ABSTRACT
BACKGROUND: Classic scrapie is a prion disease of sheep and goats that is associated with accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the prion disease of cervids. This study was conducted to determine the susceptibility of white-tailed deer (WTD) to the classic scrapie agent. METHODS: We inoculated WTD (n = 5) by means of a concurrent oral/intranasal exposure with the classic scrapie agent from sheep or oronasally with the classic scrapie agent from goats (n = 6). RESULTS: All deer exposed to the agent of classic scrapie from sheep accumulated PrPSc. PrPSc was detected in lymphoid tissues at preclinical time points, and necropsies in deer 28 months after inoculation showed clinical signs, spongiform lesions, and widespread PrPSc in neural and lymphoid tissues. Western blots on samples from the brainstem, cerebellum, and lymph nodes of scrapie-infected WTD have a molecular profile similar to CWD and distinct from samples from the cerebral cortex, retina, or the original classic scrapie inoculum. There was no evidence of PrPSc in any of the WTD inoculated with classic scrapie prions from goats. CONCLUSIONS: WTD are susceptible to the agent of classic scrapie from sheep, and differentiation from CWD may be difficult.
Subject(s)
Deer , Prion Diseases , Scrapie , Wasting Disease, Chronic , Animals , Sheep , Scrapie/metabolism , Scrapie/pathology , Deer/metabolism , Prion Diseases/metabolism , Prion Diseases/veterinary , PrPSc Proteins/metabolism , Wasting Disease, Chronic/metabolism , Goats/metabolismABSTRACT
Chronic wasting disease (CWD) is a naturally occurring, fatal neurodegenerative disease of cervids. The potential for swine to serve as hosts for the agent of CWD is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following experimental oral or intracranial inoculation. Crossbred piglets were assigned to three groups, intracranially inoculated (n = 20), orally inoculated (n = 19), and noninoculated (n = 9). At approximately the age at which commercial pigs reach market weight, half of the pigs in each group were culled ("market weight" groups). The remaining pigs ("aged" groups) were allowed to incubate for up to 73 months postinoculation (mpi). Tissues collected at necropsy were examined for disease-associated prion protein (PrPSc) by Western blotting (WB), antigen capture enzyme immunoassay (EIA), immunohistochemistry (IHC), and in vitro real-time quaking-induced conversion (RT-QuIC). Brain samples from selected pigs were also bioassayed in mice expressing porcine prion protein. Four intracranially inoculated aged pigs and one orally inoculated aged pig were positive by EIA, IHC, and/or WB. By RT-QuIC, PrPSc was detected in lymphoid and/or brain tissue from one or more pigs in each inoculated group. The bioassay was positive in four out of five pigs assayed. This study demonstrates that pigs can support low-level amplification of CWD prions, although the species barrier to CWD infection is relatively high. However, detection of infectivity in orally inoculated pigs with a mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity.IMPORTANCE We challenged domestic swine with the chronic wasting disease agent by inoculation directly into the brain (intracranially) or by oral gavage (orally). Disease-associated prion protein (PrPSc) was detected in brain and lymphoid tissues from intracranially and orally inoculated pigs as early as 8 months of age (6 months postinoculation). Only one pig developed clinical neurologic signs suggestive of prion disease. The amount of PrPSc in the brains and lymphoid tissues of positive pigs was small, especially in orally inoculated pigs. Regardless, positive results obtained with orally inoculated pigs suggest that it may be possible for swine to serve as a reservoir for prion disease under natural conditions.
