Detection of the Disease-Associated Isoform of the Prion Protein in Formalin-Fixed Tissues by Western Blot

Clinical signs of prion disease are not specific and include a variety of differential diagnoses. Serological tests and nucleic acid-based detection methods are not applicable to prion-disease-agent detection because of the unusual nature of the infectious agent. Prion-disease diagnosis is primarily conducted by means of immunodetection of the infectious agent, typically by at least 2 distinct procedures with immunohisto-chemistry and Western blot being the most informative. These approaches differ in the need for formalin-fixed and frozen or fresh tissue respectively. This work describes a method for the detection of the disease-associated isoform of the prion protein by Western blot using formalin-fixed tissues. The approach requires only minimal modification of existing Western-blot procedures and could readily be incorporated into existing detection schemes for confirmatory purposes when fresh or frozen tissues are unavailable.

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Canine ehrlichiosis: clinical, hematological, serological and molecular aspects

Ciência Rural ◽

10.1590/s0103-84782008000300027 ◽

2008 ◽

Vol 38 (3) ◽

pp. 766-770 ◽

Cited By ~ 36

Author(s):

Andréa Cristina Higa Nakaghi ◽

Rosangela Zacarias Machado ◽

Mirela Tinucci Costa ◽

Marcos Rogério André ◽

Cristiane Divan Baldani

Keyword(s):

Direct Detection ◽

Clinical Signs ◽

Chronic Phase ◽

Antibody Test ◽

Acute Stage ◽

Detection Methods ◽

Serological Tests ◽

Humoral Immune ◽

Canine Ehrlichiosis ◽

Dot Elisa

The aim of the present study was to compare the direct detection methods of Ehrlichia canis (blood smears and nested PCR), serological tests (Dot-ELISA and Immunofluorescent Antibody Test - IFAT), and demonstrate the most suitable test for the diagnosis of different stages of infection. Blood samples and clinical data were collected from 30 dogs examined at the Veterinary Teaching Hospital, UNESP, Jaboticabal, SP, Brazil. The clinical signs most frequently observed were apathy, anorexia, pale mucous membrane, fever, lymphadenopathy, splenomegaly, hemorrhages and uveitis. Evaluating the humoral immune response, 63.3% of the sera were IFAT positive, while 70% were Dot-ELISA positive. By nestedPCR 53.3% of the samples were positive. Comparing these techniques it was concluded that serology and nPCR are the most suitable tests to confirm the diagnosis of canine ehrlichiosis, however it should be always treated as a complementary data to clinical and hematological evaluation. Serology has an important role in the subclinical and in the chronic phase, nPCR is recommended in the acute stage, and, especially, to identify the ehrlichia specie.

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Enzymatic detergent treatment protocol that reduces protease-resistant prion protein load and infectivity from surgical-steel monofilaments contaminated with a human-derived prion strain

Journal of General Virology ◽

10.1099/vir.0.82961-0 ◽

2007 ◽

Vol 88 (10) ◽

pp. 2905-2914 ◽

Cited By ~ 30

Author(s):

Victoria A. Lawson ◽

James D. Stewart ◽

Colin L. Masters

Keyword(s):

Prion Protein ◽

Prion Disease ◽

Disease Transmission ◽

Prion Diseases ◽

Medical Instrument ◽

Treatment Protocol ◽

Infectious Agent ◽

Surgical Instruments ◽

Prion Strain ◽

Jakob Disease

The unconventional nature of the infectious agent of prion diseases poses a challenge to conventional infection control methodologies. The extraneural tissue distribution of variant and sporadic Creutzfeldt–Jakob disease has increased concern regarding the risk of prion disease transmission via general surgical procedures and highlighted the need for decontamination procedures that can be incorporated into routine processing. In this study, the ability of preparations of enzymatic medical instrument cleaners to reduce the infectivity associated with a rodent-adapted strain of human prion disease, previously reported to be resistant to decontamination, was tested. Efficient degradation of the disease-associated prion protein by enzymatic cleaning preparations required high treatment temperatures (50–60 °C). Standard decontamination methods (1 M NaOH for 1 h or autoclaving at 134 °C for 18 min) reduced infectivity associated with the human-derived prion strain by less than 3 log10 LD50. In contrast, a 30 min treatment with the optimized enzymatic cleaning preparation protocols reduced infectivity by more than 3 log10 LD50 and when used in conjunction with autoclave cycles eliminated detectable levels of infectivity. The development of prion decontamination procedures that are compatible with routine cleaning and sterilization of medical and surgical instruments may reduce the risk of the transmission of prion disease in general surgery.

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Goats naturally devoid of PrPC are resistant to scrapie

Veterinary Research ◽

10.1186/s13567-019-0731-2 ◽

2020 ◽

Vol 51 (1) ◽

Cited By ~ 8

Author(s):

Øyvind Salvesen ◽

Arild Espenes ◽

Malin R. Reiten ◽

Tram T. Vuong ◽

Giulia Malachin ◽

...

