We acknowledge all USDA Wildlife Services staff who currently or have previously collected enhanced rabies surveillance samples and have conducted the DRIT for rabies diagnosis. Likewise, we acknowledge the many cooperators who assist us with Enhanced Rabies Surveillance collection. We also thank the Centers for Disease Control and Prevention and The Wistar Institute for access to critical reagents required to conduct the DRIT and for providing training opportunities. In addition, we appreciate the confirmatory diagnostics and technical assistance provided by the Centers for Disease Control and Prevention and by the Wadsworth Center with the New York State Department of Health. Use of any commercial products is for comparison purposes only and does not constitute endorsement.

Each person conducting rabies diagnostic testing should receive a standard pre-exposure rabies vaccination series and undergo regular serological antibody evaluation, with booster immunizations as needed. Unimmunized individuals should not enter laboratories or areas where such work is conducted. All manipulation of tissues and slides should be conducted to not aerosolize liquids or produce airborne particles. Fume hoods are not required but when feasible they can provide extra protection from odors, ectoparasites, and bone fragments. Minimum personal protective equipment, including gloves and eye protection, should be worn at all times during sample collection and testing.
1. Brainstem Collection
<ol>
	<li>Collect brainstem samples either immediately upon carcass collection or ensure that carcasses are placed in freezers for storage until later testing. If animal carcasses are frozen, thaw at ambient temperature prior to brainstem collection for ease of collection. Under some circumstances, samples are collected directly from a frozen animal.</li>
	<li>For animals that are freshly collected or have been thawed to ambient temperature, position the animal supine on a flat surface with the cervical spine slightly rotated towards the examiner. Palpate to identify the atlanto-occipital joint on the lateral aspect of the cervical spine.
	<ol>
		<li>For carcasses that are still completely frozen, position the animal supine as described above, if practical. Use saws, knives, bone shears or similar equipment to completely separate the head from the body. 
		NOTE: All equipment used that is not one-time use should be cleaned thoroughly between samples.</li>
	</ol>
	</li>
	<li>Using a scalpel blade, make an incision at the level of the atlanto-occipital joint at the ventral aspect, cutting through all layers of muscle and soft tissue, including the trachea and esophagus. Continue until approximating anterior aspect of cervical spine vertebrae. For frozen tissue, use a scalpel blade to remove any remaining layers of muscle and soft tissue to expose the foramen magnum.</li>
	<li>Move the head of the animal into end-range extension until the brainstem is visible. Use a scalpel blade to remove all visible brainstem/central nervous system (CNS) tissue for sampling. For a frozen sample, use a scalpel blade to remove as much of the frozen brainstem/CNS tissue as possible from inside the skull.</li>
	<li>Place the brainstem samples in an unbreakable container (i.e., metal ointment tin, cryo-vial, etc.) and label accordingly.</li>
	<li>Ensure that the brainstem samples are tested immediately after collection via the DRIT test, refrigerated (4 &#176;C) for up to 24 h before testing, or frozen (-20 &#176;C) until time of testing if testing will take place more than 24 h after sample collection.</li>
</ol>
2. Preparation of Materials for DRIT
<ol>
	<li>Setting up of 10 slide staining dishes (Figure 1) 
	NOTE: Staining dishes are deep enough to allow for complete immersion of slide (diameter 96 mm, height 72 mm, depth 42 mm, volume 250 mL).6
	<ol>
		<li>Fill dish 1 with 10% phosphate buffered formalin. Replace formalin after 2 test runs or weekly.</li>
		<li>Fill dishes 2, 4, and 5 with phosphate buffered saline with 1% tween-80 (TPBS). Replace with fresh TPBS before each test.</li>
		<li>Fill dish 3 with 3% hydrogen peroxide. Replace hydrogen peroxide before each test.</li>
		<li>Fill dishes 6, 8, 9, and 10 with distilled or deionized water. Replace water before each test.</li>
		<li>Fill dish 7 with Gills hematoxylin formulation #2 diluted in a 1:1 ratio in distilled water6. Replace hematoxylin after 2 test runs or weekly. 
		NOTE: According to the original DRIT protocol developed by the CDC12, a 1:2 ratio of Gills hematoxylin to water may be used if counterstaining is too dark.</li>
