Bovine Spongiform Encephalopathy Agent Research Articles

Transcytosis of Murine-Adapted Bovine Spongiform Encephalopathy Agents in anIn VitroBovine M Cell Model

Transmissible spongiform encephalopathies (TSE), including bovine spongiform encephalopathy (BSE), are fatal neurodegenerative disorders in humans and animals. BSE appears to have spread to cattle through the consumption of feed contaminated with BSE/scrapie agents. In the case of an oral infection, the agents have to cross the gut-epithelial barrier. We recently established a bovine intestinal epithelial cell line (BIE cells) that can differentiate into the M cell type in vitro after lymphocytic stimulation (K. Miyazawa, T. Hondo, T. Kanaya, S. Tanaka, I. Takakura, W. Itani, M. T. Rose, H. Kitazawa, T. Yamaguchi, and H. Aso, Histochem. Cell Biol. 133:125-134, 2010). In this study, we evaluated the role of M cells in the intestinal invasion of the murine-adapted BSE (mBSE) agent using our in vitro bovine intestinal epithelial model. We demonstrate here that M cell-differentiated BIE cells are able to transport the mBSE agent without inactivation at least 30-fold more efficiently than undifferentiated BIE cells in our in vitro model. As M cells in the follicle-associated epithelium are known to have a high ability to transport a variety of macromolecules, viruses, and bacteria from gut lumen to mucosal immune cells, our results indicate the possibility that bovine M cells are able to deliver agents of TSE, not just the mBSE agent.

Descriptive epidemiological features of cases of bovine spongiform encephalopathy born after July 31, 1996 in Great Britain

This paper describes the results of analyses of the epidemiological features of the 164 cases of bovine spongiform encephalopathy (BSE) in Great Britain that were born after the introduction of...

Variant Creutzfeldt–Jakob disease

Variant Creutzfeldt-Jakob disease (CJD) is an emerging form of human prion disease caused by oral exposure to the bovine spongiform encephalopathy agent. Most cases have occurred in the UK, but smaller numbers of cases have been identified in 10 other countries worldwide. All confirmed cases belong to a single genetic subgroup defined by methionine homozygosity at codon 129 in the prion protein gene. Variant CJD has a widespread distribution of infectivity in the body, involving lymphoid tissues during at least the latter part of the incubation period. This is unlike other forms of human prion disease, and raised concerns that the transmissible agent might also be present in blood. To date, four probable cases of variant CJD infection have been identified following transfusion of packed red blood cells from asymptomatic donors who subsequently died from variant CJD. Recently, one case of likely transmission of variant CJD infection by UK factor VIII (FVIII) concentrates has been reported in an elderly haemophilic patient in the UK, who had been treated with FVIII produced from pooled plasma to which a donor who subsequently died from variant CJD had contributed. The recipient showed no signs or symptoms of variant CJD during life, but evidence of variant CJD infection was detected in his spleen following a postmortem examination. Continued surveillance is required to investigate the prevalence of secondary variant CJD infection in other patients with bleeding disorders who have been treated with UK-sourced pooled plasma products.

Surveillance and simulation of bovine spongiform encephalopathy and scrapie in small ruminants in Switzerland

BackgroundAfter bovine spongiform encephalopathy (BSE) emerged in European cattle livestock in 1986 a fundamental question was whether the agent established also in the small ruminants' population. In Switzerland transmissible spongiform encephalopathies (TSEs) in small ruminants have been monitored since 1990. While in the most recent TSE cases a BSE infection could be excluded, for historical cases techniques to discriminate scrapie from BSE had not been available at the time of diagnosis and thus their status remained unclear. We herein applied state-of-the-art techniques to retrospectively classify these animals and to re-analyze the affected flocks for secondary cases. These results were the basis for models, simulating the course of TSEs over a period of 70 years. The aim was to come to a statistically based overall assessment of the TSE situation in the domestic small ruminant population in Switzerland.ResultsIn sum 16 TSE cases were identified in small ruminants in Switzerland since 1981, of which eight were atypical and six were classical scrapie. In two animals retrospective analysis did not allow any further classification due to the lack of appropriate tissue samples. We found no evidence for an infection with the BSE agent in the cases under investigation. In none of the affected flocks, secondary cases were identified. A Bayesian prevalence calculation resulted in most likely estimates of one case of BSE, five cases of classical scrapie and 21 cases of atypical scrapie per 100'000 small ruminants. According to our models none of the TSEs is considered to cause a broader epidemic in Switzerland. In a closed population, they are rather expected to fade out in the next decades or, in case of a sporadic origin, may remain at a very low level.ConclusionsIn summary, these data indicate that despite a significant epidemic of BSE in cattle, there is no evidence that BSE established in the small ruminant population in Switzerland. Classical and atypical scrapie both occur at a very low level and are not expected to escalate into an epidemic. In this situation the extent of TSE surveillance in small ruminants requires reevaluation based on cost-benefit analysis.