Keyword(s):

Prion Protein ◽

Prion Disease ◽

Prion Diseases ◽

Clinical Signs ◽

Brain Regions ◽

Post Inoculation ◽

Dairy Goats ◽

Peripheral Tissues ◽

Clinical Onset ◽

Natural Mutation

AbstractPrion diseases are progressive and fatal, neurodegenerative disorders described in humans and animals. According to the “protein-only” hypothesis, the normal host-encoded prion protein (PrPC) is converted into a pathological and infectious form (PrPSc) in these diseases. Transgenic knockout models have shown that PrPC is a prerequisite for the development of prion disease. In Norwegian dairy goats, a mutation (Ter) in the prion protein gene (PRNP) effectively blocks PrPC synthesis. We inoculated 12 goats (4 PRNP+/+, 4 PRNP+/Ter, and 4 PRNPTer/Ter) intracerebrally with goat scrapie prions. The mean incubation time until clinical signs of prion disease was 601 days post-inoculation (dpi) in PRNP+/+ goats and 773 dpi in PRNP+/Ter goats. PrPSc and vacuolation were similarly distributed in the central nervous system (CNS) of both groups and observed in all brain regions and segments of the spinal cord. Generally, accumulation of PrPSc was limited in peripheral organs, but all PRNP+/+ goats and 1 of 4 PRNP+/Ter goats were positive in head lymph nodes. The four PRNPTer/Ter goats remained healthy, without clinical signs of prion disease, and were euthanized 1260 dpi. As expected, no accumulation of PrPSc was observed in the CNS or peripheral tissues of this group, as assessed by immunohistochemistry, enzyme immunoassay, and real-time quaking-induced conversion. Our study shows for the first time that animals devoid of PrPC due to a natural mutation do not propagate prions and are resistant to scrapie. Clinical onset of disease is delayed in heterozygous goats expressing about 50% of PrPC levels.

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Identification of Misfolded Proteins in Body Fluids for the Diagnosis of Prion Diseases

International Journal of Cell Biology ◽

10.1155/2013/839329 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 4

Author(s):

Francesca Properzi ◽

Maurizio Pocchiari

Keyword(s):

Prion Protein ◽

Prion Diseases ◽

Transmissible Spongiform Encephalopathy ◽

Clinical Signs ◽

Meat Products ◽

Silent Period ◽

Infectious Agent ◽

Body Fluids ◽

Cellular Prion Protein ◽

Spongiform Encephalopathy

Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal rare neurodegenerative disorders affecting man and animals and caused by a transmissible infectious agent. TSE diseases are characterized by spongiform brain lesions with neuronal loss and the abnormal deposition in the CNS, and to less extent in other tissues, of an insoluble and protease resistant form of the cellular prion protein (PrPC), namedPrPTSE. In man, TSE diseases affect usually people over 60 years of age with no evident disease-associated risk factors. In some cases, however, TSE diseases are unequivocally linked to infectious episodes related to the use of prion-contaminated medicines, medical devices, or meat products as in the variant Creutzfeldt-Jakob disease (CJD). Clinical signs occur months or years after infection, and during this silent periodPrPTSE, the only reliable marker of infection, is not easily measurable in blood or other accessible tissues or body fluids causing public health concerns. To overcome the limit ofPrPTSEdetection, several highly sensitive assays have been developed, but attempts to apply these techniques to blood of infected hosts have been unsuccessful or not yet validated. An update on the latest advances for the detection of misfolded prion protein in body fluids is provided.

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Use of TEM in Plant Disease Diagnosis: A Xylem-Limited Bacterium Associated with Diseased ‘Toronto’ Creeping Bentgrass

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098575 ◽

1981 ◽

Vol 39 ◽

pp. 414-415

Author(s):

Karen K. Baker ◽

David L. Roberts

Keyword(s):

Osmium Tetroxide ◽

Plant Disease ◽

Leaf Tissue ◽

Infectious Agent ◽

Creeping Bentgrass ◽

Disease Diagnosis ◽

Unknown Etiology ◽

Agrostis Palustris ◽

Putting Greens ◽

Plant Disease Diagnosis

Plant disease diagnosis is most often accomplished by examination of symptoms and observation or isolation of causal organisms. Occasionally, diseases of unknown etiology occur and are difficult or impossible to accurately diagnose by the usual means. In 1980, such a disease was observed on Agrostis palustris Huds. c.v. Toronto (creeping bentgrass) putting greens at the Butler National Golf Course in Oak Brook, IL.The wilting symptoms of the disease and the irregular nature of its spread through affected areas suggested that an infectious agent was involved. However, normal isolation procedures did not yield any organism known to infect turf grass. TEM was employed in order to aid in the possible diagnosis of the disease.Crown, root and leaf tissue of both infected and symptomless plants were fixed in cold 5% glutaraldehyde in 0.1 M phosphate buffer, post-fixed in buffered 1% osmium tetroxide, dehydrated in ethanol and embedded in a 1:1 mixture of Spurrs and epon-araldite epoxy resins.

