Comparison of Two US Sheep Scrapie Isolates Supports Identification as Separate Strains.

Scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats. There are different strains of sheep scrapie that are associated with unique molecular, transmission, and phenotype characteristics. However, in the United States, very little is known about the potential presence of scrapie strains. Scrapie strain and PRNP genotype could both affect susceptibility, potential for transmission, incubation period (IP), and control measures required for eliminating scrapie from a flock. The investigators evaluated 2 US scrapie isolates, No. 13-7 and x124, after intranasal inoculation to compare clinical signs, IPs, spongiform lesions, and patterns of PrPSc deposition in sheep with scrapie-susceptible PRNP genotypes (QQ171). After inoculation with x124, susceptibility and IP were associated with valine at codon 136 (V136) of the prion protein: VV136 sheep had short IPs (6.9 months), those in AV136 sheep were 11.9 months, and AA136 sheep did not develop scrapie. All No. 13-7 inoculated sheep developed scrapie, with IPs of 20.1 months for AA136 sheep, 22.8 months for AV136 sheep, and 26.7 months for VV136 sheep. Patterns of immunoreactivity in the brain were influenced by inoculum isolate and host genotype. Differences in PrPSc profiles versus isolate were most striking when examining brains from sheep with the VV136 genotype. Inoculation into C57BL/6 mice resulted in markedly different attack rates (90.5% for x124 and 5.9% for No. 13-7). Taken together, these data demonstrate that No. 13-7 and x124 represent 2 distinct strains of scrapie with different IPs, genotype susceptibilities, and PrPSc deposition profiles.

High-gamma band fronto-temporal coherence as a measure of functional connectivity in speech motor control.

The neural basis of human speech is unclear. Intracranial electrophysiological recordings have revealed that high-gamma band oscillations (70-150Hz) are observed in the frontal lobe during speech production and in the temporal lobe during speech perception. Here, we tested the hypothesis that the frontal and temporal brain regions had high-gamma coherence during speech. We recorded electrocorticography (ECoG) from the frontal and temporal cortices of five humans who underwent surgery for medically intractable epilepsy, and studied coherence between the frontal and temporal cortex during vocalization and playback of vocalization. We report two novel results. First, we observed high-gamma band as well as theta (4-8Hz) coherence between frontal and temporal lobes. Second, both high-gamma and theta coherence were stronger when subjects were actively vocalizing as compared to playback of the same vocalizations. These findings provide evidence that coupling between sensory-motor networks measured by high-gamma coherence plays a key role in feedback-based monitoring and control of vocal output for human vocalization.

Functional connection between posterior superior temporal gyrus and ventrolateral prefrontal cortex in human.

The connection between auditory fields of the temporal lobe and prefrontal cortex has been well characterized in nonhuman primates. Little is known of temporofrontal connectivity in humans, however, due largely to the fact that invasive experimental approaches used so successfully to trace anatomical pathways in laboratory animals cannot be used in humans. Instead, we used a functional tract-tracing method in 12 neurosurgical patients with multicontact electrode arrays chronically implanted over the left (n = 7) or right (n = 5) perisylvian temporal auditory cortex (area PLST) and the ventrolateral prefrontal cortex (VLPFC) of the inferior frontal gyrus (IFG) for diagnosis and treatment of medically intractable epilepsy. Area PLST was identified by the distribution of average auditory-evoked potentials obtained in response to simple and complex sounds. The same sounds evoked little if there is any activity in VLPFC. A single bipolar electrical pulse (0.2 ms, charge-balanced) applied between contacts within physiologically identified PLST resulted in polyphasic evoked potentials clustered in VLPFC, with greatest activation being in pars triangularis of the IFG. The average peak latency of the earliest negative deflection of the evoked potential on VLPFC was 13.48 ms (range: 9.0-18.5 ms), providing evidence for a rapidly conducting pathway between area PLST and VLPFC.

Acetone precipitation of the scrapie agent results in successful recovery of PrP(Sc) but decreased infectivity.

