BSE infectivity survives burial for five years with only limited spread.

The carcasses of animals infected with bovine spongiform encephalopathy (BSE), scrapie or chronic wasting disease (CWD) that remain in the environment (exposed or buried) may continue to act as reservoirs of infectivity. We conducted two experiments under near-field conditions to investigate the survival and dissemination of BSE infectivity after burial in a clay or sandy soil. BSE infectivity was either contained within a bovine skull or buried as an uncontained bolus of BSE-infected brain. Throughout the five-year period of the experiment, BSE infectivity was recovered in similar amounts from heads exhumed annually from both types of soil. Very low levels of infectivity were detected in the soil immediately surrounding the heads, but not in samples remote from them. Similarly, there was no evidence of significant lateral movement of infectivity from the buried bolus over 4 years although there was a little vertical movement in both directions. However, bioassay analysis of limited numbers of samples of rain water that had drained through the bolus clay lysimeter indicated that infectivity was present in filtrates. sPMCA analysis also detected low levels of PrPSc in the filtrates up to 25 months following burial, raising the concern that leakage of infectivity into ground water could occur. We conclude that transmissible spongiform encephalopathy infectivity is likely to survive burial for long periods of time, but not to migrate far from the site of burial unless a vector or rain water drainage transports it. Risk assessments of contaminated sites should take these findings into account.

The Risk of Prion Infection through Bovine Grafting Materials.

BACKGROUND: Bovine-derived grafting materials are frequently used in a variety of bone augmentation techniques. The aim of this paper is to assess the unique safety issue of bovine-derived grafting materials that is rarely addressed in dental literature: risk of bovine spongiform encephalopathy (BSE). METHODS: The validity of the current BSE diagnostic methods, surveillance and epidemiological trends in affected countries, and BSE infectivity in bovine bone before and after manufacturing processing were reviewed and analyzed. RESULTS: Prion screening has significant limits. Humans are not safe from the infection of prion disease of other species. Prions can and do break the species barrier. There is evidence there may be tens of thousands of infectious carriers in the western countries alone. This raises concern about the potential for perpetuation of infection via medical procedures. CONCLUSION: The limited ability to screen prions within the animal genome, along with a long latency period to manifestation of the disease (1 to over 50 years) in infected patients, provides a framework for discussing posible long-term risks of the xenografts that are used so extensively in dentistry. We suggest abolishing the use of bovine bone.

The prion protein gene polymorphisms associated with bovine spongiform encephalopathy susceptibility differ significantly between cattle and buffalo.

Prion protein, encoded by the prion protein gene (PRNP), plays a crucial role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Several polymorphisms within the PRNP are known to be associated with influencing bovine spongiform encephalopathy (BSE) susceptibility in cattle, namely two insertion/deletion (indel) polymorphisms (a 23-bp indel in the putative promoter and a 12-bp indel in intron 1), the number of octapeptide repeats (octarepeats) present in coding sequence (CDS) and amino acid polymorphisms. The domestic buffaloes, Bubalus bubalis, are a ruminant involved in various aspects of agriculture. It is of interest to ask whether the PRNP polymorphisms differ between cattle and buffalo. In this study, we analyzed the previously reported polymorphisms associated with BSE susceptibility in Chinese buffalo breeds, and compared these polymorphisms in cattle with BSE, healthy cattle and buffalo by pooling data from the literature. Our analysis revealed three significant findings in buffalo: 1) extraordinarily low deletion allele frequencies of the 23- and 12-bp indel polymorphisms; 2) significantly low allelic frequencies of six octarepeats in CDS and 3) the presence of S4R, A16V, P54S, G108S, V123M, S154N and F257L substitutions in buffalo CDSs. Sequence alignments comparing the buffalo coding sequence to other species were analyzed using the McDonald-Kreitman test to reveal five groups (Bison bonasus, Bos indicus, Bos gaurus, Boselaphus tragocamelus, Syncerus caffer caffer) with significantly divergent non-synonymous substitutions from buffalo, suggesting potential divergence of buffalo PRNP and others. To the best of our knowledge this is the first study of PRNP polymorphisms associated with BSE susceptibility in Chinese buffalo. Our findings have provided evidence that buffaloes have a unique genetic background in the PRNP gene in comparison with cattle.

