1 Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE 68178, USA
2 Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, WI 53706, USA
Correspondence
Richard Bessen
rbessen{at}montana.edu
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ABSTRACT |
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Present address: Department of Veterinary Molecular Biology, PO Box 173610, Montana State University, Bozeman, MT 59717, USA.
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INTRODUCTION |
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In flocks of sheep with endemic scrapie or in free-ranging deer populations with a high incidence of chronic wasting disease, there is an increased opportunity for exposure of an individual animal to multiple prion strains. Experimental infection of mice with two strains of scrapie at separate times (i.e. superinfection) by intraperitoneal (i.p.) inoculation can result in murine scrapie strain 22A (a long-incubation-period strain) blocking disease caused by scrapie strain 22C (a short-incubation-period strain) (Dickinson et al., 1972, 1975
). As a result, mice die from disease caused by 22A scrapie and there is no evidence of 22C scrapie infection in the brain. The basis for this blocking effect is proposed to be due to 22A scrapie replication in a majority of the limited number of replication sites in the spleen and other secondary lymphoid tissues, and as a result 22C scrapie infection is prevented upon superinfection (Dickinson & Fraser, 1979
; Dickinson et al., 1975
). This model is supported by reports that demonstrate that the outcome of superinfection depends upon the dose of the inoculated prion strains and the time interval separating the two infections. Greater blocking effects are achieved by either increasing the dose of 22A scrapie or by increasing the time interval before inoculation of 22C scrapie (Dickinson & Fraser, 1979
), indicating that this effect is mediated by a higher amount or longer period of 22A scrapie replication prior to superinfection. The ability of 22A scrapie to block 22C scrapie following extraneural infection is also dependent on inoculation of a dose of 22A that results in clinical disease, since this effect is lost upon prior heat or chemical inactivation of 22A scrapie (Kimberlin & Walker, 1985
). Both a delay and a blocking effect on a fast prion strain by a slow prion strain have been reported following superinfection by the intracerebral (i.c.) route of inoculation (Dickinson et al., 1972
; Manuelidis, 1998
). In the former case, mice inoculated with 22A scrapie agent died from a subsequent 22C scrapie superinfection but the time to onset of disease was protracted compared with a control 22C scrapie infection (Dickinson et al., 1972
).
Experimental i.c. co-infection (i.e. simultaneous inoculation) with the HY (a short-incubation-period strain) and DY (a long-incubation-period strain) strains of transmissible mink encephalopathy (TME) illustrates that the DY TME agent can block HY TME disease, and as a result hamsters develop clinical signs of DY TME (Bartz et al., 2000). Subsequent serial passage of brain tissue from co-infected animals exhibiting DY TME disease can result in either the emergence of HY TME disease or maintenance of the DY TME phenotype, depending on the doses used during the initial co-infection. In the former case, both TME strains are able to replicate during co-infection, but upon serial passage, the dose of the short incubation HY TME agent becomes greater than it was during the initial co-infection and is responsible for causing clinical disease. The strain-specific PrPSc pattern also switches from that of the DY PrPSc conformation to that of the HY PrPSc conformation during the transition from clinical symptoms of DY TME to HY TME upon serial prion passage (Bartz et al., 2000
). The blocking effects of DY TME co-infection on HY TME are only observed following i.c. inoculation and not by co-infection by the i.p. route. This latter finding is most likely due to the inability of the DY TME agent to cause clinical disease more than 300 days post-infection following i.p. inoculation (Bessen & Marsh, 1992b
).
In the current study, we investigated the ability of the DY TME agent to either delay or block HY TME infection following superinfection by intrasciatic nerve (i.n.) and i.p. routes of inoculation. We have demonstrated that prior inoculation of the DY TME agent can delay the onset of HY TME disease following i.p. inoculation, despite the inability of the DY TME agent to replicate in the lymphoreticular and nervous system tissues by this route of inoculation. Conversely, inoculation of the DY TME agent into the sciatic nerve did not delay HY TME disease upon superinfection, even though the DY TME agent can replicate and cause disease in the absence of superinfection. These findings suggest that either (i) the DY TME agent can replicate at locations other than the secondary lymphoid organs and nervous system following i.p. inoculation and that competition with the HY TME agent at these sites upon extraneural superinfection results in a delay in the progression of HY TME infection; or (ii) low levels of DY TME replication in the lymphoreticular system or nervous system, which cannot be detected by animal bioassay, are able to delay HY TME disease upon superinfection.
