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 (PrP(res)) 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 PrP(res)-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 PrP(res) 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 PrP(res). 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 PrP(res) within CNS tissues during the normal lifespan of cattle.

Transmission of sheep scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation: final outcome of the experiment.

This is a final report of an experimental transmission of sheep scrapie agent by intracerebral inoculation to Rocky Mountain elk (Cervus elaphus nelsoni). It documents results obtained in experimental (n = 6) and control (n = 2) elk. During the first 2 years postinoculation (PI), 3 animals died or were euthanized because of infection or injuries other than spongiform encephalopathy (SE). In years 3 and 4 PI, 3 other inoculated elk died after brief terminal neurological episodes. Necropsy of these animals revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of SE were seen throughout the brain and spinal cord, and the tissue was positive for proteinase K-resistant prion protein (PrPres) by immunohistochemistry (IHC) and by Western blot. Scrapie-associated fibrils (SAF) were observed by negative-stain electron microscopy in the brain of elk with neurologic signs. PrPres and SAF were not detected in the 3 inoculated elk necropsied during the first 2 years or in the 2 control animals. Retrospective analysis of the gene-encoding cervid PrP revealed a polymorphism at codon 132. The elk with SE were either homozygous (MM) or heterozygous (LM). These findings confirm that intracerebral inoculation of sheep scrapie agent results in SE with accumulations of PrPres in the central nervous system of elk. Based on morphologic and IHC findings, the experimentally induced SE cannot be distinguished from chronic wasting disease of elk with currently available diagnostic techniques.

Transmission of transmissible mink encephalopathy to raccoons (Procyon lotor) by intracerebral inoculation.

To determine the transmissibility of transmissible mink encephalopathy (TME) agent to raccoons and to provide information about clinical course, lesions, and suitability of currently used diagnostic procedures for detection of transmissible spongiform encephalopathies (TSEs) in raccoons, 4 raccoon kits were inoculated intracerebrally with a brain suspension from mink experimentally infected with TME. One uninoculated raccoon kit served as a control. All 4 animals in the TME-inoculated group showed clinical signs of neurologic disorder and were euthanized between 21 and 23 weeks postinoculation (PI). Necropsy examinations revealed no gross lesions. Spongiform encephalopathy was observed by light microscopy, and the presence of protease-resistant prion protein (PrPres) was detected by immunohistochemistry and Western blot techniques. Scrapie-associated fibrils were observed by negative-stain electron microscopy in the brains of 3 of the 4 inoculated raccoons. These findings confirm that TME is experimentally transmissible to raccoons and that diagnostic techniques currently used for TSE in livestock detect prion protein in raccoon tissue. According to previously published data, the incubation period of sheep scrapie in raccoons is 2 years, whereas chronic wasting disease (CWD) had not shown transmission after 3 years of observation. Because incubation periods for the 3 US TSEs (scrapie, TME, and CWD) in raccoons appear to be markedly different, it may be possible to use raccoons for differentiating unknown TSE agents. Retrospective genotyping of raccoons using frozen spleens showed that the raccoon PrP gene is identical to the mink gene at codons 179 and 224. Further studies, such as the incubation periods of bovine spongiform encephalopathy and other isolates of scrapie, CWD, and TME in raccoons, are needed before the model can be further characterized for differentiation of TSE agents.

Failure to detect abnormal prion protein and scrapie-associated fibrils 6 wk after intracerebral inoculation of genetically susceptible sheep with scrapie agent.

Detection of the scrapie-associated protease-resistant prion protein (PrPres) in sheep brains in the early phase after intracerebral inoculation of the scrapie agent has not been documented. Fourteen 4-mo-old, genetically susceptible lambs (QQ homozygous at codon 171 of the PrP gene) were obtained for this study. Twelve lambs were inoculated intracerebrally with a brain suspension from sheep naturally affected with scrapie, and 2 served as uninoculated controls. Two inoculated animals were euthanized at each of 6 times postinoculation (1 h to 6 wk), and their brains were collected for histopathological study, for detection of PrPres by the Western blot technique and an immunohistochemical (IHC) method, and for the detection of scrapie-associated fibrils (SAF) by negatively stained electron microscopy (EM). Microscopic lesions associated with introduction of the inoculum were seen in the brains of inoculated animals at all 6 times. However, both the Western blot and IHC techniques did not detect PrPres after the initial 3 d postinoculation, nor did EM detect SAF in any of the samples. From these findings, it is presumed that until host amplification has occurred, the concentration of PrPres in inoculum is insufficient for detection by currently available techniques.

