BSE-infected Cattle Research Articles

Analysis of German BSE Surveillance Data: Estimation of the Prevalence of Confirmed Cases versus the Number of Infected, but Non-Detected, Cattle to Assess Confidence in Freedom from Infection.

Quantitative risk assessments for Bovine spongiform encephalopathy (BSE) necessitate estimates for key parameters such as the prevalence of infection, the probability of absence of infection in defined birth cohorts, and the numbers of BSE-infected, but non-detected cattle entering the food chain. We estimated three key parameters with adjustment for misclassification using the German BSE surveillance data using a Gompertz model for latent (i.e., unobserved) age-dependent detection probabilities and a Poisson response model for the number of BSE cases for birth cohorts 1999 to 2015. The models were combined in a Bayesian framework. We estimated the median true BSE prevalence between 3.74 and 0.216 cases per 100,000 animals for the birth cohorts 1990 to 2001 and observed a peak for the 1996 birth cohort with a point estimate of 16.41 cases per 100,000 cattle. For birth cohorts ranging from 2002 to 2013, the estimated median prevalence was below one case per 100,000 heads. The calculated confidence in freedom from disease (design prevalence 1 in 100,000) was above 99.5% for the birth cohorts 2002 to 2006. In conclusion, BSE surveillance in the healthy slaughtered cattle chain was extremely sensitive at the time, when BSE repeatedly occurred in Germany (2000–2009), because the entry of BSE-infected cattle into the food chain could virtually be prevented by the extensive surveillance program during these years and until 2015 (estimated non-detected cases/100.000 [95% credible interval] in 2000, 2009, and 2015 are 0.64 [0.5,0.8], 0.05 [0.01,0.14], and 0.19 [0.05,0.61], respectively).

Low levels of classical BSE infectivity in rendered fat tissue

BSE infectivity in mesentery fat is most likely associated with embedded nervous tissue. To prove this mesentery containing celiac ganglion was taken from oral BSE infected cattle in different stages of the disease and from one control animal. Fat was rendered according to standard tallow production methods and the prion infectivity therein analysed in transgenic mouse bioassay. Rendered fat of the clinical animal revealed low infectivity levels, whereas preclinical and control animals remained negative. This study, although not representative, provides a proof of principle, indicating the potential contamination of melted mesenteric fat by embedded nervous structures during standard tallow production.

Expression of BSE-associated proteins in the CNS and lymphoreticular tissues of cattle and buffalo

Bovine spongiform encephalopathy (BSE), which has been documented in 190,000 cases of BSE-infected cattle, to our knowledge, has not been reported in buffalo. Prion protein (PrP) is critical to susceptibility and development of BSE. Moreover, a new PrP-like protein, Shadoo (Sho), has been shown to have overlapping expression patterns and shared functions with PrP. Therefore, we hypothesize that differences in expression at the transcriptional level and/or the post-transcriptional level of the two genes may be associated with these specific differences between cattle and buffalo. We compared the relative mRNA expression of the prion protein gene (PRNP) and the Shadoo gene (SPRN) in 396 RNA samples using real-time PCR. We also analyzed PrP/Sho protein from 96 samples by Western blot. Our results demonstrated significantly lower PrP expression in the cerebellum, obex, mesenteric lymph node, and bronchial lymph node tissues, but higher relative expression of Sho in the cerebrum and spleen in buffalos compared with cattles. Although these results support our primary assumption, Sho and PrP expressions did not correlate with corresponding mRNA expression, suggesting that the biological modulations of both PrP and Sho proteins are at post-translational levels. Moreover, positive correlations between PRNP and SPRN were found in cattle and buffalo cerebrum as well as in buffalo obex. Conversely, negative correlations between PrP and Sho were detected in buffalo cerebellum and obex tissues. These findings suggest that additional post-transcriptional studies are warranted to elucidate mechanisms behind prion diseases.

Whole Blood Gene Expression Profiling in Preclinical and Clinical Cattle Infected with Atypical Bovine Spongiform Encephalopathy.

Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding. Besides cBSE, two atypical forms (named H- and L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSE-infected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. The microarray analysis revealed gene expression changes in blood prior to the appearance of the clinical signs and during the progression of the disease. A set of 32 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. A 22-gene signature showed an oscillating pattern of expression, being differentially expressed in the preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was downregulated during the progression of the disease. Most of the studies performed up to date utilized various tissues, which are not suitable for a rapid analysis of infected animals and patients. Our findings suggest the intriguing possibility to take advantage of whole blood RNA transcriptional profiling for the preclinical identification of prion infection. Further, this study highlighted several pathways, such as immune response and metabolism that may play an important role in peripheral prion pathogenesis. Finally, the gene expression changes identified in the present study may be further investigated as a fingerprint for monitoring the progression of disease and for developing targeted therapeutic interventions.

Newspaper Reports on BSE around the Time of the Japan-US Trade Conflicts: Content Analysis of Japanese and US Dailies from 2002 to 2006

Mass media can affect how people understand and react to particular health risks. Reporting of health risks during the international trade disputes, resulting from the difference in safety regulations, therefore can play a pivotal role in resolving them. This study compared the newspaper reports on BSE-related events in major national dailies between Japan and the US around the period when BSE-infected cattle were discovered in the US and the import of US beef products was banned (between December 2002 and November 2006). During the study period, the number of BSE-related newspaper articles increased in both the US and Japan, but the visibility of the issue was more prominent and persistent in Japan than in the US. Geographically, most of the articles had a domestic focus, but they also reported the news of each trade partner. After the discovery of BSE cattle in the US, articles of commerce and trade issues were dominant in Japan, while the incidence of BSE, agriculture, and trade dominated in the US. Overall, the US-based newspapers carried more advocacy articles than the Japanese ones. In Japan, calls for stronger domestic policy decreased, but those for stronger foreign policy increased slightly. Meanwhile, in the US, calls for a stronger domestic policy increased slightly whereas those for weaker foreign policy dropped-both only temporarily. The major rationale for policy advocacy was the economy and health in both Japan and the US. However, the balance of competing policy objectives and the rational acceptance of BSE risks were argued more in the US papers than in the Japanese ones. In conclusion, during the BSE-related dispute on health and trade, the visibility and faces of the issues in newspapers differed between Japan and the US. Acceptance of BSE-related risks was argued differently, and those differences reflected and affected the public's perception of BSE issues, the related safety policies by the governments, and the configuration of social interests in each country. The differences evident in the media could serve as a vehicle for reappraising the existing policies as well as the possible international harmonization of risk management policies.

Quantitative analysis of wet-heat inactivation in bovine spongiform encephalopathy

The bovine spongiform encephalopathy (BSE) agent is resistant to conventional microbial inactivation procedures and thus threatens the safety of cattle products and by-products. To obtain information necessary to assess BSE inactivation, we performed quantitative analysis of wet-heat inactivation of infectivity in BSE-infected cattle spinal cords. Using a highly sensitive bioassay, we found that infectivity in BSE cattle macerates fell with increase in temperatures from 133°C to 150°C and was not detected in the samples subjected to temperatures above 155°C. In dry cattle tissues, infectivity was detected even at 170°C. Thus, BSE infectivity reduces with increase in wet-heat temperatures but is less affected when tissues are dehydrated prior to the wet-heat treatment. The results of the quantitative protein misfolding cyclic amplification assay also demonstrated that the level of the protease-resistant prion protein fell below the bioassay detection limit by wet-heat at 155°C and higher and could help assess BSE inactivation. Our results show that BSE infectivity is strongly resistant to wet-heat inactivation and that it is necessary to pay attention to BSE decontamination in recycled cattle by-products.

