Scientific
Editorial Australian
VETERINARY
JOURNAL
Prions in skeletal muscle MW BRAZIER, R CAPPAI and SJ COLLINS Department of Pathology, The University of Melbourne, Victoria, 3010
SCIENTIFIC SECTION
EDITOR COLIN WILKS
ASSOCIATE EDITOR KEITH HUGHES,
EDITORIAL COMMITTEE NORMAN ANDERSON, GLENN BROWNING, COLIN CHAPMAN, ROBIN CONDRON, TREVOR FARAGHER, STEVEN HOLLOWAY, KEITH HUGHES, TONY LEPPER, JOCK MCLEAN, CARL PETERSON, ANDREW TURNER, COLIN WILKS
JOURNAL ABSTRACTS ALAN LAWTHER
EDITORIAL ASSISTANT AND DESKTOP PUBLISHING ANNA GALLO
The Australian Veterinary Journal welcomes original contributions on all aspects of veterinary science.
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recent article by Bosque et al1 reports the presence of the disease-associated, protease-resistant, isoform of the prion protein (PrPsc) in the hind limb muscles of mice terminally ill from murine scrapie. Further, disease was transmitted to recipient mice following intracerebral (IC) inoculation of this tissue. This finding has re-ignited concerns regarding the aetiology of variant Creutzfeldt-Jakob disease (vCJD), which is believed to be zoonotically linked to bovine spongiform encephalopathy (BSE)2 through the ingestion of beef products contaminated with central nervous system (CNS) tissue. Whereas lymphatic tissues and the CNS of BSE afflicted animals have been shown to contain high titres of PrPsc and have been precluded from the human food chain, bovine skeletal muscle has been considered safe and remains a major part of the human diet. Although reticuloendothelial and peripheral nervous tissues have been shown to accumulate PrPsc in mouse models after IC or peripheral inoculation,3 there has been little conclusive data concerning skeletal muscle. Using mouse bioassays, Wells et al have reported infectivity in the distal ileum and CNS from BSE afflicted cattle.4,5 Skeletal muscles were also collected for mouse bioassay, but their results, to the best of our knowledge, have yet to be reported in peer-reviewed journals. However, negative results from these studies have been reported to government committees and used as the basis for developing public health policy. Chiesa et al6 detected a high concentration of PrPsc, by Western blot, in the heart and skeletal muscles of transgenic mice expressing a nine-octapeptide insert mutation in the prion protein gene (Prnp). These transgenic mice mimic a form of familial CJD with onset of terminal illness at around 180 days of age. Using a more sensitive Western blotting technique, Bosque et al detected PrPsc in the hind limb muscles of terminally ill, wild type mice that had received an inoculation of scrapie material. They used phosphotungstic acid to precipitate PrPsc from muscle homogenates prior to proteinase K digestion. This type of precipitation is quite selective for PrPsc7 and allows for the analysis of large volumes of a particular tissue homogenate. To achieve a PrPsc signal comparable to that of PrPc the authors required about 2000 times more homogenate of diseased muscle. These small amounts of PrPsc were, however, able to transmit disease to transgenic mice that were over expressing PrP. From these bioassays the authors determined that the skeletal muscles from terminally ill, wild type mice, contained titres of infectivity around 106 ID50 units/g; approximately 1000 times less infectivity than that contained in diseased brain. Bosque et al generated animals that express PrPc almost exclusively in muscle by introducing transgene constructs containing mouse Prnp under the control of myocyte specific promoters into mice, whose Prnp gene had been previously ablated. Following inoculation with PrPsc, the hind limb muscles harbored titres of around 107 ID50/g at the terminal stage of disease. These transgenic mice, with 100-fold higher expression of PrPc in their hind limb muscles than wild type animals, yielded only 10-fold greater PrPsc at the terminal stage of disease. Their results suggest that myocytes (and, similarly, hepatocytes) are not as efficient at propagating PrPsc as neurons and strongly support the notion of another, rate limiting factor being involved in the conversion of PrPc to PrPsc. To address the question of whether the findings of Bosque et al cause concern regarding human consumption of bovine skeletal muscle, a number of factors need to be considered. Their investigation revealed that skeletal muscles of infected wild type mice harboured around 106 ID50 units/g. This titre represents the infectivity of diseased skeletal muscle when inoculated IC into the same species. Intracerebral inoculation has been shown to be between 105 and 109 times more efficient at transmitting infection than oral ingestion.8,9 This alone suggests that the muscle would only have a
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Scientific marginal ability to transmit disease to recipient mice via the oral route. Indeed, ritualistic cannibalism of the skeletal muscle from deceased elders by the adult male members of the Fore community in Papua New Guinea resulted in only occasional transmission of Kuru whereas most transmission arose from ingestion of CNS tissue.10 Even if contaminated skeletal muscle from BSE infected cattle did contain a similar titre to that of experimentally infected mice, the enteral route would dramatically reduce the likelihood of transmission. Nevertheless, repeated oral exposure may increase the risk since Diringer et al8 have shown that multiple oral challenges have a higher rate of transmission than a single exposure. The 'species barrier' between cattle and humans, which has been estimated as an approximately 1000-fold barrier,4 would additionally reduce transmissibility. Finally, it is also likely, although unproven, that high concentrations of PrPsc and prion titres would only be present in skeletal muscle at the terminal stage of disease, and such afflicted animals are unlikely to have reached the human food chain because of routine ante- and post-mortem inspection at abattoirs. However, the types of tissues thought to be of most risk with respect to the current vCJD epidemic, those of the central nervous and reticuloendothelial systems, have been shown to contain high PrP concentrations in the subclinical phase of disease,11,12 which helps explain how high titre tissue may have entered human food supply. Although PrPsc accumulation in, and disease transmissibility from, the hind limb muscles in a murine scrapie model has been demonstrated, the specific inoculation and bioassay techniques employed raise genuine concerns regarding relevance to the vCJD epidemic. To properly address the question of whether skeletal muscle from BSE-infected (including pre-symptomatic)
