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Journal of Medical Microbiology vol. 56, part 9, pp. 1235 - 1242
Supplementary Figs S1 - S5 and Supplementary Table S1 [PDF file] (139 KB)
Supplementary Fig. S1
Schematic of experimental design for study.
Supplementary Fig. S2
Spiroplasma-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 Spiroplasma-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 Spiroplasma 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 S. mirum 16S rDNA (GenBank accession no. M24662).
Supplementary Fig. S3
The ribosomal 16S rDNA sequence of Spiroplasma sp. isolated from scrapie-affected sheep brain is compared to that of S. mirum. 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 Spiroplasma scrapie isolate shown in Fig. 1. The other sequence displayed is that portion of the GenBank oligonucleotide sequence of S. mirum 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 Spiroplasma sp. 16S rDNA (A to T at position 729 and A to G at position 779) confirming that (1) a Spiroplasma had been isolated from scrapie-affected sheep brain via passage in embryonated eggs, and (2) that the Spiroplasma scrapie isolate is closely related to S. mirum. The nucleotide substitutions seen in the scrapie Spiroplasma sp. isolate 16S rDNA are identical to previously published 16S rDNA probes of the source scrapie-affected sheep brain (Bastian et al., 2004).
Supplementary Fig. S4
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.
Supplementary Fig. S5
Spiroplasma 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) Spiroplasma were seen by negative stain electron microscopy in cell-free broth derived from scrapie-affected sheep brain after passage through an embryonated egg. The Spiroplasma sp. isolate from the scrapie preparation was morphologically identical to Spiroplasma seen in other Spiroplasma isolate preparations derived from TSE-affected brains (see Fig. 3). Magnification x50 000. The striking finding in this study is that when Spiroplasma sp. depicted in (C) were inoculated into the goat, they induced spongiform encephalopathy typical of TSE lesions seen in this natural host.
Supplementary Table S1
Efficiency of Spiroplasma spp. isolation from TSE-affected brains via passage in embryonated eggs and absence from controls. Spiroplasma spp. were isolated from all TSE-affected brains (7/7) while no Spiroplasma were discovered in normal human (0/3) or ruminant brains (0/6) or in the M1D media used to culture the Spiroplasma. The development of the molecular probe for Spiroplasma 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 et al., 2004). Such exercises could translate into identification of different strains of Spiroplasma involved in TSE and the ability to link TSE cases for epidemiological purposes.
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