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.

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

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

Is canine calprotectin in serum stabile after storage at low temperature?

BACKGROUND: In human and veterinary medicine calprotectin is most widely used in diagnosing different gastro-intestinal diseases. The aim of this study was to assess the stability of canine calprotectin (cCP) in serum after storage at low temperatures and imprecision of the method. METHODS: Blood samples were collected from dogs with different clinical diagnoses. Twenty-two dogs were included in this study. Calprotectin concentration was measured 4 hours after serum separation (T0), and after being frozen at - 80 °C for 8 (T1) and 16 weeks (T2). The maximum permissible difference (MPD) was derived from the equation for calculating total error (TE) TE = %Bias + (1.96 x %CV), where bias and coefficient of variation (CV) were defined by the manufacturer. The dogs enrolled in this study were patients admitted during the morning (9-12 a.m.), on the day the first measurement was performed. All sample analysis for determination of stability were done in duplicates. For determination of within-run precision, the two patients' serum samples were analyzed in 20 replicates. Imprecision was assessed by analyzing 20 replicates on one plate on two samples where high and low concentrations were anticipated. RESULTS: The calculated value of MPD was 32.52%. Median calprotectin concentrations were higher at T1 114.08 µg/L (IQR = 55.05-254.56) and T2 133.6 µg/L (IQR = 100.57-332.98) than at T0 83.60 µg/L (IQR = 50.38-176.07). Relative and absolute bias at T1 (49.3%; 45.98 µg/L) and T2 (109.93%; 94.09 µg /L) have shown that calprotectin concentrations increase after long term storage at - 80 °C. CONCLUSION: The results of the present study indicate that c-CP was not stable for 16 weeks at low storage temperature (- 80 °C). Considering the observed change in the concentration of c-CP at T1, a storage time of 8 weeks should be safely applied. The method imprecision was not satisfactory, especially in the lower concentration range.

Changes in Acute Phase Response Biomarkers in Racing Endurance Horses.

This study aimed to evaluate if exercise-induced acute phase response (APR) occurs in endurance horses in response to the race. The study included 23 horses competing in an endurance competition with a successfully passed clinical examination before the race. Blood samples were collected before the start and within 30 min after the end of the race. Haematological and biochemical tests were performed and correlated to acute phase biomarkers changes. Values of calprotectin and haptoglobin (Hp) decreased after the races compared to values before, while concentrations of ceruloplasmin and albumin recorded a significant increase. Greater changes in calprotectin values were noted in Arabian horses compared to other breeds. Values of Hp showed a significantly greater decrease after longer races. Based on study results, endurance racing induces APR in horses characterised by significant changes in selected acute phase biomarkers. More pronounced changes were noted at races with higher average speeds, suggesting the need for thorough horse monitoring during exhausting races.

Novel equi merozoite antigen (ema-1) gene heterogeneity in a geographically isolated Theileria equi population in Croatia.

BACKGROUND: The apicomplexan haemoparasite Theileria equi, a causative agent of equine piroplasmosis, is an established pathogen of significant welfare and economic concern within the Croatian equine population. A previous large surveillance study of T. equi has identified two distinct parasite populations, one in the north and one in the south, geographically separated by the Dinaric Alps, which traverse the country. This study aimed to further investigate the genetic diversity within these two populations, focussing on allelic variability of the equi merozoite antigen gene, ema-1. METHODS: Following nested PCR of DNA isolates, the generated ema-1 amplicons were subsequently sequenced and compared by phylogenetic analysis to available sequences representing previously described ema-1 genotypes (groups A-C). RESULTS: Isolates from the southern T. equi population clustered with the existing ema-1 groups A and B. Strikingly, isolates from the northern population clustered into two novel ema-1 genotypes, named groups D and E. CONCLUSIONS: This detection of hitherto unreported genotypes suggests that historic geographical isolation has led to a degree of divergent evolution in this northern T. equi population. Additionally, current global regulatory testing of equine piroplasmosis relies heavily on EMA-1 based immunodiagnostics, and the discovery of unique ema-1 genotypes may question the efficacy of current diagnostics in international equine movement, with ramifications for the global equine community.

