Special Veterinary Pathology

DIAGNOSIS OF RABIES

Preparation of impression smear from brain

• Flay the skin over the fore head region
• Remove the muscles to expose the skull
• Break open the skull with the help of a chisel and hammer
• Meninges are cut opened with a pair of scissors to expose the brain
• Dissect the brain into two halves
• Remove the hippocampus major by dissection
• Divide the hippocampus major transversely and hold it with the help of a pair of forceps '
• Blot out the cut surface to remove the blood, which may interfere with the examination
• Make impression smears in a clean dry glass slide by gently touching the cut surface.
• Fix the smear by dipping the slide in methanol in a coplin jar (The process also kills the organisms).
• Smears from cerebellum are preferred in cattle.

Staining the impression smear

• (William’s modification of van Gieson’s stain)
• Stock solution
• Solution 1. Saturated methanolic solution of basic fuchsin
• Solution 2. Saturated methanolic solution of methylene blue

Working solution

• This should be prepared freshly.
• To about 10mL of water in a test tube add Solution 1 – 3 drops Solution 2 – 7 drops
• Mix well (Increase or decrease the quantity of stain depending on the result)

Staining procedure

• Pour phe stain over the impression smear
• Gently heat the slide from under till steam emanates
• Allow it for a minute or two
• Wash the slide with water
• Blot.

Examination of the smear

• Screen the slide under low power to identify the areas free from erythrocytes and for groups of ganglion cells
• Place a drop of immersion oil on the smear
• Examine under oil immersion

Result

• The nuclei of nerve cells are stained blue in colour with nucleoli taking dark blue stain.
• Glial nuclei are stained in dark blue colour and nucleoli are not appreciable.
• The Negri bodies are round or oval in shape located near the nuclei of nerve cells and are magenta red in colour.
• Erythrocytes are coppery red in colour.

Interpretation

• The presence of Negri bodies is a definite evidence of rabies. While the absence of Negri bodies does not rule out rabies.

Fluroscent antibody technique

• Using the suspected hippocampus major impression smears, Fluorescent antibody technique can be applied to demonstrate the presence of antigen in the tissues.