Free-ranging wild boars exhibit elevated circulating IL-6 concentrations compared with domestic pigs from Mediterranean ecosystem

Technical Summary: Ecological Drivers of Systemic Inflammation in Sus scrofa

Problem Statement
While host ecology is recognized as a major determinant of immune function, the specific influence of environmental conditions on systemic inflammatory phenotypes remains poorly understood in closely related wild and domestic vertebrates. Previous comparative studies between domestic pigs (Sus scrofa domesticus) and wild boars (Sus scrofa) have largely focused on genetic divergence or responses to specific infectious challenges, leaving a gap in knowledge regarding circulating cytokine profiles in naturally exposed populations. This is particularly relevant in Mediterranean ecosystems where wildlife and livestock share pathogens of zoonotic importance, such as Leishmania infantum. The study addresses the need to characterize how contrasting ecological contexts (free-ranging vs. intensive rearing) shape immune phenotypes at the wildlife-livestock interface, specifically hypothesizing that free-ranging wild boars exhibit a more activated inflammatory phenotype than domestic pigs.

Methodology
The study employed a comparative cross-sectional design involving 67 animals from eastern Spain: 48 intensively reared domestic pigs and 19 free-ranging wild boars.

• Sampling: Blood samples were collected post-mortem from legally harvested wild boars and via ear vein puncture from domestic pigs during routine veterinary procedures.
• Cytokine Profiling: Plasma concentrations of nine cytokines (IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12/IL-23p40, IFN-α, IFN-γ, and TNF-α) were quantified using a multiplex immunoassay (ProcartaPlex™ Porcine Cytokine and Chemokine Panel 1) on a Luminex™ 200 platform.
• Pathogen Screening: Animals were screened for Leishmania infantum exposure via serology (ELISA) and molecular detection (real-time PCR targeting the kinetoplast minicircle region).
• Statistical Analysis: Data were analyzed using non-parametric tests (Mann–Whitney U) with Benjamini–Hochberg false discovery rate (FDR) correction. Multivariable linear models were fitted to adjust for age, sex, and infection status (PCR/ELISA). Multivariate patterns were explored using Principal Component Analysis (PCA), hierarchical clustering, and PERMANOVA. Sensitivity analyses were conducted by restricting the dataset to yearlings and PCR-characterized animals to control for age structure and infection status.

Key Results

• Cytokine Differences: Wild boars exhibited significantly higher circulating concentrations of IL-1β and IL-6 compared to domestic pigs. Conversely, domestic pigs showed higher concentrations of IL-12/IL-23p40.
• Magnitude of IL-6 Elevation: The most pronounced difference was observed in IL-6. The median concentration in wild boars was approximately 21-fold higher than in domestic pigs (92.83 pg/mL vs. 4.44 pg/mL; FDR < 0.0001). Effect size analysis (Cliff's δ = -0.760) indicated a large effect.
• Independence from Confounders: After adjusting for age, sex, and L. infantum infection status (both serological and molecular), IL-6 remained the only cytokine independently associated with host population (β = 2.475, FDR = 0.001), corresponding to an approximate 12-fold increase on the original scale. The associations for IL-1β and IL-12/IL-23p40 lost significance after adjustment, suggesting they were partially driven by demographic or infection-related factors.
• Global Cytokine Architecture: Despite the marked elevation in IL-6, multivariate analyses (PCA and hierarchical clustering) revealed substantial overlap between the two populations. PERMANOVA detected a significant but modest effect of host population on overall cytokine composition (R² = 0.063, p = 0.0035), indicating that ecological divergence affects specific inflammatory pathways rather than restructuring the entire cytokine network.
• Robustness: Sensitivity analyses restricted to yearlings and PCR-negative animals confirmed that the elevated IL-6 in wild boars was not solely attributable to age structure or active L. infantum infection.

Significance and Claims
The study identifies circulating IL-6 as the most robust immune marker associated with ecological background in suids. The authors claim that free-ranging wild boars exhibit a markedly elevated systemic inflammatory tone compared to domestic pigs, a phenotype that persists even when controlling for specific pathogen exposure (L. infantum) and demographic variables.

The findings support the ecological immunology hypothesis that animals exposed to heterogeneous environments and continuous pathogen pressure maintain a higher basal immune activation than those in controlled conditions. However, the paper modestly notes that while IL-6 is a strong differentiator, the overall cytokine architecture remains largely conserved between the species. The authors conclude that ecological background influences specific inflammatory pathways rather than generating fundamentally distinct immune architectures.

From a One Health perspective, the study highlights the potential role of ecological conditions in shaping immune phenotypes at the wildlife-livestock interface. The authors suggest that understanding these environmental influences on host immunity may improve insights into pathogen maintenance and disease emergence, though they explicitly state that whether this elevated inflammatory phenotype influences pathogen susceptibility or transmission dynamics warrants further investigation. The study does not claim to identify the specific environmental factors (e.g., nutrition, stress, specific parasite burden) responsible for the IL-6 elevation, noting this as a limitation for future research.