Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523- 1671, USA1
Department of Veterinary Sciences, University of Wyoming, 1174 Snowy Range Road, University of Wyoming, Laramie, WY 82070, USA 2
Colorado Division of Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, CO 80526-2097, USA3
Colorado State University Veterinary Diagnostic Laboratory, 300 West Drake Road, Fort Collins, CO 80523-1671, USA4
Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, 337 Bustad Hall, Washington State University, Pullman, WA 99164-7030, USA5
Author for correspondence: Edward Hoover.Fax +1 970 491 0523. e-mail ehoover{at}lamar.colostate.edu
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Abstract |
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Introduction |
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Few studies of early preclinical TSE infections have been performed in natural hosts or using probable natural routes of exposure; however, the results have been intriguing. BSE has been orally transmitted to cattle with infectivity detectable in the ileum of calves at 26 weeks post-inoculation (p.i.) (by mouse bioassay) (Wells et al., 1994 ). In another study, scrapie agent infectivity was first detected in the prescapular lymph nodes of goats at 24 weeks post- subcutaneous inoculation (Hadlow et al., 1974
). However, mice inoculated intragastrically with scrapie had detectable infectivity in Peyer's patches and cervical lymph nodes as early as 1 week p.i. (Kimberlin & Walker, 1989
). Thus, it appears that prions can cross the mucous membranes of the digestive tract to initiate infection in lymphoid tissue prior to invasion of the central nervous system and development of clinical disease.
Oral exposure is the most plausible pathway by which the CWD prion may be introduced to deer in nature. Consequently, we chose this means of inoculation in an attempt to demonstrate the feasibility of CWD transmission by this route and to study early lymphoid tissue tropism of the PrPres in deer. Each deer was repeatedly exposed to a known infectious CWD inoculum over a 5-day-period because recent results with scrapie in hamsters indicate repeated oral exposure increases the incidence of infection (Diringer et al., 1998 ). Because mice are relatively resistant to CWD (M. Bruce, personal communication) precluding bioassay, and because several studies have shown that PrPres strongly correlates with disease (McKinley et al., 1983
; Race et al. , 1998
), we employed an enhanced immunostaining method (formic acid, proteinase K and hydrated autoclaving) to detect PrPres in situ. Formic acid and hydrated autoclaving have been previously described for PrPres epitope exposure prior to immunohistochemistry (IHC) (Miller et al., 1994
; van Keulen et al., 1995
). Using these methods, we demonstrate PrPres in regional lymph nodes as early as 6 weeks after oral exposure of deer fawns to the CWD agent.
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Methods |
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Oral inoculation of deer.
Six fawns were inoculated orally with 5 ml of a 40% (w/v) brain homogenate (2 g brain) daily for 5 days using a small syringe inserted into the diastema of the oral cavity. Fawns typically licked and swallowed the material. The homogenate was prepared in normal saline solution from brains of 26 captive mule deer naturally infected with CWD. These deer had characteristic clinical signs and histological lesions of CWD in the brain. The homogenate had characteristic PrPres bands by Western blot and scrapie- associated fibrils by negative stain electron microscopy (E. Williams, personal communication). Using the same protocol, three control fawns were inoculated in a like manner with a 40% brain homogenate from free- ranging mule deer outside the CWD endemic area; these deer were collected from a heavily monitored herd with no immunohistochemical or histological lesions of CWD (M. W. Miller, unpublished data).
Necropsy and tissue collection.
Infected deer were euthanized with sodium pentobarbital given intravenously and necropsied sequentially at 10, 42, 53, 77, 78 and 80 days p.i. (n=6). Control deer were necropsied at 27, 70 and 74 days p.i. (n=3). Days p.i. were calculated from the last day of exposure. Numerous tissues were collected, including ten lymph nodes (mesenteric, ileocaecal, sublumbar, popliteal, prescapular, retropharyngeal, submandibular, parotid, ruminal and abomasal nodes), spleen, bone marrow, thymus, Peyer's patches, tonsil, conjunctiva, spinal cord and brain. Tissues were preserved in neutral- buffered 10% formalin and then trimmed, processed and embedded in paraffin blocks within 7 days.
Immunohistochemical staining.