Bioassay is considered the most sensitive method for evaluating prion inactivation procedures. Because prions are resistant to methods effective at inactivating conventional microorganisms, prion inactivation research has focused on relatively harsh alternatives, such as concentrated sodium hypochlorite or sodium hydroxide. Often, bioassay for residual infectivity in these studies requires dilution or biochemical alteration of the treated sample in order to maintain subject health and survival. Ideally, prions from treated samples could be sufficiently separated from the inactivating agent without alteration of the sample and with negligible loss of infectivity prior to inoculation into the bioassay host. The current study was designed to evaluate acetone precipitation of the disease-associated form of the prion protein (PrP(Sc)) from brain homogenate derived from mice with the RML (Rocky Mountain Laboratory) strain of scrapie. The ability to recover PrP(Sc) was evaluated by Western blot. Dilutions of acetone-precipitated RML-positive brain homogenate were compared to nonprecipitated RML homogenate, resulting in similar PrP(Sc) detection levels down to 0.025 mg equivalents of brain tissue. The impact of the method on infectivity was investigated by bioassay in intracranially inoculated tga20 mice. Additionally, contributions to infectivity from the pellet and supernatant fractions were investigated. Acetone precipitation resulted in a 1-log10 reduction in infectivity. Infectivity could not be reconstituted by the acetone soluble fraction of the infectious sample or uninfected brain. This study demonstrates that PrP(Sc) can successfully be precipitated out of infected brain homogenate using acetone but that there is a reduction in infectivity attributable to the procedure that would need to be considered when evaluating bioassay results.

Cartilaginous metaplasia in the sclera of Suffolk sheep.

Scleral cartilaginous metaplasia was detected by routine histologic examination of globes from 5 Suffolk sheep from a scrapie pathogenesis study. The extent of the metaplasia varied among the sheep but was always posterior to the tapetal fundus. The matrix surrounding chondrocytes stained intensely with alcian blue and was immunopositive for type II collagen. Retrospective evaluation of additional eyes from Suffolk and Cheviot sheep used in various scrapie pathogenesis studies at the authors' facility revealed similar histologic changes in 40% and 12.7% of eyes examined, respectively. The clinical significance of this previously unreported finding is unknown.

Detection of the abnormal isoform of the prion protein associated with chronic wasting disease in the optic pathways of the brain and retina of Rocky Mountain elk (Cervus elaphus nelsoni).

Eyes and nuclei of the visual pathways in the brain were examined in 30 Rocky Mountain elk (Cervus elaphus nelsoni) representing 3 genotypes of the prion protein gene PRNP (codon 132: MM, ML, or LL). Tissues were examined for the presence of the abnormal isoform of the prion protein associated with chronic wasting disease (PrP(CWD)). Nuclei and axonal tracts from a single section of brain stem at the level of the dorsal motor nucleus of the vagus nerve were scored for intensity and distribution of PrP(CWD) immunoreactivity and degree of spongiform degeneration. This obex scoring ranged from 0 (elk with no PrP(CWD) in the brain stem) to 10 (representing elk in terminal stage of disease). PrP(CWD) was detected in the retina of 16 of 18 (89%) elk with an obex score of > 7. PrP(CWD) was not detected in the retina of the 3 chronic wasting disease-negative elk and 9 elk with an obex score of < 6. PrP(CWD) was found in the nuclei of the visual pathways in the brain before it was found in the retina. Within the retina, PrP(CWD) was first found in the inner plexiform layer, followed by the outer plexiform layer. Intracytoplasmic accumulation of PrP(CWD) was found in a few neurons in the ganglion cell layer in the PRNP 132ML elk but was a prominent feature in the PRNP 132LL elk. Small aggregates of PrP(CWD) were present on the inner surface of the outer limiting membrane in PRNP 132LL elk but not in PRNP 132MM or 132ML elk. This study demonstrates PrP(CWD) accumulation in nuclei of the visual pathways of the brain, followed by PrP(CWD) in the retina.

