Sensitivity and specificity of monoclonal and polyclonal immunohistochemical staining for West Nile virus in various organs from American crows (Corvus brachyrhynchos).
BACKGROUND
Based on results of earlier studies, brain, heart and kidney are most commonly used for West Nile virus (WNV) detection in avian species. Both monoclonal and polyclonal antibodies have been used for the immunohistochemical diagnosis of WNV in these species. Thus far, no studies have been performed to compare the sensitivity and specificity of monoclonal and polyclonal antibodies in detecting WNV in American crows (Corvus brachyrhynchos). Our objectives were to determine 1) the comparative sensitivities of monoclonal and polyclonal antibodies for immunohistochemical (IHC) diagnosis of WNV infection in free-ranging American crows, 2) which organ(s) is/are most suitable for IHC-based diagnosis of WNV, and 3) how real-time RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tissues compared to IHC for the diagnosis of WNV infection.


METHODS
Various combinations, depending on tissue availability, of sections of heart, kidney, brain, liver, lung, spleen, and small intestine from 85 free-ranging American crows were stained using a rabbit-polyclonal anti-WNV antibody as well as a monoclonal antibody directed against an epitope on Domain III of the E protein of WNV. The staining intensity and the extent of staining were determined for each organ using both antibodies. Real-time RT-PCR on formalin-fixed paraffin-embedded tissues from all 85 crows was performed.


RESULTS
Forty-three crows were IHC-positive in at least one of the examined organs with the polyclonal antibody, and of these, only 31 were positive when IHC was performed with the monoclonal antibody. Real-time RT-PCR amplified WNV-specific sequences from tissue extracts of the same 43 crows that were IHC-positive using the polyclonal antibody. All other 42 crows tested negative for WNV with real-time PCR and IHC staining. Both antibodies had a test specificity of 100% when compared to PCR results. The test sensitivity of monoclonal antibody-based IHC staining was only 72%, compared to 100% when using the polyclonal antibody.


CONCLUSIONS
The most sensitive, readily identified, positively staining organs for IHC are the kidney, liver, lung, spleen, and small intestine. Real-time RT-PCR and IHC staining using a polyclonal antibody on sections of these tissues are highly sensitive diagnostic tests for the detection of WNV in formalin-fixed tissues of American crows.
Smedley
Rebecca C
RC
Diagnostic Center for Population and Animal Health and Department of Pathobiology and Diagnostic Investigation, MI, USA. smedley@dcpah.msu.edu <smedley@dcpah.msu.edu>
Patterson
Jon S
JS
Miller
RoseAnn
R
Massey
Jeffrey P
JP
Wise
Annabel G
AG
Maes
Roger K
RK
Wu
Ping
P
Kaneene
John B
JB
Kiupel
Matti
M
D000818:Animals; D000911:Antibodies, Monoclonal; D046368:Crows / Q000821:virology*; D003933:Diagnosis; D007150:Immunohistochemistry / Q000379:methods / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D018571:Sentinel Surveillance / Q000662:veterinary; D013048:Specimen Handling; D014481:United States; D014902:West Nile virus / Q000235:genetics / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal