Josipa Kuleš

Identification of serum biomarkers in dogs naturally infected with Babesia canis canis using a proteomic approach

BackgroundCanine babesiosis is a tick-borne disease that is caused by the haemoprotozoan parasites of the genus Babesia. There are limited data on serum proteomics in dogs, and none of the effect of babesiosis on the serum proteome. The aim of this study was to identify the potential serum biomarkers of babesiosis using proteomic techniques in order to increase our understanding about disease pathogenesis.ResultsSerum samples were collected from 25 dogs of various breeds and sex with naturally occurring babesiosis caused by B. canis canis. Blood was collected on the day of admission (day 0), and subsequently on the 1st and 6th day of treatment.Two-dimensional electrophoresis (2DE) of pooled serum samples of dogs with naturally occurring babesiosis (day 0, day 1 and day 6) and healthy dogs were run in triplicate. 2DE image analysis showed 64 differentially expressed spots with p ≤ 0.05 and 49 spots with fold change ≥2. Six selected spots were excised manually and subjected to trypsin digest prior to identification by electrospray ionisation mass spectrometry on an Amazon ion trap tandem mass spectrometry (MS/MS). Mass spectrometry data was processed using Data Analysis software and the automated Matrix Science Mascot Daemon server. Protein identifications were assigned using the Mascot search engine to interrogate protein sequences in the NCBI Genbank database.A number of differentially expressed serum proteins involved in inflammation mediated acute phase response, complement and coagulation cascades, apolipoproteins and vitamin D metabolism pathway were identified in dogs with babesiosis.ConclusionsOur findings confirmed two dominant pathogenic mechanisms of babesiosis, haemolysis and acute phase response. These results may provide possible serum biomarker candidates for clinical monitoring of babesiosis and this study could serve as the basis for further proteomic investigations in canine babesiosis.

Markers of Coagulation Activation, Endothelial Stimulation, and Inflammation in Dogs with Babesiosis

BACKGROUND Babesia infections in dogs can result in a wide range of clinical and laboratory presentations, including coagulopathy. Expression of intercellular adhesion molecule-1 (ICAM-1) and von Willebrand factor (vWF) in dogs with babesiosis is unknown. OBJECTIVES Whether inflammation in babesiosis triggers activation of ICAM-1 and the coagulation system. ANIMALS Twelve and 10 dogs with naturally occurring babesiosis before and after antiparasitic treatment, respectively, were compared with 10 healthy dogs. METHODS In this prospective study, diagnosis was made by blood smear examination and confirmed by PCR. C-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1), and von Willebrand factor (vWF) levels were measured by a canine ELISA kit, fibrinogen (FIB) and factor VIII activity levels were measured by coagulometric methods, and blood cell counts (WBC, RBC, PLT) were determined with an automatic analyzer. RESULTS Compared to healthy dogs, the CRP, sICAM-1, and FIB concentrations were significantly increased before therapy and remained high for 3 days after therapy in dogs with babesiosis. vWF activity was significantly decreased in dogs with babesiosis before treatment. FVIII activity did not differ between dogs with babesiosis and healthy dogs. WBC; RBC and PLT were significantly lower before treatment and normalized by 3 days after treatment. CONCLUSION AND CLINICAL IMPORTANCE A proinflammatory condition in babesiosis appears to influence endothelial dysfunction and hemostatic activity. Although clearly beneficial for the parasite, sequestered blood cells can obstruct blood flow in small vessels, promote an inflammatory state, and could increase the severity of babesiosis.

Serum concentrations of eicosanoids and lipids in dogs naturally infected with Babesia canis

Canine babesiosis is a tick-borne disease with world-wide significance caused by intraerythrocytic protozoa of the genus Babesia. The eicosanoids, as inflammatory mediators, are involved in the regulation of the immune response and inflammatory reaction. Metabolism of lipids is of great importance in babesiosis. In this study it was aimed to investigate the dynamics of serum concentration of prostaglandin E2 (PGE2), thromboxane B2 (TxB2), leukotriene B4 (LTB4), triglycerides, total cholesterol (Chol), HDL- and LDL-cholesterol in dogs naturally infected with Babesia canis and healthy dogs. Both groups were measured for all parameters on the admission day and on the first, second and seventh day of the disease. Dogs that were included in this study had systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). It was demonstrated that the level of LTB4, PGE2, TxB2 in dogs naturally infected with B. canis significantly changed during the disease. The level of LTB4 was significantly higher during the study, while the concentration of PGE2 was significantly higher second, third and seventh day of disease in relation with healthy dogs. The level of TxB2 was significantly lower at the beginning of the disease, but after seven days concentration was significantly higher. Both group of patients with SIRS and MODS had significantly higher level of LTB4. Substained high concentrations of PGE2 were observed in dogs with MODS after therapy but not in dogs with SIRS, and LTB4 followed a similar tendency. On the other hand, increases in TxB2 were only significant in dogs with SIRS. The lipid profile in naturally infected dogs with B. canis infection was significantly changed. Further studies are needed to assess the prognostic values of lipid mediators in dogs with B. canis infection, and the ability of these markers to predict the progress of SIRS and MODS.

