in any of the animals except for the positive control. The 16S rRNA PCR primers amplified DNA from samples from all BC source cattle, five of six of the corresponding recipient calves, and the A. marginale infected control animal. DNA sequence data indicated the presence of A. marginale only in the positive control calf. Blast analysis in GenBank showed that sequences of all other 16S rRNA PCR products clearly fit within the Ehrlichia genus in the Anaplasmataceae family which also includes members of the genus Anaplasma. Phylogenetic analyses using the 16S rRNA gene sequences strongly support the putative Ehrlichia organism as a distinct genotype with sequences of various strains of Ehrlichia canis as the closest clade. Ehrlichia ruminantium is the only other species of the genus known to naturally infect cattle, apart from the present Ehrlichia isolate. However, within the genus, E. ruminantium is phylogenetically the furthest removed species from the novel genotype. The finding of this novel Ehrlichia represents the first known natural ehrlichial infection in cattle in North America. Nevertheless, it is unclear whether cattle are an incidental or primary host, particularly since deer are recognized as reservoir hosts for other species of Ehrlichia. Although other Ehrlichia spp. are known to be pathogenic for animals and zoonotic, these features are presently unknown for this novel genotype.
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http://dx.doi.org/10.1016/j.vetpar.2010.06.034DOI Listing
October 2010

Genetic diversity of equine piroplasms in Greece with a note on speciation within Theileria genotypes (T. equi and T. equi-like).

Infect Genet Evol 2010 Oct 19;10(7):963-8. Epub 2010 Jun 19.

Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Hydrobiology, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens 11855, Greece.

Equine piroplasms in Greece were studied using the reverse line blot hybridization (RLB) assay. Three genotypes consisting of two Theileria (T. equi and T. equi-like) and one Babesia (B. caballi-like) were identified. Of 787 samples tested, 371 (47.14%) hybridised to catchall probe (probe specifically designed to capture any piroplasm species present in a sample), 346 (43.96%) to T. equi probe, 364 (46.25%) to T. equi-like probe, 0 (0%) to B. caballi probe and 3 (0.38%) to B. caballi-like probe. Seven samples gave faint signals with the catchall probe only, indicating the presence of known or unknown piroplasm species, or a novel genotype or a known genotype occurring at a very low level of parasitemia. A partial sequence (509 bp) of the V4 region of the 18S rRNA gene of a T. equi-like isolate showed only 99% similarity with the reference T. equi-like isolates from Northern Spain from which the detecting probe used in the present study was designed but showed 100% similarity with the T. equi-like variants from Southern Spain. This indicated a noticeable degree of polymorphism within the population of T. equi-like. No unusual parasites previously reported in horses, such as B. canis canis and B. bovis were detected in this study. The values of the bioclimatic variables were very similar between the geographic locations for T. equi and T. equi-like genotypes, suggesting the two are not yet different species as hypothesized by some authors but are possibly undergoing a speciation process within Theileria genotypes. Both T. equi and T. equi-like were found in predominantly forest type land cover.
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http://dx.doi.org/10.1016/j.meegid.2010.06.008DOI Listing
October 2010

A seroepidemiological study of exposure to Toxoplasma, Leishmania, Echinococcus and Trichinella in equids in Greece and analysis of risk factors.

Vet Parasitol 2010 May 12;170(1-2):170-5. Epub 2010 Feb 12.

Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Hydrobiology, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens 11855, Greece.

