Automated Spectrophotometric Assays for the Measurement of Ammonia and Bicarbonate in Saliva of Horses: Analytical Validation and Changes in Equine Gastric Ulcer Syndrome (EGUS).

Ammonia (NH3) and bicarbonate (HCO3) have been related to gastric ulcers in humans. Ammonia is considered a possible cause of gastric ulcers, whereas bicarbonate has a protective function. The presence of ulcers in the stomach of horses is defined as Equine Gastric Ulcer Syndrome (EGUS), which is a frequent disease in this species, and it has been associated with changes in saliva composition, such as in analytes related to inflammation, immune system and oxidative stress. The objectives of this study were (1) to perform an analytical validation of two automated spectrophotometric assays, one for ammonia and one for bicarbonate, in the horses' saliva and (2) to evaluate their possible variations with EGUS. Analytical validation of the automated assays for ammonia and bicarbonate in the saliva of horses showed that both assays were precise and accurate. In addition, significantly higher values of ammonia and lower values of bicarbonate were found in the saliva of horses with EGUS compared to healthy horses. It can be concluded that ammonia and bicarbonate can be measured in the saliva of horses and that ammonia increases and bicarbonate decreases in this sample type could be related to the presence of EGUS in this species.

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. .

Changes in the saliva proteome analysed by gel-proteomics in horses diagnosed with equine gastric ulcer syndrome (EGUS) at diagnosis and after successful treatment.

Equine gastric ulcer syndrome (EGUS) is currently one of the more frequent diseases in horses. We aimed to identify changes in the salivary proteome in horses with EGUS at diagnosis and after successful treatment by using gel proteomics. Saliva samples were collected from nine horses with EGUS before and after treatment and nine matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. Horses with EGUS had increases in proteins such as adenosine deaminase (ADA), triosephosphate isomerase, keratins and immuno-globulin heavy constant mu and decreases in carbonic anhydrase (CA), albumin and prolactin-induced protein. These changes would indicate various physiopathological mechanisms involved in this disease, such as the activation of the immune system, decreased stomach defence mechanisms and inflammation. The treated horses presented lower expression levels of thioredoxin (TRX) after a successful treatment, in proteomics analysis and also measured with a commercially available ELISA kit. Overall, horses with EGUS have protein changes in their saliva when measured with gel proteomics compared with healthy horses, and they also showed changes after successful treatment. These proteins could be potential biomarkers for detection and monitoring treatment response in EGUS.

Saliva Sampling Material Matters: Effects on the Results of Saliva Analysis in Pigs.

The use of saliva as a biological sample from pigs is of high practical interest because blood collection from pigs is difficult and stressful. In this study, the influence of two different materials, a cotton roll and a polypropylene sponge, in porcine saliva collection was evaluated. For this purpose, the effect of the material used for sampling was evaluated in a panel of 13 analytes, including those related to stress (cortisol and oxytocin), inflammation and immunity (adenosine deaminase, haptoglobin and myeloperoxidase), redox homeostasis (the cupric reducing ability of saliva, the ferric reducing activity of saliva, and the Trolox equivalent antioxidant capacity), and sepsis (procalcitonin), as well as other routine analytes related to metabolism and different tissues and organs, such as lactate dehydrogenase, creatine kinase, urea, and total protein concentration. The polypropylene sponge provided a higher sample volume than the cotton roll. Although the results of some salivary analytes were equivalent for both materials, other analytes, such as creatine kinase, haptoglobin and total proteins, showed significant differences depending on the material used for saliva collection. Therefore, the type of material used for salivary collection in pigs should be considered when interpreting the results of analyses of the salivary analytes.

Gaining knowledge about biomarkers of the immune system and inflammation in the saliva of pigs: The case of myeloperoxidase, S100A12, and ITIH4.

