Diagnosis Of Transmissible Spongiform Encephalopathies Research Articles

A Retrospective Immunohistochemical Study Reveals Atypical Scrapie has Existed in the United Kingdom since at Least 1987

Atypical scrapie is a relatively recent discovery, and it was unknown whether it was a new phenomenon or whether it had existed undetected in the United Kingdom national flock. Before 1998, the routine statutory diagnosis of transmissible spongiform encephalopathy (TSE) in sheep relied on the presence of TSE vacuolation in the brainstem. This method would not have been effective for the detection of atypical scrapie. Currently, immunohistochemistry (IHC) and Western blot are commonly used for the differential diagnosis of classical and atypical scrapie. The IHC pattern of PrPd deposition in atypical scrapie is very different from that in classical scrapie using the same antibody. It is thus possible that because of a lack of suitable diagnostic techniques and awareness of this form of the disease, historic cases of atypical scrapie remain undiagnosed. Immunohistochemistry was performed on selected formalin-fixed, paraffin-embedded (FFPE) blocks of ovine brain from the Veterinary Laboratories Agency archives that were submitted for various reasons, including suspect neurological disorders, between 1980 and 1989. It was found that PrPd deposits in a single case were consistent with atypical scrapie. A method was developed to obtain a PrP genotype from FFPE tissues and was applied to material from this single case, which was shown to be AHQ/AHQ. This animal was a scrapie suspect from 1987, but diagnosis was not confirmed by the available techniques at that time.

Retinal Function and Morphology Are Altered in Cattle Infected with the Prion Disease Transmissible Mink Encephalopathy

Transmissible spongiform encephalopathies (TSEs) are a group of diseases that result in progressive and invariably fatal neurologic disease in both animals and humans. TSEs are characterized by the accumulation of an abnormal protease-resistant form of the prion protein in the central nervous system. Transmission of infectious TSEs is believed to occur via ingestion of prion protein-contaminated material. This material is also involved in the transmission of bovine spongiform encephalopathy ("mad cow disease") to humans, which resulted in the variant form of Creutzfeldt-Jakob disease. Abnormal prion protein has been reported in the retina of TSE-affected cattle, but despite these observations, the specific effect of abnormal prion protein on retinal morphology and function has not been assessed. The objective of this study was to identify and characterize potential functional and morphologic abnormalities in the retinas of cattle infected with a bovine-adapted isolate of transmissible mink encephalopathy. We used electroretinography and immunohistochemistry to examine retinas from 10 noninoculated and 5 transmissible mink encephalopathy-inoculated adult Holstein steers. Here we show altered retinal function, as evidenced by prolonged implicit time of the electroretinogram b-wave, in transmissible mink encephalopathy-infected cattle before the onset of clinical illness. We also demonstrate disruption of rod bipolar cell synaptic terminals, indicated by decreased immunoreactivity for the alpha isoform of protein kinase C and vesicular glutamate transporter 1, and activation of Müller glia, as evidenced by increased glial fibrillary acidic protein and glutamine synthetase expression, in the retinas of these cattle at the time of euthanasia due to clinical deterioration. This is the first study to identify both functional and morphologic alterations in the retinas of TSE-infected cattle. Our results support future efforts to focus on the retina for the development of new strategies for the diagnosis of TSEs.

Validation of Use of Rectoanal Mucosa-Associated Lymphoid Tissue for Immunohistochemical Diagnosis of Chronic Wasting Disease in White-Tailed Deer ( Odocoileus virginianus )

