Integrated Surveillance of Viral Pathogens in Wild Boars: Serological and Molecular Insight in Southern Italy

Simple Summary

Wild animals can carry viruses that may affect both people and farm animals, so it is important to monitor their health. Wild boars are especially relevant because they live in large numbers and often come into contact with domestic pigs in rural areas. This study examined wild boars from Southern Italy for Influenza A viruses (IAVs), Circovirus, Aujeszky’s disease virus (ADV) and Flaviviruses (FLVs), which can cause illness in pigs and other wild animals. The analyses showed that influenza viruses and Circovirus type 2 (PCV-2) were actively present in wild boars, while antibodies revealed past exposure to Influenza, FLVs, and ADV. These findings show that wild boars can act as sentinels, meaning they help signal which viruses are circulating in the environment. Understanding this circulation is valuable because it helps protect farm animals and reduces risks to human health. By keeping track of viruses in wildlife, society can be better prepared to prevent outbreaks and safeguard both agriculture and public health.

Abstract

The circulation of viruses of medical and veterinary importance is monitored to evaluate risks to both human and animal health. Among the species most commonly used in surveillance programs, the wild boar (Sus scrofa) plays a key role due to its high population density and its contacts with rural swine herds in the areas under investigation. In the present study, molecular (real-time PCR) and serological analyses were conducted on wild boar samples collected in the regions of Apulia, Basilicata, Campania, and Calabria. Specifically, molecular detection was applied to IAVs, FLVs, and Porcine PCV-2, while serological testing was used to assess exposure to IAVs, FLVs, and ADV. Serological results showed an 8.31% positivity for IAVs, whereas viral presence detected by PCR was 1.17%. No active infections were detected for WNV, with only a single seropositive sample (0.30%), while ADV showed a seroprevalence of 2.61%. PCV-2 was detected exclusively by molecular analysis in 66 out of 223 tested animals (29.6%). The overall objective of the study is to deepen the understanding of virus circulation in the wildlife of Southern Italy, using the wild boar as an additional sentinel species for epidemiological surveillance activities. The distinctive feature of the project lies in its multi-pathogen approach applied to a reference population distributed across a particularly wide geographical area, allowing for the simultaneous assessment of the circulation of different viruses over a large territory.

1. Introduction

Wild boar (Sus scrofa), belonging to the Suidae family, is characterized by a generalist and opportunistic behavior [1]. Owing to its marked ecological plasticity, the species is capable of adapting to a broad spectrum of habitats, tolerating substantial anthropogenic pressures and exploiting food resources of human-made origin [1,2]. The species’ high reproductive capacity, flexible diet, and the reduced impact of hunting have enabled an exponential increase in its population over recent decades [3,4]. Recent studies have documented a substantial expansion of the species across Europe, both numerically and geographically, with increasingly significant repercussions for agriculture, the environment, wildlife–vehicle collisions, and public health [4,5]. Wild boars are animals that move in groups of variable size [6]. Specifically, females live in groups with their offspring, while males are solitary, although in autumn and winter they tend to display gregarious behavior [7]. Social interactions in this species facilitate interspecies virus transmission and contribute to increasing viral circulation. Moreover, the growing interaction between wild boars, urban environments, and domestic animals further heightens health risks, making integrated management and surveillance strategies necessary [8]. Wild boar acts as a potential reservoir for numerous zoonotic pathogens, posing a threat to both livestock and human health [6]. Its scavenger behaviour, combined with a variable diet and frequent exposure to faecal material, makes it an ideal sentinel species for monitoring zoonotic diseases. Among the most feared zoonoses, those caused by IAVs represent a concrete threat, considering their demonstrated enzootic circulation in Italy [9,10] and the ability of these viruses to cross species barriers [9,10,11]. Suids, in particular, play a crucial role as a “mixing vessel” host species, as co-infection with avian, swine and human influenza strains can promote genetic reassortment and the emergence of new viral variants with unknown antigenic and pathogenic characteristics [12,13]. In this context, virological monitoring of wild suid populations, such as wild boars, represents a strategic tool within a One Health approach, useful for the early identification of potentially zoonotic emerging strains. In light of these considerations, it is equally important to extend virological surveillance of wild boars to a panel of pathogens selected based on their documented transmissibility between wild boars and domestic pigs, the severity of the diseases they may cause, the availability of samples with matrices suitable for molecular and serological analyses, and the lack of epidemiological data in the study region. Some members of FLVs. genus, including West Nile virus (WNV) and USUTU virus (USUTUV), are known for their ability to infect a wide range of incidental hosts, including humans and wild boars [14,15]. Recent studies conducted in Italy have detected anti-Flavivirus antibodies in wild boars, suggesting previous exposure [16]. However, since this evidence derives exclusively from serological investigations, there is a clear need to deepen the research using molecular approaches and across a broader geographical scale. In this context, health surveillance of wild boars plays a strategic role for both zoonotic and non-zoonotic pathogens of veterinary relevance, highlighting the need for integrated virological monitoring, including agents with significant production impact. ADV (Suid Herpesvirus 1) is endemic in many areas of Italy and is capable of infecting various domestic and wild species [14,15,16]. Although wild boars represent the main reservoir of the virus in the wild, the increasing interaction with livestock and hunting dogs raises the risk of interspecies transmission, making monitoring necessary within the framework of veterinary public health programs [17]. In addition to the zoonotic viruses and those of veterinary relevance already discussed, Porcine PCV2 has also been frequently detected in wild boar populations in Italy and other European countries [18,19]. The circulation of PCV-2 in the wild, although it does not pose a direct zoonotic risk, represents a threat to animal health, as it may affect pig farms through possible direct or indirect interactions [20]. Several studies have confirmed the co-circulation of PCV2 with other swine viruses (e.g., SuHV-1 and PPV1) in wild boars, suggesting the possibility of mixed infections, which can potentially increase the pathogenicity and spread of viral strains [21]. In the present study, molecular and serological monitoring was conducted on a wild boar population with the aim of assessing the circulation of both zoonotic viruses (with potential direct impact on public health) and non-zoonotic viruses of veterinary relevance, which can cause significant economic losses in the swine industry. The investigation focused in particular on geographical areas where, to date, epidemiological data on the presence of these pathogens in wildlife are limited or lacking.

