Bandouchova et al. Acta Veterinaria Scandinavica 2011, 53:2 http://www.actavetscand.com/content/53/1/2 of the host-defence mechanisms appear to be intended to counteract this stress [15]. Cells are equipped with defence mechanisms that provide protection via enzy- matic activities or through low molecular weight antiox- idants (LMWAs) acting as chemical scavengers and neutralizing reactive molecular species [16]. Interest- ingly, F. tularensis is capable of utilizing glutathione pre- sent in the cytosol of infected host cells. Cleavage of this antioxidant provides the essential source of cysteine required for intracellular multiplication of Francisella [17]. It was reported recently that the biochemical responses of various hosts may vary. There were marked differences in lipid metabolism in the course of tulare- mia in BALB/c mice and common voles. Hypertriglycer- idemia and hypercholesterolemia developed in mice, while physiologically higher levels of triglycerides and cholesterol showed a decreasing tendency in common voles (Microtus arvalis). On the other hand, the total plasma antioxidant capacity gradually dropped to 81.5% in mice, while it increased to 130% after the infection in common voles. Significant correlations between tissue bacterial burdens and several biochemical parameters were found [18]. Experimental models of tularemia employ laboratory mice, in particular [15,19-23], while European brown hares have only been used exceptionally [24-26] despite their importance as a source of human infections. It was therefore the aim of the present study to investigate bio- chemical and oxidative stress responses to experimental F. tularensis infection in European brown hares. For these purposes we evaluated the dynamics of biochem- ical parameters measured in blood plasma using both standard procedures of dry chemistry and electrochemi- cal devices. Materials and methods Experimental micro-organism A wild strain of Francisella tularensis isolated from a European brown hare specimen from South Moravia in 2004 was used for experimental infections in this study. The isolate was subtyped as Francisella tularensis subsp. holarctica via the proteomic procedure [27]. Experimen- tal infections were performed using a suspension of F. tularensis cells harvested from a culture growing on blood agar supplemented with L-cysteine using sterile physiological saline solution. After thorough mixing we measured the absorbance of the suspension at 605 nm using a spectrophotometer (Unicam Helios Gamma&- Delta, Spectronic Unicam, United Kingdom) in order to determine the number of bacterial cells per unit volume according to McFarland’s standard [28]. The number obtained was only approximate and was used to esti- mate the dilution necessary to achieve the dose required. The exact infectious dose was then determined Page 2 of 13 by plating ten-fold serial dilutions and counting colony- forming units (CFU) in the suspension administered to experimental animals. Colonies were counted after 72 h of incubation at 37 °C. Virulence of the F. tularensis strain was tested by inoculation of BALB/c mice. Experimental animals One-year-old European brown hares (Lepus europaeus) were purchased from the Hare Breeders’ Association of the Czech Republic and a total of ten males were used for the study. They were fed standard granules for rab- bits (without supplementation of anticoccidials) and high quality hay, and were provided with drinking water ad libitum. At the start of the experiment the hares appeared healthy, were in an excellent nutritional state, and were certified free of tularemia and brucellosis based on agglutination tests. Experimental design Experimental hares were allocated to the control and F. tularensis-inoculated groups (five specimens each) on a random basis. Biochemical responses, lipid peroxida- tion and levels of antioxidants were studied following subcutaneous infection of the inoculated group. Hares were inoculated into the dorsal trunk area with a single dose of 2.6 × 109 CFU pro toto. Blood for plasma chem- istry profiles was collected every other day from days 0 to 16, and on days 24 and 35. The data from infected hares were then compared against values obtained from control hares from days 0 to 35 of the experiment plus healthy hares sampled prior to inoculation (n = 60). Blood was collected from the jugular vein using a hepar- inized set Omnican® 40 (Braun, Germany). Samples of blood were centrifuged immediately after collection, and the plasma was removed and frozen (-80 °C). Surviving hares were killed on day 35 post-inoculation. Necropsy was performed in hares that died or were euthanized in order to determine gross pathological findings and to collect organs aseptically (liver, spleen, lung, bone mar- row and kidney). Tissue samples were also collected to 10% buffered formalin, treated using a routine histologi- cal technique and embedded in paraffin. Sections of 5μm were made of the paraffin blocks and stained with haematoxylin and eosin. Organ and blood samples were examined for the presence of F. tularensis by culture and the mouse inoculation test. Agglutination antibody titres were examined using a commercially available antigen (Bioveta a.s., Ivanovice na Hane, Czech Republic). Experiments were performed in compliance with laws for the protection of animals against cruelty and were approved by the Ethical Committee of the University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic.