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Abstract Cardiovascular-kidney-metabolic health reflects the interactions between metabolic risk factors, chronic kidney disease, and the cardiovascular system. A growing body of literature suggests that metabolic syndrome (MetS) in individuals of normal weight is associated with a high prevalence of cardiovascular diseases and an increased mortality. The aim of this study was to establish a non-invasive preclinical model of MetS in support of future research focusing on the effects of novel antidiabetic therapies beyond glucose reduction, independent of obesity. Eighteen healthy adult Beagle dogs were fed an isocaloric Western diet (WD) for ten weeks. Biospecimens were collected at baseline (BAS1) and after ten weeks of WD feeding (BAS2) for measurement of blood pressure (BP), serum chemistry, lipoprotein profiling, blood glucose, glucagon, insulin secretion, NT-proBNP, angiotensins, oxidative stress biomarkers, serum, urine, and fecal metabolomics. Differences between BAS1 and BAS2 were analyzed using non-parametric Wilcoxon signed-rank testing. The isocaloric WD model induced significant variations in several markers of MetS, including elevated BP, increased glucose concentrations, and reduced HDL-cholesterol. It also caused an increase in circulating NT-proBNP levels, a decrease in serum bicarbonate, and significant changes in general metabolism, lipids, and biogenic amines. Short-term, isocaloric feeding with a WD in dogs replicated key biological features of MetS while also causing low-grade metabolic acidosis and elevating natriuretic peptides. These findings support the use of the WD canine model for studying the metabolic effects of new antidiabetic therapies independent of obesity.

Abstract Background Cortisone is derived from cortisol through the action of the enzyme 11β-hydroxysteroid dehydrogenase type II, and it has gained importance in recent years as a biomarker of stress. This study aimed to develop and validate an assay for the measurement of cortisone in pig saliva and evaluate whether its concentration varies in stressful situations. For this purpose, a specific immunoassay was developed and validated analytically, and a study was performed to evaluate whether cortisone concentrations in saliva can vary under heat stress conditions. Results The assay proved to be accurate, reliable, and sensitive for the measurement of cortisone in pig saliva. The limit of detection of the assay was set at 0.006 ng/ml, and the lower limit of quantification was 0.023 ng/ml. It also correlated significantly with the results obtained by LC‒MS/MS (P = 0.003; r = 0.64). In addition, the cortisone concentration in animals subjected to prolonged heat stress decreased significantly 15 days after treatment (P

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.

Equine Gastric Ulcer Syndrome (EGUS) is a widespread disease with a very high prevalence and importance in horses. It includes two conditions: Equine squamous gastric disease (ESGD), linked to acid disturbances, and Equine Glandular Gastric Disease (EGGD), associated with inflammation and immune system issues. In saliva, increased levels of immunological markers have been observed in this disease, indicating immune system involvement. In addition, changes in biomarkers of oxidative stress have been described. In horses, IgG and IgA are proteins that appear in saliva and serum and are considered to be the major proteins in specific immunity; however, their levels in horses with EGUS have not been previously studied. This report aims to evaluate IgG and IgA concentrations in the saliva and serum of horses with EGUS, measured by automated assays, and compare them with other biomarkers of the immune and oxidative stress system. This work validated the IgG and IgA assays in saliva and showed that IgA significantly increased in the saliva of horses with EGUS. When the correlation between IgG and IgA and other biomarkers of immune system issues and oxidative stress such as ADA, S100 A8-A9 (calprotectin), S100 A12 (calgranulin), uric acid, FRAS, and AOPP was studied, IgA in saliva showed a significant moderate correlation with adenosine deaminase, indicating its possible involvement in the immune reaction occurring in EGUS.

Abstract 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.

Abstract 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.

The objective of this study was to evaluate the possible use of spectrophotometric assays for the measurement of trace elements, including Zinc (Zn), Copper (Cu), Magnesium (Mg), and iron (Fe) in the saliva of horses and study their possible changes in equine gastric ulcer syndrome (EGUS). EGUS is a highly prevalent disease, with a current high incidence due to the increase in intensive management conditions. There are two EGUS diseases: equine squamous gastric disease (ESGD) and equine glandular gastric disease (EGGD), which can appear individually or together. For this purpose, automated spectrophotometric assays for measuring these analytes in horse saliva were analytically validated. Then, these analytes were measured in the saliva of horses with only ESGD, only EGGD, both ESGD and EGGD and a group of healthy horses. The methods used to measure the analytes were precise and accurate. Horses diagnosed with EGGD presented significantly lower levels of Zn and Mg. Fe concentrations were significantly lower in the saliva of horses with ESGD and EGGD. Overall, these results indicate that there are changes in trace elements in saliva in EGUS that could reflect the physiopathological mechanisms involved in this process and open the possibility of using trace elements as biomarkers of this syndrome.

