PSIII-27 Comparing Bovine salivary protein and immunoglobulin adsorption using three non-invasive collection methods.

Abstract Saliva is crucial for cattle digestion, rumen health, and immune defense, making it a valuable non-invasive source of biomarkers. Saliva contains immunoglobulins (Igs) that can modulate the ruminal microbiome. Salivary proteins are synthesized in the salivary gland, and upon secretion are exposed to proteolytic enzymes. Evidence indicates that different collection and long-term storage methods can alter the salivary proteome. While proteolytic enzyme activity may in part explain the variance in saliva proteins, another explanation could be differences in protein adsorption between sample collection methods. Protein adsorption is the process by which proteins adhere to a surface without surface penetration. Understanding the impacts of sampling method on protein adsorption will allow for more uniform sampling. We hypothesized that immunoglobulins/proteins would have different adsorption between three non-invasive sampling techniques – a cellulose sponge (SPNG), absorbent swab (SWAB), or unabsorbed (NEAT). First, “synthetic” saliva was produced using phosphate buffered saline (pH 8.8), sodium bicarbonate (80 mM), bovine mucin from the submaxillary gland (1.5 mg/mL), bovine serum albumin (BSA; 250 μg/mL), bovine IgG (10 ng/mL) and recovered with either SPNG, SWAB, or maintained NEAT. Next, 15 mL of “natural” saliva was collected from healthy steers (n = 4; 559.3 ± 234.3 kgs) via suction pipette attached to a 50 mL conical tube and motorized pump. “Natural” saliva was spiked with known concentrations of BSA (250 – 1250 μg/mL) and recovered with either SPNG, SWAB, or maintained NEAT. Following recovery, total protein was estimated using a bicinchoninic acid colorimetric assay. Next, “natural” saliva aliquots were thawed and recovered with either SPNG, SWAB, or maintained NEAT for quantification of total IgA using a commercially available ELISA. Lastly, a time course was conducted to compare the effect of storage condition on proteolysis. Aliquots were thawed and either maintained on wet ice, refrozen, maintained on wet ice with a protease inhibitor (PI), or refrozen with PI for 4 h, 2 h, 1 h, or 0.5 h. Total protein was quantified. Analyses were performed with SAS 9.4. There was no effect of collection method on total protein or IgA adsorption (P > 0.10). Regardless of collection method, there was a significant (P < 0.0001) positive correlation between the expected and measured amount of protein spiked in “natural” saliva. There was no effect (P > 0.10) of treatment, time, or the interaction on protein abundance following the time course in “natural” saliva. Collectively, the collection methods tested in the present study demonstrated minimal risk for protein adsorption and established that bovine salivary samples maintained on wet ice for up to 4 h have minimal risk for post-harvest proteolysis. These findings support the development of non-invasive sampling protocols to improve research, monitor vaccination immune responses, and promote sustainable livestock production.