Nesya Goris

Bluetongue in Belgium, 2006.

Bluetongue has emerged recently in Belgium. A bluetongue virus strain was isolated and characterized as serotype 8. Two new real-time reverse transcription–quantitative PCRs (RT-qPCRs) that amplified 2 different segments of bluetongue virus detected this exotic strain. These 2 RT-qPCRs detected infection earlier than a competitive ELISA for antibody detection.

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.

Bluetongue in Eurasian lynx.

To the Editor: Bluetongue is an infectious disease of ruminants; it is caused by bluetongue virus (BTV), has 24 known serotypes, and is transmitted by several species of Culicoides biting midges. The disease mainly affects sheep and occurs when susceptible animals are introduced to areas where BTV circulates or when BTV is introduced to naive ruminant populations. The natural host range is strictly limited to ruminants, although seroconversion without disease has been reported in carnivores (1). We report BTV infection, disease, and death in 2 Eurasian lynx (Lynx lynx) and the isolation of BTV serotype 8 (BTV-8) from this carnivorous species. The 2 Eurasian lynx, held in the same cage in a zoo in Belgium, became lethargic in September 2007; animal 1 died after 2 days, and animal 2 died in February 2008. Both had been fed ruminant fetuses and stillborns from surrounding farms in an area where many bluetongue cases had been confirmed (2). Necropsy findings for animal 1 were anemia, subcutaneous hematomas, petechial hemorrhages, and lung congestion with edema. Necropsy findings for animal 2 were emaciation, anemia, enlarged and gelatinous lymph nodes, petechial hemorrhages, and pneumonia. For each animal, microscopic examination showed edematous vascular walls; enlarged endothelial cells; and evidence of acute to subacute vasculitis in muscle, myocardium, peritoneum, and lung. Tissue samples (spleen, lung, intestine) were analyzed by using 2 real-time reverse transcriptase–quantitative PCR techniques targeting BTV segment 5 and host β-actin mRNA as a control. BTV RNA was found in all samples from animal 1; cycle threshold values (3) ranged from 28.6 to 36.2. Tissues from animal 2 were negative for BTV RNA. Although the internal control was originally designed to detect β-actin mRNA of bovine or ovine species, clear positive signals were noted in all lynx samples, which indicated that this was a reliable control procedure. Infectious virus was subsequently isolated from the lung sample of animal 1 after inoculation of embryonated chicken eggs and amplification in baby hamster kidney–21 cell cultures (4). The specificity of the cytopathic effect, observed 48 hours after passage on baby hamster kidney–21 cells, was confirmed by real-time reverse transcriptase–quantitative PCR. Virus neutralization using specific reference serum (5) proved that the isolated virus was BTV-8. Anti-BTV antibodies were detected in lung tissue fluid from animal 2 (ID Screen Bluetongue Competition assay, ID VET, Monpellier, France) (6). We describe a natural, wild-type infection of a carnivorous species. Although deaths have been documented in dogs accidentally infected with a BTV-contaminated vaccine (7), the 2 lynx in this report were neither vaccinated nor medically treated by injection. BTV-8 was first introduced to northern Europe in 2006 and has subsequently spread rapidly to many countries on that continent. During 2007, a total of 6,870 bluetongue cases were reported in Belgium (2); animal 1 died in September 2007, which corresponded to the peak of bluetongue outbreaks in that region. No deaths were reported during that period among other animals, including ruminants, held in the same zoo as the 2 lynx reported here. The time lapse between initial clinical signs and death could explain the failure to detect BTV-8 RNA in animal 2. Although speculative, the suspicion of bluetongue in this animal is based on the presence of anti–BTV-8 antibodies, vasculitis, and pneumonia, which have been found in dogs accidentally infected with BTV (7). This report raises questions about the current knowledge of the epidemiology of bluetongue. Bluetongue in lynx indicates that the list of known susceptible species must be widened, at least for serotype 8. Although infection of a susceptible host by an insect vector is the only proven natural transmission mechanism for wild-type BTV, transplacental transmission of BTV-8, resulting in the birth of seropositive (8) or virus-positive calves (9), has recently been described in cattle. Although infection by an insect vector cannot be excluded, transmission by the oral route must be strongly suspected because the lynx described in this report had been fed ruminant fetuses and stillborn animals from surrounding farms. This possibility is supported by a previous suspicion that seroconversion to BTV in carnivores was a result of oral infection (1). The possibility of oral transmission is also supported by evidence of lateral transmission of BTV infection to cattle having occurred, in the absence of insect vectors, as a result of direct contact with newborn viremic calves born to infected dams that had been imported to Northern Ireland from a bluetongue-infected region of continental Europe (S. Kennedy, unpub. data). The role of wildlife, especially carnivores, in the epidemiology of bluetongue deserves further study to elucidate their role as either dead-end hosts or new sources of infection for livestock and to help determine the risks for wildlife populations. Our findings clearly indicate that a novel transmission pathway enables the virus to cross species. Consequently, transmission to other species, including domestic animals, can no longer be excluded. Moreover, oral transmission is likely to have considerable implications for disease control, including vaccination, because BTV-8 is a fast-emerging virus with major financial consequences.