Cystatin C, ammonia, and bicarbonate have been described to be biomarkers of sepsis and inflammation in humans. The saliva of pigs can be used to detect a wide range of pathogens but also many biomarkers that can be analyzed to evaluate different conditions such as stress (i.e., cortisol and alpha amylase), immune system (i.e., ADA, S100 proteins), inflammation (i.e., acute phase proteins), redox status (i.e., various antioxidants and oxidants), and general metabolism or the status of different organs and tissues. However, there is a lack of assays for the possible measurement and use of cystatin C, ammonia, and bicarbonate in saliva as biomarkers of sepsis or inflammation in pigs. The objective of this study was to validate commercially available automated assays for the measurement of cystatin C, ammonia, and bicarbonate in the saliva of pigs, having the advantage of using a noninvasive sample that is easy to collect. The assays were precise and accurate, and the recommended storage condition for the saliva samples was −80 °C. In addition, cystatin and ammonia showed significant increases in the saliva of pigs with S. suis infection, whereas bicarbonate decreased. Further studies would be recommended to increase knowledge about the possible potential applications of the measurements of these three analytes in the saliva of pigs as biomarkers to evaluate the animals’ health and welfare.

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.

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.

Heat stress accounts for millions of dollars in losses for swine producers worldwide. The aim of the present study was to determine and evaluate cortisol and cortisone in hair as indicators of thermal stress in growing pigs reared under high environmental temperatures. The study was carried out in two independent batches of commercial crosses of Lean Duroc and Pietrain in trials 1 and 2, respectively, during the growing period (from 40 to 100 kg; 81 days in trial 1 and 77 days in trial 2) in the same commercial farm in Spain during the summers of 2020 and 2021. In both cases, four rooms were used. In Trial 1, Room 1 had cooling and 11 pigs per pen; Room 2 had no cooling and 13 pigs per pen; Room 3 had no cooling and 11 pigs per pen, and Room 4 had cooling and 13 pigs per pen. In Trial 2, Rooms 2 and 3 had cooling and rooms 1 and 4 had no cooling, and all of them had 13 pigs per pen. Mean THI value was higher (p

Abstract 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.

Maternal dietary factors have been reported to influence Clostridioides difficile colonisation in the offspring. Twenty suckling piglets from sows fed diets supplemented with high-fermentable sugar beet pulp (SBP) or low-fermentable lignocellulose (LNC) fibres during gestation and lactation were dissected in the first week after birth. Postmortem analysis included clinical mesocolon and faecal scoring, concentration of C. difficile and respective toxins in colon digesta and faeces, immunoglobulins in serum and inflammatory markers in serum and colon tissues. Sow colostrum was assessed for nutrients, immunoglobulins and biogenic amines. Toxin-neutralising IgG antibodies were measured in colostrum and serum of the sows, and in colon digesta and serum of the piglets. Mesocolonic oedema of different severity was present in most of the piglets from both sows’ feeding groups. Concentrations of C. difficile, toxins and calprotectin in colon digesta and faecal contents did not differ between the study piglets. Calprotectin correlated positively with mesocolon score (rho = 413, P = 0.07). Piglets from sows fed LNC vs SBP tended to have higher IgA (P = 0.089), IgG (P = 0.053), total Ig (P = 0.053), albumin (P = 0.075) and total protein content (P = 0.007) in serum. Colon tissues of piglets from the SFB vs LNC had upregulated expression of ZO-1 (P = 0.021), PCNA (P = 0.015) and TGF-β (P = 0.014). Titers of anti-toxin-IgG-antibodies in serum and colostrum and in piglet colon digesta and serum did not differ between sows from both dietary groups, but they all showed strong positive correlations. In conclusion, dietary sugar beet pulp or lignocellulose fed to sows did not influence the concentrations of C. difficile and toxins titers in colon digesta and faeces of neonatal piglets.

