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.

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.

Experimental transmission of bovine anaplasmosis (caused by Anaplasma marginale) by means of Dermacentor variabilis and D. andersoni (Ixodidae) collected in western Canada.

Canadian cattle are free of bovine anaplasmosis, with the exception of 4 isolated incursions since 1968, which were eradicated. It is not known why the disease has not become established in regions of Canada adjacent to the United States where it is endemic. To assess the vector competence of wild-caught ticks in cattle-rearing regions, Dermacentor variabilis and D. andersoni were collected in western Canada and fed on calves experimentally infected with Anaplasma marginale (St. Maries strain). The 2 tick species were equally competent in transmitting A. marginale to splenectomized calves, all 15 tick-exposed calves becoming infected. The prepatent periods in 13 calves ranged from 18 to 26 d and did not vary in relation to the numbers of ticks fed or the duration of transmission feedings. The unusually long prepatent periods in 2 calves (45 and 55 d) were probably due to concomitant Eperythrozoon infection. This study clearly demonstrated that tick species present in western Canada are competent vectors of bovine anaplasmosis. Potential barriers, including climate, must be considered in developing strategies to prevent A. marginale from becoming established in anaplasmosis-free regions.

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.

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.

Assessment of domestic goats as a patent host of Elaphostrongylus cervi.

A study was undertaken to determine whether domestic goats can serve as patent hosts of Elaphostrongylus cervi under natural or experimental conditions. Three-hundred and two fecal samples from 124 domestic goats raised outdoors in New Zealand, where E. cervi is enzootic, were tested for nematode larvae by the Baermann method. All samples were negative for E. cervi dorsal-spined larvae. Twenty juvenile male Nubian and Saanen goats obtained locally were assigned randomly to 5 dosage groups and were orally administered 5, 15, 35, 65, or 125 third-stage larvae of E. cervi, respectively. Two yearling female red deer (Cervus elaphus elaphus) each received 35 or 65 third-stage larvae as positive controls, and 2 uninoculated juvenile male goats served as negative controls. Fecal Baermann testing of pooled samples from the inoculated goats was conducted weekly for the first 80 days postinoculation (DPI) and daily thereafter until 250 DPI. No dorsal-spined larvae were recovered. One goat that had received 15 third-stage larvae displayed a mild transient posterior ataxia suggestive of cerebrospinal elaphostrongylosis. Gross postmortem examination did not reveal any direct evidence of nematodes in any of the goats, and only a few minor lesions were present. Histologically, these lesions were consistent with a parasite etiology. Histological evaluation of grossly normal lumbar and sacral spinal cord from 2 goats that had each received 125 third-stage larvae revealed eosinophilic meningoencephalitis and leukomyelitis, respectively, suggestive of the presence of parasites in the central nervous system. The 2 positive control red deer became patent with dorsal-spined larvae consistent with E. cervi at 131 DPI. These findings suggest that goats, at least those breeds utilized in this study, are not suitable patent hosts for E. cervi.

Recovery of putative taeniid eggs from silt in water associated with an outbreak of bovine cysticercosis.

Degenerate taeniid-like eggs consistent with Taenia saginata were recovered from sediment in the water supply of a beef feedlot under quarantine for Cysticercus bovis. Nine degenerate eggs in total were recovered from 482 modified flotation assays. Flotation controls of sediment spiked with known numbers of T. saginata eggs had poor egg recoveries, supporting the need for more sensitive assays for environmental samples.

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.

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 novel Ehrlichia genotype detected in naturally infected cattle in North America.

During a research investigation to determine if cattle from British Columbia (BC), Canada were infected with Anaplasma marginale or other related rickettsial blood parasites, a novel Ehrlichia genotype was revealed. Blood from seven BC source cattle was bioassayed by intravenous inoculation into naïve splenectomised calves. Additional splenectomised calves were used as uninoculated negative control or A. marginale-inoculated positive control. Newly designed sets of primers specific for the msp5 gene of A. marginale or for the 16S rRNA gene were used to test blood samples collected from all source cattle and from all recipient calves prior to inoculation and up to 72 days post-inoculation. Results of the PCR assays as well as microscopic examination of stained blood smears failed to demonstrate A. marginale in any of the animals except for the positive control. The 16S rRNA PCR primers amplified DNA from samples from all BC source cattle, five of six of the corresponding recipient calves, and the A. marginale infected control animal. DNA sequence data indicated the presence of A. marginale only in the positive control calf. Blast analysis in GenBank showed that sequences of all other 16S rRNA PCR products clearly fit within the Ehrlichia genus in the Anaplasmataceae family which also includes members of the genus Anaplasma. Phylogenetic analyses using the 16S rRNA gene sequences strongly support the putative Ehrlichia organism as a distinct genotype with sequences of various strains of Ehrlichia canis as the closest clade. Ehrlichia ruminantium is the only other species of the genus known to naturally infect cattle, apart from the present Ehrlichia isolate. However, within the genus, E. ruminantium is phylogenetically the furthest removed species from the novel genotype. The finding of this novel Ehrlichia represents the first known natural ehrlichial infection in cattle in North America. Nevertheless, it is unclear whether cattle are an incidental or primary host, particularly since deer are recognized as reservoir hosts for other species of Ehrlichia. Although other Ehrlichia spp. are known to be pathogenic for animals and zoonotic, these features are presently unknown for this novel genotype.