Prior to staining the fawn tissues, various pre-treatments were tested on tissue sections of obex and tonsil from a positive control CWD mule deer to produce optimal stain enhancement. This was done to maximize staining sensitivity to detect anticipated early accumulation of PrPres in tissues. Sections were treated as follows: (1) hydrated autoclaving at 121 °C for 20 min, (2) immersion of slides in 88% formic acid for 30 min followed by hydrated autoclaving for 20 min, (3) immersion in 25 µg/ml proteinase K for 10 min at 26 °C followed by hydrated autoclaving, (4) immersion in 12·5 µg/ml proteinase K for 10 min followed by hydrated autoclaving, and (5) immersion in 88% formic acid for 30 min, then 25 µg/ml proteinase K for 10 min followed by hydrated autoclaving for 20 min. Immunohistochemical staining on the treated sections followed immediately. Staining intensity and specificity was determined by light microscopy. Of these, protocol no. 5 resulted in the greatest PrPres staining.
Tissue sections were mounted onto positively charged glass slides, deparaffinized and hydrated in preparation for IHC. Tissue treatment performed prior to IHC consisted of slide immersion in 88% formic acid solution for 30 min followed by a rinse in water and immersion in 25 µg/ml proteinase K solution at 26 °C for 10 min. Tissue sections were then autoclaved for 20 min at 121 °C in Tris buffer solution and cooled for 30 min. The treatments were extensive in order to maximally expose epitopes and enhance staining.
IHC employed an automated immunostainer (Ventana Medical Systems) and PrPres monoclonal antibody (MAb) F89/160.1.5, a biotinylated secondary antibody, an alkaline phosphatasestreptavidin conjugate, a substrate chromagen (Fast Red A, naphthol, Fast Red B) and a haematoxylin and bluing counterstain (Ventana Medical Systems). MAb F89/160.1.5 recognizes a conserved epitope on the prion protein of mule deer, elk, sheep and cattle (O'Rourke et al., 1998 ). Positive and negative control tissue sections were included in each run.
Several IHC controls were performed on lymphoid tissues with MAb F89/160.1.5. Lymphoid tissues from 50 deer (collected outside the CWD endemic area) were immunostained using the same methodology as performed on the fawn tissues. IHC on known positive and negative deer tonsil sections was done using MAb F89/160.1.5 substituted by mouse serum or an irrelevant isotype-matched MAb diluted to the same protein concentration as MAb F89/160.1.5. In addition, IHC was performed on a retropharyngeal node section from each fawn with an irrelevant MAb substitution.
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Results |
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Earliest detection of PrPres in orally exposed fawns
PrPres was not detectable in any tissue of the fawn necropsied at 10 days p.i. However, in the fawn necropsied at day 42 p.i., PrPres was detected in follicular germinal centres of the retropharyngeal lymph nodes, Peyer's patches and ileocaecal nodes. Of 119 follicles examined in the retropharyngeal nodes, eight (6·7% of follicles) were PrPres-positive. PrPres also was detected in the retropharyngeal node follicles of all infected fawns examined at later time intervals p.i. (53, 77, 78 and 80 days) (Table 1).
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Clinical signs
No clinical signs of CWD occurred in any of the inoculated deer throughout the course of the study. One fawn incidentally developed severe laryngeal swelling which was resolved completely with antibiotic therapy, and two fawns developed mild diarrhoea; otherwise fawns remained healthy.
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Discussion |
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Cells associated with PrPres were within germinal centres of lymphoid follicles. The staining pattern was morphologically consistent with that of follicular dendritic cells. Experimental inoculation of mice with scrapie or CreutzfeldtJakob disease prions resulted in similar localization of PrPres to follicular dendritic cells (Kitamoto et al., 1991 ; McBride et al., 1992
). In sheep Peyer's patches are chiefly concentrated in the terminal ileum (Reynolds & Pabst, 1984
). Assuming the immunobiology of deer is similar to sheep, it seems probable that initial uptake and propagation of PrP res could occur in the ileal Peyer's patches and tonsils, and within dendritic cells emigrating via the lymphatic system to the ileocaecal and retropharyngeal lymph nodes.
Studies in mice show rapid accumulation of dendritic cells bearing antigen within regional lymph nodes hours after the skin was painted with contact allergens (Cumberbatch & Kimber, 1990 ). By analogy PrPres from inoculum would be expected in draining lymph nodes by 10 days p.i. In that PrPres was not detected in the lymphoid tissue of the day 10 fawn, the PrPres staining in fawns examined at later time-points probably represented accumulating PrPres versus residual inoculum. Interestingly, and in contrast to the sequence postulated above, PrPres was visible in the tonsil only in the two fawns with the longest p.i. intervals, 78 and 80 days. This may indicate lower initial quantities of PrPres in tonsil as compared with retropharyngeal node, perhaps due to the migration route of initially infected dendritic cells, resulting in a longer lag before PrPres accumulates in the tonsil to levels detectable with IHC.
