High Prevalence and Intensity of Trichinella Infection in Yukon American Black (Ursus americanus) and Grizzly (Ursus arctos) Bears.

Trichinella spp. nematodes are commonly found in bear species (Ursidae) and can pose severe health risks to humans when infective first-stage larvae are ingested in meat. Samples of tongue or masseter muscle from 22 male and 11 female American black bears (Ursus americanus; mean age 6.5 yr, range 1-16 yr) and 22 male, eight female, and one unknown sex grizzly bears (Ursus arctos; mean age 8.8 yr, range 2-28 yr), from Yukon, Canada, were tested to determine prevalence and intensity of Trichinella spp. infection. For black bears, prevalence was 20% and mean intensity was 401 larvae per gram of tissue (LPG), whereas for grizzly bears, prevalence was 71%, and mean infection intensity was 35 LPG. Isolates from all positive samples were identified as genotype Trichinella-T6 by multiplex PCR. For black bears, prevalence is the highest reported in Canada and infection intensity the highest recorded in North America. One black bear had a larval burden of 1,173 LPG, the second highest recorded in any host species. The prevalence in grizzly bears was the highest reported in Canada for this host. In total, 90% (27 of 30) of infected bears had infection burdens above the human food safety threshold of ≥1 LPG, reinforcing the importance of communicating the health risks to people consuming bear meat.

Early days of the International Commission on Trichinellosis (1958-1972).

Herein we describe the origin of the International Commission on Trichinellosis more than 60 years after its foundation. We attempt to clarify previous debate over the founding presidents and particularly the role of Polish parasitologist, Zbigniew Kozar. Seminal and core proceedings of the Commission published in Wiadomosci Parazytologiczne and other records were used to advance this goal. An early regional commission initially held in Budapest, Hungary at the Hungarian Meeting of Parasitologists was devoted to trichinellosis and was presided over by Kozar from 1958 to 1960. However, the official formation of the Commission did not occur until 1960 during the 1st International Conference on Trichinellosis held in Warsaw, Poland, where Witold Stefanski was elected president. During the 2nd International Conference on Trichinellosis, which was held in 1969 in Wroclaw, Poland, Samuel E. Gould was elected president until his untimely death in 1970. Zbigniew Kozar was secretary general from 1960 to 1972. Beginning with the 3rd International Conference held in Miami, Florida, USA in 1972, the activities of the Commission and the Conference became better documented.

Trichinella pseudospiralis in a wolverine (Gulo gulo) from the Canadian North.

Species of Trichinella are a globally distributed assemblage of nematodes, often with distinct host ranges, which include people, domestic, and wild animals. Trichinella spp. are important in northern Canada, where dietary habits of people and methods of meat preparation (drying, smoking, fermenting as well as raw) increase the risk posed by these foodborne zoonotic parasites. Outbreaks in the arctic and subarctic regions of Canada and the United States are generally attributed to T. nativa (T2) or the T6 genotype, when genetic characterization is performed. We report the discovery of Trichinella pseudospiralis (T4), a non-encapsulated species, in a wolverine (Gulo gulo) from the Northwest Territories of Canada. This parasite has been previously reported elsewhere from both mammals and carnivorous birds, but our findings represent new host and geographic records for T. pseudospiralis. Multiplex PCR and sequencing of fragments of Cytochrome Oxidase Subunit I (COI) and D3 rDNA confirmed the identification. Phylogenetically, Canadian isolates linked with each other and others derived from Palearctic or Neotropical regions, but not elsewhere in the Nearctic (continental USA). We suggest that migratory birds might have played a role in the dispersal of this pathogen 1000's of km to northwestern Canada. Wolverines are not typically consumed by humans, and thus should not pose a direct food safety risk for trichinellosis. However, the current finding suggests that they may serve as an indicator of a broader distribution for T. pseudospiralis. Along with infection risk already recognized for T. nativa and Trichinella T6, our observations emphasize the need for further studies using molecular diagnostics and alternative methods to clarify if this is a solitary case, or if T. pseudospiralis and other freeze susceptible species of Trichinella (such as T. spiralis) circulate more broadly in wildlife in Canada, and elsewhere.

International Commission on Trichinellosis: Recommendations on pre-harvest control of Trichinella in food animals.

Transmission of Trichinella to domestic livestock, notably pigs, is limited to certain risk factors including feeding of raw meat-containing waste products or animal carcasses and exposure to infected rodents and wildlife. Prevention of infection in food animals is dependent on eliminating risk of exposure to these potential sources of Trichinella. By implementing conditions of controlled management, pig herds can be considered to pose a negligible risk for Trichinella, and groups of herds which follow these same conditions can be considered to be a compartment with negligible risk for Trichinella. Pork from pigs originating from a negligible risk herd or compartment would not require further testing or processing to protect consumers from this parasite. Verifying the status of pigs from a controlled management negligible risk herd or compartment can be accomplished by a program of regular audits or by implementing surveillance testing of a statistical sample of pigs from the herd or compartment.

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.

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.

Detection of natural Trichinella murrelli and Trichinella spiralis infections in horses by routine post-slaughter food safety testing.

Trichinella spiralis typically infects domestic swine, wild boar and occasionally horses, has a cosmopolitan distribution, and consequently is most frequently associated with food-borne outbreaks of trichinellosis in humans. Trichinella murrelli is typically found in wild carnivores in temperate areas of North America, where it has been responsible for outbreaks of human trichinellosis due to consumption of infected wild game. There has previously been only indirect evidence of natural infection with T. murrelli in a horse originating from Connecticut and implicated in an outbreak of trichinellosis in France in 1985. We describe a T. murrelli infection detected during routine testing of a horse from the USA imported to Canada for slaughter and export to the European Union (EU). Approximately 5 or more larvae per gram were recovered from digested tongue and diaphragm samples and identified as T. murrelli by PCR. This case provides the first direct evidence of naturally acquired T. murrelli infection in a horse, and further supports the potential food safety risk posed by this parasite species. It is the first instance in Canada of the detection of a Trichinella-infected horse via routine post-mortem testing. Trichinella spiralis-infected horses have been similarly detected by regulatory testing in France, and further details of two such previously reported cases are also provided here. The cases described herein underscore the importance of continued vigilance in quality assured food safety testing of horse meat to mitigate the risk of human trichinellosis.

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.

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

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.

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.

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.

Bovine cysticercosis: preliminary observations on the immunohistochemical detection of Taenia saginata antigens in lymph nodes of an experimentally infected calf.

A newly developed immunohistochemical test was used for the first time to demonstrate the presence of Taenia saginata (Cysticercus bovis) antigens in the lymph nodes of a heifer calf experimentally inoculated with Taenia saginata eggs. The new test should aid in the differential diagnosis of eosinophilic lymphadenitis in cattle.

Malignant melanoma in a captive red deer (Cervus elaphus elaphus).

We report a case of malignant melanoma in a captive red deer (Cervus elaphus elaphus). The primary lesion was on the plantar aspect of the right pastern and infiltrated the surrounding skin, but not underlying tendons or bone. Diffuse metastasis to the regional lymph nodes had occurred. Histologically, lesions were characterized by large numbers of variably pigmented epithelioid cells with a high degree of nuclear atypia and many mitotic figures. This is the first reported case of this neoplasm in red deer.