The role of horses in the transmission of parasitic zoonoses either as a source of infection to vectors or through contamination of definitive hosts is gaining importance worldwide. For this reason sera from 773 equids including 753 horses, 13 mules and seven ponies in four regions of Greece were investigated by ELISA for the presence of IgG antibodies against Toxoplasma, Leishmania, Echinococcus and Trichinella. Anti-Toxoplasma antibodies were detected in all regions with an overall prevalence of 1.8%. In contrast, antibodies to Leishmania, Echinococcus, and Trichinella were present only in horses from the equestrian centre located in Attica region, but the status of Trichinella could not be confirmed. The seroprevalence of infection was 0.3% for Leishmania, 0.1% for Echinococcus and 0.1% for Trichinella. Only one horse was positive with a mixed infection of Toxoplasma, Leishmania and Trichinella. The following host characteristics were investigated for any significant effects on the prevalence of Toxoplasma infection: gender, age, species, origin of birth, activity, and location. The type of activity (p<0.05) and location (p<0.01) of the animals were found to be significant risk factors for Toxoplasma infection. The relative risk (RR) for Toxoplasma infection comparing the regions of Peloponnese and Thessaly to Attica were 6.92 and 6.78, respectively. Due to the very low prevalence of Echinococcus, Leishmania, and Trichinella infections, the associated risk factors were not analysed. The low seroprevalences observed suggest that the risk of infection from equids to people is very low, especially when consumption of horse meat is uncommon in this country.
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http://dx.doi.org/10.1016/j.vetpar.2010.02.004DOI Listing
May 2010

Seroprevalence of equine piroplasms and host-related factors associated with infection in Greece.

Vet Parasitol 2010 May 20;169(3-4):273-8. Epub 2010 Jan 20.

Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Hydrobiology, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens 11855, Greece.

Serum samples were collected from a total of 544 equids that included 524 horses, 13 mules, and 7 ponies from various regions of mainland Greece and were examined by competitive-inhibition ELISA (cELISA) to evaluate the level of exposure of Greek equids to Theileria (Babesia) equi and/or Babesia caballi, the causative agents of piroplasmosis. Association between seropositivity and host-related factors of species, gender, age, origin, activity and location were investigated. The overall seroprevalence was 11.6% (9.1-14.6%) with 95% confidence limit. The seroprevalence for T. equi and B. caballi was found to be 11% (8.6-14%) and 2.2% (1.2-3.9%), respectively. The animal-related factors significantly linked with seropositivity were the species, activities of farming, racing, recreation, and geographic location in Attica, Macedonia, Peloponnese and Thessaly region (p<0.05). The relative risks for the presence of T. equi, B. caballi and mixed infection in mules compared to horses was 8.39, 33.58 and 40.31, respectively. The infection level for T. equi, B. caballi and mixed infection were significantly higher in farm equids than in racing equids (p<0.05). Also, the rate of infection of T. equi was higher in farm equids than recreational equids (p<0.05). The relative risk of T. equi infection between farming equids and equids used only for recreation activity was 3.25-1, while the relative risk of B. caballi infection was 0.14-1 for racing animals relative to recreation animals. The region with the highest level of infection to both parasites was Thessaly (38.8% T. equi and 6.1% B. caballi), followed by Peloponnese (10.4% T. equi and 3.9% B. caballi), Attica region (8.3% T. equi and 0.6% B. caballi) and finally Macedonia the region with the lowest prevalence (6.6% T. equi and 4.4% B. caballi). A higher seroprevalence rate was found among local animals compared to imported equids, indicating that equine piroplasm infection is enzootic in Greece. T. equi seroprevalence was significantly different and higher among increasing age groups of equids, suggesting persistent infections or lower transmission levels whereby animals may need to be exposed longer before acquiring the infection. Competent tick vectors Rhipicephalus bursa and Rhipicephalus sanguineus for the transmission of equine piroplasmosis were recovered from horses and dogs, respectively.
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http://dx.doi.org/10.1016/j.vetpar.2010.01.011DOI Listing
May 2010

Detection of prohibited animal products in livestock feeds by single-strand conformation polymorphism analysis.

J Food Prot 2010 Jan;73(1):119-24

Centre for Food-borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada S7N 2R3.

Single-strand conformation polymorphism (SSCP) analysis of amplicons produced from a mitochondrial DNA region between the tRNA(Lys) and ATPase8 genes was applied for the detection of animal product within livestock feeds. Identification of prohibited animal (cattle, elk, sheep, deer, and goat) and nonprohibited animal (pig and horse) products from North America was possible based on the differential display of the single-stranded DNA fragments for the different animal species on SSCP gels. This method allowed specific detection and identification of mixed genomic DNA from different animal species. Trace amounts of cattle-derived materials were also detected in pig meat and bone meal and in grain-based feeds fortified with 10, 5, 1, or 0% porcine meat and bone meal. This study demonstrates the applicability of SSCP analyses to successfully identify the origin of animal species derived materials potentially present in animal feeds.
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http://dx.doi.org/10.4315/0362-028x-73.1.119DOI Listing
January 2010

A 10-year wildlife survey of 15 species of Canadian carnivores identifies new hosts or geographic locations for Trichinella genotypes T2, T4, T5, and T6.

