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Pain in people with cerebral palsy (CP) has been classically underestimated and poorly treated, particularly in individuals with impaired communication skills. To analyze changes in different salivary metabolites and pain behavior scales after a painful procedure in adults with CP and adults with typical development. Salivary levels of sTNF-α, sIgA, Cortisol, FRAP, ADA and Alpha Amylase, as well as 3 observational pain scales (Wong-Baker, Non-Communicating Adults Pain Checklist and Facial Action Coding System) were assessed before and after an intramuscular injection in 30 Individuals with CP and 30 healthy controls. Video recording of face expression was performed during the procedure for offline analysis. Pain in subjects with CP was higher than in healthy controls after the intramuscular injection as displayed by observational scales. sTNF-α experienced a significant post-stimulus increase in both groups and that increase shows a tendency to correlate with the observational scales scores. Other biomarkers classically associated with stress (cortisol, Alpha Amylase) remain stable. sTNF-α might be a promising pain indicator. Further research using controlled painful stimuli of greater intensity and pain self-reports, would be necessary to better understand its use as a pain biomarker.

Inter-alpha-trypsin inhibitor protein heavy chain 4 (ITIH4), also named porcine major acute phase protein (Pig-MAP), is a positive acute phase protein (APP) in pigs and can be measured in plasma and also in saliva to assess the animal health. The objectives of this report were (1) to evaluate if different commercially available porcine ELISA kits can measure ITIH4 concentrations in saliva samples of pigs, and (2) to study the distribution of ITIH4 components in saliva and compare it to the distribution in plasma. The results showed that two of the ELISA kits used in this report could measure salivary ITIH4 with precision and accuracy, but only one showed significant differences between pigs with tail biting and control animals without this condition. Western blotting analysis revealed the presence of a different distribution of ITIH4 bands in saliva samples compared to plasma samples. In conclusion, in saliva of pigs ITIH4 can be quantified using a commercial ELISA kit increasing its concentration in cases of tail biting. In addition, ITIH4 shows bands at western blot in saliva that are different from serum but are compatible with different ITIH4 forms. These data confirm that ITIH4 can be detected in saliva and be potentially used as a biomarker of inflammation.

The current standard treatment for canine leishmaniosis (CanL), N-methylglucamine antimoniate (MGA) given with allopurinol, is not fully effective and may cause adverse effects and drug resistance. In vitro and in vivo studies have shown that nucleotides, administered alone or with AHCC, offer benefits in the treatment of CanL. This study examines the effects of a new immunomodulatory treatment protocol in which dietary nucleotides and AHCC are added to the recommended standard treatment. Out of 160 sick dogs with naturally occurring clinical leishmaniosis recruited, 97 were randomized to a supplement (n = 47) or control (n = 50) group. All dogs received an initial 28-day course of MGA and 365-day course of allopurinol. From day 0 to day 730, dogs in the control group additionally received a placebo, while dogs in the supplement group received Impromune (Bioiberica S.A.U., Esplugues de Llobregat, Spain), an oral supplement providing 32 mg/kg nucleotides and 17 mg/kg AHCC daily. After 2 years, five dogs had relapsed in the supplement group (18.5%) while seven did so in the control group (22.6%). Over time, animals in both groups showed significant improvements in body weight, LeishVet clinical stage, clinical score, and anti- Leishmania antibodies. Adding the supplement to the standard protocol resulted in further significant improvements, namely in reducing the parasite load and urinary protein/creatinine ratio, improving IRIS stage, lowering serum creatinine levels on day 30, deceasing urine turbidity on day 365, and improving weight gain on day 545. The daily intake of the supplement over two years proved safe and well tolerated. Our study confirms the efficacy of the recommended standard treatment for CanL, but also reveals that by adding Impromune additional benefits are obtained, especially reduced parasitemia and improved renal function.

