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< > Literature Reports of DETECTION and IDENTIFICATION TECHNIQUES for Prion Protein (associated with CWD of Deer and Elk):

OVERVIEW

Editorial Summary (Editorial Overview Text Replicated on Overall Chemical page - Prion Protein)
  • For detection of prions associated with CWD the "gold standard" test is immunohistochemistry, particularly of the parasympathetic vagal nucleus at the obex, in the dorsal portion of the medulla oblongata.
  • ELISA and Western blot tests developed for BSE detection have been tested for use in detection of CWD in cervids. To date [January 2004] ELISA-based test kits from three different companies have been approved by USDA Center for Veterinary Biologicals for use as screening tests for the detection of CWD in free-ranging cervid populations, but none has been approved for use for testing of farmed cervids in regulatory programmes.

Sampling:

  • "Traditionally, confirmation of Transmissible Spongiform Encephalopathy (TSE) disease in humans or animals is by conventional light microscopy of stained tissue sections prepared from specific sites of formalin-fixed tissue after embedding in paraffin wax." (B297.5.w5)
  • For the diagnosis of CWD in deer and elk the most important site which must be examined is the dorsal portion of the medulla oblongata at the obex.  This area is involved early in infection in both deer and elk. (J40.66.w1, P10.67.w1) 
    • It is critically important that the correct portion of the brain is sampled if the results of testing are to be meaningful. (J40.66.w1)
    • It is important that specimens are preserved appropriately; the obex sample [intended for histopathology/immunohistochemistry] must be placed in 10% buffered formalin and the remainder of the brain should be frozen. (J40.66.w1)
  • In Odocoileus spp. deer samples of the tonsils, and samples of the retropharyngeal lymph nodes in Odocoileus spp. deer and in Cervus elaphus nelsoni - Rocky Mountain Elk (Cervus elaphus - Red deer), provide additional useful specimens in which PrPCWD can be detected at an early stage using immunohistochemistry (IHC).
    • Studies have shown that in Odocoileus spp. deer PrPCWD can be detected by IHC in the retropharyngeal lymph nodes before it can be detected at the obex. (P10.67.w1, J64.21.w17, J84.8.w1)
    • Studies have shown that in Cervus elaphus nelsoni - Rocky Mountain Elk (Cervus elaphus - Red deer) PrPCWD can be detected by immunohistochemistry (IHC) in the retropharyngeal lymph nodes before it can be detected at the obex. (J212.15.w2)
    • Tonsillar and retropharyngeal lymph node tissue should be preserved in 10% neutral buffered formalin for storage prior to IHC. (J40.66.w2)
    • Fresh, not formalin-preserved, tissue is used for the various "rapid tests." (P50.1.w4)

DETECTION OF ANTIBODIES

  • TSE diseases produce no known specific immune response. It is not possible to use detection of antibodies (serological response) for detection of abnormal prion proteins or TSE disease.

IMMUNOHISTOCHEMISTRY

  • Immunohistochemistry, using antibodies to PrPres (anti-scrapie amyloid antibodies), has been used for the detection of PrPres and the diagnosis of TSE diseases for many years. A variety of antibodies are used, both monoclonal antibodies (MAbs) and polyclonal antibodies. When used in immunohistochemistry such antibodies allow anatomical localisation of where the PrPres is found within the tissue being tested.
  • Immunohistochemistry is more sensitive than histopathological examination of tissues using standard stains such as haematoxylin and eosin. It can detect PrPres in mildly to moderately autolysed tissues and in tissues such as lymph nodes and tonsils where there are no visible histopathological lesions.
  • In deer and elk immunohistochemistry has been used for the detection of PrPCWD in brain, retropharyngeal lymph node, and (in deer) in tonsil and has been shown to detect PrPCWD in animals which do not yet have histopathological spongiform change in the brain.
  • Immunohistochemistry of the obex has the advantage that it allows visualisation of PrP in association with specific tissue architecture for high confidence of diagnosis.
  • The recent production of an antibody specific for the pathological form of PrP may lead to the development of new detection capabilities. 

IMMUNOBLOTTING

  • Immunoblotting, particularly Western blotting, has been used for the detection of PrPres in tissues of individuals with TSE diseases. 

  • Western blotting may be used for the detection of PrPCWD in cervids infected with CWD.

ELISA TECHNIQUES

  • Various ELISAs have been developed for the detection of TSEs, in particular for the detection of BSE.
  • To date [January 2004] ELISA-based test kits from three different companies have been approved by USDA Center for Veterinary Biologicals for use as screening tests for the detection of CWD in free-ranging cervid populations, but none has been approved for use for testing of farmed cervids in regulatory programmes. 

OTHER IMMUNOASSAYS

  • A variety of tests have been and are being developed for the detection of abnormal prion proteins.
  • One test has been described, using capillary electrophoresis and fluorescent labeled peptides, which suggested the possibility of detecting abnormal prion proteins in the blood of elk (Cervus elaphus nelsoni - Rocky Mountain Elk (Cervus elaphus - Red deer)) with CWD and sheep with scrapie. 
  • A conformation-dependent immunoassay (CDI) has been developed which may be used on a variety of ungulates and which was considered to have a sensitivity approaching that of bioassay in cattle and in transgenic mice.

ELECTRON MICROSCOPY

  • Scrapie-associated fibrils (SAF) are found only in individuals with TSE diseases.
  • In deer and elk with CWD SAF may be found in the brain and spleen.

