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.

Canine brucellosis in a Saskatchewan kennel.

Canine brucellosis is rare in Canada. This report describes an outbreak of Brucella canis infection within a kennel, emphasizing diagnostic and pathologic findings. Gender differences are described. The progestational, nongravid uterus, female spleen, and prostate gland are consistent sites of bacterial isolation.

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.

Trichinella Surveillance in Black Bears ( Ursus americanus ) from the Dehcho Region, Northwest Territories, Canada, 2002-15.

We used muscle digestion to test black bears ( Ursus americanus ) from the southwestern Northwest Territories, Canada, for Trichinella. Results showed a prevalence of 4.1%. Some bears had infection intensities of more than one larva per gram of muscle tissue; this level in meat is considered to pose a human consumption safety risk.

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.

Novel prevention program for trichinellosis in inuit communities.

Repeated outbreaks of trichinellosis caused by the consumption of Trichinella-infected walrus (Odobenus rosmarus) meat, which have sometimes led to serious morbidity, have stimulated Inuit communities in Nunavik (northern Quebec), Canada, to develop an innovative trichinellosis prevention program. The program involves preconsumption testing of meat samples from harvested walrus at a regional laboratory and the rapid dissemination of the results of such testing to communities. Local health authorities in Inukjuak conducted an epidemiological investigation after testing identified Trichinella-positive walrus meat in September 1997. This report describes the events that occurred before, during, and after the trichinellosis outbreak and also documents how the prevention program contributed to successful resolution of the outbreak.

Comparison of a modified digestion assay with trichinoscopy for the detection of Trichinella larvae in pork.

A pepsin-HCl digestion assay and two compressorium techniques (trichinoscopy) for the identification of swine muscle tissue containing low levels of Trichinella larvae were compared as part of the test validation process for quality assurance purposes. Compressoria read with a stereomicroscope detected more larvae (P < 0.0001, n = 57) and more tissues (P = 0.0047, n = 57) than did compressoria read with a projection microscope (trichinoscope). The digestion assay evaluated was 3.2 times as likely as the best compressorium technique to identify a positive tissue when these procedures were used to test 1 g of infected muscle (P < 0.001; 95% confidence interval for the odds ratio, 2.0 to 5.4; n = 161 and n = 189, respectively). Detection by trichinoscopy improved as the number of larvae in tissues increased to > 2 larvae per g, but trichinoscopy was less sensitive than the digestion assay regardless of the tissue larval load. These data indicate that the quality controlled digestion assay used in this study is more sensitive than trichinoscopic techniques in the detection of tissues containing low levels of Trichinella larvae.

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

The infectivity of Trichinella nativa larvae in three traditional northern (country) foods was assessed. Foods were prepared with meat from seals experimentally infected with Trichinella nativa and evaluated over a 317-day period during which this food was fed directly to cats while mice were orally inoculated with larvae recovered following the digestion of the food in a solution containing 1% pepsin and 1% HCl at 37 degrees C. Foods examined were igunaq (meat and blubber placed in a seal skin bag and allowed to ferment), nikku (air-dried meat), and sausage (meat, fillers, salt, and spices). Sausage was examined both in a raw state and after partial cooking. Infective T. nativa larvae survived in igunaq, nikku, raw frozen sausage, and poorly cooked sausage for at least 5 months under controlled laboratory conditions. Core temperatures of partially cooked sausage never exceeded 50 degrees C. Caution should be exercised in using these data to establish guidelines for the consumption of raw products, since the survival of infective larvae could be unpredictably extended under field conditions. These data indicate significant food safety risks associated with igunaq, nikku, and sausage prepared with Trichinella-infected meat and provide information for use in risk management and in directing future research.

Evaluation of a digestion assay and determination of sample size and tissue for the reliable detection of Trichinella larvae in walrus meat.

A digestion assay was validated for the detection of Trichinella larvae in walrus (Odobenus rosmarus) meat, and appropriate samples for testing were determined using tissues from infected walruses harvested for food. Examination of muscles from 3 walruses showed that the tongue consistently contained approximately 2-6 times more larvae than the pectoral and intercostal muscles. Comparison of numbers of larvae in the root, body, and apex of the tongue from 3 walruses failed to identify a predilection site within the tongue, but the apex was considered an optimal tissue because of the high larval density within the tongue and the ease of collection. All 31 spiked samples weighing 50 g each and containing between 0.1 and 0.4 larvae per gram (lpg) were correctly identified as infected, indicating that the sensitivity of this procedure is adequate for diagnostic use. A sample size of 10 g consistently detected larvae in 2 walrus tongues containing > or = 0.3 lpg (n = 40), and until additional data are available, sample sizes from individual walrus tongues should be a minimum of 10 g. This study provides the preliminary data that were used for the development of a food safety analytical protocol for the detection of Trichinella in walrus meat in arctic communities.

An internationally recognized quality assurance system for diagnostic parasitology in animal health and food safety, with example data on trichinellosis.

