Robbin Lindsay

Detection of polyoma and corona viruses in bats of Canada.

Several instances of emerging diseases in humans appear to be caused by the spillover of viruses endemic to bats, either directly or through other animal intermediaries. The objective of this study was to detect, identify and characterize viruses in bats in the province of Manitoba and other regions of Canada. Bats were sampled from three sources: live-trapped Myotis lucifugus from Manitoba, rabies-negative Eptesicus fuscus, M. lucifugus, M. yumanensis, M. septentrionalis, M. californicus, M. evotis, Lasionycteris (L.) noctivagans and Lasiurus (Las.) cinereus, provided by the Centre of Expertise for Rabies of the Canadian Food Inspection Agency (CFIA), and L. noctivagans, Las. cinereus and Las. borealis collected from a wind farm in Manitoba. We attempted to isolate viruses from fresh tissue samples taken from trapped bats in cultured cells of bat, primate, rodent, porcine, ovine and avian origin. We also screened bat tissues by PCR using primers designed to amplify nucleic acids from members of certain families of viruses. We detected RNA of a group 1 coronavirus from M. lucifugus (3 of 31 animals) and DNA from an as-yet undescribed polyomavirus from female M. lucifugus (4 of 31 animals) and M. californicus (pooled tissues from two females).

Identifying the last supper: utility of the DNA barcode library for bloodmeal identification in ticks

Ticks are among the most important vectors of disease in the Northern Hemisphere, and a better understanding of their feeding behaviour and life cycle is critical to the management and control of tick‐borne zoonoses. DNA‐based tools for the identification of residual bloodmeals in hematophagous arthropods have proven useful in the investigation of patterns of host use in nature. Using a blind test approach, we challenged the utility of the DNA barcode library for the identification of vertebrate bloodmeals in engorged, field‐collected Ixodes scapularis. Universal vertebrate primers for the COI barcode region successfully amplified DNA from the host bloodmeal and only rarely amplified tick DNA. Of the 61 field‐collected ticks, conclusive genus‐ and species‐level identification was possible for 72% of the specimens. In all but two cases, barcode‐based identification of the bloodmeal was consistent with the morphological identification of the vertebrate host the ticks were collected from. Possible explanations for mismatches or ambiguities are presented. This study validates the utility of the DNA barcode library as a valuable and reliable resource for the identification of unknown bloodmeals in arthropod vectors of disease. Future directions aimed at the refinement of these techniques to gain additional information and to improve the amplification success of digested vertebrate DNA in tick bloodmeals are discussed.

Does high biodiversity reduce the risk of Lyme disease invasion

BackgroundIt has been suggested that increasing biodiversity, specifically host diversity, reduces pathogen and parasite transmission amongst wildlife (causing a “dilution effect”), whereby transmission amongst efficient reservoir hosts, (e.g. Peromyscus spp. mice for the agent of Lyme disease Borrelia burgdorferi) is reduced by the presence of other less efficient host species. If so, then increasing biodiversity should inhibit pathogen and parasite invasion.MethodsWe investigated this hypothesis by studying invasion of B. burgdorferi and its tick vector Ixodes scapularis in 71 field sites in southeastern Canada. Indices of trapped rodent host diversity, and of biodiversity of the wider community, were investigated as variables explaining the numbers of I. scapularis collected and B. burgdorferi infection in these ticks. A wide range of alternative environmental explanatory variables were also considered.ResultsThe observation of low I. scapularis abundance and low B. burgdorferi infection prevalence in sites where I. scapularis were detected was consistent with early-stage invasion of the vector. There were significant associations between the abundance of ticks and season, year of study and ambient temperature. Abundance of host-seeking larvae was significantly associated with deer density, and abundance of host-seeking larvae and nymphs were positively associated with litter layer depth. Larval host infestations were lower where the relative proportion of non-Peromyscus spp. was high. Infestations of hosts with nymphs were lower when host species richness was higher, but overall nymphal abundance increased with species richness because Peromyscus spp. mouse abundance and host species richness were positively correlated. Nymphal infestations of hosts were lower where tree species richness was higher. B. burgdorferi infection prevalence in ticks varied significantly with an index of rates of migratory bird-borne vector and pathogen invasion.ConclusionsI. scapularis abundance and B. burgdorferi prevalence varied with explanatory variables in patterns consistent with the known biology of these species in general, and in the study region in particular. The evidence for a negative effect of host biodiversity on I. scapularis invasion was mixed. However, some evidence suggests that community biodiversity beyond just host diversity may have direct or indirect inhibitory effects on parasite invasion that warrant further study.

Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories

Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is <1%; however, fatality rates in those with severe clinical disease have been reported as high as 29%. The potential of RVFV as a bioterrorism agent and/or being accidentally introduced into North America is widely recognized. Currently, regional veterinary biosafety level 2 (BSL-2) diagnostic laboratories lack safe, modern, validated diagnostic tests to detect RVFV. An existing one-step real-time RT-PCR (rRT-PCR) assay was modified for quick virus inactivation for use in BSL-2 laboratories, evaluated on serum and tissue samples from experimentally infected lambs and calves, and compared to virus isolation. Viremia was detected in all inoculated sheep with titers reaching 10(6.5) plaque forming units/ml, or up to 10(10) viral RNA copies/ml. Viremia in calves was lower and not detected in all inoculated animals; however, all animals became transiently febrile and were infected as determined by rRT-PCR of tissues. Virus was isolated from rRT-PCR-positive liver and/or spleen in 33% of lamb and 41% of calf samples between 2 and 7 days post inoculation. For RVFV antigen detection, reagents are typically produced at BSL-3Ag or BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in suspected ruminant samples.

Evaluation of the efficacy, potential for vector transmission, and duration of immunity of MP-12, an attenuated Rift Valley fever virus vaccine candidate, in sheep

ABSTRACT Rift Valley fever virus (RVFV) causes serious disease in ruminants and humans in Africa. In North America, there are susceptible ruminant hosts and competent mosquito vectors, yet there are no fully licensed animal vaccines for this arthropod-borne virus, should it be introduced. Studies in sheep and cattle have found the attenuated strain of RVFV, MP-12, to be both safe and efficacious based on early testing, and a 2-year conditional license for use in U.S. livestock has been issued. The purpose of this study was to further determine the vaccines potential to infect mosquitoes, the duration of humoral immunity to 24 months postvaccination, and the ability to prevent disease and viremia from a virulent challenge. Vaccination experiments conducted in sheep found no evidence of a potential for vector transmission to 4 North American mosquito species. Neutralizing antibodies were elicited, with titers of >1:40 still present at 24 months postvaccination. Vaccinates were protected from clinical signs and detectable viremia after challenge with virulent virus, while control sheep had fever and high-titered viremia extending for 5 days. Antibodies to three viral proteins (nucleocapsid N, the N-terminal half of glycoprotein GN, and the nonstructural protein from the short segment NSs) were also detected to 24 months using competitive enzyme-linked immunosorbent assays. This study demonstrates that the MP-12 vaccine given as a single dose in sheep generates protective immunity to a virulent challenge with antibody duration of at least 2 years, with no evidence of a risk for vector transmission.

Apoptosis, autophagy and unfolded protein response pathways in Arbovirus replication and pathogenesis.

Arboviruses are pathogens that widely affect the health of people in different communities around the world. Recently, a few successful approaches toward production of effective vaccines against some of these pathogens have been developed, but treatment and prevention of the resulting diseases remain a major health and research concern. The arbovirus infection and replication processes are complex, and many factors are involved in their regulation. Apoptosis, autophagy and the unfolded protein response (UPR) are three mechanisms that are involved in pathogenesis of many viruses. In this review, we focus on the importance of these pathways in the arbovirus replication and infection processes. We provide a brief introduction on how apoptosis, autophagy and the UPR are initiated and regulated, and then discuss the involvement of these pathways in regulation of arbovirus pathogenesis.

The Accuracy of Diagnostic Tests for Lyme Disease in Humans, A Systematic Review and Meta-Analysis of North American Research.

There has been an increasing incidence of Lyme disease (LD) in Canada and the United States corresponding to the expanding range of the Ixodes tick vector and Lyme disease agent (Borrelia burgdorferi sensu stricto). There are many diagnostic tests for LD available in North America, all of which have some performance issues, and physicians are concerned about the appropriate use and interpretation of these tests. The objective of this systematic review is to summarize the North American evidence on the accuracy of diagnostic tests and test regimes at various stages of LD. Included in the review are 48 studies on diagnostic tests used in North America published since 1995. Thirteen studies examined a two-tier serological test protocol vs. clinical diagnosis, 24 studies examined single assays vs. clinical diagnosis, 9 studies examined single immunoblot vs. clinical diagnosis, 7 studies compared culture or PCR direct detection methods vs. clinical diagnosis, 22 studies compared two or more tests with each other and 8 studies compared a two-tiered serological test protocol to another test. Recent studies examining the sensitivity and specificity of various test protocols noted that the Immunetics® C6 B. burgdorferi ELISA™ and the two tier approach have superior specificity compared to proposed replacements, and the CDC recommended western blot algorithm has equivalent or superior specificity over other proposed test algorithms. There is a dramatic increase in test sensitivity with progression of B. burgdorferi infection from early to late LD. Direct detection methods, culture and PCR of tissue or blood samples were not as sensitive or timely compared to serological testing. It was also noted that there are a large number of both commercial (n = 42) and in-house developed tests used by private laboratories which have not been evaluated in the primary literature.

