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.     

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.     

Polymorphisms of the prion gene promoter region that influence classical bovine spongiform encephalopathy susceptibility are not applicable to other transmissible spongiform encephalopathies in cattle

Two regulatory region polymorphisms in the prion gene of cattle have been reported to have an association with resistance to classical bovine spongiform encephalopathy (BSE). However, it is not known if this association also applies to other transmissible spongiform encephalopathies (TSE) in cattle. In this report, we compare the relationship between these 2 polymorphisms and resistance in cattle affected with naturally occurring atypical BSE as well as in cattle experimentally inoculated with either scrapie, chronic wasting disease, or transmissible mink encephalopathy. Our analysis revealed no association between genotype and resistance to atypical BSE or experimentally inoculated TSE. This indicates the promoter polymorphism correlation is specific to classical BSE and that atypical BSE and experimentally inoculated TSE are bypassing the site of influence of the polymorphisms. This genetic discrepancy demonstrates that atypical BSE progresses differently in the host relative to classical BSE. These results are consistent with the notion that atypical BSE originates spontaneously in cattle.     

Abnormal prion protein in ectopic lymphoid tissue in a kidney of an asymptomatic white-tailed deer experimentally inoculated with the agent of chronic wasting disease

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy (TSE) of deer and elk, is one of a group of fatal, neurologic diseases that affect several mammalian species, including human beings. Infection by the causative agent induces accumulations of an abnormal form of prion protein (PrPres) in nervous and lymphoid tissues. This report documents the presence of PrPres within ectopic lymphoid follicles in a kidney of a white-tailed deer that had been experimentally inoculated by the intracerebral route with CWD 10 months previously. The deer was nonclinical, but spongiform lesions characteristic of TSE were detected in tissues of the central nervous system (CNS) and PrPres was seen in CNS and in lymphoid tissues by immunohistochemistry. The demonstration of PrPres in lymphoid tissue in the kidney of this deer corroborates a recently published finding of PrPres in lymphoid follicles of organs other than CNS and lymphoid tissues in laboratory animals with TSE (scrapie).     

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.     

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.     

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.     

Chronic wasting disease in deer and elk: scientific facts and findings

Chronic wasting disease (CWD) is a prion disease of cervids such as deer and elk in North America. Unlike other transmissible spongiform encephalopathy (TSE) such as scrapie, CWD occurs in both captive and wild ranging animals, but not in domestic ruminants such as sheep and cattle. In this paper, the history of the disease, pathogenesis of CWD, susceptibility of animals, its transmission mechanisms, potential origins of the disease, diagnostic methods in the field and laboratory tests, surveillance and survey systems in the USA and Canada, control strategies, economic impact of the disease, food and feed safety, and the risks in human and animals are reviewed and summarized. Although there is no evidence that CWD has been transmitted to humans, it may have the potential to infect humans.     

The first Canadian indigenous case of bovine spongiform encephalopathy (BSE) has molecular characteristics for prion protein that are similar to those of BSE in the United Kingdom but differ from those of chronic wasting disease in captive elk and deer

Brain tissue from a case of bovine spongiform encephalopathy (BSE) from Alberta was subjected to a Western immunoblotting technique to ascertain the molecular profile of any disease-specific, abnormal prion protein, that is, prion protein that is protease-resistant (PrP(res)). This technique can discriminate between isolates from BSE, ovine scrapie, and sheep experimentally infected with BSE. Isolates of brain tissue from the BSE case in Alberta, 3 farmed elk with chronic wasting disease (CWD) from different parts of Saskatchewan, and 1 farmed white-tailed deer with CWD from Edmonton, Alberta, were examined alongside isolates of brain tissue from BSE, ovine scrapie, and sheep experimentally infected with BSE from the United Kingdom (UK). The molecular weights of PrP(res) and the cross reactions to 2 specific monoclonal antibodies (mAbs) were determined for each sample. The BSE isolates from Canada and the UK had very similar PrP(res) molecular weights and reacted with only 1 of the 2 mAbs. The PrP(res) isolated from both elk and white-tailed deer with CWD had a higher molecular weight profile than did the corresponding PrP(res) from the scrapie and BSE isolates. The PrP(res) from CWD cases cross reacted with both mAbs, a property shared with PrP(res) in isolates from scrapie but not with PrP(res) isolates from BSE or sheep experimentally infected with BSE. The results from this study seem to confirm that the PrP(res) isolated from the BSE case in Alberta has similar molecular properties to the PrP(res) isolated from a BSE case in the UK, and that it differs in its molecular and immunological characteristics from the CWD and scrapie cases studied.     

Survey of cattle in northeast Colorado for evidence of chronic wasting disease: geographical and high-risk targeted sample.

