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Methods of diagnosing rabies

VLA uses various methods of diagnosis which include:

• The fluorescent antibody test (FAT)
• Virus isolation
• Histopathology
• In-situ hybridisation
• Molecular methods
• Serology

The fluorescent antibody test(FAT)

The fluorescent antibody test (FAT), sometimes referred to as the direct fluorescent antibody test (dFA), is the mainstay of all rabies diagnostic laboratories and is recommended by the OIE.

This test requires post mortem brain tissue from animals suspected of having rabies, and can confirm the presence of rabies virus in a brain smear.

For direct rabies diagnosis, smears prepared from the hippocampus, cerebellum or medulla oblongata are fixed in high-grade cold acetone. The fixed tissue is then stained with a drop of a specific anti-rabies antibody which is bound to a fluorescent marker.

In the FAT, the virus antigen (a nucleocapsid protein) will bind to the fluorescent antibody. These antigen-antibody complexes can be seen as apple-green fluorescent areas using a fluorescence microscope. If the tissue is not infected with rabies virus it will not fluoresce.

Virus isolation

The OIE also recommends the use of confirmatory virus isolation test, particularly when FAT results are unclear and for human exposure cases.

Virus isolation tests such as the Rabies Tissues Culture Inoculation Test (RTCIT) involve infecting neuroblastoma cells with rabies positive brain material (cortex, Ammon’s horn, cerebellum, medulla oblongata).

After 2-4 days, FAT is used to confirm the presence of rabies antigen in the cell monolayers.

Histopathology

Histological examination of biopsy or autopsy tissues, particularly fixed archive material, is occasionally useful in diagnosing rabies.

However, tests based upon the detection of aggregates of viral proteins called Negri bodies and histological tests such as Sellers’s or Mann’s tests, are no longer routinely used by the majority of diagnostic laboratories. These tests are considered unreliable, particularly for decomposed material.

In-situ hybridisation

VLA (Finnegan et al., 2004) has recently developed a protocol suitable for the detection of classical rabies virus and European bat lyssaviruses type 1 and 2.

The test is a robust, highly sensitive and specific in-situ hybridisation technique.

It is based on the specific binding of complementary riboprobes labelled with a chemical called digoxigenin, to the viral genome and messenger RNA. 

The ability to detect messenger RNA is indicative of the presence of replicating virus.

Molecular methods

The use of the polymerase chain reaction (PCR) based techniques to detect rabies virus genomic RNA is not currently recommended by the OIE or WHO for routine post-mortem diagnosis of rabies.

However, in laboratories with strict quality control procedures in place and demonstrable experience and expertise, these molecular techniques have been successfully applied for confirmatory diagnosis and epidemiological surveys.

Reverse transcription (RT)-PCR has been reported to confirm rabies diagnosis directly in suspect human cases when conventional diagnostic methods have failed and post-mortem material is not available.

Rabies virus RNA can be detected in a range of biological fluids and samples (e.g. saliva and skin biopsies of hair follicles at the nape of the neck).

For all positive PCR results, the amplified portion of the viral genome can be sequenced to confirm the origin of the virus. The resulting sequences can prove useful for molecular epidemiological and evolutionary studies.

Serology

In most animals infected with the rabies virus, anti-rabies antibodies are rarely detectable.

However, antibody detection methods such as the fluorescent antibody virus neutralisation (FAVN) can determine immunity in vaccinated animals.

Virus neutralisation tests are based on the fact that antibodies can lower the infectivity of a known dose of rabies virus in cell culture.

This test has also been used to detect anti-rabies antibodies in the cerebral spinal fluid (CSF), or serum of non-vaccinated humans, thus confirming exposure to a lyssavirus.

The FAVN is an OIE-prescribed test for international trade of companion animals. A known dose of rabies virus is mixed with different dilutions of the serum to be tested and the neutralising limit dilution is determined. The results are expressed in international units (IU) calibrated from standard reference serum.