During December 2023–February 2025, we collected samples from 644 hunted predator game (group I) and 343 hoofed game animals (groups II and III) in the context of an ongoing disease surveillance project in the federal state of Mecklenburg–Western Pomerania in northeast Germany. In particular, the offshore islands and the mainland coast form important avian migratory areas but also breeding habitats with a high overall species diversity and abundance of wild birds. Many of those species have been affected during various HPAI H5 epizootics and the recent enzootic (7).

Predator game consisted of 5 species of the taxonomic families Canidae, Procyonidae, and Mustelidae (Table). Cervidae, Bovidae, and Suidae form groups II and III with five different species in total (1 sampled individual animal remains unspecified). We screened nasal swab and, for group I, lung and brain samples (n = 980) by using quantitative reverse transcription PCR for influenza A virus (IAV) RNA (Appendix Table 1). All 980 samples tested negative.

Nucleoprotein (NP)–based ELISA indicated no seropositivity against IAV in wild ruminants, whereas 5.2% (95% CI 1.9%–11.0%) of the wild boar (Sus scrofa) and all carnivorous species, except the single tested European pine marten (Martes martes), tested positive (Table) (Appendix). The subsequent ELISA for detection of H5-specific antibodies revealed 3.5% (95% CI 0.9%–8.7%) seropositive wild boar and 12.5% (95% CI 0.3%–52.7%) to 21.9% (95% CI 12.5%–34.0%) seropositive carnivorous species (Table; Appendix Table 1), thereby confirming most of the seropositivity caused by an IAV of subtype H5. The range of non–H5 IAV antibody-positive animals ranged from 4.7% (95% CI 0.9%–13.1%) in raccoons (Procyon lotor) and 4% (95% CI 2.4%–6.9%) in (red) foxes (Vulpes vulpes) to 1.7% (95% CI 0.2%–6.1%) in wild boar and 1.1% (95% CI 0.1%–3.9%) in raccoon dogs (Nyctereutes procyonoides). We also tested a subset of NP-antibody positive or negative samples through serum neutralization test to confirm the presence or absence of antibodies against subtype H5 (Appendix). We confirmed the ELISA findings in a representative subset of 8 IAV-positive but H5-negative carnivore samples and compared them with 4 H5-positive samples, whereas H5 seropositivity in wild boar samples remained below the detection limit of the serum neutralization test.

Wild ruminants represent mammalian species with rather unlikely contact possibilities to potentially infected wild birds and, as herbivores, no apparent interface in their diet. However, direct exposure cannot be ruled out, and low susceptibility might be another plausible explanation for our consistent seronegative findings. At the other end of the food chain, our serologic results suggest previous exposure of carnivores to subtype H5 viruses. Raccoons, foxes, and raccoon dogs represent the species with the highest proportion of seropositivity (Table). Despite similar host species tested, our results only partially confirm observations of previous studies, where mainly stone martens and foxes were found to be seropositive in about 1 third of the animals tested (6). That discrepancy indicates the relevance of factors other than host susceptibility and host occurrence.

Figure

Figure. Percentage of high pathogenicity avian influenza subtype H5 seropositive juvenile and adult red foxes (Vulpes vulpes), by municipality, on the Island Rügen, northeast Germany, December 2023–February 2025. White...

Univariable regression analysis (Figure) revealed that foxes from the Island Rügen collected close to the bay coast, close to the Baltic Sea, or close to watercourses had an increased risk for H5-specific antibodies (p<0.05) (Appendix Table 2, 3). Seropositivity increased with age; 11.6% (10/86) of the juvenile and 23.5% (43/183) of the adult foxes were positive for H5-specific antibodies. A final multivariable model (Appendix Table 4) selected by stepwise forward–backward selection, which also included age, revealed that, with increasing distance to watercourses (e.g., flowing waters, water source areas, streams, ditches, rivers, and canals), the risk for foxes being positive for H5-specific antibodies decreased (p<0.0001); however, with increasing distance to shrubland (e.g., hedges, bushes, field crops, groups of trees, rows of trees, avenues, dominant single trees, and groups of shrubs), the risk increased (p<0.005). In contrast, high proportions of shrubland in a 2.5-km buffer zone around the sampling site of foxes represented a protective factor (p<0.005), supporting the assumption of less exposure to HPAIV through water-associated hosts, including Anseriformes birds, for example, that are known to graze on open lands.

The possible existence of a similar correlation for omnivorous species might serve as explanation for the 3.5% (95% CI 0.9%–8.7%) seropositivity in wild boar. In addition to their scavenging or predatory behavior (8), wild boars have been described as nest robbers of waterfowl species in other wetland areas in Europe (9). All individual animals with H5-specific antibodies belonged to family groups roaming in water-associated zones (e.g., the Island Rügen and Fischland-Darß-Zingst) (Appendix Table 1), where a higher likelihood of interaction with waterfowl species can be expected.

After the previous HPAIV H5N8 epizootic during 2016–2017, a broad serologic investigation in wild boars was conducted in southern Germany (10). Low-level prevalence of H5N8 neutralizing antibodies (1.13%) were detected; a single positive case occurred close to a prior HPAI outbreak in waterfowl (10,11). In domestic pigs the overall susceptibility in experimental settings remained low despite a high infectious dose of a single clade 2.3.4.4b strain (12); however, studies using different genotypes indicate variability in susceptibility, particularly in the extent of seroconversion (13,14).

Although little is known about the persistence of AIV antibodies in wild mammals, serologic surveillance might provide a more complete picture of past exposure to potentially infected avian (reservoir) hosts than molecular testing alone. The discrepancy in NP- and H5-antibody detection leaves the possibility of other AIV subtypes undergoing initial seroconversion, so a combination of testing approaches seems advisable.

H5-specific antibodies in carnivores and wild Suidae species indicate ongoing spillover events, especially in wetland habitats potentially shared with main (reservoir) hosts (Anseriformes and Charadriiformes). The role of wild boars warrants further attention because Suidae species are considered mixing vessels for swine-, human-, and avian-derived IAVs (15). Every such infection in mammalian species provides opportunities for virus adaptations, so continuous surveillance is recommended. Consequently, continuous surveillance also should be considered for pet animals in hotspot areas (e.g., free-ranging cats or hunting dogs) as further potential bridges for transmitting infections from wildlife to private households.