Measurement of haptoglobin in saliva of cows: Validation of an assay and a pilot study of its potential application.

In recent years, the use of saliva as a matrix for the measurement of biomarkers of health and welfare is gaining importance due to its non-invasive collection. Haptoglobin (Hp) is an acute-phase protein involved in the inflammatory response and changes in its concentration can provide information about the health status of the animals. This study aimed to develop and validate an assay based on luminescent amplification (AlphaLISA technology) for the measurement of Hp in bovine saliva and to study the possible changes in different inflammatory situations such as peripartum period and lameness. The assay proved to be accurate, reliable, and sensitive for the measurement of Hp in cow saliva (coefficient of variation (CV) 7.57%; coefficient of determination (R2) 0.992; recovery test 105.15%; lower limit of quantification (LLQ) 7.9 ng/ml). Significant differences were observed between Hp levels in saliva of cows before (13 days before) and after (7 and 20 days after) calving and at the moment of calving (p < 0.0001), and between lame and healthy cows (p < 0.008). In conclusion, this assay can detect Hp in a precise, sensitive, and accurate way in saliva of cows. Future studies with a larger population and different disease conditions should be conducted to determine the potential of Hp as an inflammatory biomarker in cow saliva.

Changes in salivary analytes in cows due to the in vitro presence of feed.

BACKGROUND: The effect in a sialochemistry profile of the presence of usually available feed in dairy cows was evaluated by an in vitro experiment. For this purpose, a pooled clean saliva from five healthy dairy cows was incubated five times with a standard feed based on a total mixed ration (F), wheat hay (H), and grass (G). The salivary panel was integrated by biomarkers of stress (cortisol -sCor-, salivary alpha-amylase -sAA-, butyrylcholinesterase -BChE-, total esterase -TEA-, and lipase -Lip-), immunity (adenosine deaminase -ADA-), oxidative status (Trolox equivalent antioxidant capacity -TEAC-, the ferric reducing ability of saliva -FRAS-, the cupric reducing antioxidant capacity -CUPRAC-, uric acid, and advanced oxidation protein products -AOPP-), and enzymes, proteins, and minerals of general metabolism and markers of liver, muscle, and renal damage (aspartate aminotransferase -AST-, alanine aminotransferase -ALP-, γ-glutamyl transferase -gGT-, lactate dehydrogenase -LDH-, creatine kinase -CK-, creatinine, urea, triglycerides, glucose, lactate, total protein, phosphorus, and total calcium). RESULTS: Most of the evaluated analytes showed a coefficient of variations (CV) higher than 15% and/or significant changes compared with the clean saliva when feed was present. Some analytes, such as the oxidative status biomarkers (CV > 80%), AST (CV > 60%), or glucose (CV > 100%), showed significant changes with all the feed types tested. Others showed significant differences only with certain types of feed, such as LDH with F (CV > 60%) or triglycerides with F (CV > 100%) and H (CV > 95%). However, sCor or gGT remained unchanged (CV < 15%, P > 0.05) in all the treatments. CONCLUSIONS: The presence of feed can produce changes in most of the analytes measured in cows' saliva, being of high importance to consider this factor when saliva is used as a sample to avoid errors in the interpretation of the results.

Basics for the potential use of saliva to evaluate stress, inflammation, immune system, and redox homeostasis in pigs.

The use of saliva as a biological sample has many advantages, being especially relevant in pigs where the blood collection is highly stressful and painful, both for the animal and the staff in charge of the sampling. Currently one of the main uses of saliva is for diagnosis and detection of infectious diseases, but the saliva can also be used to measure biomarkers that can provide information of stress, inflammation, immune response and redox homeostasis. This review will be focused on the analytes that can be used for such evaluations. Emphasis will be given in providing data of practical use about their physiological basis, how they can be measured, and their interpretation. In addition, some general rules regarding sampling and saliva storage are provided and the concept of sialochemistry will be addressed. There is still a need for more data and knowledge for most of these biomarkers to optimize their use, application, and interpretation. However, this review provides updated data to illustrate that besides the detection of pathogens in saliva, additional interesting applicative information regarding pigs´ welfare and health can be obtained from this fluid. Information that can potentially be applied to other animal species as well as to humans.

Insulin in the saliva of pigs: Validation of an automated assay and changes at different physiological conditions.

