Chronic wasting disease

Chronic wasting disease (CWD) is a unique transmissible spongiform encephalopathy (TSE) of mule deer (Odocoileus hemionus), white-tailed deer (O. virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni). The natural history of CWD is incompletely understood, but it differs from scrapie and bovine spongiform encephalopathy (BSE) by virtue of its occurrence in nondomestic and free-ranging species. CWD has many features in common with scrapie, including early widespread distribution of disease-associated prion protein (PrP(d)) in lymphoid tissues, with later involvement of central nervous system (CNS) and peripheral tissues. This distribution likely contributes to apparent efficiency of horizontal transmission and, in this, is similar to scrapie and differs from BSE. Clinical features and lesions of CWD are qualitatively similar to the other animal TSEs. Microscopically, marked spongiform lesions occur in the central nervous system (CNS) after a prolonged incubation period and variable course of clinical disease. During incubation, PrP(d) can be identified in tissues by antibody-based detection systems. Although CWD can be transmitted by intracerebral inoculation to cattle, sheep, and goats, ongoing studies have not demonstrated that domestic livestock are susceptible via oral exposure, the presumed natural route of exposure to TSEs. Surveillance efforts for CWD in captive and free-ranging cervids will continue in concert with similar activities for scrapie and BSE. Eradication of CWD in farmed cervids is the goal of state, federal, and industry programs, but eradication of CWD from free-ranging populations of cervids is unlikely with currently available management techniques.     

A histopathologic and immunohistochemical review of archived UK caprine scrapie cases

In 2005, a prion disease identified in a goat from France was reported to be consistent with disease from the bovine spongiform encephalopathy (BSE) agent. Subsequent retrospective examination of UK goat scrapie cases led to the identification of one potentially similar, but as yet unconfirmed, case from Scotland. These findings strengthened concerns that small ruminant populations exposed to the BSE agent have become infected. The lack of data relating specifically to scrapie in goats has been contributory to past assumptions that, in general, sheep and goats respond similarly to prion infections. In this study, brain material from 22 archived caprine scrapie cases from the UK was reviewed by histopathology and by immunohistochemical examination for accumulations of disease-specific prion protein (PrP(Sc)) to provide additional data on the lesions of caprine scrapie and to identify any BSE-like features. The vacuolar change observed in the goats was characteristic of transmissible spongiform encephalopathies in general. PrP(Sc) immunohistochemical morphologic forms described in scrapie and experimental BSE infections of sheep were demonstrable in the goats, but these were generally more extensive and variable in PrP(Sc) accumulation. None of the cases examined showed a PrP(Sc) immunohistochemical pattern indicative of BSE.     

Comparison of brain PrPd distribution in ovine BSE and scrapie

Scrapie and bovine spongiform encephalopathy (BSE) are both prion diseases affecting ruminants, and these diseases do not share the same public health concerns. Surveillance of the BSE agent in small ruminants has been a great challenge, and the recent identification of diverse prion diseases in ruminants has led to the development of new methods for strain typing. In our study, using immunohistochemistry (IHC), we assessed the distribution of PrP(d) in the brains of 2 experimentally BSE-infected sheep with the ARQ/ARQ genotype. Distribution of PrP(d) in the brain, from the spinal cord to the frontal cortex, was remarkably similar in the 2 sheep despite different inoculation routes and incubation periods. Comparatively, overall PrP(d) brain distribution, evaluated by IHC, in 19 scrapie cases with the ARQ/ARQ, ARQ/VRQ, and VRQ/VRQ genotypes, in some cases showed similarities to the experimentally BSE-infected sheep. There was no exclusive neuroanatomical site with a characteristic and specific PrP(d) type of accumulation induced by the BSE agent. However, a detailed analysis of the topography, types, and intensity of PrP(d) deposits in the frontal cortex, striatum, piriform cortex, hippocampus, mesencephalon, and cerebellum allowed the BSE-affected sheep group to be distinguished from the 19 scrapie cases analyzed in our study. These results strengthen and emphasize the potential interest of PrP(d) brain mapping to help in identifying prion strains in small ruminants.     