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.

LC-MS/MS analysis of the dog serum phosphoproteome reveals novel and conserved phosphorylation sites: Phosphoprotein patterns in babesiosis caused by Babesia canis, a case study.

Phosphorylation is the most commonly studied protein post-translational modification (PTM) in biological systems due to its importance in controlling cell division, survival, growth, etc. Despite the thorough research in phosphoproteomics of cells and tissues there is little information on circulating phosphoproteins. We compared serum from 10 healthy dogs and 10 dogs affected by B. canis-caused babesiosis with no organ dysfunctions by employing gel-free LC-MS/MS analysis of individual samples and tandem mass tag (TMT) label-based quantitative analyses of pools, both supported by phosphopeptide enrichment. Results showed a moderate number of phosphorylated proteins (50-55), with 89 phosphorylation sites not previously published for dogs although a number of them matched phosphorylation sites found in mammalian orthologs. Three phosphopeptides showed significant variation in babesiosis-affected dog sera compared to controls: Serum amyloid A (SAA) phosphorylated at serine 101 (up-regulation), kininogen 1 phosphorylated at threonine 326, and fibrinogen α phosphorylated at both threonine 20 and serine 22 (down-regulation). 71.9% of the detected phosphorylated sites were phosphoserine, 16.8% phosphothreonine and only 11.2% phosphotyrosine residues. TMT label-based quantitative analysis showed α-2-HS-glycoprotein / Fetuin A to be the most abundant phosphoprotein (50-70% of all phosphoproteins) followed by kininogen-1 (10-20%). The alterations of phosphorylated proteins observed in canine babesiosis caused by Babesia canis suggest new insights into the largely neglected role of extracellular protein phosphorylation in health and disease, encouraging urgent further research on this area. To the best of our knowledge the present study represents the first attempt to characterize canine serum phosphoproteome.

Glomerular and tubular kidney damage markers in canine babesiosis caused by Babesia canis.

Canine babesiosis is a tick-borne disease caused by the haemoprotozoan parasites of the genus Babesia. The aim of this study was to assess renal dysfunction in dogs with babesiosis caused by B. canis, using serum and urinary markers for both glomerular and tubular dysfunction. Assays previously not validated for use in canine samples were validated and the potential interference of haemoglobin, lipids and bilirubin with these analyses was additionally considered. In this study 42 dogs naturally infected with B. canis and 14 healthy dogs were included. Dogs with babesiosis were divided into 3 groups: group A consisted of 9 non-azotemic dogs with normal urine protein to creatinine ratio (UPC < 0.5), group B of 27 non-azotemic dogs with UPC > 0.5 and group C of 6 azotemic dogs with UPC > 2. The concentrations of urinary immunoglobin G (IgG), retinol binding protein (RBP), uromodulin, kidney injury molecule - 1 (KIM-1), and serum symmetric dimethylarginine were measured by ELISA assays, while urinary albumin and N-acetyl-b-D-glucosaminidase (NAG) were evaluated by an immunoturbidimetric and enzymatic colorimetric assay, respectively. Urinary markers were normalized to urine creatinine concentration. All tested markers, with exception of uromodulin, showed significant differences between dogs with babesiosis and healthy dogs, and also showed strong or very strong positive correlation with UPC. Increases of urinary albumin and IgG suggested glomerular damage, and increases of KIM-1, RBP and NAG proximal tubular damage in dogs with babesiosis. They demonstrated clear advantages compared to conventional parameters by showing earlier changes in detecting renal damage.

Effect of prolonged submaximal exercise on serum oxidative stress biomarkers (d-ROMs, MDA, BAP) and oxidative stress index in endurance horses.