BIOASSAY

  • Detection of the agent by bioassay- transmission of the disease from the suspect case to other animals by inoculation- is used to assay the presence of TSE-associated infectivity and to confirm that disease present in an animal is a TSE. Following inoculation the recipient animals are monitored for the development of clinical disease and examined at necropsy for the presence of lesions of spongiform encephalopathy. In most cases inoculation into rodents is used. 
  • There are limitations to bioassay:
    • Sensitivity of bioassay is optimum when the recipient is of the same species as the donor animal; this is often impractical due to prolonged incubation times and levels of biosecurity required for experimental animals. 
    • There is a limit to the amount of inoculum which can be used, particularly when using small species such as mice and inoculating by e.g. the intracerebral route. 
    • Not all TSEs will transmit to commonly used laboratory species.
  • Ferrets have been used for bioassay of CWD. 

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Detection of Antibodies 

Editorial Summary
  • TSE diseases produce no known specific immune response. It is not possible to use detection of antibodies (serological response) for detection of abnormal prion proteins or TSE disease.
Detailed Reports
  • "The absence of detectable immune responses in BSE or other transmissible spongiform encephalopathies precludes serological tests." (W31.29Mar03.w3)
  • "Scrapie infection is not known to elicit any specific immune response and there is no basis for establishing a diagnosis by detecting specific antibodies." (W31.29Mar03.w5)

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Immunohistochemistry

Editorial Summary
  • Immunohistochemistry, using antibodies to PrPres (anti-scrapie amyloid antibodies), has been used for the detection of PrPres and the diagnosis of TSE diseases for many years. A variety of antibodies are used, both monoclonal antibodies (MAbs) and polyclonal antibodies. When used in immunohistochemistry such antibodies allow anatomical localisation of where the PrPres is found within the tissue being tested.
  • Immunohistochemistry is more sensitive than histopathological examination of tissues using standard stains such as haematoxylin and eosin. It can detect PrPres in mildly to moderately autolysed tissues and in tissues such as lymph nodes and tonsils where there are no visible histopathological lesions.
  • In deer and elk immunohistochemistry has been used for the detection of PrPCWD in brain, retropharyngeal lymph node, and (in deer) in tonsil and has been shown to detect PrPCWD in animals which do not yet have histopathological spongiform change in the brain.
  • Immunohistochemistry of the obex has the advantage that it allows visualisation of PrP in association with specific tissue architecture for high confidence of diagnosis.
  • The recent production of an antibody specific for the pathological form of PrP may lead to the development of new detection capabilities. 
Detailed Reports
  • Immunohistochemistry may be used for detection of PrPres in the brains of affected and (presumably) preclinical cervids. This may be useful for diagnosis, surveillance and research. (B294.10.w10)
  • For detection of prions associated with CWD the "gold standard" test is immunohistochemistry, particularly of the parasympathetic vagal nucleus at the obex, in the dorsal portion of the medulla oblongata. (J40.66.w1, P10.67.w1)
    • Prior to IHC staining, treatment of tissue with formic acid and proteases is required to denature normal PrPC which would otherwise be detected by the antibodies used in testing, which cannot distinguish between the normal and abnormal forms of PrP. (J64.21.w17)
  • Immunohistochemistry is the main method which has been used for validated diagnosis of CWD. It is accepted as the "gold standard" for diagnosis of CWD and will be the gold standard in the long term. (D110, J1.36.w4, P40.1.w17)
    • The most important site to be examined is the parasympathetic vagal nucleus at the obex, in the dorsal portion of the medulla oblongata. (P10.67.w1)
  • The dorsal motor nucleus of the vagus (DMNV) is the area of the brain in which PrPSc is first detectable by IHC. (P46.1.w4, J40.66.w1, P10.67.w1)
  • Recent studies have shown that in deer the retropharyngeal lymph nodes and tonsils appear to be a more sensitive site than the obex for testing with immunohistochemistry. (P10.67.w1, J3.151.w3)
  • In Odocoileus spp. deer live animals may be positively diagnosed by the immunohistochemical (IHC) staining of tonsillar tissue biopsies; however a negative result may be found very early in the course of infection and the test is not suitable for diagnosis of live or preclinically infected elk ((Cervus elaphus nelsoni - Rocky Mountain Elk (Cervus elaphus - Red deer)). (J40.66.w2, J223.83.w1, P10.67.w1, P40.1.w18)
  • Immunohistochemistry detected the presence of PrPres in the brains of 10 animals in a captive elk herd, including eight animals which were not showing any clinical signs of CWD; six of these did not have any histological lesions in the brain by light microscopy. The study indicated that immunohistochemistry for PrPres was more sensitive than histopathological examination for the detection of CWD. The study also suggested that animals in the terminal stage of disease have more PrP accumulation than animals which are subclinically affected. (J212.12.w2)
  • In a survey in Colorado and Wyoming (cervids harvested 1978-1999), in mule deer only about 53% of IHC-positive deer had histological lesions of spongiform encephalopathy; for white-tailed deer the figure was 50% while in elk 0% of the IHC-positive animals had detectable histopathology of spongiform encephalopathy. (J1.36.w4)
  • The monoclonal antibody (MAb) F99/97.6.1 has been shown to be a sensitive and specific, readily standardised diagnostic test for CWD in deer. This MAb reacts with a conserved epitope on the prion protein of mule deer, rocky mountain elk, domestic sheep and domestic cattle. Used in an IHC assay this MAb demonstrated PrPres  in the brains of 100/100 mule deer Odocoileus hemionus with CWD-compatible spongiform encephalopathy and in 99/100 samples of tonsil, while no staining was seen in samples from CWD-negative deer (collected outside the CWD-endemic area). PrPres was detected in fresh, frozen and mildly to moderately autolysed brain and tonsil samples. (J212.14.w1)
  • The monoclonal antibody F89/160.1.5 recognises a conserved epitope on the PrPSc of domestic sheep, domestic cattle, mule deer and elk and was considered to be useful in both diagnosis and histopathological studies of TSEs in the ruminant species tested.. (J93.36.w1)
  • A study comparing detection of PrPCWD by immunostaining in mule deer (Odocoileus hemionus - Mule deer), using the monoclonal anitibody F99/97.6.1, in retropharyngeal lymph nodes and tonsils with detection in the obex, found a high degree of agreement for detection between the three sites. For 74 deer which were immunohistochemistry-positive at the obex, 72 (97.3%) were positive in the tonsil and 73 (98.7%) positive in the retropharyngeal lymph node; one deer positive in the obex was negative at both other sites. Seven animals from the CWD-endemic area were positive in the tonsil and retropharyngeal lymph nodes although negative in the obex and one animal was positive only in the retropharyngeal lymph nodes. All deer from areas without known CWD infection were negative in all three tissues. The data were considered to indicate that immunohistochemical staining of the tonsil and retropharyngeal lymph node were highly specific and sensitive for detection of CWD infection in mule deer and might be expected to detect about 10% more infected animals than examination of the obex using immunohistochemistry. It was considered that in most mule deer abnormal prion protein is present in lymphoid tissues (specifically, retropharyngeal lymph nodes and tonsils) at an earlier stage of infection than the point where either lesions or IHC staining are present in brain tissue. (J3.151.w3)
  • A study involving elk and mule deer found that, for individual in which both obex and retropharyngeal lymph node tissues were available, 22% of mule deer (10 of 45) which were IHC positive in the retropharyngeal lymph nodes, and 7% of deer (1#one of fifteen), were negative by IHC in the obex. (J212.15.w2)
  • It was noted that the precise tissues examined could affect the result of IHC (and of ELISA). (J212.15.w2)
  • The monoclonal antibody F99/97.6.1 is currently [2003] used most commonly for IHC for CWD (and scrapie) in the USA and Canada. This monoclonal antibody is effective on fresh and formalin-fixed tissue. (B336.78.w78)
  • IHC of the obex allows staining (detection of abnormal PrP) in association with specific tissue architecture for high confidence of diagnosis. (P50.1.w7)

