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