The studies providing the animals were funded by the UK Department for Food, Environment and Rural Affairs and the European Union (through funding of the EU TSE Reference Laboratory). We acknowledge the support of the project managers, Dr. M. Simmons and Dr. M. Jeffrey.

1. Assessment of Posture
<ol>
	<li>Assess the posture of the animal whilst undisturbed.</li>
	<li>Animals affected by TSEs may stand with wide-based limbs, may appear hunched, or may stand with a low head carriage.</li>
</ol>
2. Assessment of Behavior
<ol>
	<li>Study the animal&#8217;s behavior, when left alone, when approached, and when handled.</li>
	<li>TSE-affected animals may display altered mentation by standing separately from other sheep or not immediately following other sheep.</li>
	<li>When the animal is approached, it may run away and collapses, or it collapses to the floor when handled. Compulsive clockwise or counter-clockwise circling may be seen in sheep with atypical scrapie.</li>
</ol>
3. Menace Response Testing
<ol>
	<li>Hold the animal&#8217;s head for testing of the menace response: move the hand or several fingers several times towards each eye of the animal to induce a blink response but be careful not to touch any facial hairs or create any air turbulence that may cause the animal to blink.</li>
	<li>The menace response may be absent in scrapie-affected animals, particularly in sheep with atypical scrapie. Although testing of the menace response is the only cranial nerve testing used as part of the protocol, visual inspection of the face during this procedure may reveal other signs of neurologic deficits not normally seen in scrapie, e.g. facial paralysis. If the animal does not blink in response to the menace it is advisable to test the palpebral reflex by touching the eyelids to ensure that the animal&#8217;s inability to blink is not caused by facial paralysis.</li>
</ol>
4. Testing of the Scratch Response
<ol>
	<li>Ensure the animal is calm during this procedure because the test cannot be interpreted in a struggling animal.</li>
	<li>Scratch areas of the back at the thoracic, lumbar, and sacral regions, pausing between each scratching episode.</li>
	<li>If the animal responds with rhythmical head or body movements, or moves or licks its lips upon scratching of one particular area, repeat the scratch test to confirm reproducibility of the scratch response.</li>
	<li>A repeatable response is usually elicited in animals that are pruritic, which usually includes sheep with classical scrapie or goats with the pruritic form of scrapie, but there is generally no response in sheep with atypical scrapie unless other pruritic skin conditions or ectoparasites are present.</li>
</ol>
5. Checking for Wool Loss and Skin Lesions
<ol>
	<li>Inspect the skin.</li>
	<li>Pruritus due to scrapie usually causes hair loss, which may be on the poll, back, rump, side of chest or abdomen due to excessive rubbing or scratching where the underlying skin appears normal.</li>
	<li>Abrasions may appear later if the animal has the opportunity to rub on sharp objects. Bite wounds may appear, particularly on the limbs, due to excessive nibbling of the limbs.</li>
</ol>
6. Scoring Body Condition
<ol>
	<li>Assess the animal&#8217;s body condition by palpating the dorsal and transverse processes of the lumbar vertebrae.</li>
	<li>Scrapie may cause a progressive loss of body condition where the transverse processes of the lumbar vertebrae can be easily felt individually, and the ends of the processes appear sharp.</li>
</ol>
7. Blindfolding the Animal
<ol>
	<li>Cover the animal&#8217;s eyes with a hood, which can be made by using any nonsee-through bag where one corner is cut off to allow breathing of the animal.</li>
	<li>Release the animal only if there are no safety concerns, and there is no risk of injury due to a panicking animal; otherwise keep hold of the sheep and study animal movements whilst still in control of the animal.</li>
	<li>Blindfolding may exacerbate signs of a vestibular system or cerebellar dysfunction, which may be obscured by the animal&#8217;s ability to compensate through its vision. Loss of balance, incoordination, or circling may be elicited, particularly in otherwise clinically unremarkable sheep with atypical scrapie.</li>
</ol>
8. Assessment of the Animal&#8217;s Movement
<ol>
	<li>Study the animal&#8217;s gait and muscle movements when it moves freely.</li>
	<li>Incoordination may be characterized by wide-based movements of the hind limbs, hopping with both hind limbs, or swaying excessively in the hind quarters, which may result in stumbling or briefly sitting down.</li>
	<li>The presence of a head tremor is often subtle when the animal is undisturbed but may become more noticeable during or after completion of the clinical examination.</li>
</ol>
9. Interpretation
<ol>
	<li>Consider the significance of the findings with respect to scrapie.</li>
	<li>Although the presence of individual signs, such as a repeatable response to scratching, tremor, abnormal behavior, circling, collapsing episodes, ataxia, or an absent menace response, may have various causes, the combined occurrence of two or more of these signs is highly suggestive of scrapie in sheep or goats.</li>
</ol>

