Skip to main content
Download PDF

Suid alphaherpesvirus 1 of wild boar origin as a recent source of Aujeszky’s disease in carnivores in Germany

Virology Journal volume 20, Article number: 110 (2023) Cite this article

Abstract

Background

The high susceptibility of carnivores to Suid Alphaherpesvirus 1 [SuAHV1, synonymous pseudorabies virus (PrV)], renders them inadvertent sentinels for the possible occurrence of Aujeszky’s disease (AD) in domestic and wild swine populations. The aim of this study was to epidemiologically analyse the occurrence of PrV infections in domestic and wild animals in Germany during the last three decades and to genetically characterise the causative PrV isolates.

Methods

PrV in dogs was detected using standard virological techniques including conventional and real time PCR, virus isolation or by immunohistochemistry. Available PrV isolates were characterized by partial sequencing of the open gC reading frame and the genetic traits were compared with those of archived PrV isolates from carnivores and domestic pigs from Germany before the elimination of AD in the domestic pig population.

Results

During 1995 and 2022, a total of 38 cases of AD in carnivores, e.g. dogs and red foxes, were laboratory confirmed. Sequencing and subsequent phylogenetic analysis of PrV isolates established a strong connection between AD cases in carnivores and the occurrence of PrV infections in European wild boars in the end phase of and after elimination of AD from the domestic pig population. While PrV infections occur at low numbers but regularly in hunting dogs, interestingly, PrV was not observed in grey wolves in Germany. In none of 682 dead-found grey wolves and wolf-dog hybrids tested from Germany during 2006–2022 could PrV infection be detected by molecular means.

Conclusions

Although PrV has been eliminated from domestic pigs, spillover infections in domestic and wild carnivores should always be expected given the endemic presence of PrV in wild pig populations. Since detection of PrV DNA and virus in carnivores is sporadic even in areas with high seroprevalence of PrV in wild pigs, it may not reflect the full diversity of PrV.

Introduction

Pseudorabies virus (PrV), an enveloped double-stranded DNA virus and member of the genus Varicellovirus within the subfamily Alphaherpesvirinae of the Orthoherpesviridae family [1], is the causative agent of Aujeszky’s disease (AD), an infection of major economic impact in animal husbandry [2]. Although its taxonomic name Suid alphaherpesvirus 1 (SuAHV1) clearly indicates its natural association with swine, the virus exhibits a wide host range capable of infecting basically all mammals except higher primates and equines [2]. While only swine are able to survive a productive infection and are thus considered the only natural host, virus infections in other mammalian species, i.e. non-natural hosts, are fatal due to virus neuroinvasion and lethal inflammatory response [3, 4]. In general, such spillovers represent dead-end infections as the non-reservoir hosts are not able to independently maintain infection because of their rapid fatal outcome [5, 6].

AD has been eradicated from populations of domestic swine through use of culling and/or vaccination programs in many countries in Europe, in North America, Australia and New Zealand [2, 7,8,9] but still remains a serious problem in other parts of the world. However, PrV continues to circulate among free-roaming or farmed wild boar and feral swine, which can act as a reservoir for the virus [10,11,12,13]. PrV infections in populations of wild boar have been confirmed for several European countries [11, 13] but also a few countries in Northern Africa [14], and Asia [15,16,17,18]. PrV is also present in populations of feral swine in the United States [12] and Brazil [19]. There is evidence that PrV isolates of wild boar and feral swine origin in Europe and the US do not represent a homogenous population but rather represent several different genetic lineages [7, 20]. The geographical distribution of PrV in wild swine is rather patchy including both large-scale transboundary but also small cluster occurrence. Depending on the region, PrV seroprevalences in wild boar and feral swine can amount to 50% or even higher [11,12,13].

Because subclinical infections and nonspecific clinical signs are common in swine, the high susceptibility of carnivores renders them inadvertent sentinels for virus occurrence. Often detection of PrV in dogs (Canis lupus familaris) may be the first indication that the virus is present in a swine herd or a local wild swine population [21]. Dogs that live on pig farms may become infected after direct or indirect contact with infected swine, while hunting dogs are especially prone to infection by direct exposure to feral swine during hunting events. Consumption of uncooked offals from infected (wild) swine also plays a role as a source of infection [22,23,24]. Once infected, dogs die 6 to 96 h after the onset of clinical neurological signs [25].

Although PrV spillover infections are observed most commonly in farm dogs, in recent years cases of PrV in hunting dogs after direct contact with wild boars have been repeatedly reported from Europe [20, 26,27,28,29,30,31,32,33], the United States [34,35,36] and China [37]. PrV infections have also been reported in wildlife [38] including farmed and free-living foxes (Vulpes vulpes) [39,40,41,42], endangered carnivores such as the Florida panther (Puma concolor couguar) [43], grey wolf (Canis lupus) [44,45,46], Iberian lynx (Lynx pardinus) [47], African wild dogs (Lycaon pictus) [48] and captive brown bears (Ursus arctos) [49] after consumption of PrV-contaminated meat.

In this study, we describe occurrence of PrV infections in domestic dogs and wild carnivores in Germany during the past three decades, with a subsequent effort to genetically characterize isolates based on partial sequence analyses. Furthermore, we wanted to elucidate whether PrV infections in wild boar pose a threat to the rising, highly protected population of grey wolves in Germany.

