Sampling of free-living animals

Blood samples of 21 (13 females and 8 males) adult free-living Mongolian khulan were collected in Southern Gobi (Mongolia) in October 2015. Blood samples from 27 adult free-ranging female plains zebras were collected in 2015 in the Etosha National Park (Namibia; n = 7) and in 2016 in the Serengeti National Park (Tanzania; n = 20). All khulan and plains zebras were in good physical condition and showed no visible clinical symptoms of EHV infection. We did not see any signs of nasal, ocular or similar discharge; any signs of diarrhea or infection with ecto-parasites; and any deep open wounds or broken bones. Both electrocardiogram and body temperature measured during the anesthesia were normal. The animals were immobilized with a dart gun³⁹ and a sample of blood was drawn from the jugular vein into EDTA-tubes (S-Monovette K3E, Sarstedt, Hildesheim, Germany) approximately 15–30 minutes after darting. In order to screen for acute nasal shedding of EHV, two UTM^TM Viral Transport Media swabs (Copan Diagnostics Inc., Murrieta, California, USA) were rubbed firmly on the nasal mucosa of each animal.

Khulan samples were stored at −20 °C while on the field and were then transported to the Research Institute of Wildlife Ecology, Austria, in full compliance with the Convention on International Trade in Endangered Species (CITES), where samples were stored at −80 °C until transported to the IZW in Germany, where they were likewise stored until laboratory analyses. Zebra samples from Namibia and Tanzania were transported to the respective field laboratory in a cooling box at 4 °C and then stored and transported at −20 °C to the laboratory facilities of the IZW in Germany, where blood samples were stored at −80 °C.