Hugh W. Reid

Rabbits are not resistant to prion infection

The ability of prions to infect some species and not others is determined by the transmission barrier. This unexplained phenomenon has led to the belief that certain species were not susceptible to transmissible spongiform encephalopathies (TSEs) and therefore represented negligible risk to human health if consumed. Using the protein misfolding cyclic amplification (PMCA) technique, we were able to overcome the species barrier in rabbits, which have been classified as TSE resistant for four decades. Rabbit brain homogenate, either unseeded or seeded in vitro with disease-related prions obtained from different species, was subjected to serial rounds of PMCA. De novo rabbit prions produced in vitro from unseeded material were tested for infectivity in rabbits, with one of three intracerebrally challenged animals succumbing to disease at 766 d and displaying all of the characteristics of a TSE, thereby demonstrating that leporids are not resistant to prion infection. Material from the brain of the clinically affected rabbit containing abnormal prion protein resulted in a 100% attack rate after its inoculation in transgenic mice overexpressing rabbit PrP. Transmissibility to rabbits (>470 d) has been confirmed in 2 of 10 rabbits after intracerebral challenge. Despite rabbits no longer being able to be classified as resistant to TSEs, an outbreak of “mad rabbit disease” is unlikely.

Susceptibility to scrapie and disease phenotype in sheep: cross-PRNP genotype experimental transmissions with natural sources.

It has long been established that the sheep Prnp genotype influences the susceptibility to scrapie, and some studies suggest that it can also determine several aspects of the disease phenotype. Other studies, however, indicate that the source of infection may also play a role in such phenotype. To address this question an experiment was set up in which either of two different natural scrapie sources, AAS from AA136 Suffolk and VVC from VV136 Cheviot sheep, were inoculated into AA136, VA136 and VV136 sheep recipients (nu2009=u200952). The immunohistochemical (IHC) profile of disease-associated PrP (PrPd) accumulation in the brain of recipient sheep was highly consistent upon codon 136 homologous and semi-homologous transmission, but could be either similar to or different from those of the inoculum donors. In contrast, the IHC profiles were highly variable upon heterologous transmission (VVC to AA136 and AAS to VV136). Furthermore, sheep of the same Prnp genotype could exhibit different survival times and PrPd profiles depending on the source of infection, and a correlation was observed between IHC and Western blot profiles. It was found that additional polymorphisms at codons 112 or 141 of AA136 recipients resulted in a delayed appearance of clinical disease or even in protection from infection. The results of this study strongly suggest that the scrapie phenotype in sheep results from a complex interaction between source, donor and recipient factors, and that the Prnp genotype of the recipient sheep does not explain the variability observed upon codon 136 heterologous transmissions, arguing for other genetic factors to be involved.

Genetic variability of the prion protein gene (PRNP) in wild ruminants from Italy and Scotland

The genetics of the prion protein gene (PRNP) play a crucial role in determining the relative susceptibility to transmissible spongiform encephalopathies (TSEs) in several mammalian species. To determine the PRNP gene variability in European red deer (Cervus elaphus), roe deer (Capreolus capreolus) and chamois (Rupicapra rupicapra), the PRNP open reading frame from 715 samples was analysed to reveal a total of ten single nucleotide polymorphisms (SNPs). In red deer, SNPs were found in codons 15, 21, 59, 78, 79, 98, 136, 168 and 226. These polymorphisms give rise to 12 haplotypes, and one of which is identical to the PRNP of American wapiti (Rocky Mountain elk, Cervus elaphus nelsoni). One silent mutation at codon 119 was detected in chamois and no SNPs were found in roe deer. This analysis confirmed that European wild ruminants have a PRNP genetic background that is compatible with TSE susceptibility, including chronic wasting disease.

Disease phenotype in sheep after infection with cloned murine scrapie strains

Prion diseases exhibit different disease phenotypes in their natural hosts and when transmitted to rodents, and this variability is regarded as indicative of prion strain diversity. Phenotypic characterization of scrapie strains in sheep can be attempted by histological, immunohistochemical and biochemical approaches, but it is widely considered that strain confirmation and characterization requires rodent bioassay. Examples of scrapie strains obtained from original sheep isolates by serial passage in mice include ME7, 79A, 22A and 87V. In order to address aspects of prion strain stability across the species barrier, we transmitted the above murine strains to sheep of different breeds and susceptible Prnp genotypes. The experiment included 40 sheep dosed by the oral route alone and 36 sheep challenged by combined subcutaneous and intracerebral routes. Overall, the combined route produced higher attack rates (~100%) than the oral route (~50%) and 2–4 times shorter incubation periods. Uniquely, 87V given orally was unable to infect any sheep. Overall, scrapie strains adapted and cloned in mice produce distinct but variable disease phenotypes in sheep depending on breed or Prnp genotype. Further re-isolation experiments in mice are in progress in order to determine whether the original cloned murine disease phenotype will reemerge.

Susceptibility of European Red Deer (Cervus elaphus elaphus) to Alimentary Challenge with Bovine Spongiform Encephalopathy

European red deer (Cervus elaphus elaphus) are susceptible to the agent of bovine spongiform encephalopathy, one of the transmissible spongiform encephalopathies, when challenged intracerebrally but their susceptibility to alimentary challenge, the presumed natural route of transmission, is unknown. To determine this, eighteen deer were challenged via stomach tube with a large dose of the bovine spongiform encephalopathy agent and clinical signs, gross and histological lesions, presence and distribution of abnormal prion protein and the attack rate recorded. Only a single animal developed clinical disease, and this was acute with both neurological and respiratory signs, at 1726 days post challenge although there was significant (27.6%) weight loss in the preceding 141 days. The clinically affected animal had histological lesions of vacuolation in the neuronal perikaryon and neuropil, typical of transmissible spongiform encephalopathies. Abnormal prion protein, the diagnostic marker of transmissible encephalopathies, was primarily restricted to the central and peripheral nervous systems although a very small amount was present in tingible body macrophages in the lymphoid patches of the caecum and colon. Serial protein misfolding cyclical amplification, an in vitro ultra-sensitive diagnostic technique, was positive for neurological tissue from the single clinically diseased deer. All other alimentary challenged deer failed to develop clinical disease and were negative for all other investigations. These findings show that transmission of bovine spongiform encephalopathy to European red deer via the alimentary route is possible but the transmission rate is low. Additionally, when deer carcases are subjected to the same regulations that ruminants in Europe with respect to the removal of specified offal from the human food chain, the zoonotic risk of bovine spongiform encephalopathy, the cause of variant Creutzfeldt-Jakob disease, from consumption of venison is probably very low.

Correlation between infectivity and disease associated prion protein in the nervous system and selected edible tissues of naturally affected scrapie sheep.

The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100–1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies.

Canine distemper virus and badgers.

I WAS intrigued by the speculation from Mike Davies regarding the possible re-emergence of canine distemper virus (CDV) ( VR , February 15, 2014, vol 174, p 178), as there is a possibility that this could provide insight into another …

Deer and man

Gardens of Earthly Delight: The History of Deer Parks John Fletcher 296 pages, paperback, £25. Windgather Press. 2011. ISBN 978 1 905119 36 3 nnnnnnTHE author states that this book is a record of his ‘daydreams’ while hiding up trees waiting to tranquillise deer with a dart gun. It is, however, so much more, being a thoroughly researched and referenced analysis of not only deer and their interactions with man over the millennia, but also our relationships with animals and their domestication. As a veterinary graduate who has devoted his life to all aspects of deer management, John Fletcher is an established world authority and is pre-eminently well qualified to provide a book on ‘The History of Deer Parks’. The book explores mans relationship with …