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 work surface and use instruments to manipulate it to assess condition of the sample including evidence of decomposition, damage to skull, potential hazards (e.g., porcupine quills, scalpel blades), and the quality of the decapitation.</li>
</ol>
2. Ventral method
NOTE: When the specimen is properly decapitated at the jaw-line, the foramen magnum and the occipital condyle will be exposed. The ventral method is less complicated for retrieving the cerebellum and brain stem.
<ol>
	<li>Position the specimen with ventral side up and nose directing distally toward the back of BSC.</li>
	<li>Hold an orthopedic hammer/mallet in right hand (if right-handed) and at same time hold a councilmen chisel in left hand.</li>
	<li>Position the chisel at a 45&#176; angle with the corner point of the chisel directing between the right side of temporal bone and occipital bone making a &#8220;V&#8221; opening.</li>
	<li>Strike the top of the chisel with the hammer until the two bones separate. Make the cut to adjacent to the basisphenoid bone.</li>
	<li>Repeat on the left side of temporal bone/occipital bone (Figure 2A).</li>
	<li>Bend the &#8220;V&#8221; area of skull downward with the chisel. Expose the entire rhombencephalon area of the brain (cerebellum and brain stem) (Figure 2B).</li>
	<li>Scoop out the brain stem and cerebellum with scissors and forceps. Remove any remaining pieces from the skull if the brain stem and cerebellum did not come out in a single piece.</li>
</ol>
3. Dorsal method
NOTE: If the specimen has a poor decapitation (foramen magnum not visible) and the neck cannot be easily removed during necropsy or if damage to the cerebellum is suspected, the dorsal method should be utilized.
<ol>
	<li>Position the specimen dorsally with the nose directing distally toward the back of BSC.</li>
	<li>Using tumor tenacula, grasp the left temporal muscle with teeth of tenacula and lock by squeezing the handle.</li>
	<li>Cut the temporal muscle down to bone with sharp carving knife.</li>
	<li>Rotate the specimen 180&#176; with tenacula and knife (not hand) and repeat the process on the opposite temporal muscle. Expose the skull.</li>
	<li>Position a chisel at a 45&#176; angle with the corner point of the chisel on the center of the skull at the juncture of the parietal and intraparietal bone.</li>
	<li>Strike the top of the chisel with a hammer until a horizontal opening is made on the top half of the skull at parietal bone.</li>
	<li>Rotate the specimen 180&#176; and repeat the process on the opposite side.</li>
	<li>Insert the point of the chisel into the end of cut 1 (Figure 3A) and at 90&#176; of horizontal opening. Strike with the hammer until the opening reaches occipital bone (approximately 10 cm depending upon size of the specimen).</li>
	<li>Roll the specimen and repeat on the opposite side at the end of cut 2. 
	NOTE: With specimen dorsal and nose positioned toward the back of BSC, the openings in the skull resemble an upside-down &#8220;U&#8221;.</li>
	<li>Insert the teeth of the tenacula into the skull at the bottom of the &#8220;U&#8221; and pry towards oneself. Expose the caudal end of the cerebrum and the cerebellum (Figure 3B).</li>
	<li>Use scissors as a scoop and pry out entire cerebellum from within the cavity.</li>
	<li>Use tissue forceps to tease out the brain stem from the foramen.</li>
</ol>
4. Large animal method
<ol>
	<li>Position the specimen so that the dorsal part of the skull is in contact with the necropsy surface with the caudal portion of the skull and foramen magnum facing the technician.</li>
	<li>Insert the modified stiletto knife into the foramen magnum in between the spinal cord and spinal meninges as far as possible.</li>
	<li>Score around the spinal cord to separate the cerebellum and brain stem from the spinal meninges. After the knife is inserted through the foramen magnum, gently angle the knife to follow along the skull as much as possible.</li>
	<li>Insert a chemistry spatula or thin, long handled spoon into the space between the neural tissue and spinal meninges.</li>
	<li>Probe around the spinal cord and cerebellum to ensure the connection to the spinal meninges have been severed.</li>
	<li>Hold the brain stem with forceps. With the other hand, advance the spoon rostrally then dorsally to scoop up the cerebellum. Simultaneously pull back on the brain stem with the forceps and scoop out the cerebellum using the spoon. 
	NOTE: It may take more than one attempt to recover adequate cerebellum for rabies testing.</li>
</ol>
5. Post necropsy
<ol>
	<li>Dispose of all disposable materials (gloves, pads, work area coverings) and unused tissues in biohazardous waste.</li>
	<li>Clean and disinfect all instruments with method available (e.g., industrial dishwasher, autoclave, chemical disinfectant, boiling).</li>
	<li>Clean and disinfect all work surfaces with 20% bleach and/or 70% ethanol.</li>
</ol>