Bioassay studies support the potential for iatrogenic transmission of variant Creutzfeldt Jakob Disease through dental procedures.

BACKGROUND: Evidence is required to quantify the potential risks of transmission of variant Creutzfeldt Jakob (vCJD) through dental procedures. Studies, using animal models relevant to vCJD, were performed to address two questions. Firstly, whether oral tissues could become infectious following dietary exposure to BSE? Secondly, would a vCJD-contaminated dental instrument be able to transmit disease to another patient? METHODS: BSE-301V was used as a clinically relevant model for vCJD. VM-mice were challenged by injection of infected brain homogenate into the small intestine (Q1) or by five minute contact between a deliberately-contaminated dental file and the gingival margin (Q2). Ten tissues were collected from groups of challenged mice at three or four weekly intervals, respectively. Each tissue was pooled, homogenised and bioassayed in indicator mice. FINDINGS: Challenge via the small intestine gave a transmission rate of 100% (mean incubation 157±17 days). Infectivity was found in both dental pulp and the gingival margin within 3 weeks of challenge and was observed in all tissues tested within the oral cavity before the appearance of clinical symptoms. Following exposure to deliberately contaminated dental files, 97% of mice developed clinical disease (mean incubation 234±33 days). INTERPRETATION: Infectivity was higher than expected, in a wider range of oral tissues, than was allowed for in previous risk assessments. Disease was transmitted following transient exposure of the gingiva to a contaminated dental file. These observations provide evidence that dental procedures could be a route of cross-infection for vCJD and support the enforcement of single-use for certain dental instruments.

Transmissible spongiform encephalopathy strain-associated diversity of N-terminal proteinase K cleavage sites of PrP(Sc) from scrapie-infected and bovine spongiform encephalopathy-infected mice.

Assessment of the different conformational states of the abnormal prion protein (PrP(Sc)) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrP(Sc) conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrP(res)) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrP(res) presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography-mass spectrometry (LC-MS) was used to determine PrP(res) N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrP(res) at G(81), G(85), G(89) and G(91). Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G(91)-K(105) was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G(91)-K(105):G(89)-K(105) was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.

David Arthur John Tyrrell CBE: 19 June 1925 - 2 May 2005.

David Tyrrell is remembered by physicians and scientists principally for his discovery of the common cold viruses and elucidation of their pathogenesis, but also for his work in various other areas, including influenza, bovine spongiform encephalopathy (BSE) and chronic fatigue syndrome (CFS). David possessed a deep humanity, honesty, perseverance and a vision of collaboration as a means of making discoveries that would contribute meaningfully to the alleviation of human suffering. He also had a warmth and a mischievous sense of humour that was frequently directed at bureaucracy, which he thoroughly disliked.

[Study on bovine spongiform encephacitis and current status of safety control of medical devices deriving from animals].

OBJECTIVE: To introduce the basic information about mad cow disease and the current status of safety control of medical devices derived from mammalian animal tissues. METHODS: Literature concerned was reviewed intensively. RESULTS: Mad cow disease also known as bovine spongiform encephacitis (BSE), is generally considered from the same source of Scrapie, and they are caused by the same kind of sponginess brain tissue pathological changes. Mad cow disease is caused by the misfolding of a small protein called Prion. This disease has the character of slowly breaking down the central neuron system of animals. CONCLUSION: Further researches can provide evaluation for safety considerations of medical devices deriving from animal.

Approaches to Scrapie diagnosis by applying immunohistochemistry and rapid tests on central nervous and lymphoreticular systems.