Other TSE Diseases/General TSE Information

  • It has been shown that anti-scrapie amyloid antibody is specific for the transmissible cerebral amyloidoses (transmissible spongiform encephalopathies) and can be used to detect amyloid plaques in the brain associated with such diseases. The unlabeled secondary antibody peroxidase-antiperoxidase (PAP) technique may be used to visualise the reaction. (J225.126.w1, J224.81.w1)
  • PrPSc may be detected by immunoassay of fixed tissue; this is a useful confirmatory assay. Nearly all monoclonal and polyclonal antibodies recognise PrPC as well as PrPSc; immunodetection protocols must involve a process to selectively eliminate the reactivity of PrPC to these antibodies. Such reactivity is removed by formalin fixation and routine tissue processing procedures, therefore PrPC usually is not detectable in formalin-fixed tissues, while epitopes on the PrPSc in the same tissue samples are unmasked by pretreatment with heat, acid or enzymes. Staining of TSE-affected and healthy (control) animals in parallel is used to confirm the efficacies of the fixation and pretreatment protocols. (J93.36.w1)
  • Demonstration of PrP by immunohistochemical staining or immunoblotting is specific for the TSE diseases. Demonstration of scrapie-associated fibrils is also specific to the TSEs. (J21.53.w1)
  • Immunohistochemistry of routinely formalin-fixed lymphoid tissues with a cocktail of MAb F89/160.1.5 and MAB F99/97.6.1 would be expected to recognise "all the reported ovine alleles, as well as the reported PrP allelles of cattle, humans, deer, elk, mink, domestic cats, kudu, bison and a number of nonhuman primates, the species with naturally occurring transmissible spongiform encephalopathies (TSEs) or PrP-Sc accumulation. The single exception is the nyala (Tragelaphus angasi). Furthermore at least one of these epitopes is found in most species of domestic and nondomestic ruminants, carnivores and nonhuman primates for which PrP sequence data are available." (J93.38.w3)
    • It was considered that a cocktail of MAb F89/160.1.5 and MAb F99/97.6.1 would be suitable for immunoassay of tissues from most species with naturally occurring TSE disease as well as for surveillance of other species which may be exposed to TSEs either under field conditions or in zoological gardens. (J93.38.w3)
  • Using samples from sheep and cattle, subject to controlled autolysis (samples left at 37°C for up to 48 hours prior to fixing in 10% formal saline), it has been shown that immunohistochemistry is still capable of detecting disease specific PrP accumulation and therefore making a diagnosis with no loss of sensitivity. Staining of other tissue elements increased in autolysed tissue but this was not of a form associated with disease (J21.72.w1)
  • Recent work has produced antibodies binding selectively to the "prion protein repeat motif tyrosine-tyrosine-argenine", recognising the pathological but not the normal cellular isoform of PrP. It was suggested that this finding may lead to new therapeutics for the prion diseases as well as new diagnostic capabilities. (J242.9.w1)

Scrapie:

  • Immunohistochemistry may be used to detect the presence of PrPSc in paraffin sections of the CNS. Formalin-fixed paraffin-embedded brain sections are stained using PrPSc-specific antisera. Such antisera have been shown to stain scrapie-infected but not normal brain sections. (J64.11.w4)
  • Immunohistochemistry may be used on biopsy samples from the tonsils of sheep as a test allowing the preclinical diagnosis of scrapie in sheep. (J9.381.w1)

