Schmallenberg virus detection in bovine semen after experimental infection of bulls

Abstract

SUMMARYTo study Schmallenberg virus (SBV) excretion in bovine semen after experimental infection, two bulls were inoculated subcutaneously with a SBV isolate (1 ml Vero cell culture 106 TCID50). After inoculation (at day 0), semen was collected daily from both animals for 21 days and samples were tested for SBV by qRT–PCR assay. At 24 days post-inoculation both animals were subjected to necropsy and the genital organs and lymph nodes draining these organs were also tested for SBV RNA (qRT–PCR). After SBV infection both animals in the study showed viraemia (qRT–PCR) with fever and diarrhoea. SBV RNA could be detected in semen from both animals. The highest SBV RNA concentrations in semen were found in the first week (days 4–7 post-inoculation) but concentrations were relatively low (Ct values 30–39). Viable SBV was only isolated from blood samples and not from semen or genital tissues.

European interlaboratory comparison of Schmallenberg virus (SBV) real-time RT-PCR detection in experimental and field samples

“…Adult ruminants have short-term RNAemia during the first week after SBV infection, followed by seroconversion between 4 days and 3 weeks after a positive PCR result. 14,21,26,29 The presence of SBV RNA in spleen and mesenteric lymph nodes for up to several weeks is a common finding in most SBV-infected ruminants after RNAemia has ceased, 24,27 whereas in fetuses the detection of SBV antibodies in precolostral blood or of SBV RNA in cerebrum, brainstem, amniotic fluid, and in spinal and umbilical cords was found suitable for the confirmation of a previous SBV infection 4 (World Organization for Animal Health (OIE), 2013, http://www.oie.int/fileadmin/Home/eng/Our_scientific_ expertise/docs/pdf/A_Schmallenberg_virus.pdf). Therefore, whole blood, serum, and tissue homogenates from mesenteric lymph nodes, spleen, and different parts of the brain were chosen for the 2 independent trials ( Table 1).…”

“…The detection of infectious SBV in bovine semen by subcutaneous injection of cattle and mice with SBV RNA-positive semen and the high variability patterns of SBV RNA excretion in semen after SBV infection of bulls demands reliable methods for safe detection of SBV RNA in semen from SBVinfected bulls, although the possibility of venereal SBV transmission remains to be further investigated. 16,22,24 All extraction and RT-qPCR methods used by the 44 participating laboratories (100%) were sufficiently robust to reliably detect SBV RNA in the matrices of whole blood, RNA eluate, serum, and tissue homogenate, although a marked variety of extraction and PCR methods had been used (Table 2). More than two-thirds of the 44 laboratories also detected SBV RNA in at least 1 of the samples with a Cq value at the detection limit (Cq ≥35), whereas the proportion of SBV RNA-negative samples erroneously determined as positive or doubtful was low (DSp 95.8%).…”

“…22 The detection of infectious SBV in semen from naturally or experimentally SBV-infected fertile bulls suggested that natural and artificial insemination of dams might be an additional route of infection. 16,22,24 The SBV epizootic wave has declined (World Trade Organization [WTO], 2012, http://ec.europa.eu/food/animal/diseases/schmallenberg_ virus/docs/gen_1161_sbv_en.pdf ), 12 but insemination of SBV-naive dams with cryopreserved semen contaminated with SBV is assumed to be a possible risk for distribution of SBV to SBV-free regions. 21,22,24 The WTO removed restrictions on trade of live ruminants and their products from SBV-affected regions in July 2012 (WTO, 2012, http://ec.europa.eu/food/animal/diseases/schmallenberg_ virus/docs/gen_1161_sbv_en.pdf).…”

“…16,22,24 The SBV epizootic wave has declined (World Trade Organization [WTO], 2012, http://ec.europa.eu/food/animal/diseases/schmallenberg_ virus/docs/gen_1161_sbv_en.pdf ), 12 but insemination of SBV-naive dams with cryopreserved semen contaminated with SBV is assumed to be a possible risk for distribution of SBV to SBV-free regions. 21,22,24 The WTO removed restrictions on trade of live ruminants and their products from SBV-affected regions in July 2012 (WTO, 2012, http://ec.europa.eu/food/animal/diseases/schmallenberg_ virus/docs/gen_1161_sbv_en.pdf). Nevertheless, several SBV-free countries implemented or sustained restrictions on the import of ruminant semen 16 (ProMED-mail, 2013, http://www.promedmail.org/direct.php?id=20130131.1524123).…”

“…15,16 The high variability of excretion patterns also complicates the safe detection of SBV RNA excretion in semen from an SBV-infected bull. 16,22,24 A limited serological ring trial indicated that the enzymelinked immunosorbent assays and virus neutralization tests used in European laboratories are suitable for reliable detection of SBV antibodies in ruminant blood. 24 However, the performance characteristics of molecular diagnostic methods for the detection of SBV RNA in European laboratories have not yet been assessed, to our knowledge.…”