K. De Clercq

Transplacental infection and apparently immunotolerance induced by a wild-type bluetongue virus serotype 8 natural infection.

Until recently, bluetongue (BT) virus (BTV) serotypes reportedly causing transplacental infections were all ascribed to the use of modified live virus strains. During the 2007 BT epidemic in Belgium, a significant increase in the incidence of abortions was reported. A study including 1348 foetuses, newborns and young animals with or without suspicion of BTV infection, was conducted to investigate the occurrence of natural transplacental infection caused by wild-type BTV-8 and to check the immunocompetence of newborns. BTV RNA was present in 41% and 18.5% of aborted foetuses from dams with or without suspected BTV involvement during pregnancy, respectively. The results of dam/calf pairs sampled before colostrum uptake provide evidence of almost 10% transplacental BTV infection in newborns. Apparently immunotolerant calves were found at a level of 2.4%. The current study concludes that the combined serological and real-time PCR (RT-qPCR) result of pregnant dams gives no indication of the infection status of the offspring except in the case of a double negative result. In a group of 109 calves with clinical suspicion of BT, born during the vector-free period, 11% were found to be RT-qPCR positive. The true prevalence was estimated to be 2.3%, indicating the extent of transplacental infection in a group of 733 calves of one to 4 months of age without BT suspicion. Moreover, virus isolation was successful for two newborn calves, emphasizing the need for restricting trade to BT-free regions of pregnant dams possibly infected during gestation, even if they are BTV RT-qPCR negative.

Haemorrhagic diathesis in neonatal calves: an emerging syndrome in Europe.

In 2008 and 2009 a large number of cases of haemorrhagic diathesis (HD) in neonatal calves were reported in different European countries. In Flanders, 84 cases of neonatal HD in 30 herds were reported in this period. The disease typically affects calves younger than 1 month old from different breed and gender. Prominent clinical signs are cutaneous bleeding, petechiae on all mucosae, melena and often high fever. Early in the disease, the mental state of the animals is uncompromised. The typical haematological finding is pancytopenia, with severe to complete thrombocytopenia being the cause of the increased susceptibility to bleeding. In seven of the affected herds blood samples of calves of the same age group as the clinical case were collected and on six of those farms at least one subclinical case could be identified. Necropsy findings were generalized petechiae, ecchymoses or haemorrhages and variable lymphadenopathy. Histopathology of haemorrhagic lesions revealed multifocal extravasation of red blood cells (haemorrhage) with preservation of tissue architecture and absence of other abnormalities. Total bone marrow aplasia and depletion of all lymphoid tissue was the most prominent finding on histology. Activated macrophages and haemophagocytosis were seen on bone marrow cytology from two live calves. Polymerase chain reaction for bovine viral diarrhoea virus, bluetongue and epizootic haemorrhagic disease virus was negative. Several attempts to isolate a viral agent were unsuccessful.

Bluetongue Virus Detection by Real-Time RT-PCR in Culicoides Captured During the 2006 Epizootic in Belgium and Development of an Internal Control

After the emergence of bluetongue (BT) in Belgium in 2006, two types of entomological surveys were initiated, the one to identify the local vector species, and the other to study their population dynamics. In the vector study, Culicoides were captured near farms with recently infected cattle or sheep; in the population study Culicoides were captured in two meadows situated in the BT-affected region. A total of 130 pools of parous, non-blood engorged female midges (with a mean of 7.5 midges per pool) were analysed with real-time reverse transcription PCR (RT-qPCR) targeting bluetongue virus (BTV) segment 5. To ensure the RNA integrity of the samples, all pools were also tested in a second RT-qPCR targeting Culicoides 18S rRNA, which served as an internal control. Seventeen pools with negative results for both 18S and BTV were excluded, most of which originated from the population survey. In the vector survey near outbreak sites, female midges of the obsoletus complex, including C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, dominated the black-light trap collections with 19 of 89 pools being BTV-positive. Moreover, all the collections from the vector survey included at least one positive pool of the obsoletus complex compared with only 20% collections (C. obsoletus/C. scoticus) in the population survey. The current study also revealed the presence of BTV RNA in one of five pools of C. pulicaris females captured near recent BT outbreaks, suggesting that this species might have played a role in transmission. Finally, the use of RT-qPCR for the recognition of new potential BTV vector species and the impact of an appropriate monitoring method and internal control are discussed.

