We would like to thank S. Werner for providing the crows used in this study and USDA, APHIS, WS, NWRC animal care staff for animal care and monitoring. Mention or use of a product does not imply USDA endorsement. Funding for this study was provided by USDA, APHIS, Veterinary Services.

Our protocol is adapted from one we previously published7. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Wildlife Services (WS), National Wildlife Research Center (NWRC).
1. Crow Gavaging
<ol>
	<li>Estimate passage time of &#39;pseudo brain material&#39; through the alimentary tract of American crows.
	<ol>
		<li>Mix 5 ml of cooked and scrambled whole egg with blue dye and gavage 1 crow using a 2 in&#160;gavage needle (Figure 1).</li>
		<li>Check crow every 30 min until blue/green-stained feces are no longer being excreted.</li>
	</ol>
	</li>
	<li>Obtain uninfected and terminally ill RML (Chandler strain) -infected C57BL/6 mouse brains.</li>
	<li>Generate a 20% wt/vol normal and infected mouse-brain homogenate in a bullet blender homogenizer with 1x phosphate buffered saline (PBS) and glass beads, then centrifuge at 3,000 x g for 1 min to remove larger particulate matter. Remove supernatant and dilute with an equal volume of 1x PBS to generate a 10% wt/vol in sterile PBS. Freeze at -80 &#176;C until needed.</li>
	<li>17 hr prior to gavage remove food, but not water, from crow pens.</li>
	<li>Randomly allocate crows to treatment groups and orally gavage each crow with 5 ml of either normal or infected mouse brain homogenate using a 2 in&#160;gavage needle.</li>
	<li>Transfer crows to individual cages and collect and pool all feces within each cage for 4 hr post gavage (Figure 2).</li>
	<li>Homogenize pooled feces for each crow in a bullet blender homogenizer with glass beads until a uniform texture is achieved. *Crow feces are mostly liquid and can be mixed easily.</li>
	<li>For each crow, dilute 500 &#181;l of fecal homogenate into 9.5 ml 1x PBS for a total volume of 10 ml.</li>
	<li>Centrifuge the fecal homogenate for 15 min at 1,400 x g and extract the supernatant.</li>
	<li>To minimize the threat of a secondary microbial infection, treat supernatant with 1 &#181;l of 100 units/ml penicillin and 100 &#181;g/ml streptomycin (Invitrogen, NY) per 100 &#181;l of homogenate then place under UV light at room temperature for 20 min to reduce risk of viral and bacterial contamination. After UV exposure, sonicate samples in a 3000 Mp sonicator at setting 70 for 30 sec to disrupt the membrane of any remaining microbes.</li>
</ol>
2. Mouse Inoculation
<ol>
	<li>Randomly allocate mice to the following treatment groups (Table 1):
	<ol>
		<li>Group 1 - Positive treatment mice inoculated intraperitoneally (IP) with 1 ml of fecal homogenate from crows orally gavaged with 5ml infected mouse brains.</li>
		<li>Group 2 - Negative treatment mice inoculated IP with 1 ml of fecal homogenate from crows orally gavaged with 5ml normal mouse brains.</li>
		<li>Dilute infected and normal mouse brain homogenate for Groups 3 and 4 to 1:100 w/v in 1x PBS.</li>
		<li>Group 3 - Positive control mice inoculated IP with 1 ml of infected mouse-brain homogenate.</li>
		<li>Group 4 - Negative control mice inoculated IP with 1 ml of normal mouse-brain homogenate.</li>
	</ol>
	</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td>Treatment Group</td>
			<td>Number of Animals</td>
		</tr>
		<tr>
			<td>Group 1</td>
			<td>Scrapie + Crow Feces</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Group 2</td>
			<td>Scrapie - Crow Feces</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Group 3</td>
			<td>Scrapie + Mouse Brain</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Group 4</td>
			<td>Scrapie - Mouse Brain</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1. Scrapie status of inoculum (positive +, negative -) and number of animals used7.
<ol start="2">
	<li>Intraperitoneally inoculate mouse:
	<ol>
		<li>Scruff mouse by dorsal neck fur with thumb and index finger and gently rotate to expose ventral side.</li>
		<li>Elevate posterior end of mouse so head is slightly lower.</li>
		<li>Insert 25 G needle 1 cm through skin, 1 cm lateral of midline, and 1-2 cm anterior to the sacroiliac joint using a needle-locking syringe.</li>
		<li>Inject 1 ml inoculate slowly into mouse body cavity.</li>
	</ol>
	</li>
</ol>
3. Mouse Monitoring
<ol>
	<li>Monitor mice daily until they express clinical signs of mouse scrapie. Clinical signs may include: kyphosis, ataxia, stiff tail, lack of grooming, emaciation, and lethargy.</li>
	<li>Score mice for each of the 6 clinical signs when visible signs are evident, where 0 = none visible, 1 = moderate, and 2 = severe.</li>
	<li>Euthanize mice when total daily scores for each sign reach &#8805;8 for 1 day, &#8805; 6 continuously for 3 days, or at 365 days post inoculation (dpi).</li>
	<li>Harvest brains immediately following euthanasia and store at -70 &#176;C.</li>
	<li>To confirm a scrapie diagnosis, digest brain samples with 3 &#181;l of a 50 &#181;g/ml of proteinase-K solution (PK) diluted as follows: 3.1. &#181;l PK, 12.5 &#181;l of 500 mM EDTA, pH 8, 109.39 &#181;l of 1x PBS for 30 min at 45 &#176;C, then inactivate the PK by adding 8 &#181;l loading buffer and incubating samples at 95 &#176;C for 5 min. Load samples onto a 12% SDS-PAGE gel, electrophorese and transfer to Immobilon PVDF membrane and block with 5% nonfat milk in 0.2% Tween 20 in PBS for 1 hr at room temperature. Then probe with Bar224 anti-PrP monoclonal antibody conjugated to Horseradish peroxidase, diluted in Superblock, for 1 hr at room temperature. Rinse membrane for 1 hr with PBS-0.2% Tween 20. To visualize, incubate Western blot 5 min with chemiluminescent substrate and image on a G-box gel documentation system.</li>
</ol>

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No conflicts of interest declared.

