Changes in salivary biomarkers of stress, inflammation, redox status, and muscle damage due to Streptococcus suis infection in pigs.

BACKGROUND: Streptococcus suis (S. suis) is a Gram-positive bacteria that infects pigs causing meningitis, arthritis, pneumonia, or endocarditis. This increases the mortality in pig farms deriving in severe economic losses. The use of saliva as a diagnostic fluid has various advantages compared to blood, especially in pigs. In this study, it was hypothesized that saliva could reflect changes in different biomarkers related to stress, inflammation, redox status, and muscle damage in pigs with S. suis infection and that changes in these biomarkers could be related to the severity of the disease. RESULTS: A total of 56 growing pigs from a farm were selected as infected pigs (n = 28) and healthy pigs (n = 28). Results showed increases in biomarkers related to stress (alpha-amylase and oxytocin), inflammation (haptoglobin, inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), total protein, S100A8-A9 and S100A12), redox status (advanced oxidation protein producs (AOPP)) and muscle damage (creatine kinase (CK), CK-MB, troponin I, lactate, aspartate aminotransferase, and lactate dehydrogenase). An increase in adenosine deaminase (ADA), procalcitonin, and aldolase in infected animals were also observed, as previously described. The grade of severity of the disease indicated a significant positive correlation with total protein concentrations, aspartate aminotransferase, aldolase, and AOPP. CONCLUSIONS: This report revealed that S. suis infection caused variations in analytes related to stress, inflammation, redox status, and muscle damage in the saliva of pigs and these can be considered potential biomarkers for this disease.

Divergences between serum C-reactive protein and ferritin concentrations in canine pyometra.

The main aim of this report was to investigate and compare the response of serum C-reactive protein (CRP) and ferritin, two positive acute phase proteins (APPs) which usually show an increase in inflammatory processes, in dogs with pyometra. For this purpose, two different studies were made. In the first one , both proteins were measured together in an APPs profile in 25 dogs with pyometra, 25 dogs with pancreatitis (as an example of a positive inflammatory control group), and in 25 healthy dogs. In the second study, to advance the knowledge of the changes and evolution of serum ferritin and CRP in dogs with pyometra after treatment, the concentrations of both APPs were analyzed in 30 dogs with pyometra at diagnosis and after ovariohysterectomy and in 10 clinically healthy female dogs before and after elective spaying. In both studies, bitches with pyometra showed significant increases in serum CRP, indicating an inflammatory condition, but not in serum ferritin despite being a moderate positive APP. This divergence between the dynamics of these APPs could be a useful tool for the suspicion of cases of canine pyometra.

Measurement of anti SARS-CoV-2 RBD IgG in saliva: validation of a highly sensitive assay and effects of the sampling collection method and correction by protein.

OBJECTIVES: To develop and evaluate a new highly sensitive assay to detect IgG anti-SARS-CoV-2 RBD in saliva samples. METHODS: A two-step sandwich type immunoassay based on the amplified luminescent proximity homogeneous technology was developed and an analytical validation was performed. As a part of this validation, the influence of factors, such as different sampling conditions (stimulated saliva and passive drool) and the correction of values by total protein content, in the ability of saliva to detect increases in antibodies after an immune stimulus and be an alternative to serum, was evaluated. For this purpose, paired samples of saliva and serum at different times after vaccination were used. RESULTS: Saliva concentrations were lower than serum, but both fluids showed similar kinetics, with higher correlations when saliva was obtained by passive flow and the results were not corrected by protein. CONCLUSIONS: The developed method showed a good analytical performance and can properly measure antibody concentrations in saliva of vaccinated individuals. However, saliva could have a lower sensitivity compared to serum at initial stages of the immune response and also when the antibody response decreased after a stimulus.

Measurement of procalcitonin in saliva of pigs: a pilot study.