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METHODS |
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For the TME superinfection studies described in Fig. 2, DY TME brain homogenates were inoculated either at 30 or 60 days prior to the HY TME agent inoculation, and the incubation period was measured from the time of superinfection. The dose of the TME agent inoculated is given in i.c. LD50. Inoculation of a normal brain homogenate at 60 days prior to the HY TME agent inoculation was used as a control.
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Tissue preparation and Western blot of PrPSc.
For PrPSc analysis of brain from clinically ill hamsters, a 5 % (w/v) homogenate in Dulbecco's PBS without Ca2+ or Mg2+ (Mediatech) was digested with 0·4 U proteinase K ml-1 (Roche Diagnostics). Homogenates were incubated at 37 °C for 1 h with constant agitation followed by the addition of PMSF to a concentration of 5 mM. Proteinase K-digested brain homogenates (0·25 mg equivalents) were analysed for PrPSc content by SDS-PAGE and Western blotting as described below.
The spleen and medial iliac lymph node were homogenized in 10 mM Tris/HCl, pH 7·5, containing 5 mM MgCl2 to produce a 20 % (w/v) tissue suspension. Brain from hamsters that did not develop clinical symptoms was homogenized in PBS containing 5 mM MgCl2 to make a 10 % (w/v) homogenate. Tissue homogenates were incubated with 100 U Benzonase nuclease ml-1 (Novagen) at 37 °C for 1 h with constant agitation. An equal volume of buffer A (10 mM Tris/HCl, pH 7·5, 1 mM dithiothreitol) containing 20 % (w/v) N-lauroylsarcosine was added and the samples were mixed on a Vortex Genie (VWR Scientific) for 30 min at room temperature. Enrichment for PrPSc and proteinase K digestion were performed as previously described (Bartz et al., 2002). SDS-PAGE and Western blotting were performed as previously described (Bartz et al., 2002
; Bessen & Marsh, 1994
) using monoclonal anti-PrP antibody 3F4 ascites fluid at a 1 : 40 000 dilution (a gift from R. Kascsak, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA) or 3F4 hybridoma supernatant at a 1 : 10 000 dilution (a gift from V. Lawson, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT, USA) (Kascsak et al., 1987
).
Statistical analysis.
Incubation period data from hamsters infected with TME agents were compared using SigmaStat 2.0 software (SPSS Inc.). The t-test (paired) or the non-parametric MannWhitney U-test was used depending on whether the data had a normal distribution pattern or not. In all comparisons, a P value less than 0·01 was used to determine whether two datasets were statistically different (Fig. 2).
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RESULTS |
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In the TME superinfection experiment (Fig. 2), all hamsters that developed TME had a clinical phenotype characteristic of HY TME and also had a 21 kDa PrPSc polypeptide pattern that was characteristic of the HY TME strain (Fig. 3
). None of the hamsters exhibited symptoms of DY TME or had evidence of the 19 kDa PrPSc polypeptide pattern in the brain that is typical for DY TME infection (Fig. 3
). These results indicated that, following superinfection with the DY and HY TME agents by i.p. inoculation, hamsters acquired an HY TME infection in the brain and this strain was responsible for causing TME.
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In the absence of HY TME superinfection, DY TME infection of the sciatic nerve resulted in PrPSc deposition in the vertebral segments of the thoracic spinal cord (corresponding to the lumbar to mid-thoracic spinal cord segments) at 90 days p.i., but not at 60 day p.i., indicating that the DY TME agent entered the spinal cord from the sciatic nerve between 60 and 90 days p.i. (Fig. 4A). Following HY TME infection of the sciatic nerve, in the absence of prior DY TME infection, HY PrPSc was found in vertebral thoracic spinal cord segments 1013 at 28 days post-infection (Fig. 4B
). Therefore, the earliest time point that HY PrPSc could reach the spinal cord when i.n. inoculated at 60 days after the DY TME agent would be 88 days after the initial DY TME agent inoculation (60+28 days). The presence of DY PrPSc in the thoracic and lumbar spinal cord at the time of HY PrPSc entry into the lumbar spinal cord was not sufficient to delay or block the onset of HY TME following superinfection of the sciatic nerve.