Plasma biomarkers of SIRS and MODS associated with canine babesiosis

Canine babesiosis is a tick-borne disease caused by the haemoprotozoan parasites of the genus Babesia. Early detection of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) is of major importance in clinical practice for providing information about severity and outcomes of the disease and therapy. Plasma samples were taken at admission from five dogs with uncomplicated babesiosis caused by B. canis canis, five dogs with babesiosis and SIRS, five dogs with babesiosis and MODS, and five healthy dogs. After two-dimensional electrophoresis and capillary reversed - phase liquid chromatography coupled online with tandem mass spectrometry, 68 differentially expressed spots with level of significance P<0.05 were detected between groups. SIRS in babesiosis was characterised by increases in paraoxonase 1 and apoA-I, whereas MODS with decrease of complement inhibitors leading to prolonged complement activation and decrease of vitamin D binding protein due to haemolysis and activation of the coagulation cascade.

Relationship between paraoxonase 1 activity and high density lipoprotein concentration during naturally occurring babesiosis in dogs

Paraoxonase 1 (PON1) is a negative acute phase protein bound to high density lipoproteins (HDL) and during the acute phase response (APR) protects HDL from peroxidation. The aim of this study was to assess the relationship between PON1 and HDL in canine babesiosis, a disease characterized by oxidative damages and by an APR. PON1, HDL and C-reactive protein (CRP), were measured in blood collected from 15 controls and 29 dogs with babesiosis sampled at admission, and on days 1 and 7 after treatment. At admission, PON1 and HDL were significantly lower in affected dogs. HDL concentration increased at day 1 while PON1 increased and CRP decreased at day 7. This suggests that the decrease of PON1 at admission is in part due to an increased consumption, the decreased HDL may depend on lipid peroxidation and its rapid increase after treatment may depend on the antioxidant activity of PON1.

Blood markers of fibrinolysis and endothelial activation in canine babesiosis

BackgroundCanine 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.ResultsBlood 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.ConclusionsBiomarkers 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.

The knowledge and understanding of preanalytical phase among biomedicine students at the University of Zagreb

Introduction The educational program for health care personnel is important for reducing preanalytical errors and improving quality of laboratory test results. The aim of our study was to assess the level of knowledge on preanalytical phase in population of biomedicine students through a cross-sectional survey. Materials and methods A survey was sent to students on penultimate and final year of Faculty of Pharmacy and Biochemistry – study of medical biochemistry (FPB), Faculty of Veterinary Medicine (FVM) and School of Medicine (SM), University of Zagreb, Croatia, using the web tool SurveyMonkey. Survey was composed of demographics and 14 statements regarding the preanalytical phase of laboratory testing. Comparison of frequencies and proportions of correct answers was done with Fisher’s exact test and test of comparison of proportions, respectively. Results Study included 135 participants, median age 24 (23-40) years. Students from FPB had higher proportion of correct answers (86%) compared to students from other biomedical faculties 62%, P < 0.001. Students from FPB were more conscious of the importance of specimen mixing (P = 0.027), prevalence of preanalytical errors (P = 0.001), impact of hemolysis (P = 0.032) and lipemia interferences (P = 0.010), proper choice of anticoagulants (P = 0.001), transport conditions for ammonia sample (P < 0.001) and order of draw during blood specimen collection (P < 0.001), in comparison with students from SM and FVM. Conclusions Students from FPB are more conscious of the importance of preanalytical phase of testing in comparison with their colleagues from other biomedical faculties. No difference in knowledge between penultimate and final year of the same faculty was found.

Plasma markers of inflammation and hemostatic and endothelial activity in naturally overweight and obese dogs

BackgroundObesity is one of the most prevalent health problems in the canine population. While haemostatic parameters and markers of endothelial function have been evaluated in various disease conditions in dogs, there are no studies of these markers in canine obesity. This study was designed to evaluate the effect of naturally gained weight excess and obesity on inflammatory, hemostatic and endothelial biomarkers in dogs. A total of 37 overweight and obese dogs were compared with 28 normal weight dogs.ResultsOverweight and obese dogs had significantly elevated concentrations of serum interleukin-6 (IL-6) and C-reactive protein (hsCRP). Number of platelets, activity of factor X and factor VII were significantly higher, while activated partial thromboplastine time (aPTT) and soluble plasminogen activator receptor (suPAR) were significantly decreased. Statistical analysis of high mobility group box – 1 protein (HMGB-1), soluble intercellular adhesive molecule -1 (sICAM-1) and plasminogen activator inhibitor type 1 (PAI-1) concentrations did not show significant differences between the total overweight and obese group and the normal weight group of dogs.ConclusionsAnalytical changes in the dogs in our study reflects that weight excess in dogs can be associated with a chronic low degree of inflammation and a hypercoagulable state, where primary and secondary hemostasis are both affected. However obesity is not associated with impairment of endothelial function in dogs.

Proteomics in Veterinary Medicine and Animal Science: Neglected Scientific Opportunities with Immediate Impact

Animal/veterinary proteomics is an evolving field which holds a great promise not only for fundamental and applied discoveries regarding biology and pathology of domestic species, but can also be implemented in comparative applications of human diseases research. Experimental proteomics in domestic animals have advantages over use of rodents, such as multiple sampling in time series and availability of biological samples in sufficient volume for multiple analyses, such that both experimental and natural disease processes can be investigated. While there are certain technical limitations in the expansion of the field, they can currently be circumvented and in the future mastered with a greater participation of proteomic experts, which will in turn drive the accessibility of species‐specific reagents, data volume expansion in bioinformatic databases, and increased funding. This Viewpoint highlights some comparative proteomics studies addressing important issues and encourages readers to expand their horizons of domestic animal proteomics research. It will hopefully inspire new fruitful collaborations between veterinary and animal scientists and proteomic specialists for research in these areas that can have immediate and direct impact on health, society, and the economy.

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.