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.

Serological and molecular detection of Babesia caballi and Theileria equi in Mexico: A prospective study.

Equine piroplasmosis is a disease of horses, mules and donkeys, caused by the hemoprotozoans Babesia caballi and Theileria equi and transmitted by ticks of tropical and subtropical regions. Because the clinical signs are not specific, the diagnosis of equine piroplasmosis is difficult. In Mexico, where the environmental factors are conducive to the persistence of these pathogens, there is a lack of molecular studies to evaluate the occurrence of both parasites in horses. In the present study, matching serum and whole blood samples were obtained from 269 horses residing in 24 locations with tropical or subtropical climate and the presence of ticks. Testing of serum samples by ELISA demonstrated 55.7% seroprevalence of B. caballi and 68.4% prevalence of antibodies to T. equi. Blood samples analyzed with nPCR test were 7.8% positive to B. caballi and 78.8% positive to T. equi, while a duplex qPCR showed 15.24% positive samples to B. caballi and 59.11% to T. equi. From these results, 27 samples were sequenced for T. equi and 13 for B. caballi, confirming the presence of both horse parasites that cause equine piroplasmosis and suggesting that they are widespread in Mexico. This is the first study confirming the presence of B. caballi and T. equi in Mexico using both serological and molecular diagnostic methods. This study shows a high incidence of exposure to the etiological agents of equine piroplasmosis in horses in the studied areas.

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.

Optimization and validation of a loop-mediated isothermal amplification (LAMP) assay for detection of Giardia duodenalis in leafy greens.

Giardia duodenalis is one of the most common food and water-borne intestinal parasites of humans and animals worldwide. Fresh, ready-to-eat produce such as leafy greens and salad mixes are considered potential transmission vehicles for Giardia infection in humans. Therefore, a specific, sensitive, and reliable method for Giardia detection in leafy greens is needed. We optimized washing procedures for the recovery of Giardia cysts from leafy greens and adapted and validated an existing EF1α LAMP assay for the detection of Giardia DNA to support routine diagnostic surveillance and disease outbreak investigations. Four leafy green types (35 ±â€¯1 g) were spiked with 100 Giardia cysts and we compared washing by shaking with 1 M glycine (n = 20) or 0.1% Alconox (n = 20). DNA was extracted from washes, tested by LAMP and melt curve analysis, and time to positive (TTP) values compared. The detection limit was determined by spiking 10 (n = 40) Giardia cysts onto these same types of leafy greens and processing as above with 0.1% Alconox. Method robustness was assessed by subjecting spring mix (n = 45 total) to aging (1, 3 or 7 days) and washes to aging and freezing conditions prior to testing. Assay repeatability and specificity were evaluated, and an artificial positive control (APC) distinguishable by melt temperature (Tm) from DNA of Giardia spiked on leafy greens was designed to rule out cross-contamination from the control. Giardia detection rates were higher and TTP was lower (P < 0.05) for 0.1% Alconox (19/20, 8.85 ±â€¯0.3 min) compared with 1 M glycine (15/20, 14.53 ±â€¯7.2 min). The LAMP assay detected 10 Giardia cysts spiked on leafy greens in 13-34 min in 14/40 samples tested. Robustness assessment showed that TTP was higher (P < 0.0001) when spiked produce was stored for 7 days (13.09 ±â€¯1.14 min) compared to fresh (9.72 ±â€¯0.43 min). No unspiked samples were positive by LAMP, and the Tm for DNA of Giardia spiked on leafy greens was higher (P < 0.0001, 87.43 ±â€¯0.05 °C) than the APC (86.43 ±â€¯0.12 °C). Within-assay repeatability co-efficient of variation (CV) for TTP was 5.4% and no cross-contamination occurred when spiked and un-spiked samples were processed in alternate order. The optimized sample processing procedure combined with the EF1α LAMP assay is a sensitive, specific, labour-saving, and rapid method for the detection of Giardia cysts in leafy greens.

Trichinella nativa and Trichinella T6 in arctic foxes (Vulpes lagopus) from northern Canada.

Parasitic zoonotic nematodes of the genus Trichinella circulate in wildlife and domestic hosts worldwide through the ingestion of infected meat. Due to their role as scavengers and predators in terrestrial and marine arctic ecosystems, Arctic foxes (Vulpes lagopus) are ideal sentinels for the detection of Trichinella spp. In this study, we determined the prevalence, larval intensity, and species of Trichinella from 91 trapped Arctic foxes collected around the northern Canadian communities of Sachs Harbour (Ikaahuk) on Banks Island (n = 23), and Ulukhaktok and Cambridge Bay (Ikaluktutiak) on Victoria Island (n = 68). Using pepsin-HCl digestion, larvae of Trichinella spp. were recovered from the left forelimb muscle (flexor carpi ulnaris) in 19 of the 91 foxes (21% prevalence, 95% CI: 14-30%). For the first time in Arctic foxes in Canada, Trichinella species were identified using multiplex PCR that was followed up with PCR-RFLP to distinguish between T. nativa and T. chanchalensis. All infected foxes harbored T. nativa, and one fox was co-infected with Trichinella T6; the latter is a new host record. Age of the fox was significantly associated with Trichinella spp. infection and the odds of being infected were three times higher in foxes ≥2 years of age (p = 0.026), indicating cumulative exposure with age. While Arctic foxes are seldom harvested for human consumption, they serve as sentinel hosts of Trichinella spp., confirming the presence of the parasite in wildlife in the region.