	</ol>
	</li>
	<li>Preparation of amino-ethylcarbazole (AEC) stock solution
	<ol>
		<li>Using a glass pipette, place 5 mL of N,N-dimethylformamide in a glass container.</li>
		<li>Add one 20 mg tablet of 3-amino-9-ethylcarbazole and shake until completely dissolved. Label the jar with &#8220;AEC stock&#8221; and the date the stock was made. 
		NOTE: The AEC stock solution can be stored under refrigeration (4 &#176;C) and used for 1-2 months.</li>
	</ol>
	</li>
	<li>Preparation of AEC working dilution
	<ol>
		<li>Add 7 mL of acetate buffer to a 15 mL centrifuge tube.</li>
		<li>Using a glass pipette, add 0.5 mL of the AEC stock solution to the centrifuge tube.</li>
		<li>Add 0.075 mL of 3% hydrogen peroxide to the tube.</li>
		<li>Filter the solution with a 10 mL syringe using a 0.45 &#181;m syringe filter. 
		NOTE: The AEC working dilution should be created just prior to each DRIT test as it is only stable for 2-3 h.</li>
	</ol>
	</li>
</ol>
3. Direct Rapid Immunohistochemistry Test
<ol>
	<li>Label glass microscope slides with a unique number for each specimen using a smear-proof, waterproof, permanent ink marker.
	<ol>
		<li>Using a scalpel blade, remove the brainstem from the container and place on paper towel. Gently blot away any excess of fluid, blood, or fur with a second paper towel to reveal just the brainstem tissue13. If needed, section the brainstem tissue to reveal a cross section.</li>
		<li>Very gently touch the microscope slide to the cross section of the brainstem tissue. Touch the slide to the brainstem at several points without lateral movement to allow multiple areas of brainstem to be transferred to slide13. Ensure that only 1 or 2 layers of cells are transferred from the brain tissue to the slide with a gentle touch. No mounting agent is needed to affix the brainstem tissue to the slides. Include both a positive and negative control in each DRIT run.</li>
	</ol>
	</li>
	<li>Let the slides air-dry for approximately 5 min at room temperature.</li>
	<li>Immerse the slides in 10% buffered formalin for 10 min (dish 1).</li>
	<li>Remove the slides from formalin and dip-rinse in a solution of TPBS (dish 2).</li>
	<li>Immerse the slides in 3% hydrogen peroxide for 10 min (dish 3).</li>
	<li>Remove the slides from the hydrogen peroxide and dip-rinse in fresh TPBS (dish 4). After removing excess hydrogen peroxide, place the slides in fresh TPBS (dish 5) and work with one slide at a time while the other slides stay immersed in TPBS.</li>
	<li>Take the slides from TPBS (dish 5) one at a time, shake off and blot excess buffer, and place on a moistened paper towel on lab bench, as the basis of a &#8216;humidity chamber&#8217;. Using a pipette, drop enough primary anti-rabies virus antibody on each slide to cover the CNS tissue. Incubate the slides for 10 min in the humidity chamber (i.e., covering slides with well plates or other simple cover while they are laying on the moistened paper towel) at room temperature (Figure 2).</li>
	<li>Remove the slides from the humidity chamber, shake and blot off excess conjugate, and dip-rinse the slides in TPBS (reuse the same TPBS in dish 5).</li>
	<li>Working with one slide at a time, while others stay immersed in TPBS &#8212; use a pipette to drop enough streptavidin-peroxidase complex to cover the CNS tissue. Incubate in the humidity chamber for 10 min at room temperature.</li>
	<li>Remove the slides from the humidity chamber, shake and blot off excess complex, and dip-rinse the slides in TPBS (dish 5).</li>
	<li>Working with one slide at a time, shake off and blot excess buffer, while others stay immersed in TPBS &#8212; use a pipette to drop enough AEC (preparation is explained above and should be done immediately prior to use) to cover the CNS tissue. Incubate in the humidity chamber for 10 min at room temperature.</li>
	<li>Dip-rinse the slides in distilled water (dish 6).</li>
	<li>Place the slides in counterstain of Gills Hematoxylin (diluted 1:1 with distilled water) for 2 min (dish 7).</li>
	<li>Immediately dip-rinse all slides in distilled water (dish 8). Repeat twice with fresh water each time (dishes 9 and 10).</li>
	<li>Working with one slide at a time, while others stay immersed in distilled water (dish 10) &#8212; shake and blot off excess water and use a water-soluble mounting medium to affix a cover slip.</li>
	<li>Use a light microscope with a 20x objective to view the slides and a 40x objective if closer inspection is needed.</li>
</ol>