Blood collected on filter paper for wildlife serology: evaluating storage and temperature challenges of field collections.

Abstract Filter-paper (FP) blood sampling can facilitate wildlife research and expand disease surveillance. Previous work indicated that Nobuto FP samples from caribou and reindeer (Rangifer tarandus subspecies) had comparable sensitivity and specificity to serum samples (≥80% for both) in competitive enzyme-linked immunosorbent assays (cELISAs) for Brucella spp., Neospora caninum, and West Nile virus. The same sensitivity and specificity criteria were met in indirect ELISAs for Brucella spp., bovine herpesvirus type 1 (BHV-1), parainfluenza virus type 3 (PI-3), and bovine respiratory syncytial virus (BRSV), with adjusted FP thresholds used for PI-3 and BRSV. Comparable sensitivity and specificity values to serum were also observed for FP in virus neutralization (VN) assays for bovine viral diarrhea virus types I and II; however, reduced sensitivity is a potential limitation of FP samples in protocols that require undiluted serum (i.e., VN and N. caninum cELISA). We evaluated the performance of FP samples from reindeer and caribou in these nine assays after simulating potential challenges of high-latitude field collections: 1) different durations of storage and 2) different processing/storage regimes involving freezing or drying. Sample pairs (serum and FP) were collected from reindeer and caribou populations in 2007–10 and were tested in duplicate. Comparable performance to serum was defined as sensitivity and specificity ≥80%. In the storage experiments, FP performance was determined after 2 mo of storage dry at room temperature, and after two longer periods (variable depending on assay; up to 2 yr). After 1 yr, compared to frozen serum stored for the same period, sensitivity was ≥88% for all but two assays (68% BHV-1; 75% PI-3), and specificity remained >90%. A limited trial evaluated the effect of freezing FP samples as opposed to drying them for storage. There were no observed detrimental effects of freezing on FP sample performance, but rigorous investigation is warranted.

A review of environmental determinants and risk factors for avian-associated mosquito arboviruses in Canada

Abstract A number of mosquito-transmitted arboviruses which cause disease in humans have been isolated in Canada. Four in particular, West Nile, western equine encephalitis, eastern equine encephalitis and St. Louis encephalitis viruses have avian hosts and have caused epidemics and/or epizootics in parts of the United States and Canada over recent decades. However, with the exception of recently emerged West Nile virus, relatively little is known about the environmental drivers and risk factors which promote epidemic occurrence of mosquito-transmitted arboviruses in North America. This paper reviews environmental drivers and risk factors of those mosquito arboviruses with avian reservoirs which have had epidemic outbreaks in parts of the U.S.A. and Canada as a preliminary step towards future research on the effects of climate change and mosquito-borne arboviruses in Canada. The review highlights how the outcome of interactions between changing environmental conditions, with endemic or introduced invasive arboviruses, is likely to be strongly mediated by local abiotic and biotic conditions. It concludes that the processes linking abiotic and biotic determinants to virus transmission may not be generalizable. Continued research into the regional idiosyncrasies of the ecology of arboviruses will, therefore, be necessary in order to assess the effects of a changing climate on arboviruses in Canada.

Enhanced detection of Rift Valley fever virus using molecular assays on whole blood samples.

BACKGROUND Rift Valley fever (RVF) is an emerging arthropod-borne zoonoses of global agricultural and public health importance. In December 2006, an RVF outbreak was recognized in Kenya which led to the deployment of international response laboratory teams to the area. OBJECTIVES A field laboratory was operated in Malindi, Kenya to provide safe sample handling and molecular testing for RVF virus (RVFV) as well as selected other pathogens for differential diagnosis. STUDY DESIGN Safe sample handling was carried out using a negative pressure flexible film isolator (glovebox) and commercial reagents to inactivate clinical specimens and purify nucleic acid. Whole blood was routinely used for diagnostic testing although paired plasma samples were also tested in select cases. Subsequently, human macrophages were tested in vitro for their susceptibility to RVFV. RESULTS The field laboratory received samples from 33 individuals and a definite laboratory diagnosis was provided in 16 of these cases. Using molecular diagnostic techniques, RVFV was more consistently detected in whole blood than in plasma samples most likely due to association of RVFV with blood cells. Subsequent in vitro studies identified macrophages as a target cell for RVFV replication. CONCLUSIONS RVFV appears to replicate in blood cells such as macrophages. Thus, the sensitivity of molecular diagnostic testing is improved if whole blood is used as the clinical specimen rather than plasma or serum.