Form Of Bovine Spongiform Encephalopathy Research Articles

Discrimination of Classical and Atypical BSE by a Distinct Immunohistochemical PrPSc Profile

Bovine spongiform encephalopathy (BSE) belongs to the group of transmissible spongiform encephalopathies and is associated with the accumulation of a pathological isoform of the host-encoded glycoprotein, designated prion protein (PrPSc). Classical BSE (C-type) and two atypical BSE forms (L- and H-type) are known, and can be discriminated by biochemical characteristics. The goal of our study was to identify type-specific PrPSc profiles by using Immunohistochemistry. In our study, brain samples from 21 cattle, intracerebrally inoculated with C-, H-, and L-type BSE, were used. In addition, the corresponding samples from three orally C-type BSE infected animals were also included. From all animals, a lesion and PrPSc-profiles of six brain regions were determined. The lesion profile and the neuroanatomical distribution of PrPSc was highly consistent between the groups, but the immunohistochemical analysis revealed a distinct PrPSc profile for the different BSE-types, which included both the topographic and cellular pattern of PrPSc. This qualitative and quantitative analysis of PrPSc affected structures sheds new light into the pathogenesis of the different BSE types. Furthermore, immunohistochemical characterization is supported as an additional diagnostic tool in BSE surveillance programs, especially when only formalin-fixed tissue samples are available.

Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific reactions of real-time quaking-induced conversion

Real-time quaking-induced conversion (RT-QUIC) assays using Escherichia coli-derived purified recombinant prion protein (rPrP) enable us to amplify a trace amount of the abnormal form of PrP (PrPSc) from specimens. This technique can be useful for the early diagnosis of both human and animal prion diseases and the assessment of prion contamination. In the present study, we demonstrated that there are strain-specific differences in the RT-QUIC reactions between an atypical form of bovine spongiform encephalopathy (BSE), l-BSE, and classical BSE (C-BSE). Whereas mouse rPrP (rMoPrP) was efficiently converted to amyloid fibrils in the presence of PrPSc seed derived from either l-BSE or C-BSE, hamster rPrP (rHaPrP) was converted only in l-BSE, not C-BSE. These characteristics were preserved in the second round reaction, but gradually weakened in the subsequent rounds and were completely lost by the fifth round, most likely due to the selective growth advantage of nonspecific rPrP amyloid fibrils in the RT-QUIC. Our findings further enhance the discrimination of prion strains using RT-QUIC, and further our understanding of the molecular basis of prion strains.

Deciphering the BSE-type specific cell and tissue tropisms of atypical (H and L) and classical BSE

ABSTRACTAfter the discovery of two atypical bovine spongiform encephalopathy (BSE) forms in France and Italy designated H- and L-BSE, the question arose whether these new forms differed from classical BSE (C-BSE) in their pathogenesis. Samples collected from cattle in the clinical stage of BSE during an intracranial challenge study with L- and H-BSE were analysed using biochemical and histological methods as well as in a transgenic mouse bioassay. Our results generally confirmed what had been described for C-BSE to be true also for both atypical BSE forms, namely the restriction of the pathological prion protein (PrPSc) and BSE infectivity to the nervous system. However, analysis of samples collected under identical conditions from both atypical H- and L-BSE forms allowed us a more precise assessment of the grade of involvement of different tissues during the clinical end stage of disease as compared to C-BSE. One important feature is the involvement of the peripheral nervous and musculoskeletal tissues in both L-BSE and H-BSE affected cattle. We were, however, able to show that in H-BSE cases, the PrPSc depositions in the central and peripheral nervous system are dominated by a glial pattern, whereas a neuronal deposition pattern dominates in L-BSE cases, indicating differences in the cellular and topical tropism of both atypical BSE forms. As a consequence of this cell tropism, H-BSE seems to spread more rapidly from the CNS into the periphery via the glial cell system such as Schwann cells, as opposed to L-BSE which is mostly propagated via neuronal cells.

Molecular characterisation of atypical BSE prions by mass spectrometry and changes following transmission to sheep and transgenic mouse models.

