, 1987). Also, a disease characterized Everolimus in vitro by high mortality appeared among snakes kept in a serpentarium, and A. hydrophila was identified as the causal agent (Esterabadi et al., 1973). During 2010, a sudden mortality attributed to heat stress occurred in snakes held in the zoological gardens in Sofia, Bulgaria. This study sought to characterize the causal agent of this disease outbreak. Three newly dead snakes, that is, a Jamaican

boa (Epicrates subflavus) of 1.0 kg in weight, a yellow anaconda (Eunectes notaeus) of > 7 kg in weight and a corn snake (Pantherophis guttatus guttatus) of 1 kg in weight, were obtained within 2 h of death in 2010 from the serpentarium at the zoological gardens in Sofia, Bulgaria. Thus, the spleen, intestine, lung, kidney, liver and heart were swabbed and the material inoculated onto triplicate plates of tryptone soy agar (TSA; Merck, Sofia, Bulgaria), 5% (v/v) sheep blood agar, Endo (Merck) and MacConkey agar (Merck) with incubation at 25 and 37 °C for up to 72 h. Colonies from plates with dense pure culture growth were purified by streaking and re-streaking on fresh media, and

identified after Whitman & MacNair (2004) and Austin & Austin (2007) and with Micronaut kits (Merlin Diagnostica; Bornheim-Hersel, Germany) – Plate NF (REF E2-520-120) and Plate selleckchem E (REF E2-510-400) and with the API 50CHE system (BioMérieux, Basingstoke, UK) according to the manufacturer’s instructions. Isolates were inoculated into 10 mL volumes of brain heart

infusion broth (BHI; Oxoid, Basingstoke, UK) and incubated overnight aerobically at 25 °C, with shaking http://www.selleck.co.jp/products/Abiraterone.html at 100 r.p.m. Genomic DNA was extracted using the High Pure PCR Cleanup Micro Kit (Geneshun Biotech, Guangzhou, China) and used as the template for PCR. The 16S rRNA gene was amplified by PCR using universal primers forward (27f) 5′-AGAGTTTGATMTGGCTCAG-3′ and reverse (1492r) 5′-CGGYTACCTTGTTACGACTT-3′. The procedure used for the isolation and purification of genomic DNA from the samples involved a commercial kit bacteria genomic DNA Fast Mini Kit (Geneshun Biotech, Guangzhou, China) and agarose gel DNA Extraction Kit (Geneshun Biotech), following the manufacturers instructions. The specific region of 16S RNA was amplified by means of PCR, using the primers listed earlier. The reaction was conducted in 25 μL volumes, using USB MasterMix (USB Corporation, Cleveland, OH). The following procedure was accomplished via Thermocycler QB – 96 (Pharmacia LKB, Saint Julie, QC, Canada): denaturation at 95 °C for 5 min, followed by 30 cycles at 95 °C for 1 min., 56 °C for 1 min. and 72 °C for 2 min, with a final extension step of 72 °C for 10 min. After purification of the PCR products with a Gel DNA purification kit (GE Healthcare, Litle Chalfont, UK), the sequencing PCRs by a Thermocycler QB – 96 were applied.