Subject(s)
Brain/pathology , Disease Reservoirs/veterinary , Prion Proteins/isolation & purification , Swine Diseases/transmission , Wasting Disease, Chronic/transmission , Animals , Biological Assay/methods , Mice , Swine , Swine Diseases/diagnosis , Wasting Disease, Chronic/diagnosisABSTRACT
Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. The purpose of this experiment was to determine susceptibility of white-tailed deer to the agent of scrapie after intracerebral inoculation and to compare clinical signs and lesions to those reported for chronic wasting disease (CWD). Deer (n = 5) were inoculated with 1 mL of a 10% (wt/vol) brain homogenate derived from a sheep clinically affected with scrapie. A non-inoculated deer was maintained as a negative control. Deer were observed daily for clinical signs of disease and euthanized and necropsied when unequivocal signs of scrapie were noted. One animal died 7 months post inoculation (pi) due to intercurrent disease. Examinations of brain tissue for the presence of the disease-associated abnormal prion protein (PrP(Sc)) by western blot (WB) and immunohistochemistry (IHC) were negative whereas IHC of lymphoid tissues was positive. Deer necropsied at 15-22 months pi were positive for scrapie by IHC and WB. Deer necropsied after 20 months pi had clinical signs of depression and progressive weight loss. Tissues with PrP(Sc) immunoreactivity included brain (at levels of cerebrum, hippocampus, colliculus, cerebellum, and brainstem), trigeminal ganglion, neurohypophysis, retina, spinal cord, and various lymphoid tissues including tonsil, retropharyngeal and mesenteric lymph nodes, Peyer's patches, and spleen. This work demonstrates for the first time that white-tailed deer are susceptible to sheep scrapie by intracerebral inoculation. To further test the susceptibility of white-tailed deer to scrapie these experiments will be repeated with a more natural route of inoculation.
Subject(s)
Brain/pathology , Deer , PrPSc Proteins/metabolism , Scrapie/pathology , Wasting Disease, Chronic/pathology , Animals , Blotting, Western/veterinary , Disease Susceptibility/pathology , PrPSc Proteins/administration & dosage , Scrapie/etiology , Wasting Disease, Chronic/etiologyABSTRACT
The feasibility of exploiting fluorescence spectra of the eye for diagnosis of transmissible spongiform encephalopathies (TSEs) was examined. Retinas from scrapie-positive sheep were compared with scrapie-negative sheep using fluorescence spectroscopy, and distinct differences in the fluorescence intensity and spectroscopic signatures were observed. The characteristic fluorescent signatures are thought to be the result of an accumulation of lipofuscin in the retina. It appears that the eye, in particular the retina, is a useful tissue for noninvasive examination of some neurological pathologies such as scrapie. The development of procedures based on examinations of the eye that permit the detection of neurological disorders in animals holds great promise.
Subject(s)
Encephalopathy, Bovine Spongiform/diagnosis , Prion Diseases/diagnosis , Retina/pathology , Spectrometry, Fluorescence/methods , Animals , Brain/pathology , Cattle , Retina/physiopathology , Scrapie/diagnosisABSTRACT
Transmissible, spongiform encephalopathies including bovine spongiform encephalopathy (BSE) and scrapie are fatal neurodegenerative disorders associated with the presence of an infectious abnormal isoform of normal mammalian proteins called prions. Identification of the prion protein associated with scrapie (PrP(Sc)) in the central nervous system is typically based upon immunoassays including immunohistochemistry (IHC) using formalin-fixed tissues or Western blot (WB) assays using fresh and/or frozen, non-formalin-fixed tissues. Each assay can discriminate between BSE, classical scrapie, and a previously reported strain of scrapie recently identified in the United States named Nor98 scrapie. Different tissue samples are required from the same animal to run these 2 different immunoassays. This may result in inconsistent test results for the same animal. Sampling problems such as collecting insufficient volumes of fresh tissue or less than optimal anatomic location of brainstem for IHC can affect the ability of the test procedures to offer definitive and discriminatory results. Recently, a WB method using formalin-fixed, paraffin-embedded (FFPE) tissue to identify PrP(Sc) was developed that successfully identified PrP(Sc) in sheep affected by classical scrapie. In the current study, the use of this technique to produce discriminatory results identifying classical BSE in bovine tissue and both classical and Nor98 scrapie in ovine tissue using paraffin-embedded brain samples is described. Protein-banding patterns from WB using FFPE tissue were similar to protein-banding patterns produced by WB assays utilizing fresh tissues from the same animals, and results correlated well with the IHC PrP(Sc)-positive staining present in the cerebellum and obex regions of brain samples from these animals.
