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.

Use of acute phase proteins for the clinical assessment and management of canine leishmaniosis: general recommendations.

BACKGROUND: Dogs with canine leishmaniosis (CanL) due to Leishmania infantum can show a wide spectrum of clinical and clinicopathological findings at the time of diagnosis. The aim of this paper is to describe the possible application of acute phase proteins (APPs) for the characterization and management of this disease, based on previously published information on the utility of APPs in CanL and the experience of the authors in using APPs as analytes in the profiling of canine diseases. MAIN BODY: Dogs diagnosed with L. infantum infection by serology, polymerase chain reaction, cytological or histopathological identification, can be divided into three groups based on their clinical condition at physical examination and their APPs concentrations: Group 1: dogs with no clinical signs on physical examination and APPs in reference range; Group 2: dogs with changes in APPs but no clinical signs on physical examination; Group 3: dogs with clinical signs and changes in APPs. This report describes the main characteristics of each group as well as its association with the clinical classification schemes of CanL. CONCLUSION: APPs concentration can be a useful clinical tool to characterize and manage CanL.

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.

Validation of two ELISA assays for total ghrelin measurement in dogs.

The aim of this study was to validate two commercially available ELISA assays for total ghrelin measurement in dogs: one canine-specific and one originally designed for measuring human ghrelin. The two assays showed intra-assay coefficient of variations (CVs) lower than 10%, while the inter-assay CVs exceeded the 15% limit. Sample dilutions resulted in linear regression equations with correlation coefficients close to 1. In order to compare methods and verify ability of the ghrelin assays to differentiate between low and high levels, ghrelin concentrations were measured in plasma samples obtained before and at different times after glucose administration in five Beagle dogs. A statistically significant changes in ghrelin after glucose administration was recorded only with assay B. In conclusion, the human ELISA validated in this study showed a good intra-assay precision, accuracy, and when applied to the glucose injection study, was better in distinguishing high and low canine ghrelin levels than the canine ELISA assay.

Biomarkers of sepsis in pigs, horses and cattle: from acute phase proteins to procalcitonin.

Sepsis is a complex clinical syndrome triggered by an inflammatory host response to an infection. It is usually complicated to detect and diagnose, and has severe consequences in human and veterinary health, especially when treatment is not started early. Therefore, efforts to detect sepsis accurately are needed. In addition, its proper diagnosis could reduce the misuse of antibiotics, which is essential fighting against antimicrobial resistance. This case is a particular issue in farm animals, as antibiotics have been traditionally given massively, but now they are becoming increasingly restricted. When sepsis is suspected in animals, the most frequently used biomarkers are acute phase proteins such as C-reactive protein, serum amyloid A and haptoglobin, but their concentrations can increase in other inflammatory conditions. In human patients, the most promising biomarkers to detect sepsis are currently procalcitonin and presepsin, and there is a wide range of other biomarkers under study. However, there is little information on the application of these biomarkers in veterinary species. This review aims to describe the general concepts of sepsis and the current knowledge about the biomarkers of sepsis in pigs, horses, and cattle and to discuss possible advances in the field.

Automated assays for trace elements and ferritin measurement in saliva of pigs: Analytical validation and a pilot application to evaluate different iron status.

The aim of this study was to validate automated methods to measure iron (Fe), zinc (Zn), copper (Cu) and ferritin in pig saliva samples. A complete analytical validation was performed of all assays. In addition, these methods were applied to saliva of Fe supplemented (n = 22) and non-supplemented (n = 20) piglets. All assays were able to measure these biomarkers in pig saliva with adequate precision, accuracy and high sensitivity and, in case of trace elements without needing a deproteinization pre-process. The group of piglets supplemented with Fe presented significantly higher levels of ferritin and Zn in saliva. In conclusion, the automated assays evaluated were able to measure Fe, Zn, Cu and ferritin in saliva of pigs, and in case of trace elements, they have the advantage of not needing a deproteinization pre-treatment and thus these analytes can be measured in a simple and fast manner.

Effects of orchidectomy in selective biochemical analytes in Beagle dogs.

