Name: rabies necropsy techniques in large and small animals
Uploaded: 2019-07-30T13:00:00
Description: Watch this Scientific Journal Video about Rabies Necropsy Techniques in Large and Small Animals at JoVE.com

We are grateful to&#160;the New York State Department of Health Wadsworth Center for supporting this project. We also would like to acknowledge the support from&#160;Amy Willsey and&#160;Frank Blaisdell&#160;of Department of Health Wadsworth Center, and&#160;LL Ranch, Altamont, NY.&#160;

All methods described were approved by the Wadsworth Center Institutional Animal Care and Use Committee (IACUC).
1. Preparation
<ol>
	<li>Don PPE, at minimum eye protection (glasses or face shield), surgical or N-95 mask, and non-latex gloves.</li>
	<li>Prepare work area, ideally a bio-safety cabinet (BSC), with a disposable work surface covering (e.g., kraft paper or absorbent pads) and clean necropsy instruments (Figure 1).</li>
	<li>Place the specimen on the ...

<ol>
	<li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus+activity+-+United+States,+2009.">West Nile virus activity - United States, 2009.</a> MMWR Morbidity and Mortality Weekly Report. 59 (25), 769-772 (2010).</li><li>McDaniel, C. J., Cardwell, D. M., Moeller, R. B., Gray, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humans+and+cattle:+A+review+of+bovine+zoonoses.">Humans and cattle: A review of bovine zoonoses.</a> Vector Borne and Zoonotic Diseases. 14 (1), 1-19 (2014).</li><li>Zoonotic diseases. Merck Veterinary Manual Available from: <a target="_blank" href="https://www.merckvetmanual.com/public-health/zoonoses/zoonotic-diseases">https://www.merckvetmanual.com/public-health/zoonoses/zoonotic-diseases</a> (2019)</li><li>Wenner, L., Pauli, U., Summermatter, K., Gantenbein, H., Vidondo, B., Posthaus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerosol+generation+during+bone-sawing+procedures+in+veterinary+autopsies.">Aerosol generation during bone-sawing procedures in veterinary autopsies.</a> Veterinary Pathology. 54 (3), 425-436 (2017).</li><li>Green, F. H. Y., Yoshida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+aerosols+generated+during+autopsy+procedures+and+their+potential+role+as+carriers+of+infectious+agents.">Characteristics of aerosols generated during autopsy procedures and their potential role as carriers of infectious agents.</a> Applied Occupational and Environmental Hygiene. 5 (12), 853-858 (1990).</li><li>Barrat, J., 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=Simple+technique+for+the+collection+and+shipment+of+brain+specimens+for+rabies+diagnosis.">Simple technique for the collection and shipment of brain specimens for rabies diagnosis.</a> Laboratory techniques in Rabies 4th Edition. , 425-427 (1996).</li><li>Ness, S. L., Bain, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+perform+an+equine+field+necropsy.">How to perform an equine field necropsy.</a> American Association of Equine Practitioners. 55, 313-316 (2009).</li><li>. Sample requirements for TSE testing and confirmation &#8211; EURL guidance Available from: <a target="_blank" href="https://science.vla.gov.uk/tse-lab-net/documents/tse-oie-rl-samp.pdf">https://science.vla.gov.uk/tse-lab-net/documents/tse-oie-rl-samp.pdf</a> (2019)</li><li>. Rabies reports Available from: <a target="_blank" href="https://www.wadsworth.org/programs/id/rabies/reports">https://www.wadsworth.org/programs/id/rabies/reports</a> (2019)</li><li>. Protocol for postmortem diagnosis of rabies in animals by direct fluorescent antibody testing: A minimum standard for rabies diagnosis in the United States Available from: <a target="_blank" href="https://www.cdc.gov/rabies/pdf/rabiesdfaspv2.pdf">https://www.cdc.gov/rabies/pdf/rabiesdfaspv2.pdf</a> (2019)</li><li>Miller, L. D., Davis, A. J., Jenny, A. L., Fekadu, M., Whitfield, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveillance+for+lesions+of+bovine+spongiform+encephalopathy+in+U.S.+cattle.">Surveillance for lesions of bovine spongiform encephalopathy in U.S. cattle.</a> Developments in Biological Standardizations. 80, 119-121 (1993).</li><li>Andrews, C., Gerdin, J., Patterson, J., Buckles, E. L., Fitzgerald, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eastern+equine+encephalitis+in+puppies+in+Michigan+and+New+York+states.">Eastern equine encephalitis in puppies in Michigan and New York states.</a> Journal of Veterinary Diagnostic Investigation. 30 (4), 633-636 (2018).</li><li>Appler, K., Brunt, S., Jarvis, J. A., Davis, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+indeterminate+results+on+the+rabies+direct+fluorescent+antibody+test+using+real-time+reverse+transcriptase+polymerase+chain+reaction.">Clarifying indeterminate results on the rabies direct fluorescent antibody test using real-time reverse transcriptase polymerase chain reaction.</a> Public Health Reports. 134 (1), 57-62 (2019).</li><li>Rupprecht, C. E., Fooks, A. R., Abela-Ridder, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+7.+Brain+removal.">Chapter 7. Brain removal.</a> Laboratory techniques in Rabies 5th Edition. , 67-72 (2018).</li></ol>

Specimens submitted for rabies necropsy often have a history of clinical signs compatible with a neurological illness. The presence of clinical illness may be associated with a variety of disorders, including zoonotic diseases, increasing the risk to staff of a laboratory acquired infection. To reduce these risks, techniques have been implemented that decrease the handling and manipulation of specimens.
The methods demonstrated represent a necropsy event to remove desired tissues from a single...