Materials and methods

Epidemiological information and sampling

Because AD is a notifiable infectious disease in Germany [9], any suspect cases in animals have to be submitted to regional veterinary laboratories for laboratory diagnosis. The number of laboratories confirmed PrV cases in carnivores for the period 1995–2022 was obtained from the electronic Animal Disease Reporting System (TSN) of the competent veterinary authorities of the districts and federal states and the Federal Ministry of Food and Agriculture [50]. PrV isolates or viral DNA of PrV PCR-positive dogs and wild carnivores were submitted by regional veterinary laboratories to the national reference laboratory (NRL) for AD at the FLI for confirmation and further molecular characterization.

As part of a research project of the Leibnitz Institute for Zoo and Wildlife Research (IZW), Berlin, on the causes of mortality in grey wolves, brain samples from wolves and wolf-dog hybrids in Germany from 2006 to 2022 were submitted to the NRL at the Friedrich-Loeffler-Institut (FLI) and tested for PrV. Master data for wild carnivore samples such as date and geographical origin (Gauss-Krueger coordinates) were obtained from TSN or the IZW and DBBW (federal documentation and advisory centre on wolves) database (https://www.dbb-wolf.de/). Georeferencing of data and map visualization was done using the ArcGIS 10.8 package.

PRV diagnostics

Standard virological techniques were used to detect PrV infection [51]. Viral DNA in brain samples and/or cell culture supernatant was detected following DNA extraction using the MagNA Pure LC Total Nucleic Acid Isolation Kit for automated extraction (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) or DNA-MiniKit (Qiagen, Hilden, Germany) by both conventional PCR [52, 53] and triplex real time PCR either using the gB- and gE-gene (variant 1) specific or the UL19 (major capsid protein gene)- and gE-gene (variant 2) specific assay including respective internal control DNA (IC2) essentially as described [54]. Isolation of PrV from PCR positive brain tissues was conducted in cell cultures. To this end, brain samples were homogenized using a TissueLyser (Qiagen, Hilden, Germany), resuspended in Dulbecco’s minimum essential medium (DMEM), zentrifuged at 1000 g and the supernatant incubated at 37 °C on different cell lines. Cell lines generally used for PrV isolation at regional veterinary laboratories included rabbit kidney (RK-13), porcine kidney (PK-15), Madin–Darby bovine kidney (MDBK), bovine oesophagus (KOP), embryonic porcine kidney epithelial (SPEV) or primate Vero cells as established. Cells were observed for a minimum of seven days and passaged three times to confirm a negative result [55].

When appropriate, PrV in brain tissue was detected by immunohistochemistry (IHC) using a polyclonal PrV rabbit hyper immune serum (Vemie, Kempen, Germany) or an in-house polyclonal rabbit antibody against PrV gB [56]. Positive antigen detection was visualized via avidin-biotin-peroxidase-complex (ABC; Vector Laboratories, Burlingame, USA) with 3,3´-Diaminobenzidintetrahydrochlorid (DAB) or 3-amino-9-ethylcarbazole (AEC) as chromogen, respectively [56, 57]. Tissue sections were further stained with hematoxylin-eosin (HE) to evaluate PrV-induced lesions.

Sequencing and alignment

For the characterisation of genetic traits and for phylogenetic analysis, archived PrV isolates from carnivores and domestic pigs from Germany before the elimination of AD in 2003 were also included.

Sequencing PrV isolates or DNA was done essentially as described [20]. Briefly, DNA was prepared using commercial DNA extraction kits (Qiagen, Germany) from cell culture or brain tissue. After amplification of a 732 bp fragment comprising parts of the gC open reading frame using using pfx DNA polymerase (Invitrogen, Germany). PCR products were separated on 1% agarose gels, purified using the Genomic DNA Purification Kit (Thermo Fisher Scientific, Germany) and sequenced on both strands using the Big Dye R terminator cycle sequencing kit (Applied Biosystems, Darmstadt, Germany) with the primers used for amplification. Sequences were aligned and their evolutionary history was inferred using the Neighbor-Joining method [58] as implemented in MEGA X [59]. Nucleotide sequences generated from hunting dogs and red foxes in this study were submitted to GenBank. Accession numbers are shown in Table 1. For comparison, additional PrV sequences of domestic pigs and wild boars from previous studies were included in the phylogenetic analysis.

Table 1 Details to PrV isolates used for sequencing and subsequent phylogenetic analysis
Full size table

Results

PrV cases in carnivores

Between 1995 and 2022, PrV infections were reported in a total of 35 dogs and three foxes in Germany (Fig. 1A), with higher incidences in December and March (Fig. 2A). While the affected dogs showed an acute course of disease associated with severe neurological signs including pruritus and self-mutilation, PrV in foxes was detected during routine rabies surveillance as a differential diagnosis. Foxes originated from the federal states of Saarland, Schleswig-Holstein and Brandenburg (Fig. 1A). Twenty-seven (77.1%) and 2 (5.7%) of the 35 PrV cases in dogs could be epidemiologically linked to direct contact with wildlife and consumption of uncooked offal, respectively, while for the remaining dogs the source of infection was unclear.

Fig. 1
figure 1

A: Distribution of reported PrV-infections in dogs (dots) and foxes (rectangles) in Germany between 1995 and 2022. B: Locations of grey wolves and lynxes submitted for PrV diagnosis between 2006 and 2022. The borders of the federal states are indicated. Areas where PrV is endemic in wild boar are coloured beige and are based on a recent publication [57]

Full size image

In the frame of the research project on causes of mortality in grey wolves in Germany, during the period 2006–2022 at total of 682 grey wolves and wolf-dog hybrids, 31 lynxes (Lynx lynx) (Figs. 1B and 2B), 24 red foxes (Vulpes vulpes), three golden jackals (Canis aureus), 15 stone and pine martens (Martes foina, martes), four European polecats (Mustela putorius), four raccoons (Procyon lotor), and three badgers (Meles meles) were tested. In none of the animals could PrV be detected.

