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.

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.

A Proteomic Approach to Elucidate the Changes in Saliva and Serum Proteins of Pigs with Septic and Non-Septic Inflammation.

Sepsis is a systemic inflammatory response triggered by an infectious agent and is recognized by the World Health Organization as a global concern, since it is one of the major causes of severe illness in humans and animals. The study of the changes that can occur in saliva and serum in sepsis can contribute to a better understanding of the pathophysiological mechanisms involved in the process and also to discover potential biomarkers that can help in its diagnosis and monitoring. The objective of this study was to characterize the changes that occur in the salivary and serum proteome of pigs with experimentally-induced sepsis. The study included five pigs with sepsis induced by LPS administration and five pigs with non-septic inflammation induced by turpentine for comparative purposes. In saliva, there were eighteen salivary proteins differentially expressed in the sepsis condition and nine in non-septic inflammation. Among these, significant increments in aldolase A and serpin B12 only occurred in the sepsis model. Changes in aldolase A were validated in a larger population of pigs with sepsis due to Streptococcus suis infection. In serum, there were 30 proteins differentially expressed in sepsis group and 26 proteins in the non-septic group, and most of the proteins that changed in both groups were related to non-specific inflammation. In the saliva of the septic animals there were some specific pathways activated, such as the organonitrogen compound metabolic process and lipid transport, whereas, in the serum, one of the main activated pathways was the regulation of protein secretion. Overall, saliva and serum showed different proteome variations in response to septic inflammation and could provide complementary information about the pathophysiological mechanisms occurring in this condition. Additionally, salivary aldolase A could be a potential biomarker of sepsis in pigs that should be confirmed in a larger population.

Changes in salivary biomarkers of oxidative status in calves at weaning and grouping.

BACKGROUND: Saliva is being increasingly used as a sample for measuring biomarkers in several species and shows a high potential of use to detect and monitor stress. The weaning and grouping in dairy calves are a particularly stressful time. Therefore, the objectives of this study were to evaluate a panel of antioxidant and oxidant biomarkers in the saliva of calves on the day of weaning (W0), 2 days after weaning or milk withdrawal (W + 2), and 4 days after grouping (G + 4). In addition, to verify if cortisol and oxytocin concentrations are related to the biomarkers measured. RESULTS: Salivary cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of saliva (FRAS), Trolox equivalent antioxidant capacity (TEAC), advanced oxidation protein products (AOPP), and ferrous oxidation-xylenol orange (FOX) were significantly higher (P < 0.02) 4 days after grouping than the day of weaning and 2 days after. The increases were 50 and 54% for CUPRAC, 93 and 116% for FRAS, 117 and 135% for TEAC, 22 and 49% for AOPP and 10 and 5% for FOX in comparison with weaning and 2 days after, respectively. In addition, oxytocin and cortisol showed significant negative and positive correlations (P < 0.05) respectively with the biomarkers of oxidative status. CONCLUSIONS: Our results showed that calves after grouping show increases in antioxidants and oxidants concentrations, indicating that a balance between these molecules has been tried to maintain during this stressful situation. The dynamic changes of biomarkers of oxidative status should be explored and characterised in other stressful conditions.

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.

Oxytocin in bovine saliva: validation of two assays and changes in parturition and at weaning.

BACKGROUND: The possible use of oxytocin in saliva as an indicator of positive emotions in bovine species has been poorly investigated. In the present study, two new assays (one using a monoclonal antibody and the other using a polyclonal antibody) for the measurement of oxytocin in bovine saliva were developed and validated. Also, the changes in oxytocin in saliva were explored in two different situations. One was around parturition, and for this purpose, saliva samples from 13 cows were collected at three different times: 7 days before the parturition, the day of parturition and 7 days after the parturition. The second situation was weaning and grouping of calves, and for this purpose, saliva from 25 calves was collected at three different times: 1 day before weaning, 2 days after weaning or milk withdrawal and 4 days after grouping calves. RESULTS: In cows, oxytocin concentrations showed an increase on the day of parturition with both assays, while in calves, oxytocin concentrations showed a decrease 4 days after the grouping. CONCLUSIONS: The assays validated in this report could be used for the measurement of oxytocin in bovine saliva and detect changes in this analyte that can occur in different physiological or productive situations such as parturition and weaning.

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.

Alterations in haemolymph proteome of Mytilus galloprovincialis mussel after an induced injury.