Prion in Saliva of Bovine Spongiform Encephalopathy–Infected Cattle

Prion in Saliva of Bovine Spongiform Encephalopathy–Infected Cattle

Experimental Bovine Spongiform Encephalopathy: Detection of PrP Sc in the Small Intestine Relative to Exposure Dose and Age

European regulations for the control of bovine spongiform encephalopathy (BSE) decree destruction of the intestines from slaughtered cattle, therefore producers have been obliged to import beef casings from countries with a negligible BSE risk. This study applies immunohistochemical and biochemical approaches to investigate the occurrence and distribution of disease-associated prion protein (PrP(Sc)) in the duodenum, jejunum and ileum of cattle orally exposed to a 1 g or 100 g dose of a titrated BSE brainstem homogenate. Samples were derived from animals at various times post exposure. Lymphoid follicles were counted and the frequency of affected follicles recorded. No PrP(Sc) was detected in the duodenum or jejunum of animals exposed to a 1 g dose or in the duodenum of animals receiving a 100 g dose. PrP(Sc) was detected in the lymphoid tissue of the ileum of 1/98 (1.0%) animals receiving the 1 g dose and in the jejunum and ileum of 8/58 (13.8%) and 45/99 (45.5%), respectively, of animals receiving the 100 g dose. The frequency of PrP(Sc)- positive follicles was less than 1.5% per case and biochemical tests appeared less sensitive than immunohistochemistry. The probability of detecting lymphoid follicles in the ileum declined with age and for the 100 g exposure the proportion of positive follicles increased, while the proportion of positive animals decreased with age. Detection of PrP(Sc) in intestinal neural tissue was rare. The results suggest that the jejunum and duodenum of BSE-infected cattle contain considerably less BSE infectivity than the ileum, irrespective of exposure dose. In animals receiving the low exposure dose, as in most natural cases of BSE, the rarity of PrP(Sc) detection compared with high-dose exposure, suggests a very low BSE risk from food products containing the jejunum and duodenum of cattle slaughtered for human consumption.

Scientific Opinion on a review of the BSE‐related risk in bovine intestines

The opinion reviews a 2007 opinion of the French Food Safety Agency (AFSSA), which, referring to the current situation in France, concluded that the current removal of the whole intestine from bovine animals of all ages as specified risk material could be limited to the ileum. It is concluded that the model used in the AFSSA opinion is an appropriate tool to estimate the Classical BSE prevalence in cattle cohorts in countries with extensive surveillance systems and the maximum number of undetected Classical BSE cases that could enter into the food chain in a particular country. It is concluded that the AFSSA methodology cannot be used to make inference to situations other than the French situation to assess the Classical BSE-related risk in bovine intestines. The new scientific data available are reviewed. These data, which add some new elements, concur and confirm the presence of limited amounts of PrPSc and/or infectivity in parts of the intestine other than ileum of Classical BSE infected cattle under experimental inoculation (jejunum) and natural exposure (distal jejunum and colon). The new scientific information further confirms the presence of consistent amounts of PrPSc and infectivity in the ileum of Classical BSE infected cattle under experimental inoculation and natural exposure. Due to limitations in the data currently available, an accurate quantification of the amount of infectivity in the intestinal parts other than ileum of Classical BSE infected cattle at different stages of the incubation period cannot be provided. It is also concluded that, due to the continuous decline of the Classical BSE epidemic, the current Classical BSE exposure risk from bovine intestines has declined correspondingly. Exposure to Atypical BSE from the consumption of bovine intestines cannot be assessed at this stage and research on the pathogenesis of Atypical BSE is recommended.

Neuropathological Changes in Auditory Brainstem Nuclei in Cattle with Experimentally Induced Bovine Spongiform Encephalopathy