In the present study, molecular and serological monitoring was conducted in a wild boar population to assess the circulation of both zoonotic viruses (with potential direct impact on public health) and non-zoonotic viruses of veterinary importance, which can cause significant economic losses in the swine industry. The selection of pathogens (IAVs and FLVs) was based on their epidemiological relevance in Italy and also on their zoonotic impact, and on the need to further investigate their circulation dynamics in wild boars, since they are not included in official monitoring programs. The survey focused particularly on geographic areas where epidemiological data on the presence of these pathogens in wildlife are currently limited or lacking. We also tried to monitor the circulation of ADV and PCV2 in wild boars, since these species can play an important role in the diffusion of these pathogens in pig farms.

2. Materials and Methods

2.1. Wild Boar Sampling

The study was conducted in a geographical area including the provinces of Foggia, BAT, and Bari for Apulia; Matera and Potenza for Basilicata; Salerno for Campania; and Catanzaro and Cosenza for Calabria (Figure 1). Sampling was carried out between 2023 and 2024 using wild boars that were slaughtered as part of the activities of the ‘Extraordinary Plan for the Capture, Slaughter, and Disposal of Wild Boars’ aimed at controlling African swine fever (ASF) at the game processing center in Tito (PZ), Basilicata. The study was not part of the Plan itself, but took advantage of animals already destined for slaughter. This facility regularly receives wild boar samples culled during hunting activities from the provinces. During each sampling session (the study was conducted during the hunting periods and in the months compatible with the circulation of the diseases under investigation, i.e., from October to April biological samples were collected from animals slaughtered by personnel of the Istituto Zooprofilattico della Puglia e della Basilicata (Tito Scalo Section) and subsequently transported within 24 h by authorized personnel for the transport of biohazardous materials to the Laboratory of Virological Diagnostics and the Laboratory of Biotechnology and Vaccines (Foggia Section), where molecular and serological analyses were performed. More specifically, nasal swabs (n = 444), lung tissue (n = 346), and muscle tissue (n = 339) were collected and subsequently subjected to laboratory analyses. Overall, the study included a total of 636 individual wild boars, from which different types of biological samples were collected. Specifically, some animals were sampled exclusively for muscle tissue, others only for lung tissue, others solely for swabs, or, in some cases, a combination of these tissues. Details regarding the animals tested for each assay are provided in the Supplementary Material S1. The geographical origin, sex and weight of each animal were recorded at the time of delivery to the slaughterhouse and used for sample georeferencing. The specimens showed marked heterogeneity in terms of geographic origin, with distribution across numerous municipalities within the eight provinces under study. The distribution of the specimens was predominantly concentrated in the Basilicata region, with a high density in the province of Potenza (48%), where environmental conditions, particularly the widespread presence of forested habitats, are especially favorable for the presence and proliferation of wild boars [22]. Specifically, 115 were from the Province of Matera (MT), 115 from the Province of Foggia (FG), 5 from the Province of Bari (BA), 32 from the Province of Barletta-Andria-Trani (BAT), 43 from the Province of Catanzaro (CZ), and 8 from the Province of Salerno (SA) (Supplementary Material S1). The available data, in addition to the geographical origin of the samples, included sex and body weight. Regarding sex, of the total sample of 636 individuals, 309 were males (48.7%), 315 were females (49.5%), and 12 individuals (1.9%) had undetermined sex. The distribution between males and females was therefore essentially balanced, with an almost 50:50 ratio for each sex (Figure 2a). With respect to body weight, there was a clear predominance of adult animals, most of which weighed over 50 kg (70%) (Figure 2b).

Figure 1. Investigated area: The figure shows the geographical area covered by the study, including the regions of Southern Italy: Apulia, Basilicata, Campania, and Calabria. The municipalities where sampling was carried out are indicated by yellow dots. Data visualization generated using Flourish v19.6.5 (Flourish, a Canva UK Operations Limited brand, London, UK).