<ol>
	<li>Dean, D. J., Abelseth, M. K., Atanasiu, P., Meslin, F. X., Kaplan, M. M., Koprowski, 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> Laboratory techniques in rabies. 23, (1996).</li><li>Durr, S., 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=Rabies+diagnosis+for+developing+countries.">Rabies diagnosis for developing countries.</a> PLOS Neglected Tropical Diseases. 2 (3), e206 (2008).</li><li>Rupprecht, 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=.">.</a> Progress in the development of a direct rapid immunohistochemical test for diagnosing rabies. , (2014).</li><li>Rupprecht, C. 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=Additional+Progress+in+the+Development+and+Application+of+a+Direct,+Rapid+Immunohistochemical+Test+for+Rabies+Diagnosis.">Additional Progress in the Development and Application of a Direct, Rapid Immunohistochemical Test for Rabies Diagnosis.</a> Journal of Veterinary Science. 5 (2), (2018).</li><li>Coleman, P. G., Fevre, E. M., Cleaveland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+public+health+impact+of+rabies.">Estimating the public health impact of rabies.</a> Emerging Infectious Diseases. 10 (1), 140-142 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard Operating Procedure for the Direct Rapid Immunohistochemistry Test (DRIT) for the detection of rabies virus antigens. , (2016).</li><li>Lembo, 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=Evaluation+of+a+direct,+rapid+immunohistochemical+test+for+rabies+diagnosis.">Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis.</a> Emerging Infectious Diseases. 12 (2), 310-313 (2006).</li><li>Middel, K., Fehlner-Gardiner, C., Pulham, N., Buchanan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporating+Direct+Rapid+Immunohistochemical+Testing+into+Large-Scale+Wildlife+Rabies+Surveillance.">Incorporating Direct Rapid Immunohistochemical Testing into Large-Scale Wildlife Rabies Surveillance.</a> Tropical Medicine and Infectious Disease. 2 (3), 21 (2017).</li><li>Coetzer, A., Sabeta, C. T., Markotter, W., Rupprecht, C. E., Nel, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+biotinylated+monoclonal+and+polyclonal+antibodies+in+an+evaluation+of+a+direct+rapid+immunohistochemical+test+for+the+routine+diagnosis+of+rabies+in+southern+Africa.">Comparison of biotinylated monoclonal and polyclonal antibodies in an evaluation of a direct rapid immunohistochemical test for the routine diagnosis of rabies in southern Africa.</a> PLOS Neglected Tropical Diseases. 8 (9), e3189 (2014).</li><li>Kirby, 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=Enhanced+Rabies+Surveillance+to+Support+Effective+Oral+Rabies+Vaccination+of+Raccoons+in+the+Eastern+United+States.">Enhanced Rabies Surveillance to Support Effective Oral Rabies Vaccination of Raccoons in the Eastern United States.</a> Tropical Medicine and Infectious Disease. 2 (3), 34 (2017).</li><li>Slate, D., 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=Oral+rabies+vaccination+in+north+america:+opportunities,+complexities,+and+challenges.">Oral rabies vaccination in north america: opportunities, complexities, and challenges.</a> PLOS Neglected Tropical Diseases. 3 (12), e549 (2009).</li><li>. Direct Rapid Immunohistochemistry Test (DRIT) protocols Available from: <a target="_blank" href="https://rabiessurveillanceblueprint.org/Direct-Rapid-Immunohistochemistry?lang=fr">https://rabiessurveillanceblueprint.org/Direct-Rapid-Immunohistochemistry?lang=fr</a> (2019)</li><li>Khalid, A., Haque, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Touch Impression Cytology Versus Frozen Section as Intraoperative Consultation Diagnosis. 2, (2004).</li></ol>