The prion hypothesis proposes a causal relationship between the misfolded prion protein (PrPSc) molecular entity and the disease transmissible spongiform encephalopathy (TSE). Variations in the conformation of PrPSc are associated with different forms of TSE and different risks to animal and human health. Since the discovery of atypical forms of bovine spongiform encephalopathy (BSE) in 2003, scientists have progressed the molecular characterisation of the associated PrPSc in order to better understand these risks, both in cattle as the natural host and following experimental transmission to other species. Here we report the development of a mass spectrometry based assay for molecular characterisation of bovine proteinase K (PK) treated PrPSc (PrPres) by quantitative identification of its N-terminal amino acid profiles (N-TAAPs) and tryptic peptides. We have applied the assay to classical, H-type and L-type BSE prions purified from cattle, transgenic (Tg) mice expressing the bovine (Tg110 and Tg1896) or ovine (TgEM16) prion protein gene, and sheep brain. We determined that, for classical BSE in cattle, the G96 N-terminal cleavage site dominated, while the range of cleavage sites was wider following transmission to Tg mice and sheep. For L-BSE in cattle and Tg bovinised mice, a C-terminal shift was identified in the N-TAAP distribution compared to classical BSE, consistent with observations by Western blot (WB). For L-BSE transmitted to sheep, both N-TAAP and tryptic peptide profiles were found to be changed compared to cattle, but less so following transmission to Tg ovinised mice. Relative abundances of aglycosyl peptides were found to be significantly different between the atypical BSE forms in cattle as well as in other hosts. The enhanced resolution provided by molecular analysis of PrPres using mass spectrometry has improved insight into the molecular changes following transmission of atypical BSE to other species.

Chapter 7 - Atypical and classic bovine spongiform encephalopathy

This chapter describes the prion diseases of cattle, or bovine transmissible spongiform encephalopathies (BoTSEs). "Classic" bovine spongiform encephalopathy (C-BSE), the major prion protein disorder of Bovidae, was first described in 1986. We also describe the spatiotemporal correlation of C-BSE to a novel form of human prion disease, variant Creutzfeldt-Jakob disease (vCJD), which led to the classification of BSE as a zoonotic disease (and the "cause" of vCJD) in 1996. From isolated cases first identified retrospectively in May 1985, a major bovine spongiform encephalopathy (BSE) epidemic peaked within the British Isles in 1991, and has so far led to over 195,000 confirmed cases in cattle, and several thousand more cases within Europe and a few elsewhere. By 2017, the disease had been essentially eradicated below the level of surveillance detection in Europe, although sporadic cases are still predicted to show up for several years to come. By 2004, other forms of BSE, H-type and L-type, had been recognized and, in the absence of information to the contrary, are also included in the classification of BSE as a zoonotic prion disease of bovine origin. This chapter aims to cover the clinical, epidemiologic, neuropathologic, biochemical, biomarker, and pathogenetic aspects of the bovine prion disease and gives a brief, but important, description of the surveillance of BSE and other animal TSEs in Europe.

Chapter 25 - The zoonotic potential of animal prion diseases

Bovine spongiform encephalopathy (BSE) is the only animal prion disease that has been demonstrated to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans. The link between BSE and vCJD was established by careful surveillance, epidemiologic investigations, and experimental studies using in vivo and in vitro models of cross-species transmission. Similar approaches have been used to assess the zoonotic potential of other animal prion diseases, including atypical forms identified through active surveillance. There is no epidemiologic evidence that classical or atypical scrapie, atypical forms of BSE, or chronic wasting disease (CWD) is associated with human prion disease, but the limitations of the epidemiologic data should be taken into account when interpreting these results. Transmission experiments in nonhuman primates and human PrP transgenic mice suggest that classic scrapie, L-type atypical BSE (L-BSE), and CWD may have zoonotic potential, which for L-BSE appears to be equal to or greater than that of classic BSE. The results of in vitro conversion assays to analyze the human transmission barrier correlate well with the in vivo data. However, it is still difficult to predict the likelihood that an animal prion disease will transmit to humans under conditions of field exposure from the results of in vivo or in vitro experiments. This emphasizes the importance of continuing systematic surveillance for both human and animal prion diseases in identifying zoonotic transmission of diseases other than classic BSE.

