The experimental study showed that not all inoculated ducks developed either HI or neutralizing antibodies by the end of the trial at 34 dpi. The virus may have failed to successfully replicate at the entry point in eyes, nasal or oral cavity of these antibody-negative ducks. Alternatively, the virus may have replicated to some degree at these mucosal entries, but was unable or had no chance to stimulate a humoral response because of a strong innate immune responses such as a rapid apoptosis mechanism [41] or RIG-I pathways [42]. Another possibility is that these antibody-negative ducks developed low HI and neutralizing antibody titres below the cut-off titre. The OIE recommended cut-off titre of 4 log2 [28] was used in this study for the dichotomization of test results. A lower cut-off of 3 log2 would have resulted in nine ducks classified seropositive by both HI tests instead of eight. Using a slightly lower HI test cut-off titre may be appropriate for the early detection of H5N1 HPAI exposure in disease free regions, but further confirmatory tests supported by epidemiological data on potential exposure status of birds are required to avoid false positive results. On the other hand, a cut-off of 4 log2 is perhaps preferable for measuring the proportion of antibody positives in countries where H5N1 HPAI is endemic and major reservoirs of all AI subtypes might exist.