Canine babesiosis caused by the intraerythrocytic protozoan parasite Babesia canis is a tick-borne disease characterized by a host response that involves both cellular and humoral immunity. This study focuses on the secretion of cytokines Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Keratinocyte Chemotactic-like (KC-like), Interleukins (IL)-2, IL-7, IL-8, IL-10, IL-15, IL-18 and Monocyte Chemotactic Protein-1 (MCP-1) in babesiosis caused by Babesia canis upon treatment with Imizol®. We assessed time dependent changes in cytokine levels and tested whether these changes correlate with pathogenesis of the disease. Sixteen healthy dogs and 31 dogs infected with Babesia canis, of which 18 showed complications, were treated with Imizol®. One dog died during the study (3.2%). Longitudinal study was perfomed by monitoring dogs at the first day of presentation (day 1) and 6 days later (day 7). Our results show that higher MCP-1 levels on day 1 are positively associated with the occurrence of complications, (complicated vs. uncomplicated; p = 0.00016). A similar pattern was observed for KC-like on day 1 (p = 0.0326) and day 7 (p = 0.044). Moreover, babesiosis caused by B. canis produced a steady increase in IL-8 levels with a moderate to strong negative correlation with erythrocyte counts and hematocrit in uncomplicated diseased dogs only (Spearman's rank correlation coefficient rs = -0.582 and rs = -0.598 respectively). Like for MCP-1, KC-like levels also differed in complicated and uncomplicated diseased dogs on day 1 (p = 0.03236) and day 7 (p = 0.044). Furthermore, KC-like levels were strongly correlated with IL-8 levels (rs = 0.663-0.7) and non-segmented neutrophil counts (rs = 0.572-0.732) in both diseased groups. Analysis of ROC suggests the use of serum levels of MCP-1 and IL-7 as predictors of the occurrence of complications with an AUC of 0.906 and 0.896 respectively and linear combinations of MCP-1, KC-Like, IL-7 and GM-CSF with values up to AUC = 0.983. Cytokine cluster analysis presented in this study can contribute to a better understanding of the pathogenesis of babesiosis and serve as a prognostic tool for the early detection of cases with highest likelihood of developing complications. Overall, our studies show that infection by B. canis elicits a cytokine pattern that is distinct from that observed with B. rossi, and that some of the inflammatory mediators can be useful to predict complications. Our results also suggest targets for the development of novel therapeutic strategies in babesiosis caused by B. canis.

The objective of this research was to develop and validate two immunoassays for oxytocin measurement in human saliva, one using a monoclonal and the other a polyclonal antibody against oxytocin, whose affinity for oxytocin was tested by an antibody mapping epitope analysis. These assays were analytically validated and used to compare oxytocin concentrations with those obtained with a commercial kit before and after the extraction or reduction/alkylation (R/A) treatments to saliva samples. The assays were also used to evaluate changes in salivary oxytocin concentrations following a physical effort and an induced psychological stress, which have previously been described as situations that cause an increase in salivary oxytocin. Both assays showed to be precise and accurate in the validation studies, and the antibodies used showed a defined binding region in case of the monoclonal antibody, whereas the polyclonal antibody showed binding events through all the oxytocin sequence. Although the monoclonal and polyclonal assays showed a positive correlation, they give results in a different range of magnitude. Both assays showed significant increases in oxytocin concentrations when applied after the physical effort and the psychological stress. This study shows that a variability in the reported values of oxytocin can occur depending on the assay and indicates that the use of different types of antibodies can give a different range of values when measuring oxytocin in saliva.

African swine fever (ASF) has reached an unprecedented global spread and threatens domestic and wild pigs on all continents. In domestic pigs and wild suids outside Africa, the disease is associated with signs of a viral hemorrhagic fever, the pathogenesis of which has not been fully elucidated to date. To address these knowledge gaps, the study of biomarkers of different physiopathological pathways that can be measured in saliva, can provide important insights and contribute to a more comprehensive understanding of ASF pathogenesis. In the present study, animal experiments were performed with experimentally ASF virus (ASFV/ “Prenzlau 22”) infected domestic pigs (DP) and wild boar (WB). For this, analyses of various biomarkers in saliva from DP and WB were conducted at three different time points of ASFV infection. For comparative purposes, serum biochemistry, qPCR values, and clinical parameters (i.e., clinical score and rectal body temperature) were also assessed. Biomarker analyses in the saliva of the infected DP showed significant increases in haptoglobin, S100A8/A9, S100A12, total protein, adenosine deaminase and LDH. In WB, α-amylase was increased in saliva at 7 days post-infection. In addition, changes in biomarkers of stress and inflammation were observed in the serum of DP and WB. Overall, in this report we demonstrate notable alterations in saliva and serum analytes of ASFV-infected DP and WB, reflecting physiopathological mechanisms such as activation of the stress, immune system, and inflammation. In future studies, the potential of these analytes as biomarkers of the disease and as a tool to evaluate the response of the host to the infection should be undertaken.

