Characterization and LC-MS/MS based proteomic analysis of extracellular vesicles separated from blood serum of healthy and dogs naturally infected by Babesia canis. A preliminary study.

Canine babesiosis is a rapidly spreading tick-borne disease in Europe, which entails protozoan parasites invading red blood cells. Small extracellular vesicles (EVs) (< 200 nm) were isolated from the serum of 15 healthy and 15 by Babesia canis naturally infected dogs aimed to distinguish EV characteristics and protein profiles. There were no significant differences (P = 0.05) observed in the mean sizes and concentrations of serum EVs between the healthy and canine babesiosis groups. Despite a higher number of Canis lupus proteins detected in EVs from serum of diseased dogs, there were no statistically significant differences (P < 0.05) in the number of protein IDs between the experimental groups. We successfully identified 211 Canis lupus proteins across both experimental groups, of which 147 Canis lupus proteins were validated as being EV-associated. This data set is accessible via the ProteomeXchange PXD047647. EVs isolated from serum of B. canis infected dogs were Cd9+, Cd63+, Cd81+, and Cd82+. Furthermore, 73 Canis lupus proteins were validated as EV-associated and specific for EVs isolated from serum of B. canis-infected dogs. These were predominantly membrane and cytosolic proteins, and innate and adaptive immune system-related proteins, especially those involved in adhesion and proteoglycan mechanisms like integrins. Enrichment was also observed for proteins involved in vascular and cellular responses, including signalling pathways such as VEGF, VEGFR, and the LKB1 network. When only blood-related sites of EV expression were evaluated, the origins of EV proteins were mostly cells of immune system. These were dendritic cells, neutrophils, B cells, monocytes and platelets. In general, proteins were enriched in pathways that collectively regulate various cellular processes, including immune responses, communication, signal transduction, membrane trafficking, and apoptosis. Serum EVs and their protein cargo may have an important role in both the invasion of B. canis and the host's response to the parasitic infection, nevertheless, additional experimental research is warranted. The overall count of identified EV proteins of parasitic origin, meeting cut off criteria of two peptides and 1 % FDR, was relatively low.

Quantitative serum proteome analysis using tandem mass tags in dogs with epilepsy.

This study included four groups of dogs (group A: healthy controls, group B: idiopathic epilepsy receiving antiepileptic medication (AEM), group C: idiopathic epilepsy without AEM, group D: structural epilepsy). Comparative quantitative proteomic analysis of serum samples among the groups was the main target of the study. Samples were analyzed by a quantitative Tandem-Mass-Tags approach on the Q-Exactive-Plus Hybrid Quadrupole-Orbitrap mass-spectrometer. Identification and relative quantification were performed in Proteome Discoverer. Data were analyzed using R. Gene ontology terms were analyzed based on Canis lupus familiaris database. Data are available via ProteomeXchange with identifier PXD041129. Eighty-one proteins with different relative adundance were identified in the four groups and 25 were master proteins (p < 0.05). Clusterin (CLU), and apolipoprotein A1 (APOA1) had higher abundance in the three groups of dogs (groups B, C, D) compared to controls. Amine oxidase (AOC3) was higher in abundance in group B compared to groups C and D, and lower in group A. Adiponectin (ADIPOQ) had higher abundance in groups C compared to group A. ADIPOQ and fibronectin (FN1) had higher abundance in group B compared to group C and D. Peroxidase activity assay was used to quantify HP abundance change, validating and correlating with proteomic analysis (r = 0.8796). SIGNIFICANCE: The proteomic analysis of serum samples from epileptic dogs indicated potential markers of epilepsy (CLU), proteins that may contribute to nerve tissue regeneration (APOA1), and contributing factors to epileptogenesis (AOC3). AEM could alter extracellular matrix proteins (FN1). Illness (epilepsy) severity could influence ADIPOQ abundance.

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.

Distinguishing Natural Infections of the Bovine Mammary Gland by Staphylococcus from Streptococcus spp. Using Quantitative Milk Proteomics.

Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiology and host immune responses, independent of the mastitis level. After identification of Staphylococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantitation through the SEQUEST algorithm. Statistical analysis employing R was used to identify differentially abundant proteins between the groups. Protein classes, functions and functional-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflammation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific-pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

Serum proteome profiling of naturally acquired Babesia rossi infection in dogs.