Characterization of a US sheep scrapie isolate with short incubation time.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a previous study it was shown that sheep intracerebrally inoculated with US scrapie inoculum (No. 13-7) developed terminal disease within an average of 19 months. We have since produced an inoculum, No. x124 from pooled brains of US-origin sheep scrapie, that results in incubations nearly threefold shorter. The present study documents clinicopathologic findings and the distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemical (IHC) and Western blot (WB) techniques, in tissues of sheep inoculated with No. x124. All inoculated sheep developed clinical disease and were euthanatized within an average of 7.7 months postinoculation (MPI). Sheep that had valine/valine or alamine/valine at codon 136 of prion protein (PRNP) gene developed the disease faster and were euthanatized at an average of 4.3 and 5.6 MPI, respectively. Also, the inoculum was able to induce disease in a short time (7 MPI) in a sheep that was relatively resistant (QR at codon 171) to scrapie. This indicates that inoculum No. x124 appears to induce scrapie in shorter time than inoculum No. 13-7, especially in sheep homozygous or heterozygous for valine at codon 136.

Retinal function and morphology are altered in cattle infected with the prion disease transmissible mink encephalopathy.

Transmissible spongiform encephalopathies (TSEs) are a group of diseases that result in progressive and invariably fatal neurologic disease in both animals and humans. TSEs are characterized by the accumulation of an abnormal protease-resistant form of the prion protein in the central nervous system. Transmission of infectious TSEs is believed to occur via ingestion of prion protein-contaminated material. This material is also involved in the transmission of bovine spongiform encephalopathy ("mad cow disease") to humans, which resulted in the variant form of Creutzfeldt-Jakob disease. Abnormal prion protein has been reported in the retina of TSE-affected cattle, but despite these observations, the specific effect of abnormal prion protein on retinal morphology and function has not been assessed. The objective of this study was to identify and characterize potential functional and morphologic abnormalities in the retinas of cattle infected with a bovine-adapted isolate of transmissible mink encephalopathy. We used electroretinography and immunohistochemistry to examine retinas from 10 noninoculated and 5 transmissible mink encephalopathy-inoculated adult Holstein steers. Here we show altered retinal function, as evidenced by prolonged implicit time of the electroretinogram b-wave, in transmissible mink encephalopathy-infected cattle before the onset of clinical illness. We also demonstrate disruption of rod bipolar cell synaptic terminals, indicated by decreased immunoreactivity for the alpha isoform of protein kinase C and vesicular glutamate transporter 1, and activation of Müller glia, as evidenced by increased glial fibrillary acidic protein and glutamine synthetase expression, in the retinas of these cattle at the time of euthanasia due to clinical deterioration. This is the first study to identify both functional and morphologic alterations in the retinas of TSE-infected cattle. Our results support future efforts to focus on the retina for the development of new strategies for the diagnosis of TSEs.

Serial passage of sheep scrapie inoculum in Suffolk sheep.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a recent study, it was shown that sheep intracerebrally inoculated with a US scrapie agent (No. 13-7) developed scrapie and survived for an average of 19 months post inoculation. In the present study, when this scrapie inoculum was further passaged for 3 successive generations, the survival time was reduced by approximately 8 months in scrapie-susceptible (QQ on prion protein gene [PRNP] at codon 171) Suffolk sheep. It is concluded that inoculum No. 13-7 appears to have been stabilized in susceptible (171 QQ) Suffolk sheep and may be considered a specific isolate of sheep scrapie agent in the USA and therefore that it can be used to evaluate other isolates of sheep scrapie in this country.

Experimental transmission of chronic wasting disease (CWD) of elk (Cervus elaphus nelsoni), white-tailed deer (Odocoileus virginianus), and mule deer (Odocoileus hemionus hemionus) to white-tailed deer by intracerebral route.

To compare clinical and pathologic findings of chronic wasting disease (CWD) in a natural host, 3 groups (n = 5) of white-tailed deer (WTD) fawns were intracerebrally inoculated with a CWD prion of WTD, mule deer, or elk origin. Three other uninoculated fawns served as controls. Approximately 10 months postinoculation (MPI), 1 deer from each of the 3 inoculated groups was necropsied and their tissues were examined for lesions of spongiform encephalopathy (SE) and for the presence of abnormal prion protein (PrP(d)) by immunohistochemistry (IHC) and Western blot (WB). The remaining deer were allowed to live until they developed clinical signs of the disease which began approximately 18 MPI. By 26 MPI, all deer were euthanatized on humane grounds. Obvious differences in clinical signs or the incubation periods were not observed between the 3 groups of deer given CWD. In 1 of 3 nonclinical deer euthanatized at 10 MPI, minimal microscopic lesions of SE were seen in the central nervous system (CNS) tissues, and PrP(d) was observed by IHC in tissues of all 3 deer. In the clinical deer, CNS lesions of SE and PrP(d) accumulations were more severe and extensive. It is concluded that the 3 sources of CWD prion did not induce significant differences in time to clinical disease or qualitative differences in signs or lesions in WTD. However, this observation does not imply that these CWD agents would necessarily behave similarly in other recipient species.