Figure 2. Distribution of wild boar samples by sex and weight: (a) Pie chart showing the distribution of samples by sex: approximately half of the samples were collected from female wild boars, half from males, and a portion from individuals with indeterminate sex due to unavailable data. (b) Pie chart showing the distribution of samples by weight classes: 0–25 kg, 25–50 kg, and over 50 kg.

With respect to body weight, there was a clear predominance of adult animals, most of which weighed over 50 kg (70%) (Figure 3b). Although the literature indicates that body weight has a low correlation with age beyond 12–24 months, observed mean weights for different age classes are as follows: piglets (0–6 months) around 27.6 kg; juveniles/yearlings (6–12 months) approximately 54 kg; subadults (1–2 years), with females reaching about 90% of their final weight (mean 61–62 kg); adults (>2 years) with an average weight of 66.7 kg, while males can exceed 150–180 kg depending on habitat [23,24].

Figure 3. Geographic distribution of positive samples. (a) Locations of municipalities where wild boars tested seropositive. Colored points indicate the pathogen detected: Influenza A viruses (yellow), Flavivirus (blue), and coinfection Influenza A + Aujeszky’s disease virus (pink). (b) Locations of municipalities where wild boars tested positive by PCR. Colored points indicate the pathogen detected: Influenza A viruses (pink), PCV-2 (blue), and coinfections between Influenza A and PCV-2 (yellow).

2.2. Serological Investigation

For the serological investigation, meat juice obtained from wild boar muscle was used. Specifically, considering that at the sampling site, wild boar heads and viscera were available, the masseter muscle was collected from the head. Muscle samples were subjected to repeated freezing and thawing cycles to obtain the release of meat juice, until a minimum volume of 1 mL per sample was achieved [21]. The resulting extract represents a tissue transudate, rich in proteins and antibodies, although generally at lower concentrations compared to serum [25]. This method is particularly useful in the context of passive surveillance at slaughterhouses, due to the easy availability of muscle tissue from carcasses obtained during hunting activities or slaughter. The presence of specific antibodies against IAVs, FLVs, and ADV was investigated using competitive ELISA assays, performed according to the manufacturers’ instructions and read with a spectrophotometer. Meat juice was used as the matrix for all determinations. The following commercial kits were employed: ID Screen^® Flavivirus Competition (lot P47; IDvet, Grabels, France), based on the competition between antibodies present in the sample and a monoclonal antibody specific for the pr-E antigen of FLVs [26,27]; ID Screen^® Influenza A Antibody Competition Multi-species (lot N94; IDvet, Grabels, France), which detects antibodies directed against the nucleoprotein (NP) of Influenza A virus [28]; and IDEXX PRV/ADV gE Ab Test (lot AD11; IDEXX Laboratories, Westbrook, ME, USA), designed to detect antibodies against glycoprotein E (gE) of Aujeszky’s disease virus [29]. In all cases, the presence of antibodies in the sample resulted in a variation in the optical signal proportional to the amount of specific antibodies. The percentage of competition (S/N%) was calculated based on the optical density (OD) values obtained by spectrophotometric reading, according to the formula provided by the manufacturer.

2.3. Molecular Investigation

2.3.1. Sample Preparation

Molecular monitoring was conducted through three main steps: sample preparation, nucleic acid extraction, and PCR amplification. For preparation of samples, 0.5 g of lung tissue was homogenized in 2 mL Safe-Lock Eppendorf tubes containing 1 mL of phosphate-buffered saline (PBS, pH 7.4). Subsequently, 0.5 mL of the homogenate was transferred to a new tube containing 0.5 mL of lysis buffer and incubated on ice at 4 °C for 10 min. The samples were then centrifuged at 8000 rpm for 5 min.

2.3.2. Nucleic Acid Extraction

A volume of 300 µL of the supernatant was finally used for automated nucleic acid extraction using the TanBead^® system (TANBead^® Nucleic Acid Extraction Kit-Universal Pathogen Auto Tube M66PS46, TANBead, Taoyuan, Taiwan), following the manufacturer’s protocol. For nasal swabs, each swab was resuspended in 1 mL of PBS. A volume of 300 µL of the resulting suspension was then directly transferred to the TanBead automated extraction system, according to the manufacturer’s instructions. The extracts were stored at −80 °C until further use.

2.3.3. PCR Amplification

Viral nucleic acids were analyzed by real-time PCR assays targeting Influenza A viruses (M gene), FLVs (NS5 gene), and PCV2, using previously published protocols [18,30,31]. Amplifications were performed using commercial one-step RT-qPCR or qPCR master mixes, according to the manufacturers’ instructions. Thermal cycling conditions and positivity criteria followed the respective reference protocols. Detailed information on primers, probes, reaction mixtures, and cycling conditions is provided in Supplementary Material S2.

