Although AD has been eliminated from the domestic pig population in Germany [8], PrV infections are enzootic in European wild boar populations throughout the country [57–60] causing an estimated overall PrV seroprevalence of 12.09% (Fig. 1) [57]. Our study confirms similar studies from other European countries [19, 25–31, 61] with a sporadic number of reported PrV infections in domestic carnivores (Fig. 1A, 2A). Occasionally, PrV from wild boars caused cases in dogs, mainly hunting dogs, during the past decades as reported in the disease notification system, with a temporal association of PrV case reporting in carnivores and hunting activities. This association was confirmed by partial sequence analyses using the gC-gene (Fig. 4, 5) that had been used to characterize wild-boar derived PrV isolates from Europe [19]. In contrast, historic cases of PrV in cats and dogs prior to the elimination of AD from domestic pigs that were also included in this study, were caused by the prevailing domestic pig PrV lineages at the time (Fig. 5).
Wild boar associated PrV cases in dogs are more likely after direct contact during hunting than oral ingestion of offal. Likely, increased stress levels in latently infected wild boar may cause reactivation of virus replication and active shedding without eliciting clinical signs [62], eventually leading to an infection in hunting dogs when they actively encounter wild boar during hunting activities [10]. Up to 7% of wild swine in endemic areas of Florida were PCR positive in nasal, oral and genital swabs indicating low levels of PRV shedding [63]. Oro-nasal infection with direct brain manifestation likely only requires a low infection dose, but also ingestion of wild boar offal led to a clinical PrV- infection in captive wolves [45].
Against this background it is interesting that despite other reports none of the 682 investigated free-ranging wolves and hybrids tested positive for PrV (Fig. 2B), despite the overlap of their distribution in Germany in areas with high PrV seroprevalences in wild boars, wild boar densities [64, 65] and their natural behavior to prey on wild boars [66, 67] (Fig. 1B). Studies of scat samples from Europe indicate a certain flexibility of the wolf as a predator depending of the availbility of prey. While in northern plain lands of Europe wolves seem to rather avoid wild boar as prey [68], in the Mediterranean basin wild boars are obviously sometimes part of the prey [66, 67]. In a recent study, diet of wolves from Italy was consistently dominated by the consumption of wild boar which accounted for about two thirds of total prey biomass [69]. Data from Germany show that about 20% of total prey biomass of wolves consists of wild boar [70]. Since infection of grey wolves with PrV would inevitably lead to a clinically visible manifestation and eventually death, the absence of PrV-infected grey wolves in our sample suggests that PrV infections are very rare in wolves and the few occasional infections may have gone undetected.
In contrast, we report three cases in free-ranging red foxes. In one case, the fox was shot by a hunter as it showed atypical behavior suggestive of encephalitis, which was confirmed by immunohistopathology (Fig. 3C), while in other studies from Germany investigating hunting bags from foxes, no PrV infections were detected [71, 72]. As red foxes are known to feed on carcasses as well as to prey on European wild boar piglets [73], pointing to the oral route of infection as the most plausible.
While the virus found in one red fox (GER 641) from the western Federal state of Saarland was identical in its partial DNA sequence to other PrV isolates from the previously established “clade B” [19], isolate GER 642 form eastern Brandenburg clusters with a dog from Baden-Wurttemberg and is identical in its partial gC-gene sequence with a Belgian domestic pig isolate from 1973 (Fig. 5). This finding indicates/emphasizes that the phylogenetic clustering pattern needs to be interpreted with caution [28]. This is not only based on low bootstrap support, as indicated before [28], but also on the apparent stability of the gC-gene with limited sites of genetic diversity (Fig. 4). Whether other PrV genes are better suited for phylogenetic analysis remains to be demonstrated, if even whole-genome sequences do not provide a better resolution [74].
Furthermore, sampling biases and surveillance gaps may suggest epidemiological links where in reality there are none. As regards isolate GER 642, this red fox isolate serves as an indicator for the presence of a yet unknown PrV variant in this part of Germany bordering Poland, a country where no information on PrV characterization in wildlife is available. These results are similar to findings in Austria where the genetic diversity of PrV was only evident after investigating PrV from hunting dogs [28]. Alternative hypotheses, e.g. that the fox was PrV infected in areas endemic with a different virus, are not plausible given the short incubation period and the average home range of foxes. Also, since Germany is free of AD [8], consumption of infected offal from domestic pigs is extremely unlikely. Against the background of the limited usefulness of gC-gene-based phylogeny for epidemiological inference, PrV cases in red foxes that had been described before [39, 40] may need reconsideration.