Babesiosis is a disease of significant medically and veterinary importance with worldwide distribution. It is caused by intra-erythrocyte protozoal parasites, with Babesia rossi causing the most severe clinical signs of all the large Babesia parasites infecting dogs. The disease can be clinically classified into uncomplicated and complicated forms with a wide range of clinical presentations from a mild, subclinical illness to complicated forms and death. The aim of this study was to assess serum proteomic profiles from dogs with babesiosis and healthy dogs using a label-based proteomics approach. Altogether 32 dogs naturally infected with B. rossi (subdivided into 18 uncomplicated cases and 14 complicated cases of babesiosis) and 20 healthy dogs were included. There were 78 proteins with significantly different abundances between the three groups of dogs. Elucidation of proteins and pathways involved in canine babesiosis caused by B. rossi have revealed key differences associated with haemostasis, innate immune system, lipid metabolism and inflammation. Shotgun proteomic profiling allowed identification of potential serum biomarkers for differentiation of disease severity in canine babesiosis caused by B. rossi. These findings may be applicable to the study of host-parasite interactions and the development of novel therapeutic targets.

Serum Proteomic Profiles Reflect the Stages of Myxomatous Mitral Valve Disease in Dogs.

Canine myxomatous mitral valve disease (MMVD) is similar to Barlow's form of MMVD in humans. These valvulopathies are complex, with varying speeds of progression. We hypothesized that the relative abundances of serum proteins would help identify the consecutive MMVD stages and discover new disease pathways on a systemic level. To identify distinction-contributing protein panels for disease onset and progression, we compared the proteomic profiles of serum from healthy dogs and dogs with different stages of naturally occurring MMVD. Dogs were divided into experimental groups on the basis of the left-atrium-to-aorta ratio and normalized left ventricular internal dimension in diastole values. Serum was collected from healthy (N = 12) dogs, dogs diagnosed with MMVD in stages B1 (N = 13) and B2 (N = 12) (asymptomatic), and dogs diagnosed with MMVD in chronic stage C (N = 13) (symptomatic). Serum biochemistry and selected ELISAs (galectin-3, suppression of tumorigenicity, and asymmetric dimethylarginine) were performed. Liquid chromatography-mass spectrometry (LC-MS), tandem mass tag (TMT) quantitative proteomics, and statistical and bioinformatics analysis were employed. Most of the 21 serum proteins with significantly different abundances between experimental groups (p < 0.05, FDR ˂ 0.05) were classified as matrix metalloproteinases, protease inhibitors, scaffold/adaptor proteins, complement components, anticoagulants, cytokine, and chaperone. LC-MS TMT proteomics results obtained for haptoglobin, clusterin, and peptidase D were further validated analytically. Canine MMVD stages, including, for the first time, asymptomatic B1 and B2 stages, were successfully distinguished in dogs with the disease and healthy dogs on the basis of the relative abundances of a panel of specific serum proteins. Most proteins with significantly different abundances were involved in immune and inflammatory pathways. Their role in structural remodeling and progression of canine MMVD must be further investigated. Further research is needed to confirm the resemblance/difference with human MMVD. Proteomics data are available via ProteomeXchange with the unique dataset identifier PXD038475.

Profiling the alterations of serum proteome in dairy cows with retained placenta using high-throughput tandem mass tags quantitative approach.

BACKGROUND: Retained placenta (RP), a quite common disorder in dairy cows, shows a high negative impact on their health status and milk production. AIM: To investigate the difference in the serum proteome between the cows with RP and the physiologic puerperium (PP). MATERIAL & METHODS: Analysis of serum samples from nine cows with RP and six with PP using high-resolution liquid chromatography-tandem mass spectrometry approach. The proteins differing in the relative abundance between the PP and RP groups were classified using the Protein Analysis Through Evolutionary Relationship tool. For the pathway enrichment analysis, the REACTOME tool, with the human genome as the background, was employed. The criterion for significance was the false discovery rate corrected P-value less than 0.05. RESULTS: In total 651 proteins were identified with altered relative abundance of ten proteins. Among them, seven had higher, and three showed lower relative abundance in RP than in the PP group. The differently abundant proteins participated in 15 pathways: six related to hemostasis, three involved in lipoprotein metabolism, and the remaining ones associated with for instance redox homeostasis, post-translational modification, and scavenging. Finally, the validation of the proteomic results showed that haptoglobin and lipopolysaccharide-binding protein levels reliably differentiated between the RP and PP groups. CONCLUSION: The pattern of serum proteome alterations in the cows with RP mirrored several interplaying mechanisms underlying the systematic response to the presence of RP, therefore representing a source to mine for predictive or prognostic biomarkers.

Liver Proteome Alterations in Red Deer (Cervus elaphus) Infected by the Giant Liver Fluke Fascioloides magna.