3. Results

Serological analyses, 28 out of 337 tested subjects were positive for IAVs (Table 1) (8.31%, 95% CI: 5.81–11.75%), primarily distributed in the provinces of Potenza (18/337; 5.34%, 95%CI: 3.40–8.28%)followed by Matera (4/337; 1.19%, 95% CI: 0.46–3.01%), Foggia (4/337; 1.19%, 95% CI: 0.46–3.01), Catanzaro (1/337; 0.30%, 95% CI: 0.05–1.66%), and Salerno (1/337; 0.30%, 95% CI: 0.05–1.66%) (Figure 3a). Among the 28 positive samples, 23 (82.14%, 95% CI: 64.41–92.12%) were collected during the February–April period, in agreement with the molecular survey data. The weight of positive subjects ranged widely from 18 to 87 kg (mean ± SD: 59.6 ± 18.4 kg) and did not significantly differ from negative subjects (63.9 ± 26.9 kg; Welch’s t-test, p = 0.34). Regarding sex, 9 subjects (32.14%, 95% CI: 17.93–50.66%) were males and 19 (67.86%, 95% CI: 49.34–82.07%) females. The detection of anti-Flavivirus antibodies revealed a single positive sample out of 337 (Table 1) (0.30%, 95% CI: 0.05–1.66%), collected in the province of Foggia (Figure 3a). This sample was subsequently confirmed as positive for WNV. For Aujeszky’s disease virus (ADV), specific antibodies were detected in 4 out of 153 tested samples (Table 1) (2.61%, 95% CI: 1.02–6.53%), all of which originated from the province of Potenza (Figure 3a). Positive subjects weighed between 33 and 70 kg. Comparison with negative subjects showed no statistically significant difference in body weight (Mann–Whitney U test, p = 0.37), suggesting no significant correlation between weight and positivity. All positive subjects were male; however, the very low number of cases did not allow a statistically significant assessment of association with sex (Fisher’s exact test, p > 0.05). It should be noted that only samples from Basilicata were included for ADV analysis; consequently, no data are available from the Puglia region.

Table 1. Characteristics of wild boars testing positive in ELISA assays. S/N%: sample-to-negative ratio (%), calculated according to the manufacturer’s instructions. The table reports the animal ID, pathogen tested, ELISA quantitative result (S/N%), sex, body weight (kg), and municipality of origin. Samples were classified according to the manufacturer’s instructions for each kit as follows: (a) ID Screen^® Flavivirus Competition (IDvet, France, lot P47): S/N% ≤ 40% = positive; 40% < S/N% ≤ 50% = doubtful; S/N% > 50% = negative. (b) IDEXX PRV/ADV gE Ab Test (IDEXX Laboratories, USA, lot AD11): S/N% ≤ 60% = positive; 60% < S/N% ≤ 70% = doubtful; S/N% > 70% = negative. (c) ID Screen^® Influenza A Antibody Competition Multi-species (IDvet, France, lot N94): S/N% ≤ 45% = positive; 45% < S/N% ≤ 50% = doubtful; S/N% > 50% = negative.

Animal ID	Pathogen	Quantitative Result (S/N%)	Sex	Body Weight (kg)	Municipality
570	Flavivirus	24.8%	M	85	Rignano Garganico (FG)
1	ADV	38.10%	M	33	Muro Lucano (PZ)
12	ADV	17.50%	M	51	Castelluccio Inf. (PZ)
124	ADV	25.40%	M	49	Vietri (PZ)
139	ADV	27.70%	M	70	Muro Lucano (PZ)
616	Influenza virus	29%	F	50	S Nicandro G. (FG)
5	Influenza virus	39%	M	52	Muro Lucano (PZ)
13	Influenza virus	37%	F	66	Castelluccio Inf. (PZ)
628	Influenza virus	39.80%	F	75	Sant’Agata di Puglia (FG)
28	Influenza virus	44%	M	87	Castelluccio Inf. (PZ)
33	Influenza virus	37%	F	44	Castelluccio Inf. (PZ)
51	Influenza virus	18%	F	76	Savoia di L. (PZ)
59	Influenza virus	12.40%	M	72	Sant’Angelo (PZ)
63	Influenza virus	22.20%	F	36	Bernalda (MT)
64	Influenza virus	36.50%	F	43	Accettura (MT)
67	Influenza virus	36.30%	F	59	Bernalda (MT)
68	Influenza virus	26.70%	F	68	Pietragalla (PZ)
70	Influenza virus	5.50%	F	34	Bernalda (MT)
72	Influenza virus	6.68%	F	50	Pietragalla PZ)
73	Influenza virus	38.10%	F	87	Pietragalla (PZ)
75	Influenza virus	17.90%	M	72	Pietragalla (PZ)
76	Influenza virus	34.60%	F	18	Savoia di L. (PZ)
519	Influenza virus	37.20%	M	80	Andria (BAT)
91	Influenza virus	0.41	M	69	Muro Lucano (PZ)
92	Influenza virus	0.36	F	86	Vietri (PZ)
101	Influenza virus	0.34	M	71	Felitto Sa. (SA)
120	Influenza virus	31%	F	57	Muro Lucano (PZ)
615	Influenza virus	21.50%	M	28	S Nicandro G. (FG)
121	Influenza virus	43%	F	41	Muro Lucano (PZ)
122	Influenza virus	42%	F	67	Castelluccio Inf. (PZ) (PZ)
158	Influenza virus	18%	M	69	Muro Lucano (PZ)
386	Influenza virus	44%	M	58	Falerna (CZ)
490	Influenza virus	30.00%	F	72	Muro Lucano (PZ)