Vet Parasitol 2010 Feb 23;168(1-2):78-83. Epub 2009 Oct 23.

Centre for Food-borne & Animal Parasitology, Saskatoon Laboratory, Canadian Food Inspection Agency, Saskatchewan, Canada.

A survey of wild carnivores in Canada was conducted over a 10-year period to determine the prevalence and genotypes of Trichinella. Muscle samples collected from 1409 animals representing 15 hosts species were enzymatically digested to recover Trichinella larvae. Larvae were recovered from a total of 287 (20.4%) animals and PCR identified four genotypes of Trichinella. Trichinella nativa was found in 5 host species and was the most commonly found genotype. Trichinella T6 was present in 7 species of carnivores, and coyote and badger are new host records for this genotype. The recovery of T. pseudospiralis and T. murrelli from cougars is the first documentation of these species in Canada and in cougars. The cougar was also the only host species in which all four genotypes of Trichinella were identified. Black bears and walruses had the highest tissue levels of larvae in this study and are also the species most frequently associated with human trichinellosis in Canada. This work identifies additional host species and expanded geographic ranges for 4 genotypes of Trichinella in North America. Failure to demonstrate T. spiralis in wildlife and continued negative results from ongoing surveillance activities in swine provide additional evidence that T. spiralis is not present in Canada.
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http://dx.doi.org/10.1016/j.vetpar.2009.10.012DOI Listing
February 2010

Detection and identification of Tetratrichomonas in a preputial wash from a bull by PCR and SSCP.

Vet Parasitol 2009 Dec 4;166(3-4):199-204. Epub 2009 Sep 4.

Centre for Food-Borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan S7N 2R3, Canada.

Motile Tritrichomonas foetus-like trichomonads were found during microscopic examination of a wet mount sample of a preputial wash collected from a bull. Staining of the organisms with a modified Wright-Giemsa stain revealed that several had four anterior flagella of unequal length instead of the three anterior flagella of equal length characteristic of T. foetus. Limited propagation of these organisms was achieved in InPouch medium but no growth occurred in modified Diamond's medium. The 5.8S rRNA gene and the flanking internal transcribed spacers were amplified from the trichomonad gDNA of two preputial wash samples and a fecal sample taken from the affected bull. Amplicons were subjected to single-strand conformation polymorphism (SSCP) analyses. The SSCP banding patterns of the amplicons from gDNA of the preputial wash samples were different from those of a T. foetus control sample. These unknown trichomonads were not detected in the fecal sample. The gDNA extracted from preputial washes was also subjected to PCR using primers developed to amplify the 16S rDNA of the non-T. foetus trichomonads, Tetratrichomonas and Pentatrichomonas spp. Amplicons were produced from the gDNA of the two preputial washes but not from the T. foetus gDNA control sample. The 16S rDNA sequences obtained from the trichomonads in the two preputial washes samples were 100% similar to that of a Tetratrichomonas species previously isolated from an Angus bull from the United States.
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http://dx.doi.org/10.1016/j.vetpar.2009.08.026DOI Listing
December 2009

Trichinella diagnostics and control: mandatory and best practices for ensuring food safety.

Vet Parasitol 2009 Feb 22;159(3-4):197-205. Epub 2008 Oct 22.

Canadian Food Inspection Agency, Centre for Food-Borne and Animal Parasitology, 116 Veterinary Road, Saskatoon, Saskatchewan, Canada.

