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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 (PrPSc) 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.
La tremblante fait partie des encéphalopathies spongiformes transmissibles (TSE) et est une maladie neuro-dégénérative fatale naturelle rencontrée chez les moutons et chèvres. La présente étude fait état des résultats obtenus quant aux périodes de survie, aux trouvailles pathologiques et à la présence de protéines prions anormales (PrPSc) chez des moutons génétiquement susceptibles inoculés avec l’agent de la tremblante. Des agneaux Suffolk (AA/RR/QQ aux codons 136, 154 et 171, respectivement) âgés de 4 mo ont été injectés par voie intra-linguale (IL) ou intracérébrale (IC) avec un inoculum préparé à partir d’un pool de cerveaux de moutons souffrant de tremblante provenant des États-Unis. Les animaux ont été euthanasiés lorsque des signes cliniques avancés de tremblante étaient observés. Les lésions spongiformes dans le cerveau et des dépôts de PrPSc dans le système nerveux central (CNS) et les tissus lymphoïdes ont été détectés par immunohistochimie et immuno-buvardage (WB) chez tous les moutons présentant une maladie à prion clinique. La période moyenne de survie était de 18,3 mois pour les moutons inoculés par la voie IL et de 17,6 mois pour ceux inoculés par la voie IC. Comme la méthode d’inoculation IC est parfois associée avec des complications dues à l’anesthésie, des hématomes intracrâniens et des infections du CNS, et que la méthode IL est très efficace, il serait plus éthique d’utiliser cette dernière. Toutefois, avant que cette méthode ne soit recommandée pour l’inoculation d’agents de TSE, les recherches doivent démontrer que d’autres agents de TSE peuvent également être transmis via la langue.
(Traduit par Docteur Serge Messier)
Scrapie belongs to a group of diseases known as transmissible spongiform encephalopathies (TSEs). It is a naturally occurring, genetically influenced, fatal neurodegenerative disease of sheep and goats. Infection by the causative agent, considered to be the post-translationally modified form of the host-encoded membrane-bound prion protein (PrPc), leads to spongiform encephalopathy associated with accumulation of the abnormal form of prion protein (PrPSc) in tissues of the nervous and lymphoid systems, as well as in the placenta (1).
The most likely portal of entry in natural scrapie has been suggested to be the alimentary tract; other potential portals, such as scarified skin or the conjunctiva, have been effective experimentally (1). There is a paucity of information on experimental studies with scrapie in Suffolk sheep, the dominant sheep breed in the United States. In particular, the various routes of infection, other than oral and intracerebral (IC) with the US scrapie agent (2), have not been documented previously. This study attempted to partially fill this void by comparing intralingual (IL) and IC administration of the US scrapie agent to genetically susceptible Suffolk sheep.
Nine 4-mo-old Suffolk lambs (4 females and 5 castrated males) were obtained from a scrapie-free sheep flock at the National Animal Disease Center (NADC), Ames, Iowa. All were AA/RR/QQ at codons 136, 154, and 171, respectively, of the PRNP gene. The animals were divided into 2 groups: 4 lambs received the scrapie inoculum by the IL route, and 5 lambs received it by the IC route.
The inoculum (X124) was prepared from a pool of 7 scrapie-affected sheep brains from a single flock (3). All 7 sheep were QQ at codon 171 of the PRNP gene, and their brains were positive by Western blot (WB) analysis. The brains were sonicated, and a final concentration of 10% (w/v) was prepared with phosphate-buffered saline. The animals were each injected with a standard 1 mL of the inoculum.
For the IL route, the lambs were not given any sedation. The inoculum was injected with a 20-gauge needle into the ventral aspect of the tongue, approximately 2.5 cm from the tip. The IC method has been described previously (4). Briefly, the lambs were sedated with xylazine, a midline incision was made in the skin at the junction of the parietal and frontal bones, and a 1-mm hole was trephined through the calvarium. The inoculum was injected into the midbrain with a 22-gauge, 9-cm-long needle as the needle was withdrawn from the brain. The skin incision was closed with a single suture.
The inoculated animals were housed separately in a biosafety level 2 containment facility for 2 wk and then moved to 2 outside pens at NADC. They were fed pelleted growth and maintenance rations that contained no ruminant protein, and clean water was freely available.
