Antibody isotype epitope mapping of SARS-CoV-2 Spike RBD protein: Targets for COVID-19 symptomatology and disease control.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still poses a challenge for biomedicine and public health. To advance the development of effective diagnostic, prognostic, and preventive interventions, our study focused on high-throughput antibody binding epitope mapping of the SARS-CoV-2 spike RBD protein by IgA, IgM and IgG antibodies in saliva and sera of different cohorts from healthy uninfected individuals to SARS-CoV-2-infected unvaccinated and vaccinated asymptomatic, recovered, nonsevere, and severe patients. Identified candidate diagnostic (455-LFRKSNLKPFERD-467), prognostic (395-VYADSFVIRGDEV-407-C-KLH, 332-ITNLCPFGEV-342-C-KLH, 352-AWNRKRI-358-C-KLH, 524-VCGPKKSTNLVKN-536-KLH), and protective (MKLLE-487-NCYFPLQSYGFQPTNGVG-504-GGGGS-446-GGNYNYLYRLFRKSNLKPFERD-467) epitopes were validated with sera from prevaccine and postvaccine cohorts. The results identified neutralizing epitopes and support that antibody recognition of linear B-cell epitopes in RBD protein is associated with antibody isotype and disease symptomatology. The findings in asymptomatic individuals suggest a role for anti-RBD antibodies in the protective response against SARS-CoV-2. The possibility of translating results into diagnostic interventions for the early diagnosis of asymptomatic individuals and prognosis of disease severity provides new tools for COVID-19 surveillance and evaluation of risks in hospitalized patients. These results, together with other approaches, may contribute to the development of new vaccines for the control of COVID-19 and other coronavirus-related diseases using a quantum vaccinomics approach through the combination of protective epitopes.

Evaluation of the presence of gingivitis as confounding factor in assessing inflammatory status in serum and saliva of dogs with diabetes mellitus.

The aim of this study was to evaluate the changes in the serum and salivary inflammatory markers induced by Diabetes mellitus (DM) in dogs and to assess the possible confounding effect of gingivitis. A panel of 13 cytokines was measured in the serum and saliva of dogs diagnosed with DM and compared with healthy dogs without gingivitis (control group 1; CG1) and dogs with gingivitis but otherwise healthy (control group 2; CG2). The results of the present study showed statistically significantly higher levels of IL-8, KC-like and MCP1 in the serum of dogs with DM compared to CG1 dogs. In the case of saliva, the DM group presented statistically higher GM-CSF, IL6, IL15, and MCP1 levels compared to CG1, and lower KC-like chemokine compared to CG2. Finally, gingivitis produced changes in saliva, with salivary levels of GM-CSF, IL-6, IL-7, IL-15, IP-10, KC-like, IL-10, IL-18, MCP1, TNFα being statistically significantly higher in the saliva of CG2 dogs compared to CG1. The results of the present study indicate that dogs with DM have altered cytokine levels in serum and saliva compared to healthy dogs. In addition, this study highlights the importance of taking oral health into account when determining cytokines in dogs, as gingivitis can significantly alter their concentrations. .

Oxytocin measurements in saliva: an analytical perspective.

Oxytocin has traditionally been known for its physiological effects on muscle contraction associated with birth and lactation, but in the last years is widely used as a biomarker of "positive experiences" in psychology and behavior. Different types of samples have been used for oxytocin measurements with saliva samples having the particular advantage of an easy and non-stressful collection. However, the low concentration of oxytocin in saliva can represent a limitation for its use. For this reason, sensitive assays and even a previous sample treatment in some cases are required for saliva oxytocin quantification. In addition, the lack of standardized and generally agreed-upon approach to peripheral oxytocin measurement leads to large discrepancies between different laboratories, that use different sample treatment protocols and different assays. The main objectives of this review are to describe the current status of the use of saliva for oxytocin measurement, provide details of the different sample processing techniques that can be applied and inform about the analytical techniques and assays available in different animal species, and also in humans for comparative purposes. It is expected that this information can contribute to an increase in the knowledge about the measurements of oxytocin in saliva and to its wider use in the future.

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.

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.