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.

Saliva is gaining importance as a complementary or alternative sample to serum for biomarker analysis. However, there is still a lack of knowledge about the possible relations between saliva and serum in health and disease. In this report, a total of 21 biomarkers were studied in saliva and serum from three groups of healthy pigs with different ages (T0, recently weaned pigs, 15 females and 15 males; T1, intermediate nursery, 15 females and 15 males; and T2, fattening period, 15 females and 15 males) and in a group of animals with Actinobacillus pleuropneumoniae (A. pp.) infection (intermediate nursery, 20 females). Sex of the animals did not significantly influence the results in either saliva or serum from healthy animals. In healthy pigs, the α-amylase (AA), ferric reducing ability (FRA), Urea, Triglycerides, Calcium (Ca) and Phosphorous (P) showed a similar dynamic in saliva and serum across fattening, whereas Butyrylcholinesterase (BChE), Adenosine deaminase (ADA), Immunoglobulin G (IgG), C-reactive protein (CRP), Haptoglobin (Hp), Total protein (TP), Myeloperoxidase (MPO), Ferritin, the Advanced oxidation protein products (AOPP), Uric acid (UA), Aspartate aminotransferase (AST), Creatine kinase (CK), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and γ-glutamil transferase (gGT) showed a different dynamic between serum and saliva. In pigs with A. pp infection, saliva showed significant changes in more analytes than serum, with AA, ADA, MPO, AST, LDH and Ca showing significant increases only in saliva. Differences in sensitivity to detect A. pp. infection were found in some selected analytes between saliva and serum. Biomarkers can show different changes in saliva and serum depending on age, and they can show different responses to disease between saliva and serum.

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 R 2  > 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.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2), is usually associated with a wide variety of clinical presentations from asymptomatic to severe cases. The use of saliva as a diagnostic and monitoring fluid has gained importance since it can be used to investigate the immune response and to direct quantification of antibodies against COVID-19. Additionally, the use of proteomics in saliva has allowed to increase  our understanding of the underlying pathophysiology of diseases, bringing new perspectives on diagnostics, monitoring, and treatment. In this work, we compared the salivary proteome of 10 patients with COVID-19, (five patients with mild and five patients with severe COVID-19) and ten control healthy patients. Through the application of proteomics, we have identified 30 proteins whose abundance levels differed between the COVID-19 groups and the control group. Two of these proteins (TGM3 and carbonic anhydrase-CA6) were validated by the measurement of gGT and TEA respectively, in 98 additional saliva samples separated into two groups: (1) COVID-19 group, integrated by 66 patients who tested positive for COVID-19 (2) control group, composed of 32 healthy individuals who did not show any sign of disease for at least four weeks and were negative for COVID-19 in RT-PCR. In the proteomic study there were observed upregulations in CAZA1, ACTN4, and ANXA4, which are proteins related to the protective response against the virus disturbance, and the upregulation of TGM3, that is correlated to the oxidative damage in pulmonary tissue. We also showed the downregulation in cystatins and CA6 that can be involved in the sensory response to stimulus and possibly related to the presence of anosmia and dysgeusia during the COVID-19. Additionally, the presence of FGB in patients with severe COVID-19 but not in mild COVID-19 patients could indicate a higher viral aggregation and activation in these cases. In conclusion, the salivary proteome in patients with COVID-19 showed changes in proteins related to the protective response to viral infection, and the altered sensory taste perception that occur during the disease. Moreover, gGT and TEA could be potential biomarkers of respiratory complications that can occurs during COVID 19 although further larger studies should be made to corroborate this.