BACKGROUND: Procalcitonin (PCT) is a widely used biomarker of sepsis in human medicine and can have potential applications in the veterinary field. This study aimed to explore whether PCT could be measured in the saliva of pigs and whether its concentration changes in sepsis. Therefore, a specific assay was developed and analytically validated, and changes in PCT concentration were evaluated in two conditions: a) in an experimental model of sepsis produced by the administration of lipopolysaccharide (LPS) to pigs (n = 5), that was compared with a model of non-septic inflammation induced by turpentine oil (n = 4), and b) in healthy piglets (n = 11) compared to piglets with meningitis (n = 20), a disease that usually involves sepsis and whose treatment often requires large amounts of antibiotics in farms. RESULTS: The assay showed coefficients of variation within the recommended limits and adequate linearity after serial sample dilutions. The method's detection limit was set at 68 µg/L, and the lower limit of quantification was 414 µg/L. In the LPS experiment, higher concentrations of PCT were found after 24 h in the animals injected with LPS (mean = 5790 µg/L) compared to those treated with turpentine oil (mean = 2127 µg/L, P = 0.045). Also, animals with meningitis had higher concentrations of PCT (mean = 21515 µg/L) than healthy pigs (mean = 6096 µg/L, P value < 0.0001). CONCLUSIONS: According to these results, this assay could be potentially used as a tool for the non-invasive detection of sepsis in pigs, which is currently a topic of high importance due to antibiotic use restriction.

Analytical validation of an automated assay for the measurement of adenosine deaminase (ADA) and its isoenzymes in saliva and a pilot evaluation of their changes in patients with SARS-CoV-2 infection.

OBJECTIVES: The aim of the present study was to validate a commercially available automated assay for the measurement of total adenosine deaminase (tADA) and its isoenzymes (ADA1 and ADA2) in saliva in a fast and accurate way, and evaluate the possible changes of these analytes in individuals with SARS-CoV-2 infection. METHODS: The validation, in addition to the evaluation of precision and accuracy, included the analysis of the effects of the main procedures that are currently being used for SARS-CoV-2 inactivation in saliva and a pilot study to evaluate the possible changes in salivary tADA and isoenzymes in individuals infected with SARS-CoV-2. RESULTS: The automated assay proved to be accurate and precise, with intra- and inter-assay coefficients of variation below 8.2%, linearity under dilution linear regression with R2 close to 1, and recovery percentage between 80 and 120% in all cases. This assay was affected when the sample is treated with heat or SDS for virus inactivation but tolerated Triton X-100 and NP-40. Individuals with SARS-CoV-2 infection (n=71) and who recovered from infection (n=11) had higher mean values of activity of tADA and its isoenzymes than healthy individuals (n=35). CONCLUSIONS: tADA and its isoenzymes ADA1 and ADA2 can be measured accurately and precisely in saliva samples in a rapid, economical, and reproducible way and can be analyzed after chemical inactivation with Triton X-100 and NP-40. Besides, the changes observed in tADA and isoenzymes in individuals with COVID-19 open the possibility of their potential use as non-invasive biomarkers in this disease.

Changes of adenosine deaminase activity in serum and saliva around parturition in sows with and without postpartum dysgalactia syndrome.

BACKGROUND: Postpartum dysgalactia syndrome (PDS) is associated with a significantly higher activation of the inflammatory and stress response at parturition than in the healthy sow. Therefore, reliable and possibly non-invasive biomarkers for substantial increases of inflammation are searched to support the PDS diagnosis. This report studies the possible changes of the inflammatory marker enzyme adenosine deaminase (ADA) in serum and saliva of 38 PDS positive sows (PDS+) and 38 healthy sows (PDS-). Sampling was performed every 24 h from 60 h before to 36 h after parturition. Isoenzyme 1 (ADA1) and isoenzyme 2 (ADA2), as well as total ADA (tADA), were measured and their statistical association with several serum and saliva biomarkers of inflammation and stress was investigated. RESULTS: Compared to a baseline (60 to 36h prepartum), salivary activities of ADA1, ADA2 and tADA increased significantly over time in both PDS+ and PDS- sows, reaching their peaks after parturition. In serum from PDS- sows, no changes were observed over time in either ADA1, ADA2 or tADA. In PDS+ sows, serum ADA2 activity decreased temporarily after parturition followed by a significant increase compared to baseline. ADA1, ADA2 and tADA were all significantly associated with several inflammatory biomarkers and ADA1 in serum was associated with serum cortisol. Although serum activity was higher in PDS+ than in PDS- sows, the differences were not statistically significant. Further, no difference was noted between the groups in the analyses of saliva. CONCLUSIONS: Salivary ADA1 and ADA2 increased in all sows after parturition, potentially as a response to the postpartum inflammation. However, no difference in the activity of ADA1, ADA2 and tADA were found between PDS+ and PDS- sows indicating inability to diagnose PDS under the conditions described in this report.