Revealing the Changes in Saliva and Serum Proteins of Pigs with Meningitis Caused by Streptococcus Suis: A Proteomic Approach.

Meningitis due to Streptococcus suis causes high mortality and morbidity on pig farms and has increasing zoonotic potential worldwide. Saliva proteome analysis would potentially be useful in elucidating pathophysiological changes and mining for new biomarkers to diagnose and monitor S. suis infection. The objective of this study was to investigate the changes in the salivary and serum proteome profile of piglets with meningitis. The LC-MS/MS TMT proteomic approach was used to analyze saliva and serum samples from 20 male piglets: 10 with meningitis and 10 healthy. In saliva, 11 proteins had higher and 10 had lower relative abundance in piglets with meningitis. The proteins with the highest relative abundance were metavinculin (VCL) and desmocollin-2 (DSC2). Adenosine deaminase (ADA) was selected for validation using a spectrophotometric assay and demonstrated excellent performance in the differentiation between healthy and pigs with meningitis due to S. suis. In serum, the most protruding changes occurred for one SERPIN and haptoglobin (HP). In saliva and serum, the highest number of proteins with altered abundance were linked, via the enrichment analysis, with platelet and neutrophil pathways. Overall, meningitis caused by S. suis resulted in specific proteome changes in saliva and serum, reflecting different pathophysiological mechanisms, and marking new potential biomarkers for this infection.

Trypanosoma cruzi co-infections with other vector borne diseases are frequent in dogs from the pacific coast of Ecuador.

Dogs are a reservoir for Chagas disease, caused by Trypanosoma cruzi (T. cruzi), and other companion vector-borne diseases, including ehrlichiosis (Ehrlichia canis and Ehrlichia ewingii), anaplasmosis (Anaplasma phagocytophilum and Anaplasma platys), dirofilariasis (Dirofilaria immitis) and Lyme disease (Borrelia burgdorferi). This study has two key objectives: 1) to determine seroreactivity against T. cruzi in dogs from the town of Colón, in Portoviejo city, in the central coast of Ecuador; and 2) to establish the coinfection frequency of other companion vector-borne diseases in dogs positive for T. cruzi. Antibodies against T. cruzi were detected using two enzyme-linked immunosorbent assays. Diagnostic consensus between ELISA tests was established using the Cohen's Kappa coefficient. Other haemoparasitic diseases were detected using the IDEXX SNAP® 4Dx® kit in dogs previously diagnosed as T. cruzi-seropositive. From 84 dogs sampled, 57.14% (48/84) tested positive for T. cruzi. Co-infection analysis of 25 dogs positive for T. cruzi revealed antibodies also against Ehrlichia spp. (48%), Anaplasma spp. (28%), and Dirofilaria immitis (12%). These results provide a novel perspective regarding the status of these pathogens which co-infect dogs in Colón. Since all these pathogens are zoonotic, our findings should warn regional health authorities to implement sanitary programs, to better prevent and control vectors associated to these pathogens. On the other hand, human and veterinarian doctors, should consider that patients with a cardiac infection condition could be suffering co-infections with two or more vector transmitted pathogens.

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.

Seroprevalence of Toxoplasma gondii in outdoor dogs and cats in Bangkok, Thailand.

The aim of this study was to estimate the seroprevalence and risk factors associated with Toxoplasma gondii exposure in dogs and cats from Bangkok, Thailand. Blood samples from 318 dogs and 321 cats were tested for T. gondii antibodies by modified agglutination test (cut-off 1:25). Additionally, 18 dogs and 20 cats were longitudinally sampled for T. gondii antibodies during the same study period, between June and July 2019. The overall seroprevalence in dogs and cats was 7.9% (25/318; 95% CI 4.9­10.8%) and 18.7% (95% CI 14.4­23.0%), respectively. For dogs, risk factors identified were being a mixed-breed animal and living totally outdoors, while increasing age was shown to be a risk factor for cats. Seroconversion was not detected and titres from positive animals remained constant over longitudinal study. The present study indicates that there is a prominent presence of T. gondii in urban and peri-urban areas of Bangkok, suggesting that outdoor dogs and cats should be considered as a possible risk factor for humans.

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.

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.

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.

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.