Fluorescent Antibody Test for Rabies
Brain impression smears are prepared on microscope slides using three parts of the brain: cerebellum, hippocampus, and brain stem. The smears are fixed in acetone for at least one hour. The smears are stained using a fluorescein labeled antibody specific for rabies virus, which is diluted in a normal rabbit brain suspension, and incubated with the antibody for 30 minutes. They are then washed in PBS two times, for five minutes each, dipped in distilled water, and air-dried. The slides are mounted with buffered glycerol, pH 9.0, and examined using an epifluorescent microscope. Positive and negative control slides are included with each run.
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Monoclonal Typing of Rabies Isolates
Rabies positive specimens are tested using an indirect fluorescent-antibody procedure with a panel of 19 monoclonal antibodies. Acetone fixed touch impressions of brain material or infected cell culture monolayers are stained with each of the monoclonal antibodies for 30 minutes at 37° C, washed to remove unbound antibody, and stained with fluorescein-conjugated goat antibody to mouse IgG. The slides are mounted with buffered glycerol, pH 9.0, and examined using an epifluorescent microscope. The strains of rabies are characterized based on patterns of reaction with the panel of monoclonal antibodies.
RT-PCR and Restriction Enzyme Digestion
Several strains of rabies cannot be distinguished by monoclonal typing and must be tested further by genetic analysis to differentiate the specific types, such as the Texas fox (TF) and domestic dog/coyote (DDC) strains. In brief, RNA is extracted from positive brain material using TRIzol. Reverse transcription is performed with the extracted RNA, using primer 10g (CTA CAA TGG ATG CCG AC), complementary to the sequence encoding the amino terminus of the nucleoprotein. Amplification of reverse-transcribed cDNA is performed with the primer pair 10g and a reverse primer 304 (TTG ACG AAG ATC TTG CTC AT), complementary to the sequence encoding the amino terminus of the non-structural or phosphoprotein. The final amplified DNA, a 1449-bp fragment, is visualized by agarose gel electrophoresis with ethidium bromide staining.
A restriction endonuclease digestion is performed on DNA amplified by primer pair, 10g and 304, by the addition of restriction enzyme DdeI or HinfI to the amplicons. Distinct fragment patterns are resolved by electrophoresis for the following strains of rabies using this method: Texas fox, domestic dog/coyote, Sonora dog, hog-nosed skunk, south central skunk, north central skunk, and Arizona fox.
Monoclonal Typing Patterns of Terrestrial Rabies Strains Found in Texas
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15
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South Central skunk
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d
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San Saba skunk
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Texas fox/Domestic dog/coyote, Sonora dog
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d
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Hog-nose skunk
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Monoclonal Typing Patterns of Bat Rabies Strains
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Tadarida brasiliensis
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Lasiurus borealis, Lasiurus seminolus
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Lasiurus cinereus
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w
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Desmodus rotundus
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Eptesicus fuscus fuscus
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Eptesicus fuscus pallidus
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Lasiurus intermedius, Lasionycteris noctivagans,
Pipistrellus subflavis, Lasiurus ega
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Nycticeius humeralis
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