The objective of this study was to evaluate the possible effects of orchidectomy and associate hormonal changes on circulating concentrations of acute-phase proteins (APPs) (CRP--C-reactive protein; Hp--haptoglobin; Cp--ceruloplasmin), adiponectin and IGF-1 in dogs. For this, a total of five adult Beagle dogs were subjected to orchidectomy. Blood samples were taken before neutering, during six consecutive days and on weeks 2, 4, 8 and 12 after surgery. Appropriate diet regime was maintained to keep stable body weight of the dogs. Concentrations of APPs significantly increased on days 2-3 for CRP and 2-7 for Hp and Cp. On days 3-4 after neutering, adiponectin levels were significantly lower than before surgery (p<0.05 and <0.01, respectively). After this initial change, adiponectin did not show any significant alteration during the 3 months. Serum IGF-1 concentrations were significantly decreased at days 2-5 after neutering. In addition, on weeks 8 and 12 serum IGF-1 levels were significantly lower (p<0.001 and <0.01 respectively) in comparison with those before surgery. In conclusion, orchidectomy induced a short-term inflammatory process that was associated with the increase in serum levels of APPs and decrease in IGF-1 and adiponectin levels. However, orchidectomy did not result in long-term changes of circulating concentrations of APPs or adiponectin. Although a decrease in IGF-1 levels was recorded 2 months after surgery, possibly as a consequence of associated decrease in androgen levels or food restriction.

Effect of reduction and alkylation treatment in three different assays used for the measurement of oxytocin in saliva of pigs.

Oxytocin is a hormone that is increasingly being used for welfare evaluation in animals. Although several types of samples have been used for oxytocin measurement, saliva can be a suitable option for pigs producing less stress than blood sampling. In this study, 3 different methods for oxytocin measurements, 2 based on alphaLISA technology (one with a monoclonal and other with a polyclonal antibody) and one commercially available kit, were compared in saliva of pigs. These methods were used in saliva samples obtained from female pigs at 3 different days during gestation and lactation, with and without a reduction/alkylation (R/A), which is a procedure for breaking the links between oxytocin and proteins of the sample. The assays showed a different behavior after the R/A procedure, with no significant changes in the oxytocin results in case of the alphaLISA monoclonal method, a significant decrease with the alphaLISA polyclonal method, and a significant increase with the commercial kit. Although all assays showed a similar tendency in detecting the changes in oxytocin during gestation and lactation, they showed changes of different magnitude and statistical signification. This report indicates that different assays can measure different forms of oxytocin present in saliva and can have a different behavior after R/A of the sample and when are used to measure oxytocin in gestation and lactation.

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.

Use of proteases for the evaluation of the different adiponectin isoforms in the dog.

Adiponectin (ADP) is an adipokine secreted by adipose tissue with anti-inflammatory, antiatherogenic, and antidiabetic properties. In human serum, it is presented as three different forms: low molecular weight (LMW), medium molecular weight (MMW), and high molecular weight (HMW). High molecular weight isomer is the most active form of ADP and is more closely related with obesity-induced insulin resistance and metabolic syndrome than total ADP. Selective protease treatment can be used in humans to isolate the different ADP isoforms but this has not been applied in any veterinary species. Therefore, the objective of this study was to evaluate if the selective protease digestion is able to differentiate serum ADP isomers in dog samples, and if these isomers could change in obese dogs after a weight loss program. A Western blotting analysis confirmed that digestion with protease K showed only the HMW forms of ADP, whereas the use of protease A showed the HMW and MMW forms. This specific protease digestion was applied to serum obtained from 14 obese beagle dogs before and after a weight loss program and total ADP, HMW, and LMW forms increased significantly after the weight reduction. In conclusion, the use of selective protease digestion can be applied in canine serum as a procedure for detecting the different ADP isomers. In addition, by this procedure, it was showed that the HMW and LMW forms were increased after a weight loss program in our experimental conditions.

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.

Oxytocin in saliva of pigs: an assay for its measurement and changes after farrowing.

Oxytocin is a hormone of interest in reproduction, but also in the field of psychology and behavior, being considered as a biomarker of positive emotions. Saliva can be a noninvasive way to measure oxytocin, which is very useful in species such as the pig where blood collection can produce a high degree of stress. In this study, a new assay for oxytocin measurement was developed, analytically validated, and used to measure possible changes in oxytocin in saliva of female pigs at different days after farrowing. The assay showed an adequate accuracy and precision and does not need a previous extraction step. In addition, oxytocin concentrations were significantly higher at day 1 of lactation than at day 9 after farrowing, but levels increased at day 20 again. This assay can contribute to a wider use of oxytocin measurements in pigs as it is a noninvasive sampling procedure that minimizes stress.

C-reactive protein quantification in porcine saliva: a minimally invasive test for pig health monitoring.

