Detection of CNS and PrPSc in meat products

Several methods for the detection of tissues of the central nervous system (CNS) in meat products have been developed and partly validated for use in official food control as pertaining to human BSE-exposure risk. So far, however, methods for the detection of abnormal prion protein (PrPSc) were not evaluated for their potential applicability to the matrix of heat treated meat products. We developed a micro technological procedure for the preparation of meat products suitable for high security laboratories as masses were 6 to 8 orders of magnitude lower than in conventional meat technology. Thus it was possible to produce standard micro sausages containing defined amounts of bovine BSE-positive brain. This material showed all characteristics of normal meat products and a homogeneous distribution of brain as indicated by NSE and GFAP western immunoblotting and GFAP immunometric analyses. Using a commercially available and certified immunometric assay for detection of PrPSc in untreated brain it was possible to detect BSE-positive CNS down to a content of 0.25% in heat treated meat products. We found a high correlation between PrPSc OD-values and CNS content and linearity up to 10% CNS. In 30 samples of retail meat products no sample transgressed the official cut off value for untreated bovine brain. Further studies are needed to show whether an increase of sensitivity in PrPSc detection from the meat product matrix is possible, in particular by optimisation of the extraction procedure.     

Ten years of BSE surveillance in Italy: neuropathological findings in clinically suspected cases

Between 2001 and 2010, 244 clinically suspected cases of bovine spongiform encephalopathy (BSE) were reported in Italy. This report summarizes the neuropathological findings in cattle displaying clinical signs consistent with a diagnosis of BSE. All animal specimens were submitted for confirmatory testing; samples testing negative underwent neuropathological examination to establish the differential diagnosis. Immunohistochemistry for scrapie prion protein (PrPSc) at the level of frontal cortex was carried out to exclude atypical BSE. Neuropathological changes were detected in 34.9% of cases; no histological lesions were found in 52.3% of subjects; 12.8% of samples were found unsuitable for analysis. BSE was detected in one case, but no cases of atypical BSE were observed. This study identified the diseases most commonly encountered in the differential diagnosis of BSE; furthermore, it demonstrated that the surveillance system is necessary for monitoring neuropathological disease in cattle and for the detection of BSE cases.     

PrPSc spreading patterns in the brain of sheep linked to different prion types

ABSTRACT: Scrapie in sheep and goats has been known for more than 250 years and belongs nowadays to the so-called prion diseases that also include e.g. bovine spongiform encephalopathy in cattle (BSE) and Creutzfeldt-Jakob disease in humans. According to the prion hypothesis, the pathological isoform (PrPSc) of the cellular prion protein (PrPc) comprises the essential, if not exclusive, component of the transmissible agent. Currently, two types of scrapie disease are known - classical and atypical/Nor98 scrapie. In the present study we examine 24 cases of classical and 25 cases of atypical/Nor98 scrapie with the sensitive PET blot method and validate the results with conventional immunohistochemistry. The sequential detection of PrPSc aggregates in the CNS of classical scrapie sheep implies that after neuroinvasion a spread from spinal cord and obex to the cerebellum, diencephalon and frontal cortex via the rostral brainstem takes place. We categorize the spread of PrPSc into four stages: the CNS entry stage, the brainstem stage, the cruciate sulcus stage and finally the basal ganglia stage. Such a sequential development of PrPSc was not detectable upon analysis of the present atypical/Nor98 scrapie cases. PrPSc distribution in one case of atypical/Nor98 scrapie in a presumably early disease phase suggests that the spread of PrPSc aggregates starts in the di- or telencephalon. In addition to the spontaneous generation of PrPSc, an uptake of the infectious agent into the brain, that bypasses the brainstem and starts its accumulation in the thalamus, needs to be taken into consideration for atypical/Nor98 scrapie.     

