Pamela J. Hullinger

Airborne spread of foot-and-mouth disease - model intercomparison.

Foot-and-mouth disease virus (FMDV) spreads by direct contact between animals, by animal products (milk, meat and semen), by mechanical transfer on people or fomites and by the airborne route, with the relative importance of each mechanism depending on the particular outbreak characteristics. Atmospheric dispersion models have been developed to assess airborne spread of FMDV in a number of countries, including the UK, Denmark, Australia, New Zealand, USA and Canada. These models were compared at a Workshop hosted by the Institute for Animal Health/Met Office in 2008. Each modeller was provided with data relating to the 1967 outbreak of FMD in Hampshire, UK, and asked to predict the spread of FMDV by the airborne route. A number of key issues emerged from the Workshop and subsequent modelling work: (1) in general all models predicted similar directions for livestock at risk, with much of the remaining differences strongly related to differences in the meteorological data used; (2) determination of an accurate sequence of events on the infected premises is highly important, especially if the meteorological conditions vary substantially during the virus emission period; (3) differences in assumptions made about virus release, environmental fate and susceptibility to airborne infection can substantially modify the size and location of the downwind risk area. All of the atmospheric dispersion models compared at the Workshop can be used to assess windborne spread of FMDV and provide scientific advice to those responsible for making control and eradication decisions in the event of an outbreak of disease.

Diagnostic Evaluation of Multiplexed Reverse Transcription-PCR Microsphere Array Assay for Detection of Foot-and-Mouth and Look-Alike Disease Viruses

ABSTRACT A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true-negative samples collected from healthy animals. The mRT-PCR assay results were compared to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rRT-PCR assays.

Detection of Antibodies to West Nile Virus in Equine Sera Using Microsphere Immunoassay

One hundred and ninety-one sera from horses that recently were exposed to West Nile virus (WNV) by either vaccination or natural infection or that were not vaccinated and remained free of infection were used to evaluate fluorescent microsphere immunoassays (MIAs) incorporating recombinant WNV envelope protein (rE) and recombinant nonstructural proteins (rNS1, rNS3, and rNS5) for detection of equine antibodies to WNV. The rE MIA had a diagnostic sensitivity and specificity, respectively, of 99.3% and 97.4% for detection of WNV antibodies in the serum of horses that were recently vaccinated or naturally infected with WNV, as compared to the plaque reduction neutralization test (PRNT). The positive rE MIA results were assumed to be WNV-specific because of the close agreement between this assay and the PRNT and the fact that unvaccinated control horses included in this study were confirmed to be free of exposure to the related St Louis encephalitis virus. The NS protein–based MIA were all less sensitive than either the rE MIA or PRNT (sensitivity 0–48.0), although the rNS1 MIA distinguished horses vaccinated with the recombinant WNV vaccine from those that were immunized with the inactivated WNV vaccine (P < 0.0001) or naturally infected with WNV (P < 0.0001). The rE MIA would appear to provide a rapid, convenient, inexpensive, and accurate test for the screening of equine sera for the presence of antibodies to WNV.

The Role of Viral Population Diversity in Adaptation of Bovine Coronavirus to New Host Environments

The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were “selected” from a pre-existing pool rather than through de novo mutation and subsequent population fixation.

Application of a pathogen microarray for the analysis of viruses and bacteria in clinical diagnostic samples from pigs

Many of the disease syndromes challenging the commercial swine industry involve the analysis of complex problems caused by polymicrobial, emerging or reemerging, and transboundary pathogens. This study investigated the utility of the Lawrence Livermore Microbial Detection Array (Lawrence Livermore National Laboratory, Livermore, California), designed to detect 8,101 species of microbes, in the evaluation of known and unknown microbes in serum, oral fluid, and tonsil from pigs experimentally coinfected with Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus-2 (PCV-2). The array easily identified PRRSV and PCV-2, but at decreased sensitivities compared to standard polymerase chain reaction detection methods. The oral fluid sample was the most informative, possessing additional signatures for several swine-associated bacteria, including Streptococcus sp., Clostridium sp., and Staphylococcus sp.

Hematophagous Diptera Collected from a Horse and Paired Carbon Dioxide-Baited Suction Trap in Southern California: Relevance to West Nile Virus Epizootiology

Abstract Hematophagous Diptera landing on a horse were removed by vacuum, and their numbers were related to a paired carbon dioxide-baited suction trap at three locations in southern California where West Nile virus activity was high during the preceding year. Insects collected from the horse included mosquitoes (nine species), biting midges (Culicoides sonorensis Wirth & Jones), and black flies (Simulium bivittatum Malloch). Mosquitoes were predominantly collected from the head, crest, withers, neck, chest, and shoulders of the horse, whereas biting midges and black flies were predominantly collected from the ventral midline of the horse. Culex erythrothorax Dyar was by far the most abundant mosquito species collected overall. Frequency of engorgement for mosquitoes captured from the horse ranged by species from zero to 58.3%, with Culex pipiens quinquefasciatus Say having the lowest value (16.7% or one of six mosquitoes) of species that fed on the horse. The number of insects captured at the horse and paired CO2-baited suction trap was not different for Anopheles franciscanus McCracken, Culex tarsalis Coquillett, and S. bivittatum. Cx. p. quinquefasciatus was captured in greater numbers in the CO2-baited suction trap, whereas Anopheles hermsi Barr & Guptavanji, Cx. erythrothorax, Culiseta inornata (Williston), and Culiseta particeps (Adams) were captured in greater numbers from the horse. The horse biting rate was very low for Cx. p. quinquefasciatus, intermediate for Cx. tarsalis, and very high for Cx. erythrothorax. Both Cx. tarsalis and Cx. erythrothorax should be considered likely epizootic vectors of West Nile virus to horses in rural southern California.

Control of foot-and-mouth disease

WE refer to the recent paper in Science by Charleston and others (2011) on the relationship between clinical signs and transmission for an infection with foot-and-mouth disease virus (FMDV) and to the News report of this paper in Veterinary Record (May 14, 2011, vol 168, p 498). The results presented are interesting, and provide welcome support for conclusions derived from careful analysis of the field data from the 2001 epidemic of FMD in the UK (mentioned below), but we feel that they are not the breakthrough seemingly implied in the paper and the media reaction to it. We consider that the results must be interpreted with caution and that the conclusions to be drawn are more limited than suggested. First, the paper reports results using a single strain of FMDV (the strain responsible for the UK outbreak of 2001) to infect only one species of animal (cattle). However, and very importantly, it is well known that FMDV varies significantly between its many strains and …