Bovine Spongiform Encephalopathy (BSE):

  • Immunohistochemistry is more sensitive than conventional histopathological examination of tissue. It can also be used effectively on partially autolysed tissue for which histopathology would not be useful. (J35.161.w1)
    • "For the purpose of diagnosis, it has been agreed to simplify the protocol by focusing on the examination of a single section of the medulla oblongata taken at the level of the obex." This is the area in which the greatest frequency of both vacuolation and immunoreactivity are found. (J35.161.w1)

Creutzfeldt-Jakob Disease (CJD):

  • Immunocytochemical techniques have been developed which accurately demonstrate and localise the presence of disease-related prion protein (PrPCJD) in human cases of prion disease. (B297.4.w4)
    • A number of polyclonal and monoclonal antibodies have been developed against PrP. (B297.4.w4)
    • Antibodies raised against scrapie fibrils are noted to readily produce positive staining in humans with CJD, demonstrating cross-reactivity between species. (B297.4.w4)
    • Because antibodies do not distinguish between PrPC and PrPres it is necessary to use treatment protocols which aim to eliminate PrPC from tissue sections, leaving only PrPres, prior to exposure to the antibody, such that only disease-related, protease-resistant PrPres is visualised. (B297.4.w4)
    • For each test run it is mandatory to use positive and negative control case material and negative sections in which the primary antibody is omitted. (B297.4.w4)
    • Immunocytochemistry allows localisation of PrP at the cellular level in a way which is not possible using Western blotting or immunoblotting. (B297.4.w4)
    • Some fixed material which has been archived for a prolonged time does not respond positively with immunocytochemical techniques for detection of PrPres. (B297.4.w4)

New variant Creutzfeldt-Jakob Disease (nvCJD):

  • Using immunohistochemistry it was found that nvCJD could be correctly diagnosed in suspect cases from a tonsillar biopsy specimen as well as from tonsillar, spleen or lymph nodes obtained at necropsy. In the study, tonsillar tissue biopsies were positive in all cases (with an adequate biopsy sample) subsequently confirmed with nvCJD or with a course highly consistent with a diagnosis of nvCJD, but negative in all individuals that have subsequently been diagnosed to have other conditions. Positive staining for PrP was detected only in germinal centres. Tonsillar, spleen and lymph node tissue obtained at necropsy were positive in all cases. (J98.353.w1)
    • It was suggested that, at least in cases of advanced disease, tonsillar biopsy followed by (Western blotting and/or) immunohistochemistry was an extremely specific and sensitive test for nvCJD. (J98.353.w1)

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Immunoblotting

Editorial Summary
  • Immunoblotting, particularly Western blotting, has been used for the detection of PrPres in tissues of individuals with TSE diseases. 

  • Western blotting may be used for the detection of PrPCWD in cervids infected with CWD.

Detailed Reports
  • Western blotting may be used for detection of PrPres in the brains of affected and (presumably) preclinical cervids. This may be useful for diagnosis, surveillance and research. (B294.10.w10)
  • A monoclonal antibody (F99/97.6.1, VRMD, Inc., Pullman, WA) dot-blot assay was shown to detect PrPCWD in Odocoileus hemionus - Mule deer tonsil samples from 49 of 50 samples which were positive by immunohistochemistry (IHC) and in none of the samples which were negative by IHC. Poor trimming (i.e. lack of relevant tonsillar tissue in the tissue prepared for dot-blot) was considered the most likely reason for the single discrepant result. Quantitative analysis indicated the presence of abundant PrPCWD in the tonsils of infected mule deer; it was considered that this fact would facilitate development of high-throughput screening tests for detection of CWD in large populations of free-living deer. The discrepant result highlighted the need for standardisation of tissue collection and trimming techniques. (J212.15.w3)

Other TSE Diseases/General TSE Information

  • An improved Western blot mapping technique was described using a high-yield purification method. This method was used to detect SAF protein ( PrPres) in the brain, spinal cord and spleen of scrapie-infected hamsters, brain stem and (with a much weaker reaction) spinal cord of squirrel monkeys infected with kuru, scrapie and CJD, spinal cord of humans with sporadic CJD, spinal cord and (weakly) thalamus of a Swaledale sheep with natural scrapie and the brain and spinal cord of a cow with BSE. This method allowed detection in samples of just 1.0-10.0 µg of brain tissue from intracerebrally infected hamsters, from individual spleens of hamsters and from 20-100 mg samples of various tissues from rodent, monkey, human, ovine and bovine tissues. It was considered that the test may be suitable for use in routine diagnosis of TSE diseases and for detailed analysis of distribution patterns of TSE-specific amyloid (PrPres) in experimental investigations of TSEs. (J223.76.w1)
  • Immunoblotting may be used to detect purified, protease-resistant abnormal PrP protein by its molecular weight and reaction with specific antibodies. It was noted [1996] that immunoblotting was consistently effective for diagnosis only when used on CNS tissues in which the PrPres accumulates in large quantities, but that further technical developments might allow reliable detection of smaller amounts of PrPres in other tissues. (B297.5.w5)
    • One technique which may be used to separate PrPC from PrPres is extraction of infected brain tissue with a detergent, Sarkosyl, followed by centrifugation and digestion with proteinase K. (B297.5.w5)
    • Fresh or frozen, but not formalin-fixed, material should be used for the detection of PrPres by immunoblotting. (B297.5.w5)
    • This technique may still be used in tissues with some degree of autolysis. (B297.5.w5)
  • Western blot techniques provide sensitive and rapid tests for the detection of PrPres. They can be partially automated and the electrophoretic pattern gives qualitative results important in confirmation of the specificity of the signal. (J35.161.w1)
  • Due to the great variability in the distribution of PrPres in the brain, it has been suggested that tissue from the obex should be used preferentially for detection of PrPres in the brain by ELISA (and Western blot) techniques. This is not possible if the whole obex is fixed in formaldehyde for histopathological examination. (J35.161.w1)
  • An immunoblotting technique using a monoclonal antibody has been used to detect a protease-resistant isoform of PrP (UPrPSc) in urine from cattle with BSE, humans with CJD and hamsters with scrapie. It was noted that the UPrPSc could be detected in hamster urine some time before the onset of clinical signs. Since no UPrPSc was detected in kidney it was considered probable that it originates from other organs, arriving in the urine from blood. The molecular weight of the UPrPSc appears to be slightly higher that that of full-length and fully-glycosylated PrPC or PrPSc. (J236.276.w1)
  • Using samples from sheep and cattle, subject to controlled autolysis (samples left at up to 37°C for up to seven days prior to testing), it has been shown that Western blotting (Prionics - Check test, Prionics AG, Zurich, Switzerland) was capable of detecting PrPSc in at least one brain area (and in most cases in the medulla) even after considerable autolysis has taken place. (J21.72.w1)

