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.