A geographically targeted survey of potentially high-risk, adult cattle in chronic wasting disease (CWD)-endemic areas in Colorado was initiated to assess the possibility of the spread of CWD from deer to cattle under natural conditions. Surveyed cattle were sympatric with free-roaming deer in geographically defined areas where CWD occurs and where CWD prevalence has been estimated. To qualify for inclusion in the survey, cattle had to be at least 4 years old and had to have spent a minimum of 4 years in surveyed areas. Brains from culled cattle were examined microscopically and immunohistochemically for tissue alterations indicative of a transmissible spongiform encephalopathy (TSE). Two hundred sixty-two brains were suitable for evaluation and were found to lack changes indicative of a TSE infection. Prion deposition was not demonstrable using a method involving formic acid and proteinase-K treatment before application of monoclonal antibody to bovine prion protein (F99/97.6.1). Some incidental neuropathologic changes unrelated to those of TSEs were detected. Findings from this study suggest that large-scale spread of CWD from deer to cattle under natural range conditions in CWD-endemic areas of northeast Colorado is unlikely.     

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.     

Second passage of sheep scrapie and transmissible mink encephalopathy (TME) agents in raccoons (Procyon lotor)

To determine the transmissibility and pathogenicity of sheep scrapie and transmissible mink encephalopathy (TME) agents derived from raccoons (first passage), raccoon kits were inoculated intracerebrally with either TME (one source) or scrapie (two sources-each in separate groups of raccoons). Two uninoculated raccoon kits served as controls. All animals in the TME-inoculated group developed clinical signs of neurologic dysfunction and were euthanatized between postinoculation month (PIM) 6 and 8. Raccoons in the two scrapie-inoculated groups manifested similar clinical signs of disease, but such signs were observed much later and the animals were euthanized between PIM 12 and 18. Necropsy revealed no gross lesions in any of the raccoons. Spongiform encephalopathy was observed by use of light microscopy, and the presence of protease-resistant prion protein (PrPres) was detected by use of immunohistochemical (IHC) and Western blot analytic techniques. Results of IHC analysis indicated a distinct pattern of anatomic distribution of PrPres in the TME- and scrapie-inoculated raccoons. These findings confirm that TME and sheep scrapie are experimentally transmissible to raccoons and that the incubation periods and IHC distribution for both agents are distinct. Therefore, it may be possible to use raccoons for differentiating unknown transmissible spongiform encephalopathy (TSE) agents. Further studies, with regard to the incubation period and the pattern of PrPres deposition by use of IHC analysis in bovine spongiform encephalopathy and for other isolates of scrapie, chronic wasting disease, and TME in raccoons are needed before the model can be further characterized for differentiation of TSE agents.     

Comparison of histological lesions and immunohistochemical staining of proteinase-resistant prion protein in a naturally occurring spongiform encephalopathy of free-ranging mule deer (Odocoileus hemionus) with those of chronic wasting disease of captive mule deer

In this investigation, the nature and distribution of histologic lesions and immunohistochemical staining (IHC) of a proteinase-resistant prion protein were compared in free-ranging mule deer (Odocoileus hemionus) dying of a naturally occurring spongiform encephalopathy (SE) and captive mule deer dying of chronic wasting disease (CWD). Sixteen free-ranging deer with SE, 12 free-ranging deer without SE, and 10 captive deer with CWD were examined at necropsy. Tissue sections were stained with hematoxylin and eosin, and duplicate sections were stained with a monoclonal antibody (F89/160.1.5). Histological lesions in the free-ranging deer with SE and captive deer with CWD were found throughout the brain and spinal cord but were especially prominent in the myelencephalon, diencephalon, and rhinencephalon. The lesions were characterized by spongiform degeneration of gray matter neuropil, intracytoplasmic vacuolation and degeneration of neurons, and astrocytosis. IHC was found throughout the brain and retina of deer with SE and CWD. Positive IHC was found in lymphoid tissue of deer with SE and CWD. Histologic lesions and IHC were not found in multiple sections of integument, digestive, respiratory, cardiovascular, endocrine, musculoskeletal, and urogenital systems of deer with SE or CWD. Comparison of histologic lesions and IHC in tissues of free-ranging deer with those of captive deer provides strong evidence that these two diseases are indistinguishable morphologically.     

PrP genetics in ruminant transmissible spongiform encephalopathies

Scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) are prion diseases in ruminants with considerable impact on animal health and welfare. They can also pose a risk to human health and control is therefore an important issue. Prion protein (PrP) genetics may be used to control and eventually eradicate animal prion diseases. The PrP gene in sheep and other representatives of the order Artiodactyles has many polymorphisms of which several are crucial determinants of susceptibility to prion diseases, also known as transmissible spongiform encephalopathies (TSE). This review will present the current understanding of PrP genetics in ruminants highlighting similarity and difference between the species in the context of TSE.     

