Metabolome Profiling in the Plasma of Dogs with Idiopathic Dilated Cardiomyopathy: A Multiplatform Mass-Spectrometry-Based Approach.

Dilated cardiomyopathy is one of the important diseases in dogs and humans. The second most common cause of heart failure in dogs is idiopathic dilated cardiomyopathy (iDCM), which results in heart failure or sudden cardiac death due to arrhythmia. This study aimed to determine changes in the plasma metabolome of dogs with iDCM compared to healthy dogs. For that purpose, a multiplatform mass-spectrometry-based approach was used. In this study, we included two groups of dogs: 12 dogs with iDCM and 8 healthy dogs. A total of 272 metabolites were detected in the plasma samples of dogs by combining three approaches but four MS-based platforms (GC-MS, LC-MS (untargeted), LC-MS (targeted), and FIA-MS (targeted) methods). Our findings demonstrated changes in the canine plasma metabolome involved in the development of iDCM, including the different concentrations of amino acids, biogenic amines, acylcarnitines, triglycerides and diglycerides, sphingomyelins, and organic acids. The results of this study will enable the detection and monitoring of pathophysiological mechanisms involved in the development of iDCM in the future.

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.

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.

Data Independent Acquisition Reveals In-Depth Serum Proteome Changes in Canine Leishmaniosis.

Comprehensive profiling of serum proteome provides valuable clues of health status and pathophysiological processes, making it the main strategy in biomarker discovery. However, the high dynamic range significantly decreases the number of detectable proteins, obstructing the insights into the underlying biological processes. To circumvent various serum enrichment methods, obtain high-quality proteome wide information using the next-generation proteomic, and study host response in canine leishmaniosis, we applied data-independent acquisition mass spectrometry (DIA-MS) for deep proteomic profiling of clinical samples. The non-depleted serum samples of healthy and naturally Leishmania-infected dogs were analyzed using the label-free 60-min gradient sequential window acquisition of all theoretical mass spectra (SWATH-MS) method. As a result, we identified 554 proteins, 140 of which differed significantly in abundance. Those were included in lipid metabolism, hematological abnormalities, immune response, and oxidative stress, providing valuable information about the complex molecular basis of the clinical and pathological landscape in canine leishmaniosis. Our results show that DIA-MS is a method of choice for understanding complex pathophysiological processes in serum and serum biomarker development.

Proteomic changes associated with maternal dietary low ω6:ω3 ratio in piglets supplemented with seaweed. Part I: Serum proteomes.

This study examines whether maternal low ω6:ω3 ratio diet and offspring SW supplementation can improve offspring immunity and performance by elucidating the effects on piglet serum proteome. A total of 16 sows were given either a standard (CR, 13:1) or low ω6:ω3 ratio diet (LR, 4:1) during pregnancy and lactation and their male weaned piglets were supplemented with SW powder (4 g/kg, SW) or not (CT) in a 21-day post-weaning (PW) diet. Four PW piglet groups were then identified based on dam and piglet treatment, namely CRCT, CRSW, LRCT, and LRSW (n = 10 each). Piglet serum collected at weaning and d21 PW were analysed (n = 5 each) using TMT-based quantitative proteomics and validated by appropriate assays. The differentially abundant proteins (n = 122) displayed positive effects of maternal LR diet on anti-inflammatory properties and innate immune stimulation. Progeny SW diet activated the innate immunity and enhance the host defence during inflammation. These data demonstrate the value of decreasing ω6:ω3 ratio in maternal diet and SW supplementation in PW piglet's diet to boost their immunity and anti-inflammation properties. SIGNIFICANCE: This novel proteomic study in post-weaned piglets addresses the interplay between maternal and offspring nutritional interventions in a context of rapid and dynamic alterations in piglet metabolic status around weaning. Decreasing ω6:ω3 ratio in maternal diet and SW supplementation in PW piglet's diet can boost their immunity and anti-inflammation properties. This study also provides new insights into piglet serum proteome regulation during post-weaning, a critical development period in swine.