Subject(s)
Encephalopathy, Bovine Spongiform/diagnosis , PrPSc Proteins/analysis , Scrapie/diagnosis , Animals , Blotting, Western , Brain/pathology , Cattle , Cerebellum/pathology , Diagnosis, Differential , Encephalopathy, Bovine Spongiform/pathology , Immunoassay/methods , Immunohistochemistry , Scrapie/pathology , SheepABSTRACT
Scrapie, a transmissible spongiform encephalopathy (TSE), is a naturally occurring fatal neurodegenerative disease of sheep and goats. The current study documents incubation periods, pathologic findings, and distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemistry and Western blot in tissues of genetically susceptible and resistant neonatal lambs inoculated with pooled brain homogenates from 13 U.S. origin scrapie-affected ewes. Nine Suffolk lambs with genotypes AA/RR/QQ (n = 5) and AA/RR/QR (n = 4) at codons 136, 154, and 171, respectively) were orally inoculated, within 12 hr of birth, with 1 ml of a 10% (w/v) brain homogenate prepared from scrapie-affected sheep brains. Inoculated animals were euthanized when advanced clinical signs of scrapie were observed. All QQ sheep developed clinical signs of scrapie, with a mean survival time of 24 months. Spongiform lesions in the brains and PrP(Sc) deposits in the central nervous system and lymphoid tissues were present in these sheep. None of the QR sheep succumbed to the disease. A previous study that used a larger volume (30 ml of 10% brain suspension) of the same inoculum in 4-month-old Suffolk lambs of susceptible genotype documented longer survival periods (average 32 months), and only 5 of 9 inoculated sheep developed scrapie. Findings of this study suggest that orally exposed neonatal lambs of a susceptible (QQ) genotype exhibit a higher attack rate and shorter incubation period than older (4-month-old) lambs exposed to a larger dose (30x) of the same inoculum.
Subject(s)
Scrapie/transmission , Animals , Animals, Newborn , Brain/pathology , Female , Genetic Predisposition to Disease , Genotype , Male , Scrapie/epidemiology , Scrapie/genetics , Sheep , United States/epidemiologyABSTRACT
Bordetella hinzii is commonly acquired from the respiratory tract of diseased poultry but is generally regarded as nonpathogenic in avian hosts because attempts to demonstrate disease following experimental infection of chickens and turkeys have failed. Recently, with the availability of highly specific DNA-based methods for identification of this agent, it was recognized that some isolates used in previous studies were misidentified at the time of their acquisition as Bordetella avium, B. avium-like, or Alcaligenes faecalis type II, including a subset reported to cause disease in turkey poults. In this study six strains of B. hinzii, genetically distinct and representing all known host species, were evaluated for their ability to colonize and cause disease in turkeys following intranasal administration. Although five strains were able to colonize the tracheas of turkey poults, only a subset induced clinical signs of disease, B. hinzii-specific antibodies, or tracheal lesions. The sixth isolate was undetectable in tracheal swabs obtained 1 or 2 weeks postinfection. Birds of this group displayed no clinical signs and minimal tracheal lesions. All remained B. hinzii seronegative. Three of the six strains, differing in their capacity to colonize and/or cause disease in turkeys, were used to infect chicks intranasally. Only one was able to colonize the trachea but did not induce tracheal lesions. No clinical signs of disease were observed in any chick. These results demonstrate that some strains of B. hinzii are virulent in turkey poults and may asymptomatically colonize chicks, and suggest this agent may be of concern to poultry producers.
Subject(s)
Bordetella Infections/veterinary , Bordetella/classification , Bordetella/pathogenicity , Chickens , Poultry Diseases/microbiology , Turkeys , Animals , Antibodies, Bacterial/blood , Bordetella Infections/blood , Bordetella Infections/microbiology , Bordetella Infections/pathology , Trachea/pathology , VirulenceABSTRACT
Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies (TSE) or prion diseases. Immunoassays that identify disease-associated prion protein (PrP Sc) are integral to the diagnosis of scrapie and other prion diseases. Results obtained by either immunohistochemistry (IHC) or Western blot (WB) assay are generally adequate for the definitive diagnosis. Approved or accepted methods for WB diagnosis of TSEs requires the use of fresh or frozen nonfixed tissue samples, whereas formalin-fixed, paraffin-embedded tissue is required for the localization of PrP Sc by IHC. Because disparate processing methods are used for these accepted diagnostic techniques, separate tissue samples are collected from the same animal. Occasions arise in which there is either insufficient quantity of tissue available to complete analysis by both techniques or initial tissue processing is incompatible with one of the assays. Also, results between the assays may differ because of the vagaries of sampling, especially in case material that contains moderate-to-low levels of PrP Sc. The present article describes a method to conduct a WB assay from the same paraffin-embedded brainstem sample used for the IHC diagnosis of experimentally induced sheep scrapie.
Subject(s)
Blotting, Western/veterinary , Brain Stem/metabolism , PrPSc Proteins/isolation & purification , Scrapie/pathology , Animals , Paraffin Embedding , Sensitivity and Specificity , Sheep , Specimen HandlingABSTRACT
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.