The authors have nothing to disclose.

The DRIT is a flexible method suited for field-based surveillance for detecting the presence of rabies virus that can be used in decentralized laboratory areas. While it is possible to conduct the entire test in a field-based setting such as on the tailgate of a truck, it is ideal to have a small, indoor space dedicated for DRIT due to chemical, equipment and supply storage issues. Additionally, adherence to all applicable federal, state, and local laws, and regulations for chemical use and disposal must be considered. Currently, USDA WS has 15 DRIT facilities to test samples from 17 states. The USDA WS DRIT facilities are established in conjunction with university and state public health laboratories, in designated rooms within larger facilities and in enclosed trailers that have been retrofitted and converted to serve as mobile testing units in the event of an emergency outbreak response where enhanced rabies surveillance testing with immediate turnaround time is critical (Figure 5).
While the test has been successful using brainstem material of variable quality, fresh brainstem tissue with no tissue decomposition is optimal. As decomposition, desiccation, or liquefaction occurs, sample quality decreases and the test may detect more nonspecific staining which can confuse results. This observation is similar between DFA and DRIT2. Brainstem/CNS should be collected as soon as possible and then stored frozen (-20 &#176;C) until testing.
Typically, most USDA WS facilities process from 12 to 24 slides at once during a DRIT session, including one positive control and one negative control that have been confirmed via DFA. Positive and negative controls provide a reference point for each DRIT run to ensure the test was successful and to confirm if interpretative questions arise on the test slides. If a sample is not determined to have a clear positive or negative outcome, it is labeled as an indeterminate and tested a second time by DRIT. If this sample is not a clear positive or negative after two DRIT tests, it is sent to a reference laboratory for DFA or related testing.
As with any diagnostic test, &#8216;trouble shooting&#8217; is useful with unexpected findings. For example, if a DRIT run is unsuccessful (i.e., the positive control does not exhibit +3 or +4 staining intensity and antigen distribution), ensure all chemicals and reagents have not expired. We have found using a newly opened bottle of hydrogen peroxide at a minimum of once per week is helpful to help prevent non-specific staining through oxidation of the brain tissue. Additionally, we recommend replacing acetate buffer at least once each year at a minimum, regardless of the labeled expiration date.
There are a number of advantages of the DRIT over DFA including lower costs, ability to perform the test outside of a centralized laboratory, need for only light microscopy instead of a fluorescent microscope, and the relatively straightforward training process for people administering and reading the test2,3,4. These advantages, coupled with sensitivity and specificity of the DRIT that are comparable to DFA2,7,8,9 have already proven that the test serves as an important tool in broad scale enhanced rabies surveillance programs8,10 in North America. Additionally, the DRIT has potential to allow for increased surveillance and more prompt testing in developing nations or other areas with limited resources, especially after recent OIE/WHO guidance as a recommended test.

