Detection and Characterization of Viral Pathogens Associated with Reproductive Failure in Wild Boars in Central Italy

Simple Summary Suid herpesvirus 1, porcine circovirus 2 and porcine parvovirus are causative agents of reproductive failures in swine and are widely diffused in the wild boar population. No data describing the impact of those viruses on the reproductive performance of wild boar are so far available. We aimed to investigate the ability of the above viruses to infect foetuses of free-ranging pregnant wild boar sows living in a highly-populated area. Molecular investigation revealed that although all investigated viruses were detected in pregnant sows, only herpesvirus and circovirus were detected in the foetuses. Phylogenetic analysis revealed a close relationship between the strains circulating in wild boar and those already described in domestic swine. This study highlights the importance of monitoring the circulation of pathogens that are shared between domestic and wild pigs. This information is essential for the pig industry to avoid possible economic losses. Abstract Wild boar and domestic swine share several pathogens, including viruses responsible for reproductive failures, representing an important sanitary and economic risk for the swine industry. Among them, suid herpesvirus 1 (SuHV-1), porcine circovirus 2 (PCV2) and porcine parvovirus 1 (PPV1) are widely diffused in the wild boar population. Unfortunately, little is known about their pathogenetic mechanisms and impact on the reproductive parameters of wild animals. This study aims to investigate the presence of viruses responsible for reproductive failure in pregnant wild boar sows and their foetuses. The investigation was conducted on 46 pregnant wild boar and their foetuses by molecular analysis; a phylogenetic study was performed on the positive samples. All of the investigated pathogens were identified in sows, while only herpesvirus and circovirus were detected in the tissues of their foetuses. Phylogenetic analysis revealed that the viral sequences obtained from the positive wild boars were closely related to those previously identified in domestic swine belonging to the same study areas. The results suggest that SuHV-1 and PCV2 can infect wild boar foetuses, with a possible impact on wild boar reproductive performance. Moreover, our data highlight the importance of continuous monitoring of swine pathogens circulating in wild environments, so as to carry out adequate sanitary actions.


Introduction
Wild boar (Sus scrofa) is one of the most widely distributed ungulates, characterized by a highly adaptable capability, a high reproductive rate and the ability to assume an opportunistic feeding behaviour [1]. For these peculiarities, its number and distribution are constantly increasing [2]. In Europe, the consistency of the wild boar populations is generally high, including in many Italian regions, and often including suburban areas [2].
Wild boar and domestic swine belong to the same species and microorganism transmission between them often occurs, especially in pigs bred in extensive or semi-extensive The severity of reproductive failure depends on the virulence of the PPV1 strains. Indeed, highly pathogenic strains (e.g., Kresse and 27a) cross the placental barrier more efficiently than low pathogenic and vaccine strains (e.g., NADL-2 and MSV) [60][61][62].
Studies conducted on the wild boar populations have shown that PPV1 is also present in Italy, with a prevalence ranging from 8% to 99% according to the study areas [69][70][71].
However, despite the strong evidence of PPV1 circulating in wild boar, there is little information on the effects of the virus on wild boar health and reproductive performance; although, according to a study by Ruiz-Fons and colleagues, it seems to be associated with a decrease in the ovulation rate in female wild boar [64].
In domestic swine, SuHV-1, PCV2 and PPV1can be transmitted from pregnant sows to foetuses with several consequences on pregnancy or the piglets' health. Due to the wildness of wild boar and the difficulty to monitor their reproductive performance and parturitions, limited information is available about the pathogenesis of SuHV-1, PCV2 and PPV1 in pregnant wild boar sows and about their ability to infect foetuses, with effects on the course of pregnancy.
This study aims to investigate the ability of the main causative viral agents of reproductive failure in swine to infect foetuses in free-ranging pregnant wild boar sows living in a highly-populated area.

Sample Collection
During the 2018-2019 and 2019-2020 hunting seasons, from 1 November to 31 January, tissue samples were collected from pregnant wild boar hunted in Tuscany (Italy) in a specific area that constitutes contiguous municipalities (Pisa, Siena, Grosseto and Livorno Province), known for the copious presence of wild animals. The animals were hunted following the Regional Hunting Law (Regolamento di attuazione della legge regionale 12 gennaio 1994 no. 3 DPGR 48/R/2017). Lymph nodes and foetal specimens were sampled from 26 animals during the slaughtering procedures. The lymph nodes were sampled directly from carcasses of the animals while the pregnant uteruses were conveyed to the Department of Veterinary Science (University of Pisa) for foetus sampling. Foetuses were weighed and measured to retrieve information about their development stage, then tissue samples of the heart, lung, liver, kidney and spleen, belonging to all foetuses, were collected from a single sow and pooled for molecular analysis.