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DISCUSSION |
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These studies also demonstrate that the LRS replication-deficient DY TME agent can delay the onset of HY TME following HY TME agent superinfection by the i.p. route, but not by an i.n. route of inoculation. In previous studies with murine-adapted scrapie and CreutzfeldtJakob disease prions, the long-incubation-period prion strains could delay or block the short-incubation-period strains only if they were able to cause disease in the absence of superinfection (Dickinson et al., 1972, 1975
; Kimberlin & Walker, 1985
; Manuelidis, 1998
; Manuelidis & Yun Lu, 2000
). The current findings are paradoxical, since the DY TME agent does not cause TME or replicate in the LRS and nervous system following i.p. inoculation, yet it was able to delay HY TME following i.p. superinfection. An explanation for these findings could be that the DY TME brain inoculum induced an immune response to host components in the inoculum or to the DY TME agent, which is cross-reactive with the HY TME agent. The former outcome is unlikely since a normal brain homogenate inoculated prior to the HY TME brain homogenate did not delay disease onset. Furthermore, there was no delay in the onset of HY TME following superinfection at 30 days after DY TME agent inoculation, indicating that an adaptive immune response to normal brain components did not delay HY TME infection. The production of an immune response to the DY TME agent that can affect HY TME infection is also unlikely because a more efficacious immune response would be expected during an infection in which the DY TME agent is replicating compared with a situation where no DY TME agent is found. However, under conditions in which the DY TME agent does not replicate in the LRS and nervous system (e.g. i.p. inoculation), there was a statistically significant delay in the HY TME incubation period, but in experiments where the DY TME agent could replicate (e.g. i.n. inoculation), there was no delay following HY TME superinfection. Therefore, DY TME agent replication does not correlate with an undefined protective anti-prion immune response or the ability to delay HY TME disease onset following superinfection. We cannot exclude the possibility that i.p. inoculation of the DY TME agent induces an immune response to the TME agent in the absence of LRS replication, but other studies (Garfin et al., 1978
; Kasper et al., 1982
; Kingsbury et al., 1981
; Porter et al., 1973
) have been unsuccessful in identifying a prion-specific immune response or antibodies to PrPSc following prion infection.
In the current study, we were unable to demonstrate TME agent strain competition in the peripheral and central nervous systems following i.n. inoculation. Our findings are different from previous ones (Dickinson et al., 1972; Manuelidis, 1998
), which demonstrate that a slow prion strain can delay or block a fast prion strain following i.c. inoculation, despite the presence of DY TME replication in the spinal cord prior to the spread of the HY TME agent into the cord from the sciatic nerve. One explanation could be that there are fewer common replication sites between the TME agent strains following i.n. compared with i.c. inoculation, especially if the HY and DY TME agents use different spinal tracts to ascend to the brain. We previously demonstrated that the HY TME agent can travel in the retrograde direction within descending spinal motor tracts following i.n. inoculation (Bartz et al., 2002
) but the pathways used by the DY TME agent strain in the spinal cord are uncertain. Perhaps, if the DY TME agent is more dependent on transport within sensory spinal tracts to spread to the brain, then following superinfection with the HY TME agent, the fast TME strain can travel to the brain with minimal overlap of replication sites used by the DY TME agent. In this case, there would not be sufficient competition between the TME strains to result in a delay or block of HY TME infection. Another possible explanation for the absence of a delay in the HY TME incubation period following i.n. superinfection could be that the length of time to HY TME superinfection was too short and, as a result, there was insufficient DY TME agent replication to affect the subsequent HY TME infection. However, it is noteworthy that, in previous prion superinfection studies (Dickinson et al., 1972
; Manuelidis, 1998
) in which a slow prion strain blocked a fast prion strain, 1822 % of the incubation period of the slow prion strain elapsed before inoculation of the fast prion strain. In the present study, the HY TME agent was i.n. inoculated after 29 % of the anticipated DY TME incubation period by the i.n. route had elapsed (at day 60 of a 210 day incubation period). Therefore, a proportionally longer time period for DY TME agent replication was permitted in the sciatic nerve and/or spinal cord prior to HY TME agent superinfection but competition between the TME agent strains was not found.
