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

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.

Infectivity of Toxoplasma gondii in northern traditional (country) foods prepared with meat from experimentally infected seals.

Serological and clinical evidence of human toxoplasmosis in the Canadian Arctic indicates a food safety risk associated with the consumption of wild game meat. Such meat often is eaten raw or partially cooked in locally prepared traditional (country) foods, but no data have been collected to describe survival of Toxoplasma gondii forms in these foods. The muscle of grey seals (Halichoerus grypus) experimentally infected with T. gondii oocysts was used to prepare three country foods: igunaq, a fermented product; nikku, a dried product; and sausage, a salted and spiced product. Igunaq and nikku were stored at 4 degrees C and bioassayed in cats at 49, 95, and 140 days postpreparation (DPP) and 41, 84, and 132 DPP, respectively. Raw and cooked sausages were stored at -20 degrees C and bioassayed at 50, 92, and 141 DPP. The source seal meat was infective for cats, but none of the foods prepared with this meat were infective for cats. Some cooked sausages did not reach internal temperatures considered lethal for T. gondii. Data from studies in domestic animals suggested that the negative results in this experiment were due to temperature and duration of storage. Because of the possibility that T. gondii of arctic origin might be more freeze tolerant than the swine-origin isolate used in this experiment, additional studies are necessary to clarify the risks of toxoplasmosis associated with consumption of arctic country foods.

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

Effective performance of digestion testing methods for Trichinella, 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 Trichinella 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 Trichinella 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 Trichinella. 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.

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

Transmission dynamics of Toxoplasma gondii, 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 T. gondii in Arctic foxes (Vulpes lagopus) in the Karrak Lake region, Nunavut, Canada. Sera from adult foxes and fox pups were tested for antibodies to T. gondii 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 T. gondii 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 T. gondii 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 T. gondii, 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.

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

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.

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

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.

Validation of a new commercial serine protease artificial digestion assay for the detection of Trichinella larvae in pork.

Trichinella is a zoonotic nematode parasite transmitted by the ingestion of raw or under-cooked meat. Control of the parasite is essential to facilitate public health and trade in products from susceptible food animals, including pork and horse meat. The standard method for detecting Trichinella muscle larvae uses pepsin enzyme and hydrochloric acid (HCl) in an artificial digestion procedure. A new artificial digestion assay using serine protease was recently developed and commercialized (PrioCHECK™ Trichinella AAD) for the detection of Trichinella larvae in the muscle of infected animals. The assay uses no hazardous substances such as HCl or pepsin. Activation of the enzyme requires an elevated digestion temperature of 60 °C which kills the parasite and reduces the risk of contaminating the environment with Trichinella. Compared to the pepsin/HCl method, digestion time for the PrioCHECK Trichinella AAD assay is reduced by a third. A recent study demonstrated these features of the new assay and its suitability for digesting various muscles from domestic and wild animals. To further validate the assay's performance relative to the conventional pepsin/HCl digestion method several comparative studies were conducted using samples from different muscle sites spiked with low levels of encapsulated first stage Trichinella larvae (L1). Multiple muscle samples were collected from diaphragm, tongue, masseter, and loin of 3-4 month old pigs. Samples were spiked with 3, 4, 5, or 25 Trichinella spiralis L1. A total of 320 meat samples of 100 g each were used to compare the diagnostic proficiency of the PrioCHECK Trichinella AAD assay with the pepsin/HCl digestion method. Comparative and validation data produced from these studies showed that both methods are capable of consistently detecting Trichinella in 100 g samples which contained as few as 3 L1 or 0.03 larvae per gram of meat. Overall, the PrioCHECK Trichinella AAD assay performed satisfactorily according to international guidelines of the World Organization for Animal Health (OIE), European Union (EU) and International Commission on Trichinellosis (ICT) for the detection of Trichinella infection in pork.

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

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.

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

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.

Use of proficiency samples to assess diagnostic laboratories in France performing a Trichinella digestion assay.

Routine diagnosis of animal trichinellosis for food safety and trade relies on a method of artificial digestion to free Trichinella muscle larvae from meat for subsequent identification by microscopy. As part of a quality control system, the French National Reference Laboratory (NRL) initiated ring trials to determine the sensitivity of the test performed in the 72 routine diagnostic laboratories in France. A method was devised to obtain calibrated meat samples containing known numbers of capsules with Trichinella spiralis muscle larvae. This method was based on an incomplete artificial digestion of Trichinella-infected mice carcasses to allow the collection of intact Trichinella capsules. Capsules were placed into a meatball of 100 +/- 2 g of pork and horsemeat to produce proficiency samples. Three categories of samples were prepared: small (3 to 5 capsules), medium (7 to 10), and large (12 to 15). The sensitivity was expressed as the percentage of muscle larvae recovered from each proficiency sample. Reproducibility was tested with ring trials organized between two NRLs (France and Canada), and a reference sensitivity of 84.9% was established. National ring trials were then organized in France, with the 72 routine diagnostic laboratories each receiving four proficiency samples per session. After five sessions, an improvement in the digest test sensitivity was observed. Results at the fifth session indicated sensitivities of 78.60% +/- 23.70%, 81.19% +/- 19.59%, and 80.52% +/- 14.71% muscle larvae for small, medium, and large samples, respectively. This study supports the use of proficiency samples to accurately evaluate the performance of routine diagnostic laboratories that conduct digestion tests for animal trichinellosis diagnosis.