First and second cattle passage of transmissible mink encephalopathy by intracerebral inoculation

To compare clinicopathologic findings of transmissible mink encephalopathy (TME) with other transmissible spongiform encephalopathies (TSE, prion diseases) that have been shown to be experimentally transmissible to cattle (sheep scrapie and chronic wasting disease [CWD]), two groups of calves (n = 4 each) were intracerebrally inoculated with TME agents from two different sources (mink with TME and a steer with TME). Two uninoculated calves served as controls. Within 15.3 months postinoculation, all animals from both inoculated groups developed clinical signs of central nervous system (CNS) abnormality; their CNS tissues had microscopic spongiform encephalopathy (SE); and abnormal prion protein (PrP(res)) as detected in their CNS tissues by immunohistochemistry (IHC) and Western blot (WB) techniques. These findings demonstrate that intracerebrally inoculated cattle not only amplify TME PrP(res) but also develop clinical CNS signs and extensive lesions of SE. The latter has not been shown with other TSE agents (scrapie and CWD) similarly inoculated into cattle. The findings also suggest that the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) would detect TME in cattle should it occur naturally. However, it would be a diagnostic challenge to differentiate TME in cattle from BSE by clinical signs, neuropathology, or the presence of PrP(res) by IHC and WB.     

Development of monoclonal antibodies against the abnormal prion protein isoform (PrPres) associated with chronic wasting disease (CWD)

Monoclonal antibodies (mAbs) specific for the abnormal prion protein isoform (PrP^res) are indispensable for diagnosing chronic wasting disease (CWD). In this study, eight mAbs were developed by immunizing PrP knockout mice with recombinant elk PrP and an immunogenic PrP peptide. The reactivity of the mAbs to recombinant PrP and the PrP peptide was measured, and their isotypes were subsequently determined. Among them, four mAbs (B85-05, B85-08, B85-12, and B77-75) were shown by Western blotting to recognize proteinase K-treated brain homogenate derived from an elk suffering from CWD.     

Susceptibility of cattle to first-passage intracerebral inoculation with chronic wasting disease agent from white-tailed deer

Fourteen, 3-month-old calves were intracerebrally inoculated with the agent of chronic wasting disease (CWD) from white-tailed deer (CWDwtd) to compare the clinical signs and neuropathologic findings with those of certain other transmissible spongiform encephalopathies (TSE, prion diseases) that have been shown to be experimentally transmissible to cattle (sheep scrapie, CWD of mule deer [CWDmd], bovine spongiform encephalopathy [BSE], and transmissible mink encephalopathy). Two uninoculated calves served as controls. Within 26 months postinoculation (MPI), 12 inoculated calves had lost considerable weight and eventually became recumbent. Of the 12 inoculated calves, 11 (92%) developed clinical signs. Although spongiform encephalopathy (SE) was not observed, abnormal prion protein (PrPd) was detected by immunohistochemistry (IHC) and Western blot (WB) in central nervous system tissues. The absence of SE with presence of PrPd has also been observed when other TSE agents (scrapie and CWDmd) were similarly inoculated into cattle. The IHC and WB findings suggest that the diagnostic techniques currently used to confirm BSE would detect CWDwtd in cattle, should it occur naturally. Also, the absence of SE and a distinctive IHC pattern of CWDwtd and CWDmd in cattle suggests that it should be possible to distinguish these conditions from other TSEs that have been experimentally transmitted to cattle.     

Molecular profiling and comparison of field transmissible spongiform encephalopathy cases diagnosed in Catalunya

Molecular profiling of the proteinase K resistant prion protein (PrP(res)) is a technique that has been applied to the characterisation of transmissible spongiform encephalopathy (TSE) strains. An interesting example of the application of this technique is the ability to differentiate, at the experimental level, between bovine spongiform encephalopathy (BSE) and scrapie infection in sheep, and to distinguish between classical and atypical BSE and scrapie cases. Twenty-six BSE cases and two scrapie cases from an active TSE surveillance program and diagnosed at the PRIOCAT, TSE Reference Laboratory (Centre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona, Catalunya, Spain) were examined by Western blotting. Molecular profiling was achieved by comparing the glycosylation profile, deglycosylated PrP molecular weight and 6H4/P4 monoclonal antibody binding ratio. The results obtained during the characterisation of these field cases indicated an absence of atypical BSE cases in Catalunya.     

Validation of monoclonal antibody F99/97.6.1 for immunohistochemical staining of brain and tonsil in mule deer (Odocoileus hemionus) with chronic wasting disease.