Experimental transmission of US scrapie agent by nasal, peritoneal, and conjunctival routes to genetically susceptible sheep.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. This study documents incubation periods, pathologic findings, and distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemistry in tissues of genetically susceptible sheep inoculated with US sheep scrapie agent. Four-month-old Suffolk lambs (QQ at codon 171) were inoculated by 1 of 3 different routes (nasal, peritoneal, and conjunctival) with an inoculum (No. 13-7) consisting of a pool of scrapie-affected sheep brains. Except for 3 sheep, all inoculated animals were euthanized when advanced clinical signs of scrapie were observed between 19 and 46 months postinoculation (MPI). Spongiform lesions in the brains and labeling of PrP(Sc) in central nervous system and lymphoid tissues were present in these sheep. One intranasally inoculated sheep euthanized at 12 MPI had presence of PrP(Sc) that was confined to the pharyngeal tonsil. These results indicate that the upper respiratory tract, specifically the pharyngeal tonsil, may serve as a portal of entry for prion protein in scrapie-infected environments.

Retinal cell types are differentially affected in sheep with scrapie.

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases characterized microscopically by spongiform lesions (vacuolation) in the neuropil, neuronal loss, and gliosis. Accumulation of the abnormal form of the prion protein (PrP(Sc)) has been demonstrated in the retina of natural and non-natural TSE-affected hosts, with or without evidence of microscopically detectable retinal pathology. This study was conducted to investigate the effect of PrP(Sc) accumulation on retinal neurons in a natural host lacking overt microscopical evidence of retinal degeneration by comparing the distribution of retinal cell type-specific markers in control and scrapie-affected sheep. In retinas with PrP(Sc)-immunoreactivity, there was disruption of the normal immunoreactivity patterns of the alpha isoform of protein kinase C (PKCalpha) and vesicular glutamate transporter 1 (VGLUT1), markers of retinal bipolar cells. Altered immunoreactivity was also observed for microtubule-associated protein 2 (MAP2), a marker of a subset of retinal ganglion cells, and glutamine synthetase (GS), a marker of Müller glia. These results demonstrate alterations of immunoreactivity patterns for proteins associated with specific cell types in retinas with PrP(Sc) accumulation, despite an absence of microscopical evidence of retinal degeneration.

Progressive multifocal leucoencephalopathy in the era of natalizumab: a review and discussion of the implications.

Progressive multifocal leucoencephalopathy (PML) is an opportunistic demyelinating infection of immunocompromised patients, caused by the human polyomavirus, JC virus. Prior to 2005, PML had not been described in patients with MS. In early 2005, however, two cases of PML were reported in patients with MS treated with the alpha-antegrin inhibitor, natalizumab. A third case was subsequently described in a patient with Crohn's disease who had also received the agent. These three cases resulted in withdrawal of natalizumab from the market and have had major implications for its reintroduction into clinical use. In this review, we discuss current knowledge concerning PML and its causative agent, the possible role of natalizumab in initiating PML in patients with MS, and the challenges posed by the risk of this infection as the drug returns to clinical use.

Abnormal prion accumulation associated with retinal pathology in experimentally inoculated scrapie-affected sheep.