<ol>
	<li>Benestad, S. L., Arsac, J. N., Goldmann, W., N&#246;remark, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atypical/Nor98+scrapie:+properties+of+the+agent,+genetics,+and+epidemiology.">Atypical/Nor98 scrapie: properties of the agent, genetics, and epidemiology.</a> Vet Res.. 39 (4), 19 (2008).</li><li>D&#39;Angelo, A., Maurella, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+clinical+criteria+to+diagnose+scrapie+in+Italy.">Assessment of clinical criteria to diagnose scrapie in Italy.</a> Vet. J. 174 (1), 106-112 (2007).</li><li>Eloit, M., Adjou, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BSE+agent+signatures+in+a+goat.">BSE agent signatures in a goat.</a> Vet. Rec. 156 (16), 523-524 (2005).</li><li>Fediaevsky, A., Maurella, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+atypical+scrapie+in+sheep+from+positive+flocks+is+not+higher+than+in+the+general+sheep+population+in+11+European+countries.">The prevalence of atypical scrapie in sheep from positive flocks is not higher than in the general sheep population in 11 European countries.</a> BMC Vet. Res. 6, 9 (2010).</li><li>Healy, A. M., Weavers, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+neurology+of+scrapie+in+Irish+sheep.">The clinical neurology of scrapie in Irish sheep.</a> J. Vet. Intern. Med. 17 (6), 908-916 (2003).</li><li>Houston, E. F., Gravenor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+signs+in+sheep+experimentally+infected+with+scrapie+and+BSE.">Clinical signs in sheep experimentally infected with scrapie and BSE.</a> 152 (11), 333-334 (2003).</li><li>Kittelberger, R., Chaplin, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atypical+scrapie/Nor98+in+a+sheep+from+New+Zealand.">Atypical scrapie/Nor98 in a sheep from New Zealand.</a> J. Vet. Diagn. Invest. 22 (6), 863-875 (2010).</li><li>Konold, T., Bone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+of+clinical+signs+and+prion+protein+genotypes+in+British+sheep+with+scrapie.">Associations of clinical signs and prion protein genotypes in British sheep with scrapie.</a> Dtsch. Tier&#228;rztl. Wochenschr. 116 (11), 380-388 (2009).</li><li>Konold, T., Bone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie+in+goats.">Scrapie in goats.</a> Vet. Rec. 161 (11), 395-396 (2007).</li><li>Konold, T., Bone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pruritus+is+a+common+feature+in+sheep+infected+with+the+BSE+agent.">Pruritus is a common feature in sheep infected with the BSE agent.</a> BMC Vet. Res. 4, 16 (2008).</li><li>Konold, T., Bone, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+clinical+signs+in+goats+with+transmissible+spongiform+encephalopathies.">Monitoring of clinical signs in goats with transmissible spongiform encephalopathies.</a> BMC Vet. Res. 6, 13 (2010).</li><li>Konold, T., Davis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+findings+in+two+cases+of+atypical+scrapie+in+sheep:+a+case+report.">Clinical findings in two cases of atypical scrapie in sheep: a case report.</a> BMC Vet. Res. 3, 1 (2007).</li><li>Passler, T., Walz, P. H., Pugh, D. G., Pugh, D. G., Baird, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diseases of the Neurologic System. In: Sheep and Goat Medicine. , 361-405 (2012).</li><li>Russel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+condition+scoring+of+sheep.">Body condition scoring of sheep.</a> In Pract. 6 (3), 91-93 (1984).</li><li>Simmons, H. A., Simmons, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atypical+scrapie+in+sheep+from+a+UK+research+flock+which+is+free+from+classical+scrapie.">Atypical scrapie in sheep from a UK research flock which is free from classical scrapie.</a> BMC Vet. Res. 5, 8 (2009).</li><li>Simmons, M. M., Konold, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+atypical+scrapie+phenotype+is+preserved+on+experimental+transmission+and+sub-passage+in+PRNP+homologous+sheep.">The natural atypical scrapie phenotype is preserved on experimental transmission and sub-passage in PRNP homologous sheep.</a> BMC Vet. Res. 6, 14 (2010).</li><li>Spiropoulos, J., Lockey, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+prion+with+BSE+properties+from+farmed+goat.">Isolation of prion with BSE properties from farmed goat.</a> Emerg. Infect. Dis. 17 (12), 2253-2261 (2011).</li><li>Ulvund, M. J., H&#246;rnlimann, B., Riesner, D., Kretzschmar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical Findings in Scrapie. In: Prions in humans and animals. , 398-407 (2006).</li></ol>