Fig. 2
figure 2

A: Monthly distribution of PrV cases in dogs for the time period 1995–2022. B: Number of wolfes investigated between 2006 and 2022

Full size image

Clinical and pathomorphological findings

Two of 35 PrV positive dogs were investigated clinically and pathomorphologically. Dog 1 presented with central nervous signs including tremor, paralysis and pruritus, hypothermia and hypersalivation. Clinical and pathological investigation revealed delayed coagulation, hemolysis, hematuria and electrolyte imbalances. The dog died shortly (4 days) after onset of clinical signs. On necropsy, the dog showed hemoperitoneum, hemothorax, black-coloured stomach content, gastric mucosal hemorrhages, melaena as well as hemorrhages of the mediastinum and mesenterium. Histopathologically, moderate mixed-cellular encephalitis with vasculitis and perivasculitis, gliosis, scattered neuronal necrosis and extensive hemorrhages was found in the brainstem. Mononuclear infiltrates, neuronal necrosis and hemorrhages were also detected in the trigeminal ganglion. Immunopositive neurons and glial cells were detected within and adjacent to the affected areas (Fig. 3A). Dog 2 presented with pruritus, licking, central nervous signs, hypersalivation and somnolence leading to euthanasia. Gross pathology revealed reddened skin with dermatitis of a paw and pulmonary alveolar edema. Histopathologically, inflammation was limited to the trigeminal ganglion which showed mild mixed-cellular infiltration and occasional neuronal necrosis. Positive signals for viral antigen were only rarely detected in the trigeminal ganglion, but were more frequently found in brainstem neurons (Fig. 3B).

Fig. 3
figure 3

Histopathology of PrV-infected dogs (A and B) and a red fox (C). (A) Brainstem encephalitis with vasculitis, perivasculitis, hemorrhage and scattered PrV antigen positive neurons (inset), HE and anti-PrV gB immunohistochemistry [53]. (B) Trigeminal ganglionitis with an immunopositive neuron (inset), HE and anti-PrV gB immunohistochemistry. (C) Brain showing a PrV infected neuron in a red fox (GER 642), immunohistochemistry [54].

Full size image

One of the three PrV positive foxes from Brandenburg was found dead by a hunter in November 2017 and submitted for testing on notifiable and reportable infections including rabies, canine distemper and PrV. At necropsy, no lesions except for a few wild boar bristles in the stomach were detected suggesting acute death due to circulatory breakdown. Bacteriological investigations as well as virological and parasitological screening for rabies, canine distemper, canine adenovirus (CadV-1, CadV-2), Toxoplasma gondii, and leptospirosis yielded negative results, while the fox was infested with Toxocara canis and Echinococcus multilocularis. PrV infection was confirmed by realtime PCR and virus isolation. While histological investigations revealed no inflammatory reactions, very few PrV infected neurons were detected by subsequent IHC staining (Fig. 3C).

The PrV positive red fox from Saarland was found in January 2019 in an agonal state showing extreme pruritus, and was humanely euthanized in a local veterinary clinic. While rabies could be excluded by routine diagnostics, PrV was confirmed by standard virological methods. For the remaining PrV positive fox from Schleswig-Holstein no further information on the history and possible source of infection was available.

Phylogenetic analysis

Sequence analysis of PrV isolates from Germany identified different variants according to a limited number of sequence variations, i.e. SNPs and deletions and insertions (Fig. 4).

Fig. 4
figure 4

Symbolized alignment of the partial gC-sequence from German PrV-isolates, with identical nucleotides displayed as gray bar and SNPs and indels indicated (black)

Full size image

Phylogenetic analyses revealed that AD in carnivores in Germany was caused by different PrV strains. Carnivore PrV isolates originating from prior to the elimination of AD from domestic pig populations in Germany group together with PrV from pigs (Fig. 5). More recent PrV isolates from carnivores largely clustered closely with the prevailing PrV strains found in European wild boars. A number of PrV from dogs and one fox from Saarland are within the previously assigned Clade “B” comprising of isolates from Germany (North-Rhine Westphalia, Rhineland-Palatinate), France and Spain. The only exception is a hunting dog from Baden-Württemberg (GER 614), which has an identical sequence to a PrV from a domestic pig from Croatia (GenBank: KC865672). PrV from dogs from the eastern German federal states are identical to the identified variant from the European wild boar, which was designated as part of lineage “A”. Interestingly, a fox isolate from Brandenburg (GER 642) clusters with a domestic pig PrV isolate from Belgium. The sequence of this isolate shares two indels with a hunting dog from Baden-Wurttemberg (GER 614), but also incorporates unique SNPs (Fig. 4).

Fig. 5
figure 5

Unrooted Neighbour-Joining tree based on (gaps removed) from the PrV-gC coding region. Sequences are identified by: GenBank accession number/strain/country/species/year of isolation (if known). German PrV strains from carnivores are in highlighted (bold). Numbers along the branches represent percentages of 500 bootstrap iterations with values over 50% shown.

Full size image

Discussion

Although AD has been eliminated from the domestic pig population in Germany [9], PrV infections are enzootic in European wild boar populations throughout the country [60,61,62,63] causing an estimated overall PrV seroprevalence of 12.09% (Fig. 1) [62]. Our study confirms similar studies from other European countries [20, 26,27,28,29,30,31,32, 64] with a sporadic number of reported PrV infections in domestic carnivores (Figs. 1A and 2 A). Occasionally, PrV from wild boars caused cases in dogs, mainly hunting dogs, during the past decades as reported in the Animal Disease Reporting System (TSN), with a temporal link to hunting activities. This association was confirmed by partial sequence analyses using the gC-gene (Figs. 4 and 5) that had been used to characterize wild boar derived PrV isolates from Europe [20]. 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 [65], eventually leading to an infection in hunting dogs when they actively encounter wild boar during hunting activities [11]. 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 [66]. 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 [46].