Clinical leishmaniosis in a captive Eurasian otter (Lutra lutra) in Spain: a case report.

BACKGROUND: Captive and free-ranging wild mammals have been recognized as potential reservoirs of Leishmania infantum infection. The aim of this study was to describe the first clinical case of leishmaniosis in the Eurasian otter. CASE PRESENTATION: A case of clinical leishmaniosis is reported in a 4-year-old male Eurasian otter housed at a wildlife park (Murcia, South Eastern Spain). The Eurasian otter showed bilateral epistaxis, anorexia, apathy, and weight loss. A complete blood cell count and biochemical analyses revealed hyperproteinemia, hyperglobulinemia, decreases of paraoxonase-1, increases of haptoglobin and ferritin, and proteinuria. Bilateral nephropathy with hydronephrosis, mesenteric lymphadenomegaly, and ascites were also observed. L. infantum infection was confirmed by microscopy (amastigotes were detected in macrophages from spleen aspirate), molecular diagnosis (L. infantum DNA was detected by real-time polymerase chain reaction), and serology (anti-Leishmania IgG2 antibodies were detected by time-resolved immunofluorometry). The animal was treated with allopurinol for 3 months and gained weight, the epistaxis disappeared, and the ferritin concentration decreased. CONCLUSIONS: This is the first report of clinical leishmaniosis in the Eurasian otter. Our results suggest that Eurasian otters are susceptible to infection with L. infantum and can develop clinical leishmaniosis in endemic areas.

Characterization of total adenosine deaminase activity (ADA) and its isoenzymes in saliva and serum in health and inflammatory conditions in four different species: an analytical and clinical validation pilot study.

BACKGROUND: Measurement of adenosine deaminase (ADA) can provide information about cell-mediated immunity. This report's objective was to study the enzymatic activity of total ADA (tADA) and its isoenzymes ADA1 and ADA2 in canine, equine, porcine, and bovine serum and saliva and their changes in different inflammatory situations in each species. Besides, an automated method for ADA2 measurement was developed and validated. RESULTS: tADA was present in serum and saliva of healthy animals of the four species. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) concentration of 0.47 mM was needed for ADA1 inhibition in canine and porcine samples (serum and saliva) and bovine saliva, whereas for equine saliva 0.94 mM was needed. ADA2 activity was not detected in bovine serum and was very low or absent in equine serum and bovine saliva. An automated procedure to measure ADA2 consisting of adding EHNA to a commercial reagent for tADA measurement provided repetitive (coefficients of variation < 8.8% in serum and < 10% in saliva) and accurate (linearity of serial sample dilutions with R2 > 0.90) results, being equivalent to a manual incubation of the sample with EHNA at a similar concentration. Salivary tADA, as well as ADA1 and ADA2, were higher in dogs with leishmaniosis, horses with acute abdominal disease and pigs with lameness than in healthy animals. tADA and isoenzymes in saliva showed a positive significant correlation with serum ferritin in dogs (r = 0.602, P < 0.01; r = 0.555, P < 0.05; and r = 0.632, P < 0.01; respectively for tADA, ADA1 and ADA2) and serum C-reactive protein in pigs (r = 0.700, P < 0.01, for both tADA and ADA1; r = 0.770, P < 0.001, for ADA2), whereas salivary ADA2 significantly correlated with serum amyloid A in horses (r = 0.649, P < 0.01). In cows, salivary tADA and ADA1 significantly increased after calving, correlating with total white blood cell count (r = 0.487, P < 0.05, for both tADA and ADA1). CONCLUSIONS: The activity of total ADA and its different isoenzymes, can be measured in serum and saliva of dogs, horses, pigs and cows by a simple and fast procedure described in this report. When measured in saliva, these analytes correlated with other biomarkers of inflammation and it could potentially be used as a biomarkers of inflammation and immune activation in the species of this study.

Variation of human salivary alpha-amylase proteoforms in three stimulation models.