Subject(s)
Brain/pathology , PrPSc Proteins/administration & dosage , Scrapie/pathology , Scrapie/transmission , Tongue/pathology , Administration, Oral , Animals , Female , Genetic Predisposition to Disease , Injections/veterinary , Male , PrPSc Proteins/isolation & purification , Scrapie/genetics , Scrapie/mortality , Sheep , Survival AnalysisABSTRACT
A captive adult male white-tailed deer (Odocoileus virginianus) with wasting and neurologic signs similar to chronic wasting disease (CWD) was evaluated by histopathology, histochemistry, and immunohistochemistry (IHC) for disease-associated prion protein (PrP(d)). On histologic examination, the brainstem had areas of vacuolation in neuropil and extensive multifocal mineralization of blood vessels with occasional occlusion of the lumen. Some of the clinical and pathologic features of this case were similar to the CWD of white-tailed deer. However, the tissues were negative for PrP(d) by IHC. Because the lesions were more prominent in the obex region of the brainstem, it is speculated that this would have resulted in clinical signs similar to CWD in white-tailed deer. To our knowledge, neither cerebrovascular mineralization nor clinicopathologic changes resembling CWD have previously been described in white-tailed deer without the presence of PrP(d). Such a case should be considered in a differential diagnosis of CWD of white-tailed deer.
Subject(s)
Brain Stem/pathology , Deer , Prions/analysis , Wasting Disease, Chronic/etiology , Animals , Diagnosis, Differential , Fatal Outcome , Immunohistochemistry/veterinary , Male , Prion Diseases/diagnosis , Prion Diseases/epidemiology , Prion Diseases/veterinary , Prions/isolation & purification , Wasting Disease, Chronic/epidemiologyABSTRACT
Clinical signs of prion disease are not specific and include a variety of differential diagnoses. Serological tests and nucleic acid-based detection methods are not applicable to prion-disease-agent detection because of the unusual nature of the infectious agent. Prion-disease diagnosis is primarily conducted by means of immunodetection of the infectious agent, typically by at least 2 distinct procedures with immunohistochemistry and Western blot being the most informative. These approaches differ in the need for formalin-fixed and frozen or fresh tissue respectively. This work describes a method for the detection of the disease-associated isoform of the prion protein by Western blot using formalin-fixed tissues. The approach requires only minimal modification of existing Western-blot procedures and could readily be incorporated into existing detection schemes for confirmatory purposes when fresh or frozen tissues are unavailable.
Subject(s)
Blotting, Western/veterinary , Formaldehyde , PrPSc Proteins/analysis , Tissue Fixation/veterinary , Animals , Brain Stem/chemistry , Protein Isoforms , Scrapie/diagnosis , Sheep , Time FactorsABSTRACT
Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy of cattle, first detected in 1986 in the United Kingdom and subsequently in other countries. It is the most likely cause of variant Creutzfeldt-Jakob disease (vCJD) in humans, but the origin of BSE has not been elucidated so far. This report describes the identification and characterization of two cases of BSE diagnosed in the United States. Case 1 (December 2003) exhibited spongiform changes in the obex area of the brainstem and the presence of the abnormal form of the prion protein, PrP(Sc), in the same brain area, by immunohistochemistry (IHC) and Western blot analysis. Initial suspect diagnosis of BSE for case 2 (November 2004) was made by a rapid ELISA-based BSE test. Case 2 did not exhibit unambiguous spongiform changes in the obex area, but PrP(Sc) was detected by IHC and enrichment Western blot analysis in the obex. Using Western blot analysis, PrP(Sc) from case 1 showed molecular features similar to typical BSE isolates, whereas PrP(Sc) from case 2 revealed an unusual molecular PrP(Sc) pattern: molecular mass of the unglycosylated and monoglycosylated isoform was higher than that of typical BSE isolates and case 2 was strongly labeled with antibody P4, which is consistent with a higher molecular mass. Sequencing of the prion protein gene of both BSE-positive animals revealed that the sequences of both animals were within [corrected] the range of the prion protein gene sequence diversity previously reported for cattle.
