Novel Spiroplasma Spp. Cultured From Brains and Lymph Nodes From Ruminants Affected With Transmissible Spongiform Encephalopathy.

Abstract

Spiroplasma spp., tiny filterable wall-less bacteria, are consistently associated with the transmissible spongiform encephalopathies (TSE). Spiral forms have been transiently isolated from TSE-affected brain tissues in SP4 growth media designed for isolation of Spiroplasma spp., but the isolate could not be propagated in SP4 media. A bacterium must grow in vitro in cell-free cultures to allow full characterization of a suspect pathogen. Here, a novel Spiroplasma sp. was isolated from scrapie- and chronic wasting disease (CWD)-affected brains and lymph nodes. Filtrates of tissue homogenates inoculated into Brucella media incubated for 14 days at 35 °C resulted in high titers of spiroplasma as shown by dark-field microscopy. A drop assay of infected media on Bacto Schaedler agar showed spiroplasma isolates forming unique subsurface colonies after 21 days incubation. Spiroplasma coils, coccoid forms and clumps of entwined spiroplasma filaments were seen on the agar by scanning electron microscopy. Since Brucella media has a sodium bisulfite additive that lowers oxygen tension, TSE spiroplasma growth requires media with low oxygen tension. Brucella media allows for isolation and propagation of spiroplasma from TSE-affected tissues, which will lead to complete characterization of this TSE pathogen and determine its role as a candidate causative agent of TSE.