A quality assurance (QA) system was developed for diagnostic parasitology and implemented for several diagnostic assays including fecal flotation and sedimentation assays, trichomonad culture assay, and the testing of pork and horse meat for Trichinella to facilitate consistently reliable results. The system consisted of a validated test method, procedures to confirm laboratory capability, and protocols for documentation, reporting, and monitoring. Specific system components included a quality assurance manual, training program, proficiency panels, inter-laboratory check-sample exchange program, assay critical control points, controls, and audits. The quality assurance system of the diagnostic laboratory was audited according to ISO/IEC Standard 17025 by an international third party accrediting body and accredited as a testing laboratory for the specific parasitology tests. Test results generated from the laboratory were reliable and scientifically defensible according to the defined parameters of the tests and were therefore valid for a variety of purposes, including food safety, international trade, and declaration of disease status in an animal, herd, farm, or region. The system was applicable to various test methods for the detection of parasites in feces or other samples, and a digestion test system developed for Trichinella was used as an example. A modified tissue digestion assay was developed, validated, and implemented by the Canadian Food Inspection Agency's Centre for Animal Parasitology for efficiency and quality assurance. The details of the method were properly documented for routine testing and consisted of a homogenization process, an incubation at 45+/-2 degrees C, and two sequential sedimentations in separatory funnels to concentrate and clarify final aliquots for microscopic examination. To facilitate consistently reliable test results, 14 critical control points were identified and monitored, analysts were certified, and the test system verified through the use of validation data, proficiency samples, and training modules.

Comparison of synthetic tyvelose antigen with excretory-secretory antigen for the detection of trichinellosis in swine using enzyme-linked immunosorbent assay.

Two enzyme-linked immunosorbent assay (ELISA) systems, one using natural excretory-secretory (ES) antigens and the other a synthetic glycan antigen (3,6-dideoxy-D-arabinohexose [tyvelose, TY]), were evaluated for the serological diagnosis of trichinellosis in swine. Sensitivity was estimated using samples (n = 113) collected 3-21 wk PI from 15 experimentally infected pigs, and specificity was estimated using samples (n = 397) from a population of Trichinella spp.-free pigs. Results were analyzed using 2 cutoff values recommended in international guidelines (Office Internationale des Epizooties [OIE]) and by the optimal cutoff level as determined by receiver-operator characteristic (ROC) analysis. The ROC-optimized TY-ELISA consistently performed better than all other combinations. None of the combinations of test and cut-off detected infected pigs sooner than 35 days; however, the ROC-optimized TY-ELISA identified 8 of 15 pigs earlier than the ES-ELISA and detected 2 pigs missed by all other tests. At 49 days PI the sensitivity and specificity of the ROC-optimized TY-ELISA were 94.3 and 96.7%, respectively, as compared with the ROC-optimized ES-ELISA at 84.9 and 96.0%, respectively. The ROC-optimized TY-ELISA was 100% specific at OIE-recommended cut-offs. This study indicates that the TY-ELISA is as good or better than the ES-ELISA for the detection of trichinellosis in swine.

Evaluation of cattle for experimental infection with and transmission of Brucella suis biovar 4.

OBJECTIVE: To determine whether cattle can become persistently infected with Brucella suis biovar 4, whether the organism can be transmitted vertically or horizontally, and whether tests for bovine brucellosis are diagnostic. DESIGN: Observational study. ANIMALS: 24 pregnant cows and their calves and 6 bulls. PROCEDURE: Cows and bulls were housed separately in groups of 6 with each group consisting of 3 cattle experimentally infected with B suis biovar 4 and 3 naïve animals. Cattle were observed for clinical signs daily; blood samples were collected weekly. Clotted blood from each sample was submitted for bacterial culture. Serum was tested with an indirect ELISA and the standard tube agglutination test (STAT), buffered plate agglutination test, brucellosis card test (BCT), and complemen't fixation test (CFT). Tissues collected at necropsy were submitted for bacterial culture and histologic examination. RESULTS: All 15 inoculated cattle seroconverted on 2 or more serologic tests, and bacteria were isolated from 4 inoculated cows at necropsy. There was no bacteriologic evidence of vertical or horizontal transmission, and none of the cattle developed clinical abnormalities or gross or histologic lesions. Results of the indirect ELISA were positive for all inoculated cattle. The other tests gave variable results; the CFT, STAT, and BCT yielded negative results for at least 1 of the 4 cattle from which the organism was isolated. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cattle-to-cattle transmission of B suis biovar 4 is unlikely. Serologic tests for bovine brucellosis should be used cautiously when attempting to identify cattle with rangiferine brucellosis, as they do not discriminate between the 2 diseases and vary in their ability to detect exposed cattle.

National serologic survey for trichinellosis in sows in Canada 1996-1997.