We detected PrPres by IHC as early as 6 weeks p.i. an extraordinarily brief period. Detection of PrPres stain in lymphoid tissues by 6 weeks p.i. suggests that PrPres accumulates at early disease stages. In goats experimentally infected with scrapie, infectivity was not detected until 3 months p.i. (Hadlow et al., 1974
). Given the repeated exposure to a relatively large amount of inoculum over 5 days, it seems logical to presume that infection in these orally inoculated fawns may be accelerated, enabling earlier PrP res detection compared to naturally infected deer. Nevertheless, the present study provides proof in principle that CWD PrP res is detectable after oral exposure. Although the present study design precluded the development of clinical disease, the presence of PrPres has been shown to be strongly correlated with infectivity with other TSEs (Race et al., 1998
).
CWD in deer is similar to scrapie in that the PrPres is disseminated throughout lymphoid tissues (T. Spraker, unpublished data). This disseminated lymphoid infection is unlike some other TSEs, such as BSE, in which PrPres is detected only in the ileal Peyer's patches or not at all (Wells et al., 1998 ). Kimberlin & Walker (1989)
and Williams & Young (1992)
have made an association between infection of the lymphoreticular system and the high transmissibility of scrapie among sheep, similar to the findings described in deer and elk. It is possible that localization of PrPres to lymphoid tissues adjacent to mucosal surfaces promotes prion shedding into the environment via fluids such as saliva or faeces, although the pathway of CWD shedding and potential contagion requires further study.
The exact mode of CWD transmission in nature remains unknown. Scrapie in sheep has been demonstrated in experimental studies to be transmissible via ingestion of foetal membranes from scrapie-positive ewes (Pattison et al., 1972 ). Nevertheless, scrapie transmission in nature remains incompletely understood (Detwiler, 1992
). Understanding mechanisms of shedding and transmission will be important in management of CWD and in providing insights into the pathogenesis of other TSEs.
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Acknowledgments |
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References |
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Cumberbatch, M. & Kimber, I. (1990). Phenotypic characteristics of antigen- bearing cells in the draining lymph nodes of contact sensitized mice. Immunology 71, 404-410.[Medline]
Detwiler, L. A. (1992). Scrapie. Revue Scientifique et Technique Office International des Epizooties 11, 491-537.
Diringer, H. , Roehmel, J. & Beekes, M. (1998). Effect of repeated oral infection of hamsters with scrapie. Journal of General Virology 79, 609-612.[Abstract]
Hadlow, W. J. , Eklund, C. M. , Kennedy, R. C. , Jackson, T. A. , Whitford, H. W. & Boyle, C. C. (1974). Course of experimental scrapie virus infection in the goat. Journal of Infectious Diseases 129, 559-567.[Medline]
Hadlow, W. J. , Kennedy, R. C. & Race, R. E. (1982). Natural infection of Suffolk sheep with scrapie virus. Journal of Infectious Diseases 146, 657-664.[Medline]
Kimberlin, R. H. & Walker, C. A. (1989). Pathogenesis of scrapie in mice after intragastric infection. Virus Research 12, 213-220.[Medline]
Kitamoto, T. , Muramoto, T. , Mohri, S. , Doh-Ura, K. & Tateishi, J. (1991). Abnormal isoform of prion protein accumulates in follicular dendritic cells in mice with CreutzfeldtJakob disease. Journal of Virology 65, 6292-6295 .[Medline]
McBride, P. A. , Eikelenboom, P. , Kraal, G. , Fraser, H. & Bruce, M. E. (1992). PrP protein is associated with follicular dendritic cells of spleens and lymph nodes in uninfected and scrapie-infected mice. Journal of Pathology 168, 413-418.[Medline]
McKinley, M. P. , Bolton, D. C. & Prusiner, S. B. (1983). A protease-resistant protein is a structural component of the scrapie prion. Cell 35, 57-62.[Medline]
Marsh, R. F. & Bessen, R. A. (1993). Epidemiologic and experimental studies on transmissible mink encephalopathy. Developments in Biological Standardization 80, 111-118.[Medline]
Miller, J. M. , Jenny, A. L. , Taylor, W. D. , Race, R. E. , Ernst, D. R. , Katz, J. B. & Rubenstein, R. (1994). Detection of prion protein in formalin-fixed brain by hydrated autoclaving immunohistochemistry for the diagnosis of scrapie in sheep. Journal of Veterinary Diagnostic Investigation 6, 366-368.[Medline]
Miller, M. W. , Wild, M. A. & Williams, E. S. (1998). Epidemiology of chronic wasting disease in captive Rocky Mountain elk. Journal of Wildlife Diseases 34, 532-538.[Abstract]
O'Rourke, K. I. , Baszler, T. V. , Miller, J. M. , Spraker, T. R. , Sadler-Riggleman, I. & Knowles, D. P. (1998). Monoclonal antibody F89/160.1.5 defines a conserved epitope on the ruminant prion protein. Journal of Clinical Microbiology 36, 1750-1755 .