Characterization of Strains of Ovine Transmissible Spongiform Encephalopathy with a Short PrP d Profiling Method

Scrapie is the transmissible spongiform encephalopathy (TSE) that naturally affects sheep and goats; these species are also susceptible to experimental infection with the bovine spongiform encephalopathy (BSE) agent. Discrimination between different strains of sheep scrapie and ovine BSE has been achieved by descriptive and quantitative profiling of deposits of the disease-associated prion protein (PrPd) in different areas of the brain, but this process is time-consuming and difficult to standardize between laboratories. The present paper describes an alternative PrPd profiling method that is less demanding and addresses these difficulties. It is based on the scoring of similar 14 PrPd types in 11 precisely defined areas of the telencephalon. When applied to 48 archived cases of experimental sheep BSE, SSBP/1, CH1641 and natural scrapie, it gave comparable results to the original profiling method, previously conducted on the same brains, and allowed differentiation between the different infectious sources. This new 'short PrPd profiling' method has the advantages of being less time-consuming and easier to standardize, so that it can be readily adopted by different laboratories to provide comparable results.

Bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease risk management in Belgium

Italy experienced two imported cases of bovine spongiform encephalopathy (BSE) in 1994 and viewed the disease as a 'foreign' problem. Early, precautionary actions including: a 1989 ban on UK meat and bone meal (MBM), 1990 ban on UK beef, 1994 domestic ban on mammalian MBM to ruminants, and 1996 ban on UK live cattle, protected Italy from a much larger outbreak. In 2001 Italy implemented an effective and active BSE surveillance system in compliance with European Union regulations. Probably little MBM feeding or amplification of BSE agent occurred within Italian herds after 1996. BSE cases have declined since 2001. In 2007, the European Commission recognised Italy as a controlled BSE risk country. To date only two cases of variant Creutzfeldt-Jakob disease (vCJD) have been reported. A limited blood donor exclusion policy is in place to prevent cases of blood transfusion mediated vCJD.

Evidence-Based Semiquantitative Methodology for Prioritization of Foodborne Zoonoses

To prioritize an extended list of food- and water-borne zoonoses to allow food safety authorities to focus on the most relevant hazards in the food chain. An evidence-based semiquantitative methodology was developed. Scores were given by 35 scientific experts in the field of animal and public health, food, and clinical microbiology and epidemiology to 51 zoonotic agents according to five criteria related to public health (severity and occurrence in humans), animal health (severity of disease coupled with economic consequences and occurrence in animals), and food (occurrence in food). The scoring procedure was standardized and evidence-based as experts were provided, for each zoonotic agent, a same set of up-to-date help information data related to the five criteria. Independently, the relative importance of the five criteria was weighted by seven food chain risk managers. The zoonotic agents were ranked based on overall weighted scores and were grouped in four statistically different levels of importance. The following foodborne zoonotic pathogens were classified as "most important": Salmonella spp., Campylobacter spp., Listeria monocytogenes, and verocytotoxigenic Escherichia coli. A second group of "significant importance" included Toxoplasma gondii, the agent of bovine spongiform encephalopathy, Clostridium botulinum, Staphylococcus aureus, Cryptosporidium parvum, Mycobacterium bovis, Echinococcus granulosus, Streptococcus spp., Echinococcus multilocularis, Yersinia enterocolitica, Mycobacterium avium, Fasciola hepatica, Giardia intestinalis, and Rotavirus. This methodology allowed to rank 51 zoonotic agents with objectivity and taking account of a combined input from risk assessors and risk managers. These results support food safety policy makers to establish the multiannual monitoring program of foodborne zoonoses. They also enable to identify knowledge gaps on specific zoonotic agents and to formulate key research questions. Principally, this method of prioritization is of general interest as it can be applied for any other ranking exercise and in any country.

