L. Edwards

Seroconversion, neutralising antibodies and protection in bluetongue serotype 8 vaccinated sheep.

Bluetongue virus serotype 8 (BTV-8) has caused a major outbreak of disease in cattle and sheep in several countries across northern and western Europe from 2006 to 2008. In 2008 the European Union instigated a mass-vaccination programme in affected countries using whole virus inactivated vaccines. We evaluated vaccinal responses in sheep and the ability of the vaccine to protect against experimental challenge. Sheep vaccinated 10 months previously under field conditions were challenged with BTV-8. One of 7 vaccinated sheep became infected, as evidenced by detection of viral RNA by real-time RT-PCR and by virus isolation. The remaining 6 sheep appeared fully protected from virus replication. None of the vaccinated sheep showed clinical signs of BTV and there was a good correlation between the presence of neutralising antibodies on challenge and protection. Commercially available ELISAs were evaluated for their ability to detect antibodies in sheep vaccinated on a single occasion. The sandwich (double antigen) ELISA assays were found to be more sensitive at detecting antibodies in vaccinated sheep than the competitive ELISAs.

A real time RT-PCR assay for the specific detection of Peste des petits ruminants virus

Peste des petits ruminants virus (PPRV) causes a devastating disease of small ruminants present across much of Africa and Asia. Recent surveillance activities and phylogenetic analyses have suggested that the virus is an emerging problem as it is now being detected in areas previously free of the disease. As such, the virus not only is threatening small ruminant production and agricultural stability in the developing world, but also poses an economic threat to livestock in the European Union (EU) through introduction from European Turkey and North Africa. This report describes the development of a high throughput, rapid, real time RT-PCR method for the sensitive and specific detection of PPRV using robotic RNA extraction. This assay targets the nucleocapsid (N) gene of PPRV and has been shown to detect all four genetic lineages of PPRV in tissues, ocular and nasal swabs and blood samples collected in the field. The lowest detection limit achieved was approximately 10 genome copies/reaction, making this assay an ideal tool for the sensitive and rapid detection of PPRV in diagnostic laboratories.

Bluetongue virus serotype 26: Infection kinetics, pathogenesis and possible contact transmission in goats

The aim of this study was to assess the pathogenicity and infection kinetics of Bluetongue virus serotype 26 (BTV-26) in goats. Out of a group of six goats housed in insect free accommodation, five were experimentally infected with BTV-26 and one was kept uninfected as an in-contact control. Samples taken throughout the study were used to determine the kinetics of infection using a pan specific BTV real time RT-PCR assay and a group specific ELISA. The five infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from the blood of all 5 goats. Antibodies against BTV were first detected between 7 and 11 dpi in all 5 experimentally infected goats. Interestingly at 21 dpi viral RNA was detected in, and virus was isolated from, the blood of the in-contact control goat, which also seroconverted. These results suggest that BTV-26 replicates to high levels in goats, causing no obvious clinical disease, suggesting that goats may be the natural host for this virus. Preliminary evidence also indicates that BTV-26 may be spread by contact transmission between goats, however a more detailed study is required in order to confirm this observation.

Evaluation of the humoral immune response in adult dairy cattle three years after vaccination with a bluetongue serotype 8 inactivated vaccine

Despite the widespread use of bluetongue serotype 8 (BTV-8) inactivated vaccines across Europe from 2008 to 2011, two very practical questions remain unanswered about the length of persistence of group-specific antibodies in milk and serum post-vaccination and the duration of protection beyond one year post-vaccination. This study has firstly revealed that group-specific antibodies persist at high levels in milk and serum in the majority of cattle for at least 3 years post-vaccination, thus removing the option of using these animals in ELISA-based surveillance programmes. Secondly neutralising antibodies have been shown to persist in the majority of cattle for at least 3 years post-vaccination, indicating that the cattle are likely to be protected for this time period. This extended duration of protection may have contributed towards the rapid and efficient eradication of BTV-8 from many European countries, despite reducing levels of vaccine coverage.

Experimental infection of camels with bluetongue virus.

Three camels aged 4-5 years were experimentally infected with Bluetongue virus serotype 1 (BTV-1) and were observed for 75 days. No clinical signs of disease were observed throughout the experiment, however all three animals seroconverted and developed BTV-1 specific neutralising antibodies after challenge. All three camels developed a viraemia from 7 days post infection albeit at a lower level than that usually observed in experimental infections of sheep and cattle. Virus was isolated from the blood of all three animals suggesting that camels may act as a reservoir for BTV and play an important role in its transmission.

