Emerging Porcine Salivary Biomarkers Worth Watching

Porcine salivary biomarkers are gaining traction as non-invasive tools for monitoring swine health, immune status, and welfare. Beyond the classic serum cytokines (TNF-α, IL-6, IL-1β), six markers — ITIH4/Pig-MAP, S100A12, ADA, PCT, MPO, and LDH — now have validated ELISA or enzymatic assays in oral fluid, supported by peer-reviewed studies from 2022–2025. This guide profiles each candidate and offers practical guidance on what to confirm before you commit it to a larger study or scale your porcine ELISA workflow.

Why Move Beyond Classic Serum Cytokines in Swine Research

For years, porcine immunology studies leaned heavily on a short list of serum cytokines — TNF-α, IL-6, IL-1β — and a handful of major acute-phase proteins (APPs) such as haptoglobin (Hp) and C-reactive protein (CRP). These markers are well characterized and commercially supported, but they share one practical limitation: they require venipuncture.

Repeated blood sampling introduces its own confound. Stress responses alter the very cytokine landscape researchers are trying to measure. For longitudinal studies, welfare-compliance work, and farm-scale surveillance, a less invasive approach is increasingly attractive — and that is exactly where porcine salivary biomarkers are gaining ground.

Over the past three years, peer-reviewed studies from veterinary research groups in Spain, Sweden, and Brazil have validated assay methods for markers that are detectable in porcine oral fluid, respond robustly to inflammation, infection, and stress, and can now be quantified using commercial or in-house ELISA platforms with documented precision and accuracy.

The Six Porcine Salivary Biomarkers at a Glance

Source validated porcine ELISA kits. The biomarkers below map to off-the-shelf porcine ELISA kits available on eBioHippo. For markers without a catalog kit yet (S100A12 and ADA), our team can source or quote one on request.

ITIH4 / Pig-MAP — Salivary Assay Validation and Performance

What it is. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), also known as Pig-MAP (porcine major acute-phase protein), is the dominant positive APP in swine. In plasma, it rises sharply during infection and systemic inflammation and has been used for decades as a disease-monitoring tool in production settings.

Why saliva matters now. The practical barrier to saliva-based ITIH4 measurement has been assay performance: commercial ELISA kits are designed and validated for plasma, not oral fluid. A 2025 study in PLOS ONE (Ortín-Bustillo et al., 2025) evaluated four commercially available porcine ITIH4 ELISA kits against salivary samples from pigs with and without tail biting. Two of the four kits demonstrated acceptable precision and accuracy in saliva; one of the two also detected statistically significant differences between the tail-biting and control groups. Western blotting confirmed that ITIH4 in saliva presents in different molecular-weight forms than in plasma — an important nuance when interpreting results or cross-comparing with serum data.

An independent 2024 study in Research in Veterinary Science (Piñeiro et al., 2024) reported a sensitive in-house time-resolved immunoassay able to quantify Pig-MAP specifically in pig saliva, extending the toolkit beyond commercial options.

Practical takeaway. ITIH4 adds robust systemic-inflammation context to panels that otherwise rely on transient cytokines. Expect it to be slower-moving than IL-6 but more sustained. Not all commercial kits perform equivalently in saliva — pre-validate your specific kit lot against the matrix you plan to use.

S100A12 as a Porcine Salivary Health-Status Marker

What it is. S100A12 is a calcium-binding protein of the S100/calgranulin family, secreted predominantly by activated neutrophils. In human medicine it serves as a marker of innate immune activation and mucosal inflammation. In pigs, it has attracted increasing attention as a saliva-detectable readout of health versus disease.

The evidence base. A 2024 study in The Veterinary Journal (Gutiérrez et al., 2024) described the development and analytical validation of an immunoassay for S100A12 in porcine clinical samples, including saliva, reporting statistically robust differences between healthy cohorts and animals with active disease. A 2023 paper in Research in Veterinary Science (Botía et al., 2023) validated a spectrophotometric approach and showed that salivary S100A12 rises in response to LPS challenge, non-septic inflammation, and slaughter-transport stress, and shows circadian variation in healthy animals (peaking around 16:00 h).

Practical takeaway. Time-of-day standardization is not optional for S100A12 — the circadian signal is real and documented. Collect samples at a fixed time each day. S100A12 complements ITIH4/Pig-MAP by providing a faster-moving, neutrophil-linked signal; together they capture both acute and sustained dimensions of the inflammatory response.