Sera from 14,408 market sows from the Canadian domestic swine herd were tested for trichinellosis using an indirect-ELISA as a screening test and a competitive ELISA for confirmatory testing. Three sera (0.02%) gave low level reactions on the competitive ELISA. These reactions were considered nonspecific, and this designation was supported by data from previous and subsequent national surveys, in which serologic, trichinoscopic, and digestion test methodologies was used. This study provides additional evidence that Canada is free of trichinellosis in domestic swine.

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.

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.

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

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.

Prevalence of Trichinella spp. in black bears, grizzly bears, and wolves in the Dehcho Region, Northwest Territories, Canada, including the first report of T. nativa in a grizzly bear from Canada.

Samples of muscle from 120 black bears (Ursus americanus), 11 grizzly bears (Ursus arctos), and 27 wolves (Canis lupus) collected in the Dehcho Region of the Northwest Territories from 2001 to 2010 were examined for the presence of Trichinella spp. larvae using a pepsin-HCl digestion assay. Trichinella spp. larvae were found in eight of 11 (73%) grizzly bears, 14 of 27 (52%) wolves, and seven of 120 (5.8%) black bears. The average age of positive grizzly bears, black bears, and wolves was 13.5, 9.9, and approximately 4 yr, respectively. Larvae from 11 wolves, six black bears, and seven grizzly bears were genotyped. Six wolves were infected with T. nativa and five with Trichinella T6, four black bears were infected with T. nativa and two with Trichinella T6, and all seven grizzly bears were infected with Trichinella T6 and one of them had a coinfection with T. nativa. This is the first report of T. nativa in a grizzly bear from Canada. Bears have been linked to trichinellosis outbreaks in humans in Canada, and black bears are a subsistence food source for residents of the Dehcho region. In order to assess food safety risk it is important to monitor the prevalence of Trichinella spp. in both species of bear and their cohabiting mammalian food sources.

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.

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.

Distribution of Taenia saginata cysticerci in tissues of experimentally infected cattle.

Bovine cysticercosis caused by Taenia saginata is a zoonotic disease warranting routine inspection measures for the postmortem detection of cysticerci (cysts) in beef destined for human consumption. Detection is based on gross examination of traditional carcass predilection sites, although there is evidence to suggest that examination of other sites may offer improvements in sensitivity. In order to evaluate the efficacy of current inspection protocols, this study determined the distribution and number of cysticerci in the tissues of experimentally infected cattle. Forty-two commercial beef cattle were divided into five groups of 5-12 animals each and inoculated with either 10,000, 5000, 1000, 100 or 10 T. saginata eggs. At time points ranging from 47 to 376 days post-inoculation (DPI), 10 animals inoculated with 5000 eggs were killed and the carcasses partitioned into 31 tissue sites. These consisted of the traditionally inspected tissue sites of heart, masseter and pterygoid muscles, tongue, oesophagus, and diaphragm (membranous and crura); as well as non-traditional sites of lung, liver and an additional 20 individual muscles or muscle groups. After performing the Canadian Food inspection Agency's (CFIA) routine inspection protocol for cysticerci on traditional tissue sites, tissues from all sites were cut into approximately 0.5 cm thick slices and the total number of parasitic cysts and cyst density (number of cysts/g of tissue) determined for each site. Traditional sites were similarly evaluated for the remaining 32 animals killed between 117 and 466 DPI. Sites were ranked based on cyst density. Infection was confirmed in 37 animals, of which only 20 were detected by routine inspection, and of which 7 harboured no cysts in traditional sites. For the animals in which additional non-traditional sites were evaluated, none yielded higher cyst densities than those traditionally inspected. When only traditional sites (for all animals) were compared, the heart ranked highest overall, although it was not significantly different from the masseter muscle, and was the most frequently affected site. The traditional site of oesophagus was one of the least rewarding of all sites for detection of cysticerci. The heart was confirmed as the preferred site for detection of bovine cysticercosis based on high cyst density and frequency of infection, and greater visibility of gross lesions due to the early inflammatory response in cardiac muscle. More extensive examination of the heart is recommended to improve detection of infected animals.

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

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.

A preliminary investigation on the infectivity of Trichinella larvae in traditional preparations of walrus meat.

This study evaluated the infectivity of Trichinella nativa in freshly frozen walrus meat and traditionally aged walrus meat (igunaq) associated with two human outbreaks of trichinellosis in the Canadian Arctic. Trichinella larvae recovered from walrus meat stored at -20 degrees C for up to 20 months remained infective for guinea pigs inoculated with 135 or 716 larval doses. However, none of the 4-5 and 10-month-old igunaq preparations contained infective T. nativa larvae as measured by bioassays using mice and guinea pigs at inoculation doses ranging from 6 to 500 larvae. This indicates that the degradation process that occurred in the field can be sufficient to either kill Trichinella larvae or render them non-infective for mice and guinea pigs. Further research is needed to evaluate the food safety risk of traditional walrus igunaq aged under different field conditions and storage times.