Experimental H-type bovine spongiform encephalopathy characterized by plaques and glial- and stellate-type prion protein deposits

ABSTRACT: Atypical bovine spongiform encephalopathy (BSE) has recently been identified in Europe, North America, and Japan. It is classified as H-type and L-type BSE according to the molecular mass of the disease-associated prion protein (PrPSc). To investigate the topographical distribution and deposition patterns of immunolabeled PrPSc, H-type BSE isolate was inoculated intracerebrally into cattle. H-type BSE was successfully transmitted to 3 calves, with incubation periods between 500 and 600 days. Moderate to severe spongiform changes were detected in the cerebral and cerebellar cortices, basal ganglia, thalamus, and brainstem. H-type BSE was characterized by the presence of PrP-immunopositive amyloid plaques in the white matter of the cerebrum, basal ganglia, and thalamus. Moreover, intraglial-type immunolabeled PrPSc was prominent throughout the brain. Stellate-type immunolabeled PrPSc was conspicuous in the gray matter of the cerebral cortex, basal ganglia, and thalamus, but not in the brainstem. In addition, PrPSc accumulation was detected in the peripheral nervous tissues, such as trigeminal ganglia, dorsal root ganglia, optic nerve, retina, and neurohypophysis. Cattle are susceptible to H-type BSE with a shorter incubation period, showing distinct and distinguishable phenotypes of PrPSc accumulation.     

Glycosylation of prion strains in transmissible spongiform encephalopathies

This is a review of prion replication in the context of the cell biology of membrane proteins especially folding quality control in the endoplasmic reticulum (ER). Transmissible spongiform encephalopathies, such as scrapie and BSE, are infectious lethal diseases of mammalian neurons characterised by conversion of the normal membrane protein PrPC to the disease-associated conformational isomer called PrPSc. PrPSc, apparently responsible for infectivity, forms a number of different conformations and specific N-glycosylation site occupancies that correlate with TSE strain differences. Dimerisation and specific binding of PrPc and PrPSc seems critical in PrPSc biosynthesis and is influenced by N-glycosylation and disulfide bond formation. PrPsc can be amplified in vitro but new glycosylation cannot occur in cell free environments without the special conditions of microsome mediated in vitro translation, thus strain specific glycosylation of PrPSc formed in vitro in the absence of these conditions must take place by imprintation of PrPc from existing glycosylation site-occupancies. PrPSc formed in cell free homogenates is not infectious pointing to events necessary for infectivity that only occur in intact cells. Such events may include glycosylation site occupancy and ER folding chaperone activity. In the biosynthetic pathway of PrPSc, early acquisition of sensitivity of the GPI anchor to phospholipase C can be distinguished from the later acquisition of protease resistance and detergent insolubility. By analogy to the co-translational formation of the MHC I loading complex, it is postulated that PrPSc or its specific peptides could imprint nascent PrPc chains thereby ensuring its own folds and the observed glycosylation site occupancy ratios of strains.     

Prions as the driving force in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies are degenerative disorders affecting the central nervous system (CNS) occurring in a variety of species. The causative agent is thought to be composed of an abnormal form of the host encoded prion protein (PrPC), termed PrPSc. The conformational change of PrPC into PrPSc can occur spontaneously, however, it can also be induced by PrPSc. Prion diseases such as bovine spongiform encephalopathy (BSE), scrapie and variant Creutzfeldt-Jakob-Disease (vCJD) are most likely caused by peripheral uptake of prions. The process by which prions proceed to the CNS following peripheral uptake is referred to as neuroinvasion. Infection with prions is thought to occur in two phases: After ingestion prions first replicate in lymphatic tissue and then gain access to the CNS via peripheral nerves. Studies looking at the biochemical and clinical characteristics of BSE and vCJD demonstrated that BSE is most likely responsible for vCJD in humans.     