A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies.

BACKGROUND: Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting both humans and animals. They are associated with post-translational conversion of the normal cellular prion protein (PrPC) into a heat- and protease-resistant abnormal isoform (PrPSc). Detection of PrPSc in individuals is widely utilized for the diagnosis of prion diseases. METHODS: TSE brain tissue samples have been processed in order to quantitatively isolate PrPSc. The protocol includes an initial homogenization, digestion with proteinase K and salt precipitation. RESULTS: Here we show that over 97 percent of the PrPSc present can be precipitated from infected brain material using this simple salting-out procedure for proteins. No chemically harsh conditions are used during the process in order to conserve the native quality of the isolated protein. CONCLUSION: The resulting PrPSc-enriched preparation should provide a suitable substrate for analyzing the structure of the prion agent and for scavenging for other molecules with which it may associate. In comparison with most methods that exist today, the one described in this study is rapid, cost-effective and does not demand expensive laboratory equipment.

Resistance of domestic cats to a US sheep scrapie agent by intracerebral route.

Feline spongiform encephalopathy (FSE) is thought to have resulted from consumption of food contaminated with bovine spongiform encephalopathy and the latter is believed to result from the consumption of food contaminated with scrapie. However, no direct experimental documentation exists to indicate that the scrapie agent is capable of amplifying in cats, and, therefore, crossing the species barrier. During 1979, 6 cats ranging in age from 3.5 to 18 months were intracerebrally inoculated with sheep scrapie (inoculum G-639-PP) and were observed for an extended period. Inoculated cats did not develop neurologic disease, and microscopic lesions of spongiform encephalopathy were not evident. Immunohistochemistry and Western blot techniques failed to detect the abnormal form of prion protein (PrP(res)). These results indicate that the sheep scrapie agent (G-639-PP) used in this study was not capable of amplifying in cats and therefore was unable to cross the species barrier to produce FSE.

Neuroaxonal dystrophy in raccoons (Procyon lotor) from Iowa.

During a 12-month period (1998-1999), microscopic evidence of neuroaxonal dystrophy (NAD) in medullae oblongata of raccoons (Procyon lotor) was observed in 17/39 (47% prevalence in adults) from Iowa, USA. Three of the animals were kits (<3 months), 26 were between 1 and 2 years, and 10 were over 7 years. Lesions were not seen in the medullae of the 3 kits. In young adults, the lesions were mild and were seen in 7 animals. More severe lesions were present in the 10 older raccoons. Grossly, the brains were unremarkable. Microscopically, NAD was confined to the dorsal caudal medulla, where certain nuclei (predominantly gracilis and cuneate) were bilaterally affected. Severely affected animals had vacuolar degeneration of neurons or neuronal loss and extensive areas of spongiosis. Tests for the presence of PrP(res) in the brain were negative. Spongiotic areas often contained axonal spheroids. Degenerate neurons and axons occasionally contained amphophilic periodic acid-Schiff-positive granular material. There was a paucity of inflammatory cells in the affected areas. Since lesions were not present in kits, were either absent or mild in young adults, and were severe in older raccoons, the findings may be related to advancing age. Neuroaxonal dystrophy has not been previously reported in raccoons. Retrospective examination of raccoon brains from the eastern and northwestern areas of the country revealed very low prevalence of NAD. Because of the apparently high prevalence of this condition at this geographic location, factors other than age (genetic, nutritional, and/or environmental) may influence this degenerative process in the brains of raccoons in Iowa.