Liver fluke infections are recognised as diseases with worldwide distribution and considerable veterinary and public health importance. The giant liver fluke, Fascioloides magna, is an important non-native parasite which has been introduced to Europe, posing a threat to the survival of local wildlife populations such as red deer (Cervus elaphus). The aim of the study was to analyse differences in liver proteomes between F. magna-infected and control red deer groups using a label-based high-throughput quantitative proteomics approach. The proteomics analysis identified 234 proteins with differential abundance between the control and infected groups. Our findings showed that F. magna infection in this definitive host is associated with changes in the metabolism of proteins and fatty acids, oxidative stress, fibrosis, and signaling pathways. The identified proteins and associated biological pathways represent a valuable contribution to the understanding of host-parasite interactions and the pathogenesis of liver fluke infection.

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

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

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

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

A Proteomic Approach to Elucidate the Changes in Saliva and Serum Proteins of Pigs with Septic and Non-Septic Inflammation.

Sepsis is a systemic inflammatory response triggered by an infectious agent and is recognized by the World Health Organization as a global concern, since it is one of the major causes of severe illness in humans and animals. The study of the changes that can occur in saliva and serum in sepsis can contribute to a better understanding of the pathophysiological mechanisms involved in the process and also to discover potential biomarkers that can help in its diagnosis and monitoring. The objective of this study was to characterize the changes that occur in the salivary and serum proteome of pigs with experimentally-induced sepsis. The study included five pigs with sepsis induced by LPS administration and five pigs with non-septic inflammation induced by turpentine for comparative purposes. In saliva, there were eighteen salivary proteins differentially expressed in the sepsis condition and nine in non-septic inflammation. Among these, significant increments in aldolase A and serpin B12 only occurred in the sepsis model. Changes in aldolase A were validated in a larger population of pigs with sepsis due to Streptococcus suis infection. In serum, there were 30 proteins differentially expressed in sepsis group and 26 proteins in the non-septic group, and most of the proteins that changed in both groups were related to non-specific inflammation. In the saliva of the septic animals there were some specific pathways activated, such as the organonitrogen compound metabolic process and lipid transport, whereas, in the serum, one of the main activated pathways was the regulation of protein secretion. Overall, saliva and serum showed different proteome variations in response to septic inflammation and could provide complementary information about the pathophysiological mechanisms occurring in this condition. Additionally, salivary aldolase A could be a potential biomarker of sepsis in pigs that should be confirmed in a larger population.

Saliva changes in composition associated to COVID-19: a preliminary study.

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

Changes in Proteins in Saliva and Serum in Equine Gastric Ulcer Syndrome Using a Proteomic Approach.

Changes in the salivary proteome in 12 horses with the two diseases included in equine gastric ulcer syndrome (EGUS), equine glandular gastric disease (EGGD) (n = 6) and equine squamous gastric disease (ESGD) (n = 6), were evaluated using a high-resolution LC-MS/MS analysis of TMT-labelled peptides and compared to 10 healthy control horses. Serum was also analysed for comparative purposes. The comparison between the horses with EGGD and controls showed significant changes in 10 salivary proteins, whereas 36 salivary proteins were differently abundant between ESGD and control groups. The most upregulated proteins in the case of EGGD were related to immune activation whereas, in horses with ESGD, the most significantly changed proteins were associated with squamous cell regulation and growth. Compared to serum, saliva showed a higher number of proteins with significant changes and a different pattern of changes. The proteins identified in our study, in addition to providing new information about the pathophysiological mechanisms in these diseases, could have the potential to be novel biomarkers for the diagnosis or monitoring of EGGD and ESGD.

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.

Integrated Metabolomics and Proteomics Dynamics of Serum Samples Reveals Dietary Zeolite Clinoptilolite Supplementation Restores Energy Balance in High Yielding Dairy Cows.

Dairy cows can suffer from a negative energy balance (NEB) during their transition from the dry period to early lactation, which can increase the risk of postpartum diseases such as clinical ketosis, mastitis, and fatty liver. Zeolite clinoptilolite (CPL), due to its ion-exchange property, has often been used to treat NEB in animals. However, limited information is available on the dynamics of global metabolomics and proteomic profiles in serum that could provide a better understanding of the associated altered biological pathways in response to CPL. Thus, in the present study, a total 64 serum samples were collected from 8 control and 8 CPL-treated cows at different time points in the prepartum and postpartum stages. Labelled proteomics and untargeted metabolomics resulted in identification of 64 and 21 differentially expressed proteins and metabolites, respectively, which appear to play key roles in restoring energy balance (EB) after CPL supplementation. Joint pathway and interaction analysis revealed cross-talks among valproic acid, leucic acid, glycerol, fibronectin, and kinninogen-1, which could be responsible for restoring NEB. By using a global proteomics and metabolomics strategy, the present study concluded that CPL supplementation could lower NEB in just a few weeks, and explained the possible underlying pathways employed by CPL.

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.