<ol><li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((West+Nile+virus+activity+-+United+States%2C+2009%5BTitle%5D)+AND+%22MMWR+Morbidity+and+Mortality+Weekly+Report%22%5BJournal%5D)">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. Jr, Gray, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Humans+and+cattle%3A+A+review+of+bovine+zoonoses%5BTitle%5D)+AND+%22Vector+Borne+and+Zoonotic+Diseases%22%5BJournal%5D)">Humans and cattle: A review of bovine zoonoses.</a> Vector Borne and Zoonotic Diseases. 14 (1), 1-19 (2014).</li>
<li>Spickler, A. R. 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/pubmed?term=((Aerosol+generation+during+bone-sawing+procedures+in+veterinary+autopsies%5BTitle%5D)+AND+%22Veterinary+Pathology%22%5BJournal%5D)">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/pubmed?term=((Characteristics+of+aerosols+generated+during+autopsy+procedures+and+their+potential+role+as+carriers+of+infectious+agents%5BTitle%5D)+AND+%22Applied+Occupational+and+Environmental+Hygiene%22%5BJournal%5D)">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. Simple technique for the collection and shipment of brain specimens for rabies diagnosis. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBarrat%2C+J+%22Laboratory+techniques+in+Rabies+4th+Edition%22">Laboratory techniques in Rabies 4th Edition</a>. Meslin, F. X., Kaplan, M. M., Koprowski, H. , World Health Organization. 425-427 (1996).</li>
<li>Ness, S. L., Bain, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+perform+an+equine+field+necropsy%5BTitle%5D)+AND+%22American+Association+of+Equine+Practitioners%22%5BJournal%5D)">How to perform an equine field necropsy.</a> American Association of Equine Practitioners. 55, 313-316 (2009).</li>
<li>Animal & Plant Health Agency. Sample requirements for TSE testing and confirmation – EURL guidance. , Available from:
 <a target="_blank" href="https://protect2.fireeye.com/url?k=09f00f8d-55d40ec4-09f2f6b8-0cc47aa8d394-3f805f032cc98df8&u=https://science.vla.gov.uk/tse-lab-net/documents/tse-oie-rl-samp.pdf">https://protect2.fireeye.com/url?k=09f00f8d-55d40ec4-09f2f6b8-0cc47aa8d394-3f805f032cc98df8&u=https://science.vla.gov.uk/tse-lab-net/documents/tse-oie-rl-samp.pdf</a> (2019).</li>
<li>New York State Department of Health, Wadsworth Center. 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>CDC. 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/pubmed?term=((Surveillance+for+lesions+of+bovine+spongiform+encephalopathy+in+U.S.+cattle%5BTitle%5D)+AND+%22Developments+in+Biological+Standardizations%22%5BJournal%5D)">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/pubmed?term=((Eastern+equine+encephalitis+in+puppies+in+Michigan+and+New+York+states%5BTitle%5D)+AND+%22Journal+of+Veterinary+Diagnostic+Investigation%22%5BJournal%5D)">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/pubmed?term=((Clarifying+indeterminate+results+on+the+rabies+direct+fluorescent+antibody+test+using+real-time+reverse+transcriptase+polymerase+chain+reaction%5BTitle%5D)+AND+%22Public+Health+Reports%22%5BJournal%5D)">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>Chapter 7. Brain removal. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Laboratory+techniques+in+Rabies+5th+Edition%22">Laboratory techniques in Rabies 5th Edition</a>. Rupprecht, C. E., Fooks, A. R., Abela-Ridder, B. , World Health Organization. 67-72 (2018).</li>
</ol>

The authors have nothing to disclose.