Against this background it is interesting that despite other reports none of the 682 investigated free-ranging wolves and hybrids submitted between 2006 and 2022 tested positive for PrV (Fig. 2B), despite the overlap of their distribution in Germany in areas with high PrV seroprevalences in wild boars, high wild boar densities [67, 68] and their natural behavior to prey on wild boars [69, 70] (Fig. 1B). Certainly, one limitation is that testing on PrV only relied on brain tissues but did not include peripheral nerve system (PNS) samples, e.g. trigeminal ganglia (TG) and dorsal root ganglia (DRG). Based on the pathogenesis of PrV-induced neuropathic itch, PrV remains in the PNS and the infection will cause a systemic inflammatory response that will lead to the death of non-natural hosts. Hence, brain samples will not be systematically positive for PrV [4, 38].

Studies of scat samples from Europe, on the other hand, indicate a certain flexibility of the wolf as a predator depending of the availability of prey, which significantly increases the chances of becoming infected with PrV, as is the case with hunting dogs. While in northern plain lands of Europe wolves seem to rather avoid wild boar as prey [71], in the Mediterranean basin wild boars are obviously sometimes part of the prey [69, 70]. 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 [72]. Data from Germany show that about 20% of total prey biomass of wolves consists of wild boar [73]. According to official statistics, the main causes of death of grey wolves found dead between 2006 and 2022 are traffic accidents (74.8%), followed by illegal killing (9.1%), natural death (8.8%) and management (authorised removal of animals after incidents, 1.2%). In 5.5% and 1.2% of the cases, the cause of death is unclear and still under investigation, respectively (https://www.dbb-wolf.de/totfunde/statistik-der-todesursachen). 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 of PrV 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 hunted foxes, no PrV infections were detected [74, 75]. Since red foxes are known to feed on carcasses as well as to prey on European wild boar piglets [76], the oral route of infection is 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” [20], isolate GER 642 from eastern Brandenburg clusters with a dog from Baden-Württemberg 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 [29]. This is not only based on low bootstrap support, as indicated before [29], 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 [77].

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 [29]. 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 red foxes. Also, since Germany is free of AD [9], 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 [40, 41] may need reconsideration.

Conclusion

With their prominent clinical picture, PrV infections in carnivores are likely to be discovered and virus can be characterized, as seen in this study and others [27, 29, 31]. In contrast, even in areas with high seroprevalence of PrV in wild boar, the detection of PrV DNA and virus in carnivores is sporadic and may not disclose the full diversity of circulating PrV. Therefore, both brain and PNS samples from carnivores with suspected encephalitis should be thoroughly investigated [4], to exclude infection with rabies virus (RABV) but also to analyze for PrV, even though the infection is only regulated in domestic pigs in the EU (EU regulation (EU) 2018/1882).

Data Availability

All data generated or analysed during this study are included in this published article [and its supplementary information files].

References

  1. Davison AJ. Herpesvirus systematics. Vet. Microbiol. 2010;143:52–69. https://doi.org/10.1016/j.vetmic.2010.02.014

    Article  CAS  Google Scholar 

  2. Mettenleiter TC, Ehlers B, Müller T, Yoon K, Teifke JP. Herpesviruses. In: Zimmerman, J., Karriker, L. A., Ramirez, A., Schwartz, K. J., Stevenson. G. W., editor. Diseases of Swine: John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.; 2012. p. 421–446.

  3. Mettenleiter TC. Aujeszky’s disease (pseudorabies) virus: the virus and molecular pathogenesis - state of the art, June 1999. Vet Res. 2000;31:99–115.

    CAS  PubMed  Google Scholar 

  4. Laval K, Enquist LW. The Neuropathic Itch Caused by Pseudorabies Virus. Pathogens 2020. https://doi.org/10.3390/pathogens9040254

  5. United States Department of Agriculture, Animal and Plant Health Inspection Service. Pseudorabies (Aujeszky’s Disease) and its eradication. A review of the U.S. experience. Technical Bulletin No. 1923; 2008.

  6. Pensaert MB, Kluge P. Pseudorabies virus (Aujeszky’s disease). In: Pensaert M, editor. Virus infections of porcines. New York: Elsevier Science Publishers; 1989. pp. 39–64.

    Google Scholar 

  7. Hahn EC, Fadl-Alla B, Lichtensteiger CA. Variation of Aujeszky’s disease viruses in wild swine in USA. Vet Microbiol. 2010;143:45–51. https://doi.org/10.1016/j.vetmic.2010.02.013

    Article  PubMed  Google Scholar 

  8. MacDiarmid SC. Aujeszky’s disease eradication in New Zealand. Aust Vet J. 2000;78:470–1. https://doi.org/10.1111/j.1751-0813.2000.tb11862.x

    Article  CAS  PubMed  Google Scholar 

  9. Müller T, Bätza HJ, Schlüter H, Conraths FJ, Mettenleiter TC. Eradication of Aujeszky’s disease in Germany. J Vet Med B Infect Dis Vet Public Health. 2003;50:207–13. https://doi.org/10.1046/j.1439-0450.2003.00666.x

    Article  PubMed  Google Scholar 

  10. Müller T, Conraths FJ, Hahn EC. Pseudorabies virus infection (Aujeszky’s disease) in wild swine. Infect Dis Rev. 2000;2:27–34.

