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.

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

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

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

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

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

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

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.

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. .

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

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

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

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

Evaluation of new biomarkers of stress in saliva of sheep.

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

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.

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

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

Homocysteine measurement in pig saliva, assay validation and changes after acute stress and experimental inflammation models: A pilot study.

High homocysteine (Hcy) concentration in serum has been associated to stress and inflammation in humans, but this association has not been studied in saliva in any animal species. The purpose of this research was to study salivary Hcy levels in pigs under stressful and inflammatory conditions. A commercially available enzyme-linked immunosorbent assay specific for Hcy determination in pigs was adapted and validated in saliva, yielding reproducible and accurate results. Hcy was measured in paired serum-saliva samples and no correlation was observed between serum and salivary Hcy. Salivary Hcy was measured in two experimental models of stress induction in pigs: restraint with a nasal snare and isolation. Homocysteine concentration and the homocysteine to total protein (Hcy/TP) ratio significantly increased 15min after restraining and decreased after some days of isolation. Significant correlation was observed between Hcy and chromogranin A. After an experimentally induced inflammation by subcutaneous turpentine injection, salivary Hcy increased only 3h after turpentine administration; however, the Hcy/TP ratio did not show any change. No correlation was found between salivary Hcy and serum C-reactive protein. In conclusion, salivary Hcy concentration increased when pigs were restrained with a nasal snare or stressed by isolation, probably reflecting an increase in the sympathetic activity. On the other hand, Hcy increased after an experimental inflammation induced by turpentine administration but in this case probably reflects an increase in total protein production in saliva.

Changes in saliva biomarkers of stress and immunity in domestic pigs exposed to a psychosocial stressor.

A combination of salivary biomarkers measured at the same time, reflecting the different systems that are involved in the stress mechanism, could be the best tool for its evaluation. In this study, changes in a panel of salivary biomarkers of stress and immunity including chromogranin A (CgA), IgA, cortisol, testosterone, haptoglobin (Hp) and C-reactive protein (CRP), were evaluated. A total of 14 (7 control and 7 test) crossbred Duroc × (Landrace × Large White) males of 190 days of age were used for this experiment. The stress mechanism was evaluated after applying a psychosocial stressor model in 7 pigs, based on isolation and regrouping. Our results show that after of isolation, there was a significant (P<0.05) increase in salivary CgA and IgA. However, after regrouping, there was a significant increase (P<0.05) in salivary cortisol, testosterone and CgA. Salivary Hp and CRP concentrations did not significantly change after applying this stress model. This panel of salivary biomarkers could be used as a practical and non-invasive tool for reflecting the activity of different physiology systems involved in the stress response.

Serum biomarkers of oxidative stress in cats with feline infectious peritonitis.

The purpose of this study was to elucidate the possible presence of oxidative stress in cats naturally affected by feline infectious peritonitis (FIP) by investigating two antioxidant biomarkers in serum: paraoxonase-1 (PON1) and total antioxidant capacity (TAC). PON1 was measured by spectrophotometric assays using three different substrates: p-nitrophenyl acetate (pNA), phenyl acetate (PA) and 5-thiobutil butyrolactone (TBBL), in order to evaluate possible differences between them. The PA and TBBL assays for PON1 and the assay for TAC were validated, providing acceptable precision and linearity although PA and TAC assays showed limit of detection higher than the values found in some cats with FIP. Cats with FIP and other inflammatory conditions showed lower PON1 values compared with a group of healthy cats with the three assays used, and cats with FIP showed significant decreased TAC concentrations. This study demonstrated the existence of oxidative stress in cats with FIP.

Inflammatory and oxidative biomarkers of disease severity in dogs with parvoviral enteritis.

OBJECTIVES: To study changes in serum C-reactive protein, haptoglobin, ceruloplasmin and albumin concentration, total anti-oxidant capacity and paraoxonase-1 and butyrylcholinesterase activity in dogs with parvoviral enteritis of different degrees of clinical severity. METHODS: Prospective study of 9 healthy and 43 dogs with parvoviral enteritis that were classified into mildly, moderately and affected groups. RESULTS: Dogs with parvoviral enteritis had a significant increase in C-reactive protein compared with healthy dogs, with an increase of higher magnitude in animals with more severe clinical signs. All dogs with parvoviral enteritis had a significant increase in haptoglobin concentration compared with healthy dogs, but with no difference according to disease severity. There was a decrease in paraoxonase-1 activity in parvoviral enteritis. CLINICAL SIGNIFICANCE: Major increases of C-reactive protein concentrations in dogs with parvoviral enteritis are a marker of disease severity. In addition, higher values for anti-oxidants in severe cases compared with mild and moderate cases suggest a possible compensatory anti-oxidant mechanism.

Serum paraoxonase 1 (PON1) activity in acute pancreatitis of dogs.

OBJECTIVES: Serum paraoxonase 1 is considered a marker of inflammation and oxidative damage. The aims of this study were to evaluate changes in serum paraoxonase 1 activity in dogs with acute pancreatitis, to correlate serum paraoxonase 1 activity and other analytes known to be altered in dogs with pancreatitis and to assess the relationship between serum paraoxonase 1 activity and disease severity in dogs with acute pancreatitis. MATERIALS AND METHODS: Retrospective analysis of dogs with acute pancreatitis and healthy dogs in which serum paraoxonase 1 activity was measured were compared. RESULTS: Median serum paraoxonase 1 activity was significantly lower in dogs with pancreatitis (n = 19) compared to healthy ones (n = 19). Serum paraoxonase 1 activity was negatively correlated with serum lipase and amylase activities, and C-reactive protein and haptoglobin concentrations and was positively correlated with total cholesterol and glucose concentration. Disease severity was negatively correlated with serum paraoxonase 1 activity and positively correlated with triglyceride and C-reactive protein concentration. CLINICAL SIGNIFICANCE: Serum paraoxonase 1 activity is lower in dogs with acute pancreatitis and together with triglyceride and C-reactive protein concentrations is a potential marker of disease severity.