Transmissibility studies of vacuolar changes in the rostral colliculus of pigs

BackgroundHistopathological examinations of brains from healthy pigs have revealed localised vacuolar changes, predominantly in the rostral colliculus, that are similar to the neuropil vacuolation featured in the transmissible spongiform encephalopathies and have been described in pigs challenged parenterally with the agent causing bovine spongiform encephalopathy (BSE). Feedstuff containing BSE-contaminated meat and bone meal (MBM) may have been fed to pigs prior to the ban of mammalian MBM in feed of farmed livestock in the United Kingdom in 1996, but there is no evidence of the natural occurrence of a transmissible spongiform encephalopathy (TSE) in the domestic pig. Furthermore, experimental transmission of BSE to pigs by the oral route has been unsuccessful. A study was conducted to investigate whether the localised vacuolar changes in the porcine brain were associated with a transmissible aetiology and therefore biologically significant. Two groups of ten pigs were inoculated parenterally with vacuolated rostral colliculus from healthy pigs either born before 1996 or born after 1996. Controls included ten pigs similarly inoculated with rostral colliculus from New Zealand-derived pigs and nine pigs inoculated with a bovine BSE brain homogenate.ResultsNone of the pigs inoculated with rostral colliculus developed a TSE-like neurological disease up to five years post inoculation when the study was terminated, and disease-associated prion protein, PrPd, was not detected in the brains of these pigs. By contrast, eight of nine BSE-inoculated pigs developed neurological signs, two of which had detectable PrPd by postmortem tests. No significant histopathological changes were detected to account for the clinical signs in the PrPd-negative, BSE-inoculated pigs.ConclusionThe findings in this study suggest that vacuolation in the porcine rostral colliculus is not caused by a transmissible agent and is probably a clinically insignificant change. The presence of neurological signs in pigs inoculated with BSE without detectable PrPd raises the possibility that the BSE agent may produce a prion disease in pigs that remains undetected by the current postmortem tests.

Detection of PrP sc in Blood from Sheep Infected with the Scrapie and Bovine Spongiform Encephalopathy Agents

The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrP(sc), a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrP(sc) was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrP(sc) was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrP(sc) is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification.

Possible Case of Maternal Transmission of Feline Spongiform Encephalopathy in a Captive Cheetah

Feline spongiform encephalopathy (FSE) is considered to be related to bovine spongiform encephalopathy (BSE) and has been reported in domestic cats as well as in captive wild cats including cheetahs, first in the United Kingdom (UK) and then in other European countries. In France, several cases were described in cheetahs either imported from UK or born in France. Here we report details of two other FSE cases in captive cheetah including a 2nd case of FSE in a cheetah born in France, most likely due to maternal transmission. Complete prion protein immunohistochemical study on both brains and peripheral organs showed the close likeness between the two cases. In addition, transmission studies to the TgOvPrP4 mouse line were also performed, for comparison with the transmission of cattle BSE. The TgOvPrP4 mouse brains infected with cattle BSE and cheetah FSE revealed similar vacuolar lesion profiles, PrPd brain mapping with occurrence of typical florid plaques. Collectively, these data indicate that they harbor the same strain of agent as the cattle BSE agent. This new observation may have some impact on our knowledge of vertical transmission of BSE agent-linked TSEs such as in housecat FSE, or vCJD.

