All experiments were conducted in accordance with the guidelines of the Council European Directive (2010/63/UE). All experimental procedures were approved by the ethical review board of the Val de Loire (CEEA VdL, committee number n°19, number 2012-02-11).
Experimental design
Five adult Alpine goats, one adult Saanen buck and one adult Alpine buck were purchased from local breeders (INRA Center, Bourges, France) and were housed in the Biosafety Level 3 and insect-proof animal facilities of the National Institute of Agricultural Research (INRA), Research Loire Valley Center (PFIE, Nouzilly, France). All purchased animals were SBV-negative as determined by ELISA and RT-qPCR.
Two goats (designated A and B) were inoculated subcutaneously on day 0 with 1 mL of SBV-containing bovine serum kindly provided by the Friedrich-Loeffler-Institut (FLI), Germany [3]. Two goats (designated C and D) were inoculated on day 0 with 1 mL of SBV-containing ovine whole blood collected at the PFIE during a previous experimental infection trial [5]. One goat from each group was killed at day 7 pi and the remaining goats were killed at day 14 pi. The two bucks (designated E and F) were inoculated subcutaneously at day 0 with 1 mL of the FLI serum and killed at day 28 pi. One goat (designated G) was inoculated subcutaneously on day 0 with 1 mL of sterile saline solution and served as an in-contact negative control until it was killed at day 28 pi.
During the course of the trial, all animals were monitored twice daily, and body temperatures were recorded by telemetric measurement with rumen temperature sensors (Small Bolus®, Médria, Châteaubourg–France). After inoculation, whole blood and serum samples were collected daily during the first week and then at days 14 and 28 pi. Buck semen was collected at day 0 and then twice a week. At necropsy, all the organs were macroscopically evaluated and a panel of tissue samples was collected for histopathology and RT-qPCR (spleen, prescapular lymph node, skeletal muscle, aorta, liver, kidney, lung, small intestine, brain, skin, ovary, oviduct, uterus, testis, and epididymis).
Real-time PCR
Ovaries were dissected and follicular fluid, cumulus cells, oocytes and interstitial tissue were separated from each other prior to total RNA extraction. RNA from blood and tissue samples was extracted using the LSI MagVet™ Universal Isolation kit (Life Technologies SAS, Saint-Aubin, France) and King Fisher magnetic particle processor (Thermo Scientific™, Illkirch, France) according to the manufacturers’ instructions. RNA from semen samples was extracted with Trizol ® LS Reagent [6].
The samples were then tested for the presence of SBV RNA by RT-qPCR as previously described [10]. Quantification cycle (Cq) threshold value was 40, with higher values regarded as negative.
Serology
Serum samples were submitted to SBV specific ELISA testing (ID Screen Schmallenberg virus Indirect®, monocupule, IDvet) and virus neutralization test (VNT) [11].
Histopathological examination
After fixation in 10 % buffered formalin, tissues were routinely processed, sliced at 4 μm, stained with Hematoxylin-Eosin-Saffron (HES) and examined by light microscopy.