Identification of the agent
Clinical observation might trigger a suspicion of Rabies. But these signs are neither disease specific nor pathagnomonic, and also vary greatly from one animal to another. For a confirmative diagnosis, the virus or its antigen/antibody needs to be isolated and identified. Numerous laboratory techniques are available today, and each method varies in its efficiency, specificity and reliability. Most of them are suitable to identify the virus from a brain tissue, while the others are used detect viral particles in saliva, CSF, Cornea, Nuchal skin, serum, Kidney and Liver.
Collection of samples
For a post mortem examination, usually the brain is collected after opening up the skull in a necropsy room, and the hippocampus along with portions of medulla and cerebrum are collected for histopathology. For an ante mortem examination, saliva, swabs from oral cavity, throat, CSF, Serum and Nuchal skin might be collected.
Shipment of samples
During the shipment of the suspected material for diagnosis, no form of human or any other contamination should occur. Brain and any nerve tissues must be placed in a leak-proof rigid container as prescribed in the International Air Transport Association (IATA) and the procedures mentioned in the 'Dangerous Goods Regulations' must be followed.
Routine laboratory tests
Laboratory diagnosis can be performed employing either one or more of the three common methods.
1. Animal innoculation test.
2. Histological identification of characteristic cellular lesions: Negri bodies are pathgnomonic, and correspond to the aggregation of viral proteins, but the classical staining techniques detect only an affinity of these structures for acidophilic stains.
3. Immunohistochemical tests which are specific to detect Rabies antigens or viral particles: Viral isolates can be screened using various serological tests, Direct Fluorescent Antibody (DFA), PCR, ELIZA, and others which are detailed in the below sections.
Light microscopy or Electron microscopy, MRI Imaging, Electroencephalogram (EEG), Serological tests, Nuchal skin biopsy, Detection of RNA in saliva and CSF by PCR, Detection of antibodies from CSF and serum, Rapid tissue culture infection test (RTCIT), Viral isolation and the Mouse inoculation test (MIT).
The Mouse inoculation test (MIT), the Rapid tissue culture infection test (RTCIT) and the DFAT or FAT.
Methods for the Immunochemical identification of Rabies Viral Antigen include
Fluorescence Antibody Virus Neutralization Test (FAVN), Fluorescent Antibody Test, Corneal smear test by Fluorescent Antibody Test (FAT), Direct ImmunoFluorescent-Antibody TEST (DIFAT) , The Indirect immunofluorescence Assay (IFA) and the Latex Agglutination Test.
Detection of Rabies viral antigen in brain specimens can also be accomplished by techniques such as Rapid Rabies Enzyme Imunodiagnosis (RREID) or Dot-Blot Immunoassay. The detection of immune complexes to Rabies antigens can be also be instrumental in rapid ante-mortem diagnosis of human Rabies.
ELISA test based on monoclonal antibodies to Rabies nucleoprotein (N) and glycoprotein (G) has also been described, which is advantageous to detect immune complexes of Rabies N and G proteins. Presence of Rabies specific immune complexes in the cerebrospinal fluid (CSF) of patients with paralytic Rabies has reportedly been detected, which could also help in ante-mortem diagnosis.