Microarray Analysis of Differentially Expressed Genes from Peyer's Patches of Cattle Orally Challenged with Bovine Spongiform Encephalopathy

The most likely route of entry of infection following oral exposure to transmissible spongiform encephalopathies (TSE) is via the immunologically active Peyer's patches (PP). These secondary lymphoid organs appear to be the potential route for prion neuroinvasion. However, the molecular mechanisms involved in the uptake of the infectious prion agent and progression of disease remain still unclear. This investigation examined the changes in gene expression in PP following oral exposure of cattle to bovine spongiform encephalopathy (BSE) agents. The gene expression patterns in PP from cows 12 mo after BSE challenge were compared with controls using a microarray platform containing 24,000 oligonucleotides representing 16,846 unique gene loci and 5943 Expressed Sequence Tag (EST) from bovine genome. Between the challanged and control animals, 90 genes and 16 EST were identified as significantly differentially, expressed (>2.0-fold change): 36 were upregulated and 70 were downregulated. Of these genes, five were found to be related to immune function. Major histocompatibility complex (MHC) class II, MHC class II DQ alpha, L-RAP, and two hypothetical proteins. Differentially expressed genes related to cellular and metabolic processes including development and maturation of cells in the PP were also identified. In this context, the potential impacts of these gene expression changes in PP on BSE development are discussed.

The kuru infectious agent is a unique geographic isolate distinct from Creutzfeldt–Jakob disease and scrapie agents

Human sporadic Creutzfeldt-Jakob disease (sCJD), endemic sheep scrapie, and epidemic bovine spongiform encephalopathy (BSE) are caused by a related group of infectious agents. The new U.K. BSE agent spread to many species, including humans, and clarifying the origin, specificity, virulence, and diversity of these agents is critical, particularly because infected humans do not develop disease for many years. As with viruses, transmissible spongiform encephalopathy (TSE) agents can adapt to new species and become more virulent yet maintain fundamentally unique and stable identities. To make agent differences manifest, one must keep the host genotype constant. Many TSE agents have revealed their independent identities in normal mice. We transmitted primate kuru, a TSE once epidemic in New Guinea, to mice expressing normal and approximately 8-fold higher levels of murine prion protein (PrP). High levels of murine PrP did not prevent infection but instead shortened incubation time, as would be expected for a viral receptor. Sporadic CJD and BSE agents and representative scrapie agents were clearly different from kuru in incubation time, brain neuropathology, and lymphoreticular involvement. Many TSE agents can infect monotypic cultured GT1 cells, and unlike sporadic CJD isolates, kuru rapidly and stably infected these cells. The geographic independence of the kuru agent provides additional reasons to explore causal environmental pathogens in these infectious neurodegenerative diseases.

Transgenic Mice Expressing Porcine Prion Protein Resistant to Classical Scrapie but Susceptible to Sheep Bovine Spongiform Encephalopathy and Atypical Scrapie

How susceptible pigs are to infection with sheep prions is unknown. We show, through transmission experiments in transgenic mice expressing porcine prion protein (PrP), that the susceptibility of this mouse model to bovine spongiform encephalopathy (BSE) can be enhanced after its passage in ARQ sheep, indicating that the pathogenicity of the BSE agent is modified after passage in sheep. Transgenic mice expressing porcine PrP were, nevertheless, completely resistant to infection with a broad panel of classical scrapie isolates from different sheep PrP genotypes and with different biochemical characteristics. The atypical (Nor98 like) isolate (SC-PS152) was the only scrapie isolate capable of transmission in these mice, although with a marked transmission barrier. Unexpectedly, the atypical scrapie agent appeared to undergo a strain phenotype shift upon transmission to porcine-PrP transgenic mice and acquired new strain properties, suggesting that atypical scrapie agent may exhibit different phenotypes depending on the host cellular PrP or other genetic factors.

