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In December 2011, a previously unknown congenital syndrome of arthrogryposis and hydranencephaly in sheep and cattle appeared in the Netherlands as an emerging epizootic due to Schmallenberg virus (SBV). Gross lesions in 102 lambs and 204 calves included porencephaly, hydranencephaly, cerebellar dysplasia and dysplasia of the brainstem and spinal cord, a flattened skull with brachygnathia inferior, arthrogryposis, and vertebral column malformations. Microscopic lesions in the central nervous system showed rarefaction and cavitation in the white matter, as well as degeneration, necrosis, and loss of neurons in the gray matter. Brain and spinal cord lesions were more severe in lambs than in calves. Ovine and bovine cases examined early in the outbreak showed encephalomyelitis. SBV infection was confirmed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in brain samples in 46 of 102 lambs (45%) and in 32 of 204 calves (16%). Immunohistochemistry, performed on tissue samples from 18 RT-qPCR-positive lambs, confirmed the presence of bunyaviral antigen in neurons of the brain in 16 cases. SBV antibodies were detected by enzyme-linked immunosorbent assay in fetal blood in 56 of 61 sampled ovine cases (92%). In a virus neutralization test, all tested dams of affected newborns, 46 ewes and 190 cows, were seropositive. Compared with other teratogenic viral infections, the pathogenesis and lesions of SBV in sheep and cattle fetuses are similar to those of other ruminant orthobunyaviruses. However, the loss of spinal ventral motor neurons and their tracts, resulting in micromyelia, distinguishes SBV infection from other viral central nervous system lesions in newborn ruminants., (© The Author(s) 2014.)

At the end of 2011, a new Orthobunyavirus was discovered in Germany and named Schmallenberg virus (SBV). In the Netherlands malformations in new-born ruminants were made notifiable from the 20th of December 2011. After a notification, malformed new-borns were necropsied and brain tissue was sampled for reverse transcription-polymerase chain reaction (RT-PCR). In addition, blood samples from mothers of affected new-borns were tested for antibodies in a virus neutralization test (VNT). The aim of this study was to summarize and evaluate the diagnostic data obtained and to gain insight into the possible regional differences. In total 2166 brains were tested: 800 from lambs, 1301 from calves and 65 from goat kids. Furthermore 1394 blood samples were tested: 458 from ewes, 899 from cows and 37 from goats. Results showed that 29% of the lamb brains, 14% of the calf brains, and 9% of the goat kid brains were RT-PCR positive. The number of malformed and RT-PCR positive lambs decreased over time while the number of malformed and RT-PCR positive calves increased. In the VNT 92% of the ewes, 96% of the cows and 43% of the goats tested positive. Combining RT-PCR and VNT results, 18% of all farms tested positive in both the RT-PCR and VNT. The relative sensitivity and specificity of the RT-PCR are 19% and 97% respectively, and of the VNT 99% and 6%. The results show a widespread exposure to SBV and the regional evaluation seems to indicate an introduction of SBV in the central/eastern part., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In 1862, the veterinarian Loman reported the first sheep in The Netherlands with symptoms associated with lentiviral infection, although at the time the symptoms were ascribed to ovine progressive pneumonia. In the following century, similar cases were reported by South African, French, American, and Icelandic researchers. Extensive research into the pathology, aetiology, and epidemiology of this slowly progressive and ultimately fatal disease was initiated in several countries, including the Netherlands. Studies of the causative agents--maedi visna virus (MVV) in sheep and caprine arthritis encephalitis virus (CAEV) in goats, comprising the heterogeneous group of the small ruminant lentiviruses (SRLV)--prompted the development of diagnostic methods and the initiation of disease control programmes in many European countries including the Netherlands, as a pioneer in 1982, and in the U.S.A. and Canada.