Proteomic changes associated with maternal dietary low ω6:ω3 ratio in piglets supplemented with seaweed Part II: Ileum proteomes.

This study evaluates how long-term dietary low ω6:ω3 ratio in sows and offspring's seaweed (SW) intake affects piglet intestinal function and growth through modifying ileum proteome. Sows were assigned to either control diet (CR, ω6:ω3 ratio = 13:1) or treatment diet (LR, ω6:ω3 = 4:1) during gestation and lactation (n = 8 each). The male weaned offspring were received a basal diet with or without SW powder supplementation (4 g/kg) for 21 days, denoted as SW and CT groups, respectively. In total, four groups of weaned piglets were formed following maternal and offspring's diets combination, represented by CRCT, CRSW, LRCT, and LRSW (n = 10 each). Piglet ileum tissue was collected on day 22 post-weaning and analysed using TMT-based quantitative proteomics. The differentially abundant proteins (n = 300) showed the influence of maternal LR diet on protein synthesis, cell proliferation, and cell cycle regulation. In contrast, the SW diet lowered the inflammation severity and promoted ileal tissue development in CRSW piglets but reduced the fat absorption capacity in LRSW piglets. These results uncovered the mechanism behind the anti-inflammation and intestinal-boosting effects of maternal LR diet in piglets supplemented with SW.

Effect of dietary Spirulina (Arthrospira platensis) on the intestinal function of post-weaned piglet: An approach combining proteomics, metabolomics and histological studies.

The effect of dietary Spirulina (Arthrospira platensis) and CAZyme supplementation was assessed on the gut of weaned piglets, using an integrated NMR-metabolomics approach combined with Tandem Mass Tag labelled proteomics. Thirty weaned male piglets were assigned to one of the three following diets (n = 10): cereal and soybean meal basal diet (Control), basal diet with 10% Spirulina inclusion (SP) and SP diet supplemented with 0.01% lysozyme (SP + L). The experiment lasted 4 weeks and, upon slaughter, small intestine samples were collected for histological, metabolomic and proteomic analysis. No significant differences were found for the histology and metabolomics analysis between the three experimental groups. Lactate, glutamate, glycine and myo-inositol were the most abundant metabolites. Proteomics results showed 1502 proteins identified in the intestine tissue. A total of 23, 78, 27 differentially abundant proteins were detected respectively for the SP vs. Control, SP + L vs. Control and SP + L vs. SP comparisons. The incorporation of Spirulina and supplementation of lysozyme in the piglet's diets is associated to intestinal proteomic changes. These include increased protein synthesis and abundance of contractile apparatus proteins, related with increased nutrient availability, which has beneficial (increased glucose uptake) and detrimental (increased digesta viscosity) metabolic effects. SIGNIFICANCE: The use of conventional feedstuffs becomes increasingly prohibitive due to its environmental toll. To increase the sustainability of the livestock sector, novel feedstuffs such as microalgae need to be considered. However, its recalcitrant cell wall has antinutritional effects that can inhibit high dietary inclusion levels. The supplementation with CAZymes is a possible solution to this issue. The small intestine is a central piece in monogastric digestion and of particular importance for the weaned piglet. Studying the effect of dietary Spirulina and CAZyme supplementation on its histomorphology, metabolome and proteome allows studying relevant physiological adaptations to these diets.

Abundance of plasma proteins in response to divergent ratios of dietary ω6:ω3 fatty acids in gestating and lactating sows using a quantitative proteomics approach.