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Behavior assessment and applications for BRD diagnosis: preweaned dairy calves

Animal Health Research Reviews ◽

10.1017/s1466252320000213 ◽

2020 ◽

pp. 1-4

Author(s):

Catie Cramer ◽

Theresa L. Ollivett

Keyword(s):

Clinical Signs ◽

Sickness Behavior ◽

Behavioral Changes ◽

Dairy Calves ◽

Detection Methods ◽

Disease Detection ◽

Test Characteristics ◽

Behavioral Studies ◽

Detection Program ◽

Behavioral Assessments

Abstract Bovine respiratory disease (BRD) is an important disease in dairy calves due to its long-lasting effects. Early identification results in better outcomes for the animal, but producers struggle to identify all calves with BRD. Sickness behavior, or the behavioral changes that accompany illness, has been investigated for its usefulness as a disease detection tool. Behavioral changes associated with BRD include decreased milk intake and drinking speed, depressed attitude, and less likelihood of approaching a novel object or stationary human. Behavioral measurements are useful, as they can be collected automatically or with little financial input. However, one limitation of many BRD behavioral studies includes the use of either lung auscultation or clinical signs as reference methods, which are imperfect. Additionally, external factors may influence the expression of sickness behavior, which can affect if and when behavior can be used to identify calves with BRD. Behavioral measures available to detect BRD lack adequate sensitivity and specificity to be the sole means of disease detection, especially when detection tools, such as calf lung ultrasound, have better test characteristics. However, using behavioral assessments in addition to other detection methods can allow for a robust BRD detection program that can ameliorate the consequences of BRD.

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The First Report of the Prion Protein Gene (PRNP) Sequence in Pekin Ducks (Anas platyrhynchos domestica): The Potential Prion Disease Susceptibility in Ducks

Genes ◽

10.3390/genes12020193 ◽

2021 ◽

Vol 12 (2) ◽

pp. 193

Author(s):

Min-Ju Jeong ◽

Yong-Chan Kim ◽

Byung-Hoon Jeong

Keyword(s):

Prion Protein ◽

Dna Sequence ◽

Prion Disease ◽

Protein Gene ◽

Prnp Gene ◽

Pekin Duck ◽

Prion Protein Gene ◽

First Report ◽

Aggregation Propensity ◽

Pekin Ducks

Pathogenic prion protein (PrPSc), converted from normal prion protein (PrPC), causes prion disease. Although prion disease has been reported in several mammalian species, chickens are known to show strong resistance to prion diseases. In addition to chickens, the domestic duck occupies a large proportion in the poultry industry and may be regarded as a potential resistant host against prion disease. However, the DNA sequence of the prion protein gene (PRNP) has not been reported in domestic ducks. Here, we performed amplicon sequencing targeting the duck PRNP gene with the genomic DNA of Pekin ducks. In addition, we aligned the PrP sequence of the Pekin duck with that of various species using ClustalW2 and carried out phylogenetic analysis using Molecular Evolutionary Genetics Analysis X (MEGA X). We also constructed the structural modeling of the tertiary and secondary structures in avian PrP using SWISS-MODEL. Last, we investigated the aggregation propensity on Pekin duck PrP using AMYCO. We first reported the DNA sequence of the PRNP gene in Pekin ducks and found that the PrP sequence of Pekin ducks is more similar to that of geese than to that of chickens and mallards (wild ducks). Interestingly, Pekin duck PrP showed a high proportion of β-sheets compared to that of chicken PrP, and a high aggregation propensity compared to that of avian PrPs. However, Pekin duck PrP with substitutions of chicken-specific amino acids showed reduced aggregation propensities. To the best of our knowledge, this is the first report on the genetic characteristics of the PRNP sequence in Pekin ducks.

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Evaluation of a Preclinical Blood Test for Scrapie in Sheep Using Immunocapillary Electrophoresis

Journal of AOAC International ◽

10.1093/jaoac/89.3.720 ◽

2006 ◽

Vol 89 (3) ◽

pp. 720-727 ◽

Cited By ~ 12

Author(s):

Roy Jackman ◽

David J Everest ◽

Mary Jo Schmerr ◽

Mohammed Khawaja ◽

Pat Keep ◽

...

Keyword(s):

Capillary Electrophoresis ◽

Prion Protein ◽

Clinical Signs ◽

Test System ◽

Buffy Coat ◽

Infected Sheep ◽

Test Method ◽

Peptide Sequence ◽

Transmissible Spongiform Encephalopathies ◽

Blood Samples

Abstract An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 712 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.

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