Scrapie:

  • Immunoblotting may be used to detect PrPSc in the brain and spleen for the diagnosis of scrapie. The existence of PrPSc in clinically affected sheep is demonstrated as follows: PrPSc is extracted using detergent and purified by differential centrifugation and enzyme digestions, prior to immunoblotting (various techniques) using polyclonal antibodies raised in rabbits against mouse or sheep PrPSc. (J64.11.w4)
    • Due to limitations of sensitivity, the absence of detection of PrPSc by immunoblotting does not necessarily indicate absence of infection. (J64.11.w4)
    • Immunoblotting may be useful for detection of PrPSc in autolysed brain tissue.(J64.11.w4)
  • Western blot analysis was used to detect the presence of scrapie-associated fibrillar protein (PrPres) in the brain, spleen and lymph nodes of both clinically affected and preclinical sheep with scrapie. It was noted that for one individual with early clinical signs PrPres was detected in the lymph nodes and spleen but not in the brain. In sheep experimentally infected by intravenous inoculation, PrPres was detected in surgically obtained subiliac lymph node from one of three individuals; this animal developed clinical signs of scrapie six months after the lymph node was tested. Another individual, which had tested negative for PrPres in the lymph node, developed clinical signs five months later. (J3.128.w4)

Bovine Spongiform Encephalopathy (BSE):

Bos taurus - Domestic cattle:
  • An immunoblotting test (Prionics A.G., Switzerland) based on a Western blotting procedure using a monoclonal antibody for detection of protease-resistant fragment PrPSc (PrP 27-30), which takes eight hours to perform, was evaluated as a rapid test for the diagnosis of BSE and was found to have a sensitivity of 100% and a specificity of 100% using a predetermined cut-off point. In diluted brain homogenate samples the test detected 15 (plus two inconclusives) of 20 positive samples at a dilution of 10-1 but none at 10-1.5. The test was considered to "have excellent potential for detecting or confirming clinical BSE for diagnostic purposes or for screening dead or slaughtered animals for such cases, particularly casualty animals or carcasses sent for rendering." (J9.400.w1) The high specificity was taken to indicate also that the test might be useful for general PME screening of older cattle. (J9.400.w1, D111.7.w7)
  • An immunoblotting test (Prionics A.G., Switzerland) based on a Western blotting procedure using a monoclonal antibody for detection of protease-resistant fragment PrPSc (PrP 27-30), which takes eight hours to perform, was evaluated as a rapid test for the diagnosis of BSE and was found to have a sensitivity of 100% and a specificity of 100% using a predetermined cut-off point. In diluted brain homogenate samples the test detected 15 (plus two inconclusives) of 20 positive samples at a dilution of 10-1 but none at 10-1.5. The test was considered to "have excellent potential for detecting or confirming clinical BSE for diagnostic purposes or for screening dead or slaughtered animals for such cases, particularly casualty animals or carcasses sent for rendering." (J9.400.w1) The high specificity was taken to indicate also that the test might be useful for general PME screening of older cattle. (J9.400.w1, D111.7.w7)
  • It has been suggested than an ELISA should be used as a screening test for BSE detection with Western blot analysis for confirmation of positive cases. (J35.161.w1)

New variant Creutzfeldt-Jakob Disease (nvCJD):

  • Using Western blotting for PrPres it was found that nvCJD could be correctly diagnosed in suspect cases from a tonsillar biopsy specimen as well as from tonsillar, spleen or lymph nodes obtained at necropsy. Tonsillar, spleen and lymph node tissue obtained at necropsy were positive in all cases where fresh (unfixed) tissue were available. (J98.353.w1)
    • It was noted that while PrPres was detected in all lymphoreticular tissue examined from patients with nvCJD, PrPres  was not detected in such tissues from humans with other prion diseases such as sporadic and iatrogenic CJD, nor from control individuals without prion disease. (J98.353.w1)
    • It was suggested that, at least in cases of advanced disease, tonsillar biopsy followed by Western blotting (and/or immunohistochemistry) was an extremely specific and sensitive test for nvCJD. (J98.353.w1)
   