Working on the necropsy floor of a rabies laboratory is inherently dangerous. At times, specimens arrive with embedded porcupine quills, foreign objects including arrows/bullets/pellets or exposed bone shards that may penetrate the protective shipping wrap. Improper packaging can result in leakage, endangering individuals who unpack specimens. In addition to physical injury, necropsy technicians risk exposure to unknown zoonotic infectious agents from the CNS and body fluids of the specimens. Additionally, ectoparasites carried by the specimen may transmit other zoonotic diseases, as fleas and ticks are commonly seen on submitted animals. Depending on geographic location and species involved the diseases exposed vary. Arboviruses such as Eastern equine encephalitis virus (EEEV) or West Nile virus (WNV), tick-borne diseases including Lyme disease or tularemia, bacteria causing Q fever or tuberculosis, and infectious prions name a small number of the possible dangers1,2,3.
The purpose of these methods is to demonstrate safe and efficient necropsy techniques using instruments that minimize the potential for aerosolization unlike power tools or saws4,5. Commonly, the necropsy of small animals in the rabies laboratory requires cutting away the cranial muscles and using a hammer and chisel to open the caudal dorsal portion of the calvarium6. Removing this area of calvarium exposes the hind brain, including the entire cerebellum and cranial brain stem. Modified necropsy techniques may be performed on the ventral part of the skull, avoiding the large cranial muscles and thicker regions of the skull. However, these modified necropsy techniques are only possible when the specimen is without cervical vertebrae.
Similarly, brain tissue in large animals can be removed by separating the cranial muscles and opening the caudal dorsal portion of the skull7. Considerable effort is required to expose the cerebellum and brain stem as the skulls of larger animals are generally thicker. To avoid penetrating the skull, the head of a large animal is positioned so the ventro-caudal portion of the skull is facing the technician. Using modified instruments, the cerebellum and brain stem are removed through the foramen magnum. This is similar to the sample acquisition method recommended by the TSE European Union Reference Laboratory for Transmissible Spongiform Encephalopathy (TSE) investigations8. Cranial vertebrae should be removed beforehand to provide access to the foramen magnum.
Application of these techniques are beneficial to suitably trained technicians in rabies laboratories. As the rabies laboratory receives samples of various sizes, from juvenile bats to adult draft horses9, the technician has several methods to choose from based on the individual circumstance. The method demonstrated for a large animal is also appropriate for veterinarians who perform necropsies in the field, since shipping an entire large animal head for rabies testing is cumbersome and costly. Implementing any of these techniques will improve safety by decreasing the potential of aerosol production, reduce the handling of the specimen and save processing time. However, as the field does not have the same advantages as a laboratory set up specific for rabies testing, it is essential that any modifications made to these procedures focus on safety, especially the use of personal protective equipment (PPE).

<table>
	<tbody>
		<tr>
			<td>Chemistry spoon</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved, sharp-blunt mayo scissors</td>
			<td>Sklar</td>
			<td>14-2055</td>
			<td>Sklar Operating Scissors 5-1/2 Inch Premium OR Grade Stainless Steel Finger Ring Handle Curved Sharp/Blunt</td>
		</tr>
		<tr>
			<td>Large sharp restaurant-quality carving knife</td>
			<td>Dexter</td>
			<td>P94848</td>
			<td>8&#34; Scalloped Utility Knife, white handle</td>
		</tr>
		<tr>
			<td>Locking tumor-tenacula</td>
			<td>Diamond Scientific and Surgicals</td>
			<td>N/A</td>
			<td>Czerny Tenaculum Forcep</td>
		</tr>
		<tr>
			<td>Modified stiletto knife (6.5 inch long blade carving knife ground to 0.5 inch wide)</td>
			<td>Dexter</td>
			<td>P94848</td>
			<td>Modified 8&#34; Scalloped Utility Knife, white handle</td>
		</tr>
		<tr>
			<td>Orthopedic mallet-hammer</td>
			<td>Mortech</td>
			<td>N/A</td>
			<td>Postmortem hammer with hook</td>
		</tr>
		<tr>
			<td>Sharp councilman orthopedic bone chisel</td>
			<td>Shandon</td>
			<td>60-5</td>
			<td>Councilman&#39;s Chisel Blade: 2 in x 2.25 in standard 7 in</td>
		</tr>
		<tr>
			<td>Sharpened tablespoon or other long handled spoon</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Smooth-tipped tissue dressing forceps without teeth</td>
			<td>Shandon</td>
			<td>63-03</td>
			<td>Shandon Broad Point Dressing Thumb Forceps</td>
		</tr>
		<tr>
			<td>Powder-free non-latex gloves</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Safety glasses, goggles, or faceshield</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery or N-95 mask</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kraft paper, butcher paper, absorbent pad, etc</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

All terrestrial samples submitted with skulls between January 31, 2019 and February 28, 2019 had information regarding the presence of a neck and the method of necropsy collected. During that time, 170 heads were necropsied with 18 species represented. 52% (89/170) were properly decapitated. The remaining had at least one vertebra attached including three whole body specimens. The ventral method was used 75% (128/170) of the time, of those, necks were present on 49. Specimens submitted wi...

