Presently the gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFA) which is very expensive and requires a high level of expertise.There is a need for more economical and user friendly tests,particularly for use in developing countries.We have established one such test called the direct rapid immunohistochemical test (dRIT) for diagnosis of rabies using brain tissue.The test is based on capture of rabies nucleoprotein (N) antigen in brain smears using a cocktail of biotinylated monoclonal antibodies specific for the N protein and color development by streptavidin peroxidase-amino ethyl carbazole and counter staining with haematoxollin.The test was done in parallel with standard FAT dFA using 400 brain samples from different animals and humans.The rabies virus N protein appears under light microscope as reddish brown particles against a light blue background.There was 100 ％ correlation between the results obtained by the two tests.Also,interpretation of results by dRIT was easier and only required a light microscope.To conclude,this newly developed dRIT technique promises to be a simple,cost effective diagnostic tool for rabies and will have applicability in field conditions prevalent in developing countries.

PURPOSE: Myeloid differentiation factor 88 (Myd88), a ubiquitous Toll-like receptor adaptor molecule, has been reported to play important roles in B cell responses to infections and vaccination. The present study evaluated the effects of genetic adjuvanting with Myd88 on the immune responses to a plasmid DNA rabies vaccine. MATERIALS AND METHODS: Plasmids encoding rabies glycoprotein alone (pIRES-Rgp) or a fragment of Myd88 gene in addition (pIRES-Rgp-Myd) were constructed and administered intramuscularly or intrademally in Swiss albino mice (on days 0, 7, and 21). Rabies virus neutralizing antibody (RVNA) titres were estimated in the mice sera on days 14 and 28 by rapid fluorescent focus inhibition test. The protective efficacy of the constructs was evaluated by an intracerebral challenge with challenge virus standard virus on day 35. RESULTS: Co-expression of Myd88 increased RVNA responses to pIRES-Rgp by 3- and 2-folds, following intramuscular and intradermal immunization, respectively. pIRES-Rgp protected 80% of the mice following intramuscular and intradermal immunizations, while pIRES-Rgp-Myd afforded 100% protection following similar administrations. CONCLUSION: Genetic adjuvanting with Myd88 enhanced the RVNA responses and protective efficacy of a plasmid DNA rabies vaccine. This strategy might be useful for rabies vaccination of canines in the field, and needs further evaluation.
Animals ; Antibodies, Neutralizing ; DNA* ; Glycoproteins ; Immunization ; Mice ; Myeloid Differentiation Factor 88 ; Plasmids* ; Rabies ; Rabies Vaccines* ; Rabies virus ; Toll-Like Receptors ; Vaccination ; Vaccines, DNA

PURPOSE: Immunization against rabies in humans induces protective neutralizing antibodies; however, the induction of type 1 or type 2 cytokine mediated cellular immune responses following rabies vaccination is not understood. Hence, the present study investigated cellular cytokine responses in vaccinated individuals. MATERIALS AND METHODS: The study groups included healthy rabies antigen naive controls (n=10), individuals who received intradermal primary (n=10) or booster pre-exposure vaccination (n=20) and subjects who received postexposure rabies vaccination either by intradermal (n=18) or intramuscular (n=20) routes. The antigen specific cellular responses were analyzed by stimulating peripheral blood mononuclear cells with a rabies vaccine antigen in the interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) enzyme-linked immunospot (ELISpot) assay. These responses were compared to the rabies virus neutralizing antibody (RVNA) titers that were measured by rapid fluorescent focus inhibition test. RESULTS: We observed that cellular and humoral immune responses to primary intradermal rabies vaccination could be greatly enhanced by a booster vaccine; and both type 1 and type 2 cytokine responses were significantly elevated. The magnitude of type 1 and type 2 cytokine responses did not differ significantly among the intramuscular and intradermal routes of postexposure vaccination. The number of cells producing IFN-gamma and IL-4 correlated significantly with the levels of RVNA. CONCLUSION: Both type 1 and type 2 cellular cytokine responses are strongly induced after rabies vaccination and directly correlate with levels of RVNA titers. The neutralizing antibody as well as the type 1 and type 2 cytokine responses may be important for vaccine induced protective responses against rabies.
Antibodies, Neutralizing ; Humans ; Immunity, Cellular* ; Immunity, Humoral ; Immunization ; Interferon-gamma ; Interleukin-4 ; Rabies Vaccines ; Rabies virus ; Rabies* ; Vaccination*