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

Area of Science:

• Microbiology
• Veterinary Pathology
• Neuroscience

Background:

• Spiroplasma spp., wall-less bacteria, are consistently associated with transmissible spongiform encephalopathies (TSEs).
• Previous attempts to isolate Spiroplasma from TSE-affected tissues in SP4 media were unsuccessful for propagation.
• In vitro cell-free culture is essential for characterizing potential bacterial pathogens.

Purpose of the Study:

• To isolate and propagate a novel Spiroplasma species from TSE-affected brains and lymph nodes.
• To establish a reproducible method for culturing Spiroplasma from scrapie and chronic wasting disease (CWD) tissues.
• To facilitate the full characterization of this Spiroplasma isolate as a potential causative agent of TSE.

Main Methods:

• Tissue homogenates from TSE-affected brains and lymph nodes were filtered and inoculated into Brucella media.
• Cultures were incubated at 35°C for 14 days, and bacterial presence was assessed via dark-field microscopy.
• A drop assay on Bacto Schaedler agar and scanning electron microscopy were used for colony and morphology analysis.

Main Results:

• High titers of Spiroplasma were successfully isolated and propagated in Brucella media.
• Unique subsurface colonies were observed on agar plates after 21 days of incubation.
• Scanning electron microscopy revealed various Spiroplasma forms, including coils, coccoid shapes, and filament clumps.

Conclusions:

• Brucella media, with its low oxygen tension due to sodium bisulfite, supports the growth of TSE-associated Spiroplasma.
• This novel isolation and propagation method allows for the complete characterization of the Spiroplasma species.
• The findings establish Spiroplasma as a candidate causative agent for TSEs, warranting further investigation.