Adenosine Deaminase (ADA) — T-Cell Immune Activation Marker

What it is. Adenosine deaminase (ADA, EC 3.5.4.4) catalyzes the conversion of adenosine to inosine in the purine catabolism pathway. Because this reaction is tightly linked to lymphocyte proliferation and differentiation, ADA activity in biological fluids reflects the intensity of adaptive immune responses — particularly T-cell activation.

Published performance in pigs. A 2023 study in BMC Veterinary Research (López-Martínez et al., 2023) measured multiple salivary biomarkers in pigs with Streptococcus suis-induced meningitis. ADA showed large-magnitude increases in the saliva of infected animals compared with controls, outperforming several other markers in fold-change magnitude. A 2022 study in the International Journal of Molecular Sciences extended proteomic validation to further characterize ADA behavior in this infection model.

Practical takeaway. ADA's large signal amplitude in infectious-disease contexts makes it useful for detecting immune activation even when other markers are borderline. Its spectrophotometric or colorimetric format is accessible in most labs without specialized equipment. Confirm tolerance to lipemia and hemolysis in your specific sample matrix, as these can introduce interference in enzymatic assays.

Procalcitonin (PCT) — Bacterial Infection Severity in Pig Saliva

What it is. Procalcitonin is the peptide precursor of calcitonin, produced at high levels during systemic bacterial infection and sepsis. In human critical care, serum PCT is widely used to differentiate bacterial from viral infection and to guide antibiotic-therapy decisions. Its utility in pigs has been less explored — until recently.

The porcine data. A 2022 pilot study in BMC Veterinary Research described the first validated approach for measuring PCT in porcine saliva, confirming that the marker is detectable in oral fluid and rises in disease contexts. A 2023 study reported salivary PCT increases in pigs with S. suis infection. More recently, a 2025 study examining tail-biting pigs referenced PCT among a novel set of salivary biomarkers responsive to this chronic welfare stressor.

Practical takeaway. PCT signals systemic bacterial-infection severity. Outside strong inflammatory phenotypes, baseline concentrations can be low, placing demands on assay sensitivity. Confirm the lower limit of quantification (LLOQ) of your chosen assay against your expected range before committing PCT to a panel. Pair it with Pig-MAP and S100A12 for multi-layer characterization of infection severity.

Myeloperoxidase (MPO / Mpx) — Direct Readout of Neutrophil Activity

What it is. Myeloperoxidase is an enzyme stored in the azurophilic granules of neutrophils and monocytes. Upon activation it catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions — a key component of the neutrophil oxidative burst against pathogens. Salivary MPO activity therefore reflects the degree of local and systemic neutrophil recruitment and activation.

Published validation in pigs. The 2023 Botía et al. study in Research in Veterinary Science is the primary reference: it developed, validated, and characterized a spectrophotometric MPO assay for porcine saliva on an automated platform, with adequate analytical precision and accuracy. In 20 healthy pigs sampled every four hours, MPO activity showed significant circadian variation (peaking around 16:00 h), consistent with the pattern observed for S100A12. Following LPS administration, non-septic inflammation, and slaughter transport, MPO levels increased significantly in all challenge models.

Practical takeaway. MPO adds mechanistic resolution that APPs alone cannot provide — it distinguishes neutrophil-dominant responses from monocyte/macrophage-dominated ones. Like S100A12, it is sensitive to circadian sampling time and to handling-related processing delays. Standardize time-to-freezer and freeze-thaw cycles rigorously.

Damage and Context Markers: LDH, AST, and Total Protein

What these are. Lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and total protein are not primarily immune markers, but they provide critical interpretive context for immune panels. LDH is released from damaged or lysed cells. AST reflects hepatocellular and muscle damage. Total protein is both a physiological readout and a sample-quality indicator — unexpectedly high total protein in saliva may indicate blood contamination from oral lesions.

Why they matter. The 2023 S. suis infection study and a 2025 study on infection-associated analyte shifts in porcine saliva and serum both reported co-movement of LDH and AST alongside the immune markers above. This co-movement helps researchers judge whether an elevated cytokine or APP signal reflects true immune activation or is confounded by tissue damage and cellular lysis.

Practical takeaway. Include at least one damage marker (LDH is the most practical) in any multi-analyte porcine salivary panel. Treat these as interpretive companions and sample-quality flags, not primary endpoints. They are also useful for retrospectively identifying outlier samples before data cleaning.