Reproductive failure in sows following experimental infection with a Belgian EMCV isolate

In this study, a transplacental infection with fetal death was demonstrated following inoculation of pregnant sows with a Belgian encephalomyocarditis virus (EMCV) isolate. Eight multiparus sows were inoculated between 60 and 92 days of gestation with this EMCV-isolate to investigate its ability to cause reproductive failure in sows. Virus persistence and antibody titre in their offspring were also studied. Only the two sows inoculated at 60 days of gestation showed premature farrowing, but all sows seroconverted to EMCV. Virus was recovered from the offspring of all sows at the time of farrowing, but not from every piglet born. One month after birth EMCV could be isolated from all the piglets examined. These results can help in a better understanding of the spread of the disease in piggeries.

Experimental reproduction of bluetongue virus serotype 8 clinical disease in calves.

Cattle are commonly subclinically infected following natural or experimental infection with bluetongue virus (BTV). The introduction of BTV serotype 8 (BTV-8) in Europe has been characterized by the manifestation of clinical signs in infected cattle. In order to study the pathogenesis of BTV-8 in this host, an animal model able to reproduce the clinical manifestations of the disease is required. In this work, two calves were subcutaneously and intravenously injected with a low passage cell-adapted strain of BTV-8. Both calves showed typical bluetongue clinical signs, including pyrexia, ocular discharge, conjunctivitis, oral mucosal congestion, development of ulcers and necrotic lesions on the lips and tongue, submandibular oedema, coronitis and oedema of the coronet and pastern region. A score was assigned depending on the severity of the lesions and a total clinical score was calculated for each animal daily and at the end of the experiment. Both calves became viraemic 24h post-infection and seroconversion occurred between 7 and 11 days P.I. In this study we present the development of a protocol of infection in calves able to reproduce the severity of the lesions observed with BTV-8 in field conditions.

Vector monitoring at Belgian outbreak sites during the bluetongue epidemic of 2006

In response to the first bluetongue outbreak in Belgium a monitoring programme was started at the end of August 2006 to identify possible vectors transmitting the disease. Black light traps were deployed at 36 outbreak sites and captured 1959 Culicoides specimens belonging to 16 different species. Eighty four percent of the biting midges captured belonged to the C. obsoletus complex, among them C. obsoletus s.s., C. dewulfi and C. scoticus, three suspected bluetongue vectors. The Veterinary and Agrochemical Research Centre detected viral RNA in pools of individuals belonging to this complex. Culicoides pulicaris, a potential bluetongue vector in Italy, should yet not be excluded as a possible vector in Belgium as this species was frequently found around outbreak sites, notwithstanding this species is not easily captured with the trapping techniques used during this survey.

Bluetongue in Belgium: Episode II

Bluetongue (BT) is an arthropod-borne viral disease of ruminants. In August 2006, domestic ruminant populations in Northern Europe became infected with BT virus serotype 8 (BTV-8). The first BTV-8-case of the year 2007 in Belgium was notified in July. This case was the starting point of a second wave of BT outbreaks. The main objective of this study was to describe the evolution and the clinical impact of the second episode of BT in Belgium. In addition, the main differences with the previous episode (August-December 2006) are reported. Both outbreak and rendering plant data were analysed. Overall cumulative incidence at herd level was estimated at 11.5 (11.2-11.8) and 7.5 (7.3-7.8) per cent in cattle and sheep populations respectively. The findings went in favour of a negative association between within-herd prevalence in 2006 and the risk of showing clinical signs of BT in 2007 (via protective immunity). A high level of correlation was demonstrated between BT incidence and small ruminant mortality data when shifting the latter of 1-week backwards. This result supports the hypothesis that the high increase in small ruminant mortality observed in 2007 was the consequence of the presence of BT. For cattle, the correlation was not as high. An increase in cattle foetal mortality was also observed during the year 2007 and a fair correlation was found between BT incidence and foetal mortality.