    Google Scholar 

  11. Müller T, Hahn EC, Tottewitz F, Kramer M, Klupp BG, Mettenleiter TC, Freuling C. Pseudorabies virus in wild swine: a global perspective. Arch Virol. 2011;156:1691–705. https://doi.org/10.1007/s00705-011-1080-2

    Article  CAS  PubMed  Google Scholar 

  12. Pedersen K, Bevins SN, Baroch JA, Cumbee JC Jr, Chandler SC, Woodruff BS, et al. Pseudorabies in feral swine in the United States, 2009–2012. J Wildl Dis. 2013;49:709–13. https://doi.org/10.7589/2012-12-314

    Article  PubMed  Google Scholar 

  13. Meier RK, Ruiz-Fons F, Ryser-Degiorgis MP. A picture of trends in Aujeszky’s disease virus exposure in wild boar in the swiss and european contexts. BMC Vet Res. 2015;11:277. https://doi.org/10.1186/s12917-015-0592-5

    Article  PubMed  PubMed Central  Google Scholar 

  14. Jridi M, Bouzghaia H, Toma B. Aujeszky’s disease in wild boar in Tunisia. Epidemiol Sante Anim. 1996;30:99–105.

    Google Scholar 

  15. Kim J, Lim SI, Han GH, Song JY, Lee KW, Hyan BH. Serological survey of aujeszky’s disease in South Korean wild boar (Sus scrofa) population. In: 22nd International Pig Veterinary Society; 2012. p. 463.

  16. Albayrak H, Ozan E, Cavunt A. A serological survey of selected pathogens in wild boar (Sus scrofa) in northern Turkey. Eur J Wildlife Res. 2013;59:893–7. https://doi.org/10.1007/s10344-013-0743-6

    Article  Google Scholar 

  17. Ishiguro N, Nishimura M. Genetic profile and serosurvey for virus infections of japanese wild boars in Shikoku Island. J Vet Med Sci. 2005;67:563–8. https://doi.org/10.1292/jvms.67.563

    Article  CAS  PubMed  Google Scholar 

  18. Mahmoud HY, Suzuki K, Tsuji T, Yokoyama M, Shimojima M, Maeda K. Pseudorabies virus infection in wild boars in Japan. J Vet Med Sci. 2011;73:1535–7. https://doi.org/10.1292/jvms.11-0191

    Article  PubMed  Google Scholar 

  19. Paes Rde C, Fonseca AA Jr, Monteiro LA, Jardim GC, Piovezan U, Herrera HM, et al. Serological and molecular investigation of the prevalence of Aujeszky’s disease in feral swine (Sus scrofa) in the subregions of the Pantanal wetland. Brazil Vet Microbiol. 2013;165:448–54. https://doi.org/10.1016/j.vetmic.2013.03.028

    Article  PubMed  Google Scholar 

  20. Müller T, Klupp BG, Freuling C, Hoffmann B, Mojcicz M, Capua I, et al. Characterization of pseudorabies virus of wild boar origin from Europe. Epidemiol Infect. 2010;138:1590–600. https://doi.org/10.1017/S0950268810000361

    Article  PubMed  Google Scholar 

  21. Sykes JE, editor. Canine and Feline Infectious Diseases. 1st ed. St. Louis: Elsevier; 2013.

    Google Scholar 

  22. Acevedo P, Escudero MA, Munoz R, Gortazar C. Factors affecting wild boar abundance across an environmental gradient in Spain. Acta Theriol. 2006;51:327–36.

    Article  Google Scholar 

  23. Gore R, Osborne A, Darke P, Todd J. Aujeszky’s disease in a pack of hounds. Vet Rec. 1977;101:93–5. https://doi.org/10.1136/vr.101.5.93

    Article  CAS  PubMed  Google Scholar 

  24. Hugoson G, Rockborn G. On the occurrence of pseudorabies in Sweden. II. An outbreak in dogs caused by feeding abattoir offal. Zentralblatt Veterinarmedizin Reihe B. 1972;19:641–5. https://doi.org/10.1111/j.1439-0450.1972.tb00445.x

    Article  CAS  Google Scholar 

  25. Monroe WE. Clinical signs associated with pseudorabies in dogs. J Am Vet Med Assoc. 1989;195:599–602.

    CAS  PubMed  Google Scholar 

  26. Cay AB, Letteliert C. Isolation of Aujeszky’s disease virus from two hunting dogs in Belgium after hunting wild boars. Vlaams Diergeneeskd Tijdsch. 2009;78:194–5.

    Google Scholar 

  27. Moreno A, Sozzi E, Grilli G, Gibelli LR, Gelmetti D, Lelli D, et al. Detection and molecular analysis of Pseudorabies virus strains isolated from dogs and a wild boar in Italy. Vet Microbiol. 2015;177:359–65. https://doi.org/10.1016/j.vetmic.2015.04.001

    Article  CAS  PubMed  Google Scholar 

  28. Keros T, Jemeršić L, Brnić D, Prpić J, Dežđek D. Pseudorabies in hunting dogs in Croatia with phylogenetic analysis of detected strains. Vet Rec Case Rep. 2015;3.