The mechanism by which the DY TME agent delays the onset of clinical signs of HY TME following superinfection by the i.p. route could be dependent on replication of the two TME agent strains in a common tissue replication site. In the current study, both TME strains can replicate in the nervous system but this site is unlikely to be the location of agent strain competition since (i) there was no evidence of DY TME agent replication in the peripheral and central nervous systems following i.p. inoculation; and (ii) superinfection by the i.n. route did not result in a delay in HY TME disease onset, even though DY TME agent replication in the spinal cord preceded HY TME agent spread to the spinal cord. One possible site for TME agent strain competition following i.p. superinfection is in the LRS, even though we were unable to detect PrPSc deposition or DY TME infectivity in secondary lymphoid tissues. In this scenario, low levels of DY TME agent replication may occur in the lymph nodes since we did not directly assay for DY TME infectivity at this site. DY TME replication in lymph nodes could partially block access of the HY TME agent to replication sites and/or delay subsequent HY TME infection of peripheral nerves, which is necessary for spread to the brain. We also noticed that in HY TME-infected hamsters, the PrPSc load per weight of tissue was greater in the medial iliac lymph node than in the spleen and, although we were unable to detect PrPSc in the lymph nodes of DY TME-infected hamsters, the small size of these tissues restricted PrPSc analysis. It is also noteworthy that in Golden Syrian hamsters infected with the 263K scrapie strain, splenectomy does not delay the onset of clinical disease following extraneural inoculation (Kimberlin & Walker, 1977, 1986
), suggesting that other secondary lymphoid tissues are sufficient to support scrapie agent replication prior to neuroinvasion. Studies with immunodeficient mice have also demonstrated that scrapie infection of lymph nodes can occur in the absence of spleen infection (Prinz et al., 2002
), suggesting that within the LRS there are differences in prion targeting. Currently, we are examining lymph nodes in DY TME-infected hamsters for evidence of prion infection. Another potential explanation for our extraneural TME superinfection studies is that TME agent strain competition can occur at a site whose location precedes infection of both the LRS and nervous system. Peritoneal macrophages or other macrophage subsets are potential targets of TME agent replication that could play a role in the early pathogenesis of prion diseases.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bartz, J. C., Kincaid, A. E. & Bessen, R. A. (2002). Retrograde transport of transmissible mink encephalopathy within descending motor tracts. J Virol 76, 57595768.
Bessen, R. A. & Marsh, R. F. (1992a). Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent. J Virol 66, 20962101.[Abstract]
Bessen, R. A. & Marsh, R. F. (1992b). Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters. J Gen Virol 73, 329334.[Abstract]
Bessen, R. A. & Marsh, R. F. (1994). Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol 68, 78597868.[Abstract]
Bruce, M. E. (1985). Agent replication dynamics in a long incubation period model of mouse scrapie. J Gen Virol 66, 25172522.[Abstract]
Collis, S. C. & Kimberlin, R. H. (1985). Long-term persistence of scrapie infection in mouse spleens in the absence of clinical disease. FEMS Microbiology Lett 29, 111114.[CrossRef]
Dickinson, A. G. & Fraser, H. (1979). The scrapie replication-site hypothesis and its implications for pathogenesis. In Slow Transmissible Diseases of the Nervous System, pp. 1231. Edited by S. B. Prusiner & W. J. Hadlow. New York: Academic Press.
Dickinson, A. G., Fraser, H., Meikle, V. M. & Outram, G. W. (1972). Competition between different scrapie agents in mice. Nat New Biol 237, 244245.[Medline]
Dickinson, A. G., Fraser, H., McConnell, I., Outram, G. W., Sales, D. I. & Taylor, D. M. (1975). Extraneural competition between different scrapie agents leading to loss of infectivity. Nature 253, 556.
