PrP(Sc) detection and infectivity in semen from scrapie-infected sheep.

A scrapie-positive ewe was found in a flock that had been scrapie-free for 13 years, but housed adjacent to scrapie-positive animals, separated by a wire fence. Live animal testing of the entire flock of 24 animals revealed seven more subclinical scrapie-positive ewes. We hypothesized that they may have contracted the disease from scrapie-positive rams used for breeding 4 months prior, possibly through the semen. The genotypes of the ewe flock were highly scrapie-susceptible and the rams were infected with the 'Caine' scrapie strain having a short incubation time of 4.3-14.6 months in sheep with 136/171 VQ/VQ and AQ/VQ genotypes. PrP(Sc) accumulates in a variety of tissues in addition to the central nervous system. Although transmission of prion diseases, or transmissible spongiform encephalopathies, has been achieved via peripheral organ or tissue homogenates as well as by blood transfusion, neither infectivity nor PrP(Sc) have been found in semen from scrapie-infected animals. Using serial protein misfolding cyclic amplification followed by a surround optical fibre immunoassay, we demonstrate that semen from rams infected with a short-incubation-time scrapie strain contains prion disease-associated-seeding activity that generated PrP(Sc) in sPMCA (serial protein misfolding cyclic amplification). Injection of the ovinized transgenic mouse line TgSShpPrP with semen from scrapie-infected sheep resulted in PrP(Sc)-seeding activity in clinical and, probably as a result of the low titre, non-clinical mouse brain. These results suggest that the transmissible agent, or at least the seeding activity, for sheep scrapie is present in semen. This may be a strain-specific phenomenon.

Prion disease detection, PMCA kinetics, and IgG in urine from sheep naturally/experimentally infected with scrapie and deer with preclinical/clinical chronic wasting disease.

Prion diseases, also known as transmissible spongiform encephalopathies, are fatal neurodegenerative disorders. Low levels of infectious agent and limited, infrequent success of disease transmissibility and PrP(Sc) detection have been reported with urine from experimentally infected clinical cervids and rodents. We report the detection of prion disease-associated seeding activity (PASA) in urine from naturally and orally infected sheep with clinical scrapie agent and orally infected preclinical and infected white-tailed deer with clinical chronic wasting disease (CWD). This is the first report on PASA detection of PrP(Sc) from the urine of naturally or preclinical prion-diseased ovine or cervids. Detection was achieved by using the surround optical fiber immunoassay (SOFIA) to measure the products of limited serial protein misfolding cyclic amplification (sPMCA). Conversion of PrP(C) to PrP(Sc) was not influenced by the presence of poly(A) during sPMCA or by the homogeneity of the PrP genotypes between the PrP(C) source and urine donor animals. Analysis of the sPMCA-SOFIA data resembled a linear, rather than an exponential, course. Compared to uninfected animals, there was a 2- to 4-log increase of proteinase K-sensitive, light chain immunoglobulin G (IgG) fragments in scrapie-infected sheep but not in infected CWD-infected deer. The higher-than-normal range of IgG levels found in the naturally and experimentally infected clinical scrapie-infected sheep were independent of their genotypes. Although analysis of urine samples throughout the course of infection would be necessary to determine the usefulness of altered IgG levels as a disease biomarker, detection of PrP(Sc) from PASA in urine points to its potential value for antemortem diagnosis of prion diseases.

Spiroplasma found in the eyes of scrapie affected sheep.

OBJECTIVE: Scrapie, a transmissible spongiform encephalopathy (TSE) occurring naturally in sheep, characteristically shows a severe retinopathy that is well developed in the terminal phases of the disease. In this study, we set out to demonstrate similar retinal changes in our ruminant spiroplasmosis TSE model. PROCEDURE: The eyes from deer, sheep, and goats that were inoculated intracranially with the laboratory strain of spiroplasma (suckling mouse cataract [SMCA] strain of Spiroplasma mirum) or with Spiroplasma sp. isolated from the brains affected with scrapie or with chronic wasting disease were examined by light microscopy for pathologic changes and by immunocytochemistry for distribution of spiroplasma antigen. The eyes were also obtained from a research flock of sheep with terminal scrapie, from which the intraocular tissues were submitted aseptically for culture assay in M1D broth or as explants on bovine corneal endothelia (BCE). RESULTS: The eyes from the spiroplasmosis ruminant models showed retinopathy remarkably similar to eye lesions seen in sheep with scrapie. The spiroplasma antigen accrued in the ruminant model eye tissues, particularly in the retina, the vitreous humor, and the corneal endothelia. A Spiroplasma sp. grew out of the scrapie-affected eyes both in the M1D broth and in the BCE cultures but did not expand. These new spiroplasma isolates differed immunologically from SMCA. CONCLUSION: These data showed a clear association of spiroplasma with scrapie suggesting that these bacteria have a role in the pathogenesis of TSE and that the eye should be a research focus for future studies of TSE.

A novel method for preclinical detection of PrPSc in blood.

In this study, we demonstrate that a moderate amount of protein misfolding cyclic amplification (PMCA) coupled to a novel surround optical fibre immunoassay (SOFIA) detection scheme can be used to detect the disease-associated form of the prion protein (PrP(Sc)) in protease-untreated plasma from preclinical and clinical scrapie sheep, and white-tailed deer with chronic wasting disease, following natural and experimental infection. PrP(Sc), resulting from a conformational change of the normal (cellular) form of prion protein (PrP(C)), is considered central to neuropathogenesis and serves as the only reliable molecular marker for prion disease diagnosis. While the highest levels of PrP(Sc) are present in the central nervous system, the development of a reasonable diagnostic assay requires the use of body fluids that characteristically contain exceedingly low levels of PrP(Sc). PrP(Sc) has been detected in the blood of sick animals by means of PMCA technology. However, repeated cycling over several days, which is necessary for PMCA of blood material, has been reported to result in decreased specificity (false positives). To generate an assay for PrP(Sc) in blood that is both highly sensitive and specific, we have utilized limited serial PMCA (sPMCA) with SOFIA. We did not find any enhancement of sPMCA with the addition of polyadenylic acid nor was it necessary to match the genotypes of the PrP(C) and PrP(Sc) sources for efficient amplification.

Surround optical fiber immunoassay (SOFIA): an ultra-sensitive assay for prion protein detection.

We describe the development of a new technology (SOFIA) and demonstrate its utility by establishing a sensitive and specific assay for PrP(Sc). SOFIA is a surround optical fiber immunoassay which is comprised of a set of specific monoclonal antibodies and comprehensive capture of high energy fluorescence emission. In its current format, this system is capable of detecting less than 10 attogram (ag) of hamster, sheep and deer recombinant PrP. Approximately 10 ag of PrP(Sc) from 263 K-infected hamster brains can be detected with similar lower limits of PrP(Sc) detection from the brains of scrapie-infected sheep and deer infected with chronic wasting disease. These detection limits allow protease treated and untreated material to be diluted beyond the point where PrP(C), non-specific proteins or other extraneous material may interfere with PrP(Sc) signal detection and/or specificity. This not only eliminates the issue of specificity of PrP(Sc) detection but also increases sensitivity since the possibility of partial PrP(Sc) proteolysis is no longer a concern. SOFIA will likely lead to early antemortem detection of transmissible encephalopathies and is also amenable for use with additional target amplification protocols. SOFIA represents a sensitive means for detecting specific proteins involved in disease pathogenesis and/or diagnosis that extends beyond the scope of the transmissible spongiform encephalopathies.