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

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.

Evaluation of the PrioCHECK™ Trichinella AAD Kit for the digestion and recovery of larvae in pork, horse meat and wild meat.

The artificial digestion magnetic stirrer method using pepsin protease and hydrochloric acid is the standard assay for the detection of Trichinella larvae in muscle of infected animals. Recently, an alternative enzyme, serine protease, was employed in the development of a commercially available digestion kit (PrioCHECK™ Trichinella AAD Kit). This assay requires a higher digestion temperature of 60°C which kills the larvae during the digestion process, mitigating the risk of environmental contamination from the parasite. The present study was conducted to determine the performance of the PrioCHECK™ Trichinella AAD Kit compared to the conventional pepsin/HCl digestion. Replicate paired 115g samples of Trichinella-negative pork diaphragm and masseter, and of horse tongue and masseter, were used to compare the two methods for tissue digestibility. Similarly, paired 100g samples of pork diaphragm and horse tongue were spiked with proficiency samples containing known numbers of Trichinella spiralis first stage larvae to compare larval recoveries for the two methods. Masseter samples from wild bears and wolves naturally infected with Trichinella nativa or T6 were also used to compare the performance of the methods. The results of the study showed that the PrioCHECK™ Trichinella AAD Kit, when used according to the manufacturer's instructions, was effective in detecting Trichinella infection in all samples that contained 0.05 or more larvae per gram of tissue. Although there was no significant difference between the Kit method and the standard pepsin/HCl digestion procedure in the average number of larvae recovered from spiked pork diaphragm, 38% fewer larvae were recovered from similarly spiked samples of horse tongue by digestion using serine protease (one way ANOVA, P value <0.001). Additional clarification was also more often required for both horse meat and pork when using the Kit compared to the pepsin/HCl method. The results of testing wildlife samples were similar for the two methods. Overall, the performance of the Kit method was suitable for the digestion of muscle samples and recovery of Trichinella larvae, according to international standards. It also provides advantages of faster digestion, safer reagents and recovered parasites that are non-hazardous for analysts and the environment.

Pathology, clinical signs, and tissue distribution of Toxoplasma gondii in experimentally infected reindeer (Rangifer tarandus).

Toxoplasma gondii 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 (Rangifer tarandus) are widely harvested for food across the Canadian North, show evidence of seroexposure to T. gondii, 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 T. gondii 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 T. gondii. 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 T. gondii 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 T. gondii, and that the parasite may be associated with health effects in wildlife. The presence of T. gondii 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 T. gondii.

Single-strand conformation polymorphism (SSCP) analysis as a new diagnostic tool to distinguish dorsal-spined larvae of the Elaphostrongylinae (Nematoda: Protostrongylidae) from cervids.

Single-strand conformation polymorphism (SSCP) was used to genetically differentiate morphologically indistinguishable first-stage larvae (L(1)) of the six species of elaphostrongyline nematodes. A partial fragment (317-336bp) of the first internal transcribed spacer (pITS-1) plus 5' flanking region (76bp of the 18S gene) of the nuclear ribosomal DNA (rDNA) was amplified from individual L(1) of known identity and subjected to SSCP. The results showed that the four species of elaphostrongylines found in North American cervids, Parelaphostrongylus tenuis, P. andersoni, P. odocoilei and Elaphostrongylus rangiferi, could be distinguished from one another based on their distinct (i.e. species-specific) SSCP profiles. In addition, E. alces, a species that occurs in moose in Fennoscandinavia, also had a distinct SSCP profile with respect to the other species of elaphostrongylines. However, the SSCP profiles of E. cervi could not be distinguished from those of E. rangiferi because of a lack of interspecific sequence differences in this region of the ITS-1. The distinct SSCP profiles for the other species were consistent with the interspecific differences in ITS-1 sequences, which ranged from 2 (between P. tenuis and P. andersoni) to 59bp (between genera). The pITS-1 SSCP approach was also used to identify unknown elaphostrongyline L(1) from different hosts and localities in North America. The ability to distinguish between L(1) of the four elaphostrongyline species that occur in North American cervids has important diagnostic and epidemiological implications.

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

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.

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

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.

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

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.

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

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.

A program to accredit laboratories for reliable testing of pork and horse meat for Trichinella.

The Canadian Food Inspection Agency (CFIA) has developed a program to accredit external laboratories to conduct Trichinella digestion assays for export purposes. Accredited laboratories are responsible for staffing, equipment and operating test facilities under the auspices and guidance of the CFIA. The CFIA's Centre for Animal Parasitology provides training, proficiency samples, audits and other support for the accreditation process. The program has also been adapted for use in laboratories conducting Trichinella digestion tests for surveillance and food safety purposes and provides a useful template for others wishing to develop similar systems.