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Abstract

Spiroplasma, small motile wall-less bacteria, are linked by molecular and serological studies to the transmissible spongiform encephalopathies (TSEs), which include scrapie in sheep, chronic wasting disease (CWD) in deer and Creutzfeldt–Jakob disease in humans. In this study, two experiments were undertaken to determine the role of spiroplasma in the pathogenesis of TSE. In experiment 1, , a rabbit tick isolate that had previously been shown to experimentally induce spongiform encephalopathy in rodents, was inoculated intracranially (IC) into ruminants. -inoculated deer manifested clinical signs of TSE after 1.5 to 5.5 months incubation. The deer, as well as sheep and goats, inoculated with developed spongiform encephalopathy in a dose-dependent manner. In experiment 2, spiroplasma closely related to were isolated from TSE-affected brains via passage in embryonated eggs, and propagated in cell-free M1D media. spp. isolates from scrapie-affected sheep brain and from CWD-affected deer brain inoculated IC into sheep and goats induced spongiform encephalopathy closely resembling natural TSE in these animals. These data show spiroplasma to be consistently associated with TSE, and able experimentally to cause TSE in ruminant animal models, therein questioning the validity of studies that have concluded the prion, a miss-folded protease-resistant protein that builds up in TSE brains during the course of the disease, to be the sole causal agent. The spiroplasma infection models reported here will be important for investigating factors involved in the pathogenesis of TSE since ruminants are the natural hosts.

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2007-09-01
2019-11-22
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Schematic of experimental design for study.

-specific 16S rDNA was identified in cell-free broth containing isolate derived from scrapie-affected sheep brain passaged through embryonated eggs. The PCR probe was carried out on DNA extracted from a pellet of the cell-free culture using -specific primer sets for 16S rDNA (2). Lane 1, markers separated by 100 bp increments. Lane 2, water control negative for DNA template, lane 3, 270 bp PCR product amplified from DNA extracted from egg-passaged scrapie isolate. Later extraction of DNA from the 270 bp PCR amplified product followed by DNA sequence analysis revealed the source to be closely related to 16S rDNA (GenBank accession no. M24662).

The ribosomal 16S rDNA sequence of sp. isolated from scrapie-affected sheep brain is compared to that of . One of the sequences shown was derived from DNA sequence analysis of the 270 bp PCR amplified product of the 16S rDNA probe of the scrapie isolate shown in Fig. 1. The other sequence displayed is that portion of the GenBank oligonucleotide sequence of 16S rDNA gene (GenBank accession no. M24662) that would be amplified by the probe. Comparison of the sequences revealed 99% homology but with rare nucleotide substitutions in the sp. 16S rDNA (A to T at position 729 and A to G at position 779) confirming that (1) a had been isolated from scrapie-affected sheep brain via passage in embryonated eggs, and (2) that the scrapie isolate is closely related to . The nucleotide substitutions seen in the scrapie sp. isolate 16S rDNA are identical to previously published 16S rDNA probes of the source scrapie-affected sheep brain (Bastian , 2004).

SMCA inoculated IC into deer 1 showed spongiform encephalopathy. (A) Cerebral cortex from deer 1 shows vacuolization of neuropil with prominent perineuronal distribution of the vacuoles (indicated by arrows). Note prominence of vessels and microglial infiltrate in the cerebral cortex sections. (B) Cerebellar cortex from deer 1 shows peripheral vacuolization of Purkinje neurons with bulbous dilatation of their dendritic processes seen in the molecular layer (indicated by an arrow). (C) Higher magnification of cerebellar cortex from deer 1 showed marked dilatation of a Purkinje neuron dendritic process (indicated by arrows) and marked peripheral cytoplasmic vacuolization in Purkinje cell neurons. Dendritic swellings and loss of synapses are seen in experimental scrapie in mice (Bastian, 1991). The cerebellar cortex from deer 1 shows some of the early histological changes characteristic of experimental SMCA infection. These lesions progressed to the more severe spongiform changes seen in the cerebellar cortex of deer 2 examined after two additional months incubation (see Fig. 1c). Magnification ×400.

sp. isolated from scrapie-affected sheep brain via passage in embryonated eggs when inoculated IC into goats induced spongiform encephalopathy. (A) The goat cerebral cortex shows perineuronal and intracytoplasmic vacuolization characteristic of TSE. Magnification x400. (B) The goat brain hippocampus shows vacuolization of the neuropil involving both dentate and pyramidal neurons. There is no evidence of hypoxia or autolysis. Magnification x50. (C) were seen by negative stain electron microscopy in cell-free broth derived from scrapie-affected sheep brain after passage through an embryonated egg. The sp. isolate from the scrapie preparation was morphologically identical to seen in other isolate preparations derived from TSE-affected brains (see Fig. 3). Magnification x50 000. The striking finding in this study is that when sp. depicted in (C) were inoculated into the goat, they induced spongiform encephalopathy typical of TSE lesions seen in this natural host.

Efficiency of spp. isolation from TSE-affected brains via passage in embryonated eggs and absence from controls. spp. were isolated from all TSE-affected brains (7/7) while no were discovered in normal human (0/3) or ruminant brains (0/6) or in the M1D media used to culture the . The development of the molecular probe for displayed in Supplementary Figs S2 and S3 has allowed confirmation of the nature of the bacterial isolates from TSE-affected brain tissues. Further use of this methodology will allow documentation of nucleotide variation in the ribosomal 16S rDNA in cell-free isolates from individual TSE cases as suggested from probes of TSE-affected brain tissues (Bastian , 2004). Such exercises could translate into identification of different strains of involved in TSE and the ability to link TSE cases for epidemiological purposes.



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