cattle can cause disease in humans, the most appropriate bioassay model would utilise muscle from bovines slaughtered under normal conditions, with the tissue introduced by the oral route. The issue of species barrier would also need to be addressed by developing an accurate paradigm of the species barrier between cattle and humans.13
References 1. Bosque PJ, Ryou C, Telling G et al. Prions in skeletal muscle. Proc Natl Acad Sci 2002;99:3812-3817. 2. Hill AF, Desbruslais M, Joiner S et al. The same prion strain causes vCJD and BSE. Nature 1997;389:448-450. 3. Sy MS, Gambetti P. Prion replication-once again blaming the dendritic cell. Nat Med 1999;5:1235-1237. 4. Wells GA, Dawson M, Hawkins SA et al. Infectivity in the ileum of cattle challenged orally with bovine spongiform encephalopathy. Vet Rec 1994;135:40-41. 5. Wells GA, Hawkins SA, Green RB et al. Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Vet Rec 1998;142:103-106. 6. Chiesa R, Pestronk A, Schmidt RE et al. Primary myopathy and accumulation of PrPsc-like molecules in peripheral tissues of transgenic mice expressing a prion protein insertional mutation. Neurobiol Dis 2001;8:279-288. 7. Wadsworth JD, Joiner S, Hill AF et al. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet 2001;358:171-180. 8. Diringer H, Roehmel J, Beekes M. Effect of repeated oral infection of hamsters with scrapie. J Gen Virol 1998;79:609-612. 9. Prusiner SB, Cochran SP, Alpers MP. Transmission of scrapie in hamsters. J Infect Dis 1985;152:971-978. 10.Daniel PM. Transmissible degenerative diseases of the nervous system. Proc R Soc Med 1971;64:787-794. 11. Thackray AM, Klein MA, Aguzzi A, Bujdoso R. Chronic sub-clinical prion disease induced by low-dose inoculum. J Virol 2002;76:2510-2517. 12. Hill AF, Collinge J. Species-barrier-independent prion replication in apparently resistant species. APMIS 2002;110:44-53. 13. Collinge J, Palmer MS, Sidle KC et al. Unaltered susceptibility to BSE in transgenic mice expressing human prion protein. Nature 1995;378:779-783.
BOOK REVIEW Veterinary Parasitology, Reference Manual, 5th edn. Foreyt WJ, Iowa State University Press, Ames, 2001, 235 pages. Price US$36.95. ISBN 0 8138 2419 2. he first two chapters of this book provide brief overviews of diagnostic techniques in veterinary parasitology and the taxonomy of parasites. The bulk of the book consists of thirteen chapters on parasites in different host species; dogs, cats, ruminants, llamas, horses, swine, birds, ratites, laboratory animals, North American wildlife, marine mammals, reptiles and people. A concluding chapter illustrates some artefacts encountered in faeces. It is a soft-covered book with metal ring-binding. Each chapter on the major domestic species includes drawings illustrating faecal eggs and oocysts and a summary of the locations of the major parasites in that host. There are also tables listing antiparasitic drugs and zoonotic parasites. Brief notes on identification, clinical signs, diagnosis and drug dose rates are provided for most parasites along with an illustration of the parasite’s life cycle. Black and white photographs illustrate the important diagnostic stages. The chapters on parasites of laboratory animals and people are new to this edition. In addition, the information on anti-parasitic drugs has been updated and the chapter on marine mammals has been revised. The quality of the photographs is generally good. The life cycle diagrams are quite small and this creates problems with some of the more complex life cycles. The chapters on llamas, birds and laboratory animals offer information not readily available in many parasitology text books but, not surprisingly, provide less detail than the chapters on the more mainstream host species. For example, Trixacarus caviae, a common agent of disease in guinea pigs presented to veterinarians, is only mentioned briefly. As with other northern hemisphere texts, a number of parasites important in Australia receive little or no coverage; these include Ixodes holocyclus, Angiostrongylus cantonensis, Boophilus microplus and Haematobia irritans exigua. Not all of the information on anti-parasitic drugs is relevant to Australia. The book does not aim to provide comprehensive advice on the management of parasitic diseases. No mention is made of resistance to anti-parasitic drugs, although a table is included purporting to show the efficacy of various anthelmintics against the common nematodes of sheep and goats. The inclusion of a list of drugs after each parasite may delude some students into the belief that parasite control is simply a question of selecting appropriate drugs. Life is rarely that simple. The book is directed at practitioners, students and technicians and aims to provide a manual for routine veterinary parasitology. It does not claim to be a complete parasitology reference book and readers wishing for detailed information on specific parasitic diseases will need to look elsewhere. Its combination of concise notes and generally clear illustrations do however provide useful summaries of the kind often welcomed by students and which will be of value as a diagnostic aid for practitioners. As such, the author admirably achieves the goals he has set himself.
T
G Coleman Glen Coleman teaches veterinary parasitology at the University of Queensland.
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