Molecular Analysis
Each lymph node and the foetus samples were subjected to tissue disruption (Tissue Lyser Qiagen, Hilden, Germany) before the DNA extraction was performed, using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany).
A first set of highly sensitive PCRs was applied for diagnostic purposes to identify the positive sample; further sets of PCRs were performed to obtain the phylogenetic information from all the positive samples.
Samples that were positive after the molecular analysis were submitted to sequence analysis (BMR genomics, Padova, Italy).
In Table 1 the primer sets used for the molecular analysis are presented.

Phylogenetic Analysis
Nucleotide sequence analysis was applied to confirm the specificity of the PCR assays and to obtain phylogenetic information on the viral strains circulating in the studied areas. For each viral target investigated, a set of the most representative GenBank available sequences were identified and used to construct phylogenetic trees by maximum-likelihood methods, as available in the MEGA6 software package [76]. Phylogenetic analysis for SuHV-1 was conducted on 404 positions of the gE gene in the final dataset, for PCV2 on 431 positions of Open Reading Frame 2 (ORF2) and for PPV1 on 776 positions of Viral Protein 2 (VP2). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model.
The bootstrap test was applied to calculate the percentage of replicate trees in which the associated taxa clustered together (100 replicates).

Results
Concerning foetus sampling, they resulted in a weight median value of 169 (±71.1) grams with a median length of 138.6 (±19.3). Due to the collection sample period, the common seasonal wild boar mating season and the dimension of the collected foetuses, we can assume that the age of foetuses sampled was in the range of 50 to 70 days of gestation [77].
All the viral agents studied were found in at least one sow. The results of this investigation indicated that 1 out of 26 pregnant wild boars was positive for PCV2, 2 out of 26 were positive for parvovirus and 1 out of 26 was double-positive for SuHV-1 and PPV1.
Foetal samples collected from the pregnant wild boar positive for SuHV-1 and PPV1 were positive for SuHV-1, but not for PPV1. In addition, the foetal samples from the PPV1-positive sow were negative for the same virus. Furthermore, the pooled foetuses sampled from the PCV2-positive sow were also positive ( Table 2). The foetuses belonging to negative wild boar were negative for all studied pathogens.
All positive results were confirmed by sequence analysis. Moreover, a 100% nucleotide sequence identity was detected, comparing sows and their foetuses positive for SuHV-1 and PCV2.
Results obtained from the phylogenetic analyses performed on the single viral pathogen investigated demonstrated that the SuHV-1 strain detected in pregnant wild boar and associated foetuses (based on gE gene) was identical to a herpesvirus strain previously detected in 1996 in Italy from swine, and a strain isolated from a dog in 2010 belonging to Cluster C ( Figure 1).  All positive results were confirmed by sequence analysis. Moreover, a 100% nucleotide sequence identity was detected, comparing sows and their foetuses positive for SuHV-1 and PCV2.
Results obtained from the phylogenetic analyses performed on the single viral pathogen investigated demonstrated that the SuHV-1 strain detected in pregnant wild boar and associated foetuses (based on gE gene) was identical to a herpesvirus strain previously detected in 1996 in Italy from swine, and a strain isolated from a dog in 2010 belonging to Cluster C ( Figure 1).  A similar result, showing a correlation to previous sequences identified in Italy, was observed for swine circovirus. In this case, the Italian wild boar sequence identified in the present study was closely related to sequences derived from wild boar and domestic swine collected in Italy in 2011 and 2012, respectively. The phylogenetic analysis classifies the PCV2 sequences as belonging to PCV2d (Figure 2).
Finally, the parvovirus sequence identified from two pregnant wild boars showed a complete homology with each other and correlates with a PPV1 wild boar sequence from Romania collected in 2011, as indicated by the VP2 sequence analysis (Figure 3).
A similar result, showing a correlation to previous sequences identified in Italy, was observed for swine circovirus. In this case, the Italian wild boar sequence identified in the present study was closely related to sequences derived from wild boar and domestic swine collected in Italy in 2011 and 2012, respectively. The phylogenetic analysis classifies the PCV2 sequences as belonging to PCV2d (Figure 2).

Discussion
Numerous studies previously conducted on the Italian wild boar population have revealed the presence of several pathogens, and among them, the viral agents responsible for reproductive disorders [10,15,46,70,[78][79][80]. The present research has highlighted the circulation of SuHV-1, PCV2, and PPV1 in wild boar circulating in a research area located in central Italy. The area is characterized by a high wild boar population density in which hunting activity is widely diffused. By molecular assays, in this research, we identified the presence of all the pathogens studied, confirming their long-time persistence in the Italian wild boar population. Moreover, the identification of the genome of those viral agents in the tissue samples indicates their active circulation and has allowed us to conduct phylogenetical analysis.
The results described in the present work highlight the importance of wild boar test-