This study aimed to investigate the characteristic proteomic pattern of plasma from sows supplemented with low dietary ω6:ω3 fatty acids (FAs) ratio during gestation and lactation. Two dietary treatments (n = 8 each) comprised either a control ratio of ω6:ω3 FAs (CR, 13:1 during gestation and 10:1 during lactation) or a low ratio (LR, 4:1 during gestation and lactation) by adding soybean oil or linseed oil, respectively. High-resolution mass spectrometry-based quantitative proteomics was applied on plasma (n = 5 each) at day 108 of gestation (G108) and at the end of lactation (L-End), and a total of 379 proteins and 202 master proteins were identified. Out of these, four differentially abundant proteins between LR and CR samples at G108 may relate to serine-type endopeptidase inhibitor activity. Differentially abundant proteins in L-End versus G108 (12 up-regulated and 10 down-regulated) were positively correlated with the events that regulate plasma lipoproteins, stimulus- and defence-responses. These findings demonstrate the benefit of increased dietary ω3 FAs in modifying proteins involved in protective mechanisms against increased stresses in key life cycle phases in pigs. In addition, proteome changes from late gestation to late lactation disclosed the underlying mechanism of pigs in response to reproduction-related stimuli. SIGNIFICANCE: This study aimed to provide a proteomics insight into the beneficial effects of maternal diet supplementation with a low ω6:ω3 fatty acids ratio, based on previously reported performance and zootechnical data. The results suggest that a low dietary ω6:ω3 fatty acids ratio could enhance the cellular defence mechanisms against increased stresses and in particular to oxidative stress in sows during gestation and lactation, as reflected in proteomic changes of haptoglobin (HP), alpha-1-antitrypsin (SERPINA1) and serum amyloid P-component (APCS). Furthermore, significantly changed proteome profiles in sow plasma between late gestation and lactation phases have been revealed for the first time. This finding identified the adaptation mechanisms of sows to changing physiological events during reproduction.

Multi Platforms Strategies and Metabolomics Approaches for the Investigation of Comprehensive Metabolite Profile in Dogs with Babesia canis Infection.

Canine babesiosis is an important tick-borne disease worldwide, caused by parasites of the Babesia genus. Although the disease process primarily affects erythrocytes, it may also have multisystemic consequences. The goal of this study was to explore and characterize the serum metabolome, by identifying potential metabolites and metabolic pathways in dogs naturally infected with Babesia canis using liquid and gas chromatography coupled to mass spectrometry. The study included 12 dogs naturally infected with B. canis and 12 healthy dogs. By combining three different analytical platforms using untargeted and targeted approaches, 295 metabolites were detected. The untargeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) metabolomics approach identified 64 metabolites, the targeted UHPLC-MS/MS metabolomics approach identified 205 metabolites, and the GC-MS metabolomics approach identified 26 metabolites. Biological functions of differentially abundant metabolites indicate the involvement of various pathways in canine babesiosis including the following: glutathione metabolism; alanine, aspartate, and glutamate metabolism; glyoxylate and dicarboxylate metabolism; cysteine and methionine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. This study confirmed that host-pathogen interactions could be studied by metabolomics to assess chemical changes in the host, such that the differences in serum metabolome between dogs with B. canis infection and healthy dogs can be detected with liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) methods. Our study provides novel insight into pathophysiological mechanisms of B. canis infection.

Multi-Omics Approach to Elucidate Cerebrospinal Fluid Changes in Dogs with Intervertebral Disc Herniation.

Herniation of the intervertebral disc (IVDH) is the most common cause of neurological and intervertebral disc degeneration-related diseases. Since the disc starts to degenerate before it can be observed by currently available diagnostic methods, there is an urgent need for novel diagnostic approaches. To identify molecular networks and pathways which may play important roles in intervertebral disc herniation, as well as to reveal the potential features which could be useful for monitoring disease progression and prognosis, multi-omics profiling, including high-resolution liquid chromatography-mass spectrometry (LC-MS)-based metabolomics and tandem mass tag (TMT)-based proteomics was performed. Cerebrospinal fluid of nine dogs with IVDH and six healthy controls were used for the analyses, and an additional five IVDH samples were used for proteomic data validation. Furthermore, multi-omics data were integrated to decipher a complex interaction between individual omics layers, leading to an improved prediction model. Together with metabolic pathways related to amino acids and lipid metabolism and coagulation cascades, our integromics prediction model identified the key features in IVDH, namely the proteins follistatin Like 1 (FSTL1), secretogranin V (SCG5), nucleobindin 1 (NUCB1), calcitonin re-ceptor-stimulating peptide 2 precursor (CRSP2) and the metabolites N-acetyl-D-glucosamine and adenine, involved in neuropathic pain, myelination, and neurotransmission and inflammatory response, respectively. Their clinical application is to be further investigated. The utilization of a novel integrative interdisciplinary approach may provide new opportunities to apply innovative diagnostic and monitoring methods as well as improve treatment strategies and personalized care for patients with degenerative spinal disorders.