All procedures involving animals were carried out in accordance with the Animal (Scientific Procedures) Act 1986, under license from the UK Government Home Office. 
The authors declare that they have no competing financial interests.

Scrapie is a listed disease by the World Health Organization for Animal Health (OIE), which, in the absence of a quick and reliable ante mortem test, relies on the farmers&#8217; or veterinarians&#8217; ability to recognize the clinical signs associated with this disease. Most scrapie cases, particularly atypical scrapie cases, however, are currently found by targeted TSE surveillance, i.e. by brain examination of small ruminants subject to slaughter or fallen stock animals. Breeding sheep for resistance to classical scrapie, which reduced the number of classical scrapie cases, most likely also reduced people&#8217;s awareness for the disease so that it is less often considered in the differential diagnosis of neurologic disorders in sheep. Atypical scrapie also has a low prevalence with approximately five cases in abattoirs and eight cases in fallen stock per 10,000 tested in Europe4. In addition, pruritus, usually evident as alopecia with or without excoriation, is often considered a characteristic sign of scrapie13. Thus, a neurologic disease without concomitant pruritus as observed in atypical scrapie12 or the nonpruritic form of caprine scrapie11 may not raise suspicion of scrapie.
This examination protocol was designed to identify clinical signs of TSE in sheep by evaluating specific clinical signs associated with various types of scrapie (atypical and classical scrapie, the latter represented by an atypical BSE-inoculated sheep, which presented with classical scrapie-like signs). In classical scrapie, the percentage of cases that displayed a positive scratch response (&#8220;nibble reflex&#8221;) ranged from 50-77 in over 100 sheep studied5,8. Movement disorders can be observed in up to 81% of classical scrapie cases based on various studies18. Incoordination is also a major sign in various naturally occurring cases of atypical scrapie, often accompanied by behavioral changes1, but additional signs reported in both naturally and experimentally infected atypical scrapie sheep were circling and visual impairment12,15,16, which is why blindfolding and the testing of the menace response is included in the examination protocol. Scoring the body condition, for example using a 6 point scoring system14, enables evaluation of emaciation, which may be observed in scrapie-affected sheep5. Although clinical criteria to aid in the detection of scrapie in sheep have been proposed previously2, these only related to the detection of classical scrapie and took the clinical history into account, which may not be available if animals are presented at an abattoir.
The current protocol allows screening of many animals for signs of scrapie within a short period of time. Furthermore, the examination protocol can be applied to experimental studies of scrapie in sheep and goats to monitor clinical onset, progression, and end-point. However, it does not replace a more detailed physical and neurologic examination with appropriate recording of the clinical case history, possibly followed by ancillary diagnostic tests (examination of blood and cerebrospinal fluid; more sophisticated imaging techniques if appropriate), which may be necessary subsequently to rule out various other conditions in small ruminants that present with similar signs, such as metabolic or inflammatory neurological diseases, brain abscesses, or tumors.
Although a clinical examination is generally less sensitive in detecting TSE-affected animals than post mortem tests based on detection of prion protein2,11, particularly in an early stage of the incubation period, this is currently the only detection method in live animals and most relevant to those countries that do not have a targeted surveillance system in place to identify TSE-affected small ruminants.