OBJECTIVES: To evaluate the sAA proteoforms' expression during different stimulation situations. MATERIALS AND METHODS: This study evaluated the salivary alpha-amylase (sAA) proteoforms' behavior by western blot (WB) analysis and high-resolution mass spectrometry (LC-MS/MS) in different situations that produce increases in sAA activity. For this purpose, six healthy women with a similar body mass index, age, and fit, underwent different sAA stimulation tests, such as acetic acid stimulation, psychological stress using the standardized Trier social stress test, and physical effort using the Cooper treadmill test. RESULTS: The three models showed an increase in sAA activity. The WB demonstrated seven common bands observed in the six women (band one at 59 kDa, two at 56 kDa, three at 48 kDa, four at 45 kDa, five at 41 kDa, six at 36 kDa, and seven at 14 kDa), in which sAA protein was identified. The individual WB analysis showed that band two, which corresponded to the native non-glycosylated sAA proteoform, had a higher increase after the three sAA stimulation inducers, and this band was also the only proteoform correlated with sAA activity (r = 0.56, P = 0.001). In addition, when the label-free quantification analysis was performed, the different proteoforms showed different responses depending on the type of stimulation. CONCLUSIONS: This preliminary study showed that the diverse sAA proteoforms' expression depends on the different stimulation models. CLINICAL RELEVANCE: This study opens new perspectives and challenges for the use of the different alpha-amylase proteoforms as possible biomarkers in addition to the sAA activity.

Changes in saliva analytes in equine acute abdominal disease: a sialochemistry approach.

BACKGROUND: The biochemical components of saliva can change in certain pathologies in horses, for example in acute abdominal disease. The aim of this study was (1) to evaluate if a panel of biochemical analytes usually used in serum can be measured in saliva of horses and (2) to study the possible changes of these biochemical analytes in saliva of horses affected by acute abdominal disease. A panel of 23 analytes was analytically validated in saliva of horses and possible changes in these analytes in a pilot study with six healthy horses and six horses with acute abdominal disease were evaluated. The analytes with significant changes were then evaluated in a larger population of 20 healthy and 37 diseased horses. RESULTS: Seven analytes showed significant increases in the pilot study which were confirmed in the larger population. The analytes which showed significant changes, and their median fold increase and significance shown in the larger population were salivary γ-glutamyl transferase (gGT, 2.3 fold, P = 0.001), creatine kinase (CK, 6.2 fold, P < 0.001), urea (2.3 fold, P = 0.001), total bilirubin (2.6 fold, P < 0.001), total proteins (3.2 fold, P < 0.001), phosphorus (P, 4.5 fold, P < 0.001) and alpha-amylase (sAA, 8.5 fold, P < 0.001). Total proteins, P and sAA showed sensitivities higher than 70% at their optimal cut-off points and a specificity of 100% in differentiating between healthy horses and those with acute abdominal disease. CONCLUSIONS: A panel of 23 biochemical analytes can be measured in saliva of horses, where gGT, CK, urea, total bilirubin, total protein, P and sAA levels are raised in horses with acute abdominal disease.

Biomarkers of oxidative stress in saliva in pigs: analytical validation and changes in lactation.

BACKGROUND: Biomarkers of oxidative stress in pigs have been measured in serum/plasma samples. However, blood collection in pigs can be highly stressful to the animals. Saliva is a biological fluid with several advantages in pigs over blood, since it can be easily collected without stress to the animals, being therefore an ideal sample in this species. The objective of this study was the validation of assays for the evaluation of oxidative stress status in saliva of pigs. For this purpose, three assays commonly used to evaluate the total antioxidant capacity (TAC): trolox equivalent antioxidant capacity (TEAC), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing ability of plasma (FRAP)), one individual antioxidant (uric acid) and two assays to evaluate oxidant concentrations (advanced oxidation protein products (AOPP) and hydrogen peroxide (H2O2)) were measured and validated in porcine saliva. In addition, the possible changes of these assays in sows' saliva during lactation were be studied. RESULTS: The methods had intra- and inter-assays coefficient of variation lower than 15%. They also showed an adequate linearity and recovery, and their detection limits were low enough to detect the analytes in saliva of pigs. Overall the analytical validation tests showed that the assays used in our study are valid and reliable for the evaluation of oxidative stress in porcine saliva. In addition, it was observed that these salivary biomarkers can change in a situation of oxidative stress such as lactation in sows. CONCLUSIONS: All assays for salivary biomarkers of oxidative stress evaluated in this study have demonstrated a high analytical accuracy and low imprecision. In addition, it has been observed that these biomarkers showed significant changes in a situation of oxidative stress such as lactation in sows. Therefore, this study opens a new possibility of using saliva as a non-invasive sample to evaluate oxidative stress in pigs.