Two Unusual Bovine Spongiform Encephalopathy Cases Detected in Great Britain

Bovine spongiform encephalopathy (BSE) was first identified in Great Britain (GB) in 1986 and was subsequently detected in many other countries, worldwide. A decade after the start of the bovine epidemic, the first cases of new variant Creutzfeldt-Jakob disease (vCJD) in humans were linked to probable ingestion of BSE infected tissue, highlighting a new zoonotic disease. An abnormal protease-resistant protein (PrP(res)) in a diseased subject, derived from a post-translational change of a normal host cellular membrane protein (PrP(c)), is a reliable disease marker for the whole group of neurodegenerative transmissible spongiform encephalopathies (TSEs). Immunology-based techniques, such as Western immunoblotting, have previously indicated that BSE cases all give a uniform molecular profile for PrP(res). Periodic lesion profiling of the spongiform change throughout different brain regions of infected mice and cattle has also indicated a single agent for BSE. However, in 2001 rapid testing for PrP(res) was introduced for the active surveillance of ruminants within Europe, and approximately 40 BSE cases have now been recognized that differ in their molecular profiles from those typically found. These unusual BSE cases have been detected in several European countries, and in Japan and the USA. At present, the cases appear as two distinct types based on the molecular mass (Mm) of the unglycosylated PrP(res) protein band relative to that of classical BSE. One type is of a higher Mm (H-type) and the other shows a lower Mm (L-type). Transmission studies in mice have shown that both H-type and L-type BSE have biological characteristics that are different from those of the classical BSE agent. This study describes the prion protein (PRNP) genotype and molecular profiles of the first two cases of H-type BSE detected in GB in comparison with those obtained for classical BSE, scrapie in sheep from GB and a control H-type BSE case from France.

Transcriptional Changes in the Brains of Cattle Orally Infected with the Bovine Spongiform Encephalopathy Agent Precede Detection of Infectivity

Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. BSE can be transmitted experimentally between cattle through the oral route, and in this study, brain tissue samples from animals at different time points postinoculation were analyzed for changes in gene expression. The aims of this study were to identify differentially regulated genes during the progression of BSE using microarray-based gene expression profiling and to understand the effect of prion pathogenesis on gene expression. A total of 114 genes were found to be differentially regulated over the time course of the infection, and many of these 114 genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response, and transcription. This study also revealed a broad correlation between gene expression profiles and the progression of BSE in cattle. At 21 months postinoculation, the largest number of differentially regulated genes was detected, suggesting that there are many pathogenic processes in the animal brain even prior to the detection of infectivity in the central nervous systems of these orally infected cattle. Moreover, evidence is presented to suggest that it is possible to predict the infectious status of animals using the expression profiles from this study.

Pathogenetical significance of porencephalic lesions associated with intracerebral inoculation of sheep with the bovine spongiform encephalopathy (BSE) agent

Decreased rates of transmission of transmissible spongiform encephalopathies (TSEs) to sheep have been attributed to some polymorphisms of the prion protein (PrP) and to a 'species barrier' on interspecies experiments. In addition, the blood-brain barrier may be a further impediment to TSE neuroinvasion. The intracerebral (I/C) route is generally considered the most efficient for TSE transmission, as it may help to bypass those factors. Therefore, susceptibility of particular species to specific TSE agents is conducted by this route. This study characterizes the traumatic brain lesions associated with the I/C injection of the bovine spongiform encephalopathy agent in sheep, assesses the relevance of such lesions in the outcome of clinical disease and provides insight into the mechanisms of PrP(d) conversion and amplification following I/C challenge. A total of 27 hemibrains have been macroscopically and immunohistochemically examined to investigate the presence of lesions compatible with the needle track and the PrP(d) distribution, respectively. No residual inoculum was found and the extension and severity of the traumatic brain lesions were unrelated to the clinical outcome. Sheep with PrP(d) accumulation in the brain also showed conspicuous focal aggregates in the porencephalic lesions and in the circumventricular organs. In contrast, sheep without PrP(d) deposits in the brain were also negative in the traumatic lesions. Overall, these findings suggest that the efficiency of the I/C route is due to effective absorption and blood recirculation of infection, rather than to primary amplification at the site of injection.

Lesion Profiling at Primary Isolation in RIII Mice is Insufficient in Distinguishing BSE from Classical Scrapie

Primary isolation of bovine spongiform encephalopathy (BSE) in RIII mice generates a lesion profile believed to be reproducible and distinct from that produced by classical scrapie. This profile, which is characterized by peaks at gray matter areas 1, 4 and 7 (dorsal medulla, hypothalamus and septal nuclei), is used to diagnose BSE on primary isolation. The aim of this study was to investigate whether the BSE agent could be present in sheep diagnosed with classical scrapie, using lesion profiles in RIII mice as a discriminatory method. Sixty‐two positive scrapie field cases were collected from individual farms between 1996 and 1999 and bioassayed in RIII mice. Fifty‐five of these isolates transmitted successfully to at least one mouse. Of the 31 that produced adequate data to allow lesion profile analysis, 10 showed a consistent profile with peaks at brain areas 1, 4 and 7. All inocula for this subgroup were derived from sheep of genotype ARQ/ARQ. While the 1‐4‐7‐scrapie profile exhibited similarities to BSE in RIII mice at primary isolation, it was distinguishable based on histopathology, immunohistochemistry and cluster analysis. We conclude that caution should be taken to distinguish this profile from BSE and that additional parameters should be considered to reach a final diagnosis.