Confidence in indirect assessment of foot-and-mouth disease vaccine potency and vaccine matching carried out by liquid phase ELISA and virus neutralization tests.

The necessity of avoiding the use of animals in vaccine potency testing has been widely recognized. The repeatability and reproducibility of the Expected Percentage of Protection (EPP) as a serological potency surrogate for A24 Cruzeiro foot-and-mouth disease virus (FMDV) strain was assessed, and compared with the results obtained with challenge in the Protection against Podal Generalization (PPG) test. To determine the EPPs, the serum titers obtained by liquid phase blocking competitive ELISA (lpELISA) and virus neutralization (VNT) in 10 potency trials using the same A24 Cruzeiro vaccine, were interpolated into previously validated logit transformation curves that correlate PPG with serology. Indirect serological assessment of vaccine matching between the serotype A FMDV strains A24 Cruzeiro and A/Argentina/01 was also carried out by lpELISA and VNT. The results obtained in this study strongly support the replacement of challenge tests for vaccine potency by indirect serological assays, at least for A24 Cruzeiro FMDV strain. While determination of EPPs by lpELISA titers showed an excellent repeatability, reproducibility and concordance with PPG for vaccine potency, assessments of cross-protection by VNT titers were more consistent with the PPG outcome.

Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision

Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays are being used routinely for diagnosing foot-and-mouth disease virus (FMDV). Although most laboratories determine analytical and diagnostic sensitivity and specificity, a thorough validation in terms of establishing optimal RNA-extraction conditions, matrix effect, uncertainty of measurement and precision is not performed or reported generally. In this study, different RNA-extraction procedures were compared for two FMDV rRT-PCRs. The NucleoSpin columns available commercially combined high extraction efficiency with ease-of-automation. Furthermore, six different FMDV-negative matrices were spiked with a dilution series of FMDV SAT1 ZIM 25/89. Compared to cell-culture-spiked viral control samples, no matrix effect on the analytical sensitivity was found for blood or foot epithelium. Approximately 1log(10) reduction in detection limit was noted for faecal and tongue epithelium samples, whereas a 3log(10) decrease was observed for spleen samples. By testing the same dilution series in duplicate on 10 different occasions, an estimation of uncertainty of measurement and precision was obtained using blood as matrix. Both rRT-PCRs produced highly precise results emphasising their potential to replace conventional virological methods. The uncertainty measurement, as described in this study, proved to be a useful tool to evaluate the probability of making a wrong decision.

Indirect foot-and-mouth disease vaccine potency testing based on a serological alternative.

Foot-and-mouth disease (FMD) vaccine potency testing has historically been performed by experimentally infecting vaccinated cattle. A few alternative approaches to the in vivo challenge test based on the correlation between serum titres of primo-vaccinated cattle and protection against infection have been proposed, but none have been accepted by the European Pharmacopoeia (Ph.Eur.) due to the lack of statistical power and the pooling of data over time. The present study addresses these issues and presents data of 150 cattle vaccinated according to Ph.Eur. standards. Four laboratories took part in the serological testing and different serological assays were used, including virus neutralisation assays and ELISA formats. Models correlating specific anti-FMD virus antibody titres to protection were built using logistic regression followed by Receiver Operating Characteristic (ROC) analysis. The best models accurately predicted the in vivo protection status in 80.0% of the cases. Although differences were observed between laboratories and assays used, the majority of antibody pass-levels, determined using ROC analysis, corresponded to at least 75.0% probability of protection. The indirect potency assessment procedure proposed is at least as precise (repeatability=65.8%, reproducibility=60.7%) as the in vivo test, can be standardised and results in a quantitative PD50 value. The validity of the procedure was also demonstrated.

Classical swine fever outbreak containment using antiviral supplementation: a potential alternative to emergency vaccination and stamping-out.