TSE pathogenesis in cattle and sheep

Many studies have been undertaken in rodents to study the pathogenesis of transmissible spongiform encephalopathies (TSE). Only a few studies have focused on the pathogenesis of bovine spongiform encephalopathy (BSE) and scrapie in their natural hosts. In this review, we summarize the most recent insights into the pathogenesis of BSE and scrapie starting from the initial uptake of TSE agents and crossing of the gut epithelium. Following replication in the gut-associated lymphoid tissues (GALT), TSE agents spread to the enteric nervous system (ENS) of the gut. Infection is then carried through the efferent fibers of the post-ganglionic neurons of the parasympathetic and sympathetic nervous system to the pre-ganglionic neurons in the medulla oblongata of the brain and the thoracic segments of the spinal cord. The differences between the pathogenesis of BSE in cattle and scrapie in sheep are discussed as well as the possible existence of additional pathogenetic routes.     

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.     

Preliminary observations of genetic susceptibility of elk (Cervus elaphus nelsoni) to chronic wasting disease by experimental oral inoculation.

To compare the genetic susceptibility of elk (Cervus elaphus nelsoni) with various alleles of the PRNP gene, which encodes the normal cellular prion protein, to chronic wasting disease (CWD), eight 8-month-old elk calves of 3 genotypes (2 132MM, 2 132LM, and 4 132LL) were orally dosed with CWD-infected brain material from elk. During postinoculation (PI) month 23, both 132MM elk had lost appetite, developed clinical signs of weight loss and central nervous system (CNS) dysfunction, and were euthanized. Two other elk (both 132LM) developed similar clinical signs of disease and were euthanized during PI month 40. All 4 affected elk had microscopic lesions of spongiform encephalopathy (SE), and PrPres, the disease-associated form of the prion protein, was detected in their CNS and lymphoid tissues by use of immunohistochemical (IHC) and Western blot (WB) techniques. These findings indicate that elk with MM and LM at codon 132 are susceptible to orally inoculated CWD. All 4 LL elk are alive at PI year 4 and are clinically normal, which suggests that 132LL elk may have reduced susceptibility to oral infection with CWD-infected material or may have prolonged incubation time.     

Experimental transmission of scrapie agent to susceptible sheep by intralingual or intracerebral inoculation

Scrapie, a transmissible spongiform encephalopathy (TSE), is a naturally occurring fatal neurodegenerative disease of sheep and goats. This study documents survival periods, pathological findings, and the presence of abnormal prion protein (PrP(Sc)) in genetically susceptible sheep inoculated with scrapie agent. Suffolk lambs (AA/RR/QQ at codons 136, 154, and 171, respectively) aged 4 mo were injected by the intralingual (IL) or intracerebral (IC) route with an inoculum prepared from a pool of scrapie-affected US sheep brains. The animals were euthanized when advanced clinical signs of scrapie were observed. Spongiform lesions in the brain and PrPsc deposits in the central nervous system (CNS) and lymphoid tissues were detected by immunohistochemical and Western blot (WB) testing in all the sheep with clinical prion disease. The mean survival period was 18.3 mo for the sheep inoculated by the IL route and 17.6 mo for those inoculated by the IC route. Since the IC method is occasionally associated with anesthesia-induced complications, intracranial hematoma, and CNS infections, and the IL method is very efficient, it may be more humane to use the latter. However, before this method can be recommended for inoculation of TSE agents, research needs to show that other TSE agents can also transmit disease via the tongue.     

Preliminary findings on the experimental transmission of chronic wasting disease agent of mule deer to cattle.

To determine the transmissibility of chronic wasting disease (CWD) to cattle and to provide information about clinical course, lesions, and suitability of currently used diagnostic procedures for detection of CWD in cattle, 13 calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Between 24 and 27 months postinoculation, 3 animals became recumbent and were euthanized. Gross necropsies revealed emaciation in 2 animals and a large pulmonary abscess in the third. Brains were examined for protease-resistant prion protein (PrP(res)) by immunohistochemistry and Western blotting and for scrapie-associated fibrils (SAFs) by negative-stain electron microscopy. Microscopic lesions in the brain were subtle in 2 animals and absent in the third case. However, all 3 animals were positive for PrP(res) by immunohistochemistry and Western blot, and SAFs were detected in 2 of the animals. An uninoculated control animal euthanized during the same period did not have PrP(res) in its brain. These are preliminary observations from a currently in-progress experiment. Three years after the CWD challenge, the 10 remaining inoculated cattle are alive and apparently healthy. These preliminary findings demonstrate that diagnostic techniques currently used for bovine spongiform encephalopathy (BSE) surveillance would also detect CWD in cattle should it occur naturally.     

北美地区鹿的慢性消耗性疾病

鹿慢性消耗性疾病(CWD)是鹿类动物的传染性海绵状脑病(TSE)。它是由朊病毒(PrP)引起的,临床主要表现为慢性型消耗,体重逐渐下降,行为异常,最后致死。该病主要感染北美地区的黑尾鹿、白尾鹿和美洲马鹿,一些野生和家养的反刍动物如牛、绵羊和山羊与染病鹿直接或间接接触也可被感染。目前还不能确定CWD与人和其它动物的TSE类疾病的关系。它的起源、发病机理、传播机制和途径尚不清楚。尽管还没有证据证明CWD可传染给人类,但是它对人类有潜在威胁。