Lsi vetmax prrsv eu na

Six cross-bred pigs (Sus scrofa domesticus) of either sex, aged 6–18 months old, were used as blood donors for in vitro experiments. Pigs were housed at the Experimental Station of Istituto Zooprofilattico Sperimentale (IZS) of Sardinia (“Surigheddu”, Sassari, Italy). Animal husbandry, handling, and procedures (bleeding) were carried out according to the Italian Legislative Decree No. 26 dated 4 March 2014 and in agreement with the Guide of Use of Laboratory Animals issued by the Italian Ministry of Health (authorization No. 1232/2020-PR).
Heparinized blood samples were used for generation of monocyte-derived macrophages (moMΦ) (described in Section 4.2). Animal health was routinely monitored by trained veterinarians, and blood samples were screened for several porcine pathogens. The absence of African swine fever (ASFV), porcine parvovirus (PPV), and porcine circovirus 2 (PCV2) genome was evaluated though qualitative real-time PCR, as previously described [21 (link),60 (link)], with primers reported in the Table S1 [61 (link),62 (link),63 (link)]. The absence of the porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae was monitored using commercial real-time PCR kits (LSI VetMAX™ PRRSV EU/NA and VetMAX™-Plus qPCR Master Mix, both Thermo Fisher Scientific, respectively), following the manufacturer’s instructions [21 (link)].

Five cross-bred pigs (Sus scrofa domesticus), either male or female, 6–18 months old, were used to donate blood for macrophage generation. Animals were kept at the Experimental Station of Istituto Zooprofilattico Sperimentale (IZS) of Sardinia (Sassari, Italy). Animal husbandry, handling, and procedures (bleeding) were performed in accordance to the Italian Legislative Decree n.26 dated 4th of March 2014, as well as the Guide of Use of Laboratory Animals issued by the Italian Ministry of Health (authorization n° 1232/2020-PR). Animal health status was controlled by authorized veterinarians, and samples (EDTA blood) were routinely screened for main porcine pathogens, as previously described (22 (link)). In detail, qualitative real-time PCR was employed to exclude the presence of porcine parvovirus (PPV), porcine circovirus 2 (PCV2), and African swine fever (ASFV) genome (22 (link), 23 (link)), with primers reported in the Table S1 (24 (link)–26 (link)), whereas commercial real-time PCR kits were used to detect porcine reproductive and respiratory syndrome virus (PRRSV), and Mycoplasma hyopneumoniae genome (LSI VetMAX™ PRRSV EU/NA and VetMAX™-Plus qPCR Master Mix, both Thermo Fisher Scientific, respectively) (22 (link)).

Nucleic acids were extracted from moMΦ using a MagMax Core kit and MagMax96 extractor (Thermo Fisher) according to the manufacturer’s instructions; samples were kept at −20 °C until analyzed. The presence of PRRSV or M. hyopneumoniae genome was subsequently screened using commercial real-time PCR kits, LSI VetMAX™ PRRSV EU/NA and VetMAX™-Plus qPCR Master Mix (both Thermo Fisher Scientific), respectively. The presence of PCV2 was instead evaluated by qualitative real-time PCR, as we previously described [44 (link)], using forward primer 5′-TGGCCCGCAGTATTCTGATT-3′ and reverse primer 5′-CAGCTGGGACAGCAGTTGAG-3′ [45 (link)]. Samples with Ct values less than 40 were considered positive [44 (link)].