The examination of rectoanal mucosa-associated lymphoid tissue (RAMALT) biopsy specimens for the diagnosis of transmissible spongiform encephalopathies has been described in sheep, elk, and small numbers of mule and white-tailed deer. Previous sample numbers have been too small to validate examination of this type of tissue as a viable antemortem diagnostic test. In this study, we examined RAMALT collected postmortem from 76 white-tailed deer removed from a farm in Wisconsin known to be affected by chronic wasting disease (CWD) and from 210 free-ranging white-tailed deer harvested from an area in Wisconsin where the overall prevalence of CWD among the deer was approximately 4 to 6%. The results of immunohistochemical (IHC) staining of the RAMALT sections were compared to the results of IHC staining of sections from the brain stem at the convergence of the dorsal motor nucleus of the vagus nerve, sections of the medial retropharyngeal lymph nodes (RLNs), and sections of tonsil (sections of tonsil only from captive animals were tested). The sensitivities of the IHC staining test with RAMALT sections were 81% for the captive animals and 91% for the free-ranging animals. False-negative results were usually associated with early infection, indicated by a low intensity of immunostaining in the obex and/or a polymorphism at PRNP codon 96. While the RLN remains the tissue of choice for use for the diagnosis of CWD in white-tailed deer, the results of the present study further support the use of RAMALTs collected antemortem as an adjunct to testing of tonsil biopsy specimens and surveillance by necropsy for the screening of farmed deer which have been put at risk through environmental exposure or exposure to deer with CWD.

Disease-specific motifs can be identified in circulating nucleic acids from live elk and cattle infected with transmissible spongiform encephalopathies

To gain insight into the disease progression of transmissible spongiform encephalopathies (TSE), we searched for disease-specific patterns in circulating nucleic acids (CNA) in elk and cattle. In a 25-month time-course experiment, CNAs were isolated from blood samples of 24 elk (Cervus elaphus) orally challenged with chronic wasting disease (CWD) infectious material. In a separate experiment, blood-sample CNAs from 29 experimental cattle (Bos taurus) 40 months post-inoculation with clinical bovine spongiform encephalopathy (BSE) were analyzed according to the same protocol. Next-generation sequencing provided broad elucidation of sample CNAs: we detected infection-specific sequences as early as 11 months in elk (i.e. at least 3 months before the appearance of the first clinical signs) and we established CNA patterns related to BSE in cattle at least 4 months prior to clinical signs. In elk, a progression of CNA sequence patterns was found to precede and correlate with macro-observable disease progression, including delayed CWD progression in elk with PrP genotype LM. Some of the patterns identified contain transcription-factor-binding sites linked to endogenous retroviral integration. These patterns suggest that retroviruses may be connected to the manifestation of TSEs. Our results may become useful for the early diagnosis of TSE in live elk and cattle.

Characterisation of new monoclonal antibodies reacting with prions from both human and animal brain tissues

Post-mortem diagnosis of transmissible spongiform encephalopathies (prion diseases) is primarily based on the detection of a protease resistant, misfolded disease associated isoform (PrP Sc) of the prion protein (PrP C) on neuronal cells. These methods depend on antibodies directed against PrP C and capable of reacting with PrP Sc in situ (immunohistochemistry on nervous tissue sections) or with the unfolded form of the protein (western and paraffin embedded tissue (PET) blotting). Here, high-affinity monoclonal antibodies (mAbs 1.5D7, 1.6F4) were produced against synthetic PrP peptides in wild-type mice and used for western blotting and immunohistochemistry to detect several types of human prion-disease associated PrP Sc, including sporadic Creutzfeldt-Jakob Disease (CJD) (subtypes MM1 and VV2), familial CJD and Gerstmann-Sträussler-Scheinker (GSS) disease PrP Sc as well as PrP Sc of bovine spongiform encephalopathy (bovine brain), scrapie (ovine brain) and experimental scrapie in hamster and in mice. The antibodies were also used for PET-blotting in which PrP Sc blotted from brain tissue sections onto a nitrocellulose membrane is visualized with antibodies after protease and denaturant treatment allowing the detection of protease resistant PrP forms (PrP RES) in situ. Monoclonal antibodies 1.5D7 and 1.6F4 were raised against the reported epitope (PrP153–165) of the commercial antibody 6H4. While 1.5D7 and 1.6F4 were completely inhibitable by PrP153–165, 6H4 was not, indicating that the specificity of 6H4 is not defined completely by PrP153–165. The two antibodies performed similarly to 6H4 in western blotting with human samples, but showed less reactivity and enhanced background staining with animal samples in this method. In immunohistochemistry 1.5D7 and 1.6F4 performed better than 6H4 suggesting that the binding affinity of 1.5D7 and 1.6F4 with native (aggregated) PrP Sc in situ was higher than that of 6H4. On the other hand in PET-blotting, 6H4 reached the same level of reactivity as 1.5D7 and 1.6F4. This shows that 6H4 needs denatured PrP RES to reach maximal reactivity, confirming earlier results. As an exception, human PrP RES still reacted relatively poorly with 6H4 in PET-blotting, while 1.5D7 and 1.6F4 reacted well with PrP RES from most human CJD types. Taken together this implies that the binding epitope of 1.5D7 and 1.6F4 is accessible in the aggregates of undenatured PrP Sc (IHC) while the binding site of 6H4 is at least partly inaccessible. In techniques incorporating a denaturing and/or disaggregating step 6H4 showed good binding indicating increased accessibility of the binding site. An exception to this is human samples in PET-blotting suggesting that huPrP RES might not be as easily unfolded by denaturation as BSE and scrapie PrP RES. Also of interest was the ability of 1.5D7 and 1.6F4 to discriminate between two allelic variants of PrP CJD Sc (VV vs. MM) in immunohistochemistry as opposed to the normally used antibody 3F4.

