2.1 Sample collection and serum preparation
Blood samples were collected from 451 Iberian hares, mostly from central-south-western Iberian Peninsula, coinciding with regions that were covered by research studies in Portugal and Spain, and where the recent myxomatosis infections have been detected. Iberian hares included in the study were either live captured with long nets following drives (n=88), or post-mortem, either hunted (apparently healthy, n=313), or found dead in the field (n=50). Live captures and hunting samples were considered in this study as random sampling, contrary to the animals found dead in the field, possibly due to myxomatosis infection. Sampling took place in two time periods: 1994-1999 and 2017-2019 (Table 1). The samples from 2018 and 2019 were collected after the emergence of myxomatosis (middle and late 2018 for Spain and Portugal, respectively). Hares were hunted according to the Spanish, Portuguese and European legislation. Thus, no animal was sacrificed for the purpose of this study.
Blood collection from live hares was obtained by venepuncture of the marginal ear vein or the external jugular vein (Abade dos Santos et al., 2019), and from dead animals by cardiac puncture. Blood was transferred into plain tubes, centrifuged for 10 min at 1,500 g at 4ÂșC and the serum was preserved at -20oC until analysis.