Discussion
Numerous studies previously conducted on the Italian wild boar population have revealed the presence of several pathogens, and among them, the viral agents responsible for reproductive disorders [10,15,46,70,[78][79][80]. The present research has highlighted the circulation of SuHV-1, PCV2, and PPV1 in wild boar circulating in a research area located in central Italy. The area is characterized by a high wild boar population density in which hunting activity is widely diffused. By molecular assays, in this research, we identified the presence of all the pathogens studied, confirming their long-time persistence in the Italian wild boar population. Moreover, the identification of the genome of those viral agents in the tissue samples indicates their active circulation and has allowed us to conduct phylogenetical analysis.
The results described in the present work highlight the importance of wild boar testing for monitoring the presence of infectious diseases in a certain ecosystem. Unfortunately, due to the limited number of samples, no epidemiological information about prevalence could be inferred.
All the pathogens investigated were detected in at least one pregnant sow and SuHV-1 and PCV2 were also detected in the foetus tissues, confirming their ability to infect foetuses during the first stage of gestation. In particular, the SuHV-1 positivity has confirmed the results obtained in a previous research study conducted in the same study area [79]. It is noteworthy that, in the present study, the SuHV-1-positive pregnant wild boar was positive also for PPV1. Moreover, a second PPV1-positive pregnant wild boar was detected in the same municipality (Grosseto province). Although the presumed gestation time of 50-70 days could be considered as a susceptible period for PPV1 infection, foetal samples collected from both the PPV1-positive wild boar scored negative in the molecular assay [51,59]. Probably, the negative results in the foetuses could be justified considering that the virus in domestic swine needs 12-18 days to reach the foetus after the mother becomes infected. Moreover, the mother's immunity could be capable of protecting the foetuses from infection [51,58,59].
Finally, circovirus infection was identified in a sow and the associated foetuses, confirming the ability of the virus to cross the placenta.
For all positive cases, no macroscopic clinical evidence was recorded, neither for the adults during the standard slaughter procedures nor for foetuses during sample preparation.
The phylogenetic analysis performed on the sequences obtained for the positive samples belonging to each viral pathogen investigated revealed a close relationship to the previously detected Italian strains, confirming the continuous circulation of such viral types among the Italian wild boar population, and often with high homology to domestic animals.
In detail, the SuHV-1 gE sequences analysed cluster with suid herpesvirus Cluster C, confirming previous phylogenetic studies that identified Clusters B and C as the most diffused among domestic swine in Italy. This evidence highlights that the SuHV-1 viral type circulating in domestic swine are currently circulating also in feral animals [80].
Concerning PCV2, studies using phylogenetic analysis defined eight different genotypes of PCV2 (PCV2a to PCV2h), of which PCV2a, b and d are the most common around the world. In Italy, PCV2b is prevalent at the moment, but the PCV2d frequency is progressively rising following a stronger genotype shift from PCV2b to PCV2d, which started in 2010 and now reported on a worldwide scale [81][82][83]. The results are perfectly in line with the phenomenon described in the literature since the phylogenetic analysis of the obtained sequences identifies the detected PCV2 as belonging to Genotype D.
Concerning PPV1, the phylogenetic analysis can provide little information since the parvoviral genes are highly conserved and no PPV1 wild boar sequences are available from Italy. More recent studies revealed that the virus could be divided into distinct clusters based on some amino acid substitutions on the VP1/VP2 genes. Moreover, few residue substitutions in the VP1/VP2 proteins can lead different virus strains to different tissue tropism, virulence and pathogenetic patterns. Consequently, there are some low-virulence strains of PPV1 (i.e., NADL-2), some moderately virulent strains and some highly virulent strains (e.g., Kresse, 27a strains) [74]. The results of the phylogenetic analysis indicate an association of our sequence with the highly virulent strains. This finding should raise questions about the impact on the swine industry related to pathogen transmission from wild to domestic swine.

Conclusions
In conclusion, this work evidenced the presence and circulation of three of the most important viral agents responsible for reproductive failures in swine in wild boar in Italy. The information about the reproductive impact of such viral agents on the wild boar population is still scarce due to the difficulties in identifying the negative impact on reproduction in a wild species with a high reproductivity rate. This finding suggests that SuHV-1 and PCV2, responsible for reproductive failures in domestic swine, can maintain the same tropism for foetal tissues in wild boar. This evidence could be useful to get additional knowledge about the reproductive performance in feral swine. Noteworthy, in the studied area the presence of wild boar is abundant, but few swine industries are present. However, swine breeding is mainly based on an extensive rearing system, and animals living outdoors have the possibility to frequently encounter wild animals.
Therefore, continuous monitoring of the health status of the wild boar population is important to monitor the presence of circulating pathogens, to provide sanitary indications.  Institutional Review Board Statement: Ethical review and approval were waived for this study, since animals were sampled during hunting activities regulated by National law, and no animals were killed for research purposes.

Conflicts of Interest:
The authors declare no conflict of interest.