Bovine spongiform encephalopathy (BSE) is characterized by the appearance of spongy lesions in the brain, particularly in the brainstem nuclei. This study evaluated the degenerative changes observed in the central auditory brainstem of BSE-challenged cattle. The neuropathological changes in the auditory brainstem nuclei were assessed by determining the severity of vacuolation and the presence of disease-associated prion protein (PrP(Sc)). Sixteen female Holstein-Friesian calves, 2-4 months of age, were inoculated intracerebrally with BSE agent. BSE-challenged animals developed the characteristic clinical signs of BSE approximately 18 months post inoculation (mpi) and advanced neurological signs after 22 mpi. Before the appearance of clinical signs (i.e. at 3, 10, 12 and 16 mpi), vacuolar change was absent or mild and PrP(Sc) deposition was minimal in the auditory brainstem nuclei. The two cattle sacrificed at 18 and 19 mpi had no clinical signs and showed mild vacuolar degeneration and moderate amounts of PrP(Sc) accumulation in the auditory brainstem pathway. In the animals challenged with BSE agent that developed clinical sings (i.e. after 20 mpi), spongy changes were more prominent in the nucleus of the inferior colliculus compared with the other nuclei of the auditory brainstem and the medial geniculate body. Neuropathological changes characterized by spongy lesions accompanied by PrP(Sc) accumulation in the auditory brainstem nuclei of BSE-infected cattle may be associated with hyperacusia.

Analysis of Clusterin Glycoforms in the Urine of BSE-Infected Fleckvieh–Simmental Cows

Currently approved tests for bovine spongiform encephalopathy (BSE) monitoring in cattle are based on the detection of the disease-related isoform of the prion protein in brain tissue and consequently are only suitable for postmortem diagnosis. Previously, to meet the demand for an antemortem test based on a matrix that would permit easy access and repeated sampling, two-dimensional differential gel electrophoresis (2D-DIGE) was used to perform an unbiased screen of bovine urine. Data demonstrated the altered abundance of particular isoforms of the multifunctional glycoprotein clusterin in urine samples obtained from BSE-infected and age-matched Fleckvieh–Simmental cattle. Unfortunately, the use of particular isoforms of a relatively abundant urine protein such as clusterin for diagnosis faces many of the same challenges encountered in tests based on PrPd detection. In both instances the specific detection of the marker protein is complicated by the high background levels of proteins with identical amino acid sequences, but lacking the disease-specific posttranslational modifications or configuration. The goal of the current study was to define the distinguishing characteristics of the clusterin isoforms observed. Biochemical and mass spectrometry analyses in combination with the generation of bovine clusterin subunit-specific antibodies enabled us to demonstrate that it was β-subunits of clusterin possessing N-linked glycans of complex structure that exhibited differential abundance in response to BSE infection. The charateristic highly glycosylated clusterin β-subunit was detectable as early as 16 mo post infection (mpi) by one-dimensional (1D) Western blot analysis of urine obtained from BSE-infected cattle.

BSE infectivity in the absence of detectable PrPSc accumulation in the tongue and nasal mucosa of terminally diseased cattle

The pathogenesis of bovine spongiform encephalopathy (BSE) infections in cattle has been studied in recent years by using highly sensitive transgenic-mouse bioassays. It has been shown that in this species, the BSE agent amplifies almost exclusively in the central and peripheral nervous system. Even in animals that were killed in the clinical end stage of the disease, the lymphoreticular system was shown to be free of the infectious agent. No other animal species investigated to date exhibits such a restricted BSE-infectivity distribution pattern. However, there is growing evidence for a radial spread of infection from the central nervous system (CNS) into the periphery during the late stages of the disease. In this study, we challenged transgenic mice overexpressing the bovine prion protein with homogenates prepared from a wide variety of tissue samples collected from BSE-infected cattle. As prion infections involve the conversion of the cellular prion protein into its abnormally folded isoform (PrP(Sc)), we applied various detection methods, such as the purification of scrapie-associated fibrils, immunohistochemistry, and the protein misfolding cyclic amplification technique. Despite negative results using these highly sensitive biochemical methods, we were, for the first time, able to detect BSE infectivity in the tongue and in the nasal mucosa of terminally diseased BSE field cases as well as experimentally challenged cattle by transgenic-mouse bioassay. This shows that BSE infectivity can be present in the peripheral tissues of terminally diseased cattle, including tissues used for human consumption.