Classical swine fever (CSF) outbreaks may result in huge economic losses to countries with densely populated pig areas (DPLAs). The EU minimum control measures require depopulation of infected farms, movement restrictions, zoning and surveillance (EU Minimum strategy). Emergency vaccination is authorised for DPLAs although the EU Minimum strategy plus culling in a 1-km ring around infected premises is preferred. Nonetheless, vaccination in a 2-km ring has been found equally effective as 1-km ring culling using stochastic modelling. Alternatives control measures (e.g. antiviral agents, in particular small molecule inhibitors of the CSFV replication) are being explored. Hence, the present study was set up to simulate inter-herd CSFV spread when antiviral molecules are supplemented to pig feed in a 1-km ring around infected farms. The effectiveness of the antiviral strategy for containing CSF outbreaks was compared to six other control scenarios including the EU Minimum strategy, the EU preferred policy for DPLAs and the use of 2-km ring vaccination. The InterSpread Plus model was adapted to the 2006 Belgian pig population and outbreak simulations were performed with a fast spreading CSFV strain entering a DPLA in Belgium. Four out of the seven control strategies resulted in outbreaks that were controlled by the end of the simulation period (i.e. 365 days). The distributions of the number of infected herds and the duration of the predicted outbreaks for these four control strategies were not different. This is the first report investigating CSF outbreak containment using antiviral molecules. Although antiviral supplementation was not found to perform any better than some other conventional strategies, such as pre-emptive culling and emergency vaccination, it might be worthwhile considering it further as additional tool in a response to CSF outbreaks.

Laboratory validation of a lateral flow device for the detection of CyHV-3 antigens in gill swabs

Cyprinid herpesvirus-3 (CyHV-3) induces the highly contagious koi herpesvirus disease (KHVD) and may result in significant economic losses to the ornamental and food-producing carp industry. Suspicion of KHVD is triggered by clinical signs and confirmed using laboratory techniques. The latter are labour- and time-consuming, require specialised equipment and trained personnel. For rapid, on-site detection of CyHV-3, a lateral flow device (LFD) was developed using two monoclonal antibodies directed towards the viral glycoprotein ORF65. The LFD was highly specific with analytical and diagnostic specificities of 100%. Analytical sensitivity ranged between 1.25×10(2) and 2.40×10(4) plaque forming units per ml for isolates originating from geographically distinct regions. In experimentally infected carp, CyHV-3 was detected as early as 4-5 days post infection. Diagnostic sensitivities of 52.6% and 72.2% relative to PCR were recorded, depending on the viral isolate used. When onset of mortality was taken as reference, diagnostic sensitivities increased to 67.0% and 93.3%. The diagnostic sensitivity for freshly found-dead animals was 100%, irrespective of the virus isolate used. Given the high specificity and ease-of-use for on-site detection of CyHV-3, the LFD was regarded fit for purpose as a first-line diagnostic tool for the identification of acute CyHV-3 infections in KHVD affected (koi) carp.

The potential of antiviral agents to control classical swine fever: A modelling study.

Classical swine fever (CSF) represents a continuous threat to pig populations that are free of disease without vaccination. When CSF virus is introduced, the minimal control strategy imposed by the EU is often insufficient to mitigate the epidemic. Additional measures such as preemptive culling encounter ethical objections, whereas emergency vaccination leads to prolonged export restrictions. Antiviral agents, however, provide instantaneous protection without inducing an antibody response. The use of antiviral agents to contain CSF epidemics is studied with a model describing within- and between-herd virus transmission. Epidemics are simulated in a densely populated livestock area in The Netherlands, with farms of varying sizes and pig types (finishers, piglets and sows). Our results show that vaccination and/or antiviral treatment in a 2 km radius around an infected herd is more effective than preemptive culling in a 1 km radius. However, the instantaneous but temporary protection provided by antiviral treatment is slightly less effective than the delayed but long-lasting protection offered by vaccination. Therefore, the most effective control strategy is to vaccinate animals when allowed (finishers and piglets) and to treat with antiviral agents when vaccination is prohibited (sows). As independent control measure, antiviral treatment in a 1 km radius presents an elevated risk of epidemics running out of control. A 2 km control radius largely eliminates this risk.

The importance of quality assurance/quality control of diagnostics to increase the confidence in global foot-and-mouth disease control.

The last decade international trade in animals and animal products was liberated and confidence in this global trade can increase only if appropriate control measures are applied. As foot-and-mouth disease (FMD) diagnostics will play an essential role in this respect, the Food and Agriculture Organization European Commission for the Control of Foot-and-Mouth Disease (EUFMD) co-ordinates, in collaboration with the European Commission, several programmes to increase the quality of FMD diagnostics. A quality assurance (QA) system is deemed essential for laboratories involved in certifying absence of FMDV or antibodies against the virus. Therefore, laboratories are encouraged to validate their diagnostic tests fully and to install a continuous quality control (QC) monitoring system. Knowledge of performance characteristics of diagnostics is essential to interpret results correctly and to calculate sample rates in regional surveillance campaigns. Different aspects of QA/QC of classical and new FMD virological and serological diagnostics are discussed in respect to the EU FMD directive (2003/85/EC). We recommended accepting trade certificates only from laboratories participating in international proficiency testing on a regular basis.