Comparative study of the effects of several chemical and physical treatments on the activity of protease resistance and infectivity of scrapie strain 263K

To study the influences of chemical and physical factors on the protease resistant activity in vitro and the infectivity in vivo of scrapie strain 263K, PrPSc from the hamsters infected intracerebrally with scrapie strain 263K were treated with several commonly used disinfection methods, including sodium hydroxide (NaOH), sodium hypochlorite (NaOCl), heating or autoclaving at 80, 100, 121 and 134 degrees C in the solutions with or without 3% sodium dodecyl sulphate (SDS). The protease resistance of PrPSc was analysed by a proteinase K (PK) digesting Western blot and the infectivity of PrPSc was analysed by intracerebral (i.c.) inoculation into experimental hamsters. The results showed that PrPSc signals were removed in the preparations treated with NaOH higher than 0.05 mol/l, NaOCl higher than 0.1%, autoclaved over 121 degrees C, or heated over 80 degrees C in the presence of 3% SDS. Animal challenges revealed that mixing with 2 mol/l NaOH or 2% NaOCl, autoclaving at 134 degrees C, as well as heating at 100 degrees C or autoclaving at 121 degrees C in the solutions with 3% SDS completely blocks the transmission of scrapie 263K in this experimental situation. It is obvious that the removal of PK resistance of PrPSc happened at relatively lower concentration chemicals or lower temperature, while elimination of the infectivity needs more vigorous conditions. Our data provide the useful evidences for several commonly used methods to inactivate TSEs agent and suggest that it is inappropriate to use PrPSc as a surrogate for TSEs infectivity in inactivation experiments.     

朊蛋白与金属离子之间作用的研究进展

朊病毒(PrP^Sc)是由动物体内正常朊蛋白PrP^c构象改变形成的,PrP^Sc与PrP^C在氨基酸序列上完全一致,PrP^C中α-螺旋丰富而PrP^Sc富含β-折叠。目前,科学家还未研究清楚PrP的生理机能。人们普遍认为:人和动物感染朊病毒病时除PrP外还存在一些重要的辅助因子影响着PrP^C变构、PrP^Sc传播、PrP^Sc引起神经细胞凋亡等病变过程,因此,研究朊蛋白的各种辅助因子将有助于阐明这方面的问题,这些辅助因子包括各种金属离子,如Ca²⁺、Cu²⁺、Fe²⁺、Mn²⁺。作者概述金属离子对朊蛋白结构和功能的影响,以及它们在朊病毒病的发病过程中可能起的作用。     

Accumulation of pathogenic prion protein (PrPSc) in nervous and lymphoid tissues of sheep with subclinical scrapie

All sheep older than 1 year of age from a flock of the Rygja breed in which clinical scrapie was detected for the first time in two animals (4%) were examined for accumulation of pathogenic prion protein (PrPSc) by immunohistochemistry in the obex, the cerebellum, and the medial retrophayngeal lymph node. In addition, six lambs, 2-3 months old, all offspring of PrPSc-positive dams, were examined for PrPSc in the ileal Peyers' patch (IPP), the distal jejunal lymph node, the spleen, and the medial retropharyngeal lymph node (RPLN). In this flock, 35% (17/48) of the adult sheep showed accumulation of PrPSc, an eightfold increase compared with clinical disease. All positives carried susceptible PrP genotypes. Three sheep had deposits of PrPSc in the RPLN and not in the brain, suggesting that this organ, easily accessible at slaughter, is suitable for screening purposes. Two 7-year-old clinically healthy homozygous V136Q171 ewes showed sparse immunostaining in the central nervous system and may have been infected as adults. Further, two littermates, 86-days-old, showed PrPSc in the IPP. Interestingly, one of these lambs had the intermediate susceptible PrP genotype, VA136QR171. In addition to early immunolabeling in the dorsal motor nucleus of the vagal nerve, a few of the sheep had early involvement of the cerebellum. In fact, a 2-year-old sheep had sparse deposits of PrPSc in the cerebellum only. Because experimental bovine spongiform encephalopathy (BSE) in sheep seems to behave in a similar manner as natural scrapie, these results, particularly regarding spread of infectivity, may have implications for the handling of BSE should it be diagnosed in sheep.     