Differential acute-phase protein responses in dogs seropositive or seronegative for Neospora caninum.

Neospora caninum is one of the most prevalent Apicomplexa parasites that causes abortion in cattle, as it infects dogs as its definitive host, causing subclinical disease or active neosporosis, marked by meningoencephalitis, and myopathies with muscle and neuromuscular signs of disease. This study aimed to evaluate the acute phase protein response in dogs seropositive and seronegative for N. caninum. Serum samples of 72 dogs were tested by an immunofluorescence antibody test using N. caninum NC-1 strain, and the study population was divided into four groups: symptomatic - muscular and/or neuromuscular signs - and seropositive (n = 16); symptomatic and seronegative (n = 9); asymptomatic and seropositive (n = 34); and asymptomatic and seronegative (n = 13). C-reactive protein (CRP) was measured via immunoturbidimetric assay and serum haptoglobin (Hp) via hemoglobin-binding capacity assay. In the symptomatic groups, seropositive dogs had higher levels of Hp, but not CRP, while seronegative dogs had higher CRP levels. There was no difference in CRP concentration in asymptomatic dogs. Dogs with neuromuscular signs had higher concentrations for Hp in the group seropositive. Hp concentration did not differ between dogs seropositive and seronegative dogs for each group. Serum Hp and CRP could not sufficiently alone flag subclinical infections. Measurement of CRP and Hp concentrations could be clinically valuable to the diagnosis of neurological diseases, and their relative change may indicate the stage of the infection, although their sole use is not able to support the diagnosis of canine neosporosis. Further studies are encouraged to evaluate the specific dynamics of acute phase proteins in canine neosporosis. Serum samples of 72 dogs were tested by an immunofluorescence antibody test using N. caninum NC-1 strain, and the study population was divided into four groups: (1) dogs with muscular and/or neuromuscular signs and seropositive for N. caninum; (2) dogs with muscular and/or neuromuscular signs and seronegative for N. caninum; (3) dogs seropositive for N. caninum with no neuromuscular signs; and (4) healthy dogs and seronegative for N. caninum. The study evaluated if N. caninum infection could have pathophysiological changes activating the acute phase response and an increase in the concentration of acute phase proteins in serum.

Effects of Low ω6:ω3 Ratio in Sow Diet and Seaweed Supplement in Piglet Diet on Performance, Colostrum and Milk Fatty Acid Profiles, and Oxidative Status.