This study aimed to evaluate whether insulin could be measured in the saliva of pigs and if its concentration changes in some physiological conditions. For this purpose, a validation of an automated heterologous immunoassay for measuring insulin in the saliva of pigs was performed. In addition, the possible changes of salivary insulin concentration in sows after food intake and during gestation and lactation were studied. The evaluated immunoassay was able to detect insulin in the saliva of pigs in a precise and accurate way when species-specific calibrators were used. There was no correlation in insulin concentrations between serum and saliva. Insulin concentrations showed a significant increase in the saliva of sows after feeding. Sows at farrowing and lactation presented higher salivary insulin levels as compared with those in gestation. In conclusion, the results showed that insulin could be measured in the saliva of pigs, and changes in its concentration can be detected due to food intake and different physiological conditions.

Changes in saliva proteins in cows with mastitis: A proteomic approach.

This study aimed to evaluate the possible saliva proteome changes in cows with mastitis using a Tandem Mass Tags (TMT) proteomics approach. For this purpose, the salivary proteomes from healthy cows and cows with mastitis were analysed, and their serum proteomes were also studied for comparative purposes. A total of eight saliva and serum paired samples for each group were used for the proteomic study, and eight additional samples for each group were analysed in the analytical and overlap performance studies. In saliva samples, 2192 proteins were identified, being sixty-three differentially modulated in mastitis. In serum, 1299 proteins were identified, being twenty-nine differentially modulated in mastitis. Gamma glutamyl transferase (γGT) in saliva and serum amyloid A (SAA) were validated by commercially available automated assays. In conclusion, there are changes in protein expression and metabolic pathways in saliva and serum proteomes of cows with mastitis, showing different response patterns but complementary information.

Changes in saliva analytes during pregnancy, farrowing and lactation in sows: A sialochemistry approach.

Salivary biomarkers were studied in 17 healthy Large White sows from early gestation to the end of lactation. Saliva samples were obtained at 34 ± 3 days from insemination (G30), 24 ± 4 days before farrowing (G90), within the first 24 h after farrowing (L1) and at the end of a lactation period of 21 days (L21). The measurements in saliva included stress-related biomarkers (cortisol, chromogranin A, α-amylase, butyrylcholinesterase [BChE] and lipase [Lip]), inflammatory biomarkers (adenosine deaminase isoenzymes 1 [ADA1] and 2 [ADA2], and haptoglobin [Hp]) and oxidative stress biomarkers (cupric reducing antioxidant capacity, trolox equivalent antioxidant capacity, ferric reducing ability, uric acid, advanced oxidation protein products [AOPP] and hydrogen peroxide [H2O2]), as well as routine biochemistry analytes (aspartate aminotransferase [AST], alkaline phosphatase [ALP], γ-glutamine transferase [GGT], lactate dehydrogenase [LDH], creatine kinase [CK], urea, creatinine, triglycerides, lactate, calcium and phosphorus). The main changes were observed at farrowing, with increases in biomarkers of stress (cortisol and BChE), inflammation (ADA isoenzymes and Hp) and oxidative stress (AOPP and H2O2), as well as muscle and hepatic enzymes (CK, AST, ALP, GGT and LDH). Lactate and triglycerides increased at the end of gestation and remained at high concentrations until the end of lactation. Lip was higher in gestation than at lactation. Thus, changes in biomarkers of stress, immune function, oxidative stress, hepatic and muscle integrity, and energy mobilization occur in sow saliva during pregnancy, farrowing and lactation. These changes, caused by physiological conditions, should be taken into consideration when these biomarkers are used for the evaluation of sow health and welfare.

Changes in saliva biomarkers during a standardized increasing intensity field exercise test in endurance horses.

Salivary biomarkers could be useful to evaluate stress, fitness level, and skeletal muscle damage associated to exercise in horses in an easy and non-painful way. Therefore, this study aims to evaluate if cortisol in saliva (sCor), salivary alpha-amylase (sAMY) and butyrylcholinesterase (sBChE) and lactate (sLA) and creatine kinase (sCK) in saliva of horses can show changes during a standardized exercise test, and if they are related to heart rate variability (HRV) parameters related to sympathetic and parasympathetic tone, fitness level or skeletal muscle damage. For this purpose, ten endurance horses were submitted to a standardized exercise test in field conditions. Saliva and blood were obtained at basal time (TB), after the seven bouts of velocity (T + 01 to T + 07), and 5, 15, 30, and 45 min later (T + 5, T + 15, T + 30, and T + 45). Five endurance horses in resting condition (control group) were also enrolled. HRV and fitness level parameters, and plasma CK as a marker of muscle damage were also evaluated. Salivaryalpha-amylase increased at T + 30 (P = 0.03), sBChE at T + 5 (P = 008), and sCK at T + 07 (P = 0.009) after the exercise test, with significant differences between the exercise and control groups' results. The sCor did not show significant changes during the exercise test in the exercise group but higher concentration compared to the control horses (P < 0.001) were observed. sCor, sAMY, sBChE, and sCK showed a positive correlation (r values between 0.47 and 0.64) with the sympathetic tone and a negative correlation (r values between -0.37 and -0.56) with the parasympathetic tone. In conclusion, sAMY, sBChE, and sCK showed significant increases in ten endurance horses after an increasing intensity velocity exercise. Values of sCor, sAMY, sBChE, and sCK were associated with HRV, which is used to evaluate stress, and therefore, they could be potentially used to assess the exercise-related stress after a physical effort.

