Virus-RT-PCR protocol was determined by amplification of serial dilutions of PaBV-2 in purified fox control RNA (700 ng/l) and RNase-free water, followed by agarose gel electrophoresis. The detection limit was 0.01 ng/l PaBV-2 in 700 ng/l fox RNA.Methods used for the detection of other pathogensStatistical analysisFor comparison of detection of seropositive foxes in endemic and non-endemic administrative districts, a hypothesis test, the two-proportion z-test, with a significance level equal to 0.05 was used.ResultsOrigin of fox samplesThe State Veterinary Institutes and private hunters in Bavaria, Baden-Wuerttemberg and Hesse collected samples from 232 red foxes (59 brain, 225 blood samples) and formalin fixed tissue sections from three red foxes with encephalitis of unknown origin (#41, #42, and #43). Private hunters provided 81 frozen blood samples from 40 hunting districts in 10 administrative districts in Germany. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26398218
Sample collection was scattered geographically, depending on the location of the State Veterinary Institutes and the private hunters involved in the study including samples from known endemic and nonendemic areas. Among 232 carnivores, 64 small carnivores originated from Bavaria (Swabia, Upper Bavaria, and Middle Franconia), 132 from Baden-Wuerttemberg (Tubingen and Freiburg) and 36 from Hesse (Kassel, Giessen and Darmstadt). See Fig. 1 for further details on the sampled districts.Serology for detection of anti-BoDV-1 antibodiesThe State Veterinary Institutes screened all brain samples for the presence of rabies virus, using an immunofluorescence test (IFT) as a standard method recommended by the WHO and OIE. Immunohistochemistry was used for the presence of antigens of canine distemper virus (CDV), porcine herpesvirus 1 (SHV-1), canine adenovirus 1 (CAV-1), canine parvovirus (CPV) and of Toxoplasma gondii (Table 1). The State Veterinary Institutes Giessen, Freiburg and Aulendorf routinely conducted RT-PCR assays for CDV RNA according to established protocols . At the Institute of Veterinary Pathology in Giessen, a pan-flavivirus-RT-PCR protocol  and an adopted PCR assay for the amplification of CPV  was used.Metagenomics, next generation sequencing (NGS)Representative brain samples of three red foxes with high anti-BoDV-1 antibody titres and encephalitis were sequenced, using a MiSeq instrument [Illumina] as described before . Sequence analysis with RIEMS assembled the sequences .An indirect immunofluorescence test (IIFT) was carried out for 225/232 red foxes. Among PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27832717
225 foxes, 37 exhibited anti-BoDV-1 antibodies, representing a prevalence rate of 16.4 in the investigated districts in Germany. Sera with anti-BoDV-1 antibodies caused a brilliant granular fluorescence in the nucleus of BoDV-1 infected MDCK cells. In Bavaria, 7/63 foxes displayed antiBoDV-1 antibodies, in Baden-Wuerttemberg 23/131 and in Hesse 7/31. For an overview of results for seropositive foxes in endemic and non-endemic districts see Table 2 and Fig. 1. Statistical analysis showed the p-value (0.36) to be higher than the significance level (0.05), which is why the null hypothesis cannot be rejected. No statistically significant difference has been found between the detection of seropositive foxes in endemic and nonendemic areas in the investigated areas in Germany. Serum antibody titres in foxes ranged from 1:40 to 1:2560 (median 1:160). The red fox #21 PluriSIn 1
with a titre of 1:2560 was an adult female from the administrative district of.