Pattison, I. H. , Hoare, M. N. , Jebbett, J. N. & Watson, W. A. (1972). Spread of scrapie to sheep and goats by oral dosing with foetal membranes from scrapie- affected sheep. Veterinary Record 90, 465-468.[Medline]
Race, R. , Jenny, A. & Sutton, D. (1998). Scrapie infectivity and proteinase K- resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis. Journal of Infectious Diseases 178, 949-953.[Medline]
Reynolds, J. D. & Pabst, R. (1984). The emigration of lymphocytes from Peyer's patches in sheep. European Journal of Immunology 14, 7-13.[Medline]
Spraker, T. R. , Miller, M. W. , Williams, E. S. , Getzy, D. M. , Adrian, W. J. , Schoonveld, G. G. , Spowart, R. A. , O'Rourke, K. I. , Miller, J. M. & Merz, P. A. (1997). Spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus ), white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni) in northcentral Colorado. Journal of Wildlife Diseases 33, 1-6.[Abstract]
van Keulen, L. J. , Schreuder, B. E. , Meloen, R. H. , Poelen-van den Berg, M. , Mooij-Harkes, G. , Vromans, M. E. & Langeveld, J. P. (1995). Immunohistochemical detection and localization of prion protein in brain tissue of sheep with natural scrapie. Veterinary Pathology 32, 299-308.[Abstract]
Wells, G. A. H. , Dawson, M. , Hawkins, S. A. C. , Green, R. B. , Dexter, I. , Francis, M. E. , Simmons, M. M. , Austin, A. R. & Horigan, M. W. (1994). Infectivity in the ileum of cattle challenged orally with bovine spongiform encephalopathy. Veterinary Record 135, 40-41.[Medline]
Wells, G. A. , Hawkins, S. A. , Green, R. B. , Austin, A. R. , Dexter, I. , Spencer, Y. I. , Chaplin, M. J. , Stack, M. J. & Dawson, M. (1998). Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Veterinary Record 142, 103-106.[Medline]
Wild, M. A. & Miller, M. W. (1991). Bottle-raising wild ruminants in captivity. Colorado Division of Wildlife Outdoor Facts, 16.
Wild, M. A. , Miller, M. W. , Baker, D. L. , Gill, R. B. , Hobbs, N. T. & Maynard, B. J. (1994). Comparison of growth rate and milk intake of bottle-raised and dam-raised bighorn sheep, pronghorn antelope, and elk neonates. Journal of Wildlife Management 58, 340-347.
Williams, E. S. & Young, S. (1980). Chronic wasting disease of captive mule deer: a spongiform encephalopathy. Journal of Wildlife Diseases 16, 89-98.[Abstract]
Williams, E. S. & Young, S. (1982). Spongiform encephalopathy of Rocky Mountain elk. Journal of Wildlife Diseases 18, 465-471.[Abstract]
Williams, E. S. & Young, S. (1992). Spongiform encephalopathies in Cervidae. Revue Scientifique et Technique Office International des Epizooties 11, 551-567.
Williams, E. S. & Young, S. (1993). Neuropathology of chronic wasting disease of mule deer (Odocoileus hemionus) and elk (Cervus elaphus nelsoni). Veterinary Pathology 30, 36-45.[Abstract]
Received 1 April 1999;
accepted 15 June 1999.