Transmissible Spongiform Encephalopathies Cases Research Articles

Immunohistochemical Features of PrPd Accumulation in Natural and Experimental Goat Transmissible Spongiform Encephalopathies

Scrapie is a transmissible spongiform encephalopathy (TSE) or prion disease, which naturally affects sheep and goats. Immunohistochemical epitope mapping of abnormal PrP accumulations (PrP(d)) in brain can help in characterizing sheep TSE sources or strains and in identifying potential bovine spongiform encephalopathy (BSE) infections of sheep. Natural and experimental TSE infections of goats were examined to determine whether the epitope mapping approach could also be applied to aid recognition of BSE infection in goats. Goats experimentally infected with the SSBP/1 or CH1641 sheep scrapie strains or with cattle BSE, together with four field cases of natural TSE in goats, were examined immunohistochemically with six different antibodies. CH1641 and SSBP/1 infections in goats, as in sheep, showed PrP(d) accumulations which were mainly intracellular. Some differences in targeting, particularly of Purkinje cells, was evident in inter-species comparisons of CH1641 and SSBP/1. PrP(d) labelling of goat BSE experimental cases showed extensive intracellular and extracellular accumulations, also similar to those in sheep BSE. Intra-neuronal PrP(d) in both goat and sheep BSE was labelled only by antibodies recognizing epitopes located C-terminally of residue His99, whereas in natural sheep TSE sources, and in sheep and goat SSBP/1, PrP(d) was also detected by antibodies to epitopes located between residues Trp93 and His99. Testing of four natural goat TSE samples showed one case in which epitope mapping characteristics and the overall patterns of PrP(d) accumulation was identical with those of experimental goat BSE. The four natural goat scrapie cases examined showed some degree of immunohistochemical phenotype variability, suggesting that multiple strains exist within the relatively small UK goat population.

Comparative evidence for a link between Peyer's patch development and susceptibility to transmissible spongiform encephalopathies

BackgroundEpidemiological analyses indicate that the age distribution of natural cases of transmissible spongiform encephalopathies (TSEs) reflect age-related risk of infection, however, the underlying mechanisms remain poorly understood. Using a comparative approach, we tested the hypothesis that, there is a significant correlation between risk of infection for scrapie, bovine spongiform encephalopathy (BSE) and variant CJD (vCJD), and the development of lymphoid tissue in the gut.MethodsUsing anatomical data and estimates of risk of infection in mathematical models (which included results from previously published studies) for sheep, cattle and humans, we calculated the Spearman's rank correlation coefficient, rs, between available measures of Peyer's patch (PP) development and the estimated risk of infection for an individual of the corresponding age.ResultsThere was a significant correlation between the measures of PP development and the estimated risk of TSE infection; the two age-related distributions peaked in the same age groups. This result was obtained for each of the three host species: for sheep, surface area of ileal PP tissue vs risk of infection, rs = 0.913 (n = 19, P < 0.001), and lymphoid follicle density vs risk of infection, rs = 0.933 (n = 19, P < 0.001); for cattle, weight of PP tissue vs risk of infection, rs = 0.693 (n = 94, P < 0.001); and for humans, number of PPs vs risk of infection, rs = 0.384 (n = 46, P = 0.008). In addition, when changes in exposure associated with BSE-contaminated meat were accounted for, the two age-related patterns for humans remained concordant: rs = 0.360 (n = 46, P = 0.014).ConclusionOur findings suggest that, for sheep, cattle and humans alike there is an association between PP development (or a correlate of PP development) and susceptibility to natural TSE infection. This association may explain changes in susceptibility with host age, and differences in the age-susceptibility relationship between host species.

TSE detected in a Belgian ARR-homozygous sheep via active surveillance

It is commonly accepted that scrapie-resistance and -susceptibility in sheep are genetically controlled. Consequently, the selection of sheep with scrapie-resistant genotypes is currently one of the most important objectives of the sheep breeding associations. However, during the last two years, new data have become available on transmissible spongiform encephalopathy (TSE) cases in TSE-resistant sheep in several European Union member states. The present paper describes the first Belgian natural “atypical” TSE case in a sheep with a scrapie-resistant genotype (ARR/ARR) detected via active surveillance. No other infections or diseases were detected in the source flock. The continued finding of new “atypical” TSE cases in sheep with scrapie-resistant genotypes undermines the purpose and efficacy of the breeding programs.