Lack of germline mutation at codon 211 of the prion protein gene (PRNP) in Korean native cattle - Short communication.

Bovine prion diseases are composed of two types of bovine spongiform encephalopathy (BSE), classical BSE and atypical BSE. Recent studies have identified one case of atypical BSE with an E211K mutation. E211K is homologous to the human E200K mutation, which is related to familial Creutzfeldt-Jakob disease (CJD), one of the familial forms of human prion diseases. To date, familial forms of prion diseases have not been reported in non-human animals. Because the familial forms of human prion diseases account for more than 10% of all human prion disease cases, the detection of the E211K mutation in healthy cattle is very important for verifying the role of this mutation as a familial form of BSE. To detect putative mutations related to familial BSE, specifically E211K in Korean native cattle (Hanwoo) and Korean dairy cattle (Holstein), we performed direct sequencing targeting codon 211 and the adjacent regions of the bovine prion protein (PRNP) gene in 384 Hanwoo and 152 Holstein cattle. We did not find the E211K mutation in any of the Korean cattle. Although we did not find the E211K mutation in Korean native cattle, E211K is a postulated mutation; therefore, further screening in other countries and larger samples is highly desirable.

Biochemical Characterization of Prions.

Prion disease or transmissible spongiform encephalopathies are characterized by the presence of the abnormal form of the prion protein (PrPSc). The pathological and transmissible properties of PrPSc are enciphered in its secondary and tertiary structures. Since it's well established that different strains of prions are linked to different conformations of PrPSc, biochemical characterization of prions seems a preliminary but reliable approach to detect, analyze, and compare prion strains. Experimental biochemical procedures might be helpful in distinguishing PrPSc physicochemical properties and include resistance to proteinase K (PK) digestion, insolubility in nonionic detergents, PK-resistance under denaturing conditions and sedimentation properties in sucrose gradients. This biochemical approach has been extensively applied in human prion disorders and subsequently expanded for PrPSc characterization in animals. In particular, in sporadic Creutzfedlt-Jakob disease (sCJD) PrPSc is characterized by two main glycotypes conventionally named Type 1 and Type 2, based on the apparent gel migration at 21 and 19kDa of the PrPSc PK-resistant fragment. An additional PrPSc type was identified in sCJD characterized by an unglycosylated dominant glycoform pattern and in 2010 a variably protease-sensitive prionopathy (VPSPr) was reported showing a PrPSc with an electrophoretic ladder like pattern. Additionally, the presence of PrPSc truncated fragments completes the electrophoretic characterization of different prion strains. By two-dimensional (2D) electrophoretic analysis additional PrPSc pattern was identified, since this procedure provides information about the isoelectric point and the different peptides length related to PK cleavage, as well as to glycosylation extent or GPI anchor presence. We here provide and extensive review on PrPSc biochemical analysis in human and animal prion disorders. Further, we show that PrPSc glycotypes observed in CJD share similarities with PrPSc in bovine spongiform encephalopathy forms (BSE).

A sensitive 301V BSE serial PMCA assay

The prion strain 301V, is a mouse passaged form of bovine spongiform encephalopathy (BSE). It has been used as a model of BSE for more than 20 years, in particular in the investigation of tissue distribution of infectivity, the molecular phenotype and transmission properties of BSE, strain typing assays and prion inactivation studies. Most 301V experiments have required murine bioassay as a method for the quantitation of infectivity. To date this model strain has not been studied with the protein misfolding cyclic amplification assay (PMCA) which detects prion-associated PrPSc protein. The detection of BSE PrPSc by PMCA can be more sensitive than mouse bioassay and is carried out in a much shorter time frame of days as opposed to months/years. Here, we describe the development of a new highly sensitive and specific PMCA assay for murine 301V and assess the sensitivity of the assay in direct comparison with murine bioassay of the same material. This in vitro assay detected, in a few days, 301V at a brain dilution of at least 1x10-9, compared to bioassay of the same material in VM mice that could detect down to a 1x10-8 dilution and took >180 days. The 301V PMCA may therefore offer a faster and more sensitive alternative to live animal bioassay when studying the BSE agent in VM mice.