Western Blot Detection of PrPSc in Archived Paraffin-Embedded Brainstem from Scrapie-Affected Sheep

Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies (TSE) or prion diseases. Immunoassays that identify disease-associated prion protein (PrP Sc) are integral to the diagnosis of scrapie and other prion diseases. Results obtained by either immunohistochemistry (IHC) or Western blot (WB) assay are generally adequate for the definitive diagnosis. Approved or accepted methods for WB diagnosis of TSEs requires the use of fresh or frozen nonfixed tissue samples, whereas formalin-fixed, paraffin-embedded tissue is required for the localization of PrP Sc by IHC. Because disparate processing methods are used for these accepted diagnostic techniques, separate tissue samples are collected from the same animal. Occasions arise in which there is either insufficient quantity of tissue available to complete analysis by both techniques or initial tissue processing is incompatible with one of the assays. Also, results between the assays may differ because of the vagaries of sampling, especially in case material that contains moderate-to-low levels of PrP Sc. The present article describes a method to conduct a WB assay from the same paraffin-embedded brainstem sample used for the IHC diagnosis of experimentally induced sheep scrapie.

High Titers of Transmissible Spongiform Encephalopathy Infectivity Associated with Extremely Low Levels of PrPScin Vivo

Diagnosis of transmissible spongiform encephalopathy (TSE) disease in humans and ruminants relies on the detection in post-mortem brain tissue of the protease-resistant form of the host glycoprotein PrP. The presence of this abnormal isoform (PrP(Sc)) in tissues is taken as indicative of the presence of TSE infectivity. Here we demonstrate conclusively that high titers of TSE infectivity can be present in brain tissue of animals that show clinical and vacuolar signs of TSE disease but contain low or undetectable levels of PrP(Sc). This work questions the correlation between PrP(Sc) level and the titer of infectivity and shows that tissues containing little or no proteinase K-resistant PrP can be infectious and harbor high titers of TSE infectivity. Reliance on protease-resistant PrP(Sc) as a sole measure of infectivity may therefore in some instances significantly underestimate biological properties of diagnostic samples, thereby undermining efforts to contain and eradicate TSEs.

Les prions

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) identified twenty years ago in the British cattle herds. Creutzfeldt-Jakob disease (CJD) is a TSE that occurs in humans. In 1996, scientists found a possible link between BSE and a new variant of CJD (vCJD). The fact that the non conventional infectious agent of TSE, named prions, could cross the species barrier from cattle to human through meat consumption, raised a tremendous concern for public safety in Europe. This led to the development in the following two decades of substantial and expensive measures to contain BSE and prevent its transmission to humans. In parallel, scientific programs have been funded to progress through the comprehension of the physiopathology of these fatal disorders. In Europe, the BSE epidemics is now ending and the number of cases is decreasing thanks to the strict control of animal foodstuff that was the main source of prion contamination. Only a small number of vCJD have been detected, however, additional concerns have been raised recently for public safety as secondary transmission of CJD through medical procedure and blood transfusion is possible. In addition, the possibility that the BSE was transmitted to other animals including small ruminants is also worrisome. Research efforts are now focussing on decontamination and ante mortem diagnosis of TSE to prevent animal and human transmission. However, needs for fundamental research are still important as many questions remain to be addressed to understand the mechanism of prion transmission, as well as its pathogenesis.

