Measurement of chromogranin A in porcine saliva: validation of a time-resolved immunofluorometric assay and evaluation of its application as a marker of acute stress

Abstract

The objective of this study was to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for porcine salivary chromogranin A (CgA) measurements, using a species-specific antibody, and evaluate its behaviour in an acute stress model. Polyclonal antibodies were produced in rabbits immunized with a synthetic porcine fragment of CgA 3592379 and used to develop a sandwich TR-IFMA. This TR-IFMA was analytically validated and showed intra-and inter-assay coefficients of variation of 6.23% and 5.82%, respectively, an analytical limit of detection of 4.27 3 10 23 mg/ml and a limit of quantification of 24.5 3 10 23 mg/ml. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution (r 5 0.975) and recovery tests. When a model of experimental acute stress, in which animals were immobilized for 3 min with a nose snare (stressor stimulus), was applied, a significant increase (P , 0.05) in CgA levels in saliva was detected at 15 min post-stressor stimulus. These results indicate that the assay developed in this study could measure CgA in porcine saliva in a reliable way and that the concentrations of CgA in saliva samples of pigs increase after an acute stress situation.

Response to lipopolysaccharide in salivary components and the submandibular gland of pigs

“…A previous study showed that CgA concentrations in human saliva are elevated under stress (Nakane et al, 2002). Another study showed that CgA concentration in saliva also increased after an acute stress situation in pigs (Escribano et al, 2013). Yet Kanno et al (1999) showed that an isolated rat submandibular gland perfused with noradrenaline at 1 mM produced a marked increase of CgA concentration in saliva, whereas further increases in the noradrenaline concentration (10 or 100 mM) yielded a concentration-dependent decrease.…”

Causes, consequences and biomarkers of stress in swine: an update

“…The release of CgA in saliva is mediated by the secretion of catecholamines [75], and has been related to the activation of SAM [76]. In humans, the measurement of CgA in saliva has been used as a reliable marker of SAM activation as an alternative to adrenaline and noradrenaline [77], which have high variability in results and low stability in saliva [78–80]. …”

“…In pig, CgA has been used as a marker of SAM activation in different situations such as immobilization with a nasal snare [80], after refeeding following a period of food deprivation [25] and after isolation or regrouping [63]. CgA is not affected by age, gender or circadian rhythms [81].…”

The stress response and exploratory behaviour in Yucatan minipigs (Sus scrofa): Relations to sex and social rank

“…These gregarious, inquisitive animals demonstrate sophisticated social behaviour [36,51], as well as consistent differences between individuals in behavioural responses to novelty [26,40]. Salivary cortisol has been assayed extensively in pigs [20,32,52] and, recently, pigs have been shown to produce measurable increases in the sympathetic markers, sAA [28] and CgA [21], in response to a stressor. The aim of the current study was to investigate the existence of consistent inter-individual variation in exploratory behaviour and reactivity of the two major stress systems, and describe their relationship.…”

“…We shipped samples on dry ice to Salimetrics LLC for analysis via enzyme immunoassay for sAA, cortisol, and CgA. Saliva flow rate was not measured, as valid measurements of our analytes of interest can be obtained without the need for assessing flow rate (sAA: [65]; cortisol: [81]; CgA: [21]). …”