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 animal only. More commonly multiple specimens are processed in a shift and care is needed to ensure no cross contamination between samples. A clean worksurface (disposable kraft paper or pads), a new set of clean, disinfected instruments, and glove changes are mandatory. Once tissues are obtained, the individual laboratory&#39;s protocol for processing can be followed, including making slides for microscopy or RNA extraction for molecular methods.
There are several essential prerequisites for successfully implementing these techniques in the laboratory or field. Prior rabies vaccination and PPE are critical for anyone necropsying a rabies suspect animal. Individuals working in rabies laboratories should have their serum tested every six months to ensure an adequate level of anti-rabies antibodies are present10. It is important to remember that other zoonotic diseases, such as EEEV, WNV and Bovine Spongiform Encephalopathy (BSE), present similar signs as rabies and may also occur in rabies suspect animals11,12.
Appropriate well-maintained instruments are essential to safely perform necropsy. Once a specimen has been removed from its biohazard bag, it should only be manipulated with instruments, not hands, to decrease the potential for accidents. Prior to small animal necropsy, the technician should evaluate the condition of the specimen to determine whether a preferred ventral approach through the base of the skull is possible. With large animals it may be too cumbersome to fully evaluate the condition of the specimen as additional vertebrae may need to be removed before retrieving tissue through the foramen magnum.
Limitations present themselves in all necropsy techniques including specimen condition, tissue quality and the amount of remaining cervical vertebrae. The cervical vertebrae will not affect the results of rabies analysis, but severely decomposed tissue may result in unsatisfactory results. More sensitive molecular methods in rabies diagnostics may allow successful testing in certain samples unable to be tested by direct fluorescent antibody assay (DFA), including severely decomposed specimens13. However, no amount of sensitivity can replace the need for proper tissue sampling.
A common problem in the rabies laboratory is receiving inappropriate or inadequate brain tissue for testing when large animal necropsies are performed in the field. Without the required tissue, and if additional tissues are unavailable for resubmission, our rabies laboratory will perform testing on the tissue available but is unable to verify the specimen negative, instead it is unsatisfactory for testing. There are other published methods for field tissue collections such as the straw method or retro orbital route14. Both methods collect brain tissue without the need to open the skull. A straw or disposable pipette is inserted either through the foramen magnum or a hole created in the eye socket and pushed through the brain, essentially taking a core sample and not necessarily sampling the full cross section of the brain stem. As these field methods do not collect samples in a manner to be considered satisfactory for testing in our laboratory, these processes are not demonstrated or explored in this paper.
In the field large animal necropsy can be challenging for individuals who are not trained to remove the correct tissue for rabies testing. Instead the entire head of the animal, which can weigh between 20-45 kg, is submitted creating cumbersome transport for both the field veterinarian and rabies laboratory technicians. Frequent requests for training on large animal necropsy technique have been made to our laboratory. The objective of this manuscript is to distribute this information to individuals and groups whose work can benefit from these techniques.