How to Validate ELISA Kits in Porcine Saliva Before Scaling

The literature cited here reflects work done in specific experimental models, collection protocols, and disease contexts. Before incorporating any marker into a larger cohort study, confirm the following:

1. Matrix-matched validation. Do not assume that assay performance in plasma transfers to saliva. The ITIH4/Pig-MAP case is instructive: four commercial kits were tested, two passed the performance threshold in saliva, and only one reliably detected biological differences. Treat saliva validation as a separate analytical undertaking from plasma validation.
2. Dilution linearity. Run a wide dilution series (e.g., 1:2 to 1:16) on pooled porcine saliva. Linearity failures reveal high-background artifacts, hook effects at the top of the curve, or matrix suppression — any of which compromise field-study data.
3. Spike recovery. Add a known quantity of recombinant protein or calibrator to your matrix and calculate percent recovery. Acceptable recovery is generally 80–120%. Suppression below this range indicates the need for dilution, matrix-matched standards, or a different assay format.
4. Pre-analytical SOP. Saliva is sensitive to collection-device type (Salivette-type sponge tubes are common in published porcine studies), centrifugation speed and time, time between collection and freezing, and the number of freeze-thaw cycles. Botía et al. documented that all three of their validated analytes (MPO, S100A12, ITIH4) were affected by sampling time of day — build a fixed-hour collection protocol into your design from the start.
5. Reference-range establishment. Published baselines from the University of Murcia group and collaborators are useful benchmarks, but they reflect specific herd conditions, breed compositions, and seasonal contexts. Establish your own baseline in a representative subset before interpreting disease-group data.

Building a Practical Porcine Salivary Panel

Based on the published evidence, the following two-tier framework offers a starting point for non-invasive porcine biomarker panels:

Core panel (broadly applicable):

• ITIH4 / Pig-MAP — systemic inflammation magnitude
• S100A12 — innate immune activation, circadian-controlled
• MPO / Mpx — neutrophil activation, mechanistic readout

Supplemental panel (infection / severity contexts):

• ADA — adaptive (T-cell) immune activation, large fold-change signal
• PCT — bacterial-infection severity framing
• LDH — tissue-damage context and sample QC

This structure mirrors the stratified approach used in several referenced publications: a fast-moving neutrophil/alarmin layer (S100A12, MPO), a sustained acute-phase layer (ITIH4), and a mechanistic/severity layer (ADA, PCT, LDH). To source assays, browse our ELISA kits, porcine ELISA reagents, or explore the veterinary & agricultural research area. For markers without an off-the-shelf saliva-validated kit, request assay guidance from our team.

References

1. Ortín-Bustillo A, et al. ITIH4 (Pig-MAP) in pig saliva: evaluation of commercially available ELISA kits. PLOS ONE. 2025. https://doi.org/10.1371/journal.pone.0335133
2. Piñeiro M, Matas-Quintanilla M, Miralles A, Gutiérrez AM. A sensitive immunoassay for the quantitation of Pig-MAP in pig saliva samples. Research in Veterinary Science. 2024;180:105432. https://doi.org/10.1016/j.rvsc.2024.105432
3. Botía M, Ortín-Bustillo A, et al. Gaining knowledge about biomarkers of the immune system and inflammation in the saliva of pigs: myeloperoxidase, S100A12, and ITIH4. Research in Veterinary Science. 2023;164:104997. https://doi.org/10.1016/j.rvsc.2023.104997
4. Gutiérrez AM, et al. S100A12 as a porcine saliva health-status biomarker; immunoassay development and validation. The Veterinary Journal. 2024.
5. López-Martínez MJ, et al. Changes in salivary biomarkers of stress, inflammation, redox status, and muscle damage due to Streptococcus suis infection in pigs. BMC Veterinary Research. 2023;19:100. https://doi.org/10.1186/s12917-023-03649-0
6. Proteomics/validation context for ADA performance in meningitis. International Journal of Molecular Sciences. 2022.
7. Measurement of procalcitonin in saliva of pigs: a pilot study. BMC Veterinary Research. 2022.
8. Infection-context saliva and serum analyte shifts including S100 proteins, ADA, and LDH. Springer. 2025.

This article summarizes peer-reviewed research findings for R&D and study-planning purposes. It is not intended as a clinical diagnostic guideline. Researchers should validate all assays in their specific matrix and experimental conditions before use.