Lewerin et al. Acta Veterinaria Scandinavica 2010, 52:7 http://www.actavetscand.com/content/52/1/7 transmission, and subsequently culled. This was due to the practical difficulties in keeping them on the farm or transporting them elsewhere during the cleanup work on the holding. The animal holding, the laboratory per- forming the necropsies and the rendering plant that had received the carcasses from animals 1-7, and also received the remaining culled animals, were thoroughly cleaned and disinfected. The carcasses from animals 8- 10, plus two more animals (animals 11 and 12) that died on the farm after the diagnosis had been confirmed, were incinerated at SVA. Laboratory investigations All laboratory investigations except for the soil analyses and MLVA typing were performed at SVA. All samples analysed before the suspicion of anthrax arose were handled by routine procedures. Necropsy and histology were performed according to standard procedures. Rou- tine culture was made on blood agar plates incubated at 37°C for 24 h in aerobic conditions. The blood samples from the three suspect cases (ani- mals 8, 9 and 10) were investigated by microscopy, cul- ture, and PCR. The specimens were not entirely fresh, since the blood samples arrived to the laboratory > 24 h after the death of the animals. Smears of blood were dried, fixed and stained with polychrome methylene blue. Methylene blue solution was prepared as follows: 0.5 g of methylene blue was dissolved in 25 g of 96% ethanol; 0.01% NaOH was mixed with the methylene blue solution to a final volume of 100 ml. This was left to stand exposed to the air, with occasional shaking, for at least 1 year to oxidize and mature ("old methylene blue”). Smears were exam- ined with respect to bacterial morphology and presence of capsule. In order to demonstrate growth of B. anthracis the samples were also spread on agar (Oxoid, Cambridge, UK) supplemented with 5% horse blood as well as agar with 1.6% bromcresolepurpur (Merck, Darmstadt, Ger- many) and 20% lactose (Merck, Darmstadt, Germany) and incubated aerobically in 37°C overnight. PCR PCR on the spleen swab, blood and dust was performed at SVA. DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) was used for DNA extraction with a slight modification of the manufacturer’s protocol for isolation of DNA from gram-positive bacteria. Dust samples were cultured before extraction. Approximately 2 g of sample was added to 18 ml Luria-Bertani (LB) broth and heated at 70°C for 30 min. Dilution of the sample was done by transferring 1 ml to 10 ml LB broth. Both broths were incubated at 37°C over night. From the diluted sample 1 ml was centrifuged at 6000 × g for 2 min. The pellet was resuspended in 180 μl from the undiluted culture Page 4 of 8 and the suspension was extracted as described above. Three real-time PCR assays were used to detect B. anthracis DNA. The SYBR Green based assays target three genes; i.e. the rpoB gene on the chromosome and the virulence genes lef and cap located on the pXO1 and the pXO2 plasmids. Primers targeting cap (primers iQBa2F; 5’-CTTAAATCACTTTTGCTTGCTTTTTG and iQBa2R; 5’-TGCAGCTGAGCCATTAATCG), lef (iQBa3F; 5’-AAGAAGGATATGAACCCGTACTTGTAA and iQBa3R; 5’-AAACGTTCAGTGCCTTTTCAG- TATT) and rpoB (iQBa4F; 5’-GAAGGACGATACAGA- CATTTATTGG and iQBa4R; 5’-ACCGCAAGTTGAAT AGCAAG) for B. anthracis were used. PCR reactions were performed in a final volume of 25 μl containing 5 μl DNA template, 2 × PowerSYBR® Green PCR master mix (Applied Biosystems, Foster City, USA), forward primer 0.4 μM and reverse primer 0.4 μM and 0.2 mg/ ml BSA (Sigma, Saint Louis, USA). Temperature cycling conditions were as follows: 10 min denaturation at 95°C; 40 cycles of 95°C for 15 s, 60°C for 60 s and melting curve 95-60°C. Genetic analyses of nucleic acid extracted from soil Nucleic acid was extracted from five soil samples. Three of them were taken from a spot where carcasses of a cow and a calf (animals 11 and 12) had been left lying, and the remaining two soil samples were taken in a pad- dock next to the buildings where the animals were housed. SoilMaster DNA Extraction Kit (Epicentre Bio- technologies, Madison, Wisconsin, USA) was used for DNA extraction, following the manufacturer’s protocol. PCR analyses were done in triplicates from the extracted nucleic acid material. Primers targeting cap (primers iQBa2F and iQBa2R) and lef (primers iQBa3F and iQBa3R) were used. All soil samples were spiked with rat-DNA as a positive internal control of the DNA extraction efficiency and detected using the primers; iQFPrat36B4; 5’-GCCCAGAGGTGCTGGACAT and iQRPrat36B4; 5’-ATTGCGGACACCCTCTAGGA. PCR reactions were performed as follows; total DNA from soil was amplified in a final volume of 20 μl containing 2 × Fast Cycling SYBR® Green qPCR reaction mix (Quanta Biosciences, Gaithersburg, Maryland, USA), for- ward primer 0,4 μM and reverse primer 0,4 μM. Tem- perature cycling conditions were as follows: 10 min denaturation at 95°C; 40 cycles of 95°C for 15 s, 60°C for 60 s and melting curve 95-60°C. MLVA (Multi Locus Variable tandem repeats Analysis) typing of the three animal strains B. anthracis DNA from three isolates from animals 8, 9 and 10, respectively, were prepared as described above for blood. A MLVA using 16 markers, viz. vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3, BAMS1, BAMS3, BAMS5, BAMS13, BAMS21, BAMS25, BAMS34, BAMS44, BAMS51, and BAMS53, previously reported [15-17],