Comparative studies evaluating the performance of Transmissible Spongiform Encephalopathies (TSE) rapid tests (validated for Bovine Spongiform Encephalopathy samples) on Scrapie samples have not been reported widely, particularly those dealing with lymphoreticular system tissues to a much lesser extent. The main objective of this study was to compare the ability of two current rapid tests (Western blot and Luminiscence Immunoassay Prionics-Check; WB and LIA, respectively) to detect PrPsc using central nervous system as well as lymphoreticular system samples corresponding to naturally infected animals. Thirty-four Scrapie-affected sheep, 26 with clinical signs of the disease, were included in the study. Tonsil, retropharyngeal lymph node and medulla oblongata were assessed by three tests: immunohistochemistry (confirmatory test), WB and LIA (rapid tests). The conclusion which can be drawn from this study is the fact that all animals involved in the study, including those at a preclinical stage, could be diagnosed regardless of the test used (with immunohistochemistry consistently showing higher sensitivity) only when the analyses of both the central nervous system and the lymphoreticular system were considered. The choice of these tissues for routine diagnosis is, therefore, proposed as a valuable tool to highly reduce the number of undetected positive cases.

[The research advance of analytical methodology for detecting bovine spongiform encephalopathy].

Bovine spongiform encephalopathy (BSE) is an infectious disease which can threaten animal husbandry and human health seriously. The disease mainly violates the central neural system. This article selectively reviewed current researches on the analytical detection of BSE and transmissible spongiform encephalopathy (TSE).

A critical review of the nature of the spongiform encephalopathy agent: protein theory versus virus theory.

All spongiform encephalopathies (SEs) result in brain disorders brought about by a slow virus. Since the origin of bovine SE (BSE), the infectious nature of the disease has been firmly established. Tubulofilamentous particles/scrapie termed nemavirus (NVP) and scrapie-associated fibrils (SAF) are ultrastructural markers, whereas protease-resistant protein (PrP(sc)) is a protein marker. The PrP molecules aggregate to form SAF. Each NVP consists of three layers: an outer protein coat, an intermediate ssDNA layer, and inner PrP/SAF. Therefore, ssDNA and PrP/SAF are physically associated with each other. The existence of at least 20 stable strains of SEs implies that a nucleic acid molecule serves as the information molecule. Animals inoculated with PrP(sc) do not develop the clinical disease, however, ssDNA purified from scrapie-hamster brains by alkaline gel electrophoresis mixed with binding proteins before inoculation developed the clinical disease. It appears that an "accessory protein" coded by the ssDNA of the NVP interacts with normal PrP(c) molecules, resulting in their conversion to PrP(sc)/SAF. The pathogenesis process in the infected animal, with increasing incubation periods, reveals that larger amounts of normal PrP molecules are modified to form SAF. This interferes with the normal supply of PrP to cell membranes, which become disrupted and eventually fragment, resulting in the vacuoles typical of those found in the SEs. Critical review of scientific literature has demonstrated that the agent contains a DNA genome.

Long-term subclinical carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease in humans.

Cattle infected with bovine spongiform encephalopathy (BSE) appear to be a reservoir for transmission of variant Creutzfeldt-Jakob disease (vCJD) to humans. Although just over 100 people have developed clinical vCJD, millions have probably been exposed to the infectivity by consumption of BSE-infected beef. It is currently not known whether some of these individuals will develop disease themselves or act as asymptomatic carriers of infectivity which might infect others in the future. We have studied agent persistence and adaptation after cross-species infection using a model of mice inoculated with hamster scrapie strain 263K. Although mice inoculated with hamster scrapie do not develop clinical disease after inoculation with 10 million hamster infectious doses, hamster scrapie infectivity persists in brain and spleen for the life span of the mice. In the present study, we were surprised to find a 1-year period postinfection with hamster scrapie where there was no evidence for replication of infectivity in mouse brain. In contrast, this period of inactive persistence was followed by a period of active replication of infectivity as well as adaptation of new strains of agent capable of causing disease in mice. In most mice, neither the early persistent phase nor the later replicative phase could be detected by immunoblot assay for protease-resistant prion protein (PrP). If similar asymptomatic carriers of infection arise after exposure of humans or animals to BSE, this could markedly increase the danger of additional spread of BSE or vCJD infection by contaminated blood, surgical instruments, or meat. If such subclinical carriers were negative for protease-resistant PrP, similar to our mice, then the recently proposed screening of brain, tonsils, or other tissues of animals and humans by present methods such as immunoblotting or immunohistochemistry might be too insensitive to identify these individuals.