Measurement of cortisol, cortisone and 11ß-hydroxysteroid dehydrogenase type 2 activity in hair of sows during different phases of the reproductive cycle.

Two sensitive assays based on AlphaLISA technology were developed and validated for the measurement of cortisol and cortisone in hair of pigs, that also enabled estimation of 11ß-hydroxysteroid dehydrogenase type 2 activity. These assays were applied to hair samples from sows (n = 32) collected at 5 days before, and at 23 and 59 after farrowing, in reproductive cycles in two different periods: spring-summer (n = 16) and winter-spring (n = 16). The assays were precise (imprecision <12%) and accurate (recovery range, 80-115%) for cortisol and cortisone determination. Hair cortisone concentrations and the cortisone/cortisol ratio (an estimate of 11ß-hydroxysteroid dehydrogenase isoenzyme type 2 activity) increased after farrowing more than cortisol, being these changes of higher magnitude during periods of higher atmospheric temperature. The measurement of hair cortisone concentrations and estimations of the activity of the 11ß-hydroxysteroid dehydrogenase isoenzyme type 2, measured by the assays developed in this study, are complementary biomarkers to hair cortisol, and can increase at periods associated with stress, such as farrowing and lactation, especially at high atmospheric temperatures. .

Salivary D-dimer in pigs: Validation of an automated assay and changes after acute stress.

D-dimer is a peptide found in serum and is derived from the degradation of blood clots. Even though it has been analysed in human saliva, D-dimer has not been previously evaluated in the saliva of any veterinary species, and its source and role remain unknown. The objectives of this research were firstly, to validate the use of an automated method for the measurement of D-dimer in porcine saliva, and secondly, to evaluate whether D-dimer concentration changes in pig saliva after an acute stress stimulus. For this purpose, a complete analytical validation of a commercially-available immunoturbidimetric assay was carried out. In addition, an experimental acute stress model was induced in 11 pigs based on a technique involving restraint by nose-snare immobilisation for 1 min. Saliva samples were subsequently collected at different times and D-dimer, salivary alpha-amylase (sAA) and cortisol were assessed in order to evaluate changes in its concentrations after the stress induction. The D-dimer automated assay showed adequate reproducibility and sensitivity, with coefficients of variation below 10% and a limit of quantification of 0.167 µg/mL fibrinogen equivalent units (FEU). It also showed a high accuracy, determined by linearity under dilution and recovery tests. In the stress model, a significant increase (P < 0.05) in salivary D-dimer 15 min after the stress stimulus and a positive correlation between D-dimer and sAA (r = 0.51; P < 0.001) were observed. These results indicate that D-dimer can be measured in porcine saliva with an automated method and suggest that its concentration can be influenced by stressful conditions.

Biochemical changes in saliva of cows with inflammation: A pilot study.

Saliva contains a variety of compounds that can change in local and systemic pathologies including inflammation. Although changes in acute phase proteins and markers of oxidative stress in saliva during inflammation in humans and different animal species have been described, no data exist about possible changes during inflammation in analytes in saliva of cows. The aim of the present study was to evaluate changes in selected salivary biomarkers of stress, inflammation and immune system, and oxidative stress in cows with inflammation. For this purpose, bovine mastitis was used as model. Saliva and serum from 18 clinically healthy cows and 18 cows with clinical mastitis were used in the study. A panel of analytes integrated by alpha-amylase, cortisol, haptoglobin, adenosine deaminase, cholinesterase, total antioxidant capacity, lactate, and uric acid was measured in all samples and differences between the two groups of animals were evaluated. Significant increases in cortisol, alpha-amylase, uric acid, lactate and significant decreases in cholinesterase were detected in saliva of cows with mastitis. These results indicate that that cows with mastitis show changes in salivary biomarkers that reflect presence of stress, inflammation and oxidative stress in the animals.

Salivary alpha-amylase activity and concentration in horses with acute abdominal disease: Association with outcome.

