Within-farm transmission characteristics of bluetongue virus serotype 8 in cattle and sheep in the Netherlands, 2007-2008.

In 2006 and 2007, sheep and cattle farms in the Netherlands were affected by an epidemic of bluetongue virus serotype 8 (BTV-8). In order to obtain insight into the within-farm spread of the virus, five affected cattle and five affected sheep farms were longitudinally monitored between early 2007 and mid or late 2008. The farms were visited between four and seven times to collect blood samples. During each visit, all animals present in the flock or herd were sampled. The samples were analysed for the presence of BTV-8 antibodies (ELISA) and BTV-8 antigen (rRT-PCR). The observed patterns of RT-PCR positives indicate a rapid within-farm virus spread during the vector season. During vector-free periods we observed a complete rRT-PCR positivity decline within a few months. During the vector season a lower bound estimate of the basic reproduction number (R0) ranges from 2.9-6.9 in the cattle herds (one herd not analysed), and from 1.3-3.2 in the sheep flocks in this study.

Case series: periocular habronemiasis in five horses in the Netherlands.

In tropical and subtropical climates, infection of periocular tissue by Habronema larvae is a recognised cause of conjunctivitis or blepharitis. To the authors' knowledge, only a few cases of habronemiasis have been described in Western Europe, and it has not been documented previously in the Netherlands. The objective of this report is to describe the occurrence of five cases of (peri)ocular habronemiasis in the Netherlands, of which four date from the past few years. The diagnosis was based on the history, clinical signs and histopathologic examination of biopsy specimens. A granulomatous conjunctivitis/dermatitis and sulphur-like granules were present in all cases. Histopathology showed an eosinophilic granulomatous inflammation, and three out of five (60 per cent) samples revealed one or more nematodes on section. Treatment combinations with surgical excision, local corticosteroid and/or anthelmintic drugs were used. Furthermore, all horses received ivermectin or moxidectin. Treatment resulted in healing of the lesions in four horses. One case, which was refractory to treatment, resolved spontaneously after the onset of colder weather. This case series suggests an increased prevalence of (peri)ocular habronemiasis in the Netherlands. This diagnosis should therefore be considered when being presented with a horse with granulomatous conjunctivitis/dermatitis in Western Europe, especially during the summer months.

Potential role of ticks as vectors of bluetongue virus.

When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not to survive the northern European winter, and transovarial transmission in Culicoides is not recorded, we examined the potential vector role of ixodid and argasid ticks for bluetongue virus. Four species of ixodid ticks (Ixodes ricinus, Ixodes hexagonus, Dermacentor reticulatus and Rhipicephalus bursa) and one soft tick species, Ornithodoros savignyi, ingested BTV8-containing blood either through capillary feeding or by feeding on artificial membranes. The virus was taken up by the ticks and was found to pass through the gut barrier and spread via the haemolymph into the salivary glands, ovaries and testes, as demonstrated by real-time reverse transcriptase PCR (PCR-test). BTV8 was detected in various tissues of ixodid ticks for up to 21 days post feeding and in Ornithodoros ticks for up to 26 days. It was found after moulting in adult Ixodes hexagonus and was also able to pass through the ovaries into the eggs of an Ornithodoros savignyi tick. This study demonstrates that ticks can become infected with bluetongue virus serotype 8. The transstadial passage in hard ticks and transovarial passage in soft ticks suggest that ticks have potential vectorial capacity for bluetongue virus. Further studies are required to investigate transmission from infected ticks to domestic livestock. This route of transmission could provide an additional clue in the unresolved mystery of the epidemiology of Bluetongue in Europe by considering ticks as a potential overwintering mechanism for bluetongue virus.

Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection.

Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven out of fifteen multi-parous cows eight months in gestation, each newborn calf was tested prior to colostrum intake for transplacental transmission of BTV by RRT-PCR. If transplacental transmission was not established the calves were fed colostrum from infected dams or colostrum from non-infected dams spiked with BTV-8 containing blood. One calf from an infected dam was born RRT-PCR positive and BTV-specific antibody (Abs) negative, BTV was isolated from its blood. It was born with clinical signs resembling bluetongue and lived for two days. Its post-mortem tissue suspensions were RRT-PCR positive. Of the seven calves fed colostrum from infected dams, none became infected. Of the six calves fed colostrum from non-infected dams spiked with infected blood, one calf became PCR-positive at day 8 post-partum (dpp), seroconverted 27 days later, and remained RRT-PCR and Abs positive for the duration of the experiment (i.e., 70dpp). This work demonstrates that transplacental transmission in late gestation and oral infection of the neonate with wild-type BTV-8 is possible in cattle under experimental conditions.

A cross-sectional study to determine the seroprevalence of bluetongue virus serotype 8 in sheep and goats in 2006 and 2007 in the Netherlands.

BACKGROUND: In August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe. In the course of 2007 it became evident that BTV8 had survived the winter in North-West Europe, re-emerged and spread exponentially. Recently, the European Union decided to start vaccination against BTV8. In order to improve the understanding of the epidemiological situation, it was necessary to execute a cross-sectional serological study at the end of the BT vector season. Cattle were the target species for cross-sectional serological studies in Europe at the end of 2006 and 2007. However, there was no information on the BTV8-seroprevalence in sheep and goats. RESULTS: On the basis of our cross-sectional study, the estimated seroprevalence of BTV8-exposed locations in the Netherlands in 2006 was 0% for goats (95% confidence interval: 0 - 5.6%) and 7.0% for sheep (95% confidence interval: 3.5 - 12.9%). The estimated seroprevalence of BTV-8 exposed locations in 2007 was 47% for goats (95% confidence interval: 36 - 58%) and 70% for sheep (95% confidence interval: 63 - 76%). There was a wide range in within-location seroprevalence in locations with goats and sheep (1 - 100%). A gradient in seroprevalence was seen, with the highest level of seroprevalence in the southern Netherlands, the area where the epidemic started in 2006, and a decreasing seroprevalence when going in a northern direction. CONCLUSION: There is a much higher estimated seroprevalence of locations with goats exposed to BTV8 than can be inferred from the rather low number of reported clinical outbreaks in goats. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep. The wide range in within-location seroprevalence observed means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between flocks. This should be taken into account when vaccinating animals.

Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains.

During 2006 the first outbreak of bluetongue ever recorded in northern Europe started in Belgium and the Netherlands, spreading to Luxemburg, Germany and north-east France. The virus overwintered (2006-2007) reappearing during May-June 2007 with greatly increased severity in affected areas, spreading further into Germany and France, reaching Denmark, Switzerland, the Czech Republic and the UK. Infected animals were also imported into Poland, Italy, Spain and the UK. An initial isolate from the Netherlands (NET2006/04) was identified as BTV-8 by RT-PCR assays targeting genome segment 2. The full genome of NET2006/04 was sequenced and compared to selected European isolates, South African vaccine strains and other BTV-8 strains, indicating that it originated in sub-Saharan Africa. Although NET2006/04 showed high levels of nucleotide identity with other 'western' BTV strains, it represents a new introduction and was not derived from the BTV-8 vaccine, although its route of entry into Europe has not been established.

Avian influenza (H5N1) susceptibility and receptors in dogs.

Inoculation of influenza (H5N1) into beagles resulted in virus excretion and rapid seroconversion with no disease. Binding studies that used labeled influenza (H5N1) showed virus attachment to higher and lower respiratory tract tissues. Thus, dogs that are subclinically infected with influenza (H5N1) may contribute to virus spread.

Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant.

To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and sulfolipo-cyclodextrin in squalene in water. We demonstrate that a DNA vaccine combined with DDA, but not with the other adjuvants, induced significantly stronger immune responses than plasmid vaccination alone. Moreover, pigs vaccinated in the presence of DDA were protected against clinical disease and shed significantly less PRV after challenge infection. This is the first study to demonstrate that DDA, a conventional adjuvant, enhances DNA vaccine-induced antiviral immunity.