Discriminating Scrapie and Bovine Spongiform Encephalopathy Isolates by Infrared Spectroscopy of Pathological Prion Protein

Abstract

For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27–30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27–30. However, FT-IR spectroscopy revealed that PrP27–30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in β-sheet and other secondary structure elements of PrP27–30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27–30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species. For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27–30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27–30. However, FT-IR spectroscopy revealed that PrP27–30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in β-sheet and other secondary structure elements of PrP27–30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27–30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species. Transmissible spongiform encephalopathies (TSEs) 1The abbreviations used are: TSE, transmissible spongiform encephalopathy; BSE, bovine spongiform encephalopathy; FT-IR, Fourier-transform infrared; mAb, monoclonal antibody; TBS, Tris-buffered saline; BE, brain equivalents; TME, transmissible mink encephalopathy; dpi, days postinfection. such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans are invariably fatal neurodegenerative disorders of the central nervous system. After the initial reports on the emergence of BSE and variant Creutzfeldt-Jakob disease, in 1986 and 1996, respectively, compelling evidence has gradually accumulated that the latter can most likely be attributed to transmissions, presumably via contaminated food, of BSE agent from cattle to man (1Bruce M.E. Will R.G. Ironside J.W. McConnell I. Drummond D. Suttie A. McCardle L. Chree A. Hope J. Birkett C. Cousens S. Fraser H. Bostock C.J. Nature. 1997; 389: 498-501Crossref PubMed Scopus (1727) Google Scholar, 2Cousens S.N. Linsell L. Smith P.G. Chandrakumar M. Wilesmith J.W. Knight R.S.G. Zeidler M. Stewart G. Will R.G. Lancet. 1999; 353: 18-21Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 3Hill A.F. Desbruslais M. Joiner S. Sidle K.C. Gowland I. Collinge J. Doey L.J. Lantos P. Nature. 1997; 389: 448-526Crossref PubMed Scopus (1208) Google Scholar, 4Scott M.R. Will R. Ironside J. Nguyen H.O.B. Tremblay P. DeARmond S.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15137-15142Crossref PubMed Scopus (462) Google Scholar). Therefore, effective infection control measures for the containment and repression of BSE have become a matter of crucial importance to public health. According to the present state of knowledge, the countermeasures implemented in response to the BSE epidemic are expected to minimize or even eliminate the risk of new primary variant Creutzfeldt-Jakob disease infections of humans directly originating from bovines (5Bradley R. Rabenau H.F. Cinatl J. Doerr H.W. Prions: A Challenge for Science, Medicine, and the Public Health System. S. Karger AG, Basel, Switzerland2004: 146-185Google Scholar). However, further challenges in the area of infection control arise from the hypothetical risk that the BSE agent might have spread via contaminated feed such as meat and bone meal to sheep (5Bradley R. Rabenau H.F. Cinatl J. Doerr H.W. Prions: A Challenge for Science, Medicine, and the Public Health System. S. Karger AG, Basel, Switzerland2004: 146-185Google Scholar) and that BSE, like scrapie, might now be sustained in the ovine population. The clinical symptoms of scrapie, which has been endemic in sheep for centuries without any apparent association with human disease, cannot be reliably distinguished from those exhibited by experimentally challenged BSE-infected ovines. “While it is possible to demonstrate the presence of a TSE by several laboratory techniques using microscopy, electron microscopy, or immunological methods which detect the abnormal form of the prion protein, distinguishing between one strain of scrapie and another, and between BSE and scrapie, is not straightforward” (6, Spongiform Encephalopathy Advisory Committee (1999) http://www.seac.gov.uk/publicats/sub-rep.pdf,Google Scholar). So far, reliable differentiation of BSE and scrapie in sheep has required time-consuming and expensive strain-typing in mice using lesion profiles (7Fraser H. Dickinson A.G. J. Comp. Pathol. 1973; 83: 29-40Crossref PubMed Scopus (279) Google Scholar). Therefore, the development of new methods for an inexpensive, robust, and rapid discrimination between BSE and scrapie constitutes a topical challenge in the surveillance of ovine TSEs addressed in a variety of studies (8Baron T.G. Madec J.Y. Calavas D. J. Clin. Microbiol. 1999; 37: 3701-3704Crossref PubMed Google Scholar, 9Hope J. Wood S.C. Birkett C.R. Chong A. Bruce M.E. Cairns D. Goldmann W. Hunter N. Bostock C.J. J. Gen. Virol. 1999; 80: 1-4Crossref PubMed Scopus (145) Google Scholar). During the past few years, considerable progress has been achieved in this field of TSE research, predominantly by using immunobiochemical techniques (10Kuczius T. Groschup M.H. Mol. Med. 1999; 5: 406-418Crossref PubMed Google Scholar, 11Stack M.J. Chaplin M.J. Clark J. Acta Neuropathol. 