BACKGROUND: Salivary biomarkers could be useful to objectively evaluate critical illness and prognosis for survival in horses with acute abdominal disease. OBJECTIVES: To compare salivary alpha-amylase (sAA) activity and concentration in healthy horses and horses with acute abdominal disease, and evaluate the association between sAA activity and concentration with disease severity and outcome. STUDY DESIGN: A prospective cohort. METHODS: sAA activity, measured using a colorimetric commercial kit, and concentration, measured using a Time-resolved immunofluorometric assay, in 25 healthy horses and in 33 horses with acute abdominal disease was compared using an ANOVA. Associations between survival to discharge and sAA activity and concentration and other clinical parameters were examined using univariable logistic regression and Spearman correlation. RESULTS: sAA activity and concentration were different between healthy (median = 4.3 [2.6-11.2] IU/L and 58.4 [53.4-80.6] ng/mL, respectively) and diseased (median = 29.8 [14.2-168.9] IU/L and 388.3 [189.1-675.8] ng/mL, respectively) (P<0.001). The sAA activity was higher in non-survivors (median = 479.0 [78.7-2064.0] IU/L, n = 8) compared to survivors (median = 19.3 [12.1-103.7] IU/L, n = 25, P<0.001) and sAA activity and concentration correlated (P<0.001) moderately with HR (r = 0.66 and r = 0.61, respectively). sAA activity correlated weakly with salivary cortisol (r = 0.45, P<0.001) and systemic inflammatory response syndrome score (r = 0.43, P<0.05), while activity and concentration correlated (P<0.001) moderately with plasma lactate concentration (r = 0.57 and r = 0.60, respectively). The sAA activity was significantly (P = 0.01) associated with increased risk of nonsurvival. MAIN LIMITATIONS: Pain scores were not recorded. The sample population was small. CONCLUSIONS: The sAA activity, but not concentration, shows potential as a biomarker of prognosis for survival in horses with acute abdominal disease. The summary is available in Spanish - see Supporting Information.

Evaluation of new biomarkers of stress in saliva of sheep.

Some routine handling procedures can produce stress in farm animals, and an adequate control of these stressors is important to avoid the negative effects on animal health and production. The measurement of biomarkers in saliva can be a suitable tool for the evaluation and control of stress. In this report, lipase, butyrylcholinesterase (BChE), total esterase (TEA) and adenosine deaminase (ADA) activities in the saliva of sheep were evaluated as biomarkers of stress. For this purpose, they were measured after inducing stress by facing a dog (experiment 1) and shearing (experiment 2), and comparing them to other stress salivary biomarkers such as α-amylase (sAA) and cortisol, as well as heart rate (HR). Each analyte was measured at the basal time, and during and just after the end of the stressful stimulus, and at various times for the first hour after the period of stress induction. Values were compared with those obtained from a control group. Lipase was the only analyte that showed significant changes between the stress and the control group in both experiments. Although TEA and ADA increased after stress, no significant differences were seen compared with the control group. Lipase was correlated highly with sAA and HR, in experiment 1; and correlated moderately with cortisol and HR in experiment 2. Lipase showed the greatest percentage increase after the stressful stimuli and less overlap with the control group in the two experiments. From the results of this study it can be concluded that lipase, TEA, BChE and ADA are enzymes present in the saliva of sheep and that they can be measured by using simple and fast colorimetric methods. Further studies should be undertaken with regard to the possible application of lipase as a biomarker of stress in sheep.

Evaluation of adenosine deaminase in saliva and serum, and salivary α-amylase, in canine pyometra at diagnosis and after ovariohysterectomy.

An assay for adenosine deaminase (ADA) was validated in serum and saliva in dogs. Changes in ADA and salivary α-amylase activities were analysed in 26 bitches diagnosed with pyometra and compared with activities in 19 healthy bitches. All animals were classified according to the American Society of Anaesthesiologists (ASA) scoring for physical status. In the validation study, the ADA assay had an imprecision<12% and determination coefficients>0.90 in linearity under dilution experiments, with recoveries of 99.2-114.4%. On the day of presentation, salivary ADA activity was significantly higher in dogs with pyometra than in healthy dogs (median values 7.1IU/L vs. 0.8IU/L, respectively; P<0.01). ADA had a moderate positive correlation with leucocyte and band neutrophil counts, haptoglobin, salivary α-amylase and ASA score, and a low positive correlation with C-reactive protein. There were no significant differences in salivary α-amylase activity between dogs with pyometra and healthy dogs (57.3IU/L vs. 27.4IU/L, respectively). Salivary α-amylase had a low correlation with ASA grade, and leucocyte and band neutrophil counts. In 7/26 bitches with pyometra that were sampled 3 and 10days after ovariohysterectomy, there were no significant changes in α-amylase or ADA activities. These results indicate that ADA activity is increased in the saliva of bitches with pyometra, probably related to systemic inflammation.