One lamb, injected by the IL route, was euthanized 9 d after inoculation for immunohistochemical (IHC) detection of inoculated material in the tongue tissue; at necropsy, only the brain (for negative-control samples) and the tongue were obtained. The other 8 animals were euthanized when advanced clinical signs of scrapie developed. In these 8, a detailed gross examination did not show any lesions. Two sets of representative tissue samples were collected. One set of tissues included representative sections of liver, kidney, spleen, skin, striated muscles (heart, tongue, diaphragm, masseter), tonsils (pharyngeal, palatine), thyroid gland, turbinates, lung, tonsils, intestines (ileum), adrenal gland, lymph nodes (retropharyngeal, mesenteric), pituitary gland, Gasserian ganglion, brain (hemisections of cerebral cortex, cerebellum, superior colliculi, and brainstem, including obex), and eye (retina). For the tongue, a minimum of 8 cross-sections from the site of inoculation of each animal that received an IL injection were obtained. These tissues were fixed in 10% buffered formalin, embedded in paraffin wax, sectioned at 5 μm, and stained with hematoxylin and eosin (H–E) for light microscopy. The 2nd set of tissues was frozen.
All paraffin-embedded tissues and negative-control sections were labeled by an automated IHC method for detection of PrPSc as described previously (2). For WB detection of PrPSc in the brainstem, a commercial kit (Bio-Rad, Marnes-La-Coquette, France) with monoclonal antibody P4 was used as described previously (5).
Except for the lamb euthanized 9 d after inoculation, the animals showed clinical signs of scrapie before euthanasia. Initial signs were a progressive decrease in appetite and associated weight loss. Later signs were fine head tremors, listlessness, progressive problems with locomotion, and terminal sternal recumbency. None of the sheep exhibited obvious pruritus or loss of wool.
Table I shows the survival period after inoculation for the individual animals. The mean was 18.3 mo for the group inoculated by the IL route and 17.6 mo for the group inoculated by the IC route; analysis of variance did not show a significant difference (P = 0.76). Table I also shows the distribution of histologic, IHC, and WB findings. In all the sheep except the lamb euthanized 9 d after inoculation (sheep 3511), microscopic spongiform lesions characteristic of prion disease (Figure 1) were observed, and characteristic PrPSc deposition was present in the CNS samples. In addition, PrPSc deposition was present in the retropharyngeal lymph node and the tonsils (Figure 2), as well as all the tested retinas (Figure 3), of the sheep with clinical scrapie. A single submucosal lymphoid aggregate was detectable in the 3rd-eyelid samples from 1 of the sheep with an IHC-positive brain; however, this follicle contained no detectable PrPSc. Submucosal lymphoid follicles were not present in the other examined 3rd eyelids.
In sheep 3511, a locally extensive cellular infiltrate was noted at the site of inoculation in the tongue. The inflammatory cells were predominantly epithelioid macrophages arranged in broad sheets (Figure 4). At scattered sites within the infiltrate there were small numbers of multinucleated giant cells and multiple small areas of mineralization (Figure 5). In some areas away from the main focus of inflammation, muscle fibers were separated by edema fluid and lesser numbers of macrophages. The IHC-stained section of this area revealed foci of PrPSc within the macrophages (Figure 6).
Except for the tongue of sheep 3511, the IHC-stained sections of non-CNS and nonlymphoid tissues, including striated muscle (heart, diaphragm, and masseter muscle), did not reveal PrPSc. The adrenal medulla of sheep 3514 revealed PrPSc in the cytoplasm of multiple endocrine cells in a focally restricted area.
In the WB analysis with monoclonal antibody P4, brainstem samples from all the sheep except the negative-control lamb were positive for PrPSc, showing the typical profile of 3 bands of proteinase- K-resistant isoforms of PrPSc (Figure 7), which represent the diglycosylated, monoglycosylated, and unglycosylated polypeptides. The molecular pattern of the 3 isoforms in the samples from the animals inoculated by the IC route was similar to the pattern for the animals inoculated by the IL route.
In this study, the localization of PrPSc deposits in brain, lymphoid tissues, and retina in the scrapie-positive sheep was similar to that previously seen in sheep with scrapie after inoculation by the oral or IC route (2). Labeling of variable intensity was seen in various sections of the brain. The labeling was predominantly either particulate and diffuse or multifocal and extensive but also appeared as scattered small aggregates of consolidated plaques. Labeling predominated in the grey matter neuropil as perineuronal accumulations and in the perikaryon. Staining in the white matter was markedly less intense and appeared as scattered aggregates of particles. Labeling of the inner and outer plexiform layers of the retina was characterized by diffuse coalescing particles forming roughly uniform sheets. In IHC-positive lymphoid organs, PrPSc labeling was principally confined to the germinal centers of follicles and was either particulate or appeared as small aggregates of particles. Sparse punctate labeling of cells in the paracortex was noted in some sections.