A proteomic and biochemical approach was performed to assess the effects of an induced muscle injury on the haemolymph of bivalve molluscs. For this purpose, Mytilus galloprovincialis were exposed to puncture of adductor muscle for three consecutive days, and their haemolymph proteome was then compared to healthy animals using 2-dimensional electrophoresis (2-DE) to identify proteins that differed significantly in abundance. Those proteins were then subjected to tandem mass spectrometry and 6 proteins, namely myosin, tropomyosin, CuZn superoxide dismutase (SOD), triosephosphate isomerase, EP protein and small heat shock protein were identified. SOD and tropomyosin changes were verified by spectrophotometric measurements and western blotting, respectively. As some of the proteins identified are related to muscular damage and oxidative stress, other biomarkers associated with these processes that can be evaluated by automatic biochemical assays were measured including troponin, creatine kinase (CK), and aspartate aminotransferase (AST) for muscle damage, and SOD, trolox equivalent antioxidant capacity (TEAC) and esterase activity (EA) for oxidative stress. Significantly higher concentrations of troponin, CK, AST, and TEAC were observed in mussels after puncture, being also possible biomarkers of non-specific induced damage.

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.

Inflammatory markers before and after farrowing in healthy sows and in sows affected with postpartum dysgalactia syndrome.

BACKGROUND: The pathogenesis of postpartum dysgalactia syndrome (PDS) in sows is not fully elucidated and affected sows often present vague clinical signs. Accurate and timely diagnosis is difficult, and PDS is often recognized with a delay once piglets begin to starve. Increased rectal temperature of the sow is an important diagnostic parameter, but it may also be influenced by a number of other parameters and is thus difficult to interpret. Inflammatory markers may be important adjuncts to the clinical assessment of sows with PDS, but such markers have only been studied to a limited extent. The objective was to characterize the inflammatory response in healthy sows and in sows suffering from PDS, and to identify biomarkers that may assist in early identification of PDS-affected sows. RESULTS: Thirty-eight PDS-affected (PDS+) and 38 healthy (PDS-) sows underwent clinical examination and blood sampling every 24 h, from 60 h before the first piglet was born to 36 h after parturition. In both groups, inflammatory markers changed in relation to parturition. Most inflammatory markers changed 12-36 h after parturition [white blood cell counts (WBC), neutrophil counts, lymphocyte counts, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), serum amyloid A (SAA), C-reactive protein (CRP), haptoglobin (Hp), iron (Fe) and albumin (ALB)]. Changes in neutrophil counts, lymphocyte counts, CRP, Fe and ALB were observed -12 to 0 h before parturition. WBC, neutrophil and lymphocyte counts, serum concentrations of TNF-α, IL-6, Hp and Fe differed between PDS+ and PDS- sows. These differences were mainly apparent 12 to 36 h after parturition, but already at 12 h before parturition, PDS+ sows had lower lymphocyte counts than PDS- sows. CONCLUSIONS: Parturition itself caused significant inflammatory changes, but PDS+ sows showed a more severe response than PDS- sows. WBC, neutrophil and lymphocyte counts, and concentrations of TNF-α, IL-6, Hp and Fe can be potential biomarkers for PDS. Lymphocyte counts may be used to detect PDS at pre-partum. To assess their diagnostic potential, these markers must be investigated further and most likely combined with assessment of clinical parameters and other biomarkers for improved identification of sows at risk of developing PDS.

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.

Hormonal and metabolic indicators before and after farrowing in sows affected with postpartum dysgalactia syndrome.

BACKGROUND: Postpartum dysgalactia syndrome (PDS) in sows is difficult to diagnose and the pathogenesis is obscure. Hormonal changes related to the disease are often difficult to distinguish from those found in the normal transition period from gestation to lactation. The study aimed to investigate metabolic and hormonal changes related to PDS with the goal of identifying potential biomarkers in sows suffering from PDS (PDS+). Selected biomarkers were examined by comparing 38 PDS+ sows with 38 PDS negative (PDS-) sows. The sows were sampled every 24 h from 60 h ante partum (a.p.) to 36 h post partum (p.p.). RESULTS: Compared to the baseline (60 to 36 h a.p.), cortisol in serum and saliva and fasting blood glucose concentrations increased in PDS+ as well as PDS- sows. C-peptide decreased relative to the baseline in PDS+ sows, and prolactin and 8-epi prostaglandin F2 alpha (8-epi-PGF2α) decreased in PDS- sows. Concentrations of cortisol in serum and saliva, salivary chromogranin A (CgA), fasting blood glucose, C-peptide, and 8-epi-PGF2α differed significantly between PDS+ and PDS- sows, with levels of cortisol in serum and saliva, salivary CgA, and 8-epi-PGF2α in serum being different in the two groups already before parturition. Concentrations of salivary CgA were significantly lower in PDS- sows than in PDS+ sows during the entire study period. CONCLUSIONS: The results suggest that salivary CgA, cortisol and serum 8-epi-PGF2α may potentially serve as early diagnostic indicators for PDS. The consistently higher salivary CgA concentration in PDS+ sows compared to PDS- sows may indicate that homeostatic disturbances are present between 36 to 60 h before parturition in sows developing PDS. The higher serum and saliva cortisol concentration in PDS+ sows compared to PDS- sows could reflect an early sign of inflammation or stress. The significantly lower C-peptide in PDS+ sows compared to PDS- sows may reflect a lower food intake. Our results contribute to the understanding of the pathogenesis of PDS, and the homeostatic disturbances detected before parturition warrants further investigation. The diagnostic potential of the markers identified in this study should be investigated further in a larger population of sows.

