Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

The DRIT is significant because it provides a rabies diagnostic test that can be performed in decentralized laboratories in the field or in areas without routine access to electron microscopy. The main advantage of the DRIT is that it utilizes only simple light microscopy and the results can be obtained in approximately one hour. The DRIT provides a powerful economical tool for rabies diagnosis that can be used by laboratorians and field biologists to improve rabies surveillance, prevention and control programs globally.

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. 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 the anterior aspect of cervical spine vertebrae. Then move the head of the animal into an end range extension until the brain stem is visible. Use a scalpel blade to remove all visible brainstem and central nervous system tissue for sample.

Place the brainstem samples into an unbreakable container and label accordingly. First, set out staining dishes that are deep enough to allow for complete immersion of a sample slide. Fill dish one with 10%phosphate buffered formalin.

Fill dishes two, four and five with TPBS. Fill dish three with 3%hydrogen peroxide. Then fill dishes six, eight, nine and 10 with distilled or deionized water.

Fill dish seven with Gill's hematoxylin formulation number two diluted in a 1:1 ratio with distilled water. To prepare the amino-ethyl-carbazole stock solution, using a glass pipette, place five milliliters of N, N-dimethylformamide in a glass container. Add 120 milligram tablet of 3-amino-9-ethylcarbazole to the glass container and shake until completely dissolved.

To prepare the AEC working dilution, add seven milliliters of acetate buffer to a 15 milliliter centrifuge tube. Use a glass pipette to add 0.5 milliliters of AEC stock solution to the centrifuge tube. Add 0.75 milliliters of 3%hydrogen peroxide solution.

Use a 10 milliliter syringe with a 0.45 micrometer syringe filter to filter the solution. To begin, use a smear-proof waterproof permanent ink marker to label glass microscope slides with a unique number for each specimen. Use a scalpel blade to remove the brainstem from the container and place it on a paper towel.

Use a second paper towel to gently blot away any excess fluid, blood or fur to reveal just the brainstem tissue. If needed, section the brainstem tissue to reveal a cross-section. Very gently touch the microscope slide to several points of the brainstem without lateral movement to allow multiple areas of brainstem to be transferred to the slide making sure that only one or two layers of cells are transferred from the brain tissue to the slide.

Let the slides air dry for approximately five minutes at room temperature. Then immerse the slides in the 10%buffered formalin in dish one for 10 minutes. Remove the slides from the formalin and dip rinse them in the TPBS solution in dish two.

Immerse the rinsed slides in the hydrogen peroxide solution contained in dish three for 10 minutes. After this, remove the slides from the hydrogen peroxide and dip rinse them in the fresh TPBS contained in dish four. After removing any excess hydrogen peroxide, place the slides in the fresh TPBS contained in dish five.

Take the slides one at a time from dish five, shake off and blot any excess buffer and place the slide on a moistened paper towel on the lab bench. When all the slides have been transferred, use a dropper or pipette to drop enough primary anti-rabies virus antibody on each slide to cover the CNS tissue. Cover the slides with well plates or another simple cover to create a humidity chamber and incubate the slides at room temperature in the chamber for 10 minutes.

After this, remove the slides from the humidity chamber, shake and blot off any excess conjugate and dip rinse the slides in the TPBS in dish five. Working with one slide at a time, remove a slide from the TPBS and use a dropper or pipette to drop enough Streptavidin peroxidase complex to cover the CNS tissue. Once all of the slides have been covered, incubate them in the humidity chamber for 10 minutes at room temperature.

Then remove the slides from the humidity chamber, shake and blot off any excess complex and dip rinse the slides in the TPBS contained in dish five. Working with one slide at a time, remove a slide from the TPBS and use a pipette to drop enough AEC working solution to cover the CNS tissue. Incubate the slides in the humidity chamber for 10 minutes at room temperature.

After this, dip rinse the slides in the distilled water contained in dish six and place them in a counterstain of Gill's hematoxylin in dish seven for two minutes. Immediately dip rinse all of the slides in the distilled water in dish eight. Repeat this twice using the freshwater in dishes nine and 10 for each wash.

Working one slide at a time, remove a slide from the water and shake and blot off any excess water. Use a water soluble mounting medium to affix a coverslip to the slide. Then use a light microscope with a 20X objective to view the slides.

Positive results from the DRIT show red intracytoplasmic viral inclusions that can vary in shape and size within the cytoplasm of the bluish cell bodies. The inclusions appear smooth with very bright margins and a less intensively stained central area. The antigen distribution is graded from plus four to plus one with plus four representing antigen distribution comprised of an abundance of large and small inclusions varying in size and shape and present in every field of view within the CNS tissue touch impression.

A distribution of plus three is 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 to 50%of the microscope fields, a plus two antigen distribution is assigned while plus one is assigned when inclusions are found in less than 10%of the fields. Most CNS tissues with rabies virus present exhibit typical viral inclusions graded as plus three or plus four intensity and antigen distribution.

If the results indicate a plus one or plus two in either intensity or antigen distribution, the sample is declared an indeterminate and repeat testing is warranted. A test sample using DRIT is considered negative for rabies virus antigens after the slide containing the CNS tissue is scanned at a magnification of 200X or greater and no typical virus inclusions are detected. Negative samples exhibit bluish cell bodies with little or no known specific staining.

Each person conducting rabies diagnostic testing should receive standard pre-exposure rabies vaccinations and undergo regular serological antibody evaluation. Unimmunized individuals should not enter areas where such work is being conducted. In addition to the precautions taken when working with live rabies virus, several of the reagents used in the test are hazardous.

Refer to the safety data sheets for each reagent.