S100A12 protein as a porcine health status biomarker when quantified in saliva samples.

The S100A12 protein was validated as a biomarker of health status in porcine saliva samples using a semi-quantitative approach based on Western blotting in four healthy and sixteen diseased animals, and in four animals with severe respiratory disease during three days of antibiotic therapy. Afterwards, a non-competitive sandwich immunoassay was then developed, validated, and used to quantify S100A12 in clinical porcine samples, using 14 healthy and 25 diseased pigs. Finally, the S100A12 concentrations in the saliva of ten pigs with respiratory disease were monitored during antibiotic therapy. Diseased animals showed higher concentrations of S100A12 than healthy animals, and the high concentrations of S100A12 in pigs with respiratory distress were reduced after antimicrobial therapy. The assay developed showed good precision and accuracy, as well as a low limit of detection of 3.19 ng/mL. It was possible to store saliva samples at -20 °C, or even at 4 °C, for two weeks before analysis without losing the validity of the results. The concentrations of S100A12 observed in serum and saliva samples showed a moderately positive association with a correlation coefficient of 0.48. The concentrations of the new validated biomarker S100A12 are highly associated with the novel salivary biomarker of inflammation, adenosine deaminase, and moderately to highly associated with the total oxidant status. The results reported in this study provide a new way of evaluating inflammatory diseases in pigs using saliva samples, which should be further explored for disease prevention and monitoring in the field.

Influence of the circadian cycle, sex and production stage on the reference values of parameters related to stress and pathology in porcine saliva.

BACKGROUND: The concentration of biomarkers in saliva could be influenced by several factors not related to the specific condition under analyses, which should be considered for proper clinical interpretation. In the present study, the circadian rhythm of C-reactive protein (CRP), haptoglobin (Hp), Pig-MAP, S100A12, Cu, Zn, Adenosine deaminase (ADA), total protein (TP), total antioxidant capacity (TAC), total oxidant status (TOS), oxidative stress index (OSI), cortisol and α-amylase in saliva of 20 female and 20 male pigs was investigated. Moreover, the influence of sex and production phase (post-weaning, fattening and finishing) on the concentrations of biomarkers in a total of 414 healthy pigs was studied and the reference intervals for all salivary biomarkers were calculated accordingly. RESULTS: All parameters except Pig-MAP, OSI and α-amylase varied significantly along the daytime, and most of them peak around early afternoon (13-15 h). The cosinor analysis described the temporal dynamics of circadian rhythms for all parameters. The range values showed differences between male and female pigs in 8 out of the 13 biomarkers, with higher concentrations in females in comparison to male pigs. The influence of the production phase on the salivary concentrations was observed for all the biomarkers. The highest concentrations were observed for Pig-MAP, S100A12 and α-amylase in post-weaning animals, for TP in growing pigs and for OSI in finishing animals. Most of the sex-influenced biomarkers showed the highest concentrations at growing stages with some exceptions such as ADA or Hp that showed the peak at finishing and post-weaning stages respectively. CONCLUSIONS: It is necessary to establish the optimal daytime for routine saliva sampling to avoid circadian variations and for that end, the time interval between 10:00 a.m. to 12:00 a.m. is highly recommended. The factors sex and production phase influence the concentration of biomarkers and should be considered for proper biomarker interpretation. The reference intervals presented here for each salivary biomarker will help to correctly interpret the results of these analytes and contribute to the use of saliva as a non-invasive sample for the diagnosis and monitoring of the health status of swine farms.

The Connection Between Stress and Immune Status in Pigs: A First Salivary Analytical Panel for Disease Differentiation.

This paper analyzes the association between stress and immune response activations in different diseases, based on the salivary analytics. Moreover, a first attempt to discriminate between diseases was performed by principal component analysis. The salivary analytics consisted of the measurement of psychosocial stress (cortisol and salivary alpha-amylase) indicators, innate (acute phase proteins: C-reactive protein and haptoglobin), and adaptive immune (adenosine deaminase, Cu and Zn) markers and oxidative stress parameters (antioxidant capacity and oxidative status). A total of 107 commercial growing pigs in the field were divided into six groups according to the signs of disease after proper veterinary clinical examination, especially, healthy pigs, pigs with rectal prolapse, tail-biting lesions, diarrhea, lameness, or dyspnea. Associations between stress and immune markers were observed with different intensities. High associations (r = 0.61) were observed between oxidative stress markers and adaptive immune markers. On the other hand, moderate associations (r = 0.31-0.48) between psychosocial stress markers with both innate and adaptive immune markers were observed. All pathological conditions showed statistically significant differences in at least 4 out of the 11 salivary markers studied, with no individual marker dysregulated in all the diseases. Moreover, each disease condition showed differences in the degree of activation of the analyzed systems which could be used to create different salivary profiles. A total of two dimensions were selected through the principal component analysis to explain the 48.3% of the variance of our data. Lameness and rectal prolapse were the two pathological conditions most distant from the healthy condition followed by dyspnea. Tail-biting lesions and diarrhea were also far from the other diseases but near to healthy animals. There is still room for improvements, but these preliminary results displayed a great potential for disease detection and characterization using salivary biomarkers profiling in the near future.

A multi-herd study shows that saliva is more than a reflection of serum biomarkers in pigs.

This study evaluates if biomarkers of porcine health status in saliva samples is a mere reflection of serum to detect disease in pigs under field conditions. Four farms from the same commercial company were included to obtain samples from animals with different pathological conditions. A total of 10 healthy animals and 10-15 animals from each farm with clinical symptoms of the disease were sampled for paired saliva and blood during a veterinary clinical visit. The biomarker panel included acute-phase proteins (APPs), C-reactive protein (CRP), haptoglobin (Hp), an inflammatory marker, adenosine deaminase (ADA), the total antioxidant capacity (TAC), the levels of essential trace elements, copper (Cu) and zinc (Zn), and the measurement of the total protein content (TP). After detailed statistical analysis, the results showed that saliva could replace serum for APP measurements since a good agreement has been observed between the concentrations of APPs in both body fluids. For any other biomarker, no agreement between the concentrations quantified in serum and saliva samples was observed visually. However, salivary ADA and TP concentrations were statistically significantly higher in the diseased, whereas the statistical tests with serum concentrations were inconclusive. Furthermore, greater differentiation between healthy and diseased animals could be observed when the distribution of biomarkers was analysed in saliva than in other serum samples. The diagnostic power to discriminate between healthy and diseased pigs is similar in saliva and in serum samples. Preliminary regression models may offer an optimal combination of biomarkers for disease detection in saliva (Hp, CRP, and TAC) and serum (Hp, CRP, and Cu), which demands less labour, sample, and financial cost for saliva determinations. The contradictory results observed for TAC, Cu, and Zn levels between body fluids indicate a need for further studies. To sum up, saliva-based biomarkers instead of serum-based biomarkers could contribute to more efficient detection of diseased animals.