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&quot; 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&quot; 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&#039;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 with a neck will have it removed during necropsy to allow for the ventral method whenever possible. Three large animals (cow, deer, pig) were submitted and in two cases the large animal protocol was used. The large animal protocol was not used on the pig because extra brain tissue samples were required for additional testing. A squirrel was submitted with a crushed skull and simply cutting away the skin exposed brain tissue, thus none of the above methods were used (Table 1).
On fresh intact submissions, all three methods will result with the required tissue for reliable rabies diagnostic test results. Occasionally, the cerebellum and brain stem cannot be removed intact, although after removing all tissue from the hindbrain these tissues can be identified and processed accordingly.
These three valuable methods cannot compensate for poor specimen quality caused prior to receipt at the lab. Trauma, decomposition and poor decapitation methods can affect the outcome regardless of how efficiently the samples are collected.
<img alt="Dissection tools set, including scissors, forceps, scalpel, and chisel on brown background." class="xfigimg" src="/files/ftp_upload/59574/59574fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Instruments used in rabies necropsy. Curved sharp-blunt mayo scissors, smooth-tipped tissue dressing forceps without teeth, councilman orthopedic bone chisel, orthopedic mallet-hammer, locking tumor-tenacula, restaurant-quality carving knife, modified stiletto knife, chemistry spoon, and sharpened tablespoon. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59574/59574fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Dissection diagram showing brain tissue identification and comparison in labeled regions." class="xfigimg" src="/files/ftp_upload/59574/59574fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Ventral method of necropsy. (A) Location of cuts: Place point of a chisel at base of arrow, cut in direction of green arrow and repeat following the yellow arrow forming a "V" around the foramen magnum. Pry "V" down to expose the brain stem and cerebellum. (B) Brain stem (green) and cerebellum (blue) when exposed using the ventral method of necropsy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59574/59574fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Dissection technique diagram: feline cranial procedure showing incisions and exposed brain structure." class="xfigimg" src="/files/ftp_upload/59574/59574fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Dorsal method of necropsy. (A) Location of cuts: Place point of a chisel at base of arrow and cut in direction of arrow in the order noted forming a "U". Pry "U" down to expose the cerebellum with the brain stem below it. (B) Cerebellum (circled) when exposed using the dorsal method. Brain stem lies directly beneath and is not visible until cerebellum is removed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59574/59574fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>species</td>
			<td>vertebrae attached</td>
			<td>method used V=ventral, D=dorsal, LA=large animal</td>
			<td>total</td>
			<td>comments</td>
		</tr>
		<tr>
			<td>bear</td>
			<td>no</td>
			<td>V</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>cat</td>
			<td>no</td>
			<td>V</td>
			<td>32</td>
			<td></td>
		</tr>
		<tr>
			<td>cat</td>
			<td>no</td>
			<td>D</td>
			<td>3</td>
			<td>2 small enough to open skull with scissors, 1 had abcess that was being investigated exposing top of skull</td>
		</tr>
		<tr>
			<td>cat</td>
			<td>yes</td>
			<td>V</td>
			<td>11</td>
			<td></td>
		</tr>
		<tr>
			<td>cat</td>
			<td>yes</td>
			<td>D</td>
			<td>8</td>
			<td></td>
		</tr>
		<tr>
			<td>cow</td>
			<td>no</td>
			<td>LA</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>coyote</td>
			<td>no</td>
			<td>V</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>coyote</td>
			<td>yes</td>
			<td>V</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>coyote</td>
			<td>yes</td>
			<td>D</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>deer</td>
			<td>no</td>
			<td>LA</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>dog</td>
			<td>no</td>
			<td>V</td>
			<td>19</td>
			<td></td>
		</tr>
		<tr>
			<td>dog</td>
			<td>no</td>
			<td>D</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>dog</td>
			<td>yes</td>
			<td>V</td>
			<td>2</td>
			<td>1 was small dog</td>
		</tr>
		<tr>
			<td>dog</td>
			<td>yes</td>
			<td>D</td>
			<td>18</td>
			<td></td>
		</tr>
		<tr>
			<td>ferret</td>
			<td>no</td>
			<td>V</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>fisher</td>
			<td>no</td>
			<td>V</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>flying squirrel</td>
			<td>yes</td>
			<td>D</td>
			<td>1</td>
			<td>whole body</td>
		</tr>
		<tr>
			<td>grey fox</td>
			<td>no</td>
			<td>V</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>grey fox</td>
			<td>yes</td>
			<td>V</td>
			<td>4</td>
			<td></td>
		</tr>
		<tr>
			<td>pig</td>
			<td>yes</td>
			<td>V</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>porcupine</td>
			<td>no</td>
			<td>V</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>raccoon</td>
			<td>no</td>
			<td>V</td>
			<td>16</td>
			<td></td>
		</tr>
		<tr>
			<td>raccoon</td>
			<td>no</td>
			<td>D</td>
			<td>1</td>
			<td>frozen</td>
		</tr>
		<tr>
			<td>raccoon</td>
			<td>yes</td>
			<td>V</td>
			<td>26</td>
			<td></td>
		</tr>
		<tr>
			<td>red fox</td>
			<td>no</td>
			<td>V</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>skunk</td>
			<td>no</td>
			<td>V</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>skunk</td>
			<td>yes</td>
			<td>V</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>squirrel</td>
			<td>no</td>
			<td>V</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>squirrel</td>
			<td>yes</td>
			<td>D</td>
			<td>1</td>
			<td>full body, crushed skull, used scissors to cut away skin to exposed brain cavity</td>
		</tr>
		<tr>
			<td>weasle</td>
			<td>no</td>
			<td>D</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>woodchuck</td>
			<td>yes</td>
			<td>D</td>
			<td>1</td>
			<td>whole body</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>170</td>
			<td></td>
		</tr>
		<tr>
			<td>Breakdown of total</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>with neck</td>
			<td></td>
			<td>81</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>no neck</td>
			<td></td>
			<td>89</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>ventral method</td>
			<td></td>
			<td>128</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>dorsal method</td>
			<td></td>
			<td>39</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>large animal method</td>
			<td></td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>other method</td>
			<td></td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>ventral method with neck</td>
			<td></td>
			<td>49</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Breakdown of specimens requiring tissue removal from the skull submitted from January 31, 2019 through February 28, 2019 at the New York State Department of Health Rabies Laboratory.