Labeling of the tongue with PrPSc was documented in 7 of 10 sheep with naturally occurring scrapie in European breeds of sheep (6). However, in the present study, except for the sheep euthanized 9 d after inoculation, which had IHC staining of the tongue, PrPSc labeling was not observed in striated muscles of the scrapie-affected animals. This observation is in accordance with previous findings in 20 animals (cattle, sheep, elk, and raccoons) that had a TSE after experimental inoculation: PrPSc was found by IHC examination in the CNS but not in striated muscle tissues (7). Recent investigations with an enriched WB technique (8,9) have enabled the detection of PrPSc in the tongues of some sheep and elk experimentally infected with the agents of scrapie and chronic wasting disease (CWD), respectively (Richard Bessen, Montana State University, Bozeman, Montana: personal communication, 2006). This technique, however, failed to detect PrPSc in tongues of cattle in which a TSE developed after inoculation with the agent of CWD or that of transmissible mink encephalopathy (TME) (Richard Bessen, Montana State University: personal communication, 2006). The contrast in IHC results between the present study and the European study (6) could be due to differences between scrapie strains, breeds of sheep, natural and experimental inoculation, or the IHC procedure.
Inoculation route apparently did not influence the molecular phenotype of the PrPSc observed in the CNS tissues in our study. Alternative routes of infection and spread of prion disease have been suggested as being responsible for different disease phenotypes, as in cattle with typical and atypical bovine spongiform encephalopathy (9).
In general, the IC route of inoculation is considered to be the fastest means of transmitting TSE agents to susceptible hosts. In a study of sheep injected by the IC route with a different US scrapie inoculum (no. 13-7), the average incubation time was 18.8 mo (2); in the present study, with injection by this route of a different US scrapie inoculum (no. X124), the mean time to development of advanced clinical signs of scrapie was 17.6 mo. The sheep in both studies had clinical scrapie, lesions of spongiform encephalopathy, and PrPSc deposits in their tissues. Although sheep 3504 in our study had a significantly longer survival than the other sheep inoculated by the IC route, no difference in survival time was noted between the IL and IC groups. Recently, hamsters in which TME agent was inoculated into the tongue and 4 other non-neuronal anatomic sites were found to have PrPSc in submandibular lymph nodes and the hypoglossal nucleus in the brain within 2 wk after inoculation (10).
The IC route of inoculation is an invasive surgical procedure that involves the use of light anesthesia and carries the risks of intracranial hematoma, CNS infection, and complications of anesthesia. Aside from being safer, the IL method may mimic the natural route of infection for prion diseases. Development of an inoculation method that has an incubation time comparable to that of the IC method and is safer for the recipient animals is desirable. Although the numbers of animals used in this study were small, the results of the IL method were comparable to those of the IC method, and the former can be performed without general anesthesia. However, before the IL method of inoculation can be recommended for general use in TSE studies, similar studies need to be done with other TSE agents and need to involve larger numbers of susceptible hosts to validate the efficacy of the IL inoculation method.
Results of this study show that the transmission of scrapie via the IL route is possible. In comparison, oral inoculation required an average survival time of 32 mo and resulted in a lower attack rate (2). In another study (9), the IC route required a smaller volume of inoculum than the oral route (1 mL versus 30 mL of a 10% brain suspension), yet had a higher attack rate (100% versus 56%). It therefore appears that under field conditions the oral cavity in general, and the tongue in particular, may serve as a portal of entry for prions to gain access to the host’s PrPSc-susceptible cell population.
We thank Dr. Marcus Kehrli for statistical analysis, Dr. Katherine O’Rourke for the prion antibodies for IHC testing, Drs. Lawayne Nusz and Jean Laufer for clinical assistance, and James Fosse for the photomicrographs. Expert technical assistance was provided by Martha Church, Dennis Orcutt, Joseph Lesan, Deb Clouser, Trudy Tatum, Sharla Van Roekel, Ginny Montgomery, Micky Fenneman, and the TSE animal caretakers.
A grant from the US Department of Defence and a contract from the National Institutes of Health (NO1-NS-0-2327) supported the laboratories of Drs. Bulgin, Rowher, and Gregori.
This study was carried out under the guidelines of the NADC Institutional Animal Care and Use Committee. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.