Scrapie is a transmissible spongiform encephalopathy (TSE) of sheep and goats, which belongs to the group of protein misfolding disorders, like bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in deer, and Creutzfeldt-Jakob disease in humans. The diagnosis is made post mortem by detecting the abnormal form of the prion protein, which is proteinase-resistant, usually in the brain. Two major naturally occurring scrapie forms can be distinguished, which are epidemiologically, pathologically, and biochemically different: classical scrapie and atypical scrapie. In addition, a neurological disease with classical scrapie-like clinical signs can be induced in sheep and goats by experimental infection with the BSE agent6,10-11. In fact, naturally occurring BSE has been diagnosed in goats3,17. Control of classical scrapie (and BSE in sheep if it occurred naturally) has been achieved by breeding for genetic resistance targeting the prion protein gene, but this is more difficult for atypical scrapie, which is believed to be a sporadic disease and has been diagnosed in countries considered to be free from classical scrapie, e.g. New Zealand7.
Scrapie is a neurologic disease of adult sheep and goats resulting in changes in behavior, sensation, and movement, but the disease is slowly progressive, and clinical signs are often not recognized because the animal keeper or veterinary surgeon may not be familiar with the clinical signs, or scrapie is not considered at all in the differential diagnosis of neurologic diseases in small ruminants.
The proposed examination protocol was designed to aid in the clinical diagnosis of scrapie with two purposes in mind: it should be considerably brief to allow scanning of many animals within a short period of time but specific enough to allow detection of scrapie-affected animals, which is achieved by assessing the presence of clinical signs frequently encountered in scrapie-affected animals. This protocol is a slight modification from the one used to monitor goats for signs of classical scrapie11 and includes blindfolding of the animal, which may exacerbate clinical signs not normally associated with scrapie9.

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.

Clinically healthy sheep. Poll Dorset wether from a scrapie-free flock intracerebrally inoculated with bovine brain of New Zealand origin and examined at 49 months post inoculation. This sheep has a normal posture, behaves normally, displays a normal menace response (Figure 1), does not react to the scratch test, has an intact fleece, and a very good body condition. It is clinically unremarkable when blindfolded and shows no signs of a movement disorder.
Atypical scrapie sheep presenting with the nonpruritic form of TSEs in sheep. Cheviot wether from a scrapie-free flock intracerebrally inoculated with brain homogenate from an atypical scrapie field case of the same prion protein genotype (AHQ/AHQ) and examined at 36 months post inoculation. This sheep also has a normal posture but behaves differently to the other sheep in the pen and circles in clockwise direction. It does not blink when the menace response is tested. Scratching does not elicit any response, and no fleece changes are apparent, but the body condition is only fair. It circles clockwise when blindfolded (Figure 2) and loses balance. The gait is ataxic, and a fine head tremor is visible.
Atypical BSE sheep presenting with the pruritic form of TSEs in sheep, which is clinically similar to the clinical presentation of classical scrapie-affected sheep. For this reason, a classical scrapie case is not included. Poll Dorset wether from a scrapie-free flock intracerebrally inoculated with bovine atypical (L-type) BSE brain homogenate and examined at 50 months post inoculation. This sheep stands slightly hunched, tends to keep the hind limbs wide-based and falls on the floor when caught. The menace response is weak. Scratching of various areas of the back elicits head or lip movements (Figure 3), and there is wool loss with minor skin trauma on the poll. The body condition is good. When blindfolded, it is reluctant to move and rests its rear end on the hurdle to maintain balance. The sheep is ataxic and displays fore limb hypermetria.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51101/51101fig1highres.jpg" src="/files/ftp_upload/51101/51101fig1.jpg" /> 
Figure 1.&#160;Menace response testing. Menacing gesture towards the animal&#8217;s eye induces a blink response. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51101/51101fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51101/51101fig2highres.jpg" src="/files/ftp_upload/51101/51101fig2.jpg" /> 
Figure 2.&#160;Blindfolding. Clockwise circling elicited by blindfolding the sheep with a hood. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51101/51101fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51101/51101fig3highres.jpg" src="/files/ftp_upload/51101/51101fig3.jpg" /> 
Figure 3.&#160;Testing of the scratch response. Scratching of the back elicits lip licking with sideward movements of the head <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51101/51101fig3highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep at JoVE.com

