Validation of an assay for quantification of alpha-amylase in saliva of sheep.

The objective of this study was to develop a time-resolved immunofluorometric assay (TR-IFMA) for quantification of salivary alpha-amylase in sheep. For that purpose, after the design of the assay, an analytical and a clinical validation were carried out. The analytical validation of the assay showed intra- and inter-assay coefficients of variation (CVs) of 6.1% and 10.57%, respectively and an analytical limit of detection of 0.09 ng/mL. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution. For clinical validation, a model of acute stress testing was conducted to determine whether expected significant changes in alpha-amylase were picked up in the newly developed assay. In that model, 11 sheep were immobilized and confronted with a sheepdog to induce stress. Saliva samples were obtained before stress induction and 15, 30, and 60 min afterwards. Salivary cortisol was measured as a reference of stress level. The results of TR-IFMA showed a significant increase (P < 0.01) in the concentration of alpha-amylase in saliva after stress induction. The assay developed in this study could be used to measure salivary alpha-amylase in the saliva of sheep and this enzyme could be a possible noninvasive biomarker of stress in sheep.

L'objectif de la présente étude était de développer un test immunofluorométrique en temps résolu (TIMF-TR) pour la quantification de l'alpha-amylase salivaire chez le mouton. À cette fin, suite au design du test, une validation analytique et clinique fut effectuée. La validation analytique du test a montré des coefficients de variation (CV) intra- et inter-tests de 6,1 % et 10,57 %, respectivement, et une limite de détection analytique de 0,09 ng/mL. Le test a également montré un haut niveau de précision, tel que déterminé par la linéarité suite aux dilutions. Pour la validation clinique, un modèle de test de stress aigu a été mené afin de déterminer si des changements significatifs attendus de l'alpha-amylase étaient détectés dans le nouveau test développé. Dans ce modèle, 11 moutons étaient immobilisés et confrontés avec un chien de berger afin d'induire le stress. Des échantillons de salive ont été obtenus avant l'induction du stress et 15, 30, et 60 min par la suite. Le cortisol salivaire a été mesuré à titre d'indicateur de référence du stress. Les résultats du TIMF-TR ont montré une augmentation significative (P < 0,01) de la concentration d'alpha-amylase dans la salive après l'induction du stress. Le test développé au cours de cette étude pourrait être utilisé afin de mesurer l'alpha-amylase salivaire dans la salive de mouton et cet enzyme pourrait être un biomarqueur non-invasif du stress chez le mouton.(Traduit par Docteur Serge Messier).

Porcine salivary analysis by 2-dimensional gel electrophoresis in 3 models of acute stress: a pilot study.

The purpose of this research was to study changes in the salivary proteome of healthy pigs in stressful situations to identify any potential new salivary biomarker of stress. Three groups of animals were subjected to 3 stress models: snaring restraint followed by simulated sampling of vena cava blood; brief transport by road; and restriction of movement in a digestibility cage. Saliva was obtained from each animal before and 15 and 30 min after the induction of stress. The samples from the animals that showed the greatest increase in salivary cortisol concentration were pooled and run on 2-dimensional gels. Coomassie Brilliant Blue R-250 was used for spot detection and mass spectrometry for spot identification. Statistical analyses showed that 2 proteins had significant differences in expression before and after the induction of stress. These proteins were identified as odorant-binding protein and fragments of albumin. Further studies will be necessary to confirm the value of using these proteins as salivary biomarkers of stress in pigs.

L'objectif de la présente recherche était d'étudier les changements dans le protéome salivaire de porcs en santé dans des situations de stress afin d'identifier de nouveaux biomarqueurs de stress potentiels. Trois groupes d'animaux ont été soumis à 3 modèles de stress : contention au moyen d'un lasso suivie par simulation d'une ponction sanguine de la veine cave; bref transport sur route; et restriction des mouvements dans une cage à digestibilité. De la salive fut obtenue de chaque animal avant, ainsi que 15 et 30 minutes suivant l'induction du stress. Les échantillons provenant des animaux qui présentaient la plus grande augmentation de concentration de cortisol salivaire ont été regroupés et analysés sur gels en 2-dimensions. Le Bleu Brillant de Coomassie R-250 fut utilisé pour détection de taches et la spectrométrie de masse pour identification des taches. Les analyses statistiques ont montré que deux protéines avaient des différences significatives dans leur expression avant et après l'induction du stress. Ces protéines ont été identifiées comme étant une protéine de transport des odorants et des fragments de l'albumine. Des études ultérieures seront nécessaires pour confirmer la valeur d'utiliser ces protéines à titre de biomarqueurs salivaires du stress chez les porcs.(Traduit par Docteur Serge Messier).