Abstract Background Postpartum dysgalactia syndrome (PDS) is associated with a significantly higher activation of the inflammatory and stress response at parturition than in the healthy sow. Therefore, reliable and possibly non-invasive biomarkers for substantial increases of inflammation are searched to support the PDS diagnosis. This report studies the possible changes of the inflammatory marker enzyme adenosine deaminase (ADA) in serum and saliva of 38 PDS positive sows (PDS+) and 38 healthy sows (PDS-). Sampling was performed every 24 h from 60 h before to 36 h after parturition. Isoenzyme 1 (ADA1) and isoenzyme 2 (ADA2), as well as total ADA (tADA), were measured and their statistical association with several serum and saliva biomarkers of inflammation and stress was investigated. Results Compared to a baseline (60 to 36h prepartum), salivary activities of ADA1, ADA2 and tADA increased significantly over time in both PDS+ and PDS- sows, reaching their peaks after parturition. In serum from PDS- sows, no changes were observed over time in either ADA1, ADA2 or tADA. In PDS+ sows, serum ADA2 activity decreased temporarily after parturition followed by a significant increase compared to baseline. ADA1, ADA2 and tADA were all significantly associated with several inflammatory biomarkers and ADA1 in serum was associated with serum cortisol. Although serum activity was higher in PDS+ than in PDS- sows, the differences were not statistically significant. Further, no difference was noted between the groups in the analyses of saliva. Conclusions Salivary ADA1 and ADA2 increased in all sows after parturition, potentially as a response to the postpartum inflammation. However, no difference in the activity of ADA1, ADA2 and tADA were found between PDS+ and PDS- sows indicating inability to diagnose PDS under the conditions described in this report.

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.

Abstract Background Acute and chronic besnoitiosis in extensive natural-service herds can have relevant effects in the health of bulls and negative consequences in their productive performance. Recent progress has been made in order to elucidate the pathogenesis of this disease. In this context, the study of biomarkers of inflammation in serum would contribute to gaining knowledge about the physiopathology of bovine besnoitiosis. Serological biomarkers could help in early diagnosis and prognosis, as seropositive bulls may have mild or severe testicular lesions. Methods Herein, we have investigated the diagnostic and/or prognostic value of a panel of serum (serological) biomarkers related to inflammation, including total protein, globulin and albumin, haptoglobin (Hp), adenosine deaminase (ADA) paraoxonase-1 (PON-1) and acetylcholinesterase (AChE) in naturally and experimentally B. besnoiti-infected males classified according to different clinical phases of the disease (acute, chronic and subclinical besnoitiosis). Results Results showed a similar response pattern in these biomarkers for naturally and experimentally infected cattle, with a few relevant variations. Most significant changes occurred during the acute phase of infection, although significant changes in a few biomarkers were also observed during the chronic infection. Haptoglobin, albumin, PON-1 and ADA were identified as the biomarkers that showed changes of higher magnitude in the acute phase of the infection, whereas high total protein and globulin values were found in chronically infected cattle. We have described the changes of a panel of inflammatory biomarkers of acute and chronic bovine besnoitiosis. Conclusions In summary, several biomarkers with promising diagnostic value have been identified. The biomarkers associated with acute infection are related to previously reported molecular biomarkers in testicular parenchyma of infected bulls and could help in the diagnosis of early infections and complement results from specific immunoglobulin M (IgM) detection. Graphical abstract

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.

Acute phase proteins have been used as tools for the diagnosis, monitoring, and prognosis of several diseases in domestic animals. However, the dynamics of these proteins in infection by Trypanosoma cruzi, the causative agent of Chagas disease in dogs, is still unknown. The aim of this study was to determine concentrations of acute phase proteins (C-reactive protein, haptoglobin, ferritin and paraoxonase-1) in dogs in a coastal town of Ecuador, with natural Trypanosoma cruzi infection with or without seroreactivity of Ehrlichia canis, Ehrlichia ewingii, Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi and Dirofilaria immitis. For the detection of Trypanosoma cruzi serum antibodies, two different antigen-based enzyme-linked immunosorbent assay tests were implemented. For the detection of seroreactivity of Ehrlichia canis, Ehrlichia ewingii, Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi and Dirofilaria immitis, an IDEXX SNAP® 4Dx® test was used. To determine the concentration of C-reactive protein and ferritin, an immunoturbidimetric assay was used; haptoglobin concentration was measured using a commercial colorimetric method validated in dogs; a spectrophotometric method was used to determine the serum concentration of paraoxonase-1. Results showed a reduction in the serum levels of paraoxonase-1 in Trypanosoma cruzi-seroreactive dogs, either with or without seroreactivity to other vector-borne diseases. A serum ferritin increment was observed in Trypanosoma cruzi-seroreactive dogs with seroreactivity to any other vector-borne diseases. Our findings suggest that paraoxonase-1 levels are reduced in Trypanosoma cruzi-seroreactive dogs without evident clinical signs of Chagas disease, despite their seroreactivity to the other vector-borne diseases studied. These findings could indicate an oxidative stress response in Trypanosoma cruzi-seroreactive dogs with no evident signs of inflammation.

