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United States Department of Agriculture

Agricultural Research Service

Research Project: APPLICATION OF BIOLOGICAL AND MOLECULAR TECHNIQUES TO THE DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA AND OTHER EMERGING POULTRY PATHOGENS

Location: Exotic and Emerging Avian Viral Diseases Research Unit

Title: Development and evaluation of an avian influenza (AI) neuraminidase subtype 1 (N1) based serological ELISA for poultry using the differentiation of infected and vaccinated animals (DIVA)approach

Authors
item Liu, Yuru - UNIV GA, PDRC
item Sylte, Matt - UNIV GA, VET MED
item Swayne, David
item Mundt, Egbert - UNIV GA, PDRC
item Garcia, Maricarmen - UNIV GA, PDRC

Submitted to: International Symposium on Avian Influenza
Publication Type: Abstract Only
Publication Acceptance Date: January 20, 2009
Publication Date: April 5, 2009
Citation: Liu, Y., Sylte, M., Swayne, D.E., Mundt, E., Garcia, M. 2009. Development and evaluation of an avian influenza (AI) neuraminidase subtype 1 (N1) based serological ELISA for poultry using the differentiation of infected and vaccinated animals (DIVA)approach [abstract]. Abstracts of the 7th International Symposium on Avian Influenza, April 5-8, 2009, Athens, Georgia. p. 67.

Technical Abstract: An indirect enzyme-linked immunosorbent assay (ELISA) was developed using baculovirus expressed N1 protein from the A/CK/Indonesia/PA/2003 (H5N1) virus. The specificity of the assay was tested with a panel of chicken anti-sera raised against N1 to N9 virus subtypes. The N1-ELISA was specific for the detection of N1 antibodies in chicken serum raised against North America H1N1 viruses with a percentage of agreement of 70 to 73% when compared to commercially available ELISAs. The ability of the N1-ELISA to detect vaccinated/infected birds in specific pathogen free (SPF) chickens vaccinated with H5N2, H5N9, and infected with Asian H5N1 viruses; and chickens vaccinated with Pox-H7 vector vaccine and infected with highly pathogenic H7N1 virus. Serum samples were collected pre-challenge (n=45), 14 days post-challenge (n=45), and tested by hemagglutining 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 analytical specificity (Sp) of the N1-ELISA was estimated at a ratio of 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 on 40% and 73% of the samples tested, respectively. Although the N1-ELISA showed lower sensitivity, it was shown that screening for 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. However, the N1-ELISA should be utilized and interpreted as a flock assay, and should be complemented with other rapid diagnostic assay.

Last Modified: 8/22/2014