Subject(s)
Brain/pathology , Encephalopathy, Bovine Spongiform/diagnosis , Amino Acid Sequence , Animals , Base Sequence , Cattle , DNA/genetics , DNA/isolation & purification , Encephalopathy, Bovine Spongiform/pathology , Goats , Molecular Sequence Data , Polymerase Chain Reaction , Prions/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , Sheep , United StatesABSTRACT
This communication documents age-associated pathologic changes and final observations on experimental transmission of chronic wasting disease (CWD) by the intracerebral route to raccoons (Procyon lotor). Four kits were inoculated intracerebrally with a brain suspension from mule deer with CWD. Two uninoculated kits served as controls. One CWD-inoculated raccoon was humanely killed at 38 months after inoculation, and 1 control animal died at 68 months after inoculation. Both animals had lesions that were unrelated to transmissible spongiform encephalopathy. Six years after inoculation, none of the 3 remaining CWD-inoculated raccoons had shown clinical signs of neurologic disorder, and the experiment was terminated. Spongiform encephalopathy was not observed by light microscopy, and the presence of abnormal prion protein (PrP(d)) was not detected by either immunohistochemistry or Western blot techniques. Age-related lesions observed in these raccoons included islet-cell pancreatic amyloidosis (5/6), cystic endometrial hyperplasia (3/4), cerebrovascular mineralization (5/6), neuroaxonal degeneration (3/6), transitional-cell adenoma of the urinary bladder (1/6), and myocardial inclusions (4/6). The latter 2 pathologic conditions were not previously reported in raccoons.
Subject(s)
Aging , Deer , Raccoons , Wasting Disease, Chronic/pathology , Wasting Disease, Chronic/transmission , Animals , PrionsABSTRACT
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.
Subject(s)
Deer , Sheep Diseases/etiology , Wasting Disease, Chronic/transmission , Animals , Autopsy/veterinary , Blotting, Western , Central Nervous System/chemistry , Central Nervous System/pathology , Cerebellum/chemistry , Cerebellum/pathology , Immunity, Innate/genetics , Immunohistochemistry , Medulla Oblongata/pathology , Palatine Tonsil/chemistry , Palatine Tonsil/pathology , Prions/analysis , Scrapie/genetics , Scrapie/immunology , Sheep , Sheep Diseases/pathology , Wasting Disease, Chronic/pathologyABSTRACT
Amino acid polymorphisms of the prion protein (PrP) greatly influence the susceptibility of sheep to scrapie. Selective breeding to increase the prevalence of PrP gene alleles associated with scrapie resistance is a flock management practice that is important for scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. We describe application of a DNA extraction procedure for formalin-fixed, paraffin-embedded tissues (PET) for the purpose of PCR amplification and nucleotide sequencing of relevant codons (136-171) of the sheep PrP gene. Tissues derived from 96 sheep were studied. The DNA sequence identity was confirmed in 87 of 94 matched samples of PET and frozen tissue specimens. DNA from brainstem PET of 2 sheep, from which fresh tissue was not available, was amplified and sequenced after formalin fixation for 7-70 days. This method will allow retrospective analysis of PrP genetics of sheep subsequent to postmortem diagnosis of scrapie when nonfixed tissue is unavailable for DNA extraction; however, it is not recommended that submission of fixed tissue supplant collection of fresh tissues for the purpose of determining PrP gene polymorphisms.
Subject(s)
Prions/genetics , Scrapie/genetics , Sheep/genetics , Animals , Brain Stem/chemistry , DNA/genetics , DNA/isolation & purification , Genetic Predisposition to Disease , Genotype , Paraffin Embedding/veterinary , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/veterinary , Polymorphism, Single Nucleotide , Retrospective StudiesABSTRACT
A naturally occurring prion disease has not been recognized in swine, but the agent of bovine spongiform encephalopathy does transmit to swine by experimental routes. Swine are thought to have a robust species barrier when exposed to the naturally occurring prion diseases of other species, but the susceptibility of swine to the agent of sheep scrapie has not been thoroughly tested. We conducted this experiment to test the susceptibility of swine to U.S. scrapie isolates by intracranial and oral inoculation. Scrapie inoculum was a pooled 10% (w/v) homogenate derived from the brains of clinically ill sheep from the 4th passage of a serial passage study of the U.S scrapie agent (No. 13-7) through susceptible sheep (homozygous ARQ at prion protein residues 136, 154, and 171, respectively). Pigs were inoculated intracranially (n=19) with a single 0.75â mL dose or orally (n=24) with 15â mL repeated on 4 consecutive days. Necropsies were done on a subset of animals at approximately six months post inoculation (PI): the time the pigs were expected to reach market weight. Remaining pigs were maintained and monitored for clinical signs of transmissible spongiform encephalopathies (TSE) until study termination at 80 months PI or when removed due to intercurrent disease (primarily lameness). Brain samples were examined by immunohistochemistry (IHC), western blot (WB), enzyme immunoassay (EIA), and for a subset of pigs in each inoculation group, bioassay in mice expressing porcine prion protein. At six-months PI, no evidence of scrapie infection was noted by any diagnostic method. However, at 51 months of incubation or greater, 5 animals were positive by one or more methods: IHC (n=4), WB (n=3), or EIA (n=4). Furthermore, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study) suggesting that swine are potential hosts for the agent of scrapie.