Salivary adiponectin, but not adenosine deaminase, correlates with clinical signs in women with Sjögren's syndrome: a pilot study.

OBJECTIVES: To evaluate salivary adiponectin and adenosine deaminase (ADA) in women suffering from Sjögren's syndrome (SS). METHODS: Salivary adiponectin and ADA were measured in patients with SS (n = 17) and compared to their values in healthy controls (n = 13) and patients suffering from drug-induced xerostomia (non-SS sicca group; n = 19). A clinical history was made for each patient, patients were examined clinically, and xerostomia inventory (XI) was performed. RESULTS: Salivary adiponectin corrected by total protein was higher in patients with SS than in healthy individuals (P < 0.05) or patients with non-SS sicca (P < 0.01) and correlated with XI (r = 0.555; P < 0.05). Salivary ADA was higher in patients with SS and non-SS sicca compared to controls (P < 0.05 in both cases). CONCLUSION: The results of the present study indicate that adiponectin and ADA are increased in the saliva of patients with SS. CLINICAL RELEVANCE: Salivary adiponectin corrected by total protein can be a potential biomarker of SS. TRIAL REGISTRATION: NCT03156569.

Salivary alpha-amylase activity and cortisol in horses with acute abdominal disease: a pilot study.

BACKGROUND: The aim of this study was to evaluate salivary alpha-amylase (sAA), considered a non-invasive biomarker for sympathetic nervous system (SNS) activity, and salivary cortisol as possible pain-induced stress biomarker, in horses with acute abdominal disease. Therefore, a prospective observational study was performed in which both biomarkers were analyzed in a group of horses with acute abdomen syndrome, and compared with a group of healthy control horses by an unpaired Student's t-test. In addition, the possible relationship between both biomarkers, the score in Equine Acute Abdominal Pain scales version 1 (EAAPS-1 scale), Heart Rate (HR) and Respiratory Rate (RR), plasma lactate, the systemic inflammatory response syndrome (SIRS) score and serum amyloid A (SAA) concentration was assessed by a Spearman correlation test. RESULTS: A total of 30 horses were included in the study, 19 with acute abdominal disease diagnosed as large colon displacements, simple impactions of the pelvic flexure, spasmodic colics and enteritis and 11 healthy ones. sAA activity (24.5 median-fold, P <  0.0001) and salivary cortisol (1.7 median-fold, P <  0.01) were significantly higher in horses with acute abdomen than in healthy horses. sAA activity was significantly correlated with EAAPS-1 scale (r = 0.78, 95% confidence interval [CI] 0.38-0.89, P < 0.001) and SIRS score (r = 0.49, 95% CI 0.03-0.78, P < 0.05). Neither sAA nor salivary cortisol correlated with HR, RR, plasma lactate and SAA. CONCLUSIONS: Although this study should be considered as preliminary one, alpha-amylase measurements in saliva could be a biomarker of pain-induced stress in horses with acute abdominal disease.

Changes in alpha-amylase activity, concentration and isoforms in pigs after an experimental acute stress model: an exploratory study.

BACKGROUND: Salivary alpha-amylase (sAA) is considered a non-invasive biomarker of acute stress that can be evaluated by changes in activity and concentration, and also by changes in its isoforms, although this last way of evaluation has never been used in veterinary medicine. This research evaluated the changes of sAA by three different ways in which sAA can be evaluated in an experimental acute stress model in six pigs based in a technique of temporarily restraining. These ways of evaluation were 1) activity by a spectrophotometric assay, 2) concentration by a fluorometric assay, and 3) isoforms of the enzyme by a Western blot. RESULTS: Although salivary cortisol significantly increased due to the stimulus of stress and all the pigs manifested signs of stress by high-pitched vocalization, sAA activity showed an increase of different degree in the six pigs after the stress stimulus, while sAA concentration showed decreases in four of the six pigs. sAA activity did not correlate with sAA concentration or salivary cortisol, and a low correlation was observed between sAA concentration and salivary cortisol (r = 0.48, p = 0.003). The inter-individual variability was higher in sAA activity than in sAA concentration and salivary cortisol. Finally, three possible isoforms of sAA at 154-160 kDa, 65-66 kDa and 59-60 kDa were observed that showed different dynamics after the stress induction. CONCLUSIONS: Although this pilot study's results should be taken with caution due to the low sample size, it reveals a different behavior between sAA activity and concentration in pig after an acute stressful stimulus leading to evident external signs of stress by high-pitched vocalization, and opens a new field for the evaluation of possible selected isoforms of sAA as potential biomarkers of stress.