Experimental Transmission of H-Type Bovine Spongiform Encephalopathy to Bovinized Transgenic Mice

To characterize the biological and biochemical properties of H-type bovine spongiform encephalopathy (BSE), a transmission study with a Canadian H-type isolate was performed with bovinized transgenic mice (TgBoPrP), which were inoculated intracerebrally with brain homogenate from cattle with H-type BSE. All mice exhibited characteristic neurologic signs, and the subsequent passage showed a shortened incubation period. The distribution of disease-associated prion protein (PrP(Sc)) was determined by immunohistochemistry, Western blot, and paraffin-embedded tissue (PET) blot. Biochemical properties and higher molecular weight of the glycoform pattern were well conserved within mice. Immunolabeled granular PrP(Sc), aggregates, and/or plaque-like deposits were mainly detected in the following brain locations: septal nuclei, subcallosal regions, hypothalamus, paraventricular nucleus of the thalamus, interstitial nucleus of the stria terminalis, and the reticular formation of the midbrain. Weak reactivity was detected by immunohistochemistry and PET blot in the cerebral cortex, most thalamic nuclei, the hippocampus, medulla oblongata, and cerebellum. These findings indicate that the H-type BSE prion has biological and biochemical properties distinct from those of C-type and L-type BSE in TgBoPrP mice, which suggests that TgBoPrP mice constitute a useful animal model to distinguish isolates from BSE-infected cattle.

Target cattle age of post-slaughter testing for bovine spongiform encephalopathy and infectivity entering the human food chain in Japan

A model of possible outcomes of bovine spongiform encephalopathy (BSE)-infected cattle in Japan was constructed, and the expected proportion of BSE-infected cattle entering the human food chain and the expected infectivity remaining in edible meat per capita by breed variety and sex were calculated, while changing target ages of BSE testing. As widely accepted, BSE testing for all ages could reduce the proportion of infected cattle entering the human food chain and their infectivity. It was especially effective among female dairy cattle, but the resulting reduction was quite limited. The effectiveness of BSE testing on food safety hardly differs between the target age of ⩾21 months and all ages.

Simulation-based estimation of BSE infection in Japan

The first case of bovine spongiform encephalopathy (BSE) in Japan was found in September 2001. As a result, national BSE surveillance systems in slaughterhouses and farms were introduced between October 2001 and April 2004. All cattle, with the exception of those under 24 months of age that die at farms, now undergo compulsory testing when they die or are slaughtered. The removal of specified risk material (SRM) from all slaughtered cattle and a ban on the feeding of meat-and-bone meal to all farm animals were implemented in October 2001. However, infected cattle that died or were slaughtered before these measures were put into practice could have been a source of infection to other cattle through the rendering process. The slaughtered cattle could also have been a source of infection to humans via SRM that entered the food chain. The purpose of this study was to estimate the number of BSE-infected cattle that could have been a source of infection to cattle and humans before October 2001. Since all typical cases were dairy cattle, this study focused on the dairy cattle population. We developed a simulation model to obtain the year of death and the final disposition of infected cows born in each year from 1996 to 2001. In this model, the dairy cattle population was divided into birth cohorts, and parameters regarding its population dynamics were assumed to be constant. Using this model, the total number of infected cattle in each birth year was estimated by maximum likelihood estimation using data on the number of detected cases from 2002 to 2006. Finally, the number of infected cattle that died or were slaughtered each year was estimated by Monte-Carlo simulation using the same model with the total number of infected cattle estimated by maximum likelihood estimation. It was estimated that the majority of infected cattle that could have been sources of infection before 2001 were born in 1996. The total number born in 1996 was estimated to be 155 (95% confidence interval: 90–275). Of these 155 cattle, 56 died or were slaughtered before October 2001, after the accumulation of infectious agent in their bodies. Only 5 of these 56 cattle were estimated to have been slaughtered. Therefore, the number of infected cattle that could have served as a source of human infection would appear to have been a very limited subset of the BSE-infected cattle in Japan.

