8 ± 0.2 °C with a 12:12 h light/dark cycle. After 3 days acclimatization, the frogs were anaesthetized in MS222 (Sigma-Aldrich) before subgroups of three animals were injected intramuscularly (i.m.) in the flank and intraperitoneally (i.p.) with 0.2 mL volumes of the bacterial suspensions to

achieve 2.8 × 105, 2.8 × 106 Selleck SRT1720 and 2.8 × 107 CFU per frog amounts. Negative controls received a similar volume of 0.85% (w/v) physiological saline. The frogs were monitored daily for 14 days and mortalities subjected to bacteriological examination to confirm the presence of A. hydrophila. The survivors were sacrificed and also examined bacteriologically. The specimens were among a group of different snake species that died in the serpentarium at the zoological gardens in Sofia, and autopsies were performed at the National Veterinary Medical Institute, Sofia. The snakes were housed in separate enclosures within a single serpentarium, with the first incidence of disease noted in the boa. The deaths were attributed to a temperature irregularity leading to a sudden drop from the norm of c. 38–40 to c. 18–20 °C because of a broken temperature regulator. All three snakes examined here developed PXD101 order petechial haemorrhages in the mouth and gums, and haemorrhages occurred in the lung, spleen and intestines. The

abdomen and anus were swollen with bloody-tinged mucus in the colon. A total of 18 isolates were recovered from the internal organs of the dead snakes and were tentatively identified as A. hydrophila based on the key phenotypic characteristics and by the Micronaut automatic system. Thus, cultures comprised facultatively anaerobic motile Gram-negative rods that produced arginine dihydrolase, catalase, β-galactosidase oxidase, phosphodiesterase and phospholipase, produced acid from maltose, mannitol, mannose and sucrose, and degraded aesculin and chitin but not urea. These matched the overall phenotypic

characteristics of A. hydrophila (Martin Carnahan & Joseph, 2005). All isolates were β-haemolytic for sheep blood. The identity was confirmed by sequencing of the 16S rRNA gene. blast analysis provided an ID-8 identity value of 99% with A. hydrophila (Fig. 1). The phylogenetic tree constructed for the three strains, which were recovered from the heart of diseased snakes, confirmed the phylogenetic position of the snake isolates in the genus Aeromonas (Fig. 1). The nucleotide sequences of the three isolates were compared with each other, and it was determined that there were not any differences between the sequences. The 16S rRNA gene sequences have been deposited in NCBI GenBank under the accession numbers BankIt 1524667 A. hydrophila OSA1-11 JQ818547, BankIt 1524671 A. hydrophila OSB1-11 JQ818548 and BankIt 1524673 A. hydrophila OSG1-11 JQ818549.