Because of its role in human disease, there are increasing global requirements for reliable diagnostic and control methods for Trichinella in food animals to ensure meat safety and to facilitate trade. Consequently, there is a need for standardization of methods, programs, and best practices used in the control of Trichinella and trichinellosis. This review article describes the biology and epidemiology of Trichinella, and describes recommended test methods as well as modified and optimized procedures that are used in meat inspection programs. The use of ELISA for monitoring animals for infection in various porcine and equine pre- and post-slaughter programs, including farm or herd certification programs is also discussed. A brief review of the effectiveness of meat processing methods, such as freezing, cooking and preserving is provided. The importance of proper quality assurance and its application in all aspects of a Trichinella diagnostic system is emphasized. It includes the use of international quality standards, test validation and standardization, critical control points, laboratory accreditation, certification of analysts and proficiency testing. Also described, are the roles and locations of international and regional reference laboratories for trichinellosis where expert advice and support on research and diagnostics are available.
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http://dx.doi.org/10.1016/j.vetpar.2008.10.063DOI Listing
February 2009

Rapid discrimination of Salmonella isolates by single-strand conformation polymorphism analysis.

J Food Prot 2008 Oct;71(10):1960-6

Centre for Food-Borne and Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada.

A molecular typing technique was developed for the differentiation of Salmonella isolates based on single-strand conformation polymorphism (SSCP) analysis of amplicons generated by PCR. Amplicons from parts of the fimA (both the 5' and 3' ends), mdh, invA, and atpD genes were generated separately from a panel of Salmonella strains representing Salmonella bongori, and four subspecies and 17 serovars of Salmonella enterica. These amplicons were subjected to SSCP analysis for differentiation of the salmonellae on the basis of different conformational forms arising due to nucleotide sequence variations in the target genes. Several distinct SSCP banding patterns (a maximum of 14 each for atpD and fimA 3' end) were observed with this panel of Salmonella strains for amplicons generated from each target gene. The best discrimination of Salmonella subspecies and serovar was achieved from the SSCP analysis of a combination of at least three gene targets: atpD, invA, and either mdh or fimA 3' end. This demonstrates the applicability of SSCP analysis as an important additional method to classical typing approaches for the differentiation of foodborne Salmonella isolates. SSCP is simple to perform and should be readily transferable to food microbiology laboratories with basic PCR capability.
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http://dx.doi.org/10.4315/0362-028x-71.10.1960DOI Listing
October 2008

Highly sensitive and specific PCR assay for reliable detection of Cyclospora cayetanensis oocysts.

Appl Environ Microbiol 2008 Jul 23;74(14):4354-8. Epub 2008 May 23.

Canadian Food Inspection Agency, Saskatoon Laboratory, Centre for Food-borne and Animal Parasitology, 116 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 2R3.

Multiple outbreaks of food-borne gastroenteritis caused by the coccidian parasite Cyclospora cayetanensis have been reported annually in North America since 1995. Detection of C. cayetanensis contamination typically relies on laborious and subjective microscopic examination of produce washes. Molecular detection methods based on nested PCR, restriction fragment length polymorphism, or multiplex PCR have been developed for C. cayetanensis; however, they have not been adequately validated for use on food products. Further challenges include reliably extracting DNA from coccidian oocysts since their tough outer wall is resistant to lysis and overcoming PCR inhibitors in sample matrices. We describe preliminary validation of a reliable DNA extraction method for C. cayetanensis oocysts and a sensitive and specific novel PCR assay. The sensitivity and repeatability of the developed methods were evaluated by multiple DNA extractions and PCR amplifications using 1,000-, 100-, 10-, or 1-ooycst aliquots of C. cayetanensis oocysts in water or basil wash sediment. Successful PCR amplification was achieved on 15 and 5 replicates extracted from aliquots containing 1,000 oocysts in water and basil wash, respectively. All 45 replicates of the 100-oocyst aliquots in water and 5 in basil wash were amplified successfully, as were 43/45 and 41/45 of the 10- and 1-oocyst aliquots in water and 9/15 and 2/15 in basil wash, respectively. The developed primers showed no cross-reactivity when tested against bacteria, nematodes, and protozoans, including Eimeria, Giardia, and Cryptosporidium. Our results indicate that these methods are specific, can reliably detect a single oocyst, and overcome many of the limitations of microscopic diagnosis.
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http://dx.doi.org/10.1128/AEM.00032-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493149PMC
July 2008

Experimental transmission of bovine anaplasmosis (caused by Anaplasma marginale) by means of Dermacentor variabilis and D. andersoni (Ixodidae) collected in western Canada.