Comparison of strategies for substantiating freedom from scrapie in a sheep flock

BackgroundThe public health threat represented by a potential circulation of bovine spongiform encephalopathy agent in sheep population has led European animal health authorities to launch large screening and genetic selection programmes. If demonstrated, such a circulation would have dramatic economic consequences for sheep breeding sector. In this context, it is important to evaluate the feasibility of qualification procedures that would allow sheep breeders demonstrating their flock is free from scrapie. Classical approaches, based on surveys designed to detect disease presence, do not account for scrapie specificities: the genetic variations of susceptibility and the absence of live diagnostic test routinely available. Adapting these approaches leads to a paradoxical situation in which a greater amount of testing is needed to substantiate disease freedom in genetically resistant flocks than in susceptible flocks, whereas probability of disease freedom is a priori higher in the former than in the latter. The goal of this study was to propose, evaluate and compare several qualification strategies for demonstrating a flock is free from scrapie.ResultsA probabilistic framework was defined that accounts for scrapie specificities and allows solving the preceding paradox. Six qualification strategies were defined that combine genotyping data, diagnostic tests results and flock pedigree. These were compared in two types of simulated flocks: resistant and susceptible flocks. Two strategies allowed demonstrating disease freedom in several years, for the majority of simulated flocks: a strategy in which all the flock animals are genotyped, and a strategy in which only founders animals are genotyped, the flock pedigree being known. In both cases, diagnostic tests are performed on culled animals. The less costly strategy varied according to the genetic context (resistant or susceptible) and to the relative costs of a genotyping exam and of a diagnostic test.ConclusionThis work demonstrates that combining data sources allows substantiating a flock is free from scrapie within a reasonable time frame. Qualification schemes could thus be a useful tool for voluntary or mandatory scrapie control programmes. However, there is no general strategy that would always minimize the costs and choice of the strategy should be adapted to local genetic conditions.

Transmission of Atypical Bovine Prions to Mice Transgenic for Human Prion Protein

To assess risk for cattle-to-human transmission of prions that cause uncommon forms of bovine spongiform encephalopathy (BSE), we inoculated mice expressing human PrP Met129 with field isolates. Unlike classical BSE agent, L-type prions appeared to propagate in these mice with no obvious transmission barrier. H-type prions failed to infect the mice.

Developing PCR Primers Using a New Computer Program for Detection of Multiple Animal-Derived Materials in Feed

To reduce the risk of the bovine spongiform encephalopathy agent being recycled to cattle through animal feed, in October 2001 Japan introduced a feed ban prohibiting the use of animal proteins in feed. PCR identification of feed ingredients is part of the audit program to ensure the proper implementation of the feed ban. For efficient analysis, screening of feed products for materials from multiple species is essential. In our study, we developed a computer program GSPRIMER (http://www.famic.go.jp/ffis/feed/gsprimer/) that facilitates development of PCR primers specific to multiple species. The most important feature of GSPRIMER is its ability to estimate the specificity and homology of a potential primer in incremental steps from the 3′ terminal. We analyzed all regions of mitochondrial DNA from the target and nontarget species using GSPRIMER. We designed species-specific primer sets for three animal species (sheep, goats, and swine) and group-specific primer sets for ruminants and animals susceptible to transmissible spongiform encephalopathy. The primers were efficiently screened by the PCR protocol using a mixture of mitochondrial DNA from nontarget species as a template. As a result, one primer set each for sheep and goats, two for swine, and three for a group of ruminant species were developed. The detection limit of one of the ruminant primer sets ranged from 0.05 to 0.01% bovine meat and bone meal and 0.1pg of bovine DNA. We also successfully applied the primer set to 17 commercial feed samples that were known to be free from ruminant-derived materials. No false-positive results were found.