Materials and Methods
The calves were either stillborn or were euthanized for animal welfare
reasons on the farms in connection to the delivery, which occurred in a
narrow time period from April 12 – 26, 2021. The calves were submitted
for necropsy as part of the Danish surveillance system for bovine
genetic diseases. Two cases were frozen to - 20 °C before being shipped
and were necropsied after thawing, while the other five cases were
shipped directly to the laboratory and necropsied within 2-8 days after
parturition. The calves underwent a necropsy and samples from cerebrum
and cerebellum were taken as was a pleural effusion fluid. A blood
sample from the dam was taken in a plain vacutainer tube and either
followed the carcass or was requested afterwards. The blood was
centrifuged at 1160 g for 15 min to separate the serum. All
samples were stored at - 20 °C in plastic tubes before being shipped for
virological and serological analyses at the Friedrich-Loeffler-Institut.
Upon arrival at the Friedrich-Loeffler-Institut, tissue samples were
homogenized in 1 ml serum-free Minimum Essential Medium (MEM). Nucleic
acid was extracted from 140 µl of the tissue homogenates, serum or
pleural effusion fluid by using the QIAamp Viral RNA Mini kit (Qiagen,
Hilden, Germany) and subsequently tested by an SBV S-segment based
real-time RT-PCR (Bilk et al., 2012). To exclude an infection with
further viral pathogens, especially viruses with a teratogenic
potential, the samples have been additionally tested by a multiplex
real-time RT-PCR for the detection of bovine viral diarrhea virus
(BVDV), bluetongue virus (BTV), foot-and-mouth disease virus (FMDV),
epizootic hemorrhagic disease virus (EHDV), and Rift Valley fever virus
(RVFV) (Wernike, Hoffmann, & Beer, 2015).
In addition, sera and pleural effusion fluids were analyzed by a
commercially available enzyme linked immunosorbent assay (ELISA) for the
detection of anti-SBV antibodies (ID Screen® Schmallenberg virus
Competition, ID.vet, France).
Data on calving outcomes in the herds +/- 3 months around the date for
birth of the malformed calf were obtained from the Danish Cattle
Database.