Title : Live test for chronic wasting disease based on consistent association with an extreme thermoacidphilic bacterium
Abstract:
The problem with controlling chronic wasting disease (CWD) infection in ruminants is there is no reliable live test for this devastating fatal encephalopathy. Our strategy is to use a disease biomarker to develop a live test for CWD. The most recognized biomarker of CWD and other transmissible spongiform encephalopathies (TSE) is prion amyloid that accumulates in brain and spinal cord during the course of the disease. However, attempts developing a live test for CWD based upon detection of the prion amyloid protein has been troublesome since the quaking test for identification of prion biomarker has proved to be less than satisfactory as the test is only 50% accurate. As used in the diagnosis of CJD in humans, the test shows numerous false positives. A problem with this approach is the prion amyloid is absent in 5% of Creutzfeldt-Jakob disease (CJD) cases, and the new variant of scrapie in sheep does not produce prion amyloid. The concept of the quaking test does not fit with the prion theory since severe shaking of the unknown brain sample in the presence of normal prion isoform may lead to increased prion amyloid, likely by selfassembly, while there is no increase in infectivity. Nonetheless, much of the monies appropriated by the USDA go to efforts of improving the quaking test rather than trying to develop alternate methodology. Since the protein sequence of the normal prion protein isoform on the cell surface is identical to that of the disease-associated prion amyloid, a credible diagnostic test based on this strategy will likely not be practical. A second biomarker is the scrapie-associated fibril (SAF) that are present in 100% of
TSE cases, even in absence of the prion amyloid. However, the use of this biomarker is impractical since SAF is identified by negative stained electron microscopy in synaptosomal ultracentrifuge fractions. Prion researchers have declared SAF to be the morphological marker of prion amyloid, but this is total conjecture. It is noteworthy that SAF are identical to fibril proteins within a third bacterial biomarker of TSE infection and show immune cross-reactivity. Our laboratory has found the consistent association of a wall-less bacterium with CWD and other TSEs, documented by morphological and molecular studies. In fact, we are able to isolate this novel spiroplasma bacterium from 100% of CWD-affected tissues. The biological properties of this isolate are identical to those of the transmissible TSE agent including surviving boiling for one-hour, massive doses of gamma irradiation, formalin exposure for 18 hours, standard autoclaving and hyperacidity (pH 2). This novel bacterial isolate grows in cell free media and forms subsurface colonies on special agar plates prepared with Brucella media. These extreme thermoacidophiles represent a third biomarker of CWD infection and provide the best opportunity to develop a live test since they are foreign to the animals. Our strategy is to identify a protein epitope on the bacterial surface that can be used as a diagnostic serological test. That protein may also provide immune protection as a vaccine against CWD and other TSEs.