  29. Steinrigl A, Revilla-Fernandez S, Kolodziejek J, Wodak E, Bago Z, Nowotny N, et al. Detection and molecular characterization of Suid herpesvirus type 1 in austrian wild boar and hunting dogs. Vet Microbiol. 2012;157:276–84. https://doi.org/10.1016/j.vetmic.2011.12.033

    Article  CAS  PubMed  Google Scholar 

  30. Schoniger S, Klose K, Werner H, Schwarz BA, Muller T, Schoon HA. Nonsuppurative encephalitis in a dog. Vet Pathol. 2012;49:731–4. https://doi.org/10.1177/0300985811432349

    Article  CAS  PubMed  Google Scholar 

  31. Deblanc C, Oger A, Simon G, Le Potier M-F. Genetic Diversity among Pseudorabies Viruses Isolated from Dogs in France from 2006 to 2018. Pathogens 2019. https://doi.org/10.3390/pathogens8040266

  32. Engelhardt S, Schneider S, Buder A, Aupperle-Lellbach H, Pfeil I. Magnetresonanztomographie des Gehirns bei einem Hund mit nachgewiesenem Morbus Aujeszky. [MRI in a dog with confirmed pseudorabies infection]. Tierarztl Prax Ausg K Kleintiere Heimtiere. 2019;47:272–81. https://doi.org/10.1055/a-0948-8760

    Article  PubMed  Google Scholar 

  33. Černe D, Hostnik P, Toplak I, Juntes P, Paller T, Kuhar U, Ren L-Z. Detection of Pseudorabies in Dogs in Slovenia between 2006 and 2020: from clinical and diagnostic features to Molecular Epidemiology. Transbound Emerg Dis. 2023;2023:4497806. https://doi.org/10.1155/2023/4497806

    Article  Google Scholar 

  34. Pedersen K, Turnage CT, Gaston WD, Arruda P, Alls SA, Gidlewski T. Pseudorabies detected in hunting dogs in Alabama and Arkansas after close contact with feral swine (Sus scrofa). BMC Vet Res. 2018;14:388. https://doi.org/10.1186/s12917-018-1718-3

    Article  PubMed  PubMed Central  Google Scholar 

  35. Cramer SD, Campbell GA, Njaa BL, Morgan SE, Smith SK 2, et al. Pseudorabies virus infection in Oklahoma hunting dogs. J Vet Diagn Invest. 2011;23:915–23. https://doi.org/10.1177/1040638711416628

    Article  PubMed  Google Scholar 

  36. Buergelt C, Romero C, Chrisman C. Pseudorabies in two dogs. Vet Med. 2000;95:439–42.

    Google Scholar 

  37. Minamiguchi K, Kojima S, Sakumoto K, Kirisawa R. Isolation and molecular characterization of a variant of chinese gC-genotype II pseudorabies virus from a hunting dog infected by biting a wild boar in Japan and its pathogenicity in a mouse model. Virus Genes. 2019;55:322–31. https://doi.org/10.1007/s11262-019-01659-x

    Article  CAS  PubMed  Google Scholar 

  38. Sehl J, Teifke JP. Comparative Pathology of Pseudorabies in Different Naturally and Experimentally Infected Species-A Review. Pathogens 2020. https://doi.org/10.3390/pathogens9080633

  39. Bitsch V, Munch B. On pseudorabies in carnivores in Denmark. I: the red fox (Vulpes vulpes). Acta Vet Scand. 1971;12:274–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Moreno A, Chiapponi C, Sozzi E, Morelli A, Silenzi V, Gobbi M, et al. Detection of a gE-deleted Pseudorabies virus strain in an italian red fox. Vet Microbiol. 2020;244:108666. https://doi.org/10.1016/j.vetmic.2020.108666

    Article  CAS  PubMed  Google Scholar 

  41. Caruso C, Dondo A, Cerutti F, Masoero L, Rosamilia A, Zoppi S, et al. Aujeszky’s disease in red fox (Vulpes vulpes): phylogenetic analysis unravels an unexpected epidemiologic link. J Wildl Dis. 2014;50:707–10. https://doi.org/10.7589/2013-11-312

    Article  PubMed  Google Scholar 

  42. Jin HL, Gao SM, Liu Y, Zhang SF, Hu RL. Pseudorabies in farmed foxes fed pig offal in Shandong province. China Arch Virol. 2016;161:445–8. https://doi.org/10.1007/s00705-015-2659-9

    Article  CAS  PubMed  Google Scholar 

  43. Glass CM, McLean RG, Katz JB, Maehr DS, Cropp CB, Kirk LJ, et al. Isolation of pseudorabies (Aujeszky’s disease) virus from a Florida panther. J Wildl Dis. 1994;30:180–4.

    Article  CAS  PubMed  Google Scholar 

  44. Lian K, Zhang M, Zhou L, Song Y, Wang G, Wang S. First report of a pseudorabies-virus-infected wolf (Canis lupus) in China. Arch Virol. 2020;165:459–62. https://doi.org/10.1007/s00705-019-04502-8

    Article  CAS  PubMed  Google Scholar 

  45. Amoroso MG, Di Concilio D, D’Alessio N, Veneziano V, Galiero G, Fusco G. Canine parvovirus and pseudorabies virus coinfection as a cause of death in a wolf (Canis lupus) from southern Italy. Vet Med Sci. 2020. https://doi.org/10.1002/vms3.270

    Article  PubMed  PubMed Central  Google Scholar 

  46. Verpoest S, Cay AB, Bertrand O, Saulmont M, de Regge N. Isolation and characterization of pseudorabies virus from a wolf (Canis lupus) from Belgium. Eur J Wildlife Res. 2014;60:149–53. https://doi.org/10.1007/s10344-013-0774-z

    Article  Google Scholar 

  47. Masot AJ, Gil M, Risco D, Jiménez OM, Núñez JI, Redondo E. Pseudorabies virus infection (Aujeszky’s disease) in an Iberian lynx (Lynx pardinus) in Spain: a case report. BMC Vet Res. 2017;13:6. https://doi.org/10.1186/s12917-016-0938-7

    Article  PubMed  PubMed Central  Google Scholar 

  48. Haddane B, Essalhi A. An outbreak of Aujeszky’s disease in the hunting dog (Lycaon Pictus). In: European Association of Zoo-and Wildlife Veterinarians (EAZWV), Second Scientific Meeting, May 21–24, 1998.