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).

A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis.

BACKGROUND: Zoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp. METHODS: We compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites. RESULTS: The protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile. CONCLUSION: The new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

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.

Bovine herpesvirus-1 VP8 interacts with DNA damage binding protein-1 (DDB1) and is monoubiquitinated during infection.

VP8 is the most abundant tegument protein of bovine herpesvirus-1 (BHV-1). In the present study DNA damage binding protein 1 (DDB1) was identified as interacting partner of VP8. MALDI-TOF mass spectroscopy analysis of proteins co-immunoprecipitated with VP8 identified DDB1 as a protein interacting with VP8. The interaction between VP8 and DDB1 was confirmed based on co-immunoprecipitation and co-localization in both VP8-transfected and BHV-1 infected cells. DDB1 was distributed both in the nucleus and the cytoplasm with some nuclear speckles prior to BHV-1 infection, became perinuclear by 4h and was predominantly nuclear at 5h post infection, where it co-localized with VP8. In contrast, in cells infected with a U(L)47 deletion mutant DDB1 remained cytoplasmic throughout the course of infection. This suggests that VP8 mediates nuclear re-localization of DDB1. Finally, VP8 was shown to be monoubiquitinated both in VP8-transfected and BHV-1-infected cells. These data suggest that BHV-1 VP8 interacts with DDB1-CUL4 E3 ubiquitin ligase, which correlates to monoubiquitination of this viral protein.

Serological cross-reactivity between Anaplasma marginale and an Ehrlichia species in naturally and experimentally infected cattle.

Seroconversion and cross-reactivity in cattle infected with Anaplasma marginale or a recently described Ehrlichia species (BOV2010 from British Columbia, Canada) were investigated. The study used 76 samples from 20 animals, a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for bovine anaplasmosis, and an indirect fluorescent antibody test (IFAT). Blood smear examination and/or polymerase chain reaction assay were performed to confirm or rule out the presence of Anaplasma or Ehrlichia. Samples comprised 3 groups. Group 1 consisted of 24 samples from 9 cattle naturally infected with Ehrlichia sp. BOV2010. Group 2 had 13 samples from 3 A. marginale-infected cattle from Manitoba, Canada. Group 3 had 39 samples, consisting of 26 from 5 calves experimentally infected with Ehrlichia sp. BOV2010, 10 from 2 calves experimentally infected with A. marginale from cattle (Manitoba) or bison (Saskatchewan), and 3 from an uninfected calf. All samples from cattle naturally or experimentally infected with Ehrlichia sp. BOV2010 or A. marginale were seropositive for A. marginale by both cELISA and IFAT, except 3 calves euthanized at 28 and 33 days post-inoculation (DPI) that did not seroconvert. Antibodies were detected in 2 experimental animals inoculated with Ehrlichia sp. BOV2010, as early as 28 and 33 DPI by the cELISA and IFAT, respectively, and by 42 DPI for both tests. The current study demonstrates that the specificity of the recombinant major surface protein 5 (MSP5) antigen is not restricted to Anaplasma spp., which reduces the utility of the test for serological diagnosis of bovine anaplasmosis in regions where Ehrlichia sp. BOV2010-infected cattle might exist.

A UL47 gene deletion mutant of bovine herpesvirus type 1 exhibits impaired growth in cell culture and lack of virulence in cattle.

Tegument protein VP8 encoded by the U(L)47 gene of bovine herpesvirus type 1 (BHV-1) is the most abundant constituent of mature virions. In the present report, we describe the characterization of U(L)47 gene-deleted BHV-1 in cultured cells and its natural host. The U(L)47 deletion mutant exhibited reduced plaque size and more than 100-fold decrease in intracellular and extracellular viral titers in cultured cells. Ultrastructural observations of infected cells showed normal maturation of BHV-1 virions in the absence of VP8. There was no evidence for a change in immediate-early gene activator function of VP16 in the U(L)47 deletion mutant virus-infected cells, since bovine ICP4 mRNA and protein levels were similar to those in the wild-type and revertant virus-infected cells throughout the course of infection. Whereas VP16, glycoprotein C (gC), gB, and VP5 were expressed to wild-type levels in the U(L)47 deletion mutant-infected cells, the gD and VP22 protein levels were significantly reduced. The reduction in gD protein was associated with increased turnover of the protein. Furthermore, some of the analyzed early and late proteins were expressed with earlier kinetics in the absence of VP8. Extracellular virions of the U(L)47 deletion mutant contained reduced amounts of gD, gB, gC, and VP22 but similar amounts of VP16 compared to those of wild-type or revertant virus particles. In addition, the U(L)47 gene product was indispensable for BHV-1 replication in vivo, since no clinical manifestations or viral shedding were detected in the U(L)47 deletion mutant-infected calves, and the virus failed to induce significant levels of humoral and cellular immunity.

Intracellular trafficking of VP22 in bovine herpesvirus-1 infected cells.

The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 (131)PRPR(134) NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.