2.7.3. Nucleic Acid Amplification

For amplification of DNA from SuHV-1, CaHV-1, CPV-2, FoxCV, and N. caninum, QuantiTect^® Multiplex PCR NoROX Kit (Qiagen, Hilden, Germany), and for amplification of RNA from WNV, BoDV-1, and CDV QuantiFast^® Pathogen RT-PCR + IC Kit (Qiagen, Hilden, Germany) were used according to the manufacturer's instructions. Detailed (RT-)qPCR conditions for each pathogen are listed in Supplementary Table S3. All (RT-)qPCR reactions were run on AriaMx Real-time PCR Systems (Agilent Technologies, Santa Clara, USA). Samples were considered positive if the cycle threshold was less than or equal to 40. Results considered positive were repeated individually using a new sample from the individual animal for confirmation. In the following, these positive samples are referred to as infection as described in Pschyrembel (40) even if there was no histomorphological correlate in the examined brain sections. Positive controls included material from infected cell cultures (BoDV-1 and WNV, kindly provided by colleagues from the Friedrich-Loeffler-Institute, Germany; CaHV-1 and CPV-2, kindly provided by colleagues from the University of Leipzig, Germany) or organ samples from naturally infected animals (FoxCV, kindly provided by colleagues Department of Virology, Erasmus MC, Rotterdam, Netherlands Virology Department of the Netherlands as well as the Virology Department of the Istituto Zooprofilattico Sperimentale dell'Abruzzo e Molise (IZSAM), Teramo, Italy; CDV, SuHV-1, and N. caninum, own sample material whose validity was confirmed by other laboratories). Two individually confirmed negative brain samples and RNase-free water were used as no template control in each individual (RT-)qPCR run.