A new monoclonal antibody (MAb), F99/97.6.1, that has been used to demonstrate scrapie-associated prion protein PrP(Sc) in brain and lymphoid tissues of domestic sheep with scrapie was used in an immunohistochemistry assay for diagnosis of chronic wasting disease (CWD) in mule deer (Odocoileus hemionus). The MAb F99/97.6.1 immunohistochemistry assay was evaluated in brain and tonsil tissue from 100 mule deer that had spongiform encephalopathy compatible with CWD and from 1,050 mule deer outside the CWD-endemic area. This MAb demonstrated abnormal protease-resistant prion protein (PrP(res)) in brains of all of the 100 mule deer and in 99 of the 100 tonsil samples. No immunostaining was seen in samples collected from deer outside the endemic area. MAb F99/97.6.1 demonstrated excellent properties for detection of PrP(res) in fresh, frozen, or mildly to moderately autolytic samples of brain and tonsil. This immunohistochemistry assay is a sensitive, specific, readily standardized diagnostic test for CWD in deer.     

Variable patterns of distribution of PrP(CWD) in the obex and cranial lymphoid tissues of Rocky Mountain elk (Cervus elaphus nelsoni) with subclinical chronic wasting disease

Sections of medulla oblongata, taken at the level of the obex, palatine tonsil and medial retropharyngeal lymph node from 10,269 captive Rocky Mountain elk (Cervus elaphus nelsoni), were examined by immunohistochemical staining with monoclonal antibody for the prion protein associated with the transmissible spongiform encephalopathy of cervids, chronic wasting disease (PrP(CWD)). The protein was detected in 226 of them. On the basis of the anatomical location of the deposits in the brainstem of 183 elk, four distinct patterns of distribution of PrP(CWD) within the parasympathetic region of the dorsal motor nucleus of the vagus nerve and the adjacent nuclei were observed. Mild gross lesions of chronic wasting disease (serous atrophy of fat) were observed in only three elk, all with spongiform degeneration; the other elk were considered to be in the preclinical stage of the disease. In contrast with the relatively predictable distribution of prion protein (PrP) in the brain and cranial nodes of sheep and mule deer, the distribution of PrP(CWD) in the brain and nodes of the elk was more variable and unrelated to their PrP genotype. One hundred and fifty-five of the 226 positive elk had deposits of PrP(CWD) in the brainstem and lymphoid tissues, 43 had deposits only in the lymphoid tissue and 28 had deposits only in the brainstem.     

Immunohistochemical studies of scrapie archival material from Irish ARQ/ARQ sheep for evidence of bovine spongiform encephalopathy-derived disease

Since scrapie and bovine spongiform encephalopathy (BSE) in sheep are clinicopathologically indistinguishable, BSE in sheep may have been misdiagnosed as scrapie. Disease-specific prion protein (PrP(d)) patterns in archival tissues of 38 Irish ARQ/ARQ sheep diagnosed as scrapie-affected were compared to those in four Dutch BSE-challenged sheep. When medulla oblongata was immunolabelled with an antibody directed against amino acids 93-99 of ovine prion protein (ovPrP), intraneuronal PrP(d) was apparent in all 38 Irish sheep but was absent in BSE-challenged sheep. When lymphoid follicles were immunolabelled with antibodies directed against amino acids 93-106 of ovPrP, granule clusters of PrP(d) were seen in 34 of the 38 Irish sheep. Follicles of the remaining four archive sheep contained either no PrP(d) or single PrP(d) granules, similar to follicles from BSE-challenged sheep. Based on the medulla results, none of the archival cases had BSE-derived disease. The identification of some scrapie sheep with little or no intrafollicular PrP(d) suggests that this technique may be limited in discriminating between the two diseases.     

Atypical BSE in Germany--proof of transmissibility and biochemical characterization

Intensive active surveillance has uncovered two atypical German BSE cases in older cattle which resemble the two different atypical BSE phenotypes that have recently been described in France (designated H-type) and Italy (designated L-type or BASE). The H-type is characterized by a significantly higher molecular size, but a conventional glycopattern of the proteinase K treated abnormal prion protein (PrP(Sc)), while the L-type PrP(Sc) has only a slightly lower molecular size and a distinctly different glycopattern. In this paper we describe the successful transmission of both German atypical BSE cases to transgenic mice overexpressing bovine PrP(C). Upon challenge with the L-type, these mice developed BSE after a substantially shorter incubation period than any classical BSE transmission using these mice to date. In contrast, the incubation period was distinctly prolonged when these mice were challenged with the H-type. PrP(Sc) accumulated in the brains of these mice were of the same atypical BSE type that had been used for the transmission. These atypical cases suggest the possible existence of sporadic BSE cases in bovines. It is thus feasible that the BSE epidemic in the UK could have also been initiated by an intraspecies transmission from a sporadic BSE case.     

