Identification of Trichinella taxa by ITS-1 amplicon next-generation sequencing with an improved resolution for detecting underrepresented genotypes in mixed natural infections.

BACKGROUND: Amplicon-based next-generation sequencing (NGS) has rapidly gained popularity as a powerful method for delineating taxa in complex communities, including helminths. Here, we applied this approach to identify species and genotypes of zoonotic nematodes of the Trichinella genus. A known limitation of the current multiplex PCR (mPCR) assay recommended by the International Commission on Trichinellosis is that it does not differentiate Trichinella nativa from T. chanchalensis. METHODS: The new assay entails deep sequencing of an amplified variable fragment of the ribosomal cistron's (rDNA) internal transcribed spacer 1 using the Illumina platform. The assay was evaluated using first-stage larvae (L1) of select laboratory strains of various Trichinella taxa mixed in known proportions and then validated using archived L1 from 109 wildlife hosts. The species/genotypes of these L1 isolates from wildlife were previously determined using mPCR. RESULTS: NGS data analysis for Trichinella laboratory strains selected as representative of North American fauna revealed a sequence representation bias. Trichinella pseudospiralis, a non-encapsulated species, was the most underrepresented when mixed with T. spiralis, T. murrelli, T. nativa and Trichinella T6 in equal quantities. However, five L1 of T. pseudospiralis were readily revealed by NGS in a mix with 2000 L1 of T. nativa (1:400 ratio). From naturally infected wildlife, all Trichinella taxa revealed by mPCR were also identified by NGS in 103 of 107 (96.3%) samples amplified on both assays. NGS identified additional taxa in 11 (10.3%) samples, whereas additional taxa were revealed by mPCR in only four (3.7%) samples. Most isolates comprised single or mixed infections of T. nativa and Trichinella T6. On NGS, T. chanchalensis (T13) was detected in combination with Trichinella T6 in a wolverine (Gulo gulo) and in combination with T. nativa and Trichinella T6 in a marten (Martes americana) from the Northwest Territories, Canada. CONCLUSIONS: This new NGS assay demonstrates strong potential as a single assay for identifying all recognised Trichinella taxa as well as improved sensitivity for detecting under-represented and novel genotypes in mixed infections. In addition, we report a new host record for T. chanchalensis in American marten.

Performance of indirect enzyme-linked immunosorbent assay using Trichinella spiralis-derived Serpin as antigen for the detection of exposure to Trichinella spp. in swine.

Indirect enzyme-linked immunosorbent assay (ELISA) utilizing excretory-secretory (E-S) antigens of Trichinella spiralis is currently the method of choice for testing pigs and wild boars for exposure to Trichinella spp. The E-S proteins are released by first-stage larvae (L1) of this parasitic nematode maintained in vitro. However, the production of these antigens is cumbersome and time-consuming. The process requires animals to be experimentally infected with the parasite as the source of L1. Antigen production using recombinant technology would be more time- and cost-effective. In this study, we produced a Serpin of T. spiralis as a recombinant protein secreted by the yeast Pichia pastoris. The diagnostic performance of indirect ELISA with purified Serpin antigen was compared to that of E-S ELISA. Both Serpin ELISA and E-S ELISA demonstrated 98 % diagnostic specificity in testing 1056 pigs from the Canadian Trichinella-free commercial herd. Twenty of 21 pigs with non-negative test results in E-S ELISA tested negative by the confirmatory Western blot (WB) assay. Therefore, the diagnostic specificity of combined E-S ELISA and WB was 99.9 %. Forty-five sera collected at or after six weeks from 34 pigs experimentally infected with various numbers of T. spiralis L1 produced positive results in both E-S and Serpin ELISA, resulting in 100 % diagnostic sensitivity. However, testing of sera serially collected from four pigs experimentally infected with various low doses of T. spiralis L1 demonstrated a delayed Serpin-specific antibody response compared to seroconversion detected by E-S ELISA in three animals. Moreover, Serpin ELISA demonstrated significantly lower sensitivity for detecting antibodies induced by experimental infections of pigs with T. britovi, T. nativa, Trichinella T6 and T. pseudospiralis, suggesting that it will not provide consistent detection of exposure to sylvatic Trichinella spp. The validation data support the application of Serpin ELISA in seroepidemiological surveys for detecting exposure to T. spiralis in swine.