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ELISA Techniques

Editorial summary
  • Various ELISAs have been developed for the detection of TSEs, in particular for the detection of BSE. 
  • To date [January 2004] ELISA-based test kits from three different companies have been approved by USDA Center for Veterinary Biologicals for use as screening tests for the detection of CWD in free-ranging cervid populations, but none has been approved for use for testing of farmed cervids in regulatory programmes. 
Detailed Reports
  • ELISAs are being used widely for diagnosis [fall 2003]. (V.w49)
  • The Bio-Rad test kit has been approved by APHIS for use in mule deer (Odocoileus hemionus - Mule deer), white tailed deer (Odocoileus virginianus - White-tailed deer) and elk ((Cervus elaphus nelsoni - Rocky Mountain Elk (Cervus elaphus - Red deer)) by testing specific lymph nodes. (W30.09Jun03.CWD1, V.w48)
  • The Bio-Rad test "combines a purification protocol for increased sensitivity with rapid detection by ELISA. Results are available in 4 hours." Two specific monoclonal antibodies are used in the test. (W473.Jan04.CWD1)
  • The Bio-Rad ELISA CWD antigen test kit was validated using 4,175 retropharyngeal lymph node or obex samples, with the results of the ELISA being compared with immunohistochemistry (IHC) findings (IHC-positive individuals being taken as being CWD-infected and IHC-negative individuals being taken as uninfected). The relative specificity was 99.9-100% and the sensitivity was 98.3-100% for retropharyngeal lymph node samples and 92.1-93.3% sensitive for obex samples. Over all there was agreement for at least 97.6% of lymph node and at least 95.7% of obex samples where values could be calculated. ELISA optical density (OD) values were at least 46 times higher for IHC-positive than for IHC-negative samples. Discrepancies between IHC and ELISA results were found only for early-stage CWD cases. In the field-application stage, 20,875 retropharyngeal lymph node samples were screened with the ELISA. The 155/8,877 mule deer, 33/11,731 elk and 9/267 white-tailed deer with ELISA OD values greater than 0.1 (value based on data from the validation phase of the study) were classified as "CWD-suspect" and evaluated by IHC; 143/155 mule deer, 29/33 elk and 9/9 white-tailed deer were IHC-positive and mean ELISA OD values were comparable to those measured during the validation stage. It was considered that the Bio-Rad ELISA was "an excellent rapid test for screening large numbers of samples in surveys designed to detect CWD infection in deer and elk populations."  It was noted that "selection and sub-sampling of RLN tissue may influence the outcome of both brELISA and IHC." From the study an OD cut-off of greater than or equal to 0.1 was recommended for screening retropharyngeal lymph node tissues of deer and elk in large-scale surveys to minimise the risk of false-negative results, giving a screening test with about 99.6% sensitivity, with a follow-up of IHC, carried out by a reliable experienced laboratory, to remain as the final determination test for CWD on ELISA-positive samples. (J212.15.w2)
    • It was noted that the precise tissues examined could affect the result of both ELISA and IHC. (J212.15.w2)
  • The VMRD dot blot ELISA uses retropharyngeal lymph node tissue from deer (Odocoileus hemionus - Mule deer or Odocoileus virginianus - White-tailed deer). It utilises a prion-specific primary antibody and a labeled secondary antibody which is detected by means of a colour change. Its advantage is "its ability to process a large number of samples in a short period of time." A positive test, indicating the presence of CWD prions in the animal from which the sample was taken, should be confirmed by immunohistochemistry; a negative test indicates either absence of CWD prions or that such prions, if present are at a level below the detection limits of the test. (W474.Jan04.CWD1, W474.Jan04.CWD1)
  • ELISA-based test kits from Bio-Rad and VMRD, which can be used on tissue from the brainstem or lymph nodes have been approved by USDA for use for surveillance of free-ranging cervids but have not yet been approved for use in regulatory programmes for farmed cervids. (W253.Jan04.CWD1)
  • "A dot blot ELISA test for CWD, developed by VMRD, Inc., has been licensed for CWD testing. The test uses the retropharyngeal lymph nodes and has a turnaround time of approximately 24 hours. Cost for a test kit that will run approximately 960 samples is $4,300. However, the sensitivity of this test is only 91.5%, which means that about 1 in ten positives may be missed. This is not close enough for accurate monitoring of CWD in populations of wild cervids in the opinion of many wildlife managers. Also, the certification was based on only 298 samples." (W399.08Jan04.CWD1)
  • The HerdChek® test kit from IDEXX has been approved by USDA. (W475.Jan04.CWD1)
  • The HerdChek test uses homogenised lymph node tissue. The process takes a total of three-and-a-half hours and "offers 98.8% sensitivity and 100% specificity, validated through IHC confirmation testing." Sensitivity was 98.8% (80 of 81 samples) and specificity 100% (248 samples) as confirmed by IHC on samples from Odocoileus virginianus - White-tailed deer. (W475.Jan04.CWD2, W475.Jan04.CWD3)
  • A second Bio-Rad ELISA-based test (TeSeE® has been approved by USDA. (W399.08Jan04.CWD2)
  • ELISA-based test kits have the advantage that they allow rapid testing of large numbers of samples. (P50.1.w7)

Other TSE Diseases/General TSE Information

  • Due to the great variability in the distribution of PrPres in the brain, it has been suggested that tissue from the obex should be used preferentially for detection of PrPres in the brain by ELISA (and Western blot) techniques. This is not possible if the whole obex is fixed in formaldehyde for histopathological examination. (J35.161.w1)

Bovine Spongiform Encephalopathy (BSE):