An assay for the measurement of myeloperoxidase (Mpx) in porcine saliva was developed and validated, and factors influencing Mpx and another two biomarkers of inflammation and immune system, the protein S100A12 and the inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), were studied. The spectrophotometric method for Mpx measurement validated in this assay showed an adequate analytical performance including precision and accuracy. When a group of twenty healthy pigs was sampled every 4 h from 8 a.m. until 8 p.m., Mpx and S100A12 showed significant increases at 4 p.m., whereas ITIH4 concentration showed a significant decrease at 12 a.m. Increases were also seen in salivary Mpx, S100A12, and ITIH4 levels 24 h after the intramuscular administration of Escherichia coli lipopolysaccharide in five pigs; whereas in a non-septic inflammation after the subcutaneous administration of turpentine oil to five pigs changes were seen in S100A12 at 3 h and in ITIH4 at 48 h. When a stressful situation consisting of the transportation and stay of 4 h to a slaughterhouse of 24 pigs was performed, all analytes were increased after 4 h of lairage in the slaughterhouse compared with the values that were obtained the day before at the same time of the day. Mpx can be measured in the saliva of pigs with the automated assay described in this report. Mpx, S100A12, and ITIH4 salivary levels can change depending on the hour of the day in which the sample is taken, and increases can be produced due to sepsis, non-septic inflammation and stress.

Changes in S100A8/A9 and S100A12 and Their Comparison with Other Analytes in the Saliva of Pigs with Diarrhea Due to E. coli.

The family of calgranulins includes S100A8 (calgranulin A), S100A9 (calgranulin B), which can appear as a heterodimer known as S100A8/A9 or calprotectin, and S100A12 (calgranulin C). These proteins are related to different inflammatory conditions, immune-mediated diseases, and sepsis and are considered biomarkers of potential interest. This study aims to evaluate if S100A8/A9 and A12 could change in pigs with diarrhea due to E. coli and to compare the changes of S100A8/A9 and A12 with other analytes in order to explore the possible causes or mechanisms involved. For this purpose, a panel integrated by analytes related to inflammation (haptoglobin, inter-alpha trypsin inhibitor 4 (ITIH4), and total protein); immune system (adenosine deaminase, ADA); stress (alpha-amylase); tissue damage (lactate and lactate dehydrogenase (LDH)); sepsis (aldolase) and redox status (ferric-reducing ability of saliva (FRAS) and advanced oxidation protein products (AOPP)) was evaluated. S100A8/A9 and A12 and the other analytes measured in this study showed increases in the saliva of pigs with diarrhea due to E. coli. S100A8/A9 and/or A12 showed a significant correlation of different magnitude with some of the other analytes evaluated. Further studies should be conducted to gain knowledge about the possible practical applications as biomarkers of the measurements of S100A8/A9 and A12 in the saliva of pigs.

Serum Ferritin in Obese Dogs: Changes and Comparison with Other Analytes.

Canine obesity is the most common nutritional disorder and is associated with decreased quality of life and longevity as well as comorbidities including cardiorespiratory, endocrine, oncologic, or orthopaedic disorders. Ferritin is a major acute-phase protein in dogs, increasing during inflammation; however, it could also be affected by other conditions, including trauma, iron metabolism dysregulations, neoplasia, or hypoxia. Higher ferritin levels have been reported in obese humans, but ferritin has not been explored in canine obesity. To evaluate the possible changes in serum ferritin in canine obesity, ferritin levels from lean/normal weight (CG, n = 55) and overweight/obese dogs (OG, n = 37) were measured, together with complete hemogram and biochemical analyses. Statistically significant higher ferritin levels (1.2-fold) were found in OG (median, (interquartile range), 204 (166-227.5) µg/L) in comparison to CG animals (172 (137-210) µg/L)), with median levels of ferritin in OG dogs above the reference range for healthy animals in our laboratory (60-190 µg/L). In addition, statistically significant higher mean corpuscular volume (MCV), mean cell haemoglobin concentration (MCHC), total proteins, globulins, haptoglobin, total ferric fixation capacity (TIBC), alkaline phosphatase (ALP), butyrylcholinesterase (BChE), triglycerides, and calcium were observed in OG in comparison to CG. The higher levels in ferritin, together with higher TBIC, haematocrit, and MCV, could indicate tissue hypoxia in obese dogs.

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.

S-100 Proteins: Basics and Applications as Biomarkers in Animals with Special Focus on Calgranulins (S100A8, A9, and A12).