Long-term increases in pathogen seroprevalence in polar bears (Ursus maritimus) influenced by climate change.

The influence of climate change on wildlife disease dynamics is a burgeoning conservation and human health issue, but few long-term studies empirically link climate to pathogen prevalence. Polar bears (Ursus maritimus) are vulnerable to the negative impacts of sea ice loss as a result of accelerated Arctic warming. While studies have associated changes in polar bear body condition, reproductive output, survival, and abundance to reductions in sea ice, no long-term studies have documented the impact of climate change on pathogen exposure. We examined 425 serum samples from 381 adult polar bears, collected in western Hudson Bay (WH), Canada, for antibodies to selected pathogens across three time periods: 1986-1989 (n = 157), 1995-1998 (n = 159) and 2015-2017 (n = 109). We ran serological assays for antibodies to seven pathogens: Toxoplasma gondii, Neospora caninum, Trichinella spp., Francisella tularensis, Bordetella bronchiseptica, canine morbillivirus (CDV) and canine parvovirus (CPV). Seroprevalence of zoonotic parasites (T. gondii, Trichinella spp.) and bacterial pathogens (F. tularensis, B. bronchiseptica) increased significantly between 1986-1989 and 1995-1998, ranging from +6.2% to +20.8%, with T. gondii continuing to increase into 2015-2017 (+25.8% overall). Seroprevalence of viral pathogens (CDV, CPV) and N. caninum did not change with time. Toxoplasma gondii seroprevalence was higher following wetter summers, while seroprevalences of Trichinella spp. and B. bronchiseptica were positively correlated with hotter summers. Seroprevalence of antibodies to F. tularensis increased following years polar bears spent more days on land, and polar bears previously captured in human settlements were more likely to be seropositive for Trichinella spp. As the Arctic has warmed due to climate change, zoonotic pathogen exposure in WH polar bears has increased, driven by numerous altered ecosystem pathways.

Hiding in plain sight: discovery and phylogeography of a cryptic species of Trichinella (Nematoda: Trichinellidae) in wolverine (Gulo gulo).

Understanding parasite diversity and distribution is essential in managing the potential impact of parasitic diseases in animals and people. Imperfect diagnostic methods, however, may conceal cryptic species. Here, we report the discovery and phylogeography of a previously unrecognized species of Trichinella in wolverine (Gulo gulo) from northwestern Canada that was indistinguishable from T. nativa using the standard multiplex PCR assay based on the expansion segment 5 (ESV) of ribosomal DNA. The novel genotype, designated as T13, was discovered when sequencing the mitochondrial genome. Phylogenetic analyses of the mitochondrial genome and of 15 concatenated single copy orthologs of nuclear DNA indicated a common ancestor for the encapsulated clade is shared by a subclade containing Trichinella spiralis and Trichinella nelsoni, and a subclade containing T13 and remaining taxa: T12 + (T2 + T6) + [(T5 + T9) + (T3 + T8)]. Of 95 individual hosts from 12 species of mammalian carnivores from northwestern Canada from which larvae were identified as T. nativa on multiplex PCR, only wolverines were infected with T13 (14 of 42 individuals). These infections were single or mixed with T. nativa and/or T6. Visual examination and motility testing confirmed that T13 is encapsulated and likely freeze-tolerant. We developed a new Polymerase Chain Reaction-Restriction Fragment Length Polymorphism which unequivocally distinguishes between T13 and T. nativa. We propose Trichinella chanchalensis n. sp. for T13, based on significant genetic divergence from other species of Trichinella and broad-based sampling of the Trichinella genome. Exploration of Alaskan and Siberian isolates may contribute to further resolution of a phylogeographically complex history for species of Trichinella across Beringia, including Trichinella chanchalensis n. sp. (T13).

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.