Bos taurus - Domestic cattle:
  • A chemiluminescent ELISA has been developed (Enfer BSE Testing system, Enfer Technology Ltd., Ireland), using a polyclonal anti-PrP antibody. This test, which takes four hours to perform, was evaluated as a rapid test for the diagnosis of BSE and was found to have a sensitivity of 100% and a specificity of 100% using a predetermined cut-off point. In diluted brain homogenate samples the test detected positive samples at a dilution of 10-1.5 but not at 10-2.0. The test was considered to "have excellent potential for detecting or confirming clinical BSE for diagnostic purposes or for screening dead or slaughtered animals for such cases, particularly casualty animals or carcasses sent for rendering." (J9.400.w1) The high specificity was taken to indicate also that the test might be useful for general PME screening of older cattle. (J9.400.w1, D111.7.w7)
  • A sandwich immunoassay for PrPSc (Commissariat ą l'Energie Atomique (CEA), France), which uses two monoclonal antibodies, is carried out following denaturation and concentration steps, and takes less than 24 hours to perform was evaluated as a rapid test for the diagnosis of BSE and was found to have a sensitivity of 100% and a specificity of 100% using a predetermined cut-off point. In diluted brain homogenate samples the test detected positive samples at a dilution of 10-1.5 and 10-2.0, with 18/20 positives detected at 10-2.5 but only 1/20 positives detected at 10-3.0 and none at 10-3.5. The test was considered to "have excellent potential for detecting or confirming clinical BSE for diagnostic purposes or for screening dead or slaughtered animals for such cases, particularly casualty animals or carcasses sent for rendering." (J9.400.w1) The high specificity was taken to indicate also that the test might be useful for general PME screening of older cattle. (J9.400.w1, D111.7.w7)
  • The BSE Bio-Rad test, developed by CEA and commercialized in kit form by the Bio-Rad company, which is designed for detection of PrP in ruminant CNS tissue taken post mortem, was evaluated in samples from animals at different stages of BSE infection (following a single oral challenge with BSE-infected tissue), including samples taken before the onset of clinical signs. The test detected PrPSc in most BSE-exposed animals killed at 32 months or later after exposure to the agent - before clinical signs were first recorded at 35 months after exposure. Two animals in which the test did not detect PrPSc were also negative by other tests (detection of SAF, immunohistochemistry). There were not enough animals in the study to determine at what stage the test kit could be expected to detect infected individuals. It was noted that the kit allowed testing to be completed in less than five hours. The assay was considered to be at least as sensitive as mouse bioassay for detection of BSE. (J3.149.w2)
  • It has been suggested than an ELISA should be used as a screening test for BSE detection with Western blot analysis for confirmation of positive cases. (J35.161.w1)

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Other Immunoassays

Editorial summary
  • A variety of tests have been and are being developed for the detection of abnormal prion proteins.
  • One test was able to detect abnormal prion protein in the blood of elk with CWD and sheep with scrapie.
  • A conformation-dependent immunoassay (CDI) has been developed which may be used on a variety of ungulates and which was considered to have a sensitivity approaching that of bioassay in cattle and in transgenic mice.
Detailed Reports
  • An assay has been developed using capillary electrophoresis and fluorescent labelled peptides to detect abnormal prion protein in blood following treatment with proteinase K (to destroy normal PrP). This assay has a high sensitivity and detected the abnormal protein in blood from an elk with CWD. In a further five elk which had been exposed to CWD, testing two blood samples from each elk, taken with a three month interval between bleedings, one animal was positive with the test on the second blood sample, which may have indicated development of infection. It was noted that the assay could also be used for detecting abnormal prion protein in blood from other species. (J241.853.w1)

Other TSE Diseases/General TSE Information

Scrapie:

  • An assay has been developed using capillary electrophoresis and fluorescent labeled peptides to detect abnormal prion protein in blood following treatment with proteinase K (to destroy normal PrP). This assay has a high sensitivity and detected the abnormal protein in blood from sheep with scrapie. It was noted that the assay could also be used for detecting abnormal prion protein in blood from other species. (J241.853.w1)

Bovine Spongiform Encephalopathy (BSE):

  • A two-site non-competitive immunometric test (WALLAC DELFIA test, E.G. & G Wallac Ltd, UK) using two different monoclonal antibodies and DELFIA technology to generate the reading signal was evaluated as a rapid test for the diagnosis of BSE. This test, which takes less than 24 hours to perform, was found to have a sensitivity of 70% and a specificity of 90%. It did not detect positive samples in diluted brain homogenate at a dilution of 10-1. (J9.400.w1, D111.7.w7)
  • A conformation-dependent immunoassay (CDI) has been developed, including an automated form of the assay (aCDI). The CDI uses recombinant fluorescent antibodies (recFabs) binding to conserved residues of ungulate PrP. The CDI showed sensitivity equal to or greater than that for detection by inoculation of transgenic mice. The aCDI sensitivity was considered to approach that of bioassay in cattle and transgenic (Tg(BoPrP)Prnp0/0) mice. (J243.20.w1)

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Electron microscopy: Presence of Scrapie-Associated Fibrils

Editorial Summary
  • Scrapie-associated fibrils (SAF) are found only in individuals with TSE diseases.
  • In deer and elk with CWD SAF may be found in the brain and spleen.
Detailed Reports
  • Scrapie-associated fibrils may be found. (J64.21.w17)
  • Scrapie-associated fibrils were detected in the brains of 12 free-living animals by immunohistochemical staining and in a further six by Western blot [in a paper on findings in free-ranging deer and elk in Colorado]. (P4.1.w4)
  • SAFs are readily identified in the brain and spleen of clinically affected individuals. (B294.10.w10)
  • "Presence of scrapie-associated fibrils in the spleen and/or brain of both affected deer and elk." (J64.11.w3)