Emergence of bluetongue serotypes in Europe, part 1: description and validation of four real-time RT-PCR assays for the serotyping of bluetongue viruses BTV-1, BTV-6, BTV-8 and BTV-11.

The control of bluetongue virus (BTV) in Central-Western Europe is greatly complicated by the coexistence of several BTV serotypes. Rapid, sensitive and specific assays are therefore needed to correctly identify the currently circulating BTV serotypes in field samples. In the present study, four serotype-specific real-time RT-PCR assays (RT-qPCR) are described for the detection of the BTV-1, BTV-6, BTV-8 and BTV-11 serotypes. The analytical sensitivity of the BTV-1/S2, BTV-6/S2, BTV-8/S2 and BTV-11/S2 serotype-specific RT-qPCR assays is comparable to the earlier described serogroup-specific pan-BTV/S5 RT-qPCR assay. In silico and in vitro analyses indicated that none of the assays cross-react with viruses which are symptomatically or genetically related to BTV and only detect the intended BTV serotypes. All assays exhibited a linear range of at least 0.05-3.80 log(10) TCID(50) ml(-1) and a PCR-efficiency approaching the ideal amplification factor of two per PCR cycle. Both intra- and inter-run variations were found to be low with a total coefficient of variation of 1-2% for clear positive samples and <10% for very weak positive samples. Finally, the performance of the described assays was compared with commercially available kits for the detection of BTV-1, BTV-6 and BTV-8. Three in-house assays gave exactly the same diagnostic result (positive/negative) as the commercial assays and can thus be used interchangeably. Together with the earlier described serogroup-specific pan-BTV/S5, the serotype-specific RT-qPCR assays form a flexible and properly validated set of tools to detect and differentiate the BTV serotypes currently circulating in Central-Western Europe.

Clinical Pattern Characterization of Cattle Naturally Infected by BTV-8

Forty-one cattle from seven Belgian farms and two French farms confirmed as infected with bluetongue virus serotype 8 (BTV-8) were monitored from the onset of clinical signs to describe the disease pattern and estimate the duration of blood RT-qPCR and competitiveELISA positivity under field conditions. On each visit, blood samples were taken, and a standardized clinical form was filled in for each animal. A clinical score was calculated for every week until the end of clinical signs. A classification and regression tree (CART) analysis was conducted to determine the most important clinical signs every week for the first 7 weeks. The highest scores were recorded within 2 weeks of clinical onset. The first recorded clinical signs were quite obviously visible (lethargy, conjunctivitis, lesions of nasal mucosa, nasal discharge). Skin lesions, a drop in milk production and weight loss appeared later in the course of the disease. A biphasic pattern regarding nasal lesions was noticed: the first peak concerned mainly congestive and ulcerative lesions, whereas the second peak mainly concerned crusty lesions. The median time estimated by survival analysis to obtain negative RT-qPCR results from the onset of clinical signs was 195 days (range 166-213 days) in the 23 cattle included in the analysis. Serological results remained strongly positive until the end of the study. These results should ensure more accurate detection of an emerging infectious disease and are of prime importance in improving the modelling of BTV-8 persistence in Europe.

Bluetongue Virus RNA Detection by Real-Time RT-PCR in Post-Vaccination Samples from Cattle

Bluetongue virus serotype 8 (BTV-8) was responsible for a large outbreak among European ruminant populations in 2006-2009. In spring 2008, a massive vaccination campaign was undertaken, leading to the progressive disappearance of the virus. During surveillance programmes in Western Europe in 2010-2011, a low but significant number of animals were found weakly positive using BTV-specific real-time RT-PCR, raising questions about a possible low level of virus circulation. An interference of the BTV-8 inactivated vaccine on the result of the real-time RT-PCR was also hypothesized. Several studies specifically addressed the potential association between a recent vaccination and BTV-8 RNA detection in the blood of sheep. Results were contradictory and cattles were not investigated. To enlighten this point, a large study was performed to determine the risks of detection of bluetongue vaccine-associated RNA in the blood and spleen of cattle using real-time RT-PCR. Overall, the results presented clearly demonstrate that vaccine viral RNA can reach the blood circulation in sufficient amounts to be detected by real-time RT-PCR in cattle. This BTV-8 vaccine RNA carriage appears as short lasting.