Abstract 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.

Abstract 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 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

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.

The purpose of this trial was to evaluate serum levels of oxidative stress biomarkers and biochemical analytes in crossbred lambs during the rearing phase in an integrated crop-livestock system (ICLS) to control gastrointestinal parasites. The experiment used 36 crossbred lambs (cross: Ile de France × White Dorper × Texel) divided into two groups. The WCS group was supplemented with whole cottonseed (WCS), and controls had no supplementation. Body weight, blood collection, and fecal analysis of nematode eggs and Eimeria oocysts counting per gram of feces were performed for each animal within 84 days of experiment. The following serum analytes were determined: total protein, albumin, globulin, cholesterol, haptoglobin, and 10 oxidative stress biomarkers: cupric reducing antioxidant capacity, ferric reducing ability of plasma, trolox equivalent antioxidant capacity, thiol, uric acid, paraoxonase-1, total oxidant status, ferric-xylenol orange, advanced oxidation protein products, and reactive oxygen metabolites derived compounds. The inclusion of WCS suggested the benefit in controlling infection as well as inducing an increase in antioxidants and a decrease in oxidants in lambs naturally infected by gastrointestinal parasites. The combination of WCS and ICLS could be a useful tool in controlling gastrointestinal parasite infection without affecting the production performance.

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.

Abstract 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.

Saliva from pigs is gaining attention as an easy sample to obtain, being a source of biomarkers that can provide information on animal health and welfare. This study aimed to evaluate the changes that can occur in salivary biomarkers of the redox status of pigs with an experimentally induced sepsis. For that, the cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of saliva (FRAS), Trolox equivalent antioxidant capacity (TEAC), advanced oxidation protein products (AOPP), ferrous oxidation-xylenol orange (FOX), peroxide activity (POX-Act), and reactive oxygen-derived compounds (d-ROMs) were measured in the saliva of pigs with experimentally induced sepsis by endotoxin lipopolysaccharide (LPS), non-septic inflammation induced by turpentine, and in healthy individuals before and after 3 h, 6 h, 24 h, and 48 h. AOPP, POX-Act, and d-ROMs in the sepsis group were higher than in the control from 3 h to 24 h after the inoculation. CUPRAC, FRAS, and TEAC were higher in sepsis than the control group at 24 h. These changes were of higher magnitude than those that occurred in the turpentine group. In conclusion, our findings reveal that sepsis produces changes in salivary biomarkers of redox status, which opens the possibility of using them as potential biomarkers in this species.

The evaluation of the biomarkers of oxidative status is usually performed in serum, however, other samples, such as red blood cells (RBCs) lysates or whole blood (WB), can be used. The objective of this study was to evaluate if a comprehensive panel of redox biomarkers can be measured in the WB and RBCs of dogs, and their possible changes “in vitro” after the addition of different concentrations of ascorbic acid. The panel was integrated by biomarkers of the antioxidant status, such as cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), Trolox equivalent antioxidant capacity (TEAC), thiol and paraoxonase type 1 (PON-1), and of the oxidant status, such as total oxidant status (TOS), peroxide-activity (POX-Act), reactive oxygen-derived compounds (d-ROMs), advanced oxidation protein products (AOPP) and thiobarbituric acid reactive substances (TBARS). All the assays were precise and accurate in WB and RBCs lysates. In addition, they showed changes after ascorbic acid addition that are in line with previously published results, being WB more sensitive to detect these changes in our experimental conditions. In conclusion, the panel of assays used in this study can be measured in the WB and RBCs of the dog. In particular, the higher sensitivity to detect changes in our experimental conditions and its easier sample preparation makes WB a promising sample for the evaluation of redox status in dogs, with also potential applications to other animal species and humans.