Measurement of urea and creatinine in saliva of dogs: a pilot study.

BACKGROUND: Urea and creatinine in saliva have been reported to be possible markers of chronic kidney disease (CKD) in humans. The aim of this study was to assess if urea and creatinine could be measured in canine saliva, and to evaluate their possible changes in situations of CKD. RESULTS: The spectrophotometric assays for urea and creatinine measurements in saliva of dogs showed intra- and inter-assay imprecision lower than 12% and coefficients of correlation close to 1 in linearity under dilution tests. Healthy dogs showed median salivary concentrations of urea of 39.6 mg/dL and creatinine of 0.30 mg/dL, whereas dogs with CKD showed median salivary urea of 270.1 mg/dL and creatinine of 1.86 mg/dL. Positive high correlations were found between saliva and serum activities of the two analytes (urea, r = 0.909; P < 0.001; creatinine, r = 0.819; P < 0.001). CONCLUSIONS: Urea and creatinine concentrations can be measured in canine saliva with commercially available spectrophotometric assays. Both analytes showed higher values in saliva of dogs with CKD compared with healthy dogs and their values were highly correlated with those in serum in our study conditions.

Leptin and NGF in saliva of patients with diabetes mellitus type 2: A pilot study.

AIMS: This study aimed to measure salivary levels of leptin and nerve growth factor (NGF) in patients with type 2 diabetes (T2D) and to compare with healthy subjects. In addition, markers previously evaluated in diabetes, including insulin, hepatocyte growth factor (HGF), and monocyte chemoattractant protein-1 (MCP-1), and markers of inflammation interleukin ([IL]-1b, IL-6, IL-8, Tumor necrosis factor alpha [TNF-α]), were also measured in saliva to evaluate possible relationship of these markers with the new analytes evaluated in the study. METHODS: Unstimulated whole saliva was collected by passive drooling from a total of 65 individuals (34 controls and 31 with T2D) and used for leptin, NGF, HGF, MCP-1, insulin, IL-1b, IL-6, IL-8, and TNF-α determination. RESULTS: Salivary leptin was 2.1 higher in T2D than in healthy controls (P<.001), while no statistically significant differences were detected between the two groups in salivary concentrations of NGF. Salivary IL-6, TNF-α, insulin, and MCP-1 were higher in DM in comparison with controls (P<.05 in all cases). Leptin showed positive significant correlations with MCP-1, IL-6, TNF-α, and insulin, while NGF positively correlated with HGF, MCP-1, IL-1 ß, IL-6, and TNF-α. CONCLUSIONS: This pilot study indicates that salivary leptin is increased in patients with T2D being positively correlated with insulin and pro-inflammatory cytokines and should be further explored as a non-invasive biomarker of T2D. In addition, salivary NGF was positively correlated with pro-inflammatory cytokines and further studies should be performed to evaluate whether it could be useful to detect diabetic neuropathy in T2D patients.

Influence of different sample preparation strategies on the proteomic identification of stress biomarkers in porcine saliva.

BACKGROUND: The influence of two different sample treatments comprising the enrichment of glycoproteins by boronic acid and dynamic range compression by hexapeptide libraries, on the detection of stress markers in saliva of pigs was evaluated in this study. For this purpose, saliva samples collected before and after the application of an acute stress model consisting of nasal restraining in pigs were processed without any treatment and with the two different treatments mentioned above. Protein separation by two-dimensional gel electrophoresis (2-DE) followed by identification of proteins using MALDI-TOF/TOF mass spectrometry (MS) was used as proteomic technique. RESULTS: The application of each of the two different sample treatment protocols allowed the identification of unique proteins that could be potential salivary acute stress markers in pigs: lipocalin 1, protein S100-A8 and immunoglobulin M by enrichment of glycoproteins; protein S100-A9, double headed protease inhibitor submandibular gland, and haemoglobin by dynamic range compression; and protein S100-A12 by both protocols. Salivary lipocalin, prolactin inducible protein, light chain of immunoglobulins, adenosine deaminase and carbonic anhydrase VI were identified as potential markers in untreated saliva as well as one of the other treatments. CONCLUSION: The use of different procedures allowed the detection of different potential stress markers. Although from a practical point of view, the use of saliva without further treatment as well as the enrichment of glycoproteins are less expensive and easy to do procedures.

