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Title: Detection of Newcastle disease virus RNA by reverse transcription polymerase chain reaction using formalin-fixed, paraffin-embedded tissue and comparison with immunohistochemistry and in situ hybridization

Author
item WAKAMATSU, NOBUKO - UNIVERSITY OF GEORGIA
item King, Daniel
item Seal, Bruce
item BROWN, CORRIE - UNIVERSITY OF GEORGIA

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/7/2007
Publication Date: 7/1/2007
Citation: Wakamatsu, N., King, D.J., Seal, B.S., Brown, C.C. 2007. Detection of Newcastle disease virus RNA by reverse transcription polymerase chain reaction using formalin-fixed, paraffin-embedded tissue and comparison with immunohistochemistry and in situ hybridization. Journal of Veterinary Diagnostic Investigation. 19:396-400.

Interpretive Summary: Newcastle disease (ND) continues to be a world wide problem. The diagnosis of a Newcastle disease virus (NDV) infection may be missed because infections of some bird species with viruses that are virulent for poultry may not produce a clinical manifestation typical of ND. Formalin fixed tissues saved as diagnostic pathology specimens from those disease outbreaks can serve as a retrospective sample source for those cases where ND was not considered in the initial diagnosis. A RT-PCR test was developed for detection of NDV genes in formalin fixed tissues and that assay was compared with the previously used immunohistochemistry (IHC) and in situ hybridization (ISH) tests of those tissues. Results of this study demonstrated a greater sensitivity of the RT-PCR procedure than the IHC and ISH assays. An additional benefit of the RT-PCR procedure is that the nucleotide sequence can also be determined from the amplified genes and that information can be compared with the sequence of other NDV strains to provide epidemiological information about the potential source of the virus that caused the outbreak.

Technical Abstract: The usefulness of reverse transcription polymerase chain reaction (RT-PCR) from formalin-fixed, paraffin-embedded (FFPE) tissues was examined and compared to the immunohistochemistry (IHC) and in situ hybridization (ISH) assays for detection of Newcastle disease virus (NDV). Spleen and lung tissues were collected from chickens experimentally infected with either of two NDV isolates, a low virulent virus (LaSota) and a virulent virus (from the 2002-2003 California outbreak). The tissues were harvested immediately postmortem and fixed in 10% neutral buffered formalin for approximately 52 hours. Also, just prior to euthanasia, oral and cloacal swabs were collected for virus isolation. RNA was obtained from the FFPE tissues by digestion with proteinase K and subsequent extraction with phenol, chloroform, and isoamyl alcohol. By semi-nested RT-PCR with primers for the NDV matrix gene, a 232-base pair (bp) product was generated and visualized by electrophoresis. The results of PCR were compared to those of IHC for viral nucleoprotein and ISH for matrix gene (850 bp) on 3-µm sections. Results of virus isolation from swabs were also compared to RT-PCR data. All samples from infected chickens were positive by RT-PCR, including samples that were negative by both IHC and ISH. The RT-PCR positives included tissue from chickens no longer shedding virus detectable by virus isolation. The RT-PCR was found to be an effective and sensitive method to detect NDV in FFPE tissues, and to our knowledge, this is the first report of NDV detection in FFPE tissues as a diagnostic approach possibly suitable for archival materials.