Absence of Evidence for a Causal Link between Bovine Spongiform Encephalopathy Strain Variant L-BSE and Known Forms of Sporadic Creutzfeldt-Jakob Disease in Human PrP Transgenic Mice.

Since the quasi-extinction of classical BSE, atypical BSE forms are the sole BSE variants circulating in cattle worldwide. They are observed in rare cases of old cattle, making them difficult to detect. Extrapolation of our results suggests that L-BSE may propagate in humans as an unrecognized form of CJD, and we urge both the continued utilization of precautionary measures to eliminate these agents from the human food chain and active surveillance for CJD phenotypes in the general population.

Atypical variants of bovine spongiform encephalopathy: rare diseases with consequences for BSE surveillance and control.

Occurring for the first time in 1986 in the United Kingdom, bovine spongiform encephalopathy (BSE), the so-called "mad-cow disease", has had unprecedented consequences in veterinary public health. The implementation of drastic measures, including the ban of meat-and-bone-meal from livestock feed and the removal of specified risk materials from the food chain has eventually resulted in a significant decline of the epidemic. The disease was long thought to be caused by a single agent, but since the introduction of immunochemical diagnostic techniques, evidence of a phenotypic variation of BSE has emerged. Reviewing the literature available on the subject, this paper briefly summarizes the current knowledge about these atypical forms of BSE and discusses the consequences of their occurrence for disease control measures.

Intra- and Interspecies Transmission of Atypical BSE - What Can We Learn from It?

After the detection of the first cases of atypical bovine spongiform encephalopathy (BSE) more than ten years ago, the etiology, pathogenesis and agent distribution of these novel BSE forms in cattle were completely unknown. Many studies have been performed in the meantime to elucidate the pathogenic mechanisms of these diseases. A wealth of data has been accumulated regarding the distribution of the abnormal isoform of the prion protein, PrPSc, in tissues of affected cattle, confirming the general restriction of the PrPSc and agent distribution to the central and peripheral nervous system, albeit at slightly higher levels as compared to classical BSE. However, due to lack of data, the assumptions regarding the spontaneous etiology of both atypical BSE forms (H-BSE and L-BSE) and also the origin of the classical BSE epidemic are still mainly speculative. By performing subpassage experiments of both the atypical BSE forms in a variety of conventional and transgenic mice and Syrian Gold hamsters, we aimed to improve our understanding of the strain stability of these BSE forms. It turned out that under these experimental conditions, both the atypical BSE forms may alter their phenotypes and become indistinguishable from classical BSE. Information about the classical and atypical BSE strain characteristics help to improve our understanding of the correlation between all three BSE forms.

Methods for Differentiating Prion Types in Food-Producing Animals.

Prions are an enigma amongst infectious disease agents as they lack a genome yet confer specific pathologies thought to be dictated mainly, if not solely, by the conformation of the disease form of the prion protein (PrPSc). Prion diseases affect humans and animals, the latter including the food-producing ruminant species cattle, sheep, goats and deer. Importantly, it has been shown that the disease agent of bovine spongiform encephalopathy (BSE) is zoonotic, causing variant Creutzfeldt Jakob disease (vCJD) in humans. Current diagnostic tests can distinguish different prion types and in food-producing animals these focus on the differentiation of BSE from the non-zoonotic agents. Whilst BSE cases are now rare, atypical forms of both scrapie and BSE have been reported, as well as two types of chronic wasting disease (CWD) in cervids. Typing of animal prion isolates remains an important aspect of prion diagnosis and is now becoming more focused on identifying the range of prion types that are present in food-producing animals and also developing tests that can screen for emerging, novel prion diseases. Here, we review prion typing methodologies in light of current and emerging prion types in food-producing animals.

Detection and discrimination of classical and atypical L-type bovine spongiform encephalopathy by real-time quaking-induced conversion.