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

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 ...

clinical-examination-protocol-to-detect-atypical-classical-scrapie

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.9K Views.  Animal Health and Veterinary Laboratories Agency Weybridge. A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

Video: Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

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 ...

Immunology and Infection

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.

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 ...

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 ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, there is a need to validate additional diagnostic techniques to improve rabies surveillance, particularly in developing countries. Here, we present a standard protocol for the DRIT as an alternative, laboratory or field-based testing option that uses light microscopy as compared to the DFA. Touch impressions of brain tissue collected from suspect animals are fixed in 10% buffered formalin. The DRIT uses rabies virus-specific monoclonal or polyclonal antibodies (conjugated to biotin), a streptavidin-peroxidase enzyme, and a chromogen reporter (such as acetyl 3-amino-9-ethylcarbazole) to detect viral inclusions within infected tissue. In approximately 1 h, a brain tissue sample can be tested and interpreted by the DRIT. Evaluation of suspect animal brains tested from a variety of species in North America, Asia, Africa, and Europe have illustrated high sensitivity and specificity by the DRIT approaching 100% with results compared to DFA. Since 2005, the United States Department of Agriculture&#8217;s Wildlife Services (USDA WS) program has conducted large-scale enhanced rabies surveillance efforts using the DRIT to test &#62;94,000 samples collected from wildlife in strategic rabies management areas. The DRIT provides a powerful, economical tool for rabies diagnosis that can be used by laboratorians and field biologists to improve current rabies surveillance, prevention and control programs globally.

The direct rapid immunohistochemical test (DRIT) offers a World Organization for Animal Health and World Health Organization (OIE/WHO) recognized alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis. This test allows for field-based applications that can be performed in approximately 1 h upon brain impressions using light microscopy.

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, ...

Rabies Necropsy Techniques in Large and Small Animals

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the entire state, with the exception of New York City. The Rabies laboratory necropsies a variety of animals ranging in size from bats to bovids. Most of these specimens are animals exhibiting neurological signs, however, less than 10% actually test positive for rabies; implying trauma, lesions or other infectious agents as the cause of these symptoms. Due to the risk of aerosolizing undiagnosed infectious agents, the Rabies Laboratory does not use power tools or saws. Three necropsy techniques will be presented for animals whose skulls are impenetrable with scissors. The laboratory has implemented these techniques to decrease potential exposure to infectious agents, eliminate unnecessary manipulation of the specimen and reduce processing time. The advantages of a preferred technique opposed to another is subject to the trained individual processing the specimen.

The goal of this protocol is to demonstrate safe necropsy techniques in small and large animals to obtain satisfactory tissue samples for rabies testing.

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the ...

Neuroscience

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.

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform ...

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

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Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

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

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

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

Rabies Necropsy Techniques in Large and Small Animals

Prion Safety Laboratory Swipe Test

Detection of Abnormal Prion Protein by Immunohistochemistry

Modeling Multiple Sclerosis in the Two Sexes: MOG_35-55-Induced Experimental Autoimmune Encephalomyelitis