Rabies Necropsy Techniques in Large and Small Animals

Summary

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

Abstract

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.

Introduction

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 dangers^1,2,3.

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 saws^4,5. 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).

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Protocol

All methods described were approved by the Wadsworth Center Institutional Animal Care and Use Committee (IACUC).

1. Preparation

1. Don PPE, at minimum eye protection (glasses or face shield), surgical or N-95 mask, and non-latex gloves.
2. 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).
3. 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.

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.

1. Position the specimen with ventral side up and nose directing distally toward the back of BSC.
2. Hold an orthopedic hammer/mallet in right hand (if right-handed) and at same time hold a councilmen chisel in left hand.
3. Position the chisel at a 45° angle with the corner point of the chisel directing between the right side of temporal bone and occipital bone making a “V” opening.
4. Strike the top of the chisel with the hammer until the two bones separate. Make the cut to adjacent to the basisphenoid bone.
5. Repeat on the left side of temporal bone/occipital bone (Figure 2A).
6. Bend the “V” area of skull downward with the chisel. Expose the entire rhombencephalon area of the brain (cerebellum and brain stem) (Figure 2B).
7. 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.

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.

1. Position the specimen dorsally with the nose directing distally toward the back of BSC.
2. Using tumor tenacula, grasp the left temporal muscle with teeth of tenacula and lock by squeezing the handle.
3. Cut the temporal muscle down to bone with sharp carving knife.
4. Rotate the specimen 180° with tenacula and knife (not hand) and repeat the process on the opposite temporal muscle. Expose the skull.
5. Position a chisel at a 45° angle with the corner point of the chisel on the center of the skull at the juncture of the parietal and intraparietal bone.
6. 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.
7. Rotate the specimen 180° and repeat the process on the opposite side.
8. Insert the point of the chisel into the end of cut 1 (Figure 3A) and at 90° of horizontal opening. Strike with the hammer until the opening reaches occipital bone (approximately 10 cm depending upon size of the specimen).
9. 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 “U”.
10. Insert the teeth of the tenacula into the skull at the bottom of the “U” and pry towards oneself. Expose the caudal end of the cerebrum and the cerebellum (Figure 3B).
11. Use scissors as a scoop and pry out entire cerebellum from within the cavity.
12. Use tissue forceps to tease out the brain stem from the foramen.

4. Large animal method

1. 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.
2. Insert the modified stiletto knife into the foramen magnum in between the spinal cord and spinal meninges as far as possible.
3. 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.
4. Insert a chemistry spatula or thin, long handled spoon into the space between the neural tissue and spinal meninges.
5. Probe around the spinal cord and cerebellum to ensure the connection to the spinal meninges have been severed.
6. 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.

5. Post necropsy

1. Dispose of all disposable materials (gloves, pads, work area coverings) and unused tissues in biohazardous waste.
2. Clean and disinfect all instruments with method available (e.g., industrial dishwasher, autoclave, chemical disinfectant, boiling).
3. Clean and disinfect all work surfaces with 20% bleach and/or 70% ethanol.

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Results

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...

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Discussion

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...

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Materials

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