Absence of Spiroplasma or Other Bacterial 16S rRNA Genes in Brain Tissue of Hamsters with Scrapie

Abstract

Spiroplasma spp. have been proposed to be the etiological agents of the transmissible spongiform encephalopathies (TSEs). In a blind study, a panel of 20 DNA samples was prepared from the brains of uninfected hamsters or hamsters infected with the 263K strain of scrapie. The brains of the infected hamsters contained >10 10 infectious doses/g. The coded panel was searched for bacterial 16S rRNA gene sequences, using primers selective for spiroplasma sequences, primers selective for mollicutes in general, and universal bacterial primers. After 35 PCR cycles, no samples were positive for spiroplasma or any other bacterial DNA, while control Spiroplasma mirum genomic DNA, spiked at 1% of the concentration required to account for the scrapie infectivity present, was readily detected. After 70 PCR cycles, nearly all samples yielded amplified products which were homologous to various bacterial 16S rRNA gene sequences, including those of frequent environmental contaminants. These sequences were seen in uninfected as well as infected samples. Because the concentration of scrapie infectivity was at a known high level, it is very unlikely that a bacterial infection at the same concentration could have escaped detection. We conclude that the infectious agent responsible for TSE disease cannot be a spiroplasma or any other eubacterial species.The identity of the causal agents of the transmissible spongiform encephalopathies (TSEs)-neurodegenerative diseases which include scrapie in sheep, Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer and elk-has been a major source of controversy since these diseases were shown to be transmissible (41,43,17,38). Early experiments showing that TSE infectivity passed through bacterial filters (54) and was resistant to disinfection by common bacteriocides (27,10,41,42,9) seemingly eliminated bacteria as the agent. However, the discovery in the 1970s of spiroplasmas, very small, thermostable, wall-less, helical-fibrillar bacteria that pass through 0.2-m bacterial filters and show remarkable resistance to many common biocides including heat (47), provided a possible bacterial candidate for the infectious agent of TSEs. There have also been reports of spiral structures resembling spiroplasmas in brain tissue from CJD patients (2, 23, 39, 33), although other investigators have suggested these structures might be artifactual (24, 28). Moreover, no spiroplasmas could be cultured from CJD brain tissue (32). Intracranial inoculation of Spiroplasma mirum into new-born hamsters (31), suckling rats (5), or suckling mice (16) produces central nervous system disease, though not a progressive spongiform encephalopathy (16).More recently, Bastian et al. (3,4) have reported the presence of spiroplasma-specific 16S rRNA genes in brain tissue taken at autopsy/necropsy from TSE-infected humans and animals. PCR amplification of 16S rRNA genes, or "ribotyping," is a powerful method for detecting and identifying microbial agents in environm...

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