Lorry B. Forbes

A validated Trichinella digestion assay and an associated sampling and quality assurance system for use in testing pork and horse meat.

A revised digestion method, developed for efficiency and quality assurance, was validated for the detection of Trichinella larvae in pork and horse meat to meet requirements for food safety testing and facilitate access to international markets. The method consisted of a tissue homogenization step and a spin bar digestion procedure conducted at 45 degrees C to free larvae from muscle tissue, followed by two sequential separatory funnel steps to concentrate the larvae for detection using a stereomicroscope. Critical control points were determined for the method and monitored during testing. Under conditions of a defined protocol, test capacity was suitable for industrial applications, since multiples of up to 100 g of tissue could be analyzed at one time. The overall sensitivity of the test system depended on the size and origin of the sample taken from individual infected carcasses. Data from swine indicated that the currently accepted sample size of 1 g from individual carcasses consistently detected larval loads of > or =3 larvae per gram. Larval loads of 1.0 to 1.9 larvae per gram required 3- to 5-g samples of muscle tissue for reliable detection. Five-gram samples were considered optimal, because they consistently detected more tissues than 3-g samples, although the difference was not statistically significant. Tissue localization studies in experimental pigs indicated that the tongue and diaphragm were the tissues of choice for the most sensitive larval recovery. A system of analyst training, laboratory certification based on ISO guide 25, and on-site proficiency panel testing was used to ensure that external laboratories would consistently produce reliable test results. The system developed for pork was successfully modified for the testing of horse meat.

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.

The occurrence and ecology of Trichinella in marine mammals.

Trichinella in marine mammals has a circumpolar arctic distribution and a narrow range of host species. It is commonly found in polar bears (Ursus maritimus), and increasingly in walruses (Odobenus rosmarus) where it presents a significant zoonotic hazard. This has resulted in the implementation of food safety programs in some arctic communities to test harvested walrus meat for Trichinella larvae prior to consumption. Trichinella has been reported infrequently in bearded seals (Erignathus barbatus) and ringed seals (Phoca hispida), and was once observed in a Beluga whale (Delphinapterus leucas). Cannibalism is probably the most important factor in maintaining a Trichinella cycle in polar bears. Arctic carnivores such as polar bears, arctic foxes (Alopex lagopus) and domestic dogs (Canis familiaris) have a high prevalence of Trichinella infection and the carcasses of at least some of these animals are deposited in the ocean. Scavenging of these carcasses by walruses probably occurs, but may not account for the high prevalence the parasite seen in this host species. Predation, carrion feeding and cannibalism have been documented for walruses and a sylvatic cycle similar to that of bears may exist in walrus populations. Seals and whales are likely infected through infrequent exposure to infected carcasses, either directly by scavenging or indirectly by consuming amphipods or fish that have fed on infected carcasses. The inefficiency of this mechanism may account for the low prevalence of Trichinella in seals and whales. It is known that isolates from marine mammals are cold tolerant, and infectious for man, and have been identified as Trichinella nativa (T2). Molecular and other phylogenetic studies would be useful to facilitate studies on the inter-relationship of Trichinella cycles involving marine and terrestrial mammals in the arctic and subarctic, and in the investigation of human outbreaks of trichinellosis in these areas.

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 Agencys (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.

EXPERIMENTAL TOXOPLASMA GONDII INFECTION IN GREY SEALS (HALICHOERUS GRYPUS)

Laboratory-reared animals were used to assess the susceptibility of seals (Halichoerus grypus) to Toxoplasma gondii infection. Four seals were each orally inoculated with 100 or 10,000 oocysts of T. gondii (VEG strain), and another 4 seals served as negative controls. Occasionally, mild behavioral changes were observed in all inoculated seals but not in control animals. A modified agglutination test revealed the presence of antibodies to T. gondii in sera collected from inoculated seals and mice inoculated as controls. No evidence of the parasite was found on an extensive histological examination of seal tissues, and immunohistochemical staining of tissue sections from inoculated seals revealed a single tissue cyst in only 1 seal. Control mice inoculated with 10 oocysts from the same inoculum given to seals became serologically and histologically positive for T. gondii. Cats that were fed brain or muscle tissue collected from inoculated seals passed T. gondii oocysts in feces. This study demonstrates that T. gondii oocysts can establish viable infection in seals and supports the hypothesis that toxoplasmosis in marine mammals can be acquired from oocysts in surface water runoff and sewer discharge.

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.

Proficiency samples for quality assurance in Trichinella digestion tests.

A reliable method to produce proficiency samples containing known numbers of Trichinella spiralis cysts for use in quality assurance systems for Trichinella digestion tests was developed and validated. A filtrate containing Trichinella cysts was produced by homogenizing and filtering the muscles of an experimentally infected rat. Using a stereomicroscope and micropipette, intact cysts were removed from the filtrate and were transferred onto an agar substrate to allow accurate counting and subsequent transfer into a sample matrix. The proficiency sample matrix consisted of 20-g balls of lean ground beef and was combined with 80 g of a Trichinella-free muscle tissue to obtain the required 100-g sample weight for the assay. The mean overall larval recovery from 404 proficiency samples was 93.0%. Larval recoveries > or = 95, 85, and 75% occurred in 52.4, 84.4, and 94.3%, respectively, of the 404 samples tested. Results indicated that, after a short training period, technicians with no prior experience in digestion techniques performed as well as experienced technicians. The maximum shelf life of proficiency samples was not determined but was at least 3 weeks. Validation data were used to develop panels composed of proficiency samples prepared as described above and to establish guidelines for the interpretation of proficiency panel results.

Experimental Trichinella infection in seals

The susceptibility of seals to infection with Trichinella nativa and the cold tolerant characteristics of muscle larvae in seal meat were evaluated. Two grey seals, Halichoerus grypus, were inoculated with 5000 (100 larvae/kg) T. nativa larvae and two grey seals with 50000 (1000 larvae/kg). One seal from each dose group and two control seals were killed at 5 and 10 weeks post-inoculation (p.i.). At 5 weeks p.i., infection was established in both low and high dose seals with mean larval densities of 68 and 472 larvae per gram (lpg), respectively, using eight different muscles for analyses. At 10 weeks p.i., mean larval densities were 531 and 2649 lpg, respectively, suggesting an extended persistence of intestinal worms. In seals with high larval density infections, the distribution of larvae in various muscles was uniform, but in one seal with a low larval density infection, predilection sites of larvae included muscle groups with a relative high blood flow, i.e. diaphragm, intercostal and rear flipper muscles. Trichinella-specific antibody levels, as measured by ELISA, increased during the 10 week experimental period. Infected seal muscle was stored at 5, -5 and -18 degrees C for 1, 4 and 8 weeks. Muscle larvae released from stored seal muscle by artificial digestion were inoculated into mice to assess viability and infectivity. Larvae from seal muscle 10 weeks p.i. tolerated -18 degrees C for 8 weeks but larvae from seal muscle 5 weeks p.i. tolerated only 1 week at -18 degrees C, supporting the hypothesis that freeze tolerance increases with the age of the host-parasite tissue complex. The expressed susceptibility to infection, extended production of larvae, antibody response and freeze tolerance of T. nativa in seals are new findings from the first experimental Trichinella infection in any marine mammal and suggest that pinnipeds (phocids, otariiids or walrus) may acquire Trichinella infection by scavenging even small amounts of infected tissue left by hunters or predators.

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.