A very robust VNT was developed with a very high specificity and sensitivity, both close to 100%, in sheep and cattle. The assay was shown to be highly repeatable and reproducible. The VNT is easy to perform and can be read macroscopically as a result of the amido black staining. With the current, optimized protocol, this staining results most of the times in a very sharp transition between wells with and without CPE. This makes it easy and very fast to read the plates, with a high reliability and reproducibility. A serum control for each individual sample was not included during our evaluation of the test. No evidence for a toxic effect of serum on the cells was ever noticed, but to avoid false negative results, routine use of a serum control could be considered.
In infected herds, i.e. confirmed by RT-PCR, more than 94% of the sheep and 99% of the cows tested seropositive. The ewes and cows tested were sampled because of congenitally malformed offspring. In lambs, approximately 30% of these malformations were confirmed to be positive for SBV by RT-PCR on brain tissue. However, in many cases it was not possible to trace the confirmed SBV-positive lambs back to the exact mother, so the serum blood sample was sometimes obtained from other ewes in the herd. Nevertheless, more than 94% of these ewes tested seropositive, suggesting that at least within these sheep herds with congenital malformations, the infection is widespread with a high within-herd seroprevalence.
Calves with malformations, on the other hand, could usually be traced back to the mother and from these cows serum blood samples were collected for serology. More than 99.3% of these mothers were seropositive. These cattle sera originated from cows that gave birth to malformed calves that were positive in the RT-PCR for SBV and are currently the most well-defined serum samples from previously infected animals. The sensitivity of the VNT can be estimated most reliably from these samples and is therefore >99%, which is in line with the estimation during the initial evaluation of the test, based on the distribution of titres in a smaller set of field samples.
In suspect herds, SBV was not confirmed by RT-PCR and in some cases RT-PCR was not carried out at all. It is likely that the virus, after infecting the foetus and causing the malformations, was cleared during the last part of the gestation period, at least from the brain, which was the most common sampling site. The fact that calves were more often RT-PCR negative than lambs can then be explained by the longer gestation period in cows and supports this explanation. The fact that seroprevalences in suspect herds were slightly lower than in infected herds, suggests that also malformed offspring with defects unrelated to a SBV infection were submitted for testing. Given the high alertness for congenital malformations, this can be expected to happen. In any case it shows that serological assays are needed to reliably detect infections.
High seroprevalences, both on a regional level, but also within herds, with and without obvious clinical signs, are not uncommon for viruses related to SBV. In Australia, seroprevalence studies into the related Akabane virus (AKAV), revealed within-herd seroprevalences of 77% in 1964
, up to 89% in 1971
 and 99% in 1988 in the New South Wales area
. Furthermore, in Japan, 74% of apparently healthy cattle cohabitated with cows showing clinical signs from an AKAV infection were seropositive also
. Finally, a very high seroprevalence is reported in the Netherlands, both within the Netherlands as a whole as in a few individual herds
In goats the number of seropositive animals is far lower than in sheep and cattle, although less samples from goats were tested and the estimated seroprevalence is therefore less precise. Most of the malformations in goats therefore likely had another cause, as no infection with SBV could be proven in most of the mothers of the affected kids, neither by RT-PCR, nor by serology. Probably these affected kids were submitted and tested for SBV due to the ongoing outbreak and the high alertness for congenital malformations. These results may reflect a lower seroprevalence in goats in general. Given that goats, in contrast to cattle and sheep, are often housed indoors, and the SBV is supposedly transmitted by Culicoides vectors, like other viruses of the Simbu serogroup
[2, 13, 14], this lower seroprevalence would not be surprising.
Many countries outside the EU have closed their borders for cattle and sheep from SBV infected countries. Importing countries may require testing of animals originating from infected countries, which could include RT-PCR and/or serological testing. Although ELISA’s are being developed, and one became recently available on the market, the VNT that was developed, is a possible alternative. It is easy to carry out, and even though the incubation period is quite long with 5 days, hands-on time is relatively short. This means that the VNT is relatively cheap, and the total test capacity for an average laboratory to carry out the VNT could be quite high. Depending on the number of dilutions to be tested, whether samples will be tested in duplicate or not, and based on existing experience and logistics within a laboratory, thousands of samples could be tested per week. Costs of the VNT are mainly related to labour, while costs of an ELISA are for a large part expenditures on materials. Furthermore, the VNT could be used as a confirmation test and probably even a gold standard if further evaluated and validated. For (semi)quantitative studies, either in animal experiments or field studies, a VNT also has its advantages.