Pathological characterization of TgElk mice injected with brain homogenate from elk with chronic wasting disease

Chronic wasting disease (CWD) is classified as a transmissible spongiform encephalopathy or prion disease that affects cervids. CWD has been reported in 15 US states, two Canadian provinces, and in imported elk on several farms in Korea. This study was conducted to examine the molecular biological and pathogenic characteristics of a CWD-associated prion isolated in Korea. The epidemiological origin of this pathogen was also determined. Homozygous TgElk mice were infected with a CWD-affected elk brain pool prepared from the brain of an imported Canadian elk. We measured the incubation time of the pathogen, neuropathological changes by immunohistochemical staining, the pattern(s) of scrapie prion protein (PrP^Sc) deposition, and PrP^Sc protein profiles by Western blotting. We found that TgElk mice infected with brain homogenate from the elk suffering from CWD showed incubation times, vacuolar degeneration, and PrP^Sc accumulation similar to those previously reported in the literature. Our results suggest that homozygous TgElk mice efficiently transmit CWD with short incubation times and that this animal can serve a valuable research model and reliable in vivo diagnostic tool.     

A case of chronic wasting disease in an elk imported to Korea from Canada

A seven-year-old male elk (Cervus elaphus nelsoni) was euthanized and necropsied after having a 3-week history of body weight loss, emaciation, excessive salivation, teeth grinding, fever, anorexia, and respiratory distress. The elk was imported into Korea from Canada on March 9, 1997. Gross pathologic findings were restricted to a diffuse fibrinous pneumonia. Microscopic lesions included mild neuronal vacuolation and spongiform change in the neuropil of selected brain stem nuclei and generalized astrocytosis. Immunohistochemistry for protease-resistant prion protein (PrP(res)) was positive in all brain sections but more pronounced in the section of the obex of the medulla. And the PrP(res) was also detected by western immunoblotting in the brain and spinal cord. All the remaining elk and deer that had been in contact with this elk were destroyed and negative for chronic wasting disease (CWD). To our knowledge, this is the first case of CWD occurring outside of the U.S.A. and Canada.     

Preliminary observations on the experimental transmission of scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation

To determine the transmissibility of scrapie to Rocky Mountain elk (Cervus elaphus nelsoni), six elk calves were inoculated intracerebrally with brain suspension from sheep naturally affected with scrapie. One elk developed a brain abscess and was euthanatized at 7 weeks postinoculation (PI), and two others died at 6 and 15 months PI because of physical injuries. At 25 and 35 months PI, two other elk died after brief terminal neurologic episodes. Necropsy of these revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of spongiform encephalopathy were seen throughout the brains and the spinal cords, and in both cases these tissues were positive for PrP(res) by immunohistochemistry. Brains of both animals were positive for PrP(res) by western blot and for scrapie-associated fibrils (SAFs) by negative stain electron microscopy. PrP(res) and SAFs were not detected in the three elk that died or were euthanatized because of coincidental causes. Over 3.5 years after initiation of this experiment, the one remaining inoculated elk and two uninoculated (control) elk are alive and apparently healthy. These preliminary findings demonstrate that 1) sheep scrapie agent can be transmitted to elk by intracerebral inoculation; 2) the infection can result in severe, widely distributed spongiform change and accumulations of PrP(res) in the central nervous system (CNS); and 3) based on the examination of a limited number of CNS sections from two cases, this condition cannot be distinguished from chronic wasting disease with currently available diagnostic techniques.     

In situ detection of cellular and abnormal isoforms of prion protein in brains of cattle with bovine spongiform encephalopathy and sheep with scrapie by use of a histoblot technique.

To detect prion protein, brains from 5 cattle naturally affected with bovine spongiform encephalopathy (BSE) and 3 sheep naturally affected with scrapie were examined and compared with brains of normal cattle and sheep using a histoblot technique. The technique enabled the in situ distinctive detection of the cellular (PrP(C)) and abnormal (PrP(Sc)) isoforms of the prion protein. In BSE- or scrapie-affected brains, the Prp(C) signal decreased, especially in those areas where the PrP(Sc) signal was detected.     