While the DFA is the most widely used test for routine rabies diagnosis1, the cost of purchasing and maintaining a fluorescent microscope can be limiting to developing nations2,3,4 and for broad, large-scale enhanced rabies surveillance programs3,4. In addition, the DFA requires the ability to refrigerate samples during fixation and to incubate samples above ambient temperature during the antibody-antigen reactions, which can be a significant hurdle in countries without suitable infrastructure. Due in part to the limitations associated with modern DFA testing, the global impact of rabies has long been underestimated5.
In this regard, there is a need to validate additional diagnostic techniques to improve rabies surveillance globally, particularly in developing countries. The DRIT protocol presented here offers a laboratory or field-based testing alternative to the DFA that uses light microscopy and does not require refrigeration or laboratory incubation during the test6. The DRIT and DFA are similar in that both techniques use touch impressions of brain samples collected from potentially rabid animals. However, the first step of the DRIT uses formalin as an historical fixative for samples, which inactivates rabies virus. This provides a substantial biosafety improvement over the acetone used to fix samples in the DFA protocol3, which is important not only in the laboratory but perhaps even more so in field-based or decentralized lab environments. To date, the DRIT shows sensitivity and specificity equal to the DFA2,7,8,9.
Since 2005, the USDA WS program has conducted large-scale enhanced rabies surveillance efforts in North America using the DRIT as part of a comprehensive wildlife rabies management program10. Enhanced rabies surveillance is used as a complement to exposure-based public health surveillance and tests primarily wildlife meso-carnivore species, including raccoons (Procyon lotor), striped skunks (Mephitis mephitis), gray foxes (Urocyon cinereoargenteus), red foxes (Vulpes vulpes), and coyotes (Canis latrans) that have not been involved in a human or domestic animal exposure. Animal carcasses used in testing were submitted to USDA WS through enhanced rabies surveillance efforts and comprised rabies vector species that are: ill or strange acting; found dead, road-killed, or a nuisance; and are not associated with a human or domestic animal exposure. The DRIT provides a rabies diagnostic test that can be employed by trained field biologists with little or no laboratory experience to improve rabies surveillance and reduce the financial burden and workload of public health diagnostic laboratories10. The basic DRIT training for USDA WS field employees typically takes 1.5 to 2 days, which includes approximately 4 h of classroom instruction covering fundamental principles of biosafety, methods for sample collection and the DRIT processes as well as &#62;8 h of laboratory time conducting the test. Training opportunities have been available to USDA WS and program cooperators through the Centers for Disease Control and Prevention (CDC), LYSSA LLC, and The Wistar Institute.
Within strategic management areas, USDA WS has tested over 94,000 wildlife samples using the DRIT and has detected more than 1,850 rabid samples that would have likely gone undetected relying only upon exposure based public health testing of suspect animals. Enhanced rabies surveillance data provided by DRIT results are critical to providing a more complete temporal and spatial representation of rabies on the landscape in support of oral rabies vaccination programs for wildlife throughout the USA10,11. Similarly, Provincial wildlife agencies in Canada have incorporated DRIT into large scale wildlife rabies surveillance efforts in tandem with their public health programs with success8. Both USDA WS and Canadian DRIT surveillance programs have used national reference laboratories to confirm rabies by DFA and to perform viral variant typing as appropriate8. Moreover, USDA WS field biologists send 10% of negative specimens for DFA confirmation to reference laboratories and since 2008, have participated in biannual DRIT proficiency testing provided by the Wisconsin State Laboratory of Hygiene, as part of standard quality assurance measures.
The goal of this method is to offer an alternative approach for rabies diagnostic testing that can be done in decentralized laboratories, in the field, or in areas without routine access to electron microscopy.

