No abstract

No abstract

Bovine herpesvirus type 1 (BHV-1) is an alphaherpesvirus which is an important pathogen of cattle, causing a variety of clinical manifestations in its natural host (46). BHV-1 virions have a typical herpesvirus structure characterized by the presence of a double-stranded DNA genome enclosed in an icosahedral capsid, the tegument surrounding the capsid, and the outer host-derived lipid envelope bearing virus-encoded glycoproteins. While the major constituents of the viral envelope have been extensively studied (reviewed in reference 17), the proteins present in the tegument and nucleocapsid of BHV-1 have been poorly characterized. Compositionally, the tegument is the most complex compartment of the virion, containing more than 15 viral gene products (32). In addition to their structural role, various regulatory functions, including modulation of transcription (34, 47), kinase activity (39), RNase activity (41), and DNA packaging (43), have been assigned to some tegument proteins, suggesting that these virion constituents function at several stages during virus infection, establishing conditions for efficient viral replication and promoting virus assembly and egress.Although the U L 47 gene product, tegument protein VP8, is the most abundant component of mature BHV-1 virions (5), its function is unknown. Like its herpes simplex virus type 1 (HSV-1) homologue (31), VP8 is posttranslationally modified by phosphorylation (5,23) and by the addition of O-linked carbohydrates (49). Both HSV-1 and BHV-1 U L 47 homologues possess nuclear localization and nuclear export signatures (7,51,53,56), enabling them to shuttle between the nucleus and cytoplasm when expressed in transiently transfected cells (51,56) or during viral infection (52, 53). Furthermore, both proteins exhibit a steady-state nuclear localization at early stages of infection and during transient expression (6,35,49,51,52,56), suggesting a functional role for these homologues in the nucleus. Nucleocytoplasmic shuttling of VP8 is sensitive to treatment with a RNA polymerase II inhibitor, actinomycin D (52). This observation coupled with recently demonstrated RNA binding activity of the HSV-1 and BHV-1

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.

Open reading frame 9b (ORF 9b) encodes a 98 amino acid group-specific protein of severe acute respiratory syndrome (SARS) coronavirus (CoV). It has no homology with known proteins and its function in SARS CoV replication has not been determined. The N-terminal part of the 9b protein was used to raise polyclonal antibodies in rabbits, and these antibodies could detect 9b protein in infected cells. We analyzed the sub-cellular localization of recombinant 9b protein using fluorescence microscopy of live transfected cells and indirect immunofluorescence of transfected fixed cells. Our findings indicate that the 9b protein is exported outside of a cell nucleus and localizes to the endoplasmic reticulum. Our data also suggest that the 46-LRLGSQLSL-54 amino acid sequence of 9b functions as a nuclear export signal (NES).

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.

Twenty-three free-ranging white-tailed deer (WTD; Odocoileus virginianus) and six mule deer (MD; Odocoileus hemionus) from south-central British Columbia, Canada, were tested for Anaplasma marginale by msp5 gene-specific PCR and Ehrlichia spp. by 16S rRNA or citrate synthase (gltA) gene-specific PCR, as well as by PCR with universal 16S rRNA primers detecting a wide range of bacteria. No deer tested positive for A. marginale. Amplification with universal 16S rRNA primers followed by sequencing of cloned fragments detected an Anaplasma sp. in one of 23 (4.3%) WTD and six of six (100%) MD and Bartonella sp. in four of 23 (17.4%) WTD. The Anaplasma sp. was genetically distinct from A. marginale and all other recognized members of the genus. Four of six (66.7%) MD and 0 of 23 (0%) WTD were Ehrlichia positive by PCR with primers for 16S rRNA and gltA genes. The sequences of gltA PCR fragments were identical to each other and to the respective region of the gltA gene of an Ehrlichia sp. which we detected previously in naturally infected cattle from the same area, suggesting the possibility of biological transmission of this rickettsia between cattle and wild cervids. Antibodies reactive with the MSP5 protein of A. marginale were detected using a competitive enzyme-linked immunosorbent assay in two of six (33.3%) MD, but not in WTD. The two seropositive MD were PCR positive for both the Anaplasma sp. and Ehrlichia sp. detected in this study, suggesting a reaction of antibodies against one or both of these rickettsias with the MSP5 antigen.

BackgroundEquine 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.ResultsWe 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.ConclusionsThe 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 ...