Identification of a proteinase K resistant protein for use as an internal positive control marker in PrP Western blotting

The routine use of an internal positive control (IPC) marker could prove useful in the diagnosis of transmissible spongiform encephalopathy (TSE) diseases, particularly in surveillance programmes where large numbers of negative results are reported. Detection of an endogenous IPC protein in a negative sample adds confidence to the correct sample processing throughout the analytical procedure and could avoid the reporting of false negative diagnoses. Proteinase K (PK) resistance is one of the key diagnostic determinants of the disease-associated form of PrP (PrP Sc), the only disease-specific macromolecule currently associated with TSE disease. Additional PK resistant proteins, endogenous to TSE-suspect diagnostic tissue samples, were therefore assessed for use as IPC markers in the Western blot diagnosis of BSE and scrapie. Results indicated that, whilst essentially maintaining a standard PrP extraction and detection protocol, a ferritin heavy chain sub-unit of approximately 22 kDa, was consistently detected in all PK treated TSE positive and negative tissue samples tested. Its presence in a range of sample types, any of which could be submitted under BSE and scrapie surveillance programmes, confirmed it as a suitable protein for an IPC marker in PrP Sc Western blotting.

Ultra-efficient Replication of Infectious Prions by Automated Protein Misfolding Cyclic Amplification

Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrPSc starting from a 1x10(-12) dilution of scrapie hamster brain, which contains the equivalent of approximately 26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as one oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrPSc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.

Presymptomatic Detection of Prions in Blood

Prions are thought to be the proteinaceous infectious agents responsible for transmissible spongiform encephalopathies (TSEs). PrP(Sc), the main component of the infectious agent, is also the only validated surrogate marker for the disease, and its sensitive detection is critical for minimizing the spread of the disease. We detected PrP(Sc) biochemically in the blood of hamsters infected with scrapie during most of the presymptomatic phase of the disease. At early stages of the incubation period, PrP(Sc) detected in blood was likely to be from the peripheral replication of prions, whereas at the symptomatic phase, PrP(Sc) in blood was more likely to have leaked from the brain. The ability to detect prions biochemically in the blood of infected but not clinically sick animals offers a great promise for the noninvasive early diagnosis of TSEs.

Stressing out the ER: a role of the unfolded protein response in prion-related disorders.

Transmissible Spongiform Encephalopathies are fatal and infectious neurodegenerative diseases characterized by extensive neuronal apoptosis and the accumulation of an abnormally folded form of the cellular prion protein (PrP), denoted PrP(SC). Compelling evidence suggests the involvement of several signaling pathways in prion pathogenesis, including proteasome dysfunction, alterations in the protein maturation pathways and the unfolded protein response. Recent reports indicate that endoplasmic reticulum stress due to the PrP misfolding may be a critical factor mediating neuronal dysfunction in prion diseases. These findings have applications for developing novel strategies for treatment and early diagnosis of transmissible spongiform encephalopathies and other neurodegenerative diseases.