Statutory surveillance of bovine spongiform encephalopathy (BSE) indicates that cattle are susceptible to both classical BSE (C-BSE) and atypical forms of BSE. Atypical forms of BSE appear to be sporadic and thus may never be eradicated. A major challenge for prion surveillance is the lack of sufficiently practical and sensitive tests for routine BSE detection and strain discrimination. The real-time quaking-induced conversion (RT-QuIC) test, which is based on prion-seeded fibrillization of recombinant prion protein (rPrPSen), is known to be highly specific and sensitive for the detection of multiple human and animal prion diseases but not BSE. Here, we tested brain tissue from cattle affected by C-BSE and atypical L-type bovine spongiform encephalopathy (L-type BSE or L-BSE) with the RT-QuIC assay and found that both BSE forms can be detected and distinguished using particular rPrPSen substrates. Specifically, L-BSE was detected using multiple rPrPSen substrates, while C-BSE was much more selective. This substrate-based approach suggests a diagnostic strategy for specific, sensitive, and rapid detection and discrimination of at least some BSE forms.

Presence of subclinical infection in gene-targeted human prion protein transgenic mice exposed to atypical bovine spongiform encephalopathy

The transmission of bovine spongiform encephalopathy (BSE) to humans, leading to variant Creutzfeldt-Jakob disease has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health. Until recently, TSE disease in cattle was thought to be caused by a single agent strain, BSE, also known as classical BSE, or BSE-C. However, due to the initiation of a large-scale surveillance programme throughout Europe, two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H have since been discovered. To model the risk to human health, we previously inoculated these two forms of atypical BSE (BASE and BSE-H) into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP) (HuTg) but were unable to detect any signs of TSE pathology in these mice. However, despite the absence of TSE pathology, upon subpassage of some BASE-challenged HuTg mice, a TSE was observed in recipient gene-targeted bovine PrP Tg (Bov6) mice but not in HuTg mice. Disease transmission from apparently healthy individuals indicates the presence of subclinical BASE infection in mice expressing human PrP that cannot be identified by current diagnostic methods. However, due to the lack of transmission to HuTg mice on subpassage, the efficiency of mouse-to-mouse transmission of BASE appears to be low when mice express human rather than bovine PrP.

Biochemical Characteristics and PrPSc Distribution Pattern in the Brains of Cattle Experimentally Challenged with H-type and L-type Atypical BSE

Besides the classical form of bovine spongiform encephalopathy (BSE) that has been known for almost three decades, two atypical forms designated H-type and L-type BSE have recently been described. While the main diagnostic feature of these forms is the altered biochemical profile of the accumulated PrPSc, it was also observed in the initial analysis that L-type BSE displays a distribution pattern of the pathological prion protein (PrPSc), which clearly differs from that observed in classical BSE (C-type). Most importantly, the obex region in the brainstem is not the region with the highest PrPSc concentrations, but PrPSc is spread more evenly throughout the entire brain. A similar distribution pattern has been revealed for H-type BSE by rapid test analysis. Based on these findings, we performed a more detailed Western blot study of the anatomical PrPSc distribution pattern and the biochemical characteristics (molecular mass, glycoprofile as well as PK sensitivity) in ten different anatomical locations of the brain from cattle experimentally challenged with H- or L-type BSE, as compared to cattle challenged with C-type BSE. Results of this study revealed distinct differences in the PrPSc deposition patterns between all three BSE forms, while the biochemical characteristics remained stable for each BSE type among all analysed brain areas.

Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein

The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several novel TSEs in sheep, cattle and deer have been described and the risk posed to humans by these agents is currently unknown. In this study, we inoculated two forms of atypical BSE (BASE and H-type BSE), a chronic wasting disease (CWD) isolate and seven isolates of atypical scrapie into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP). Upon challenge with these ruminant TSEs, gene-targeted Tg mice expressing human PrP did not show any signs of disease pathology. These data strongly suggest the presence of a substantial transmission barrier between these recently identified ruminant TSEs and humans.