Combined Untargeted and Targeted Metabolomics Approaches Reveal Urinary Changes of Amino Acids and Energy Metabolism in Canine Babesiosis With Different Levels of Kidney Function.

Canine babesiosis is a tick-borne disease with a worldwide distribution, caused by the haemoprotozoan parasites of the genus Babesia. One of the most prevalent complication is acute kidney injury, and an early diagnosis of altered kidney function remains a challenge for veterinary practice. The aim of this study was to assess the urine metabolic profile from dogs with babesiosis and different degree of kidney function using untargeted and targeted MS-based metabolomics approaches. In this study, 22 dogs naturally infected with Babesia canis and 12 healthy dogs were included. Untargeted metabolomics approach identified 601 features with a differential abundance between the healthy group and groups of dogs with babesiosis and different level of kidney function, with 27 of them identified as a match to known standards; while targeted approach identified 17 metabolites with significantly different concentrations between the groups. A pattern of significantly altered metabolites referring to the inflammatory host response, oxidative stress, and energy metabolism modulation in babesiosis was presented. Our findings have demonstrated that kidney dysfunction accompanying canine babesiosis was associated with changes in amino acid metabolism, energy metabolism, fatty acid metabolism, and biochemical pathways such as urea cycle and ammonia detoxication. These findings will enable the inclusion of urinary markers for the detection and monitoring of renal damage in babesiosis, as well as in other similar diseases.

Evaluation of Changes in Metabolites of Saliva in Canine Obesity Using a Targeted Metabolomic Approach.

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.

Complementary liver and serum protein profile in wild boars infected by the giant liver fluke Fascioloides magna using tandem mass tags quantitative approach.

Liver fluke, Fascioloides magna, is an important non-native parasite introduced to Europe, posing a threat to survival of local wildlife populations. The aim of this study was to assess the serum and liver protein profile of control and F. magna infected wild boars, by means of shotgun tandem mass tag - based quantitative high resolution proteomics approach. In serum, 4 differentially abundant proteins were found out of total 1073 identified, while in liver from 3520 identified proteins, 116 were differentially abundant between healthy and F. magna infected wild boars. Pathway analysis revealed that most of the proteins differing in abundance are involved in metabolism, biological oxidations, cellular responses to stimuli, fatty acid metabolism, and others. Validation of proteomic results was performed for paraoxonase-1, ceruloplasmin, glutathione S-transferase and liver enzymes by ELISA and automated assays. Complementary analysis of liver and serum in F. magna infection enabled insight into changes of proteome profile of the host at local and sistemic level. Our findings showed that chronic infection with F. magna is associated with immune response in host, oxidative stress and metabolomic changes in liver. SIGNIFICANCE: Liver fluke infections are recognised as worldwide neglected diseases with considerable veterinary and public health importance. Pathological changes, clinical signs and outcome of F. magna infection are strongly related to the type of final hosts and their different tolerance to infection. In order to gain insight into host-parasite interactions in wild boars, dead-end host for F. magna, we assessed proteomics profile of serum and liver of control animals and those infected with F. magna. Proteomics analysis of serum and liver in parallel showed as advantageous and beneficial, demonstrating protein alterations mainly at local level. Bioinformatics analysis enabled elucidation of molecular pathways associated with F. magna infection. Identification and validation of proteins associated with infection may have added value to current tools for efficient liver fluke control.

Metabolic profiling of serum from dogs with pituitary-dependent hyperadrenocorticism.

Hyperadrenocorticism (HAC) is one of the most common endocrine diseases in dogs characterized by excessive cortisol production caused by an adrenocorticotropic hormone (ACTH)-secreting tumor, namely pituitary-dependent HAC (PDH) or cortisol-secreting adrenal tumor. Metabolomics presents the ability to identify small molecule metabolites. Thus, the use of metabolomics techniques in canine PDH can provide information about the pathophysiology and metabolic changes in this disease. This study aimed to identify and compare differences in serum metabolites between dogs with PDH and healthy dogs. The metabolomic profile of 20 dogs diagnosed with PDH was compared with 20 healthy dogs using liquid chromatography/mass spectrometry (LC/MS), and metabolite discrimination was performed using partial least squares-discriminant analysis (PLS-DA), the variable important in projection (VIP) and fold changes (FC) group-wise comparisons. The hypergeometric test identified the significantly altered pathways. A total of 21 metabolites were found to be significantly different between the two groups. The major alterations were found in arachidonic and decanoic acid, and phospholipids related to phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI). These metabolites are related to insulin resistance and other complications (i.e. hypertension). Our results indicate that PDH produces changes in serum metabolites of dogs, and the knowledge of these changes can aid to better understanding of pathophysiological processes involved and contribute to potentially detect new biomarkers for this disease.