<table>
	<tbody>
		<tr>
			<td>3-Amino-9-Ethylcarbazole (AEC tablets; 50 count)</td>
			<td>Sigma Aldrich (https://www.sigmaaldrich.com/)</td>
			<td>A6926</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate Buffer, 0.1M, 5.2 pH, 32oz</td>
			<td>Poly Scientific R&#38;D Corp. (https://www.polyrnd.com/)</td>
			<td>s140</td>
			<td></td>
		</tr>
		<tr>
			<td>Ag Tek MiniScalpel, PN110, Non-sterile #10, 40 per package</td>
			<td>Patterson Veterinary (https://www.pattersonvet.com/)</td>
			<td>PN110</td>
			<td>Supplemental equipment for sample touch impressions; Also available through Clipper Distributing (http://www.clipperdist.net/)</td>
		</tr>
		<tr>
			<td>BD Luer-Lok Disposable Syringe without needle, 10cc</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>14-823-2A</td>
			<td>BD Manufacturer Number 309604</td>
		</tr>
		<tr>
			<td>Binocular Light Microscope with Seidentopf Head or equivalent</td>
			<td>Multiple Vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Rectangular UN-rated Disposal Container, 5G</td>
			<td>Berlin Packaging (https://www.berlinpackaging.com/)</td>
			<td>1147T01BLU</td>
			<td>Supplemental equipment for chemical waste storage/disposal (Gill hematoxylin and AEC solution)</td>
		</tr>
		<tr>
			<td>Corning Square and Rectangular Cover Glass, 24x60</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>12-553-465</td>
			<td>Corning Manufacturer Number 2975246</td>
		</tr>
		<tr>
			<td>Corning Universal Fit Pipet Tips: Racked, Nonsterile (1-200ul)</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>07-200-300</td>
			<td>Corning Manufacturer Number 4863</td>
		</tr>
		<tr>
			<td>Falcon 15mL Conical Centrifuge Tubes, polypropylene</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>14-959-70C</td>
			<td>Corning Manufacturer Number 352097</td>
		</tr>
		<tr>
			<td>Falcon 96-Well Assay Plates (Tissue culture plate lids)</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>08-772-5</td>
			<td>Corning Manufacturer Number 353910</td>
		</tr>
		<tr>
			<td>Fisher Brand 25mm Syringe Filter, Nylon, 0.45um, Sterile</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>09-719D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher Chemical Gill Method Hematoxylin Stain (Gill-2), 4L</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>CS401-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Sharps-A-Gator Point-of-Use Sharps Containers, 5qt</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>14-827-122</td>
			<td>Supplemental equipment for proper BSL-2 Laboratory Set-Up</td>
		</tr>
		<tr>
			<td>Fluoro-Gel with Tris Buffer (Gel/Mount Media), 20mL</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>102092-122</td>
			<td>Fluoro-Gel Substitute for BioMeda&#8482; Gel-Mount, Electron Microscopy Sciences</td>
		</tr>
		<tr>
			<td>Formalin, Buffered, 10%, 4L</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>SF100-4</td>
			<td>Available each or in case of 4</td>
		</tr>
		<tr>
			<td>Gilson Pipetman P200 Pipet, 50-200uL</td>
			<td>Daigger Scientific (https://www.daigger.com)</td>
			<td>EF9930E</td>
			<td></td>
		</tr>
		<tr>
			<td>Hy-Clone Phosphate Buffered Saline, 1X Solution, 1L (PBS)</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>SH30256LS</td>
			<td>Alternative dry powder product can be used</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide, 3%</td>
			<td>Multiple Vendors</td>
			<td></td>
			<td>Any commercially available source, such as pharmacy or store brands, etc.</td>
		</tr>
		<tr>
			<td>Lens Microscope Objective 20X and 40X</td>
			<td>Multiple Vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lysol IC Quarternary Disinfecting Cleaner, 1G</td>
			<td>Daigger Scientific (https://www.daigger.com)</td>
			<td>EF8481</td>
			<td>Supplemental materials for proper BSL-2 Laboratory disinfection</td>
		</tr>
		<tr>
			<td>Miltex brand Disposable Scalpel Size 22 (alternative size to MiniScalpel)</td>
			<td>AMD Next (www.amdnext.com)</td>