Regarding molecular analysis, the presence of IAVs was detected in 5 out of 429 animals examined (Table 2) (1.17%, 95% CI: 0.50–2.70%). The positive subjects had a body weight ranging from 44 to 60 kg, corresponding to an estimated age of approximately 8 to 14 months [31]. However, the very low number of positive cases does not allow a statistical confirmation of any association between age and susceptibility to infection. Regarding sex, three females and two males tested positive, suggesting a balanced distribution between the two genders, with no apparent correlation. All infected individuals originated from the Basilicata region, with a territorial distribution of 2 animals from the province of Matera (40%) and 3 from the province of Potenza (60%) (Figure 3b). Concerning FLVs, no positive cases were detected in the analyzed samples, indicating an absence of detectable viral circulation during the study period and in the areas considered. Regarding PCV2, 66 out of 223 examined subjects tested positive (Table 2) (29.6%, 95% CI: 23.99–35.89%). The geographical distribution of positive cases showed a higher concentration in the province of Potenza (41/66; 62.12%, 95% CI: 50.06–72.85%), followed by Matera (15/66; 24.2%, 95% CI: 15.51–35.81%) and Catanzaro (10/66; 16.7%, 95% CI: 9.57–27.43%) (Figure 3b). The sex distribution revealed a substantial parity between females (32/66; 48.5%, 95% CI: 36.85–60.29%) and males (34/66; 51.52%, 95% CI: 39.71–63.15%), and the binomial test confirmed that the difference was not statistically significant (p = 0.85), suggesting that sex does not represent a significant risk factor for infection. The weight of positive subjects ranged from 28 to 121 kg, with no apparent correlation between body mass and the likelihood of positivity.

Table 2. Characteristics of wild boars testing positive by PCR assays. The table reports the animal ID, the tested pathogen, the quantitative result expressed as Cycle threshold (Ct), sex, body weight (kg), and municipality of origin.

Animal ID	Pathogen	Quantitative Result (Ct)	Sex	Body Weight (kg)	Municipality
33	Influenza virus	30	F	44	Castelluccio Inf. (PZ)
141	Influenza virus	34.16	M	49	Bernalda (MT)
152	Influenza virus	31.57	M	52	Vietri (PZ)
155	Influenza virus	32.52	F	52	Castelluccio Inf. (PZ)
178	Influenza virus	33.33	F	60	Tricarico (MT)
203	PCV-2	32	M	52	Picerno (PZ)
207	PCV-2	30	M	41	Baragiano (PZ)
214	PCV-2	34	M	41	Ruoti (PZ)
217	PCV-2	30	F	58	Ruoti (PZ)
222	PCV-2	31	F	39	Bernalda (MT)
225	PCV-2	28	M	43	Tito (PZ)
230	PCV-2	28	F	52	Pietragalla (PZ)
240	PCV-2	31	F	73	Picerno (PZ)
246	PCV-2	30	M	39	Ruoti (PZ)
280	PCV-2	34	F	38	Pietragalla (PZ)
282	PCV-2	32	M	40	Pietragalla (PZ)
283	PCV-2	33	F	34	Muro Lucano (PZ)
288	PCV-2	34	F	44	Ruoti (PZ)
293	PCV-2	34	F	70	Ruoti (PZ)
294	PCV-2	24	M	75	Ruoti (PZ)
295	PCV-2	34	M	38	Castelluccio Inf. (PZ)
297	PCV-2	28	M	35	Castelluccio Inf. (PZ)
298	PCV-2	29	F	33	Castelluccio Inf. (PZ)
299	PCV-2	28	M	93	Castelluccio Inf. (PZ)
302	PCV-2	33	F	45	Pietragalla (PZ)
303	PCV-2	31	F	50	Castelluccio Inf. (PZ)
304	PCV-2	31	M	60	Vietri (PZ)
305	PCV-2	30	F	49	Martirano L. (CZ)
306	PCV-2	34	M	121	Martirano L. (CZ)
318	PCV-2	29	F	40	Martirano L. (CZ)
320	PCV-2	26	F	58	Martirano L. (CZ)
321	PCV-2	34	M	44	Ruoti (PZ)
326	PCV-2	24	M	53	Pietragalla (PZ)
327	PCV-2	32	M	75	Bernalda (MT)
328	PCV-2	32	M	69	Ruoti (PZ)
329	PCV-2	22	F	78	Albano di L. (PZ)
330	PCV-2	34	M	63	Pietragalla (PZ)
331	PCV-2	31	M	80	Bernalda (MT)
335	PCV-2	28	M	35	Bernalda (MT)
338	PCV-2	34	F	51	Bella San Cataldo (PZ)
340	PCV-2	30	F	74	Bella San Cataldo (PZ)
347	PCV-2	28	F	51	Pietragalla (PZ)
348	PCV-2	32	F	58	Calciano (MT)
349	PCV-2	32	M	70	Bella San Cataldo (PZ)
350	PCV-2	31	F	55	Calciano (MT)
353	PCV-2	34	F	28	Pisticci (MT)
355	PCV-2	34	F	62	Pisticci (MT)
357	PCV-2	29	F	36	Pomarico (MT)
361	PCV-2	32	F	56	Pisticci (MT)
365	PCV-2	29	F	59	Pisticci (MT)
370	PCV-2	34	M	50	Motta S. Lucia (CZ)
373	PCV-2	33	F	47	Motta S. Lucia (CZ)
385	PCV-2	33	F	66	Nocera Terinese (CZ)
386	PCV-2	34	M	58	Falerna (CZ)
387	PCV-2	25	F	61	Conflenti (CZ)
390	PCV-2	33	M	60	Falerna (CZ)
427	PCV-2	34	M	79	Baragiano (PZ)
429	PCV-2	32	M	73	Pietragalla (PZ)
430	PCV-2	32	F	65	Castelluccio Inf. (PZ)
434	PCV-2	28	M	46	Pietragalla (PZ)
442	PCV-2	34	M	93	Castelluccio Inf. (PZ)
448	PCV-2	30	F	53	Pietragalla (PZ)
465	PCV-2	28	M	52	Satriano Di Lucania (PZ)
466	PCV-2	31	M	66	Satriano Di Lucania (PZ)
469	PCV-2	26	F	67	Castelluccio Inf. (PZ)
478	PCV-2	31	M	48	Accettura (MT)
484	PCV-2	28	M	59	Rapone (PZ)
491	PCV-2	33	M	60	Calciano (MT)
493	PCV-2	29	M	35	Vietri (PZ)
497	PCV-2	25	F	32	Accettura (MT)
500	PCV-2	24	M	44	Accettura (MT)