The ratio of omega-6 (ω6) to omega-3 (ω3) polyunsaturated fatty acids (PUFAs) in the diet contributes to animal health and performance modulations because they have mostly opposite physiological functions. Increasing ω3 PUFAs content in the maternal diet can stimulate antioxidative capacity in sow and piglets; however, the optimal ratio of ω6 and ω3 PUFAs in the sow diet is still under discussion. Rich sources of bioactive constituents such as brown seaweed are an excellent supplementation to promote animal health and antioxidant status. However, the knowledge of the effects of this compound, specifically in post-weaning piglets, is still limited. Moreover, the combined effect of a low ω6:ω3 PUFAs ratio in sow diet and seaweed supplementation in post-weaning piglets' diet has never been studied. This research aims to assess the combined effect of a low ω6:ω3 ratio in sow diets and seaweed supplementation in piglet diets on their growth and oxidative status. We also assessed the impact of a low ω6:ω3 ratio in the maternal diet on reproduction, milk fatty acid (FA) profile, and plasma leptin concentration. Two sow diets (n = 8 each) contained either a control ratio (CR, 13:1 during gestation, starting from day 28 (G28) and 10:1 during lactation) or a low ratio (LR, 4:1 from G28 until the end of lactation (L-End)) of ω6:ω3 FA by adding soybean oil or linseed oil, respectively. Reproductive performance was evaluated. Colostrum and milk at lactation day 7 (L7) and L-End were collected to analyze FA profile. Plasma was collected at G28, G79, G108, L7, L14, and L-End for determination of leptin and oxidative status. At weaning, 20 male piglets were selected per sow group to form 4 diet treatments (n = 10 each), which were supplemented with or without 4 g/kg seaweed. Recording of growth performance and collection of blood were performed at days 0, 7, 15, and 21 of post-weaning for oxidative status. LR diet increased (p < 0.05) the survival rate of piglets at weaning, and individual and litter weight gains. Colostrum and milk at L7 and L-End had lower (p < 0.05) ω6:ω3 ratio in LR sows. Interaction between dietary treatments on sows and piglets was revealed for all examined growth parameters at most time points (p < 0.05). LR diet did not affect plasma leptin levels and oxidative status. These findings suggest that the seaweed supplement during post-weaning could not improve growth rate and oxidative status of piglets born from mothers receiving a low dietary ω6:ω3 ratio (4:1) during gestation and lactation. However, this low ratio was beneficial for weaning survival rate, sucking piglets' weight gain, and ω3 enrichment in colostrum and milk.

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes.

BACKGROUND: Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. METHODS: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. RESULTS: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. CONCLUSIONS: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.

Cardiovascular-renal axis disorder and acute-phase proteins in cats with congestive heart failure caused by primary cardiomyopathy.

BACKGROUND: Currently, the pathogenesis of congestive heart failure (CHF) in cats is not fully understood. OBJECTIVE: To identify novel biomarkers for CHF in cats caused by primary cardiomyopathy, particularly related to cardiovascular-renal axis disorder and systemic inflammatory response. ANIMALS: Twenty-five cats in CHF caused by primary cardiomyopathy, 12 cats with preclinical cardiomyopathy, and 20 healthy controls. METHODS: Case control and observational case series. The following serum biomarkers were compared among the 3 cat groups: a cardiorenal profile that included N-terminal pro-brain natriuretic peptide (NT-proBNP), symmetric dimethylarginine (SDMA), and creatinine and an inflammatory profile that included 7 acute-phase proteins (APPs). Survival analyses and longitudinal studies were performed in CHF cats. RESULTS: All cardiorenal biomarkers were positively correlated and higher in CHF cats, and high NT-proBNP and SDMA were associated with poor clinical outcome. Cats with CHF had significantly higher leucine-rich alpha-2-glycoprotein 1, serum amyloid A, and ceruloplasmin, and these APPs were positively correlated with NT-proBNP and left atrial size. In a multivariable survival analysis, alpha-1-acid glycoprotein concentration (P = .01), body weight (P = .02) and left atrial-to-aortic root ratio (P = .01) were independent prognostic factors for CHF in these cats. CONCLUSIONS AND CLINICAL IMPORTANCE: In cats, CHF is an inflammatory disorder and outcome in CHF may be determined by the extent of inflammation and possibly the amount of residual renal function. These novel biomarkers have potential use for the clinical management, prognosis, and future research into CHF and cardiomyopathy in cats.

Use of Saliva for Diagnosis and Monitoring the SARS-CoV-2: A General Perspective.

