Bandouchova et al. Acta Veterinaria Scandinavica 2011, 53:2 http://www.actavetscand.com/content/53/1/2 Assay of low molecular weight antioxidants by square wave voltammetry Square wave voltammetry (SWV) was used to estimate low molecular weight antioxidants (LMWAs) in plasma samples as described previously [29]. The anodic current was measured in order to estimate the occurrence of compounds that are able to donate electrons, i.e., anti- oxidants [16]. The device EmStat (PalmSens, Houten, Netherlands) and screen-printed strips with platinum working (1 mm diameter, dot-shaped), silver/silver chloride reference and platinum auxiliary electrodes on a ceramic support (BVT, Brno, Czech Republic) were used throughout the experiments. The strips were washed with ethanol and water prior to use. EmStat was adjusted to the following parameters: applied potential in the range 0 - 1 V; potential step as well as potential amplitude 0.01 V; frequency 1 Hz. Measurement began by spreading 20 μl of plasma over the electrodes. Each strip was only used for one measurement in order to avoid hysteretic influences. Thiobarbituric acid reactive substances assay Total thiobarbituric acid reactive species (TBARS) in plasma were assayed as described previously [30]. A stock solution of thiobarbituric acid (TBA) was prepared by diluting 67 mg of TBA in 1 ml dimethylsulphoxide and subsequently adding 9 ml of deionized water. One hundred μl of plasma were mixed with 200 μl ice cold 10% trichloroacetic acid and incubated in an ice bath for 15 minutes. The mixture was centrifuged at 3000 × g for 15 minutes in order to displace precipitated pro- teins. After centrifugation, 200 μl of supernatant were injected into a new tube and the same volume of TBA solution was added. Finally, the mixture was incubated in a boiling water bath for 10 minutes. A blank was pre- pared using the above-mentioned protocol with plasma replaced by physiological solution. After cooling to laboratory temperature, absorbance was measured against the blank at 532 nm. Biochemistry Within a few days of collection, plasma was analysed using an automated analyser (SPOTCHEM™ EZ SP- 4430, ARKRAY, Japan) for total proteins and albumin (g/l), creatinine (μmol/l), urea (mmol/l), uric acid (mmol/l), aspartate aminotransferase (μkat/l), alkaline phosphatase (μkat/l), alanine aminotransferase (μkat/l), lactate dehydrogenase (μkat/l), creatine kinase (μkat/l), total cholesterol (mmol/l), triglycerides (mmol/l), glucose (mmol/l) and total bilirubin (μmol/l). Statistical analysis Statistical analyses were performed using Statistica for Windows 7.0 (StatSoft, Tulsa, OK, USA). Data normality Page 3 of 13 and homogeneity of variances were evaluated by the Kolmogorov-Smirnov test and the Levene’s test, respec- tively. One-way analysis of variance (ANOVA) and the nonparametric Kruskal-Wallis test were used for statisti- cal comparisons. In the case of non-normal data distri- bution, nonparametric statistical analysis also included the Mann-Whitney U test. Values of p < 0.05 and p < 0.01 were considered statistically significant and highly significant, respectively, for all tests. Spearman rank order correlation analysis was employed to examine the relationship between low molecular weight antioxidants and plasma chemistry profiles. Results and discussion There was no mortality in the control group during the study, while two of the five European brown hares from the F. tularensis-inoculated group died. Clinical signs of tularemia started to develop one day post-inoculation and included fever as high as 41 °C, lethargy and anor- exia. Hares succumbed to the infection on days 5 and 9 post-inoculation. There was splenomegaly and micro- scopic examination of tissue slides revealed diffuse necroses in the spleen, focal necroses in the liver and moderate vacuolization of hepatocytes. Blood culture yielded positive results in samples collected from three, four and one hares on days 2, 4 and 6 post-inoculation, respectively. Bacteraemia was also confirmed using the above samples and the mouse inoculation test. Positive cultures were obtained from liver, spleen, lung, bone marrow and kidney tissues in the hare that died on day 5, while only spleen and bone marrow tissues were bur- dened by bacteria in the hare dying on day 9 post- inoculation. The remaining three hares were killed on day 35 post-inoculation. There were no gross and microscopic pathological findings in the surviving hares, and organs collected aseptically (liver, spleen, lung, bone marrow and kidney) were free of F. tularensis based on culture and the mouse inoculation test. Tube agglutina- tion antibodies first occurred between days 8 to 10 and amounted up to the titre of 1:640 on day 35 post- inoculation. Since we used only a single dose with approximately 2.6 × 109 colony forming units (CFU) pro toto, it was not the purpose of the present study to determine the LD50 of the F. tularensis infection in hares. However, the selected dose resulted in the death of two out of five inoculated hares and it seems that it was close to the LD50 for this mammalian species and the subcutaneous route of infection. The European brown hare may thus be considered a species of relatively low susceptibility to tularemia when exposed via this route because, for example, in the highly susceptible BALB/c mice and common voles the LD50 was calculated to be about 1 and 38 CFU, respectively [19]. Similar results of lower