22L毒株朊蛋白错误折叠循环扩增方法的建立

参考Saborio和Soto创建的名为蛋白质错误折叠循环扩增(protein misfolding cyclic amplification,PMCA)的方法建立一种22L毒株系朊蛋白错误折叠循环扩增方法,以便研究22L毒株系异常朊蛋白(scrapie PrP,PrPSc)的合成机制。在阴离子去污剂十二烷基硫酸钠(SDS)存在的条件下,这种方法可以通过超声降解从而重复扩增产物,并在仓鼠脑匀浆中快速有效的模仿体内朊病毒PrPSc的复制,因此可以在几个小时内高浓度扩增PrPSc。已有试验结果发现,PrPSc体外转化率在使用粗提的脑组织匀浆时比使用纯化重组PrP更高,这个发现暗示着高效率的PrPSc转化可能还需要探索其他未知的宿主因素。研究结果表明,建立的这种改良的PMCA技术可用于研究PrPSc合成的生物化学机制,以此来进行诊断与治疗方法的研究。     

Immunohistochemical detection and distribution of prion protein in a goat with natural scrapie.

Formalin-fixed, paraffin-embedded tissue sections from a 3-year-old female Angora goat suffering from clinical scrapie were immunostained after hydrated autoclaving using a monoclonal antibody (mAb, F99/97.6.1; IgG1) specific for a conserved epitope on the prion protein. Widespread and prominent deposition of the scrapie isoform of the prion protein (PrPSc) was observed in the brain, brainstem, spinal cord, retina, postganglionic neurons associated with parasympathetic ganglia of myenteric and submucosal plexuses, Peyer's patches, peripheral lymph nodes, and pharyngeal and palatine tonsils. The goat was homozygous for PrP alleles encoding 5 octapeptide repeat sequences in the N-terminal region of the prion protein and isoleucine at codon 142, a genotype associated with high susceptibility and short incubation times in goats. The results of this study indicate that mAb F99/97.6.1 is useful for detection of PrPSc deposition, and this is a specific and reliable immunohistochemical adjunct to histopathology for diagnosis of natural caprine scrapie, although precise determination of the diagnostic sensitivity and specificity of the assay as a diagnostic test for scrapie in goats will require examination of a sufficiently large sample size. As with ovine scrapie, prion protein is widely distributed in the central and peripheral nervous systems, gastrointestinal tract, and lymphoid tissues in natural caprine scrapie.     

Feline spongiform encephalopathy: first clinical case in Switzerland

A six-year-old female Birman cat was referred to our clinic because of chronic progressive changes in behavior. Additionally, generalized vestibular ataxia and psychomotor seizures were noticed. A multifocal lesion in the forebrain as well as brainstem was suspected. Ancillary investigations such as complete blood cell count, serum biochemistry profile, urinalysis and cerebrospinal fluid examination revealed no significant abnormalities. Electroencephalography showed diffuse changes in the cortical activity. Feline spongiform encephalopathy was confirmed by histological brain examination and positive immunohistochemistry for PrPSc. This is the first time that a case of feline spongiform encephalopathy is diagnosed in Switzerland.     

The use of non-prion biomarkers for the diagnosis of Transmissible Spongiform Encephalopathies in the live animal

Scrapie and bovine spongiform encephalopathy (BSE) are major global concerns and the emergence of variant Creutzfeldt-Jakob disease (vCJD) has caused turmoil for blood transfusion services and hospitals worldwide. Recent reports of iatrogenic CJD (iCJD) cases following blood transfusions from Transmissible Spongiform Encephalopathies (TSE)-infected donors have fuelled this concern. Major diagnostic tests for BSE and scrapie are conducted post-mortem from animals in late stages of the disease. Although the lymphoreticular system is involved in the earlier pathogenesis of some forms of sheep scrapie and vCJD, which presents great opportunity for diagnostic development, other TSE diseases (some strains of scrapie, sporadic CJD (sCJD) and bovine BSE) do not present such a diagnostic opportunity. Thus, there is an urgent need for pre-mortem tests that differentiate between healthy and diseased individuals at early stages of illness, in accessible samples such as blood and urine using less invasive procedures. This review reports on the current state of progress in the development and use of prion and non-prion biomarkers in the diagnosis of TSE diseases. Some of these efforts have concentrated on improving the sensitivity of PrPSc detection to allow in vivo diagnosis at low abundances of PrPSc whilst others have sought to identify non-prion protein biomarkers of TSE disease, many of which are still at early stages of development. In this review we comment upon the limitations of prion based tests and review current research on the development of tests for TSE that rely on non-prion disease markers in body fluids that may allow preclinical disease diagnosis.     

Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review

Postweaning multisystemic wasting syndrome, caused by porcine circovirus type 2 (PCV2), is a recently described clinical condition which affects nursery and growing pigs. PMWS was initially recognized in Canada in 1991 and nowadays is considered to be worldwide distributed. Clinically, PMWS most representative symptoms include wasting, unthriftness, paleness of the skin, respiratory distress, diarrhea and sometimes icterus. PCV2 infection occurs in both PMWS affected and non-affected farms, and viral seroconversion shows a typical pattern, with declining of colostral antibodies during the lactating and nursery periods, with the lowest levels at the end of the nursery period, and active seroconversion of almost all pigs during the grower period. Although antibodies to PCV2 have been detected as early as 1969, no explanation for the emergence of this disease in the 90s has been established. Macroscopic lesions associated with PMWS are quite unspecific, but histopathological lesions in lymphoid tissues (lymphocyte depletion with histiocytic infiltration) are almost unique for this disease. These lesions together with other clinical and laboratorial findings suggest that severely affected pigs may be immunosuppressed. The criteria used for the diagnosis of PMWS include the existence of compatible clinical signs, presence of characteristic microscopic lesions and detection of PCV2 within these lesions. Because of the lack of appropriate treatment or vaccination against PCV2, zootechnical changes have been proposed in affected farms to reduce the so-called "infection pressure" due to PCV2 as well as to any other pathogen.     

Trace elements and prion diseases: a review of the interactions of copper, manganese and zinc with the prion protein

Transmissible spongiform encephalopathies (TSEs) are a family of neurodegenerative diseases characterized by their long incubation periods, progressive neurological changes, and spongiform appearance in the brain. There is much evidence to show that TSEs are caused by an isoform of the normal cellular surface prion protein PrPC. The normal function of PrPC is still unknown, but it exhibits properties of a cupro-protein, capable of binding up to six copper ions. There are two differing views on copper's role in prion diseases. While one view looks at the PrPC copper-binding as the trigger for conversion to PrPSc, the opposing viewpoint sees a lack of PrPC copper-binding resulting in the conformational change into the disease causing isoform. Manganese and zinc have been shown to interact with PrPC as well and have been found in abnormal levels in prion diseases. This review addresses the interaction between select trace elements and the PrPC.     

Emerging therapeutic agents for transmissible spongiform encephalopathies: a review

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative disorders associated with misfolding of prion protein, from PrPC to PrPSc. Different types of experimental studies have resulted in a better understanding of the pathogenesis of the prion diseases. Genetic and molecular properties of PrP isoforms have been explained but the conformational conversion of the PrPC isoform to the PrPSc isoform has not yet been entirely elucidated. However, a number of possible therapeutic agents have been tried and some have proven to be effective against TSEs but most have limitations in terms of toxicity and pharmacokinetics. Congo red (CR), anthracyclines, and polyanionic dextran sulfate have limited ability to cross the blood-brain barrier and may be toxic. The efficacy of polyene antibiotics seems to be restricted to certain scrapie strains. Tetrapyrroles and tetracyclines with low toxicities and favorable pharmacokinetics could be useful in preventing PrPSc accumulation. Compounds like branched polyamines, Cp-60, analogs of CR, quinacrine and chlorpromazine, beta-sheet breaker peptides and inhibitory peptides, active immunization using recombinant PrP and passive immunization with anti-PrP antibodies, have potential use as therapeutic agents but would need further research and clinical trials.     