2002; 104: 279-286Crossref PubMed Scopus (182) Google Scholar, 12Lezmi S. Martin S. Simon S. Comoy E. Bencsik A. Deslys J.P. Grassi J. Jeffrey M. Baron T. J. 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Therefore, the of the of the prion the secondary structure the of as for the phenotypic characteristics of TSE During the past few it has been that several different TSE strains can be distinguished by immunobiochemical typing of the electrophoretic mobilities and glycosylation characteristics of in the J. Sidle K.C. J. Ironside J. A.F. Nature. PubMed Scopus Google Scholar, P. R. Capellari S. Ghetti B. M. Gambetti P. PubMed Scopus Google Scholar, P. Capellari S. Gambetti P. N. P. T. J. Giese A. Kretzschmar H. Nature. 1997; PubMed Scopus Google Scholar, Chong A. Birkett C.R. Wood S.C. Hope J. Nature. 1997; PubMed Scopus Google Scholar). strains of hamster-adapted transmissible mink encephalopathy and the of PrP27–30 from and by with exhibited different apparent molecular of and J. Virol. PubMed Google Scholar). evidence for the presence of in and most be for by in the of the prion protein that were from with an with these a has further evidence that from and as as from other hamster-adapted TSE strains have in the structure of J. H. D. H. M. Prusiner S.B. Med. PubMed Scopus Google Scholar). Fourier transform-infrared (FT-IR) spectroscopy used to directly the structure of prion with different TSE the were and substantially by that from and in different β-sheet B. G.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). as as to phenotypic information of TSE agents or mirrored in the secondary structure of FT-IR structural of or PrP27–30, a that in the secondary structure of the protein, as a tool for the molecular typing and differentiation of TSE agents. this the in this to in laboratory animals TSE agents that TSEs and the challenge of distinguishing isolates that can be by the clinical symptoms or neuropathological by the immunobiochemical of For this Syrian were as animals as have the between different TSE strains and and of such isolates in the it is possible to reliably and other TSE agents by FT-IR structural characterization of PrP27–30 from the the on an that FT-IR of PrP27–30 potentially a for the differentiation of TSE agents, those that are or even to by a variety of approaches for strain the animals used in our study have information this may be of a that the rapid and reliable discrimination of strains in nonexperimental TSEs of animals and humans. TSE and of hamster-adapted scrapie strains ME7-H, and 22A-H and of a new hamster-adapted BSE by infection of Syrian with of in from scrapie strain J. Gen. Virol. PubMed Scopus Google Scholar) by R. H. and has been passaged for in our and 22A-H by and in Fraser H. J. Gen. Virol. PubMed Scopus Google Scholar), were by the for of and of 22A-H were used for the of these TSE agents the in our laboratory one of BSE agent from cattle in mice and to hamsters. H. and M. in of brain in from a BSE in a from the were between and days to fatal disease with clinical symptoms of transmissible spongiform of brain in from a were hamsters. symptoms between and in For the present all were on the of ME7-H, and 22A-H in our which showed of and days as the of disease, respectively, and on a of with an of in further not of ME7-H, and 22A-H the in our hamsters. were for clinical symptoms and by the of After the were and further as were in for with for to in for and After in an were in from the were and in for The of as (7Fraser H. Dickinson A.G. J. Comp. Pathol. 1973; 83: 29-40Crossref PubMed Scopus (279) Google Scholar). of S. N. W. D. Giese A. Groschup M.H. Kretzschmar H.A. J. Pathol. Full Text Full Text PDF PubMed Scopus Google Scholar) of brain as M. Bruce M. H. Kretzschmar H.A. M. J. Virol. PubMed Scopus Google Scholar). were in for with for to in for and using a brain After in an were in were on and for For to S. N. W. D. Giese A. Groschup M.H. Kretzschmar H.A. J. Pathol. Full Text Full Text PDF PubMed Scopus Google Scholar), were in TBS, with and with in a and for were in in in and with R. M. R. H. J. Virol. PubMed Google After with the secondary for were with and to the The were for using a For brain were in of to with secondary of of brain in TBS, were with of and of and for M. E. S. H. P. P. J. Gen. Virol. PubMed Scopus Google Scholar). The by of in and and for of the to of brain were in a Nature. PubMed Scopus Google Scholar) or in and using the The were with in for and with in bovine in After in and for with the secondary in bovine in a for used After the in TBS, to using a of and as The of and in each were by for a of the glycosylation were in for each with from four animals with 22A-H, and the of to Different of brain in from with the four different TSE strains the of were in to of and were with as a of for After the by and for were to and with as FT-IR of and prion protein PrP27–30 from the of Syrian the of disease, using