Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, nonhazardous transportation of samples from FMD endemic countries to international reference laboratories.

BACKGROUND:Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly.METHODS:In this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe.RESULTS:A total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction.CONCLUSIONS:The results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

The control of foot-and-mouth disease (FMD) in vaccinated populations relies upon surveillance activities such as clinical inspections (CI) and serological monitoring. New evidence to refine current surveillance guidelines has been provided by evaluating (1) the diagnostic performance of CI and serological tests for detection of FMD virus (FMDV) non-structural proteins (NSP), and (2) the within-herd transmission of the virus in partially immune cattle. Data came from 23 affected herds during an epidemic of FMDV type 0 in Bolivia, in 2007. All cattle (n = 957) in these herds were clinically inspected and serum samples were collected one month after the last animal with clinical signs was detected. Samples were tested for the presence of antibodies against NSP using the PANAFTOSA 3ABC-ELISA test and a subset of samples were tested using the enzyme-linked immunoelectrotransfer blot assay (EITB). Data from clinical and serological diagnoses were analysed using a Bayesian model. The sensitivity Se and specificity Sp of the tests, as well as the prevalence and the within-herd reproduction ratio R of FMDV were estimated. In addition, risk factors for infection were identified. The Se of CI, the 3ABC-ELISA and the EITB tests were estimated to be 0.30, 0.88 and 0.96 respectively. The estimated Sp, in the same order, were 0.88, 0.93 and 0.97. The within-herd prevalence of infected animals ranged from 0.04 to 0.91 and R ranged from 1.02 to 2.68. It was observed that cattle coming from areas with high vaccination coverage had a lower risk of becoming infected than home-bred cattle from the affected herds, where vaccination coverage was thought to be low. Although these estimates come from herds kept under specific conditions, they provide a reference for future surveillance design and can inform simulation models for surveillance and control of FMD in similar cattle populations.

In the summer of 2011 Schmallenberg virus (SBV), a Culicoides-borne orthobunyavirus, emerged in Germany and The Netherlands and subsequently spread across much of Europe. To draw inferences about the transmission of SBV we have developed two models to describe its spread within and between farms. The within-farm model was fitted to seroprevalence data for cattle and sheep farms in Belgium and The Netherlands, with parameters estimated using approximate Bayesian computation. Despite the short duration of viraemia in cattle and sheep (mean of 3–4 days) the within-farm seroprevalence can reach high levels (mean within-herd seroprevalence >80%), largely because the probability of transmission from host to vector is high (14%) and SBV is able to replicate quickly (0.03 per day-degree) and at relatively low temperatures (threshold for replication: 12.3 °C). Parameter estimates from the within-farm model were then used in a separate between-farm model to simulate the regional spread of SBV. This showed that the rapid spread of SBV at a regional level is primarily a consequence of the high probability of transmission from host to vector and the temperature requirements for virus replication. Our results, obtained for a region of the UK in a typical year with regard to animal movements, indicate that there is no need to invoke additional transmission mechanisms to explain the observed patterns of rapid spread of SBV in Europe. Moreover, the imposition of movement restrictions, even a total movement ban, has little effect on the spread of SBV at this scale.