			<td>999112314</td>
			<td>Supplemental equipment for sample touch impressions; Alternative size to Ag Tek MiniScalpel</td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide, Amber Glass Packaging, 500mL</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>D119-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase Labeled Streptavidin, 50mL</td>
			<td>SeraCare (https://www.seracare.com/)</td>
			<td>5550-0001</td>
			<td>KPL Immunoassay and Kits Reference Number 71-00-38</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline Powder (alternative to Fisher liquid PBS)</td>
			<td>Sigma Aldrich (https://www.sigmaaldrich.com/)</td>
			<td>P3813</td>
			<td>Must be prepared in 1L distilled water; Available in quantities of 1, 10 and 50 packs</td>
		</tr>
		<tr>
			<td>Primary antibody: Polyclonal anti-nucleoprotein or cocktail of anti-lyssavirus biotinylated antibodies</td>
			<td></td>
			<td></td>
			<td>Store at 4 degrees C</td>
		</tr>
		<tr>
			<td>PYREX Disposable Serological Pipets, Glass, Sterile, Plugged, Corning, 1.0mL</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>7078D-1</td>
			<td>VWR Manufacturer Number 89091-220</td>
		</tr>
		<tr>
			<td>PYREX Disposable Serological Pipets, Glass, Sterile, Plugged, Corning, 10.0mL</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>7078D-10</td>
			<td>VWR Manufacturer Number 89091-106</td>
		</tr>
		<tr>
			<td>PYREX Disposable Serological Pipets, Glass, Sterile, Plugged, Corning, 5.0mL</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>7078D-5</td>
			<td>VWR Manufacturer Number 89091-484</td>
		</tr>
		<tr>
			<td>Richard-Allan Scientific Gills Hematoxylin Stain No. 2, 1PT (alternative to above)</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>22-050-201</td>
			<td>Thermo Scientific Manufacturer Number 72504</td>
		</tr>
		<tr>
			<td>Slide Holders, 24-place</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>25608-868</td>
			<td>Sakura&#174;Finetek Supplier Number 4465; Available through multiple vendors</td>
		</tr>
		<tr>
			<td>Specimen Tin Boxes, 1/2oz</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>101412-452</td>
			<td>Supplemental equipment for storage of brain tissue samples</td>
		</tr>
		<tr>
			<td>Taylor 2-Event Digital Timer/Clock</td>
			<td>Multiple Vendors</td>
			<td></td>
			<td>Supplemental equipment</td>
		</tr>
		<tr>
			<td>Tissue-Tek Slide Staining Kit</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>25608-902</td>
			<td>Sakura&#174;Finetek Supplier Number 4551; Available through multiple vendors</td>
		</tr>
		<tr>
			<td>TWEEN 80, Polyethylene glycol, 500mL</td>
			<td>Sigma Aldrich (https://www.sigmaaldrich.com/)</td>
			<td>P1754</td>
			<td>Also available in 25mL, 1L and 1G volumes</td>
		</tr>
		<tr>
			<td>VWR FLIP Pipette Filler (0.05-100mL)</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>53497-055</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Soft Nitrile Examination Gloves, L (100 per box)</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>89038-272</td>
			<td>Supplemental equipment for proper PPE</td>
		</tr>
		<tr>
			<td>Water, Deionized (20L)</td>
			<td>VWR, part of Avantor (https://vwr.com)</td>
			<td>10806-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheaton Clear Glass Sample Vials, 8mL</td>
			<td>Fisher Scientific (part of Thermo Fisher Scientific https://www.fishersci.com/us/en/home.html)</td>
			<td>06-408C</td>
			<td>DWK Life Sciences Manufacturer Number 224884</td>
		</tr>
		<tr>
			<td>White Coated, Double Well Pattern Microscope Slides, 14mm</td>
			<td>Tekdon Incorporated (https://www.tekdon.com/coated-microscope-slides.html)</td>
			<td>2-140</td>
			<td></td>
		</tr>
		<tr>
			<td>White Rectangular UN-rate Disposal Container, 5G</td>
			<td>Berlin Packaging (https://www.berlinpackaging.com/)</td>
			<td>1147T01WHT</td>
			<td>Supplemental equipment for chemical waste storage/disposal (Formalin)</td>
		</tr>
	</tbody>
</table>