Molecular Pathogenesis of Virus Infections

Molecular Pathogenesis of Virus Infections describes our current understanding of the pathogenesis of selected virus and prion infections. The innate response is an early barrier to virus spread. In this context, O. Haller et al. describe the antiviral activity of type I interferons and the various virus-encoded countermeasures. R.P. van Rij and R. Andino review the role of RNAi as a therapeutic antiviral agent and its use by the host and virus during viral infections. J.L. Whitton gives an overview of the adaptive CD8+ T-cell immune response in the context of virus infections. G. Screaton and J. Mongkolsapaya explain potential roles of T-cell responses in dengue hemorrhagic fever. E. Turnbull and P. Borrow provide a detailed description of the ineffectual roles of the innate and immune responses in the control of HIV and the long road ahead for development of either a prophylactic or therapeutic vaccine. Transmissible spongiform encephalopathies have perhaps the most unconventional natural history of any infectious agent. J.C. Manson and R.M. Barron describe the diagnosis of transmissible spongiform encephalopathies, the appearance of new strains, and the nature of host susceptibility to disease. C.M. Dixon et al. depict the special problems presented to the host by certain RNA viruses that are maintained and persist in human populations through avoidance or inhibition of apoptosis, innate immune response, and adaptive immune response. Other viruses infect humans only as incidental hosts and cause epizootics of varying degrees. A.L. Hartman et al. review our current understanding of the pathogenesis of Ebola and Marburg filoviruses, paying particular attention to the factors that contribute to lethal disease. C. Dye and S. Siddell discuss the pathogenesis of feline coronavirus, an animal disease model that has provided insights into the study of the newly recognized disease, severe acute respiratory syndrome. R.G. Webster et al. enumerate the key influenza genes responsible for human pathogenicity, their roles in past pandemics, and the potential of avian influenza virus strains to evolve into highly pathogenic and transmissible viruses for human populations. Many viruses modify host metabolism and innate/immune responses to their own ends. L. Gray et al. describe the impact of human papillomaviruses on cell cycle and apoptosis. S.M. Lemon and K. Li review the data documenting hepatitis C virus disruption of innate intracellular antiviral defenses, including interferons and toll-like receptors. M.B. Ruiz-Arguello et al. enumerate the multiple, distinct receptor homologs and binding proteins encoded by poxviruses that target tumor necrosis factor. L.K. Dixon summarizes the multiple host pathways that are targeted at multiple levels by African swine fever virus. J.P. Stewart et al. describe the pathogenesis of murid herpesvirus 4 that supports its use as model for gammaherpesviruses. M.L. Freeman et al. provide an overview of the potential role of the immune system in the latency of the alphaherpesvirus, herpes simplex virus 1. This book is suitable for the serious student and professional and is well referenced for further reading.

Ovine Infection with the Agents of Scrapie (CH1641 Isolate) and Bovine Spongiform Encephalopathy: Immunochemical Similarities can be Resolved by Immunohistochemistry

Immunochemical ("rapid") tests, which recognize a partly protease-resistant conformer of the prion protein (PrP(res)) are now widely used in Europe for the diagnosis of transmissible spongiform encephalopathies (TSEs). Some of these tests can be used to distinguish natural scrapie from experimental bovine spongiform encephalopathy (BSE) in sheep, on the basis of migration pattern differences of PrP(res) in Western immunoblots. However, PrP(res) from sheep inoculated with CH1641 scrapie gives an immunoblot profile similar to that of sheep inoculated with BSE. Therefore, field scrapie strains similar to CH1641 might be misclassified as ovine BSE in the rapid tests currently employed. This study confirmed that the Western blot similarities (size of the unglycosylated band and distinct reactivity with 6H4 and P4 antibodies) between CH1641 and BSE remained consistent regardless of the PrP genotype of the sheep, but the two infections resulted in accumulation of disease-associated PrP (PrP(d)) that could easily be distinguished by the immunohistochemical "peptide mapping" method. This method, which reveals conformational differences of PrP(d) by the use of a panel of antibodies, indicated that PrP(d) from the CH1641 isolate was truncated further upstream in the N terminus than was PrP(d) from other ovine TSEs, including experimental BSE. In addition, the immunohistochemical "PrP(d) profile method", which defines the phenotype of PrP(d) accumulation in the brain of affected sheep, showed that CH1641 infection leads to much more intra-neuronal and considerably less extracellular PrP(d) than does experimental BSE. The overall results demonstrate that a combined Western blotting and immunohistochemical approach is required to discriminate between different TSE strains in sheep.