4. Discussion

Viral circulation in wildlife populations plays a crucial role in emerging epidemiological systems, as these populations can act as reservoirs, sentinel indicators, and bridges to livestock populations [32,33]. In particular, the analysis of viral presence in wildlife species is strategically important from both an animal health and a public health perspective. For this reason, the combined use of molecular and serological investigations allows not only the detection of active infections but also the reconstruction of past exposure within the population, highlighting temporal dynamics that are often invisible through virological analysis alone [34]. Numerous studies report that specific antibodies can persist in animal serum for months or longer, supporting the use of serology to track previous viral circulation [35].

Moreover, the seroprevalence of the viruses analyzed in this study has been reported in other Italian regions, specifically: for IAVs in Emilia-Romagna and Tuscany [36,37]; for FLVs in Lazio, Friuli Venezia Giulia, Veneto, and Lombardy [34,38]; and for ADV, the presence of antibodies has been documented in both Campania and Piedmont [14,39]. Similarly, viral circulation of these viruses has been demonstrated in other regions: for IAVs in Emilia-Romagna and Tuscany [37,40,41], for PCV2 in Campania [42], whereas viral presence of FLVs has not been detected in wild boars in Italy [34].

However, no studies have previously addressed the regions investigated in this research. In our study, PCR analyses confirmed the active circulation of influenza viruses in hunted wild boars, particularly during the February–April 2024 period. Serological testing, however, detected a higher number of positive animals for influenza viruses (28 seropositive subjects compared to 5 PCR-positive cases) during the same period and in the same geographic areas. Overall, the data on IAVs positivity indicate a correlation with the seasonality of viral circulation, mainly concentrated in February–April. Additionally, the distribution of positive individuals by weight suggests that infection predominantly affects sub-adult animals. From a geographical perspective, most positive samples originated from the Basilicata region, especially from the province of Potenza; however, this finding may reflect the higher number of samples collected from this area rather than a real higher viral circulation. It should be emphasized that the wild boar organ samples analyzed in this study were obtained through an opportunistic sampling approach, based on the availability of biological materials at the slaughterhouses. As a consequence, the geographical distribution of samples is unbalanced, with a higher proportion originating from the province of Potenza, so this his sampling strategy can represent a limitation of data representativeness. Therefore, the results should be interpreted as an estimation deriving from the analyzed samples rather than a measure of the true regional prevalence.

However, regarding FLVs, no positive cases were detected by PCR, while serological analysis revealed past exposure in a single individual (antibodies directed against Flaviviruses), ruling out the possibility of maternal or colostral antibodies, since the animal weighed 85 kg. This pattern is fully consistent with previous reports in the literature [34]. The data therefore indicate past exposure to the virus but do not provide evidence of active viral circulation during the sampling period. The observed seropositivity should be interpreted as reflecting prior contact with the pathogen, while the absence of molecular detections does not allow the demonstration of ongoing viral replication.

