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Prion Diseases pp 159-172 | Cite as

Methods for Isolation of Spiroplasma sp. from Prion-Positive Eye Tissues of Sheep Affected with Terminal Scrapie

  • Frank O. BastianEmail author
Protocol
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Part of the Neuromethods book series (NM, volume 129)

Abstract

Spiroplasma, a tiny wall-less bacterium, has been consistently found in tissues affected with transmissible spongiform encephalopathy (TSE), and a closely related spiroplasma laboratory strain isolated from rabbit ticks experimentally induces TSE-like clinical and pathological findings in rodents and ruminants. The consistent presence of a Spiroplasma sp. in naturally occurring TSE has been documented by morphological and molecular studies, and the direct isolation of these spiroplasma from TSE-affected tissues into cell-free media confirms our hypothesis that spiroplasma is a candidate causal agent of TSE. In this treatise, we show evidence of isolation of Spiroplasma sp. from scrapie-affected eyes and note that the isolate is immunologically distinct from other spiroplasma species. It is noteworthy that experimental inoculation of these scrapie spiroplasma isolates intracranially (IC) into sheep and goats induced the classic vacuolar neuropathology of naturally occurring scrapie in the obices of these ruminants. The growth characteristics of this isolate differ from other spiroplasma species in that the scrapie spiroplasma isolate is extremely fastidious and cannot be propagated beyond a few passages in the special medias designed for spiroplasma growth. Herein we describe the protocols that we have used in our laboratory for isolation of the scrapie-related spiroplasma from scrapie-affected eyes. The tissues are homogenized in special media with high osmolality especially designed for spiroplasma growth; after low speed centrifugation, the supernatant is passed through a 0.45 μm membrane filter and overlaid onto a confluent monolayer of mosquito larvae cells (Aedes albopictus clone C6/36). The presence of spiroplasma on the surface of mosquito cells is documented by scanning electron microscopy (SEM) and can be shown as well by dark field microscopy and transmission electron microscopy (TEM). There is minimal cytopathogenic effect (CPE) with some vacuolization of mosquito cells, apoptosis, and multi-nucleation as seen by phase microscopy. The methodology is applicable for use in mammalian cell culture lines, such as bovine corneal endothelial (BCE) cells and mouse neuroblastoma (Neuro-2a) cells, the latter commonly used in prion research. In our hands this methodology has been successful in recovering spiroplasma from the TSE tissue >80% of the time based upon recognition of spirals by dark field microscopy while not from control tissues. These procedures along with future efforts to optimize the specialized growth medias should allow characterization of the scrapie spiroplasma isolates and determine the interrelationship with the prion.

Key words

Spiroplasma TSE Prion Scrapie CJD CWD BSE TME 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Animal ScienceLouisiana State University Agricultural CenterBaton RougeUSA

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