BACKGROUND: Oxidative stress (OS) associated with an intense exercise may have a negative influence on equine health. The aim of this study was to determine the effects of endurance races on oxidative and antioxidative status of horses by evaluating changes in reactive oxygen metabolites (d-ROMs), malondialdehyde (MDA), biological antioxidant potential (BAP) and oxidative stress index (OSI) values. The study was carried out on 53 race starts (28 individual horses) competing at different endurance races according to distance (40 and 80 km) and difficulty (easy and demanding). Blood samples were taken before and after the race. RESULTS: Compared to levels of OS serum biomarkers before the race, an increase in values of d-ROMs (P <  0.01), MDA (P <  0.01), and BAP (P <  0.001), and a decrease in OSI (P <  0.001) have been noted after the race. Contrary to other measured biomarkers, BAP did not show significant individual effects of horses. Horses competing at shorter races have shown a significant change in d-ROMs (P = 0.002), BAP (P <  0.001) and OSI (P = 0.004), whereas those competing at longer races in MDA (P = 0.002), BAP (P <  0.001) and OSI (P <  0.001) post-race values. Endurance racing induced changes in values of d-ROMs, BAP and OSI during both easy and demanding races. CONCLUSIONS: Changes in all measured OS biomarkers indicate that prolonged aerobic exercise during endurance race could contribute to the imbalance between oxidants and antioxidants in horses, mainly characterised by a pronounced antioxidant response. Biological antioxidant potential was found to be the most reliable biomarker of OS in endurance horses in the present study.

Alteration of haemostatic parameters in uncomplicated canine babesiosis.

Babesiosis is a tick-borne zoonotic disease caused by haemoprotozoan parasites. The aim of this study were to assess markers of coagulation pathways in 25 dogs with naturally occurring babesiosis caused by B. canis, compared to 10 healthy controls. Protein C (PC) and antithrombin III (AT III) activity were assessed using a chromogenic substrate test, while levels of thrombin-antithrombin (TAT) complexes, activated protein C (APC) and endothelial protein C receptor were assessed using canine-specific ELISA. AT III activity was decreased as a result of a negative acute phase response, degradation by elastase, reduced availability of glycosaminoglycans, and, most importantly, consumption as a consequence of thrombin formation. Procoagulant state and haemostatic shift towards thrombin formation are also demonstrated by elevated TAT levels. Regarding PC pathway only significant difference was found for APC. Taken together, haemostatic alterations in uncomplicated babesiosis represent a procoagulant state that is mostly reversed during treatment.

Prevalence and Geographic Distribution of Vector-Borne Pathogens in Apparently Healthy Dogs in Croatia.

Vector-borne pathogens (VBPs) are a group of globally extended and quickly spreading pathogens that are transmitted by various arthropod vectors. The aim of the present study was to investigate the seroprevalence against Babesia canis, Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Leishmania infantum, Dirofilaria immitis, and Ehrlichia canis in dogs in Croatia. We investigated 435 randomly selected apparently healthy dogs in 13 different locations of Croatia for antibodies to B. canis by indirect immunofluorescence using a commercial IFA IgG Antibody Kit. All samples were also tested for qualitative detection of D. immitis antigen and for antibodies to A. phagocytophilum, B. burgdorferi sensu lato, L. infantum, and E. canis with two point-of-care assays. Overall, 112 dogs (25.74%, 95% confidence interval [CI] 21.70-30.12) were serologically positive for one or more of the pathogens. B. canis was the most prevalent pathogen (20.00%, 95% CI 16.34-24.07), followed by A. phagocytophilum (6.21%, 95% CI 4.12-8.90), L. infantum, (1.38%, 95% CI 0.51-2.97), and B. burgdorferi sensu lato (0.69%, 95% CI 0.01-2.00). The lowest seroprevalence was for D. immitis and E. canis (0.46%, 95% CI 0.01-1.65). Coinfection was determined in 12 dogs (2.76%, 95% CI 1.43-4.77), of which 10 were positive to two pathogens (7 with B. canis and A. phagocytophilum and 1 B. canis with B. burgdorferi sensu lato or L. infantum or E. canis). One dog was positive to three pathogens and another dog to four pathogens. Seroprevalence for babesia was age, breed, and lifestyle/use dependent. Purebred dogs had almost half the chance of developing disease than crossbred (OR = 0.58, p < 0.026, 95% CI 0.37-0.94). Seropositivity to B. canis was 3.41 times higher for dogs that lived outdoors/shelter (p < 0.006) or 4.57 times higher in mixed/hunting (p < 0.001) compared to indoor/companion dogs. This is the first comprehensive survey of VBP seropositivity conducted in Croatia. Some of these VBPs are zoonotic and represent a potential risk to public health.