A proteomic approach to porcine saliva.

This paper reviews recent progress in salivary animal proteomics, with special reference to the porcine proteome. Until fairly recently, most studies on saliva as a diagnostic fluid have focused on humans, primates and rodents, and the development of salivary analysis in monitoring health in farm animals including pigs has received only limited consideration. The porcine salivary proteome has been characterised by 2D-electrophoresis followed by mass spectrometry. Major and minor proteins have been identified. The use of saliva as a non-invasive biological fluid in monitoring health and disease in pigs will be reviewed, together with the potential use of proteomics for the development of biomarkers. In this review, methods of collection and the composition of porcine saliva will be considered, together with saliva handling and analysis. The overall findings indicate that there is considerable potential for the development of salivary analysis as a non-invasive diagnostic fluid in the pig, and that it offers advantages over other body fluids in this animal.

Saliva chromogranin A in growing pigs: a study of circadian patterns during daytime and stability under different storage conditions.

Salivary chromogranin A (CgA) is considered to be a biomarker of activation of the sympatho-adrenomedullary system, and has recently been proposed as a useful indicator of the acute stress response in pigs. The aim of the present study was to determinate whether salivary CgA concentrations in healthy growing pigs exhibits any circadian pattern during the daytime, and to evaluate its stability under different storage conditions. A total of 80 pigs (40 in spring and another 40 in autumn) of two different ages and genders were used. To establish the circadian pattern, saliva samples were collected at 07.00, 11.00, 15.00 and 19.00 h on two consecutive days. Pooled samples were used for the stability study and were measured on the day of sampling and periodically for up to 360 days later. Samples were stored at 4 °C, -20 °C or -80 °C and the effect of repeated freezing and thawing was also evaluated. No circadian pattern was detected for salivary CgA in either season and there were no significant effects of gender or age. However, mean salivary CgA concentrations were significantly higher (P<0.0001) in the pigs sampled in autumn, compared to those sampled in the spring. Short term storage at 4 °C is recommended for up to 2 days, whereas frozen samples can be stored for 1 year at -20 °C or -80 °C, without substantial reduction in CgA values. In addition, samples can be frozen and thawed up to seven times without significant loss of the biomarker.

Assessment of stress associated with an oral public speech in veterinary students by salivary biomarkers.

In this report, salivary cortisol, alpha-amylase activity, and testosterone were measured to assess stress associated with a public oral presentation in Veterinary Clinical Pathology students. Stimulated saliva samples were collected before and directly after a 5-minute oral presentation and at 20 and 35 minutes after the beginning of the presentation. Cortisol peaked 20 minutes after the beginning of the presentation, whereas salivary alpha-amylase (sAA) peaked at 5 minutes, just at the end of the speech. These changes were not related to the level of stress that was indicated by the student in a questionnaire, the student's sex, or the quality of the presentation. No changes were detected in testosterone levels during the study. Saliva biomarkers evaluated in this research could be extended to other stress-producing situations in the university life of veterinary students.

Validation of an automated chemiluminescent immunoassay for salivary cortisol measurements in pigs.

The aim of the current study was to validate an automated immunoassay for cortisol quantification in the saliva of pigs. The assay had intra- and interassay coefficients of variation lower than 16%, in all cases. The limit of detection was 0.016 µg/dl, and the lower quantification limit was 0.197 µg/dl. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution and recovery tests. In addition, this assay was used to quantify cortisol in 2 models of stress: 1 in which animals were immobilized with a nose-snare and 1 in which pigs were transported for a duration of 30 min. In both cases, a significant increase (P < 0.01) in salivary cortisol was detected after the stressful situation. Overall, the assay validated in the present study could be used for the evaluation of cortisol changes in stressful situations.