Pennsylvania Animal Diagnostic Laboratory System
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Immunohistochemical Detection of West Nile Virus in Crows

H. Kim*, D. Tewari, B. Corson and P. Miller

PADLS, Pennsylvania Veterinary Laboratory
Harrisburg, Pennsylvania

During the summer of 2000, the Pennsylvania Veterinary Laboratory (PVL) received a total of 315 birds, mainly crows for diagnosis of West Nile Virus. Brain, heart, lung, liver, spleen, kidney and intestine specimen were collected and processed for immunohistochemical (IHC) staining, histology, pcr and virus isolation.

For IHC testing, an automated immunostainer (Leica ST 5050) was used. Paraffin blocks of the bird tissues were sectioned at 5 micrometer, and the sections were mounted on positively charged glass slides. These sections were heated and deparaffiniezed. All cases were analyzed by IHC for WNV using an immunoperoxidase method (Envsion system; DAKO Corporation, Carpinteria, CA), performed according to the manufacture's instructions. The primary antibody was a rabbit hyperimmune polyclonal antiserum prepared against WNV (provided by Dr. Keith Steele, USAMRIID). Prior to immunostaining, the tissue sections were incubated with proteinase at room temperature for 6 minutes, followed by peroxidase blocking in 0.03% H2O2 at room temperature for 5 minutes. The primary antibody (1:500) and the peroxidase-conjugated secondary antibody were incubated with the tissues sections at room temperature for 30 minutes each. Color was developed with aminoethylcarbazole (AEC). All immunostained samples were counter-stained with hematoxylin.

Four out of 315 birds were positive for West Nile Virus antigen on IHC and confirmed by PCR test. The WNV was mainly detected from the heart, spleen, kidney and intestine. Cellular targets included myocardial fibers, macrophages, renal tubular epithelium, intestinal crypt epithelium, fibroblast and smooth muscle cells.

Immumohistochemistry was an efficient and reliable method for identifying WNVinfections, but the polyclonal antibody cross-reacted with St. Louis encephalitis virus and other flaviviruses1.

Reference:

  1. Steele KE, Linn MJ, Schoepp N, et al: 2000, Pathology of Fatal West Nile Infections in Native and Exotic Birds during the 1990 Out break in New York City, New York. Vet Patholo 37:208-224.

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