a by H. M. M. Simon D. I. M. Ironside J.W. 1997; PubMed Scopus Google Scholar) with some were used of as each of the that required of new were used of by in of in to of the protein with from the The in of to with and and and of brain and in a using a for The and the protein were For of the protein were with of and for The protein by with as M. E. S. H. P. P. J. Gen. Virol. PubMed Scopus Google Scholar). The of the protein by and as M. E. S. H. P. P. J. Gen. Virol. PubMed Scopus Google Scholar), and PrP27–30 also by using as The of protein in the in the of FT-IR of PrP27–30 were with a FT-IR applied for a and a of used an of For each spectrum were and FT-IR were and between and the with to The FT-IR were in the of an FT-IR with a For FT-IR of in the protein from and using a for in of in to a protein to of from the of the were used to for from the protein The of PrP27–30 were from in with a of were to an from in one of which a with a PrP27–30 from of ME7-H, 22A-H, and were each For and the were between and to of the were using a the study on approaches for the characterization and discrimination of TSE agents in would also be for a strain differentiation field such as the or techniques such as strain typing by lesion in mice (7Fraser H. Dickinson A.G. J. Comp. Pathol. 1973; 83: 29-40Crossref PubMed Scopus (279) Google Scholar, M.E. Med. PubMed Scopus Google Scholar) as and the for and with scrapie showed and of animals were and in by and which and and from a animals challenged with or 22A-H scrapie agent exhibited with and scrapie and animals were and not by or and to from a of or as in scrapie were not but the animals showed of with were also to and in a and However, in to the and apparent to by and as in and 22A-H scrapie, exhibited from a and an to to the which showed of and without the scrapie were to from a and not and and symptoms to scrapie and from each other as as from and However, a discrimination between the latter not clinical the lesion profiles caused by ME7-H, 22A-H, and profiles provide a tool for the typing of TSE strains in mice by the and the of in brain (7Fraser H. Dickinson A.G. J. Comp. Pathol. 1973; 83: 29-40Crossref PubMed Scopus (279) Google Scholar). 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A for all TSE agents the but the β-sheet for each of the individual isolates for for ME7-H, for and for the β-sheet were in the of and ME7-H, for the is only for the 22A-H, the as a of the β-sheet these that the PrP27–30 from the different TSE agents in β-sheet and respectively, TSE agents (263K, and which to PrP27–30 from and showed also and respectively, which might be attributed to the of β-sheet J. PubMed Scopus Google Scholar, H. PubMed Scopus Google Scholar). strains showed a with which is to D. PubMed Scopus Google Scholar, H. of Scholar). and were for 22A-H and such be in the of and The of this have been to from structure in several H. PubMed Scopus Google Scholar). However, the might also an structure to by between and or by the of to S. J. Chem. PubMed Scopus Google Scholar, D. 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The for this is but might be for by of the agents to our of Syrian hamsters. with the or immunobiochemical methods all four and 22A-H, be by FT-IR characterization of pathological prion on the that PrP27–30 from or showed attributed to in β-sheet structure but also to in other secondary structure TSE with in the past few a of has several of evidence that with TSE agents in the and with different J. Sidle K.C. J. Ironside J. A.F. Nature. PubMed Scopus Google Scholar, P. R. Capellari S. Ghetti B. M. Gambetti P. PubMed Scopus Google Scholar, J. Virol. PubMed Google Scholar, J. H. D. H. M. Prusiner S.B. Med. PubMed Scopus Google Scholar). has also been by FT-IR on PrP27–30 from different TSE strains B. G.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to the study by B. G.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the of PrP27–30 from our this has the of the of to secondary structure may be by the of A. D. 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PubMed Scopus Google Scholar) or the of an as or in the agent to be of this study the of FT-IR spectroscopy for TSE strain that structural characterization of pathological prion protein is to of PrP27–30 from different TSE agents, which can be by immunobiochemical The and of FT-IR spectroscopy in this have to be by of the to a spectrum of TSE agents, host species, and possible in this to scrapie and BSE agents from sheep, such studies also isolates from with different of or human the emergence of new BSE in cattle has been from and A.G. Ryder S. Baron T. 2004; 5: PubMed Scopus Google Scholar, C. G. P. S. L. S. M. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar) and the as as public FT-IR of pathological prion protein a tool that not only to the further of the isolates these BSE cases but would potentially improve the surveillance of TSE agents in as as We for and this by and We also and A. for for in to scrapie strains and 22A-H and for the BSE to mice and hamsters.