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 ...

rabies-necropsy-techniques-in-large-and-small-animals

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.7K Views.  New York State Department of Health. Weefselverzameling voor rabiës testen heeft zowel duidelijke als onaangekeurde gevaren. De technicus moet de veiligste beschikbare methoden gebruiken en de juiste persoonlijke beschermingsmiddelen dragen. Deze protocollen bieden veilige obductietechnieken zonder gebruik te maken van elektrisch gereedschap of zagen, waardoor de productie van aerosolen wordt geminimaliseerd.Het grote dierprotocol kan ook in het veld worden uitgevoerd. Het gebruik van de grote methode voor het terughalen...

Video: Rabies Necropsy Techniques in Large and Small Animals

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Genetics

Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are natural reservoirs of most lyssaviruses, a lyssavirus surveillance program of bats has been conducted in Taiwan since 2008 to understand the ecology of these viruses in bats. In this program, non-governmental bat conservation organizations and local animal disease control centers cooperated to collect dead bats or bats dying of weakness or illness. Brain tissues of bats were obtained through necropsy and subjected to direct fluorescent antibody test (FAT) and reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus antigens and nucleic acids. For the FAT, at least two different rabies diagnosis conjugates are recommended. For the RT-PCR, two sets of primers (JW12/N165-146, N113F/N304R) are used to amplify a partial sequence of the lyssavirus nucleoprotein gene. This surveillance program monitors lyssaviruses and other zoonotic agents in bats. Taiwan bat lyssavirus is found in two cases of the Japanese pipistrelle (Pipistrellus abramus) in 2016&#8211;2017. These findings should inform the public, health professionals, and scientists of the potential risks of contacting bats and other wildlife.

This protocol introduces a standard laboratory operating procedure for diagnostic testing of lyssavirus antigens in bats in Taiwan.

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are ...

Environment

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Biology

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 ...

Immunology and Infection

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, ...

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, ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

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 ...

Postmortem Diagnosis of Rabies in Animals by the Updated, Multiplexed LN34 Real-Time Reverse Transcription-Polymerase Chain Reaction Assay

Rabies is a fatal zoonotic disease caused by Lyssavirus rabies (RABV) and related negative strand RNA viruses from the Lyssavirus genus (family Rhabdoviridae). The LN34 assay targets the highly conserved leader region and nucleoprotein gene of the lyssavirus genome and utilizes degenerate primers and a TaqMan probe containing locked nucleotides to detect RNA across the diverse Lyssavirus genus. A negative finding for rabies should be made only if a full cross-section of the brain stem and three lobes of the cerebellum are examined; however, identification of lyssavirus RNA in any tissue is diagnostic of rabies infection. Tissue is collected and homogenized in TRIzol reagent, which also inactivates the virus. RNA extraction is performed using a spin column-based commercial extraction kit. Master mixes are prepared in a clean space and aliquoted into a 96 well plate before adding sample RNA. In clinical settings, each sample is tested by real-time RT-PCR for the presence of lyssavirus RNA in triplicate and singly for host &#946; -actin mRNA. Positive and negative controls are included at extraction and real-time RT-PCR steps of the protocol. Data analysis involves manual adjustment of the thresholds to standardize Ct values across instrument runs. Positive results are determined by the presence of typical amplification in the pan-lyssavirus assay (Ct &#8804; 35). Negative results are determined by the absence of typical amplification in the pan-lyssavirus assay and detection of host &#946;-actin mRNA (Ct &#8804; 33). Observation of values outside of these ranges or failure of the assay controls can invalidate the run or result in inconclusive results for a specimen. The protocol should be closely followed to ensure high assay sensitivity and specificity. Procedural modifications can affect assay performance and lead to false positive, false negative, or uninterpretable results.

This protocol demonstrates the pan-lyssavirus LN34 real-time reverse transcription-polymerase chain reaction (RT-PCR) assay from tissue collection to result interpretation, including updates to primer sequences and formulations to improve assay performance for some non-rabies lyssaviruses and lagomorphs. We also demonstrate the assay setup for a single-well LN34 multiplexed (LN34M) format.

Rabies is a fatal zoonotic disease caused by Lyssavirus rabies (RABV) and related negative strand RNA viruses from the Lyssavirus genus (family ...

Diagnostic Necropsy and Tissue Harvest

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Many animal experiments rely on final data collection time points that are gathered from the harvesting and testing of organs and tissues. The use of appropriate methods for the collection of organs and tissues can impact the quality of the samples and the analysis of the data that is gleaned for the testing of the tissues. The method of euthanasia of the animal can also impact the quality of the samples. This manuscript will outline proper necropsy techniques for rats.

Rabies Necropsy Techniques in Large and Small Animals

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