ABSTRACT
Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent on the genetic makeup of the host. This study documents clinicopathological findings and the distribution of abnormal prion proteins (PrPres) by immunohistochemical and Western blot techniques, in tissues of genetically susceptible sheep inoculated with US sheep scrapie agents. Four-month-old Suffolk lambs (QQ or HQ at codon 171) were inoculated (5 intracerebrally and 19 orally) with an inoculum (#13-7) consisting of a pool of scrapie-affected sheep brains. Intracerebrally inoculated animals were euthanized when advanced clinical signs of scrapie were observed. Orally inoculated animals were euthanized at predetermined time points (4, 9, 12, 15, and 21 months postinoculation [PI]) and thereafter when the animals had terminal signs of disease. All intracerebrally inoculated animals exhibited clinical signs of scrapie and were euthanized between 13 and 24 months PI. Spongiform lesions in the brains and PrPres deposits in central nervous system and lymphoid tissues were present in these sheep. In orally inoculated sheep, clinical signs of scrapie were seen between 27 and 43 months PI in 5/9 animals. The earliest detectable PrPres was observed in brainstem and lymphoid tissues of a clinically normal, orally inoculated sheep at 15 months PI. Three of the 4 clinically normal sheep were positive at 15, 20, and 49 months PI by PrPres immunohistochemistry.
Subject(s)
Scrapie/genetics , Scrapie/transmission , Administration, Oral , Animals , Brain/pathology , Female , Genetic Predisposition to Disease , Injections/veterinary , Lymph Nodes/pathology , Male , Prions , Scrapie/pathology , Sheep , United StatesABSTRACT
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.
Subject(s)
Cattle Diseases/transmission , Deer , Wasting Disease, Chronic/transmission , Animals , Cattle , Cattle Diseases/pathology , Medulla Oblongata/pathology , Wasting Disease, Chronic/pathologyABSTRACT
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.
Subject(s)
Deer , Scrapie/transmission , Animals , Autopsy/veterinary , Brain/pathology , Case-Control Studies , Diagnosis, Differential , Immunohistochemistry , Prions/analysis , Sheep , Wasting Syndrome/veterinaryABSTRACT
Sheep scrapie is a transmissible spongiform encephalopathy that can be transmitted horizontally. The prion protein gene (PRNP) profoundly influences the susceptibility of sheep to the scrapie agent and the tissue levels and distribution of PrPSc in affected sheep. The purpose of this study was to compare the survival time and PrPSc tissue distribution in sheep with highly resistant and highly susceptible PRNP genotypes after intracranial inoculation of the agent of scrapie. Five sheep each of genotype VRQ/VRQ, VRQ/ARR or ARQ/ARR were inoculated. Sheep were euthanized when clinical signs of scrapie became severe. Clinical signs, microscopic lesions, and western blot profiles were uniform across genotypes and consistent with manifestations of classical scrapie. Mean survival time differences were associated with the 171 polymorphic site with VRQ/VRQ sheep surviving 18 months, whereas VRQ/ARR and ARQ/ARR sheep survived 60 and 56 months, respectively. Labeling of PrPSc by immunohistochemistry revealed similar accumulations in central nervous system tissues regardless of host genotype. Immunoreactivity for PrPSc in lymphoid tissue was consistently abundant in VRQ/VRQ, present but confined to tonsil or retropharyngeal lymph node in 4/5 VRQ/ARR, and totally absent in ARQ/ARR sheep. The results of this study demonstrate the susceptibility of sheep with the ARQ/ARR genotype to scrapie by the intracranial inoculation route with PrPSc accumulation in CNS tissues, but prolonged incubation times and lack of PrPSc in lymphoid tissue.