Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/9/2007
Publication Date: 9/1/2007
Citation: Swayne, D.E., Avellaneda, G.E., Mickle, T.R., Pritchard, N., Cruz, J., Bublot, M. 2007. Improvements to the hemagglutination inhibition test for serological assessment of recombinant Fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals. Avian Diseases. 51:697-704.
Interpretive Summary: Differentiating vaccinated from infected animals (DIVA) is a recurring problem in avian influenza (AI) control. To eliminate this problem, we vaccinated chickens with a recombinant fowlpox vaccine containing an AI H5 gene insert and examined them before and after challenge with high pathogenicity (HP) AI virus for antibodies that would indicate vaccination with and without infection. Using the hemagglutination inhibition (HI) test with the test hemagglutinin protein identical to that produced by the vaccine, we consistently detect HI antibodies in all vaccinated chickens, but no antibodies against the nucleoprotein. In addition, after HPAI virus challenge, antibodies against both the hemagglutinin and nucleoprotein were detected. This methodology will be an effective DIVA strategy in AI vaccination and control.
Technical Abstract: Avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, respiratory and intestinal replication of an AI challenge virus. Therefore, surveillance programs must be developed to differentiate infected from non-infected flocks of vaccinated poultry (DIVA) in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for protective immunological response by hemagglutination inhibition (HI) test and determination of infection status following highly pathogenic AI virus challenge by agar gel immunodiffusion (AGID) test. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes (NP/M), but 0-100% had HI antibodies depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure and whether the birds had been pre-immunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) high pathogenicity (HP) AI virus strain as the Beta-propiolactone (BPL)-treated HI test antigen which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether/BPL-treated antigen was used in place of the BPL-treated antigen. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from non-infected birds in vaccinated populations of chickens when using standard AGID and HI tests.