engleski

A proteomic analysis of canine serum during the course of babesiosis

Objectives Canine babesiosis is a tick-borne disease that is caused by the haemoprotozoan parasites of the genus Babesia (Taboada and Merchant, 1991). There are limited data of serum proteomics in dogs, and none of babesiosis. The aim of this study was to identify the potential serum biomarkers using proteomic techniques and to increase our understanding about disease pathogenesis. Materials and methods Serum samples were collected from 25 dogs of various breeds and sex with naturally occurring babesiosis caused by B. canis, admitted to Clinic for Internal Diseases, Faculty of Veterinary Medicine, University of Zagreb, Croatia. Blood was collected on the day of admission (day 0), and subsequently on the 1st and 6th day of treatment. Serum was also collected from 10 healthy dogs. Serum samples were pooled into 4 groups (day 0, day 1, day 6 and control). The protein concentrations of the serum samples were measured using the Bradford assay (Biorad Ltd, Hemel Hempstead UK). Sera from each pool were suspended in rehydration buffer in order to archive final protein concentration of 200 μg/μl. The mixture was loaded onto a 11 cm IPG strip (immobilized pH gradient, pH 3–10, linear, Biorad Ltd, Hemel Hempstead UK). Isoelectric focusing (IEF) was performed (Protean IEF Cell, Biorad Ltd, Hemel Hempstead UK) according to the manufacturers instruction. Electrophoresis in the second dimension was carried out using Criterion precast gels (XT Bis-Tris Gel, 4-12% polyacrylamide gel, IPG+1 well, 11 cm IPG strip ; Biorad Ltd, Hemel Hempstead UK) at 200 V for 45-50 minutes. 2-DE gels were then stained with colloidal Coomassie blue G250 and destained in 5% v/v acetic acid. Each sample was analyzed in triplicate. The stained gels were scanned and digitized images of gels were analyzed using the Progenesis Same Spot software (Nonlinear Dynamics Ltd, Newcastle, UK) to identify protein spots which were differentially expressed through time (power >0.8 and ANOVA significance score of <0.05 between replicate gels). Six selected spots were excised manually and subjected to trypsin digest prior to identification by electrospray ionisation (ESI) mass spectrometry on an Amazon ion trap MS/MS (Bruker Daltonics Ltd, UK). MS data was processed using Data Analysis software (Bruker) and the automated Matrix Science Mascot Daemon server (v2.1.06). Protein identifications were assigned using the Mascot search engine to interrogate protein sequences in the NCBI Genbank database, allowing a mass tolerance of 0.4 Da for both MS and MS/MS analyses. Results and discussion 2D electrophoresis of pooled serum samples of dogs with naturally occurring babesiosis (day 0, day 1 and day 6) and healthy dogs were run in triplicate. 2D image analysis showed 64 differentially expressed spots with ANOVA p≤0.05 and 49 spots with fold change ≥2. All together, there were 37 spots with p≤0.05 and fold change ≥2, with power of >0.8. Six spots (Mascot score ˃ 100 and sequence coverage ˃ 10%) were selected for running on mass spectrometry. In this study, we used a proteomic approach to analyze the alterations in canine serum proteome due to B. canis infection. 2DE gels on three replicates generated from the infected serum samples were compared with the gels from healthy serum samples, followed by MS/MS analysis, which revealed several significantly differential serum proteins. 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. Our findings confirmed two dominant pathogenic mechanisms of babesiosis, haemolysis and acute phase response (Reyers et al., 1998 ; Taboada and Lobetti, 2006). Three APP involved in haemoglobin and iron metabolism and transport, haptoglobin, hemopexin and serotransferrin, indicate the role of haemolysis in the course of babesiosis. Tissue hypoxia, which is a common feature in babesiosis, is probably one of the major causes for the release of cytokines, oxygen free radicals, nitric oxide and other inflammatory mediators (Crnogaj et al., 2010 ; Matijatko et al., 2007). As a consenquence, acute phase response was triggered and demonstrated throughout wide variety of acute phase proteins (alpha-1-acid glycoprotein, alpha-1-antitrypsin, inter-alpha-trypsin inhibitor H4 (ITIH4), leucine-rich-alpha-2-glycoprotein, haptoglobin, hemopexin, serotransferrin, alpha-2-HS-glycoprotein, albumin). Also, release of ROS and consequent oxidative damage, lead to increased expression of clusterin and apolipoproteins (apoA-I and apoA-IV) with antioxidative activity. Impairment of coagulation and fibrinolytic system was demonstrated throughout consumption of AT III due to the coagulation activation. Complement activation was confirmed by clusterin and C3 increased expression. And finally, vitamin D binding protein, as a novel biomarker for MODS in different conditions, can be a possible target for further validation in babesiosis. These results may provide possible serum biomarker candidates for clinical monitoring and prognosis of babesiosis. The major limitation of this study is insensitivity of 2-DE gel methods used for proteome analysis. Thus, the depletion of major proteins has been suggested to be a potential strategy for enhancing detection sensitivity in serum. However, this study could serve as the basis for further proteomic investigations in canine babesiosis.

proteomics; dog; babesiosis

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