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Title: Development and evaluation of an avian influenza, neuraminidase subtype 1, indirect enzyme-linked immunosorbent assay for poultry using the differentiation of infected from vaccinated animals control strategy

Author
item LIU, YING - University Of Georgia
item MUNDT, EGBERT - University Of Georgia
item MUNDT, ALICE - University Of Georgia
item SYLTE, MATTHEW - University Of Georgia
item Suarez, David
item Swayne, David
item GARCIA, MARICARMEN - University Of Georgia

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/1/2009
Publication Date: 3/1/2010
Citation: Liu, Y., Mundt, E., Mundt, A., Sylte, M.J., Suarez, D.L., Swayne, D.E., Garcia, M. 2010. Development and evaluation of an avian influenza, neuraminidase subtype 1, indirect enzyme-linked immunosorbent assay for poultry using the differentiation of infected from vaccinated animals control strategy. Avian Diseases. 54:613-621.

Interpretive Summary: Identifying infected chickens within a vaccinated population is critical in avian influenza control programs. A specific serological test, the indirect ELISA, was developed and bench validated to detect antibodies against a neuraminidase type 1 protein. The test was negative for N1 antibodies after vaccination with H5N2 and H5N9 vaccines, but was positive for N1 antibodies after challenge with highly pathogenic H5N1 and H7N1 avian influenza viruses. This test will be an effective and rapid assay to identify exposure to H5N1 field virus during a differentiation of infected from vaccinated animals (DIVA) vaccination strategy.

Technical Abstract: An indirect ELISA was developed using baculovirus expressed N1 protein from the A/chicken/Indonesia/7/2003 (H5N1) virus. The specificity of the assay was tested with a panel of chicken antisera raised against N1 to N9 virus subtypes. The N1-ELISA was specific for the detection of N1 antibodies in chicken and turkey sera and had a percentage of agreement of 64 to 67% when compared to commercially available ELISAs. The ability of the N1-ELISA to discriminate vaccinated from subsequently challenged specific pathogen free (SPF) chickens was tested. The chickens were either vaccinated with H5N2, H5N9, and challenged with highly pathogenic H5N1 viruses; or vaccinated with recombinant poxvirus vaccine encoding H7 and challenged with highly pathogenic H7N1 virus. Serum samples were collected pre-challenge (n=45), 14 days post-challenge (n=45), and tested by hemagglutinion inhibition (HI), quantitative neuraminidase inhibition (NI), and the N1-ELISA. At two days post-challenge oropharyngeal swabs were collected for virus isolation (VI) to confirm infection. As compared to VI, the overall analytical specificity (Sp) of the N1-ELISA was 1.00, and the analytical sensitivity (Se) was estimated at a ratio of 0.51. N1 antibodies were detected by N1-ELISA and NI assay in 40% and 73% of the samples tested, respectively. Although the N1-ELISA showed a lower sensitivity it was demonstrated that screening of N1 antibodies by ELISA was an effective and rapid assay to identify exposure to the challenge virus during a differentiation of infected from vaccinated animals (DIVA) vaccination strategy. The N1-ELISA should be utilized and interpreted as a flock assay and should be complemented by other rapid diagnostic assays.