Relation of antioxidant status at admission and disease severity and outcome in dogs naturally infected with Babesia canis canis.

BACKGROUND: Canine babesiosis is caused by species of the Babesia genus and has become an emerging disease worldwide. To the authors' knowledge there are no reports in which antioxidants have been analyzed in different presentations of canine babesiosis or in which the prognostic value of antioxidants has been studied. The aim of this study was to evaluate whether oxidative stress could be related to the severity and outcome of canine babesiosis. For this purpose a profile consisting of four antioxidant biomarkers (superoxide dismutase - SOD, glutathione peroxidase - GPx, catalase, total antioxidant status - TAS) and malondialdehyde - MDA as an oxidant biomarker (previously evaluated, here studied for comparative purposes) were evaluated in dogs with canine babesiosis of different clinical severity and outcomes. RESULTS: The study was conducted with a sample of 40 dogs suffering from babesiosis (further divided into uncomplicated, one complication and multiple organ dysfunction syndrome - MODS group) and 30 healthy dogs (control group). Additionally, the babesiosis group was divided according to the anaemia into non-anaemic, mildly anaemic, moderately anaemic and severely anaemic dogs. The results of our study showed significantly decreased SOD, catalase and TAS values in diseased dogs compared to controls, while there were no significant differences in GPx between these groups. Dogs that developed MODS showed lower activities of SOD and GPx and higher MDA values compared to dogs with uncomplicated babesiosis as well as with dogs that developed one complication. Superoxide dismutase, catalase and GPx were negatively correlated whereas MDA was positively correlated with the lethal outcome of the disease. Furthermore, this study detected more pronounced decrease in antioxidant biomarkers (SOD, GPx and catalase) in dogs with moderate anaemia compared to those with mild anaemia. CONCLUSIONS: The results of this study showed changes in biomarkers related to the antioxidant status of dogs naturally infected with B. canis canis. These biomarkers could be used as indicators of disease severity and outcome in dogs suffering from babesiosis.

Blood markers of fibrinolysis and endothelial activation in canine babesiosis.

BACKGROUND: Canine babesiosis is a tick-borne disease caused by hemoprotozoan parasites of the genus Babesia. The disease can be clinically classified into uncomplicated and complicated forms. The aim of this study was to assess the level of endothelial activation and alterations in the fibrinolytic pathway during canine babesiosis. RESULTS: Blood samples were collected on the day of admission and on the 6th day after treatment with imidocarb propionate, from 30 dogs of various breeds and of both sexes with naturally occurring babesiosis caused by B. canis. In this prospective study, plasminogen activity was assessed using a chromogenic assay, and concentrations of high mobility group box-1 protein (HMGB-1), intercellular adhesive molecule-1 (ICAM-1), vascular adhesive molecule-1 (VCAM-1), soluble urokinase receptor of plasminogen activator (suPAR), thrombin activatable fibrinolysis inhibitor (TAFI), soluble thrombomodulin (TM) and plasminogen activator inhibitor-1 (PAI-1) were determined using a canine specific ELISA. Concentrations of TM, HMGB-1, VCAM-1 and suPAR were increased in dogs with babesiosis at admission compared to healthy dogs. After treatment, concentrations of TM were lower in infected dogs compared to healthy dogs. Dogs with babesiosis also had increased concentrations of TM, ICAM-1 and HMGB-1 and decreased plasminogen and PAI-1 at presentation compared to day 6 after treatment. Dogs with complicated babesiosis had higher concentrations of TM, HMGB1 and TAFI at admission compared to the 6th day. CONCLUSIONS: Biomarkers of endothelial activation and fibrinolysis were altered in dogs with babesiosis. Further studies into their usefulness as biomarkers of disease severity or prognosis is warranted.