Open Access Highly Accessed Research article

Comparison of serological assays for detecting antibodies in ducks exposed to H5 subtype avian influenza virus

Hendra Wibawa123, Joerg Henning2*, Dessie Eri Waluyati3, Tri Bhakti Usman3, Sue Lowther1, John Bingham1, Akhmad Junaidi34 and Joanne Meers2

Author affiliations

1 CSIRO-Australian Animal Health Laboratory, Geelong, VIC, Australia

2 School of Veterinary Science, The University of Queensland, Gatton Campus, Gatton, QLD, Australia

3 Disease Investigation Centre Wates, Yogyakarta, Indonesia

4 Present address: Directorate General of Livestock and Animal Health Services, Ministry of Agriculture, Jakarta, Indonesia

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Citation and License

BMC Veterinary Research 2012, 8:117  doi:10.1186/1746-6148-8-117

Published: 23 July 2012



Chicken red blood cells (RBCs) are commonly used in hemagglutination inhibition (HI) tests to measure hemagglutinating antibodies against influenza viruses. The use of horse RBCs in the HI test can reportedly increase its sensitivity when testing human sera for avian influenza antibodies. This study aims to compare the proportion of positives detected and the agreement between two HI tests using either chicken or horse red blood cells for antibody detection in sera of ducks experimentally infected or naturally exposed to Indonesian H5 subtype avian influenza virus. In addition, comparison with a virus neutralisation (VN) test was conducted with the experimental sera.


In the experimental study, the proportion of HI antibody-positive ducks increased slightly, from 0.57 when using chicken RBCs to 0.60 when using horse RBCs. The HI tests indicated almost perfect agreement (kappa = 0.86) when results were dichotomised (titre ≥ 4 log2), and substantial agreement (weighted kappa = 0.80) for log titres. Overall agreements between the two HI tests were greater than between either of the HI tests and the VN test. The use of horse RBCs also identified a higher proportion of antibody positives in field duck sera (0.08, compared to chicken RBCs 0.02), with also almost perfect agreements for dichotomized results (Prevalence and bias adjusted Kappa (PABAK) = 0.88) and for log titres (weighted PABAK = 0.93), respectively. Factors that might explain observed differences in the proportion of antibody-positive ducks and in the agreements between HI tests are discussed.


In conclusion, we identified a good agreement between HI tests. However, when horse RBCs were used, a higher proportion of sera was positive (titre ≥ 4 log2) than using chicken RBCs, especially during the early response against H5N1 virus. The HRBC-HI might be more responsive in identifying early H5N1 HPAI serological response and could be a recommended assay for avian influenza sero-surveillance in both wild and domestic birds.

Avian influenza; H5N1; Hemagglutination inhibition test; Virus neutralization test; Horse red blood cells; Duck; Kappa