Lewerin et al. Acta Veterinaria Scandinavica 2010, 52:7 http://www.actavetscand.com/content/52/1/7 was done on the genetic material from all three animal isolates with some modifications compared to Lista [17]. Briefly, singleplex PCR reactions were performed as fol- lows; 10 ng DNA were amplified in a final volume of 25 μl containing 1xBuffer for DyNAzyme DNA polymerase (Finnzymes, Espoo, Finland), dNTP 0,15 mM (Finn- zymes, Espoo, Finland), DyNAzyme DNA polymerase II 0,6 U (Finnzymes, Espoo, Finland), forward primer 0,4 μM and reverse primer 0.4 μM. The thermal cycling conditions were initial step, 96°C, 3 min for polymerase activation; PCR (40 cycles), 95°C, 20 s for denaturation, 60°C, 30 s for annealing and 65°C, 2 min for extension. The reactions were terminated by a final incubation at 65°C for 5 min. After diluting the PCR products 1/5, 1 μl was added to 40 μl of Sample Loading Solution (Beckman-Coulter, Fullerton, California, USA) containing 0.32 μl MapMar- ker 1000 (Bioventures, Inc., Murfreesboro, Tennessee, USA). The samples were separated on a CEQ 8800 automatic DNA Analysis System (Beckman-Coulter, Fullerton, California, USA) with the following condi- tions: denaturation 90°C for 120 s, inject 2.0 kV for 30 s, separation 6.0 kV for 60 min. The MLVA profiles were compared, using a web- based tool, to a large global MLVA database (http:// minisatellites.u-psud.fr/MLVAnet/) containing typing data from B. anthracis strains. Antimicrobial susceptibility testing Susceptibility to antimicrobials was tested following the standards for microdilution of the Clinical and Labora- tory Standards Institute [18,19]. Minimum inhibitory concentration (MIC) was recorded as the lowest concen- tration of the antimicrobial that inhibits bacterial growth. The antimicrobials tested were: ampicillin (representative for penicillin), ciprofloxacin, gentamicin, streptomycin and tetracycline, based on the EMEA/ CPMP guidelines [20]. Results of investigations Macroscopic examination (in the local veterinary clinic) of the organs of animal 5 No specific findings were observed, apart from bleeding in an area of the inner wall of the left chamber and atrium of the heart, some bleeding in the lungs and fat deposition in one liver lobule. The blood appeared nor- mal in colour and coagulation. Histology and culture (at SVA) on organs from animal 7 No specific histological lesions were seen, only haemor- rhages in examined organs. Routine culture from the lung revealed no bacterial growth. Bacteriological examinations (at SVA) of spleen swab from animal 2 Routine culture from the swab revealed a mixed flora with no specific growth. PCR on the swab, performed later, was positive for pXO1, pXO2 and the Page 5 of 8 chromosomal markers. The Ct values were in the range of 21-23 for all three targets. Necropsy findings (in the regional laboratory) in animal 8 The carcass appeared normal before opening, with no extensive bleeding form orifices. The necropsy revealed massive internal bleeding with non-coagulated blood in almost every organ. Typical signs were seen such as petechia in mucuous membranes, connective tissue oedema and a large and friable spleen with a dark cut surface reminiscent of blackberry jam. Severe subsero- sal bleedings were noticed on the diaphragm, as well as subpleural bleedings on the lung surface. The blood was non-coagulated and dark. The content of the jeju- num was watery and blood-stained. On the ventral side of the neck there was a large haemorrhagic oedema. Bacteriological examination (at SVA) of blood from animals 8, 9 and 10 Direct smears of blood showed numerous bacillus- shaped rods and sparse occurrence of other bacteria. However, the presence of capsule could not be demon- strated. Cultures from all three animals showed heavy growth of B. anthracis mixed with contaminating flora. The colonies were typical for B. anthracis; grey, non- haemolytic, with a ground-glass moist surface. Micro- scopy revealed spore forming rods, and a capsule could be visualised after culture for 5 h in horse serum and staining with polychrome methylene blue.The real-time PCR assay was positive for B. anthracis since all three genes were detected. The Ct values from animal 8, 9 and 10 were in the range of 11-18 for all three targets. The Ct values indicated a high concentration of B. anthracis cells in the blood. DNA was sent to the Cen- tre for Microbiological Preparedness at SMI for a sec- ond real-time PCR confirmation and the result showed positive results for B. anthracis DNA. According to the MIC interpretive standard from CLSI for potential agents of bioterrorism the isolates were found to be susceptible to ciprofloxacin and tet- racycline with MIC-values of 0.12 μg/ml and 0.25 μg/ ml, respectively [19]. The MIC-value for ampicillin was 0.25 μg/ml, indicating that the anthrax strain was sus- ceptible using the MIC interpretive standard for peni- cillin. The MIC-value for gentamicin was 0.25 μg/ml and for streptomycin 2 μg/ml. For these two antimi- crobials there is no data available for interpretation of susceptibility. Bacteriological examination of environmental samples None of the dust samples were positive. Two out of three soil samples taken practically on the same spot (where animals 11 and 12 had been lying) were positive for both the cap and the lef gene, while the third sample was negative. Two other soil samples taken on another spot were both negative.