Infectivity in Skeletal Muscle of Cattle with Atypical Bovine Spongiform Encephalopathy

The amyloidotic form of bovine spongiform encephalopathy (BSE) termed BASE is caused by a prion strain whose biological properties differ from those of typical BSE, resulting in a clinically and pathologically distinct phenotype. Whether peripheral tissues of BASE-affected cattle contain infectivity is unknown. This is a critical issue since the BASE prion is readily transmissible to a variety of hosts including primates, suggesting that humans may be susceptible. We carried out bioassays in transgenic mice overexpressing bovine PrP (Tgbov XV) and found infectivity in a variety of skeletal muscles from cattle with natural and experimental BASE. Noteworthy, all BASE muscles used for inoculation transmitted disease, although the attack rate differed between experimental and natural cases (∼70% versus ∼10%, respectively). This difference was likely related to different prion titers, possibly due to different stages of disease in the two conditions, i.e. terminal stage in experimental BASE and pre-symptomatic stage in natural BASE. The neuropathological phenotype and PrPres type were consistent in all affected mice and matched those of Tgbov XV mice infected with brain homogenate from natural BASE. The immunohistochemical analysis of skeletal muscles from cattle with natural and experimental BASE showed the presence of abnormal prion protein deposits within muscle fibers. Conversely, Tgbov XV mice challenged with lymphoid tissue and kidney from natural and experimental BASE did not develop disease. The novel information on the neuromuscular tropism of the BASE strain, efficiently overcoming species barriers, underlines the relevance of maintaining an active surveillance.

Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases

BackgroundThree distinct forms of bovine spongiform encephalopathy (BSE), defined as classical (C-), low (L-) or high (H-) type, have been detected through ongoing active and passive surveillance systems for the disease.The aim of the present study was to compare the ability of two sets of immunohistochemical (IHC) and Western blot (WB) BSE confirmatory protocols to detect C- and atypical (L- and H-type) BSE forms.Obex samples from cases of United States and Italian C-type BSE, a U.S. H-type and an Italian L-type BSE case were tested in parallel using the two IHC sets and WB methods.ResultsThe two IHC techniques proved equivalent in identifying and differentiating between C-type, L-type and H-type BSE. The IHC protocols appeared consistent in the identification of PrPSc distribution and deposition patterns in relation to the BSE type examined. Both IHC methods evidenced three distinct PrPSc phenotypes for each type of BSE: prevailing granular and linear tracts pattern in the C-type; intraglial and intraneuronal deposits in the H-type; plaques in the L-type.Also, the two techniques gave comparable results for PrPSc staining intensity on the C- and L-type BSE samples, whereas a higher amount of intraglial and intraneuronal PrPSc deposition on the H-type BSE case was revealed by the method based on a stronger demasking step.Both WB methods were consistent in identifying classical and atypical BSE forms and in differentiating the specific PrPSc molecular weight and glycoform ratios of each form.ConclusionsThe study showed that the IHC and WB BSE confirmatory methods were equally able to recognize C-, L- and H-type BSE forms and to discriminate between their different immunohistochemical and molecular phenotypes. Of note is that for the first time one of the two sets of BSE confirmatory protocols proved effective in identifying the L-type BSE form. This finding helps to validate the suitability of the BSE confirmatory tests for BSE surveillance currently in place.

Classical Bovine Spongiform Encephalopathy by Transmission of H-Type Prion in Homologous Prion Protein Context

Bovine spongiform encephalopathy (BSE) and BSE-related disorders have been associated with a single major prion strain. Recently, 2 atypical, presumably sporadic forms of BSE have been associated with 2 distinct prion strains that are characterized mainly by distinct Western blot profiles of abnormal protease-resistant prion protein (PrPres), named high-type (BSE-H) and low-type (BSE-L), that also differed from classical BSE. We characterized 5 atypical BSE-H isolates by analyzing their molecular and neuropathologic properties during transmission in transgenic mice expressing homologous bovine prion protein. Unexpectedly, in several inoculated animals, strain features emerged that were highly similar to those of classical BSE agent. These findings demonstrate the capability of an atypical bovine prion to acquire classical BSE-like properties during propagation in a homologous bovine prion protein context and support the view that the epidemic BSE agent could have originated from such a cattle prion.