High incidence of genetic human transmissible spongiform encephalopathies in Italy

To assess the incidence and mortality rates of genetic transmissible spongiform encephalopathy (TSE) diseases in Italy. The authors have sequenced the prion protein gene (PRNP) in 643 patients referred to the Italian Registry of Creutzfeldt-Jakob disease (CJD) and related disorders between 1993 and 2002. Crude age- and sex-specific incidence and mortality rates were calculated. Differences in morbidity from genetic TSE diseases in the 20 Italian regions were assessed by the standardized morbidity ratio (SMR). A total of 130 cases were classified as genetic TSE diseases with a mean yearly incidence rate of 0.28 cases per million people. Genetic TSE diseases represent 17.7% of all TSE diseases, including sporadic, iatrogenic, and variant CJD. The most frequent mutation was the V210I (n = 54), and the second most common the E200K (n = 42). Mortality rates for genetic TSE diseases did not increase in any of the age groups under examination over the 10 years of surveillance. The analysis of regional distribution of genetic cases by place of birth revealed that in Campania and Calabria regions the number of genetic TSE cases was higher than in other regions. In Italy the incidence of genetic transmissible spongiform encephalopathy (TSE) diseases is the second highest among European countries. Genetic analysis is important for a correct classification of patients with TSE.

Mortality from Human Transmissible Spongiform Encephalopathies: A Record Linkage Study

To evaluate the ability of the Italian Creutzfeldt-Jakob (CJD) register to detect human transmissible spongiform encephalopathy (TSE) cases we compared mortality data from the CJD register with those obtained from death certificates collected by the Italian National Census Bureau (ISTAT) between 1993 and 1999. We used the method of record linkage to compare and integrate data from these two sources. The integrated estimate of TSE deaths was 457: 183 deaths recorded by the CJD register and ISTAT, 210 cases only by the CJD register, and 64 cases only by ISTAT. The average integrated estimated mortality rate was 1.58 deaths per million people per year over the study period and peaked in 1999 at 2.13. This figure is similar to that obtained from data from the CJD register alone in the years 2000–2002. The increase in mortality rates is likely due to an improvement in case ascertainment. The misclassification of cases by ISTAT was above 50% from 1996 onward, suggesting that using only death certificates is not a reliable way to monitor TSE cases in Italy.

Strain Typing of German Transmissible Spongiform Encephalopathies Field Cases in Small Ruminants by Biochemical Methods

Following the implementation of a large scale transmissible spongiform encephalopathies (TSE) surveillance programme of small ruminants, evidence for a natural transmission of bovine spongiform encephalopathy (BSE) to a French goat has been found. During the years 2002-2004, a massive TSE rapid testing programme on >250,000 small ruminants was carried out in Germany. In this national survey, 186 scrapie-affected sheep were found which originated from 78 flocks. The majority of these cases were of the classical TSE type (115 sheep belonging to 14 outbreaks). However, 71 cases coming from 64 flocks were of the novel atypical scrapie type. According to the regulation EU 999/2001, all TSE cases in small ruminants have to be examined by strain typing methods to explore any possibility of the existence of BSE cases in the field sheep population. Here we report on a biochemical typing strategy (termed FLI-test), which includes the determination of molecular masses, antibody binding affinities and glycosylation pattern of the TSE induced abnormal prion protein. Based on this typing approach none of the analysed German classical TSE outbreaks (total number of analysed sheep: 36) displayed biochemical features indicative for a BSE infection. However, in two cases distinct but BSE-unrelated PrP(Sc) types were found, which alludes to the existence of different scrapie strains in the German sheep population.

Population-level retrospective study of neurologically expressed disorders in ruminants before the onset of bovine spongiform encephalopathy (BSE) in Belgium, a BSE risk III country.

A retrospective epidemiological study (n = 7,875) of neurologically expressed disorders (NED) in ruminants before the onset of the bovine spongiform encephalopathy epidemic (years studied, 1980 to 1997) was carried out in Belgium. The archives of all veterinary laboratories and rabies and transmissible spongiform encephalopathy (TSE) epidemiosurveillance networks were consulted. For all species, a significantly higher number of NED with virological causes (rabies) was reported south of the Sambre-Meuse Valley. During the period 1992 to 1997, for which the data were complete, (i) the predicted annual incidence of NED varied significantly as a function of species and area (higher numbers in areas where rabies was present) but was always above 100 cases per million, and (ii) the mean incidence of suspected TSE cases and, among them, those investigated by histopathological examination varied significantly as a function of species and area. The positive predictive value of a presumptive clinical diagnosis of NED ranged from 0.13 (game) to 0.63 (sheep). Knowledge of the positive predictive value permits the definition of a reference point before certain actions (e.g., awareness and training campaigns) are undertaken. It also shows the usefulness of a systematic necropsy or complementary laboratory tests to establish an etiological diagnosis. TSE analysis of a small, targeted historical sampling (n = 48) permitted the confirmation of one case and uncovered another case of scrapie. The results of the present study help to develop and maintain the quality of the worldwide clinical epidemiological networks for TSE, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries with TSE cases.

Linking chronic wasting disease to scrapie by comparison of Spiroplasma mirum ribosomal DNA sequences

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases of man and animals and are transmitted by a filterable pathogen whose identity is currently unresolved. Our data indicates that Spiroplasma, a wall-less bacterium, is involved in the pathogenesis of TSE. We searched for Spiroplasma ribosomal gene sequences in 10 scrapie-infected sheep brains and 10 normal sheep brains, 7 cervid samples infected with chronic wasting disease (CWD), and 7 normal cervid brains. DNA was extracted from these tissue samples and amplified by polymerase chain reaction (PCR) using primers specific for Spiroplasma-specific 16S rDNA. Specificity of the amplicon was determined by Southern blotting and DNA sequence analyses. Spiroplasma 16S rDNA was found in 8 of 10 scrapie-infected sheep brains and 6 of 7 CWD-infected tissue samples. All normal animal brain samples were negative. Spiroplasma 16S rDNA was also found in two human Creutzfeldt-Jakob diseased (CJD) brains but not in two age-matched normal human brains. DNA sequence analyses of the amplified PCR products from human and animal TSE cases revealed greater than 99% nucleotide sequence homology with Spiroplasma mirum. The presence of Spiroplasma DNA in TSE-infected tissues supports our hypothesis that Spiroplasma may be involved in the pathogenesis of these diseases.

Primer extension assay for prion protein genotype determination in sheep

Scrapie is a transmissible spongiform encephalopathy (TSE) which affects sheep and goats. TSEs are characterised by the conversion of the cellular prion protein (PrP C) into the pathological form PrP Sc. The occurrence of scrapie in sheep is influenced by polymorphisms in the PrP gene; in particular, three codons (136, 154 and 171) are important in conditioning the susceptibility/resistance of sheep to the disease, with the Val/Val 136 Arg/Arg 154 Gln/Gln 171 genotype being the most susceptible and the Ala/Ala 136 Arg/Arg 154 Arg/Arg 171, the most resistant one. The latter genotype seems to confer, in sheep, resistance to the oral infection with bovine spongiform encephalopathy, as well. The selection of genetically resistant sheep populations represents the basis of the recent strategies against ovine TSE in the European Union (EU). Herein, we describe a rapid and simple method, based on the primer extension technique, for PrP genotype determination at codons 136, 154 and 171. Intra-laboratory validation of the method showed accuracy levels comparable to those of sequencing analysis. Such method could be used for both the application of the EU policies requiring PrP genotype analysis in all ovine TSE cases, and the large-scale genotyping claimed by the implementation of breeding programmes for genetic resistance to TSE in sheep.

The challenge of prion decontamination.

Prions are a class of proposed proteinaceous infectious agents that cause similar fatal brain diseases. These diseases are known as transmissible spongiform encephalopathies (TSEs), and include Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler syndrome (GSS) in humans, bovine spongiform encephalopathy (BSE) in cattle (also variant CJD [vCJD] in humans), and scrapie in sheep. latrogenic transmission of these diseases via contaminated medical devices is considered rare, but it has been documented. In an experimental case, transmission of the disease was observed despite cleaning and decontamination of the medical device with formaldehyde [1]. Clinically, 2 cases of TSEs have been confirmed in which transmission of the disease was due to the use of implanted depth electrodes that had been previously used on a patient with CJD and were inadequately decontaminated by cleaning with benzene and disinfecting with 70% alcohol and formaldehyde [2]. Other cases have been linked to the use of neurosurgical instruments [3]. These reports highlight the need for safe and effective prion decontamination methods. Recommended prion decontamination

Risk management of transmissible spongiform encephalopathies in Europe.