Evaluation of a rapid western immunoblotting procedure for the diagnosis of bovine spongiform encephalopathy (BSE) in the UK.

Bovine brain tissue samples from 625 UK cattle, clinically suspected as bovine spongiform encephalopathy (BSE) cases, were used in a blind analysis to assess a rapid Western immunoblotting technique (Prionics Check; Prionics AG, Zurich), which detects bovine disease-specific protease-resistant prion protein (PrP(Sc)). By means of statutory histopathological examination, 599 of the 625 cattle were confirmed as BSE cases by the demonstration of spongiform encephalopathy, the remaining 26 being classified as negative. Duplicate samples from the same animals were also examined by electron microscopy for the presence of abnormal brain fibrils (scrapie-associated fibrils; SAFs). The Prionics technique showed a high sensitivity, particularly when compared with the fibril detection test; the detection rates were 99.3% and 92.0% respectively, with histopathology being used as the "gold standard". The false negative results by the Prionics test were possibly related to the sampling procedure. Analysis of 50 BSE-positive samples revealed similar glycoprofiles, the majority of PrP(Sc)isoforms being di-glycosylated protein. The Prionics test also detected PrP(Sc)in the four brain samples from the 26 histopathologically negative animals, apparently reducing the specificity of the test to 84.6%; however, confirmatory positive results in these samples were obtained by demonstrating SAF or by immunohistochemical examination, or both. It was concluded that the Prionics test detected PrP(Sc)in a small percentage (0.64%) of clinically suspected BSE cases showing no spongiform change. Since January 2000, the Prionics Western blot test has been introduced as one of the statutory tests for the diagnosis of clinically suspected BSE and scrapie cases in the UK.

BSE as an organizational construction: a case study on the globalization of risk.

This article examines the BSE problem as an example of the 'globalization of risk'. In order to determine whether the 'globalization of risk' is a social construction depending on the context, the paper emphasizes the particular role of organizations. It makes an empirical comparison of the BSE-related risk-constructions of five business associations in the German meat industry sector. The results show that the associations construct the risk in close relation to their horizons of globalization, thereby reflecting provision problems, which the companies they are representing face. While the main organizational domains in the sector tried to cope with the risk problem by different means of local market 'closure', one association, founded in reaction to the BSE problem, took over a 'reflexive' role with regard to the emerging risk communication on BSE in Germany.

Cellular prion protein expressed by bovine squamous epithelia of skin and upper gastrointestinal tract.

Routes of transmission of bovine spongiform encephalopathy have not yet been determined. We show that bovine squamous epithelia of the skin and upper gastrointestinal tract express Prp(c), suggesting that these epithelia may be a target for prion entry and replication.

Recent developments in the epidemiology of virus diseases and BSE.

There is a continuous change in viral epidemics with respect to clinical symptoms, their duration or disappearance and the emergence of new diseases. This can be observed both in human and animal diseases. This evolution of virus diseases is mainly related to three factors: etiological agent, host and environment. As far as genetic alterations of the virus are concerned, two major mechanisms are involved: 1) mutations such as recombination and reassortment; 2) selection for resistance or susceptibility. The epidemiology of newly emerged virus diseases in man and animals, such as AIDS and hemorrhagic fevers, and bovine spongiform encephalopathy (BSE), canine hemorrhagic gastroenteritis or respiratory syndrome in horses will be discussed.

[Effects of prion protein on central nervous system and the mechanisms of action].

The following questions are discussed in this paper: (1) Can the bovine prion protein PrpRES infect a man? (2) What is the route of PrPRES infection? (3) In what condition the PrPRES may infect animals, (4) What are the normal functions of PrPc at the surface of neurons and neuroglia cells, (5) What is the structure of the prion protein, (6) What is possible mechanism of the PrPRES destroying neurons. Finally, the two possible mechanisms of the PrPRES to affect central nervous system are analysed. One is to destroy the normal function of the PrPC, the other is to raise the sensibility of the neuron to the free radical and increase the content of the oxygen radical.