Study objectives were to investigate whether C-reactive protein (CRP) in pig saliva could be quantified using an adapted, time-resolved immunofluorometry assay (TR-IFMA), and to determine whether the assay could distinguish healthy from diseased animals. The test method had intra- and inter-assay coefficients of variation of 5.75% and 9.73%, respectively, the limit of detection was 0.47ng/mL and the coefficient of determination was 0.98. Analysis of CRP concentrations in paired serum and saliva samples from 50 pigs gave a positive correlation (r=0.702, P<0.01) and the salivary CRP concentration was able to distinguish healthy from diseased animals in 62 samples from pigs with naturally occurring or experimentally-induced inflammation. The results suggest that this minimally invasive, straightforward and sensitive assay may be useful in pig health and welfare monitoring.

A new highly sensitive immunoassay for the detection of adiponectin in serum and saliva of dogs and its application in obesity and canine leishmaniosis.

Adiponectin is an adipokine that exerts insulin-sensitizing and antiinflammatory properties. The aim of this study was to develop and validate a new heterologous ultrasensitive assay based on amplified luminescent technology for adiponectin determination in serum and saliva of dogs. A complete analytical validation of the assay was made in these fluids, and also this assay was applied to quantify adiponectin in serum and saliva of obese and lean dogs and dogs with leishmaniosis. These conditions were selected because in obesity there is a controversy about how adiponectin concentrations change in dogs, and in case of canine leishmaniosis, although it is described a decrease in serum adiponectin, there are not studies about how adiponectin changes after treatment. A total of 11 dogs were used in the validation and 26 dogs with different body condition and 8 with canine leishmaniosis were used for the clinical evaluation of the new assay for adiponectin quantification in serum and saliva of dogs. The analytical evaluation showed that the developed method could measure adiponectin in serum and saliva of dogs with high repeatability and sensitivity, adding a limit of quantification lower than commercially available ELISAs. In addition, significantly higher adiponectin concentrations were found in lean dogs and a correlation between serum and saliva was observed (P < .01). Moreover, dogs with leishmania presented reduced levels of adiponectin in serum. In conclusion, a new assay has been developed for adiponectin measurements which is more sensitive and faster than the traditional ELISA assays requiring less sample volume.

Glucose, fructosamine, and insulin measurements in saliva of dogs: variations after an experimental glucose administration.

The aim of this study was to evaluate if glucose, fructosamine, and insulin levels can be measured in saliva of dogs and assess the changes in these compounds after an experimental glucose administration. Automated spectrophotometric assays for glucose and fructosamine and an ELISA assay for insulin measurements were validated in saliva of dogs, by evaluating precision, accuracy, and limits of detection. In addition, an intravenous glucose bolus was administrated to 10 beagles and fasting serum and saliva samples were obtained immediately before and 5, 10, 20, 30, and 45 min after glucose infusion. The results of the between-run imprecision gave mean CVs of 6.16, 9.40, and 3.10% for glucose, fructosamine, and insulin, respectively. Linearity under dilution showed coefficient of correlation of 0.999, 0.994, and 0.990 for glucose, fructosamine, and insulin, respectively. The LDs were 0.04 mg/dL, 4.08 µmol/L, and 0.02 µg/mL for glucose, fructosamine, and insulin, respectively. The glucose administration caused an increase in serum and salivary levels of glucose with a peak in salivary levels at 30 min and of insulin with a peak in salivary levels at 45 min, while fructosamine did not change. No correlations between serum and salivary concentrations were found for any compound. It is concluded that glucose, fructosamine, and insulin can be measured in saliva of dogs, and an experimental administration of glucose in this species can lead to increases in glucose and insulin in saliva.

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.

Serum haptoglobin response in red deer naturally infected with tuberculosis.

The analysis of haptoglobin (Hp) serum concentration is a very sensitive, but non-specific, indicator of inflammation or infection. Methods to accurately diagnose infection in vivo in wildlife are usually constrained by low sensitivity due to the effects of stress on individual immune response and the challenging logistics of performing tests in the wild. Firstly, we sought to determine serum Hp concentration in red deer (Cervus elaphus) naturally infected with bovine tuberculosis (TB). Secondly, we assessed the complementary diagnostic value of serum Hp levels in conjunction with the cervical comparative skin test (CCT) performed in a subsample (n = 33). Serum Hp concentrations were significantly higher in TB-infected individuals (based on the presence of macroscopic lesions confirmed by culture) compared to those uninfected. In addition, serum Hp significantly changed with the type of animal handling, with captured and handled animals showing higher levels of Hp than hunted animals. Four out of 6 TB positive individuals that tested negative to the CCT (false negatives) showed Hp levels higher than the 95th percentile of healthy animals. These findings indicate that an acute phase response develops in animals with TB. In this paper, we demonstrate for the first time that an acute phase protein can provide a complementary assessment for specific diagnosis tests in wild species.

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.

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.