Abstract Background The prevalence of Leishmania infantum infection in clinically healthy dogs can be several times higher than that of clinical disease in endemic areas. Although treatment is not recommended in dogs with subclinical infection, these animals should be managed to prevent disease progression and parasite transmission to human beings or to other dogs. Dietary nucleotides and active hexose correlated compound (AHCC) have been shown to modulate the immune response. A recent study in dogs with clinical leishmaniosis receiving an initial 28-day course of methylglucamine antimoniate showed that six-month administration of a dietary supplement containing nucleotides plus AHCC achieves similar efficacy to allopurinol. Since the type of immune response plays a key role in the evolution of patients with leishmaniosis, the present study was aimed at evaluating the preventive effect of this supplement in avoiding or delaying disease progression in clinically healthy Leishmania-infected dogs. Methods Forty-six dogs were included in this multicenter, randomized, double-blind, placebo-controlled trial. Dogs received once-daily oral administration of a placebo or a dietary supplement containing nucleotides plus AHCC. Disease progression was monitored throughout the study in both groups. At 0, 60, 180 and 365 days of treatment, clinical signs were evaluated using a validated clinical scoring system, and several analytes were measured from blood, urine, and bone marrow samples. Results During the study, a significantly lower (P = 0.047) proportion of dogs changed their clinical status and became sick in the supplement group (3/20; 15%), compared to the placebo group (10/22; 45.5%). ELISA-determined antibody titers were significantly reduced compared to baseline at all time points with the supplement (P

Obesity is a common problem in pet dogs, affecting half of the general population in some countries. Excess body weight causes several disorders and has a negative impact on dogs’ quality of life. The use of metabolomics allows the identification of metabolite traces from the metabolic pathways involved in pathological processes. This study aimed to evaluate salivary metabolite variations in dogs with obesity. The salivary samples of 19 dogs were analyzed using a targeted metabolomic approach, through which 234 metabolites were quantified. Of these, multivariate analysis identified 27 different metabolites altered in dogs with obesity compared with control dogs. These metabolites were mainly classified as amino acids, glycerides, sphingolipids, glycerophospholipids, and acylcarnitines. Some of the changes in these metabolites reflect the insulin resistance status related to obesity in dogs. Overall, it can be concluded that the salivary metabolome of obese dogs reflects the metabolic changes occurring in obesity and could be a source of potential biomarkers for this complex condition.

Abstract Background The influence of two different sample treatments comprising the enrichment of glycoproteins by boronic acid and dynamic range compression by hexapeptide libraries, on the detection of stress markers in saliva of pigs was evaluated in this study. For this purpose, saliva samples collected before and after the application of an acute stress model consisting of nasal restraining in pigs were processed without any treatment and with the two different treatments mentioned above. Protein separation by two-dimensional gel electrophoresis (2-DE) followed by identification of proteins using MALDI-TOF/TOF mass spectrometry (MS) was used as proteomic technique. Results The application of each of the two different sample treatment protocols allowed the identification of unique proteins that could be potential salivary acute stress markers in pigs: lipocalin 1, protein S100-A8 and immunoglobulin M by enrichment of glycoproteins; protein S100-A9, double headed protease inhibitor submandibular gland, and haemoglobin by dynamic range compression; and protein S100-A12 by both protocols. Salivary lipocalin, prolactin inducible protein, light chain of immunoglobulins, adenosine deaminase and carbonic anhydrase VI were identified as potential markers in untreated saliva as well as one of the other treatments. Conclusion The use of different procedures allowed the detection of different potential stress markers. Although from a practical point of view, the use of saliva without further treatment as well as the enrichment of glycoproteins are less expensive and easy to do procedures.