We demonstrate a procedure to document passage of PrPRes material through the digestive system of crows. We used conventional methods to determine if crows have the ability to translocate PrPRes material to disease-free geographic areas. Others have evaluated resistance of PrPRes to ruminant19-21 and rodent22,23 digestive fluids, both of which failed to eliminate it. Future application of these techniques should be applied to other carnivores24 as they cou...

Transmissible spongiform encephalopathies (TSE) are fatal infectious neurodegenerative disorders that affect wildlife, domestic animals, and humans. The infectious agent of TSE diseases appears to be misfolded or pathogenic isoforms (PrPRes) of prion proteins1. Animal TSE diseases include chronic wasting disease (CWD) in mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), elk (Cervus elaphus), and moose (Alces alces); scrapie in sheep and goats; bovine spongiform encephalopathy (BSE) in domestic cattle; transmissible mink encephalopathy in farmed mink; feline spongiform encephalopathy in cats; exotic ungulate spongiform encephalopathy in exotic zoo ruminates of the family Bovidae; and spongiform encephalopathy in nonhuman primates8. The single human TSE disease, variant Creutzfeldt-Jakob disease, is rare and thought to be acquired by consuming PrPRes-contaminated food9. Similarly, BSE can infect humans if contaminated beef is consumed10. Of all the TSE diseases, scrapie and CWD are the only two with self-sustaining epidemics and sources for infection are presumed to be from animal to animal2,3,11 as well as from environmental sources4-6. Research suggests that most TSE diseases require notable extended incubation periods from natural exposure events of PrPRes material to manifestation of clinical signs2-4,6,8 and apparent species barriers minimize, but do not eliminate the potential for, interspecies transmission12-14.
Identifying mechanisms for the spread of infectious prion (PrPRes) material is extremely important for answering questions about how TSE diseases move across the landscape. Experimental investigations have suggested that insects15,16, poultry and pigs17, and American crows (Corvus brachyrhynchos)7,18 are passive carriers or dispersers of PrPRes material. Passage of PrPRes material through the digestive system of crows has recently been documented, demonstrating the role they may play in dispersal of TSE diseases7. These results make it plausible that crows, a scavenger, could encounter, consume, and transport infectious material via feces deposition, to disease-free areas.
The procedures we demonstrate here were used to document passage of PrPRes material through the digestion system of crows and will greatly facilitate the application of these methods to other scavenger and carnivore species-specific models in related future research. In this study conventional methods were used to investigate an unconventional means of trafficking PrPRes material, which could contribute to the spread and overall burden of PrPRes material.

<table><tbody><tr><td>RML Chandler strain mouse-adapted scrapie</td><td>Rocky Mountain Laboratories</td><td></td><td></td></tr><tr><td>RC57BL/6 mice</td><td>Hilltop Lab Animals</td><td></td><td></td></tr><tr><td>American crows</td><td>wild captured</td><td></td><td></td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Phosphate buffered Saline</td><td>Invitrogen</td><td>70011-044</td><td></td></tr><tr><td>Sonicator</td><td>Misonix</td><td></td><td></td></tr><tr><td>Proteinase-K solution</td><td>Roche</td><td>3115887001</td><td></td></tr><tr><td>Loading buffer</td><td>Invitrogen</td><td>NP0007 and 0009</td><td></td></tr><tr><td>Bis-tris SDS PAGE 12% gel</td><td>Invitrogen</td><td>NP0342</td><td></td></tr><tr><td>Immobilon PVDF membrane</td><td>Millipore</td><td>1SEQ00010</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P2287</td><td></td></tr><tr><td>Bullet blender homogenizer</td><td>Braintree Scientific</td><td>BBX24B</td><td></td></tr><tr><td>2.3 mm Zirconia/silica beads</td><td>BioSpec Products</td><td>11079125Z</td><td></td></tr><tr><td>Bar224 anti-PrP monoclonal antibody</td><td>Cayman Chemical</td><td>10009035</td><td></td></tr><tr><td>Superblock</td><td>Thermo Scientific</td><td>37517</td><td></td></tr><tr><td>chemiluminescent substrate</td><td>Millipore</td><td>WBKLS0500</td><td></td></tr><tr><td>G-box gel documentation system</td><td>Syngene</td><td></td><td></td></tr></tbody></table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

The procedures used demonstrate that the digestive system of crows does not eliminate PrPRes infectivity 4 hr after oral gavage of scrapie brain homogenate7. All twenty crows that were gavaged with PrPRes material subsequently transmitted PrPRes material via feces to mice. Diseased mice were identified by manifestation of clinical mouse-scrapie signs and disease confirmation was completed by Western blot analysis.
Investigation of the retention time ...

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

procedures-for-identifying-infectious-prions-after-passage-through

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

11.0K Views.  USDA. Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Video: Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

Medicine

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

Neuroscience

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

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Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Avian Influenza Surveillance with FTA Cards: Field Methods, Biosafety, and Transportation Issues Solved

Protein Misfolding Cyclic Amplification of Prions

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

Prion Safety Laboratory Swipe Test

Detection of Abnormal Prion Protein by Immunohistochemistry