Because clinical diagnosis of rabies is not reliable, a definitive diagnosis can only be obtained by laboratory investigations post-mortem.
However, serological tests are used for surveys and post-vaccinal control in order to test immunity level in vaccinated animals, especially in the context of international movements.
The recommendations from the experts of the OIE First International Conference “Rabies in Europe” Kiev, Ukraine, 15-18 June 2005 are the following:
- Routine laboratory diagnosis should be undertaken using only the techniques specified by the OIE (Terrestrial Manual)[OIE 2011] and the WHO (Laboratory Techniques in Rabies).
- The FAT (Fluorescent antibody test) is the primary method recommended.
- The confirmation test should use rabbit tissue culture inoculation test (RTCIT). The mouse inoculation test can be used only if rabbit tissue culture is not available.
- PCR is presently not recommended for routine diagnosis but may be useful for epidemiological studies or for confirmatory diagnosis only in reference laboratories.
Reference laboratories: The list of reference experts and laboratories can be found on the OIE web site (http://www.oie.int/en/our-scientific-expertise/reference-laboratories/list-of-laboratories/).
Virus detection methods
Only direct detection methods are recommended to confirm rabies in human beings and animals.
Samples (animal heads, brain tissues or other organs) should be sent according to the national and international shipping regulations and care should be taken in order to avoid potential human contamination. Because rabies virus is rapidly inactivated, the specimen should be shipped (preferably) refrigerated or at room temperature in 50% glycerine in phosphate buffered saline solution.
Brain tissue (especially thalamus, pons and medulla) is the preferred sample for postmortem diagnosis but other organs such as salivary glands can also be used.
Fluorescent antibody test
Fluorescent antibody test (FAT) is the method recommended by WHO and OIE for fresh or glycerol samples [Bourhy et al, 1989, Birgham & van der Merwe 2002], but is less sensitive in formalin-fixed tissues. FAT provides a reliable diagnosis in 95% to 99% of cases for all lyssaviruses in fresh samples. It can also be used for rabies detection in cell cultures and in brain tissue of mice that have been inoculated for diagnosis.
Other methods available include immunochemical tests (e.g. avidin-biotin peroxydase system, ELISA, direct blot enzyme immunoassay). The rapid rabies enzyme immunodiagnosis (RREID) is an alternative to FAT but detects only the rabies virus. Correlation between FAT and RREID is between 96 and 99% [Barrat 1993].
Inoculation to laboratory animals and cell cultures
These tests are used to confirm inconclusive results with FAT in organs or when FAT is negative if human exposure has been reported.
Intracerebral inoculation of mice is performed in newborn or 3 to 4 week-old mice. FAT is used to detect virus 5 days to 11 days post-inoculation. Ideally, these inoculation tests should be replaced by cell culture tests, which are as sensitive, less time-consuming and more ethical. Neuroblastoma cell lines may be used and presence of the rabies virus is revealed by FAT, with results being available within 2 to 4 days.
Histology – Immunochemistry
Since histology and immunohistochemistry to detect Negri bodies are less sensitive
than FAT, especially in autolysed tissues, these methods are not recommended
for routine diagnosis.
Other direct methods
Reference laboratories may identify rabies virus, and especially some variants, using
monoclonal antibodies, nucleic probes or PCR and sequencing. These techniques can
distinguish vaccine and field strains and may identify the geographic origin of the isolate .
Seroneutralisation tests in cell cultures, such as fluorescent antibody virus neutralisation
(FAVN) or rapid fluorescent focus inhibition test (RFFIT) are widely used to confirm immunity induced by vaccination.
The principle of FAVN is neutralisation in vitro of a rabies CVS strain before inoculating BHK-21 C13 cells. The titer is expressed in IU/ml and is the reciprocal value of the dilution at which 100% of the virus is neutralised in 50% of the wells. RFFIT and FAVN give equivalent results. A titer of 0.5 IU/ml of serum antibodies is an internationally accepted threshold titer.
ELISA has recently been developed and is used for testing vaccinated animals [Servat et al, 2007]. Commercial kits are now available for the detection of antibodies in sera from vaccinated cats and dogs. ELISA tests do not require the culture of live virus and the result can be obtained within 4 hours. The sensitivity and specificity of ELISA tests still need to be confirmed before it can be accepted as an official method [Servat et al, 2006]. For further details, refer to Barrat et al,  and the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [OIE 2011].