In this report, updated information and future perspectives about the use of saliva as a sample for laboratory analysis of the Covid-19 are highlighted. Saliva can be used for the direct detection of the SARS-CoV-2 virus, the quantification of the specific immunoglobulins produced against it, and for the evaluation of the non-specific, innate immune response of the patient. Moreover, a deeper knowledge of potential changes in the saliva proteome in this disease may allow the identification of new diagnostic and prognostic biomarkers, or even help our understanding of the mechanisms associated with the disease. With the development of appropriate sample collection and processing methods and the use of adequate assays, saliva can provide useful clinical information about the disease and could be potentially included in guidelines for sample collection for the diagnosis, disease management, and control of Covid-19.

Serum proteome profiling in canine chronic valve disease using a TMT-based quantitative proteomics approach.

Chronic valve disease (CVD) is the most common clinically significant heart disease of dogs, affecting 20 to 40% of dogs. The aim of this study was to evaluate the serum protein profile of healthy and CVD affected dogs, by means of an isobaric tandem mass tag (TMT) label-based high-resolution quantitative proteomic approach. Additionally, conventional cardiac biomarkers were measured in the serum, functional bioinformatics analysis was employed for elucidating molecular mechanisms and pathways associated with CVD, and validation of proteomic results was performed by immunoassays and Western blotting. Of 290 identified and quantified proteins, 15 proteins showed significantly different abundances (p < .05), including antithrombin-III, alpha-2-antiplasmin, tetranectin, apolipoprotein M, adiponectin, inter-alpha-trypsin inhibitor heavy chain H1, gelsolin and apolipoprotein B-100. The identified proteins with differently abundances are involved in a number of pathways, such as complement and coagulation cascades, haemostasis, regulation of actin cytoskeleton, lipid metabolism and transport. We found comparative similarities with human disease in terms of identified proteins and GO pathways, which confirmed similar pathophysiology of this disease, but also differences, mainly in lipid metabolism. SIGNIFICANCE: There have been few investigations of canine serum proteome despite the potential for biomarker discovery and comparative disease analysis. Establishing serum proteomic signatures in healthy dogs and dogs with CVD will benefit for understanding the aetiology of disease in dogs, identify putative biomarkers and provide models of comparative human disease. Circulating biomarkers are important for understanding of the mechanisms of cardiovascular disease and high incidence of CVD in dogs prioritizes the search for novel biomarkers.

Plasma proteomic profiling and pathway analysis of normal and overconditioned dairy cows during the transition from late pregnancy to early lactation.

This study applied a quantitative proteomics approach along with bioinformatics analyses to investigate changes in the plasma proteome of normal and overconditioned dairy cows during the transition period. Fifteen weeks before their anticipated calving date, 38 multiparous Holstein cows were selected based on their current and previous body condition scores (BCS) and allocated to either a high or a normal BCS group (19 cows each). They received different diets until dry-off to reach targeted differences in BCS and back fat thickness (BFT) until dry-off. At dry-off, normal BCS cows had a BCS <3.5 (minimum, 2.75) and BFT <1.2 cm (minimum, 0.58), and the high BCS cows had a BCS >3.75 (maximum, 4.50) and BFT >1.4 cm (maximum, 2.90). The proteomics study used a subset of 5 animals from each group. These cows were selected based on their circulating concentrations of fatty acids (FA) on d 14 postpartum and ß-hydroxybutyrate (BHB) on d 21 postpartum, representing the greater or the lower extreme values within their BCS group, respectively. The high BCS subset (HE-HBCS) had 4.50 < BCS > 3.75, FA = 1.17 ± 0.46 mmol/L, and BHB = 2.15 ± 0.42 mmol/L (means ± SD), and the low BCS subset (LE-NBCS) had 3.50 < BCS > 2.75, FA = 0.51 ± 0.28 mmol/L, and BHB = 0.84 ± 0.17 mmol/L. Plasma samples from d -49, +7, and +21 relative to parturition were used for proteome profiling by applying the quantitative tandem mass tags (TMT) approach. Nondepleted plasma samples were subjected to reduction and digestion and then labeled with TMT 10plex reagents. High-resolution liquid chromatography-tandem mass spectrometry analysis of TMT-labeled peptides was carried out, and the acquired spectra were analyzed for protein identification and quantification. In total, 254 quantifiable proteins (criteria: 2 unique peptides and 5% false discovery rate) were identified in the plasma samples. From these, 24 differentially abundant proteins (14 more abundant, 10 less abundant) were observed in the LE-NBCS cows compared with the HE-HBCS cows during the transition period. Plasma α-2-macroglobulins were more abundant in HE-HBCS versus LE-NBCS cows at d +7 and +21. Gene Ontology enrichment analyses of differentially abundant proteins revealed that the acute inflammatory response, regulation of complement activation, protein activation cascade, and regulation of humoral immune response were the most enriched terms in the LE-NBCS group compared with the HE-HBCS group. In addition, we identified 24 differentially abundant proteins (16 in the LE-NBCS group, and 8 in the HE-HBCS group) during the transition period. The complement components C1q and C5 were less abundant, while C3 and C3d were more abundant in LE-NBCS compared with HE-HBCS cows. Overall, overconditioning around calving was associated with alterations in protein pathways related to acute inflammatory response and regulation of complement and coagulation cascades in transition cows.