Changes in serum proteins in dogs with Ehrlichia canis infection.

The aim of this study was the identification of proteins differentially represented in the serum proteome of seropositive dogs with (Group 1) and without (Group 2) clinical-pathologic signs consistent with ehrlichiosis compared to healthy control dogs. Serum samples were collected from 20 dogs of various breeds with naturally occurring ehrlichiosis (10 dogs belonged to Group 1 and 10 to Group 2) and 10 healthy dogs. Two-dimensional electrophoresis (2DE) of pooled serum for each of the group of dogs were run in triplicate. 2D image analysis showed 39 spots differently expressed between Group 1 and Group 2 compared with healthy ones. Mass spectrometry analysis allowed identification of 6 proteins: albumin, haptoglobin (Hp), alpha-1-antitrypsin (AAT), Retinol Binding Protein 4 (RBP-4), alpha-1-acid glycoprotein (AGP) and vitamin D-binding protein (VDBP). When a confirmatory study was performed for albumin, Hp, AAT and RBP-4 by using different assays, significant differences (P < 0.05) between diseased and healthy groups were observed. It can be concluded that there are significant changes in the serum proteome of dogs with ehrlichiosis with modifications in proteins related with the acute phase response such as Hp, albumin and AGP, with vitamin A transport such as RBP-4, with inhibitors of serine proteases and anti-inflammatory proteins such as AAT, and vitamin D metabolism and actin scavengers such as VDBP.

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.

Growth promotion in pigs by oxytetracycline coincides with down regulation of serum inflammatory parameters and of hibernation-associated protein HP-27.

The growth promoting effect of supplementing animal feed with antibiotics like tetracycline has traditionally been attributed to their antibiotic character. However, more evidence has been accumulated on their direct anti-inflammatory effect during the last two decades. Here we used a pig model to explore the systemic molecular effect of feed supplementation with sub therapeutic levels of oxytetracycline (OTC) by analysis of serum proteome changes. Results showed that OTC promoted growth, coinciding with a significant down regulation of different serum proteins related to inflammation, oxidation and lipid metabolism, confirming the anti-inflammatory mechanism of OTC. Interestingly, apart from the classic acute phase reactants also down regulation was seen of a hibernation associated plasma protein (HP-27), which is to our knowledge the first description in pigs. Although the exact function in non-hibernators is unclear, down regulation of HP-27 could be consistent with increased appetite, which is possibly linked to the anti-inflammatory action of OTC. Given that pigs are good models for human medicine due to their genetic and physiologic resemblance, the present results might also be used for rational intervention in human diseases in which inflammation plays an important role such as obesity, type 2 diabetes and cardiovascular diseases.

Oral lichen planus: salival biomarkers cortisol, immunoglobulin A, adiponectin.

BACKGROUND: Oral lichen planus (OLP) is a chronic mucocutaneous disease, inflammatory and autoimmune in character, in which the pathogenesis is not fully understood. Psychological stress has also been implicated in triggering or exacerbating the disease. OBJECTIVE: The aim of this study was to evaluate the psychological profile, sleep, and salivary biological markers-cortisol, immunoglobin A (IgA), and adiponectin - in patients with oral lichen planus (OLP). METHODS: The sample consisted of 65 patients (33 with OLP and 32 control patients). Patients completed hospital anxiety and depression scales (HADD, HADA). Questionnaires were used to assess sleepiness: the Pittsburgh sleep quality index (PSQI) and the Epworth sleepiness scale (ESS). A visual analog scale (VAS) was used for rating pain. Unstimulated whole saliva was evaluated, together with total proteins: cortisol, IgA, and adiponectin. RESULTS: Patients with OLP obtained significantly higher HADA and PSQI scores than control subjects (P = 0.001, P = 0.012, respectively). Total salivary protein (flow at rest) analysis found that total proteins were higher in the OLP group (P = 0.001). In the OLP group, IgA was 80.3 ± 51.3 vs. the control group 48.9 ± 32.8 (P = 0.005). Mean cortisol was 0.5 ± 0.3 µg/dl in the OLP group vs. 0.4 ± 0.2 µg/dl in the control group (P = 0.010). The OLP group showed a correlation between the HADA variable and pain (r = 0.358; P = 0.041), HADD (r = 0.568; P = 0.001), and PSQI (r = 0.537; P = 0.001). CONCLUSIONS: OLP patients presented worse psychological profiles and sleep disturbances, as well as higher values for IgA, cortisol, and total proteins than control subjects.