Abstract Background Canine heartworm infection is characterized by pulmonary endarteritis and pulmonary hypertension (PH). The aim of the present study was to evaluate the relationship between PH with the concentrations of different positive (C-reactive protein [CRP] and haptoglobin [Hp]) and negative (albumin and paraoxonase-1 [PON-1]) acute phase proteins (APP), as well as the oxidative stress, by measuring glutathione peroxidase (GPx) and total antioxidant capacity (TAC) in 27 heartworm-infected dogs on Day 0 (diagnosis) and Day 120 (1 month after the last adulticide injection). Presence/absence of PH was determined by the Right Pulmonary Artery Distensibility (RPAD) Index. Results On Day 0, 62.9% of the dogs showed PH. Concentrations of CRP and Hp were higher in dogs with PH, especially in dogs with moderate-severe PH (P

There is growing interest in oxytocin as a biomarker of stress and welfare. The objective of this study was to develop and validate a procedure based on a highly sensitive immunoassay to measure oxytocin in the hair of pigs. In addition, a pilot study to apply this procedure to evaluate possible changes in concentrations of oxytocin in hair during the reproductive cycle of pigs at different periods of the year was conducted. This procedure used methanol for sample extraction, since it offered better recoveries than acetonitrile, and the immunoassay developed was precise and accurate for the quantification of the oxytocin in the hair. When this procedure was applied to hair collected at different times of the reproductive cycle and season, higher values were found at days 23 and 59 after farrowing in the winter–spring period. In addition, higher oxytocin values in the spring–summer period were found in hair collected 5 days before farrowing compared to winter–spring. Oxytocin in hair showed moderate and low correlations with cortisone and cortisol in hair, respectively. This study represents the first report in which oxytocin was measured in hair and could open new lines for future research about the measurement of oxytocin in pigs and other biological species as a biomarker of stress.

The aim of this study was to investigate the changes in the salivary proteome in horses with acute abdominal disease (AAD) using a tandem mass tags (TMT)-based proteomic approach. The saliva samples from eight horses with AAD were compared with six healthy horses in the proteomic study. Additionally, saliva samples from eight horses with AAD and eight controls were used to validate lactoferrin (LF) in saliva. The TMT analysis quantified 118 proteins. Of these, 17 differed significantly between horses with AAD and the healthy controls, 11 being downregulated and 6 upregulated. Our results showed the downregulation of gamma-enteric smooth muscle actin (ACTA2), latherin isoform X1, and LF. These proteins could be closely related to an impaired primary immune defense and antimicrobial capacity in the mucosa. In addition, there was an upregulation of mucin 19 (MUC19) and the serine protease inhibitor Kazal-type 5 (SPINK5) associated with a protective effect during inflammation. The proteins identified in our study could have the potential to be novel biomarkers for diagnosis or monitoring the physiopathology of the disease, especially LF, which decreased in the saliva of horses with AAD and was successfully measured using a commercially available immunoassay.

This study aims to evaluate the changes in salivary and serum proteomes that occur in canine diabetes mellitus type-1 (DM) through a high-throughput quantitative proteomic analysis. The proteomes of 10 paired serum and saliva samples from healthy controls (HC group, n = 5) and dogs with untreated DM (DM group, n = 5) were analyzed using Tandem Mass Tags (TMT)-based proteomic approach. Additionally, 24 serum samples from healthy controls and untreated DM were used to validate haptoglobin in serum. The TMT analysis quantified 767 and 389 proteins in saliva and serum, respectively. Of those, 16 unique proteins in serum and 26 in saliva were differently represented between DM and HC groups. The verification of haptoglobin in serum was in concordance with the proteomic data. Our results pointed out changes in both saliva and serum proteomes that reflect different physiopathological changes in dogs with DM. Although some of the proteins identified here, such as malate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase, were previously related with DM in dogs, most of the proteins modulated in serum and saliva are described in canine DM for the first time and could be a source of potential biomarkers of the disease. Additionally, the molecular function, biological process, pathways and protein class of the differential proteins were revealed, which could improve the understanding of the disease’s pathological mechanisms.

Tick vaccination is an environmentally friendly alternative for tick control, pathogen infection, and transmission. Tick vaccine protection is sometimes incomplete, which may be due to problems in the stability, conformation, and activity of antibodies. This might be related to oxidative stress, but more studies are needed about the possible relationships between oxidative stress and immune function. The objective of this study was to evaluate and compare various serum biomarkers of antioxidant response and oxidative damage in cattle vaccinated with two recombinant antigens, the chimera of Subolesin- BM95 (homologue antigen of BM86)-MSP1a and BM86, and a control consisting in the adjuvant of the vaccines. Cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of the plasma (FRAP), trolox equivalent antioxidant capacity (TEAC), total thiol concentrations, and uric acid were evaluated in serum to determine the antioxidant response. To evaluate oxidative status, ferrous oxidation-xylenol orange (FOX), total oxidant status (TOS), advanced oxidation protein products (AOPP) and hydrogen peroxide (H2O2) concentrations in serum were determined. In addition, correlations between biomarkers of oxidative stress and antibody titers were evaluated. A significant decrease in all antioxidant biomarkers, with exception of thiol, and also a decrease in the oxidant markers TOS, AOPP and H2O2 was observed in cattle vaccinated with BM86, that also showed the highest antibody titers response whereas no significant differences in any of the biomarkers were detected in the Subolesin-Bm95-MSP1a and control groups. In addition, the dynamics of Cuprac and H2O2 with time showed significant differences between the groups. Although this is a pilot study and the results should be interpreted with caution and corroborated by studies involving a large number of animals, our results indicate that, in our experimental conditions, those vaccines able to induce a lower oxidative stress produce a higher concentration of antigen-specific antibodies. Overall, the results of the study provided information on the behavior of different biomarkers related to antioxidant defense, and the oxidative damage in cattle in response to vaccination.