Can J Vet Res 2007 Oct;71(4):271-7

Canadian Food Inspection Agency (CFIA), Saskatoon Laboratory, Centre for Food-Borne & Animal Parasitology, 116 Veterinary Road, Saskatoon, Saskatchewan S7N 2R3.

Canadian cattle are free of bovine anaplasmosis, with the exception of 4 isolated incursions since 1968, which were eradicated. It is not known why the disease has not become established in regions of Canada adjacent to the United States where it is endemic. To assess the vector competence of wild-caught ticks in cattle-rearing regions, Dermacentor variabilis and D. andersoni were collected in western Canada and fed on calves experimentally infected with Anaplasma marginale (St. Maries strain). The 2 tick species were equally competent in transmitting A. marginale to splenectomized calves, all 15 tick-exposed calves becoming infected. The prepatent periods in 13 calves ranged from 18 to 26 d and did not vary in relation to the numbers of ticks fed or the duration of transmission feedings. The unusually long prepatent periods in 2 calves (45 and 55 d) were probably due to concomitant Eperythrozoon infection. This study clearly demonstrated that tick species present in western Canada are competent vectors of bovine anaplasmosis. Potential barriers, including climate, must be considered in developing strategies to prevent A. marginale from becoming established in anaplasmosis-free regions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1940274PMC
October 2007

Bighorn sheep, a new host record for Parelaphostrongylus odocoilei (Nematoda: Protostrongylidae).

J Wildl Dis 2006 Oct;42(4):877-82

Centre for Animal Parasitology, Canadian Food Inspection Agency, Saskatoon, Saskatchewan S7N 2R3, Canada.

Larval nematodes with a dorsal spine on the tail were recovered from fecal samples of California bighorn sheep (Ovis canadensis californiana) in northeastern Washington State, USA. The identity of these dorsal-spined larvae (DSL) was established by single-strand conformation polymorphism (SSCP) analyses of a partial fragment of the first internal transcribed spacer of the ribosomal DNA. The SSCP profiles of individual DSL from bighorn sheep were compared with those of DSL of five protostrongylid species (Parelaphostrongylus andersoni, P odocoilei, P. tenuis, Elaphostrongylus rangiferi, and Muellerius capillaris) but were identical to only those of P. odocoilei. This study represents the first confirmed identification of P. odocoilei in bighorn sheep.
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http://dx.doi.org/10.7589/0090-3558-42.4.877DOI Listing
October 2006

A method for extracting genomic DNA from individual elaphostrongyline (Nematoda: Protostrongylidae) larvae and differentiation of Elaphostrongylus spp. from Parelaphostrongylus spp. by PCR assay.

J Vet Diagn Invest 2005 Nov;17(6):585-8

Department of Biology, University of Saskatchewan, Saskatoon, Canada.

This article reports a rapid and effective method for the extraction and purification of genomic DNA (gDNA) from individual first-stage larvae (L1) of elaphostrongyline nematodes that had been stored frozen or fixed in 95% ethanol for 1 to 5 years. The method was highly effective for L1s of all 6 species of elaphostrongylines, based on polymerase chain reaction (PCR) amplification of a partial fragment of the first internal transcribed spacer (ITS-1) of the ribosomal DNA. Differences were detected in the sizes of partial ITS-1 amplicons between the 2 elaphostrongyline genera, Elaphostrongylus and Parelaphostrongylus. The reliability of the ITS-1 PCR assay was tested by using L1s of unknown identity from Newfoundland and Labrador, Canada. The ability to consistently isolate gDNA from individual L1s, together with a simple PCR-based method to distinguish between Parelaphostrongylus and Elaphostrongylus, have important implications for diagnostic testing and for conducting epizootiological studies on these parasites of veterinary importance.
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http://dx.doi.org/10.1177/104063870501700612DOI Listing
November 2005
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