Regarding PCV-2, recent studies conducted in the Basilicata and Campania regions reported a prevalence of 27% in wild boars in Basilicata [19] and 47.3% in Campania [18], with evidence of the dominant genotype 2. Our findings are fully consistent with these studies, confirming that genotype 2 is widespread in southern Italy and that the wild population may represent an important reservoir. The agreement between our results and the literature indicates that viral circulation is stable and well-established in the area. Similarly, to what was observed for IAVs, a higher number of PCV-2 positive samples were detected in the province of Potenza. As previously discussed, this finding may be related to the larger number of samples analysed from this province. Regarding antibodies detected through the serological component, our study revealed seropositivity for ADV. Previous investigations in Italy reported, for example, a seroprevalence of 23.85% for ADV in wild boars in the Campania region during the 2016–2017 hunting season [14]. Our results confirm the circulation of these agents in the study area and indicate a territorial correlation, as all positive samples originated from the province of Potenza. Additionally, all positive subjects were male. The overall analysis did not reveal any significant associations among the investigated pathogens. Positive cases were mutually exclusive, and no coinfections were detected in any of the examined animals. The operational choice to include four regions of Southern Italy (Apulia, Basilicata, Campania, and Calabria) and to simultaneously investigate multiple viral diseases gives our study a distinctive contribution. Sampling wild boars across a wide geographic area—characterized by diverse environmental conditions, altitudes, and hunting pressures—allows for a more representative snapshot of viral circulation in wildlife in Southern Italy. Our findings highlight the importance of an integrated multi-pathogen molecular and serological approach, which is also valuable in studying potential overlaps and interactions between viral agents. It is important to note that not all samples were tested for the full panel of pathogens due to insufficient biological material. Nevertheless, the results obtained provide a solid foundation for future prospective investigations, which could include studies on the genetic characterization and variability of the detected pathogens, and highlight the importance of an integrated multipathogen approach based on molecular and serological analyses to assess potential overlaps and interactions among different viral agents.

This multi-pathogen investigation provides updated evidence of viral circulation in wild boars from four regions of Southern Italy, revealing heterogeneous epidemiological patterns depending on the pathogen considered. By integrating molecular and serological data, our study contributes to a broader understanding of viral dynamics at the wildlife–livestock interface and places local findings within a wider European and multi-host ecological framework.

The detection of Influenza A virus (IAV) RNA (1.17%) alongside a higher seroprevalence (8.31%) suggests intermittent viral circulation combined with wider past exposure. This discrepancy between molecular and serological findings is consistent with surveillance conducted in Northern Italy [40], where virological detection remained sporadic while serological evidence indicated sustained exposure over time. Comparable epidemiological trends have been documented in Spain, where longitudinal monitoring between 2015 and 2023 demonstrated low but continuous viral circulation in wild boars, with increasing seroprevalence and evidence of subtype shifts, including potential H5N1 spillover risk [43]. Similar patterns were reported in Germany, where low PCR detection rates contrasted with measurable seroprevalence across multiple subtypes [44].

Importantly, recent findings from other wild species reinforce the interpretation of spillover-driven dynamics. In Europe, IAV exposure has been documented in wild carnivores such as red foxes and mustelids, often temporally associated with outbreaks in domestic pigs or wild birds. Wild avifauna remain the primary natural reservoir, and terrestrial mammals—including wild boars—appear to act as incidental or bridge hosts rather than autonomous maintenance populations. The seasonal clustering of PCR-positive cases in our study (February–April) mirrors the winter–spring peaks of influenza activity in domestic swine, further supporting the hypothesis of recurrent spillover from livestock or avian sources. The higher frequency of infection in subadults aligns with age-related susceptibility patterns described in other wild pig populations, where immunological naivety increases the probability of active infection.

Overall, the concordance between our findings and those reported in other wild mammals suggests that IAV circulation in wild boars is unstable and largely dependent on external epidemiological pressure rather than sustained endemic transmission. Only one animal tested seropositive for flavivirus antibodies, confirmed as West Nile virus (WNV), while no molecular detection was recorded. This pattern—serological evidence without PCR positivity—is consistent with the short viremic phase typical of flaviviral infections in mammals. Similar discrepancies have been reported in Italian surveillance programs, including investigations conducted in the Latium region [34]. Recent European studies further clarify the epidemiological role of wild boars in WNV ecology. Integrated surveillance systems in Germany combining dogs and wild boars demonstrated that seropositivity in these mammals effectively tracked viral circulation in endemic areas without evidence of amplification [45]. Comparable serosurveys in the Netherlands detected flavivirus antibodies in wild boars, dogs, and horses, again without indication of sustained mammalian transmission [46].

Beyond wild boars, WNV antibodies have also been reported in other wild ungulates such as roe deer and red deer, as well as in wild carnivores across Europe. These species, like wild boars, are considered incidental or dead-end hosts within a transmission cycle maintained primarily between birds and mosquitoes. The agreement between our findings and those observed in other mammalian wildlife species supports the interpretation that wild boars function as serological sentinels rather than amplification hosts. Their value lies in mapping spatial and temporal patterns of viral activity, particularly in areas where entomological and avian surveillance are already implemented.