The effects of two different concentrations of micro capsuled oregano essential oil (OEO) and purple garlic powder on biomarkers of oxidative status, stress, and inflammation, as well as on average daily gain (ADG) and feed conversion ratio (FCR), were evaluated in piglets during the postweaning period. The trial was carried out with 300 crossbred pigs of 21 days of age fed with different concentrations of OEO and purple garlic powder and ZnO. Saliva and serum samples were taken to evaluate a panel of biomarkers of oxidative status, stress, and inflammation. OEO and garlic powder at 0.4% did not produce significant changes in C-reactive protein (CRP) and cortisol and yielded higher levels of the antioxidant biomarker CUPRAC in serum than higher doses (p < 0.01); they yielded a better ADG than the control and ZnO diets. OEO and garlic powder at higher concentrations than 0.4% showed higher concentrations of CRP (p < 0.05). Overall, doses of OEO and garlic powder at 0.4% did not lead to inflammation, stress, or negative changes in oxidative biomarkers in piglets during the postweaning period and gave better productive performance than the control and ZnO diets. High doses of OEO and garlic powder were ineffective and could negatively affect the animals. Therefore, our results highlight the importance of the dose used when OEO or garlic are supplemented to piglets.

Adiponectin is an abundant plasma protein that is closely related to obesity and obesity-related pathologies. The molecule can be found in three different isoforms, each with different biological activities. Studies on canine obesity have suggested that adiponectin concentrations are decreased in obesity; however, no canine meta-analyses have been performed that feature all the required data. The aim of this study is to perform a systematic review and meta-analysis of studies that pertain to total and high molecular weight (HMW) adiponectin in relation to canine obesity. From 20 different studies, a total of 366 dogs with obesity and 349 normal weight dogs are included in the meta-analysis. Client-owned dogs were most represented, accounting for 54.3% of the dogs used, while experimental dogs enrolled in the studies made up the remaining 45.7%. The concentrations of total adiponectin in dogs with obesity were significantly lower compared with normal weight dogs. Additionally, adiponectin concentrations were significantly higher in dogs after a successful weight loss protocol compared to the start of the protocol and were significantly lower in dogs after gaining weight. In conclusion, although caution should be taken due to the relatively low number of studies that exist and the high heterogeneity between them, this meta-analysis indicates that adiponectin is decreased in obese dogs.

A single-dose disposition kinetics for tildipirosin was evaluated in clinically healthy ewes (n = 6) after intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration of a commercial formulation. Tildipirosin concentrations were determined by high-performance liquid chromatography with ultraviolet detection. Plasma concentration-time data was calculated by non-compartmental pharmacokinetic methods. The apparent volume of distribution (Vz) of tildipirosin after IV administration was 5.36 ± 0.57 L/kg suggesting a wide distribution in tissues and inside the cells. The elimination half-life (t½λz) was 17.16 ± 2.25, 23.90 ± 6.99 and 43.19 ± 5.17 h after IV, IM and SC administration, respectively. Following IM administration, tildipirosin was rapidly absorbed (tmax = 0.62 ± 0.10 h) even to a greater extent than after SC administration. Time to reach peak concentration (tmax) and peak plasma concentrations (Cmax) differed significantly, but both parameters showed a more significant variability after SC than after IM administration. Bioavailabilities after extravascular administration were high (>70%). Therefore, given general adverse reactions that were not observed in any ewe and favourable pharmacokinetics, tildipirosin could be effective in treating bacterial infections in sheep.