Diagnosis of Transmissible Spongiform Encephalopathies in Animals: A Review

Transmissible spongiform encephalopathies (TSEs) in animals include, among others, bovine spongiform encephalopathy (BSE), scrapie, chronic wasting disease, and atypical forms of prion diseases. Diagnosis of TSEs is based on identification of characteristic lesions or on detection of the abnormal prion proteins in tissues, often by use of their partial proteinase K resistance property. Correctly sampling of target tissues is of utmost importance as this has a considerable effect on test sensitivity. Most of the rapid or screening tests are based on ELISA or Western immunoblot (WB) analysis, and many are officially approved. Confirmatory testing is normally performed by use of histologic examination, immunohistochemical analysis, certain WB protocols, or detection of prion fibrils by use of electron microscopy (scrapie-associated fibril). The discriminatory methods for diagnostic use are mostly based on WB technology and provide initial identification of the prion strain, particularly for differentiation of BSE from scrapie in small ruminants. Definitive prion strain characterization is performed by use of bioassays, usually in mice. A burgeoning number of transgenic mice have been developed for TSE studies. Development of new tests with higher sensitivity and of more reliable diagnostic applications for live animals tested for food safety reasons is a rapidly developing field. Ultimately, the choice of a test for TSE diagnosis depends on the rationale for the testing.

Scientific Report of the European Food Safety Authority on the Evaluation of two Rapid post mortem BSE Tests

EFSA JournalVolume 3, Issue 9 48r StatementOpen Access Scientific Report of the European Food Safety Authority on the Evaluation of two Rapid post mortem BSE Tests European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author First published: 16 September 2005 https://doi.org/10.2903/j.efsa.2005.48r Adoption date: 2 September 2005 Published date: 16 September 2005 Question number: EFSA-Q-2003-084F On request from: European Commission AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References EC, (1999). DG XXIV Consumer Policy and Consumer Health Protection Report on “The evaluation of tests for the diagnosis of Transmissible Spongiform Encephalopathy in bovines” on 8 July 1999. EC, (2001). Regulation No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication of certain Transmissible Spongiform Encephalopathies. O. J. L 147 of 31.05.2001, pp. 1– 40. EC, (2002). DG-JRC Report on the “Evaluation of five rapid tests for the diagnosis of Transmissible Spongiform Encephalopathy in bovines (2nd study) on 27 March 2002, IRMM. www.irmm.jrc.be. EC, (2003a). Regulation No 1053/2003 of the European Parliament and of the Council of 19 June 2002 amending Regulation (EC) No 999/2001 as regards rapid tests. O. J. L 152 of 20.06.2003, pp. 8– 9. EC, (2003b). Call for expression of an interest to participate in a programme for the evaluation of tests for the diagnosis of transmissible spongiform encephalopathies (TSE) in ruminants O. J. C15 of 22.01.2003, pp. 13– 14. EFSA (2004a). Scientific Report of the European Food Safety Authority on the Evaluation of Seven New Rapid post mortem BSE Tests. EFSA Journal 31, 1– 17. EFSA, (2004b). 1, 1–10 on the “Design of a field trial for the evaluation of new rapid BSE post mortem tests”. EFSA Journal 1, 1– 10. SSC, (2002). Opinion of the Scientific Steering Committee on “Design of a field trial for the evaluation of new rapid BSE post mortem tests” adopted on 22 February 2002. SSC, (2003). Opinion of the Scientific Steering Committee on “The field trial evaluation of two new rapid BSE post mortem tests” adopted on 6 March 2003. Volume3, Issue9September 200548r ReferencesRelatedInformation

An Alternative Pretreatment Procedure in Animal Transmissible Spongiform Encephalopathies Diagnosis Using PrPsc Immunohistochemistry

Because of its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used more often in the diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE). PrPsc IHC requires a combination of pretreatments (chemical, heating, and enzymatic). The method of application may depend on the anti-prion antibody considered. If these pretreatments are efficient for diagnostic purpose, it may, however, be interesting to use an alternative method to efficiently detect PrPsc IHC immunohistochemically using chemical pretreatments solely. Here we describe such pretreatments reporting the difficulty (section adhesion) but also the potential advantages of such methods (easy, quick, inexpensive, and amplifying effect).