Watch this Scientific Journal Video about Rabies Necropsy Techniques in Large and Small Animals at JoVE.com

Rabies Necropsy Techniques in Large and Small Animals

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 ...

Tissue collection for rabies testing has both obvious and unapparent dangers. The technician must use the safest methods available and wear proper personal protective equipment. These protocols provide safe necropsy techniques without using power tools or saws, minimizing the production of aerosols.The large animal protocol can also be performed in the field. Using the large animal sample retrieval method in the field eliminates the challenge of transporting an animal head weighing upwards of 45 kilograms to the laboratory. The large animal method is similar to the European protocol for transmissible spongiform encephalopathy sample retrieval and can be used for any investigation using the brainstem and cerebellum.To retrieve the cerebellum and brainstem by the ventral method, position the specimen ventral side up with the nose directed distally toward the back of the biosafety cabinet. Holding an orthopedic mallet in the dominant hand and a councilman chisel in the other hand, position the chisel at a 45 degree angle with the corner point to the chisel in the V shaped opening between the right side of the temporal and occipital bones. Strike the top of the chisel with the mallet until the two bones separate.And make a cut adjacent to the basisphenoid bone. Separate the temporal and occipital bones on the left side of the specimen as just demonstrated. And use the chisel to bend the V region of the skull downward to expose the entire rhombencephalon area of the brain.Then, use scissors and forceps to scoop out the brainstem and cerebellum, removing any remaining tissue pieces from the skull as necessary. If the specimen does not permit ventral retrieval position the sample dorsally, with the nose directed distally toward the back of the biosafety cabinet. And use a tumor tenacula to grasp the left temporal muscle with the teeth of the tenacula.Squeeze the handle to lock the teeth around the tissue and use a sharp carving knife to cut the temporal muscle down to the bone. Rotate the specimen 180 degrees with the tenacula and the knife and repeat the incision on the opposite temporal muscle to expose the skull. Return the head to its original orientation and place the chisel at a 45 degree angle with the corner point to the chisel on the center of the skull at the juncture of the parietal and intraparietal bones.And strike the top of the chisel with the mallet until a horizontal opening is made on the top half of the skull at the parietal bone. Rotate the specimen 180 degrees and make an opening on the opposite side of the skull as just demonstrated. Position the head as needed to insert the point of the chisel into the end of the first cut at a 90 degree angle to the horizontal opening and strike the chisel until the opening reaches the occipital bone.Roll the specimen to make an opening on the opposite occipital bone in the same manner. The openings in the skull should resemble an upside down U.Insert the teeth of the tenacula into the skull at the bottom of the U and pull the bone backward to expose the caudal end of the cerebrum and the cerebellum. Then use scissors as a scoop to pry out the entire cerebellum from within the cavity.And use tissue forceps to tease out the brainstem from the foramen. For large animal cerebellum and brainstem retrieval, position the specimen so that the dorsal part of the skull is in contact with the necropsy surface. With the caudal part of the skull and foramen magnum facing the front of the biosafety cabinet.Grasp the brainstem with forceps. Insert a modified stiletto knife into the foramen magnum between the spinal cord and the spinal meninges as far as possible and scour around the spinal cord to separate the cerebellum and the brainstem from the spinal meninges. Then, pull the stem out of the foramen with forceps.When the knife has been inserted through the foramen magnum, gently angle the blade to follow along the skull as much as possible and insert a metal spatula or knife with a long enough handle to reach under the brain into the space between the neural tissue and the spinal meninges. Probe around the cerebellum to ensure that connections to the spinal meninges have been severed. When the meninges have been completely severed advance the spatula rostrally and dorsally to scoop up the cerebellum.Then scoop out the cerebellum with the spatula. All terrestrial samples submitted with skulls between January 31st, 2019 and February 28th, 2019 had information regarding the presence of a neck and the method of necropsy collected. During that time, 170 heads were necropsied with 18 species represented.52%were properly decapitated. The remaining had at least one vertebrae attached including three whole body specimens. The ventral method was used a 75%of the time.Of those, necks were present on 49. Three large animals were submitted and in two cases, the large animal protocol was used. One squirrel was submitted with a crushed skull and simply cutting away the skin exposed the brain tissue so none of the demonstrated methods were used.The tissues used to make microscopy slides and or extracted from molecular methods when processing multiple animals avoid cross contamination by using new gloves instruments and a clean work area. Be cautious when performing these protocols as the specimens may be infected with rabies virus or other zoonotic diseases. Note that sharp objects may also be present within the specimens.

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