Garfin, D. E., Stites, D. P., Perlman, J. D., Cochran, S. P. & Prusiner, S. B. (1978). Mitogen stimulation of splenocytes from mice infected with scrapie agent. J Infect Dis 138, 396400.[Medline]
Kascsak, R. J., Rubenstein, R., Merz, P. A., Tonna-DeMasi, M., Fersko, R., Carp, R. I., Wisniewski, H. M. & Diringer, H. (1987). Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J Virol 61, 36883693.[Medline]
Kasper, K. C., Stites, D. P., Bowman, K. A., Panitch, H. & Prusiner, S. B. (1982). Immunological studies of scrapie infection. J Neuroimmunol 3, 187201.[CrossRef][Medline]
Kimberlin, R. H. & Walker, C. (1977). Characteristics of a short incubation model of scrapie in the golden hamster. J Gen Virol 34, 295304.[Abstract]
Kimberlin, R. H. & Walker, C. A. (1985). Competition between strains of scrapie depends on the blocking agent being infectious. Intervirology 23, 7481.[Medline]
Kimberlin, R. H. & Walker, C. A. (1986). Pathogenesis of scrapie (strain 263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly. J Gen Virol 67, 255263.[Abstract]
Kingsbury, D. T., Smeltzer, D. A., Gibbs, C. J., Jr & Gajdusek, D. C. (1981). Evidence for normal cell-mediated immunity in scrapie-infected mice. Infect Immun 32, 11761180.[Medline]
McBride, P. A., Schulz-Schaeffer, W. J., Donaldson, M., Bruce, M., Diringer, H., Kretzschmar, H. A. & Beekes, M. (2001). Early spread of scrapie from the gastrointestinal tract to the central nervous system involves autonomic fibers of the splanchnic and vagus nerves. J Virol 75, 93209327.
Manuelidis, L. (1998). Vaccination with an attenuated CreutzfeldtJakob disease strain prevents expression of a virulent agent. Proc Natl Acad Sci U S A 95, 25202525.
Manuelidis, L. & Yun Lu, Z. (2000). Attenuated CreutzfeldtJakob disease agents can hide more virulent infections. Neurosci Lett 293, 163166.[CrossRef][Medline]
National Research Council (1996). Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press.
Porter, D. D., Porter, H. G. & Cox, N. A. (1973). Failure to demonstrate a humoral immune response to scrapie infection in mice. J Immunol 111, 14071410.[Medline]
Prinz, M., Montrasio, F., Klein, M. A., Schwarz, P., Priller, J., Odermatt, B., Pfeffer, K. & Aguzzi, A. (2002). Lymph nodal prion replication and neuroinvasion in mice devoid of follicular dendritic cells. Proc Natl Acad Sci U S A 99, 919924.
Prusiner, S. B. (1991). Molecular biology of prion diseases. Science 252, 15151522.[Medline]
Schulz-Schaeffer, W. J., Fatzer, R., Vandevelde, M. & Kretzschmar, H. A. (2000). Detection of PrP(Sc) in subclinical BSE with the paraffin-embedded tissue (PET) blot. Arch Virol Suppl 16, 173180.[Medline]
Somerville, R. A., Birkett, C. R., Farquhar, C. F. & 8 other authors (1997). Immunodetection of PrPSc in spleens of some scrapie-infected sheep but not BSE-infected cows. J Gen Virol 78, 23892396.[Abstract]
Spongiform Encephalopathy Advisory Council (2001). Statement on Infectivity in Bovine Tonsil. London: DEFRA, DH, FSA.
Terry, L. A., Marsh, S., Ryder, S. J., Hawkins, S. A., Wells, G. A. & Spencer, Y. I. (2003). Detection of disease-specific PrP in the distal ileum of cattle exposed orally to the agent of bovine spongiform encephalopathy. Vet Rec 152, 387392.[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. Vet Rec 142, 103106.[Medline]
Received 4 June 2003;
accepted 1 October 2003.