  49. Zanin E, Capua I, Casaccia C, Zuin A, Moresco A. Isolation and characterization of Aujeszky’s disease virus in captive brown bears from Italy. J Wildl Dis. 1997;33:632–4.

    Article  CAS  PubMed  Google Scholar 

  50. Probst C, Klöss D, Schröder R, Micklich A, Conraths FJ. The new animal disease notification and crisis management system in Germany. Tierärztl Umschau. 2010:311–9.

  51. OIE. Chapter 3.1.2. Aujeszky’s disease (infection with Aujeszky’s disease virus) (NB: Version adopted in May 2018). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2019:312–37.

  52. Mengeling WL, Lager KM, Volz DM, Brockmeier SL. Effect of various vaccination procedures on shedding, latency, and reactivation of attenuated and virulent pseudorabies virus in swine. Am J Vet Res. 1992;53:2164–73.

    CAS  PubMed  Google Scholar 

  53. Schang L, Osorio FA. A quantative technique for the studies of the latency of Aujeszky virus. Rev Sci Tech. 1993;12:505–21.

    Article  CAS  PubMed  Google Scholar 

  54. Wernike K, Beer M, Freuling CM, Klupp B, Mettenleiter TC, Muller T, Hoffmann B. Molecular double-check strategy for the identification and characterization of Suid herpesvirus 1. J Virol Methods. 2014;209:110–5. https://doi.org/10.1016/j.jviromet.2014.08.022

    Article  CAS  PubMed  Google Scholar 

  55. Müller T, Klupp B, Zellmer R, Teuffert J, Ziedler K, Possardt C, et al. Characterisation of pseudorabies virus isolated from wild boar (Sus scrofa). Vet Rec. 1998;143:337–40. https://doi.org/10.1136/vr.143.12.337

    Article  PubMed  Google Scholar 

  56. Sehl J, Hölper JE, Klupp BG, Baumbach C, Teifke JP, Mettenleiter TC. An improved animal model for herpesvirus encephalitis in humans. PLoS Pathog. 2020;16:e1008445. https://doi.org/10.1371/journal.ppat.1008445

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Grieco V, Gelmetti D, Finazzi G, Brocchi E, Finazzi M. Immunohistologic diagnosis of pseudorabies (Aujeszky’s disease) using monoclonal antibodies. J Vet Diagn Invest. 1997;9:326–8.

    Article  CAS  PubMed  Google Scholar 

  58. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406–25. https://doi.org/10.1093/oxfordjournals.molbev.a040454

    Article  CAS  PubMed  Google Scholar 

  59. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018;35:1547–9. https://doi.org/10.1093/molbev/msy096

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Müller T, Teuffert J, Ziedler K, Possardt C, Kramer M, Staubach C, Conraths FJ. Pseudorabies in the european wild boar from eastern Germany. J Wildl Dis. 1998;34:251–8. https://doi.org/10.7589/0090-3558-34.2.251

    Article  PubMed  Google Scholar 

  61. Pannwitz G, Freuling C, Denzin N, Schaarschmidt U, Nieper H, Hlinak A, et al. A long-term serological survey on Aujeszky’s disease virus infections in wild boar in East Germany. Epidemiol Infect. 2012;140:348–58. https://doi.org/10.1017/S0950268811000033

    Article  CAS  PubMed  Google Scholar 

  62. Denzin N, Conraths FJ, Mettenleiter TC, Freuling CM, Müller T. Monitoring of Pseudorabies in Wild Boar of Germany-A Spatiotemporal Analysis. Pathogens. 2020. https://doi.org/10.3390/pathogens9040276

    Article  PubMed  PubMed Central  Google Scholar 

  63. Lutz W, Junghans D, Schmitz D, Müller T. A long-term survey of pseudorabies virus infections in european wild boar of western Germany. Z fur Jagdwissenschaft. 2003;49:130–40.

    Google Scholar 

  64. Čonková-Skybová G, Ondrejková A, Mojžišová J, Bárdová K, Reichel P, Korytár Ľ, et al. Herpesvirus diseases of domestic animals and game species in the Slovak Republic. Acta Virol. 2020;64:409–16. https://doi.org/10.4149/av_2020_404

    Article  PubMed  Google Scholar 

  65. Müller T, Teuffert J, Zellmer R, Conraths FJ. Experimental infection of european wild boars and domestic pigs with pseudorabies viruses with differing virulence. Am J Vet Res. 2001;62:252–8. https://doi.org/10.2460/ajvr.2001.62.252

    Article  PubMed  Google Scholar 

  66. Hernandez FA, Sayler KA, Bounds C, Milleson MP, Carr AN, Wisely SM. Evidence of Pseudorabies Virus Shedding in Feral Swine (Sus Scrofa) populations of Florida, USA. J Wildl Dis. 2018;54:45–53. https://doi.org/10.7589/2017-04-071

    Article  CAS  PubMed  Google Scholar 

  67. Massei G, Kindberg J, Licoppe A, Gacic D, Sprem N, Kamler J, et al. Wild boar populations up, numbers of hunters down?: a review of trends and implications for Europe. Pest Manag Sci. 2015;71:492–500. https://doi.org/10.1002/ps.3965

    Article  CAS  PubMed  Google Scholar 

  68. Pittiglio C, Khomenko S, Beltran-Alcrudo D. Wild boar mapping using population-density statistics: from polygons to high resolution raster maps. PLoS ONE. 2018;13:e0193295. https://doi.org/10.1371/journal.pone.0193295

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Meriggi A, Brangi A, Matteucci C, Sacchi O. The feeding habits of wolves in relation to large prey availability in northern Italy. Ecography;1996:287–95.