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.

Positive results from the DRIT show red intracytoplasmic viral inclusions that can vary in shape and size (Figure 3) within the cytoplasm of bluish cell bodies. The inclusions appear smooth with very bright margins and a less intensively stained central area. Intensity and antigen distribution are recorded when inclusions are detected. Intensity is graded from +4 to +1. The positive control slide should have an intense, glaring magenta brilliance which is referred to as a +4 intensity. A slight loss of color can occur especially when sample handling has not been optimal (i.e., sample tissue has decomposed slightly) and these should be graded as +3. Noticeably dull stain is graded as a +2 to +1, is not considered diagnostic for rabies virus infection and is labeled as indeterminate.
Additionally, antigen distribution is graded from +4 to +1 with +4 representing antigen distribution comprised of an abundance of large and small inclusions varying in size and shape, and present in every field (or almost every field) of view within the CNS tissue touch impression. The positive control typically has a +4 antigen distribution. An antigen distribution of +3 would be assigned when there are inclusions in a variety of sizes in most but not all of the fields of view. If inclusions are found in 10%-50% of the microscope fields, a +2 antigen distribution is assigned. When inclusions are found in &#60;10% of the microscope fields, a +1 antigen distribution is assigned.
Most CNS tissue with rabies virus present exhibit typical viral inclusions graded as +3 or +4 intensity and antigen distribution. If results indicate a +2 or +1 intensity or a +2 or +1 antigen distribution, the sample is declared an &#8220;indeterminate&#8221; and repeat testing is warranted. If the same sample has a repeat indeterminate test result, the sample should be sent to a reference laboratory for DFA or related confirmatory testing.
A test sample using the DRIT is considered negative for rabies virus antigens after the slide containing the CNS tissue has been scanned at a magnification of 200X or greater and no typical virus inclusions are detected (Figure 4). Negative samples exhibit bluish cell bodies with little or no nonspecific staining.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59416/59416fig1.jpg" /> 
Figure 1: Setup of 10 slide staining dishes with reagents for testing. The dishes are labeled with reagent name in the order needed to follow the protocol. <a href="https://www.jove.com/files/ftp_upload/59416/59416fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59416/59416fig2.jpg" /> 
Figure 2: Simple humidity chamber created with a wet paper towel and cell culture plates. &#160;A simple humidity chamber using a wet paper towel and cell culture plates allows for field-based application.&#160; <a href="https://www.jove.com/files/ftp_upload/59416/59416fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59416/59416fig3.jpg" /> 
Figure 3: Representative slides of rabies positive viral inclusions with +4 intensity and +4 antigen distribution.&#160;(A&#160;and B) show positive rabies viral inclusions at 200x magnification. (C and D) show positive rabies viral inclusions at 400x magnification. Scale bars = 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59416/59416fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59416/59416fig4.jpg" /> 
Figure 4: Representative slides of negative samples with no rabies viral inclusions. (A and B) show samples negative for rabies viral inclusions at 200x magnification. (C and D) show samples negative for rabies viral inclusions at 400x magnification. Scale bars = 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59416/59416fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59416/59416fig5.jpg" /> 
Figure 5: Representative photographs of DRIT testing facilities used by USDA WS. (A) Mobile testing facility in an enclosed trailer for transport. (B) Recreational vehicle retrofitted for DRIT testing. (C) DRIT testing facility in conjunction with university laboratory. <a href="https://www.jove.com/files/ftp_upload/59416/59416fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test at JoVE.com

Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

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, ...

enhanced-rabies-surveillance-using-direct-rapid-immunohistochemical

Research

JoVE Journal

Immunology and Infection

8.7K Views.  United States Department of Agriculture (USDA). 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.

Video: Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

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 ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Quantitation of Rabies Virus in Various Bovine Brain Structures

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon&#8217;s horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major ...