Comparison of retropharyngeal lymph node and obex region of the brainstem in detection of chronic wasting disease in white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) in Wisconsin was first identified in February 2002. By April 2005, medial retropharyngeal lymph node (RLN) tissues had been examined from over 75,000 white-tailed deer for the presence of CWD by either immunohistochemical (IHC) staining for the prion protein associated with CWD (PrP(res)) or by using enzyme-linked immunosorbent assays with confirmation of positives by IHC staining and had been detected in 469 animals. Obex tissue was also available from 438 of the CWD-positive animals and was CWD positive by IHC staining in 355 (81%). To verify whether false-negative results were possible examining only RLN, both obex and RLN samples were examined for CWD by IHC staining from 4,430 of the white-tailed deer harvested from an area in Wisconsin where the overall deer CWD prevalence was approximately 6.2%. Two hundred and fourteen of the 269 positive deer (79.6%) had deposits of PrP(res) in both obex and lymphoid tissues, 55 (20.4%) had deposits only in lymphoid tissue, and there were no deer that had deposits only in obex.     

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.     

Transmission of chronic wasting disease of mule deer to Suffolk sheep following intracerebral inoculation.

To determine the transmissibility of chronic wasting disease (CWD) to sheep, 8 Suffolk lambs of various prion protein genotypes (4 ARQ/ARR, 3 ARQ/ARQ, 1 ARQ/VRQ at codons 136, 154, and 171, respectively) were inoculated intracerebrally with brain suspension from mule deer with CWD (CWDmd). Two other lambs were kept as noninoculated controls. Within 36 months postinoculation (MPI), 2 inoculated animals became sick and were euthanized. Only 1 sheep (euthanized at 35 MPI) showed clinical signs that were consistent with those described for scrapie. Microscopic lesions of spongiform encephalopathy (SE) were only seen in this sheep, and its tissues were determined to be positive for the abnormal prion protein (PrP(res)) by immunohistochemistry and Western blot. Three other inoculated sheep were euthanized (36 to 60 MPI) because of conditions unrelated to TSE. The 3 remaining inoculated sheep and the 2 control sheep did not have clinical signs of disease at the termination of the study (72 MPI) and were euthanized. Of the 3 remaining inoculated sheep, 1 was found to have SE, and its tissues were positive for PrP(res). The sheep with clinical prion disease (euthanized at 35 MPI) was of the heterozygous genotype (ARQ/VRQ), and the sheep with subclinical disease (euthanized at 72 MPH) was of the homozygous ARQ/ARQ genotype. These findings demonstrate that transmission of the CWDmd agent to sheep via the intracerebral route is possible. Interestingly, the host genotype may play a notable part in successful transmission and incubation period of CWDmd.     

Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats

This paper compares the dinical signs, histopathology, detection of PrPSc protein and PrP genetics of the transmission of BSE to sheep and goats, with the effects of the transmission of natural scrapie from a brain homogenate from a single sheep. After intracerebral and oral inoculations there were similarities in the clinical signs due to the two sources of infection, but there were differences in pathology at the end stage of disease and in the genotypes of the sheep which succumbed to the challenges. The incubation period of BSE was associated with the sheep PrP codon 171 genotype, but the natural scrapie source, despite inducing disease only in known susceptible genotypes, showed no clear association with PrP genotype.     

Evaluation of Western blotting methods using samples with or without sodium phosphotungstic acid precipitation for diagnosis of scrapie and chronic wasting disease

The purpose of this study was to enhance the sensitivity of the Western blot (WB) test for use as an alternative and confirmatory method for the diagnosis of scrapie and chronic wasting disease (CWD) in Canada by comparing 2 sample preparation procedures: an abnormal prion protein (PrPSc) concentration procedure using sodium phosphotungstic acid (PTA) precipitation and a procedure using crude sample without precipitation. A total of 100 cerebrum samples (52 sheep and 48 elk), including 66 negative (31 sheep, 35 elk) and 34 positive (21 scrapie and 13 CWD positive) samples diagnosed by using immunohistochemistry (IHC) on retropharyngeal lymph node (RPLN) and medulla oblongata at obex, were tested by using WB with the 2 sample preparation procedures. The WB using non-PTA enriched sample (crude extract) detected, on average, only 71.7% (9 of 15, 60.0% for scrapie, 5 of 6, 83.3% for CWD) of the samples that tested positive by using WB with PTA enriched samples. No case was positive by WB using crude extract but negative by WB using PTA enriched sample. No false positive was found. Serial dilution of PTA precipitated samples demonstrated that the technique increases the detection limit approximately 100 fold. Additionally, the comparison of the WB and IHC on cerebrum from all the positive cases demonstrated that WB following PTA precipitation and IHC had 100% agreement by detecting 6 positive for CWD on cerebrum; while IHC detected scrapie in only 14 out of 15 positive cerebrum samples by using WB following PTA precipitation. Phosphotungstic acid precipitation is therefore a useful adjunct to WB analysis of scrapie and CWD and tissues.