Abstract
Brucellosis is a zoonosis found globally and is the result of infection by members of the genus Brucella. Twelve species of Brucella have been formally described, with the more recently described species significantly expanding the range of mammalian hosts. Furthermore, a growing number of phenotypically and genetically atypical strains have been reported from non-mammalian hosts, most notably amphibians. Reports of human infection with atypical Brucella sp. strains raise questions regarding the incidence and impact of emerging species. Initial bacterial identifications from the post mortem of three captive Amazon milk frogs (Trachycephalus resinifictrix) indicated Ochrobactrum anthropi or unspecified intralesional Gram negative bacteria. The tissues were sent to the UK Reference Laboratory for Brucellosis. The excised tissues were macerated prior to the inoculation of solid and liquid media. Supportive and selective (Farrell’s) solid media and Brodie and Sinton broths were used for the isolation and propagation of potential colonies. The growth characteristics and morphology of target isolates from all three amphibians resembled members of the atypical Brucella group (e.g. B. inopinata). The isolates grew readily within 24 hours of inoculation at 37°C with 5-10% CO2 on all media types. However, traditional methods of classical biotyping to characterise members of the Brucella genus did not identify a specific known species. Positive results of PCR targeting Brucella specific genes indicated that these isolates represent members of the emerging atypical Brucella group. Further analysis of the isolates was performed using whole genome sequencing and multi-locus sequence typing approaches, to characterise the isolates relative to other members of the atypical group. The primary isolation and characterisation of these isolates provides the opportunity to evaluate the potential clinical and diagnostic significance of emerging Brucella species. Accelerated growth characteristics coupled with negative monospecific sera reactions, used to identify the A and M epitopes of the O-polysaccharide, suggests that infection with these isolates may go undetected through the use of current serological and bacterial identification methods. These isolations emphasise the need for innovation in routine approaches to aid in the detection and identification of Brucella.
