Evaluation of antibody-ELISA and real-time RT-PCR for the diagnosis and profiling of bluetongue virus serotype 8 during the epidemic in Belgium in 2006

Abstract

In 2006 bluetongue (BT) emerged for the first time in North-Western Europe. Reliable diagnostic tools are essential in controlling BT but data on the diagnostic sensitivity (Se) and specificity (Sp) are often missing. This paper aims to describe and analyse the results obtained with the diagnostics used in Belgium during the 2006 BT crisis. The diagnosis was based on a combination of antibody detection (competitive ELISA, cELISA) and viral RNA detection by real-time RT-PCR (RT-qPCR). The performance of the cELISA as a diagnostic tool was assessed on field results obtained during the epidemic and previous surveillance campaigns. As the infectious status of the animals is unknown during an epidemic, a Bayesian analysis was performed. Both assays were found to be equally specific (RT-qPCR: 98.5%; cELISA: 98.2%) while the diagnostic sensitivity of the RT-qPCR (99.5%) was superior to that of the cELISA (87.8%). The assumption of RT-qPCR as standard of comparison during the bluetongue virus (BTV) epidemic proved valid based on the results of the Bayesian analysis. A ROC analysis of the cELISA, using RT-qPCR as standard of comparison, showed that the cut-off point with the highest accuracy occurred at a percentage negativity of 66, which is markedly higher than the cut-off proposed by the manufacturer. The analysis of the results was further extended to serological and molecular profiling and the possible use of profiling as a rapid epidemiological marker of the BTV in-field situation was assessed. A comparison of the serological profiles obtained before, during and at the end of the Belgian epidemic clearly showed the existence of an intermediate zone which appears soon after BTV (re)enters the population. The appearance or disappearance of this intermediate zone is correlated with virus circulation and provides valuable information, which would be entirely overlooked if only positive and negative results were considered.

Evaluation of commercial ELISA kits for the detection of antibodies against bluetongue virus

“…All ELISA kits used were easy and quick to perform, especially the VMRD, ID VET and Pourquier taking about 1 hour to complete. The relative specificity of ID VET kit was similar to its performance reported by Vandenbussche et al (2008) and specificity of PrioCHECK and Pourquier kits was comparable to the earlier reported results (http://www.eavld2010.org, http://www.institut-pourquier.fr). Our results of analytical sensitivity of c-ELISAs evaluated on BTV "post-infection" sera correspond with the previously reported findings (Vandenbussche et al 2008, http://www.institut-pourquier.fr).…”

“…The relative specificity of ID VET kit was similar to its performance reported by Vandenbussche et al (2008) and specificity of PrioCHECK and Pourquier kits was comparable to the earlier reported results (http://www.eavld2010.org, http://www.institut-pourquier.fr). Our results of analytical sensitivity of c-ELISAs evaluated on BTV "post-infection" sera correspond with the previously reported findings (Vandenbussche et al 2008, http://www.institut-pourquier.fr). Moreover, the c-ELISA was a superior to the other ELISA assays in the detection of anti-BTV antibody in the sera samples from cattle and sheep early after infection with BTV (Afshar et al 1987).…”

Experimental reproduction of bluetongue virus serotype 8 clinical disease in calves

“…The cELISA was performed following manufacturer's instructions. Besides the kit controls, a twofold dilution series of an anti-BTV antibody positive reference serum was included and the results were expressed as % negativity (PN) as described by Vandenbussche et al (2008).…”

“…These samples were used to detect the levels of viraemia by real-time RT-PCR (RT-qPCR) according to Vandenbussche et al (2008). In order to amplify BTV genome, a RT-qPCR amplifying a region of BTV segment 5 (RT-qPCR_S5) was used (Toussaint et al, 2007a).…”

Bluetongue in Eurasian Lynx

“…Risk for vector transmission is highest in the peripheral quarters of the city and towards late summer (6). Besides, Buenos Aires, like other Latin American metropolitan areas, is undergoing demographic changes that convey further risk for mosquito-borne disease transmission, namely, accelerated population growth mainly caused by informal settlements, defi cient public health infrastructure and basic services, unregulated immigration from neighboring countries, and increased international mobility especially in or from neighboring countries (1).…”