An automated turbidimetric method for fibrinogen determination in dogs.

BACKGROUND: Several methods have been used for fibrinogen determination in dogs, but to the authors' knowledge, methods based on ammonium sulfate precipitation have not yet been reported in this species. OBJECTIVES: The aim of this study was to develop and validate an automated method based on ammonium sulfate precipitation for canine fibrinogen determination. METHODS: A reagent containing ammonium sulfate, sodium chloride, and K2 EDTA was used to precipitate fibrinogen at a final ammonium sulfate concentration of 0.57 M and final turbidity was measured on a Cobas Mira Plus autoanalyzer. Analytic validation included imprecision, accuracy, comparison with reference method (Clauss), limits of detection and quantification, and the evaluation of the influence of different anticoagulants. For diagnostic validation, fibrinogen was determined in a group of Beagle dogs before and after neutering, and in dogs affected by diseases known to produce low fibrinogen plasma concentration, such as liver insufficiency, disseminated intravascular coagulation, and protein-losing enteropathy. RESULTS: Low imprecison (<4%), excellent recovery (>90%), and low bias (0.092 g/L) with respect to Clauss method indicated a high reproducibility and accuracy. Limits of detection and quantification were 0.01 and 0.22 g/L, respectively. The method was applicable in plasma samples anticoagulated with EDTA, heparin, or sodium citrate. The fibrinogen concentration in Beagle dogs after neutering was increased, and decreased in animals with disseminated intravascular coagulation, liver insufficiency, or gastrointestinal protein loss. CONCLUSIONS: The automated method validated in this study represents a rapid, cheap, and easy protocol to quantify canine fibrinogen in routine practice.

Serum ferritin and paraoxonase-1 in canine leishmaniosis.

Ferritin and paraoxonase-1 (PON-1) were measured in dogs experimentally infected by Leishmania infantum (during experimental infection and following treatment) and also in naturally-infected dogs which presented different degrees of proteinuria. Experimentally-infected dogs were monitored for 7 months post-infection, then treated for 3 months with allopurinol, and their response to therapy was followed for 11 additional months. Naturally-infected dogs were staged based on the urine protein/creatinine (UPC) ratio into three groups as follows: group 1 (non-proteinuric; UPC ratio: <0.2), group 2 (borderline proteinuric; UPC ratio: 0.2-0.5) and group 3 (proteinuric; UPC ratio>0.5). An increase in serum ferritin values and a decrease in PON-1 activity were observed 2 months after infection. Both analytes returned to preinfection values following treatment. Significantly higher concentrations of ferritin were observed in dogs classified as either borderline or proteinuric when compared with non-proteinuric dogs whereas serum PON-1 activity was decreased only in proteinuric dogs.

Comparative study of clinical courses, gross lesions, acute phase response and coagulation disorders in sheep inoculated with bluetongue virus serotype 1 and 8.

Bluetongue virus serotypes 1 (BTV-1) and 8 (BTV-8) have been described as the most prevalent in Europe during recent outbreaks displaying intense virulence, sheep being among the most severely affected livestock species. However, BTV pathogenesis is still unclear. This study sought to elucidate differences in the pathogenetic mechanisms of BTV-1 and -8 in sheep. For this purpose, a time-course study was carried out, with sequential sacrifices in order to relate pathological lesions to changes in a range of virological and serological parameters. A greater virulence of BTV-1 was probed. BTV-1 infected sheep showed a longer clinical course, with a significant increase of clinical signs and more severe gross lesions than BTV-8 infected sheep. These differences appear not to be attributable to greater virus replication, suggesting viral loads did not influence in the pathogenicity of these serotypes. While both groups displayed an early, intense antibody response, they still developed clinical signs and lesions characteristic of bluetongue, indicating a lack of correlation between antibody levels and protection against the disease. Both acute phase response (APR) and thrombocytopenia induced by BTV-1 in sheep were more intense. Furthermore, an association between acute phase proteins (APPs) concentrations and the evolution of clinical signs and gross lesions was also observed, suggesting the existence of a direct link between the pathogenicity of BTV serotypes, the severity of vascular lesions and the serum concentrations of APPs. To our knowledge, this is the first verification of a measurable APR in sheep with both experimental and naturally occurring bluetongue.

A proteomic analysis of serum from dogs before and after a controlled weight-loss program.

The objective of this study was to investigate how weight-loss program would alter the proteome of the serum of Beagle dogs. For this purpose, serum samples from 5 Beagle dogs, before and after weight loss, were analyzed using 2-dimensional electrophoresis. Protein profiles of all samples were obtained, divided into 2 classes (obese and lean), and compared using specific 2-dimensional software, giving a total of 144 spot matches. Statistical analysis revealed 3 spot matches whose expressions were modulated in response to weight loss: 2 protein spots were upregulated and 1 protein spot was downregulated in the obese state in comparison with the lean state of the dogs. Mass spectrometric identification of differentially regulated spots revealed that these protein spots corresponded to retinol-binding protein 4, clusterin precursor, and α-1 antitrypsin, respectively, which could be considered potential markers of obesity and obesity-related disease processes in dogs.