Bluetongue virus serotype 26: Infection kinetics and pathogenesis in Dorset Poll sheep

Bluetongue virus serotype 26 (BTV-26) has recently been isolated from sheep in Kuwait. The aim of this study was to assess the pathogenicity and infection kinetics of BTV-26 in Dorset Poll sheep. Six sheep were experimentally infected with BTV-26 and samples taken throughout the study were used to determine the kinetics of infection using a pan specific BTV real time RT-PCR assay and two group specific ELISAs. Five of the six sheep showed mild clinical signs characteristic of bluetongue including conjunctivitis, reddening of the mouth mucosal membranes, slight oedema of the face and nasal discharge. Viral RNA was detected in 5 of the 6 sheep by real time RT-PCR, however the levels of viral RNA detected in the samples were lower and of shorter duration than seen with other field strains of BTV. Virus was isolated from the blood of infected animals at the peak of viraemia at around 9 dpi. Antibodies against BTV were first detected by 7 dpi using the early detection BTV ELISA and a little later (7-14 dpi) using a BTV specific competitive ELISA. Four of the five remaining sheep developed neutralising antibodies to BTV-26, measured by a serum neutralisation test (SNT), with titres (log(10)) ranging from 1.40 to 2.08.

Colostral antibody protection and interference with immunity in lambs born from sheep vaccinated with an inactivated Bluetongue serotype 8 vaccine

Widespread vaccination programmes against Bluetongue virus serotype 8 (BTV-8), using inactivated vaccines, are being carried out across many countries in northern, western and southern Europe. This study investigates the extent and length of colostral antibody protection, as well as the degree of colostral antibody induced interference of the immune response to BTV-8, in sheep. Significantly lower titres of neutralising antibodies were transferred in colostrum to lambs born from sheep vaccinated once as opposed those vaccinated twice (single vaccine in the first year and a booster vaccine in the second year). On BTV-8 challenge, lambs born from sheep vaccinated on two occasions, with the second booster vaccine given approximately 1 month prior to lambing, were protected from clinical disease for up to 14 weeks. BTV-8 was isolated from 5 of the 22 challenged lambs, although only one of these lambs showed a transient rise in body temperature with no other clinical signs. Lambs born from ewes given a second booster vaccine 1 month prior to lambing, are likely to be protected from clinical disease for at least 14 weeks, whereas lambs born from ewes vaccinated once are likely to be protected for a shorter time. Colostral antibodies present in the 13-14-week-old lambs appeared to interfere with the humoral response to challenge virus. These results suggest that colostral antibodies may interfere with vaccination in lambs up to at least 14 weeks of age.

Epizootic hemorrhagic disease virus serotype 7 in European cattle and sheep: diagnostic considerations and effect of previous BTV exposure.

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus (family Reoviridae), is an emerging pathogen of wild and domestic ruminants that is closely related to bluetongue virus (BTV). The present study examines the outcome of an experimental EHDV-7 infection of Holstein cattle and East Frisian sheep. Apart from naïve animals that had not been exposed to BTV, it included animals that had been experimentally infected with either BTV-6 or BTV-8 two months earlier. In addition, EHDV-infected cattle were subsequently challenged with BTV-8. Samples were tested with commercially available ELISA and real-time RT-PCR kits and a custom NS3-specific real-time RT-PCR assay. Virus isolation was attempted in Vero, C6/36 and KC cells (from Culicoides variipennis), embryonated chicken eggs and type I interferon receptor-deficient IFNAR(-/-) mice. EHDV-7 productively infected Holstein cattle, but caused no clinical signs. The inoculation of East Frisian sheep, on the other hand, apparently did not lead to a productive infection. The commercial diagnostic kits performed adequately. KC cells proved to be the most sensitive means of virus isolation, but viremia was shorter than 2 weeks in most animals. No interference between EHDV and BTV infection was observed; therefore the pre-existing immunity to some BTV serotypes in Europe is not expected to protect against a possible introduction of EHDV, in spite of the close relation between the viruses.

Detection of capripoxvirus DNA using a novel loop-mediated isothermal amplification assay

BackgroundSheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA.ResultsA single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/μl which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device.ConclusionsThis study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.

Infection kinetics of Epizootic Haemorrhagic Disease virus serotype 6 in Holstein-Friesian cattle

Epizootic Haemorrhagic Disease virus serotype 6 (EHDV-6) has recently caused serious outbreaks of Epizootic Haemorrhagic Disease (EHD) on the edges of Europe, in Turkey, Israel and Morocco. The aim of this study was to assess the pathogenicity and infection kinetics of EHD in Holstein-Friesian cattle infected with the two distinct strains of EHDV-6 isolated from the recent Turkish and Moroccan outbreaks. Samples taken throughout the study were used to validate two recently developed diagnostic assays that detect EHDV antibodies and viral genome. Two groups of five Holstein-Friesian cattle were experimentally infected with either the Moroccan or the Turkish isolate of EHDV-6. Cattle in both groups remained clinically unaffected throughout the study, but displayed high levels of viral RNA and virus in their blood, confirming that sub-clinical infection of cattle is likely to play an important role in EHDV transmission. A recently developed and commercialised real-time RT-PCR assay detected viral RNA as early as 2 days post infection (dpi) in both infection studies and viral RNA persisted for the course of the study. Antibodies against EHDV were first detected by 9dpi using a recently developed EHDV blocking ELISA and antibodies persisted up to the end of the study. All animals developed high levels of neutralising antibodies to EHDV-6, measured by a serum neutralisation test (SNT), with titres (log(10)) ranging from 2.20 to 2.38 at the end of the study. Virus was isolated from the blood of infected animals from as early as 2dpi up to 28dpi.