S100 proteins are a group of calcium-binding proteins which received this name because of their solubility in a 100% saturated solution of ammonium sulphate. They have a similar molecular mass of 10-12 KDa and share 25-65% similarity in their amino acid sequence. They are expressed in many tissues, and to date 25 different types of S100 proteins have been identified. This review aims to provide updated information about S100 proteins and their use as biomarkers in veterinary science, with special emphasis on the family of calgranulins that includes S100A8 (calgranulin A; myeloid-related protein 8, MRP8), S100A9 (calgranulin B; MRP14), and S100A12 (calgranulin C). The proteins SA100A8 and S100A9 can be linked, forming a heterodimer which is known as calprotectin. Calgranulins are related to the activation of inflammation and the immune system and increase in gastrointestinal diseases, inflammation and sepsis, immunomediated diseases, and obesity and endocrine disorders in different animal species. This review reflects the current knowledge about calgranulins in veterinary science, which should increase in the future to clarify their role in different diseases and potential as biomarkers and therapeutic targets, as well as the practical use of their measurement in non-invasive samples such as saliva or feces.

Changes in the Saliva Proteome of Pigs with Diarrhoea Caused by Escherichia coli.

Escherichia coli represents the main cause of diarrhoea in pigs. Saliva can provide information about the pathophysiology of diseases and be a source of biomarkers. We aimed to identify changes in the salivary proteome of pigs with diarrhoea caused by E. coli. Saliva samples were collected from 10 pigs with this disease and 10 matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. For validation, adenosine deaminase (ADA) was measured in 28 healthy and 28 diseased pigs. In 1DE, increases in lipocalin and IgA bands were observed for diseased pigs, whereas bands containing proteins such as odorant-binding protein and/or prolactin-inducible protein presented decreased concentrations. Two-dimensional gel electrophoresis (2DE) results showed that saliva from E. coli animals presented higher expression levels of lipocalin, ADA, IgA and albumin peptides, being ADA activity increased in the diseased pigs in the validation study. Spots containing alpha-amylase, carbonic anhydrase VI, and whole albumin were decreased in diseased animals. Overall, pigs with diarrhoea caused by E. coli have changes in proteins in their saliva related to various pathophysiological mechanisms such as inflammation and immune function and could potentially be biomarkers of this disease.

Measurement of Calprotectin (S100A8/A9) in the Saliva of Pigs: Validation Data of A Commercially Available Automated Assay and Changes in Sepsis, Inflammation, and Stress.

Calprotectin (CALP, S100A8/A9), also named myeloid-related protein 8/14, is a dimer complex of S100A8 and S100A9 that belongs to the S-100 protein family. It is involved in inflammation and has a wide range of proinflammatory functions, such as cytokine production and regulation of leukocyte adhesion, migration, and phagocytosis. In humans, CALP traditionally can be measured in faeces, serum, and saliva as a biomarker of inflammation and sepsis. The objective of this study was to validate an automated assay for CALP measurements in the saliva of pigs, having the advantage of the use of a non-invasive sample that is easy to collect. The assay was precise and accurate. CALP in saliva measured by this assay showed significant changes depending on the hour of the day. It also showed significant increases in the saliva of pigs after the administration of lipopolysaccharide (LPS), and showed a rise, although with increases of lower magnitude, after a stressful stimulus. Further studies should be made to gain knowledge about the possible practical applications of the measurements of CALP in the saliva of pigs as a biomarker to evaluate the animals' health and welfare.

Changes in Calprotectin (S100A8-A9) and Aldolase in the Saliva of Horses with Equine Gastric Ulcer Syndrome.

Equine gastric ulcer syndrome (EGUS) is a highly prevalent disease that affects horses worldwide. Within EGUS, two different forms have been described: equine squamous gastric disease (ESGD) and equine glandular gastric disease (EGGD). The associated clinical signs cause detrimental activity performance, reducing the quality of life of animals. Saliva can contain biomarkers for EGUS that could be potentially used as a complementary tool for diagnosis. The objective of this work was to evaluate the measurements of calprotectin (CALP) and aldolase in the saliva of horses as potential biomarkers of EGUS. For this purpose, automated assays for the quantification of these two proteins were analytically validated and applied for detecting EGUS in a total of 131 horses divided into 5 groups: healthy horses, ESGD, EGGD, combined ESGD and EGGD, and horses with other intestinal pathologies. The assays showed good precision and accuracy in analytical validation, and they were able to discriminate between horses with EGUS and healthy horses, especially in the case of CALP, although they did not show significant differences between horses with EGUS and horses with other diseases. In conclusion, salivary CALP and aldolase can be determined in the saliva of horses and further studies are warranted to elucidate the potential of these analytes as biomarkers in EGUS.

Trace Elements and Ferritin in Pig Saliva: Variations during Fattening, Time of Sampling, Effect of Dirtiness and Stability under Different Storage Conditions.