Differential expression of erythroid genes in prion disease

We previously reported reduced expression of erythroid-associated factor (ERAF) within haematopoietic tissues of rodent scrapie models, suggesting an unrecognized role for the erythroid lineage in prion disease. In the present study, we compared the expression of a panel of erythroid genes within four murine scrapie models and five virus infection models with parallels to prion disease pathogenesis. We report that differential expression of erythroid genes is not limited to ERAF, and is a common feature of murine scrapie, dependent on host expression of cellular prion protein. In contrast, erythroid gene expression was not altered following virus infection. Whilst these results further implicate cells of the erythroid lineage in the peripheral pathogenesis of prion disease, analysis of blood from BSE-infected cattle and scrapie-infected sheep reveals that the extent of differential expression of erythroid genes within peripheral blood is not sufficient to provide a discriminatory diagnostic test.

Plasma glucose concentration is maintained during TSE infection of cattle and sheep

Keywords BSE.Plasmaglucoseconcentration.Scrapie.TSEdiseaseAbbreviationsABTS 2, 2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)BSE Bovine Spongiform EncephalopathyTSE Transmissible Spongiform EncephalopathyVLA Veterinary Laboratories AgencyIntroductionPrevious studies on cattle incubating BSE have suggested that the energy metabolism ofthese animals is disrupted during the preclinical stages of the disease, particularly at timesassociated with nutritional stress such as early lactation (Moorby et al. 2000). Thisdisruption is manifest as increased plasma concentrations of lactate, β-hydroxybutyrate,alanine and leucine in conjunction with decreased concentrations of serine and glutamicacid (Moorby et al. 2002). The hypothesis suggested was that animals become moredependent upon glycolysis as TSE disease progresses. This hypothesis is supported bymore recent studies with dairy cattle infected with BSE, that showed similar increases inplasma alanine and demonstrated decreased ratios of muscle glycogen to plasma lactatecompared to healthy control cattle, suggesting that BSE infected cattle have altered energymetabolism (Allison et al. 2007). Furthermore, NMR studies have suggested that changes in

The Chickens Come Home to Roost

The Chickens Come Home to Roost

Counting of single prion particles bound to a capture-antibody surface (surface-FIDA)

Hitherto accredited prion tests use the PK resistance of PrP Sc, the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP Sc. Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP Sc was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.

Shedding a negative image

Although the idea of a protein as an infectious agent emerged almost 40 years ago, the hypothesis only became mainstream after Stanley Prusiner's seminal paper describing how proteins could cause disease (Prusiner, 1982). Prusiner, now a professor of neurology at the University of California, San Francisco, USA, won the 1997 Nobel Prize in Physiology or Medicine for his discovery of these proteinaceous infectious particles, known as prions. After some initial resistance, the scientific community has now largely accepted that prions, by misfolding and aggregating into larger clumps, cause neurodegenerative diseases such as scrapie in sheep, bovine spongiform encephalitis (BSE) in cattle and Creutzfeldt–Jakob Disease (CJD) in humans. However, it is only in the past few years that scientists have begun to understand the molecular mechanisms behind infectious proteins. Recent work on how prions propagate their structural conformation by recruiting other proteins helps to explain the pathology behind the neurodegenerative diseases that they cause, and also expands the concept to other proteins and diseases. As scientists gain a better understanding of prions, these proteins are shedding some of their predominantly negative image as pathological aberrations of nature. In fact, prions might have important biological roles in both prokaryotic and higher organisms. An exciting development came earlier this year with the discovery that the ability of prion particles to spread infection depends on their brittleness (Tanaka et al , 2006). Jonathan Weissman, from the Howard Hughes Medical Institute at the University of California, San Francisco, USA, and colleagues showed that the more brittle—and therefore less stable—prion particles break into smaller fragments more frequently. This creates new seeding particles that, in turn, grow into larger aggregates by recruiting new molecules of the same protein. Weissman and his colleagues conducted their studies on yeast prions; although their ability to cause infection is doubted by many …