White-tailed deer are susceptible to the agent of sheep scrapie by intracerebral inoculation

ABSTRACT: Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. The purpose of this experiment was to determine susceptibility of white-tailed deer to the agent of scrapie after intracerebral inoculation and to compare clinical signs and lesions to those reported for chronic wasting disease (CWD). Deer (n = 5) were inoculated with 1 mL of a 10% (wt/vol) brain homogenate derived from a sheep clinically affected with scrapie. A non-inoculated deer was maintained as a negative control. Deer were observed daily for clinical signs of disease and euthanized and necropsied when unequivocal signs of scrapie were noted. One animal died 7 months post inoculation (pi) due to intercurrent disease. Examinations of brain tissue for the presence of the disease-associated abnormal prion protein (PrPSc) by western blot (WB) and immunohistochemistry (IHC) were negative whereas IHC of lymphoid tissues was positive. Deer necropsied at 15-22 months pi were positive for scrapie by IHC and WB. Deer necropsied after 20 months pi had clinical signs of depression and progressive weight loss. Tissues with PrPSc immunoreactivity included brain (at levels of cerebrum, hippocampus, colliculus, cerebellum, and brainstem), trigeminal ganglion, neurohypophysis, retina, spinal cord, and various lymphoid tissues including tonsil, retropharyngeal and mesenteric lymph nodes, Peyer's patches, and spleen. This work demonstrates for the first time that white-tailed deer are susceptible to sheep scrapie by intracerebral inoculation. To further test the susceptibility of white-tailed deer to scrapie these experiments will be repeated with a more natural route of inoculation.     

Cases of scrapie with unusual features in Norway and designation of a new type,Nor98

Five cases of scrapie with unusual features have been diagnosed in Norway since 1998. The affected sheep showed neurological signs dominated by ataxia, and had the PrP genotypes homozygous A136 H154 Q171/ A136H154Q171 or heterozygous A136H154Q171/A136R154Q171, which are rarely associated with scrapie. Brain histopathology revealed neuropil vacuolisation essentially in the cerebellar and cerebral cortices; vacuolation was less prominent in the brainstem, and no lesions were observed at the level of the obex. The deposits of PrPSc were mainly in the cortex of the cerebellum and cerebrum, and no PrPSC was detectable by immunohistochemistry and ELISA in the lymphoid tissues investigated. Western blot analysis showed that the glycotype was different from other known scrapie strains and from the BSE strain. From a diagnostic point of view, these features indicate that this type of scrapie, designated Nor98, could have been overlooked and may be of significance for sampling in scrapie surveillance programmes.     

Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats

This paper compares the dinical signs, histopathology, detection of PrPSc protein and PrP genetics of the transmission of BSE to sheep and goats, with the effects of the transmission of natural scrapie from a brain homogenate from a single sheep. After intracerebral and oral inoculations there were similarities in the clinical signs due to the two sources of infection, but there were differences in pathology at the end stage of disease and in the genotypes of the sheep which succumbed to the challenges. The incubation period of BSE was associated with the sheep PrP codon 171 genotype, but the natural scrapie source, despite inducing disease only in known susceptible genotypes, showed no clear association with PrP genotype.     

Magic angle magnetic resonance imaging of diode laser induced and naturally occurring lesions in equine tendons

Magic angle magnetic resonance (MR) imaging consists of imaging tendons at 55° to the magnetic field. In people, magic angle MR imaging is valuable for detection of chronic tendon lesions and allows calculation of tendon T1 values. Increased T1 values occur in people with chronic tendinopathy. The T1 values of normal equine tendons have been reported but there are no available data for abnormal equine tendons. Twelve limbs were studied. Two limbs had diode laser tendon lesions induced postmortem, four limbs had diode laser tendon lesions induced in vivo and six limbs had naturally occurring tendon lesions. The limbs were imaged at 1.5 T using both conventional MR imaging and magic angle MR imaging. The post-mortem laser induced lesions were identified only with magic angle MR imaging. The in vivo induced lesions and naturally occurring lesions were identified with both techniques but had a different appearance with the two imaging techniques. Magic angle imaging was helpful at identifying lesions that were hypointense on conventional imaging. Increased T1 values were observed in all abnormal tendons and in several tendons with a subjectively normal MR appearance. The increased T1 value may reflect diffuse changes in the biochemical composition of tendons. Magic angle imaging has potential as a useful noninvasive tool to assess the changes of the extracellular tendon matrix using T1 values.