The objective of this study was to evaluate the possible changes of zinc (Zn), copper (Cu), iron (Fe) and ferritin during the entire productive cycle in fattening pigs and at different diurnal sampling times. Moreover, the possible effects of the presence of pen contaminants and storage stability at different temperature conditions were assessed. The analytes changed along the different phases of the fattening productive cycle, showing, in general, higher values at the initial phases. In addition, statistically significant variations were found in Zn and Cu measurements at different sampling times of the day. In the spectrophotometric assays, the values of all analytes significantly increased after adding high concentrations of feces or feed. However, when low concentrations of feces or feed were added, only Cu showed a significant increase. Overall, the salivary levels of Zn, Cu, Fe and ferritin in pigs can change during different fattening phases and the different hours of the day. These analytes were more stable at -80 °C and, if saliva is contaminated with feces or feed, it can lead to an increase in these analytes.

Serum and urine profiling by high-throughput TMT-based proteomics for the investigation of renal dysfunction in canine babesiosis.

Canine babesiosis is a tick-borne disease caused by Babesia canis, with acute kidney injury as one of the common complications. In the study 8 healthy control dogs and 22 dogs with naturally occurring babesiosis were enrolled, with the aim to analyse differences in serum and urinary proteomes between healthy dogs and dogs with different degree of renal dysfunction in babesiosis using a label-based high-throughput quantitative proteomic approach. In serum, 58 proteins were found differentially abundant between healthy controls and groups of dogs with different degrees of renal dysfunction in babesiosis, while in urine there were 259 differentially abundant proteins. In addition, altered biological pathways were detected in the diseased dogs using bioinformatics tools and validation of several candidate biomarkers was performed. SIGNIFICANCE: The main aim of this comprehensive study was to perform analyses of serum and urinary proteomes of dogs with renal dysfunction in babesiosis compared to healthy dogs using, for the first time, a high-throughput proteomic method and functional enrichment analyses. Serum and urine samples of the same dogs were investigated in order to gain a more complete picture of pathologic changes taking place in renal dysfunction in babesiosis. We highlighted two putative biomarkers validated herein which could be of importance for early diagnosis of renal dysfunction in canine babesiosis, as they are easily accessible from urine and their concentration rises before the appearance of azotaemia: urinary neutrophil gelatinase-associated lipocalin (NGAL) and urinary liver-type fatty acid-binding protein (L-FABP).

Evaluation of a Comprehensive Profile of Salivary Analytes for the Diagnosis of the Equine Gastric Ulcer Syndrome.

In this report, the measurement of salivary biomarkers as an aid for diagnosis of equine gastric ulcer syndrome (EGUS) was studied. A comprehensive panel of 23 salivary analytes was measured in the saliva of horses affected by EGUS and compared to healthy animals and horses with other diseases clinically similar to EGUS but with a negative diagnosis at gastroscopic examination. A total of 147 horses were included in the study and divided into heathy population (n = 12), the EGUS group (n = 110), and the group of horses with other diseases (n = 25). From the 23 analytes studied, 17 showed increased values in EGUS horses when compared to healthy ones, and uric acid, triglycerides, and calcium were significantly increased in horses with EGUS compared to the group of other diseases. The receiver operating characteristic curve analyses showed a modest but significant discriminatory power of those three analytes to identify EGUS from other diseases with similar symptoms. The discriminatory power enhanced when the results of the three analytes were combined. In conclusion, the results showed that selected salivary analytes could have potential use as biomarkers in horses with EGUS.

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.

Changes in the salivary metabolome in canine hypothyroidism: A pilot study.

Hypothyroidism is the most commonly diagnosed endocrine disorder in dogs. It produces a deficiency of thyroid hormones which impacts negatively the dog's quality of life. The objective of this study is to evaluate the possible changes in the salivary metabolic profile in dogs with hypothyroidism. For this purpose, targeted metabolomics analysis performed by LC/MS analysis was made in saliva samples from a group of dogs with hypothyroidism and a group of healthy dogs. Twenty-three metabolites showed a significant decrease between hypothyroid and healthy dogs, most of these associated with thyroid hormone synthesis, catecholamine synthesis, and tyrosine and phenylalanine metabolism. Based on the results, it can be stated that hypothyroidism produces changes in the metabolome of saliva and some of them can reflect the metabolic changes presented in the disease and could serve as a potential biomarker of this condition.

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.

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.