Bovine spongiform encephalopathy (BSE) was first described in the United Kingdom (UK) in November 1986. After the introduction of an active surveillance system, most countries in Europe have reported BSE cases in the cattle population. This indicates that the use of active surveillance in addition to passive surveillance is important to assess the true BSE status in a country. Scrapie, a transmissible spongiform encephalopathy (TSE) in sheep and goats, has been reported in countries throughout the world with a few notable exceptions. Concern was expressed that BSE could have been introduced into sheep and goats. Currently, distinguishing between scrapie and BSE in small ruminants is only possible through lengthy experiments in mice. Preliminary results of active surveillance, introduced in 2002, show that significant under-reporting occurred. The history of BSE in cattle shows that risk assessments concerning the risk in a given country were often ignored and subsequent risk management decisions were inaccurate, i.e. although the risk was probable, no measures were taken in terms of either animal or human health. Furthermore, the effect of the measures taken was often overestimated and these had to be amended several times. The most important action to prevent new cases of TSEs occurring is by banning the feeding meat-and-bone meal (MBM) to ruminants. Further measures to be considered are the exclusion of specified risk material and carcasses from rendering, the process parameters for rendering of animal waste and the prevention of cross-contamination of feed with MBM. The most important measures to protect the consumer are the ban on specified risk material, such as brain and spinal cord, which may contain particularly high concentrations of the BSE agent, and the ban on mechanically recovered meat. The most important measures taken in Europe and the scientific background thereof are described and discussed.

Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States.

Transmissible spongiform encephalopathies (TSEs) attracted increased attention in the mid-1980s because of the emergence among UK cattle of bovine spongiform encephalopathy (BSE), which has been shown to be transmitted to humans, causing a variant form of Creutzfeldt-Jakob disease (vCJD). The BSE outbreak has been reported in 19 European countries, Israel<b>,</b> and Japan, and human cases have so far been identified in four European countries, and more recently in a Canadian resident and a US resident who each lived in Britain during the BSE outbreak. To monitor the occurrence of emerging forms of CJD, such as vCJD, in the United States, the Centers for Disease Control and Prevention has been conducting surveillance for human TSEs through several mechanisms, including the establishment of the National Prion Disease Pathology Surveillance Center. Physicians are encouraged to maintain a high index of suspicion for vCJD and use the free services of the pathology center to assess the neuropathology of clinically diagnosed and suspected cases of CJD or other TSEs.

Dendritic and synaptic alterations of hippocampal pyramidal neurones in scrapie-infected mice.

Neurone damage and eventual loss may underlie the clinical signs of disease in the transmissible spongiform encephalopathies (TSEs). Although neurone death appears to be through apoptosis, the trigger for this form of cell death in the TSEs is not known. Using two different murine scrapie models, hippocampal pyramidal cells were studied through microinjection of fluorescent dye, and synaptic integrity, using p38-immunoreactivity (p38-IR), both visualized using confocal laser scanning microscopy. Intradendritic distensions and dendritic spine loss were found to co-localize to areas of vacuolar and prion protein pathology in the hippocampus of mice infected with ME7 or 87 V scrapie. A significant reduction in p38-IR was found concomitantly in the hippocampus in ME7 scrapie mice. These results indicate that both pre- and post-synaptic sites are altered by scrapie infection; this would disrupt neuronal circuitry and may initiate apoptotic cell death, giving rise to the neurological disturbances manifested in clinical TSE cases.

Immunodetection of PrPSc in spleens of some scrapie-infected sheep but not BSE-infected cows.

The development of diagnostic tools for transmissible spongiform encephalopathies (TSEs) would greatly assist their study and may provide assistance in controlling the disease. The detection of an abnormal form of the host protein PrP in noncentral nervous system tissues may form the basis for diagnosis of TSEs. Using a new antibody reagent to PrP produced in chickens, PrP can be readily detected in crude tissue extracts. PrP from uninfected spleen had a lower molecular mass range than PrP from brain, suggesting a lower degree of glycosylation. A simple method for detecting the abnormal form of the protein, PrPSc, in ruminant brain and spleen has been developed. PrPSc was detected in sheep spleen extracts from a flock affected by natural scrapie and was also found in spleens from some, but not all, experimental TSE cases. In spleens from cattle with bovine spongiform encephalopathy (BSE) no PrPSc was detected. It is therefore suggested that there is differential targeting of PrPSc deposition between organs in these different types of TSE infection which, with other factors, depends on strain of infecting agent.