Detection and measurement of alpha-amylase in canine saliva and changes after an experimentally induced sympathetic activation.

BACKGROUND: Salivary alpha-amylase (sAA) is considered a biomarker of sympathetic activation in humans, but there is controversy regarding the existence of sAA in dogs. The hypothesis of this study was that sAA exists in dogs and it could change in situations of sympathetic stimulation. Therefore, the aims of this study were: 1) to demonstrate the presence of alpha-amylase in saliva of dogs by Western-Blot, 2) to validate an spectrophotometric method for the measurement of sAA activity and 3) to evaluate the possible changes in sAA activity after the induction of an ejaculation in dogs which is known to produce a sympathetic activation. RESULTS: Western-Blot demonstrated a band in dog saliva specimens between 60 kDa and 50 kDa, similar to purified sAA. The spectrophotometric assay validated showed an adequate inter- and intra-assay precision, and a high correlation coefficient (r = 0.999) in the linearity under dilution study. sAA median activity significantly increased just after ejaculation compared with just before the ejaculation (2.06-fold, P = 0.005). CONCLUSIONS: This study demonstrated the existence of alpha-amylase in saliva of dogs and that this enzyme can be measured by a spectrophotometric assay. In addition, results showed that sAA increase after a sympathetic activation and could be potentially used as non-invasive biomarker of sympathetic activity in this species.

Measurement of Creatine kinase and Aspartate aminotransferase in saliva of dogs: a pilot study.

BACKGROUND: Muscle enzymes in saliva have been reported to be possible markers of heart and muscle damage in humans. The aim of this study was to assess if Creatine kinase (CK) and Aspartate aminotransferase (AST) activities could be measured in canine saliva, and to evaluate their possible changes in situations of muscle damage. RESULTS: The spectrophotometric assays for CK and AST measurement in saliva of dogs showed intra- and inter-assay imprecision lower than 1 and 16% and coefficients of correlation close to 1 in linearity under dilution tests. Healthy dogs showed activities in saliva of CK between 27 and 121 U/L and AST between 46 and 144 U/L, whereas in saliva of dogs with muscle damage CK ranged between 132 and 3862 U/L and AST between 154 and 4340 U/L. Positive moderate correlations were found between saliva and serum activities of the two enzymes (CK, r = 0.579; P = 0.001; AST, r = 0.674; P = 0.001). CONCLUSIONS: CK and AST activities can be measured in canine saliva with commercially available spectrophotometric assays. In addition these enzymes show higher values in saliva of dogs with muscle damage and their values are moderately correlated with those of serum.

Development and validation of an assay for measurement of leptin in pig saliva.

BACKGROUND: Leptin has been measured in human in saliva samples. However, the low leptin concentration found in this biological fluid makes necessary the use of high sensitive methods. To the authors' knowledge, leptin has not been measured in porcine saliva. This study aimed to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for salivary leptin measurements in pigs, using a species-specific antibody, and to evaluate how salivary leptin changes with body weight, food ingestion, and in experimental models of stress and inflammation. Polyclonal antibodies were produced in rabbits immunized with recombinant porcine leptin and used to develop a sandwich TR-IFMA. RESULTS: The method had intra-assay and inter-assay coefficients of variation lower than 10 and 16 %, respectively. The assay was accurate and the low limit of detection allowed detection of leptin in all analyzed samples. Salivary leptin concentration was positively correlated to body weight (r = 0.58, P = 0.01) and increased after food ingestion (P < 0.001) and after 24 h of applying a model of experimental inflammation by turpentine injection (P < 0.05). However, it did not significantly change after a model of acute stress consisting of a nose snare restraining. CONCLUSION: These results indicate that the developed assay can measure leptin in porcine saliva in a reliable way and that leptin in saliva is influenced by body weight, food ingestion and inflammation.