The purpose of this study was to characterize the patterns of PrP(Sc) immunoreactivity in the retinae of scrapie-affected sheep and to determine the extent of retinal pathology as indicated by glial fibrillary acidic protein immunoreactivity (GFAP-IR) of Müller glia. Sections from the retina of 13 experimentally inoculated scrapie-affected and 2 negative control sheep were examined with immunohistochemical staining for PrP(Sc), GFAP, and PrP(Sc)/GFAP double staining. GFAP-IR of Müller glia is suggestive of retinal pathology in the absence of morphologic abnormality detected by light microscopy. Sheep with the least amount of PrP(Sc) in the retina have multifocal punctate aggregates of prion staining in the outer half of the inner plexiform layer and rarely in the outer plexiform layer. In these retinae, GFAP-IR is not localized with prion accumulation, but rather is present in moderate numbers of Müller glia throughout the sections of retina examined. The majority of sheep with retinal accumulation of PrP(Sc) have intense, diffuse PrP(Sc) staining in both plexiform layers, with immunoreactivity in the cytoplasm of multiple ganglion cells and lesser amounts in the optic fiber layer and between nuclei in nuclear layers. This intense PrP(Sc) immunoreactivity is associated with diffuse, intense GFAP-IR that extends from the inner limiting membrane to the outer limiting membrane. This is the first report of a prion disease in a natural host that describes the accumulation of PrP(Sc) in retina associated with retinal pathology in the absence of overt morphologic changes indicative of retinal degeneration.

Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle.

To compare clinicopathological findings in first and second passage chronic wasting disease (CWD(mule deer)) in cattle, six calves were inoculated intracerebrally with brain tissue derived from a first-passage CWD-affected calf in an earlier experiment. Two uninoculated calves served as controls. The inoculated animals began to lose both appetite and weight 10-12 months later, and five subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months, all cattle had been subjected to euthanasia because of poor prognosis. None of the animals showed microscopical lesions of spongiform encephalopathy (SE) but PrP(res) was detected in their CNS tissues by immunohistochemistry (IHC) and rapid Western blot (WB) techniques. Thus, intracerebrally inoculated cattle not only amplified CWD PrP(res) from mule deer but also developed clinical CNS signs in the absence of SE lesions. This situation has also been shown to occur in cattle inoculated with the scrapie agent. The study confirmed that the diagnostic techniques currently used for diagnosis of bovine spongiform encephalopathy (BSE) in the US would detect CWD in cattle, should it occur naturally. Furthermore, it raised the possibility of distinguishing CWD from BSE in cattle, due to the absence of neuropathological lesions and to a distinctive multifocal distribution of PrP(res), as demonstrated by IHC which, in this study, appeared to be more sensitive than the WB technique.

Optic neuritis: a novel presentation of Schilder's disease.

The clinical features of a 7-year-old girl who presented with unilateral optic neuritis are presented. Magnetic resonance imaging (MRI) showed lesions in the affected optic nerve and the centrum semiovale bilaterally. Biopsy of one of the cerebral lesions was consistent with a diagnosis of Schilder's disease. Visual acuity returned to normal, and the demyelinating MRI lesions improved markedly with corticosteroid treatment. Optic neuritis is a novel mode of presentation in Schilder's disease.

Antibody types and IgG subclasses in paraneoplastic neurological syndromes.

Three major patterns of antineuronal antibody response have been identified in patients with paraneoplastic neurological syndromes: Type I ('Anti-Yo'), associated with cerebellar degeneration in the setting of breast or gynecological cancer, Type IIa ('anti-Hu') associated with encephalomyeloneuritis in patients with small cell carcinoma of the lung, and Type IIb ('anti-Ri') associated with breast cancer. We have employed immunofluorescence methods to determine the antibody classes and the IgG subclasses which react with neurons in each of these patterns of paraneoplastic antibody response. In this study, IgG was the only antibody class identified; IgM and IgA antibodies were not found. IgG1 was the major subclass represented and was found in 9/9 patients with Type I antibody response, 26/27 patients with Type IIa antibody response, and 3/3 patients with Type IIb antibody response. Many patients also exhibited positive staining for IgG2 and IgG3. Trace amounts of IgG4 antineuronal antibodies were detected in a single patient with Type I antibody response; IgG4 antibodies were not found in other patients. Patients with paraneoplastic neurological syndromes exhibit an antibody response which is overwhelmingly IgG and is comprised predominantly of IgG subclasses capable of fixing complement. The role of these antibodies in the pathogenesis of paraneoplastic neurological disease remains uncertain.