Failure to detect prion protein (PrPres) by immunohistochemistry in striated muscle tissues of animals experimentally inoculated with agents of transmissible spongiform encephalopathy.

Transmissible spongiform encephalopathies (TSEs) are fatal neurologic diseases. Infection by the causative agent, a prion, induces accumulations of an abnormal form of prion protein (PrP(res)) in tissues of nervous and lymphoid systems. Presence of characteristic histopathologic changes (spongiform encephalopathy) and detection of protease-resistant PrP(res) in neural and lymphoid tissues are the basis of currently available methods for diagnosis of TSEs. In this study, samples of striated muscle tissues (tongue, heart, diaphragm, and masseter muscle) from 20 animals (cattle, sheep, elk, and raccoons) were examined for PrP(res) by immunohistochemistry (IHC). All the animals had developed a TSE after experimental inoculation. PrP(res) was found by IHC in the brain but not in the muscle tissues of all the animals examined. These findings are contradictory to recently published reports of laboratory animals with TSEs, where these altered prion proteins were detected in tongue and other striated muscles. Further testing of muscle tissues is needed to confirm the findings of the present study.

Comparison of PrP transcription and translation in two murine myeloma cell lines

The generation of monoclonal antibodies (MAbs) to the prion protein, PrP, is important in order to establish a large repertoire of useful reagents for the diagnosis of transmissible spongiform encephalopathies (TSE), or prion diseases. However, the presence of PrP on the surface of all mammalian cells (PrP C) causes self-recognition, thereby restricting the ability of mice to produce an immune response to the PrP immunogen. Although this problem has been alleviated with the generation and use of PrP-knockout mice, the production of MAbs has continued to be severely hampered presumably since the fusion partner for spleen-derived lymphocytes was PrP C-containing myeloma cell lines. The availability of a mouse myeloma cell line expressing little or no PrP C on the surface would therefore be useful for MAb generation. Our data indicate that cells differ in their levels of PrP C expression and suggest that not all murine myeloma cell lines are equally useful for obtaining hybridomas secreting anti-PrP MAbs.

Identification and Detection of Transmissible Spongiform Encephalopathies

Scrapie is a slowly developing transmissible spongiform encephalopathy (TSE) occurring naturally in sheep and goats. At present, there is no efficient antemortem diagnostic test for TSEs. Electroencephalograms (EEGs), electrocardiograms (ECGs), magnetic-resonance imaging (MRI), positron emission tomography (PET), single photo emission computed tomography (SPECT), as well as conformation-dependent immunoassay (CDI), which show patterns characteristic of prion diseases, can obtain diagnostic information for TSEs. Other methods include testing cerebrospinal fluid for the presence of 14-3-3 proteins, Fourier transformation infrared spectroscopy (FTIR), brain biopsy, nasal biopsy and tonsil biopsy. For better TSE diagnosis, most TSEs need to be confirmed by the presentation of abnormal prion protein by Western blot, ELISA immunochemical methods, or immunocytochemistry methods, or by bioassays, a more sensitive but time-consuming method, which involves inoculating suspended homogenates into susceptible hosts and waiting for a long incubation period to conclude. An antemortem diagnostic test for TSEs can be done from spleen or lymph node biopsy by evaluating PrPSc presence. Since it has been suggested that the buffy coat of TSE animals may carry infectivity, a method for presymptomatic diagnosis of TSE from blood is clearly possible and desirable. At present, antibodies against prion protein cannot distinguish normal prion protein (PrP C) and abnormal prion protein (PrPSc); there is no reliable antibody to detect PrPSc only. In this review, we propose that the development of gene chips and / or protein chips for assays of TSE diseases might prove very useful. A great deal of basic research on TSE diagnosis, prevention, and treatment remains to be conducted. Keywords: transmissible spongiform encephalopathies, prion diseases, tse, prion protein, abnormal prion protein