Serological evidence of Suid herpesvirus 1 (SuHV-1; Aujeszky’s disease virus, ADV) was detected in 2.61% of animals, confirming ongoing viral circulation in Basilicata. Although lower than the 23.85% seroprevalence reported in Campania during the 2016–2017 hunting season, the presence of seropositive animals remains epidemiologically significant within the context of Italy’s eradication program in domestic pigs. Across Europe, wild boars are widely recognized as a stable reservoir of SuHV-1. In France, seroprevalence values between 5% and over 30% have been documented depending on population density and hunting intensity [47]. In Spain, exposure has been consistently demonstrated in free-ranging wild boars [8], while in Croatia, seroprevalence frequently ranges from 10% to 40% [48]. Molecular detection is generally sporadic, but recurrent seropositivity confirms long-term maintenance in wildlife independent of domestic swine. Recent evidence from other wild species underscores the broader ecological impact of ADV circulation. Fatal pseudorabies cases have been described not only in hunting dogs in Italy [49] but also in wild carnivores such as wolves and foxes in several European countries, following predation or scavenging of infected wild boars. These spillover events highlight the ecological consequences of persistent infection in wild suids and confirm their role as a long-term reservoir capable of affecting both domestic animals and wildlife conservation. Thus, even moderate seroprevalence values, as observed in our study, warrant continued wildlife monitoring, particularly in regions characterized by intense hunting activity and frequent dog–wild boar interactions.

PCV-2 exhibited the highest molecular prevalence (29.6%) among the investigated pathogens, indicating stable endemic circulation. This result is comparable with previous findings from Southern Italy, including reports from Campania (47.3%) and Basilicata (~27%), supporting the hypothesis of long-term viral establishment in the region. Similar epidemiological patterns have been documented across Europe. In Spain, Vicente et al. (2004) [50] demonstrated widespread PCV-2 infection in Iberian wild boars, with prevalence levels comparable to those in domestic pigs, suggesting endemicity in wildlife. In Central Europe and the Western Balkans, PCV-2 has been repeatedly detected in wild boars, sometimes in co-infection with other swine viruses such as PPV and PCMV [51], further confirming sustained transmission. Importantly, PCV-2 has also been identified in other wild ungulates, including roe deer, red deer, and mouflon in several European countries. Although the clinical relevance in non-suid hosts remains uncertain, these findings indicate a degree of interspecies transmission and ecological plasticity. Unlike IAV, PCV-2 in our study showed no clear seasonal clustering or association with sex or body weight, reinforcing the interpretation of widespread, stable endemic circulation within wild boar populations. Given the economic impact of PCV-2 in domestic swine production, the confirmed presence of the virus in free-ranging wild boars raises concerns regarding indirect transmission, particularly in areas with outdoor or backyard farming systems. A notable strength of this study is the simultaneous investigation of zoonotic and non-zoonotic viral agents across multiple regions of Southern Italy (Apulia, Basilicata, Campania, and Calabria). Compared with previous Italian investigations typically focused on single pathogens or restricted geographic areas, this integrated approach provides a broader ecological snapshot.

The absence of detected coinfections may reflect true epidemiological independence among pathogens or may be influenced by sample size and testing scope. Additionally, the opportunistic sampling design—based on animals culled under the African swine fever control plan—introduced some geographic imbalance, particularly the overrepresentation of the province of Potenza. Therefore, prevalence estimates should be interpreted cautiously.

Overall, our findings delineate two distinct epidemiological patterns. Influenza A virus and West Nile virus appear to follow spillover-driven dynamics within complex multi-host systems, where wild boars function primarily as incidental or sentinel hosts. In contrast, Suid herpesvirus 1 and PCV-2 demonstrate characteristics of sustained maintenance within wild boar populations, with potential spillover to other wildlife species and domestic animals.

This broader multi-species perspective reinforces the necessity of coordinated surveillance strategies integrating wildlife, livestock, and public health sectors under a One Health framework. Future research should prioritize molecular characterization of detected strains, longitudinal sampling, and quantitative risk-factor analyses to better elucidate interspecies transmission dynamics at the wildlife–livestock–ecosystem interface.

5. Conclusions

The present study documents the circulation of IAVs, FLVs, ADV, and the persistent presence of PCV-2 in wild boar populations, reflecting patterns already observed in wildlife surveillance programs across Europe. The strengths of the study lie in its broad spatial coverage, the simultaneous assessment of multiple viral pathogens, and the combined use of serological and molecular diagnostic approaches. It is important to note that sampling was performed opportunistically, based on the availability of biological material at slaughterhouses, and that not all samples could be tested for the full panel of pathogens due to limited material. Moreover, the study did not include sequencing or genetic characterization of viral strains, representing a methodological limitation. Nevertheless, the results provide a solid foundation for future prospective studies aimed at addressing these gaps through more representative sampling, comprehensive molecular analyses, and investigations into the genetic variability of isolated viral strains. Overall, this study makes a significant contribution to the understanding of pathogen ecology in free-ranging Suidae populations and provides a basis for future research on long-term epidemiological dynamics and interspecific transmission risks at the wildlife–livestock interface.