Experimental transmission of chronic wasting disease agent from mule deer to cattle by the intracerebral route

This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle by the intracerebral route. Thirteen calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years after inoculation. During that time, abnormal prion protein (PrPres) was demonstrated in the central nervous system (CNS) of 5 cattle by both immunohistochemistry and Western blot. However, microscopic lesions suggestive of spongiform encephalopathy (SE) in the brains of these PrPres-positive animals were subtle in 3 cases and absent in 2 cases. Analysis of the gene encoding bovine PRNP revealed homozygosity for alleles encoding 6 octapeptide repeats, serine (S) at codon 46, and S at codon 146 in all samples. Findings of this study show that although PrPres amplification occurred after direct inoculation into the brain, none of the affected animals had classic histopathologic lesions of SE. Furthermore, only 38% of the inoculated cattle demonstrated amplification of PrPres. Although intracerebral inoculation is an unnatural route of exposure, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum but also may not result in amplification of PrPres within CNS tissues during the normal lifespan of cattle

Experimental transmission of chronic wasting disease agent from mule deer to cattle by the intracerebral route

This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle by the intracerebral route. Thirteen calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years after inoculation. During that time, abnormal prion protein (PrPres) was demonstrated in the central nervous system (CNS) of 5 cattle by both immunohistochemistry and Western blot. However, microscopic lesions suggestive of spongiform encephalopathy (SE) in the brains of these PrPres-positive animals were subtle in 3 cases and absent in 2 cases. Analysis of the gene encoding bovine PRNP revealed homozygosity for alleles encoding 6 octapeptide repeats, serine (S) at codon 46, and S at codon 146 in all samples. Findings of this study show that although PrPres amplification occurred after direct inoculation into the brain, none of the affected animals had classic histopathologic lesions of SE. Furthermore, only 38% of the inoculated cattle demonstrated amplification of PrPres. Although intracerebral inoculation is an unnatural route of exposure, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum but also may not result in amplification of PrPres within CNS tissues during the normal lifespan of cattle

Ablation of prion protein immunoreactivity by heating in saturated calcium hydroxide

<p>Abstract</p> <p>Background</p> <p>Prions, the infectious agents that cause transmissible spongiform encephalopathies (TSEs), are relatively resistant to destruction by physical, enzymatic, and chemical treatments. Hydrolysis in boiling saturated calcium hydroxide (limewater) utilizes inexpensive chemicals to digest protein components of offal. The purpose of this work was to determine if incubating brain material from scrapie-infected sheep in near-boiling saturated calcium hydroxide solution (Ca(OH)₂) would abolish immunoreactivity of the infectious prion (PrP^Sc) as determined by western blot.</p> <p>Findings</p> <p>After incubating for as few as 10 minutes in saturated calcium hydroxide at 99°C, immunoreactivity of protease resistant bands by western blot analysis is completely lost.</p> <p>Conclusion</p> <p>Boiling in limewater may offer an alternative for disposal of carcasses and enable alternative uses for rendered products from potentially infected carcasses.</p

Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases

<p>Abstract</p> <p>Background</p> <p>Three distinct forms of bovine spongiform encephalopathy (BSE), defined as classical (C-), low (L-) or high (H-) type, have been detected through ongoing active and passive surveillance systems for the disease.</p> <p>The aim of the present study was to compare the ability of two sets of immunohistochemical (IHC) and Western blot (WB) BSE confirmatory protocols to detect C- and atypical (L- and H-type) BSE forms.</p> <p>Obex samples from cases of United States and Italian C-type BSE, a U.S. H-type and an Italian L-type BSE case were tested in parallel using the two IHC sets and WB methods.</p> <p>Results</p> <p>The two IHC techniques proved equivalent in identifying and differentiating between C-type, L-type and H-type BSE. The IHC protocols appeared consistent in the identification of PrP^Scdistribution and deposition patterns in relation to the BSE type examined. Both IHC methods evidenced three distinct PrP^Scphenotypes for each type of BSE: prevailing granular and linear tracts pattern in the C-type; intraglial and intraneuronal deposits in the H-type; plaques in the L-type.</p> <p>Also, the two techniques gave comparable results for PrP^Scstaining intensity on the C- and L-type BSE samples, whereas a higher amount of intraglial and intraneuronal PrP^Scdeposition on the H-type BSE case was revealed by the method based on a stronger demasking step.</p> <p>Both WB methods were consistent in identifying classical and atypical BSE forms and in differentiating the specific PrP^Scmolecular weight and glycoform ratios of each form.</p> <p>Conclusions</p> <p>The study showed that the IHC and WB BSE confirmatory methods were equally able to recognize C-, L- and H-type BSE forms and to discriminate between their different immunohistochemical and molecular phenotypes. Of note is that for the first time one of the two sets of BSE confirmatory protocols proved effective in identifying the L-type BSE form. This finding helps to validate the suitability of the BSE confirmatory tests for BSE surveillance currently in place.</p

PrP^Scdetection in formalin-fixed paraffin-embedded tissue by ELISA

<p>Abstract</p> <p>Background</p> <p>Formalin-fixed paraffin-embedded tissue is regularly employed in the diagnosis of transmissible spongiform encephalopathies (TSE) by immunohistochemistry (IHC), the standard by which all other TSE diagnostic protocols are judged. While IHC affords advantages over diagnostic approaches that typically utilize fresh or frozen tissue, such as Western blot and ELISA, the process of fixing, staining, and analyzing individual sections by hand does not allow for rapid or high throughput screening. However, preservation of tissues in formalin is not dependent upon the availability of refrigeration.</p> <p>Findings</p> <p>Formalin-fixed paraffin-embedded tissues from TSE transmission studies of scrapie in sheep, chronic wasting disease in white-tailed deer or transmissible mink encephalopathy in cattle were cut at 5 μm thickness. Samples containing the tissue equivalent of as little as one 5 μm section can be used to readily discriminate positive from negative samples.</p> <p>Conclusions</p> <p>This approach cannot replace IHC but may be used along with IHC as both a more rapid and readily high throughput screen where fresh or frozen tissues are not available or impractical.</p