Metabolome and proteome changes in skeletal muscle and blood of pre-weaning calves fed leucine and threonine supplemented diets.

In pre-weaning calves, both leucine and threonine play important roles in growth and muscle metabolism. In this study, metabolomics, proteomics and clinical chemistry were used to assess the effects of leucine and threonine supplementation added to milk replacer on 14 newborn Holstein male calves: 7 were fed a control diet (Ctrl) and 7 were fed the Ctrl diet supplemented with 0.3% leucine and 0.3% threonine (LT) from 5.6 days of age to 53.6 days. At this time, blood and semitendinosus muscle biopsies were collected for analysis. Integrated metabolomics and proteomics showed that branched-chain amino acids (BCAA) degradation and mitochondrial oxidative metabolism (citrate cycle and respiratory chain) were the main activated pathways in muscle because of the supplementation. BCAA derivatives and metabolites related to lipid mobilization showed the major changes. The deleterious effects of activated oxidative phosphorylation were balanced by the upregulation of antioxidant proteins. An increase in protein synthesis was indicated by elevated aminoacyl-tRNA biosynthesis and increased S6 ribosomal protein phosphorylation in skeletal muscle. In conclusion, LT group showed greater BCAA availability and mitochondrial oxidative activity; as the muscle cells undergo greater aerobic metabolism, antioxidant defenses were activated to compensate for possible cell damage. Data are available via ProteomeXchange (PXD016098). SIGNIFICANCE: Leucine and threonine are essential amino acids for the pre-weaning calf, being of high importance for growth. In this study, we found that leucine and threonine supplementation of milk replacer to feed pre-weaning calves led to differences in the proteome, metabolome and clinical chemistry analytes in skeletal muscle and plasma, albeit no differences in productive performance were recorded. This study extends our understanding on the metabolism in dairy calves and helps optimizing their nutritional status.

Biomarkers of health and welfare: A One Health perspective from the laboratory side.

A biomarker is any measurement reflecting an interaction between a biological system and a potential hazard, which may be chemical, physical, or biological. The One World, One Health concept established that human and animal health and the environmental state are highly interconnected, sharing common aspects that can be applied globally in these three components. In this paper, we review how the concept of One Health can be applied to biomarkers of health and welfare, with a special focus on five points that can be applied to any biomarker when it is expected to be used to evaluate the human, animal or environmental health. Three of these points are: (1) the different biomarkers that can be used, (2) the different sample types where the biomarkers can be analysed, and (3) the main methods that can be used for their measurement. In addition, we will evaluate two key points needed for adequate use of a biomarker in any situation: (4) a proper analytical validation in the sample that it is going to be used, and (5) a correct selection of the biomarker. It is expected that this knowledge will help to have a broader idea about the use of biomarkers of health and welfare and also will contribute to a better and more accurate use of these biomarkers having in mind their One Health perspective.