Cervus elaphus nelsoni - Rocky Mountain Elk (Cervus elaphus - Red deer):

  • Scrapie-associated fibrils were present in extracts of brain from affected elk. (J239.34.w1)
  • Scrapie-associated fibrils (SAFs) were visible by electron microscopy in brain tissue from elk with CWD. (J1.33.w10)
  • In the one individual examined by electron microscopy the presence of scrapie-associated fibrils was demonstrates in brain tissue. (J1.34.w6)

Other TSE Diseases/General TSE Information

  • Scrapie-associated fibrils or prion rods are a unique fibrillary structure which have been described in preparations from the brains of animals affected with scrapie, kuru or CJD and shown to be specific to these diseases, not being present in the brains of individuals suffering from other conditions with similar clinical courses, histopathological findings or ultrastructural findings. (J22.225.w1)
    • It was noted that the presence of SAF could not be demonstrated in all individuals with TSE diseases. (J22.225.w1)
    • It was suggested that the SAF represent either the agent, a component of the agent, or a product of the pathological process of the TSEs. (J22.225.w1)
    • SAF are not found in mice with virus- or chemical-induced CNS lesions of spongiform change or gliosis. (J22.225.w1)
    • SAF may be found in the spleen of TSE-affected animals, in the absence of histopathological change.(J22.225.w1)
    • SAF may be demonstrated in the CNS of TSE-affected animals prior to the onset of clinical signs and histopathological change. (J22.225.w1)
  • Electron microscopy can be used for the detection of scrapie associated fibrils (SAF) in the brains of sheep, cattle and other species affected by TSEs, including scrapie, BSE and chronic wasting disease. (B297.5.w5)
    • Fresh or frozen, but not formalin-fixed, material should be used for the detection of SAF by transmission electron microscopy. (B297.5.w5)
    • This technique may still be used in tissues with some degree of autolysis. (B297.5.w5)

Scrapie:

  • Negative stain electron microscopy may be used to show the presence of SAF in brain extracts. Frozen tissue (ideally stored at -70°C and kept frozen during transport to the laboratory) may be used but fixed tissue is not suitable for use. The fibrils are detected in detergent-extracted brain tissue and other tissue such as spleen. SAF have not been found in brain extracts from normal sheep or rodents, nor from rodents affected by other agents such as conventional viruses or chemical insults. (J64.11.w4)
    • It is noted that due to questions of sensitivity, while the presence of SAF indicates the presence of scrapie-associated protein and therefore presumably infection with scrapie, the absence of SAF should not be taken necessarily to mean that the individual is free from scrapie infection. (J64.11.w4)

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Bioassay

Editorial Summary
  • Detection of the agent by bioassay - transmission of the disease from the suspect case to other animals by inoculation - is used to assay the presence of TSE-associated infectivity and to confirm that disease present in an animal is a TSE. Following inoculation the recipient animals are monitored for the development of clinical disease and examined at necropsy for the presence of lesions of spongiform encephalopathy. In most cases inoculation into rodents is used. 
  • There are limitations to bioassay:
    • Sensitivity of bioassay is optimum when the recipient is of the same species as the donor animal; this is often impractical due to prolonged incubation times and levels of biosecurity required for experimental animals. 
    • There is a limit to the amount of inoculum which can be used, particularly when using small species such as mice and inoculating by e.g. the intracerebral route. 
    • Not all TSEs will transmit to commonly used laboratory species.
  • Ferrets have been used for bioassay of CWD. 
Detailed Reports
  • Ferrets have been used for bioassay of CWD. (P47.1.w3)

Other TSE Diseases

  • Detection of the infectious agent, usually by inoculation of rodents with a tissue homogenate from a suspect case, with clinical disease resulting and examination of the tissues at necropsy, is the 'gold standard' reference method for confirmation of TSEs however such bioassay techniques have limitations: sensitivity is dependent on the species barrier between the donor and recipient animals and they are slow and unsuitable for either large-scale epidemiological studies or systematic testing of bovine carcasses prior to meat from the carcasses being passed as safe for human consumption. (J3.149.w2)
  • The only way to assay the presence of TSE-associated infectivity is by inoculation of laboratory animals. (J35.161.w1)
    • For optimal sensitivity the donor and recipient should be of the same species. The use of cattle for detection of BSE is precluded by long incubation times and by the level of safety required. (J35.161.w1)
    • The rodent model is limited by the amount of inoculum which can be tested, the long incubation period and the species barrier. (J35.161.w1)

Transmissible Mink Encephalopathy:

  • Transmissibility to mink has been used as a means of definitive diagnosis of TME. (J64.11.w5)

Scrapie:

Ovis aries - Domestic sheep:

  • Mouse bioassay for transmissibility of the disease is a sound method for confirmation of scrapie. The long incubation period and the possibility of false-negative results (since not all attempts to transmit scrapie to mice are successful) make it impractical for general diagnostic purposes, however it may be useful for confirmation of the first introduction of the disease into indigenous animals in a country previously free of scrapie. (J64.11.w4)
  • It has been noted that one reason why some sources of scrapie failed to infect mice could be that, prior to recognition of the fact that the titre of infectivity varies though the brain, being highest in areas with greatest vacuolation, samples for inoculation were sometimes taken from brain areas with relatively low titres of infectivity. (B298.10.w10)

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Authors & Referees

Authors Dr Debra Bourne MA VetMB PhD MRCVS (V.w5)
Referee Suzanne I Boardman BVMS MRCVS (V.w6)

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