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Novel Spiroplasma Spp. Cultured From Brains and Lymph Nodes From Ruminants Affected With Transmissible Spongiform
Frank O Bastian1, James Lynch1, Sue Hagius1
1Department of Animal Sciences, Louisiana State University Agricultural Center; Department of Comparative Biomedical Sciences, Department of Pathobiology and National Hansen's Disease Laboratory, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana.
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.
Insights
Researchers successfully isolated and propagated a novel Spiroplasma species from transmissible spongiform encephalopathy (TSE) affected tissues. This breakthrough in culturing Spiroplasma spp. from TSEs paves the way for pathogen characterization.
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.