  70. Mattioli L, Capitani C, Gazzola A, Scandura M, Apollonio M. Prey selection and dietary response by wolves in a high-density multi-species ungulate community. Eur J Wildlife Res;2011:909–22.

  71. Okarma H. The trophic ecology of wolves and their predatory role in ungulate communities of forest ecosystems in Europe. Acta Theriol. 1997:335–87.

  72. Davis ML, Stephens PA, Willis SG, Bassi E, Marcon A, Donaggio E, et al. Prey selection by an apex predator: the importance of sampling uncertainty. PLoS ONE. 2012;7:e47894. https://doi.org/10.1371/journal.pone.0047894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Holzapfel M, Kindervater J, Wagner C, Ansorge H. Nahrungsökologie des Wolfes in Sachsen von 2001 bis 2016; 2017.

  74. Bourg M, Nobach D, Herzog S, Lange-Herbst H, Nesseler A, Hamann H-P, et al. Screening red foxes (Vulpes vulpes) for possible viral causes of encephalitis. Virol J. 2016;13:151. https://doi.org/10.1186/s12985-016-0608-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Lempp C, Jungwirth N, Grilo ML, Reckendorf A, Ulrich A, van Neer A, et al. Pathological findings in the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides), with special emphasis on infectious and zoonotic agents in Northern Germany. PLoS ONE. 2017;12:e0175469. https://doi.org/10.1371/journal.pone.0175469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Lanszki J, Nagyapáti N, Kurys A. Long-term changes in the Diet of the Red Fox in an agricultural area. Mammal Study. 2019. https://doi.org/10.3106/ms2018-0048

    Article  Google Scholar 

  77. Hu R, Wang L, Liu Q, Hua L, Huang X, Zhang Y, et al. Whole-genome sequence analysis of Pseudorabies Virus Clinical isolates from Pigs in China between 2012 and 2017 in China. Viruses. 2021. https://doi.org/10.3390/v13071322

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors are grateful to the environmental ministries of the German federal states for providing wolf carcasses to the IZW. Zoltán Mezö's technical assistance in necropsy of grey wolves and other wildlife and in sample collection and preparation is greatly appreciated. The authors would also like to thank Jeannette Kliemt for her excellent technical support in performing diagnostic confirmatory tests, propagation of PrV isolates and subsequent PCR sequencing.

Funding

Open Access funding enabled and organized by Projekt DEAL. This research was funded by the German Federal Ministry of Food and Agriculture (BMEL). The BMEL did not play any role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Author information

Authors and Affiliations

  1. Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493, Greifswald- Insel Riems, Germany

    Conrad M. Freuling & Thomas Müller

  2. Berlin-Brandenburg State Laboratory, 15236, Frankfurt (Oder), Germany

    Andreas Hlinak & Christoph Schulze

  3. Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institut, 17493, Greifswald- Insel Riems, Germany

    Julia Sehl-Ewert

  4. Friedrich-Loeffler-Institut, Institute of Epidemiology, 17493, Greifswald- Insel Riems, Germany

    Patrick Wysocki

  5. IZW - Leibniz Institute for Zoo and Wildlife Research, 10315, Berlin, Germany

    Claudia A. Szentiks

  6. Landesamt für Verbraucherschutz Saarland, 66115, Saarbrücken, Germany

    Klaus Schmitt

  7. Department of Pathology, University of Veterinary Medicine Hannover, Foundation, 30559, Hannover, Germany

    Peter Wohlsein

  8. LUPUS – German Institute for Wolf Monitoring and Research, 02826, Görlitz, Germany

    Gesa Kluth & Ilka Reinhardt

  9. Friedrich-Loeffler-Institut, 17493, Greifswald- Insel Riems, Germany

    Thomas C. Mettenleiter

Authors
  1. Conrad M. Freuling
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas Hlinak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christoph Schulze
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Julia Sehl-Ewert
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Patrick Wysocki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Claudia A. Szentiks
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Klaus Schmitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peter Wohlsein
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gesa Kluth
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ilka Reinhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Thomas C. Mettenleiter
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Thomas Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

CMF, TM and TCM conceived the ideas, planned and designed the study and were major contributors in writing the manuscript and substantiall revised the work. AH, ChS and KS, were responsible for the acquisition of clinical data, virus isolates or tissue samples from PrV positive dogs and red foxes. CS, GK and IR were responsible for collecting, submitting of as well as obtaining master data on dead-found grey wolves in Germany. CS, JS and PW performed the macroscopic and histological examinations of PrV positive carnivores and interpreted related findings. PW was mainly responsible for data reconciliation, the spatio-temporal analysis and creation of maps. CMF and TM performed the sequencing and phylogenetic analysis of the PrV isolates. All authors read, subtantially revised and approved the final manuscript.

Corresponding author

Correspondence to Thomas Müller.

Ethics declarations

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Freuling, C.M., Hlinak, A., Schulze, C. et al. Suid alphaherpesvirus 1 of wild boar origin as a recent source of Aujeszky’s disease in carnivores in Germany. Virol J 20, 110 (2023). https://doi.org/10.1186/s12985-023-02074-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12985-023-02074-3

Keywords

Virology Journal

ISSN: 1743-422X