The objective of the study was to evaluate the diagnostic performances of the ELITEST-MVV ELISA for detection of antibodies against small ruminant lentiviruses and of two recently published PCRs for the detection of proviral DNA of SRLV in blood and corresponding individual milk samples. In addition, the feasibility of bulk milk testing was investigated by titrating ELISA positive pooled milk samples in negative milk, and by investigating bulk milk samples by ELISA and PCR in relation to the SRLV-status of the flocks. The results show that plasma and milk are suitable replacements for serum. For sheep, both PCRs showed a better diagnostic performance than for goats. ELISA results for bulk milk samples were promising with a putative detection limit of <3% within-herd prevalence using 1/10 pre-diluted samples and even <1% within-herd prevalence when samples were tested undiluted. In a panel of 249 bulk milk samples, all samples from SRLV free flocks (n=138) tested negative in the ELISA, while 50% of the samples from flocks with an unknown SRLV-status (n=111) were positive. For a subset of 59 bulk milk samples, agreement between ELISA results and leader-gag PCR results was almost 100%. These results demonstrate the potential of bulk milk testing as a cost effective tool for early detection of infection in dairy flocks, which is essential for SRLV-monitoring programs., (Copyright 2009 Elsevier B.V. All rights reserved.)

A SRLV-free sheep flock incurred infection which led to an SRLV infection rate of over 50% of the ewes (34/64) within a 30 months period, indicating that environmental conditions were favourable to transmission. An intensive regimen of sampling at short intervals and testing for SRLV antibodies and proviral DNA combined with strict management was implemented for the entire flock, lambs and yearlings included. This resulted in eradication of the infection within two testing and culling rounds with a 3 months interval. The additional value of the proviral DNA detection by PCR in identifying infected animals was clear in that nine infected animals were found that would have been missed if tested by serology alone. PCR also saved two lambs from being culled; they were sero-positive probably due to maternal antibodies, but not infected., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Real-time polymerase chain reaction (RT-PCR) detection of proviral nucleic acid sequences of small ruminant lentiviruses (SRLV) in blood samples was developed and evaluated. Priming oligonucleotides were designed on the highly conserved 5' untranslated leader-gag region while those on the long terminal repeat (LTR) assay were derived from literature. DNA was extracted from the buffycoat interlayer of centrifuged blood samples. Real-time PCR was performed by means of LightCycler technology (Roche Applied Science) using melting temperature analysis (SYBR Green I) for detection. Results were compared with those of serology using samples from Dutch sheep and goat flocks with known SRLV statuses, with sequential samples from a natural transmission experiment and samples from different regions in Norway, France, Spain and Italy. Real-time PCR testing, especially the application of oligonucleotides for priming the leader-gag region appeared promising in detecting SRLV specific proviral DNA in blood samples from both sheep and goats.

In the framework of the Dutch control program for small ruminant lentiviral (SRLV) infections, too many drawbacks were encountered with respect to serological testing. To improve the quality of testing, five enzyme-linked immunosorbent assays (ELISAs) and an agar gel immunodiffusion test (AGIDT) were evaluated. The focus was on the sensitivity, specificity, and variances of the commercially available tests. Clear differences were found among the tests in analytical and diagnostic sensitivity and overall diagnostic performance, whereas no significant differences in specificity were found. For serodiagnosis of sheep with clinical symptoms of maedi-visna virus (MVV) (histopathologically confirmed), one ELISA was significantly more sensitive than the other ELISAs and than the AGIDT, while for asymptomatic sheep originating from infected flocks, three ELISAs and the AGIDT demonstrated similar performance. The diagnostic performance appeared to be related to animal species and virus infection (MVV or caprine arthritis encephalitis virus [CAEV]) as well as the phase of infection/progression of disease. Receiver operating characteristic analysis, demonstrating the diagnostic potential of tests irrespective of defined cutoffs, again revealed clear differences between tests with respect to diagnostic performance for detection of antibodies against CAEV or MVV. An indirect ELISA, of which the solid phase is sensitized with a combination of the core protein p27 of MVV produced in Escherichia coli and a peptide derived from the transmembrane protein gp46, appeared to be the test of choice for serodiagnosis of SRLV infections in sheep and goats.