Nasopharyngeal swabs (all dogs) and rectal swabs (sick dogs only) were collected and analyzed by Laboklin GmbH & Co. (Bad Kissingen, Germany) using conventional PCR or real-time PCR (qPCR and RT-qPCR). Swabs were incubated in 750 µL MagNA Pure DNA Tissue Lysis Buffer (Roche Diagnostics GmbH, DE-Mannheim, Germany) plus 75 µL Proteinase K (Carl Roth GmbH + Co. KG, DE-Karlsruhe, Germany) for 1 h at 65 °C. Automated isolation of nucleic acids (RNA and DNA) was performed with the MagNA Pure 96 system from Roche Diagnostics GmbH according to manufacturer's instructions. Nasopharyngeal swabs from sick dogs were tested for canine adenovirus type 2 (CAV-2) [16 (link)], Bordetella bronchiseptica [17 (link)], CDV [18 (link)], canine parainfluenza virus (CPIV) (in-house method), canine influenza A virus (CIV) [19 (link)] and canine herpesvirus-1 (Canid alphaherpesvirus-1: CaHV-1) [20 (link)] by Taqman real-time PCR on a LightCycler®96 (Roche Diagnostics, Basel, Switzerland) and for Mycoplasma spp. [21 (link)] by conventional PCR. Swabs from all sick dogs, and swabs from healthy dogs that presented α-SARS-CoV-2 IgG, were also tested for SARS-CoV-2 [22 ] by Taqman real-time PCR on a LightCycler®96 (Roche Diagnostics).
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