The objective of the present study was to evaluate the possible changes of oxytocin concentrations in saliva during and after ejaculate collection in breeding boars usually used in artificial insemination programs. Saliva samples of 33 boars were collected the day before ejaculate collection (DB), during the ejaculation time (T0) and two hours after ejaculate collection (T2). Free oxytocin and oxytocin linked to proteins concentrations were measured by two methods previously developed and validated for saliva of pigs. Younger boars, boars with higher libido intensity and boars of the Pietrain breed showed higher values of oxytocin in saliva during ejaculation than the day before. In addition, boars with higher libido showed higher concentrations two hours after ejaculate collection than during the day before. These changes were of higher magnitude and significance when oxytocin linked to proteins was measured. In conclusion, this study demonstrated for the first time that ejaculation influences the salivary oxytocin concentrations in breeding boars, although this influence varies according to age, libido and breed.

The aim of this study was to evaluate changes in serum and saliva proteomes in canine mammary tumors (CMT) using a high-throughput quantitative proteomic analysis in order to potentially discover possible biomarkers of this disease. Proteomes of paired serum and saliva samples from healthy controls (HC group, n = 5) and bitches with CMT (CMT group, n = 5) were analysed using a Tandem Mass Tags-based approach. Twenty-five dogs were used to validate serum albumin as a candidate biomarker in an independent sample set. The proteomic analysis quantified 379 and 730 proteins in serum and saliva, respectively. Of those, 35 proteins in serum and 49 in saliva were differentially represented. The verification of albumin in serum was in concordance with the proteomic data, showing lower levels in CMT when compared to the HC group. Some of the modulated proteins found in the present study such as haptoglobin or S100A4 have been related to CMT or human breast cancer previously, while others such as kallikrein-1 and immunoglobulin gamma-heavy chains A and D are described here for the first time. Our results indicate that saliva and serum proteomes can reflect physiopathological changes that occur in CMT in dogs and can be a potential source of biomarkers of the disease.

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.

Osteoarthritis (OA) is one of the most significant joint diseases worldwide. There are different therapies for OA treatment, and a relatively new strategy is the use of plasma rich in growth factors (PRGF), a platelet rich plasma (PRP) derivative. The objective of this study was to objectively assess the efficacy and duration of the effect of an intraarticular injection of PRGF and a combination of PRGF + physical therapy. The objective assessment was provided using a force platform. The obtained parameters were peak vertical force (PVF) and vertical impulse (VI). A total of 24 dogs with lameness and pain associated to OA attributable to bilateral hip dysplasia were included in the study. Animals were divided into two study groups and evaluated at baseline and at 30, 90, and 180 days after intraarticular PRGF or PRGF + physical therapy. Significant differences were observed at every checkpoint with respect to basal time in both groups. However, after 180 days, the PRGF group showed a decrease in PVF and VI with respect to the values obtained at 90 days. However, the PRGF + physical therapy group maintained increased values of both PVF and VI values during the 180-day study period.

Some positive effects regarding the use of enrichment material on the stimulation of pig exploration and a reduction in redirected behaviour was reported. This study aims to evaluate the effects of four enrichment materials on the behaviour, physiology/health, performance and carcass and meat quality in pigs kept in Spanish production conditions. Ninety-six male pigs (six pigs/pen) ranging from 70 to 170 days old were used. Chains were used for the control group (CH), and wooden logs (W), straw in a rack (S) or paper (P) were also used. The pigs were subjected to two pre-slaughter treatments: 0 or 12 hours of fasting. Their behaviour was observed for 12 weeks using scan and focal sampling. Samples of the Neutrophil: Lymphocyte (N:L) ratio and lactate were obtained from the pigs at 66 and 170 days old. Saliva samples for Chromogranin-A (CgA) were obtained at 67, 128, 164 and 170 days old. The weight, skin lesions and feed intake of the pigs were recorded. S triggered more exploratory behaviour than W and CH (P < 0.001). Skin lesions and redirected behaviour were lower for pigs with S (P < 0.01 and P < 0.05, respectively). The pigs offered S presented lower CgA after no fasting than pigs with P or CH (P = 0.055). Lactate was higher in pigs with W and CH treatments, regardless of fasting (P < 0.05). The N:L ratio increased over time (P < 0.05). No other significant effects were found. Overall, straw in a rack was the enrichment material that enhanced pig inherent behaviour.