This study presents the first report of employing horse RBCs in the HI test for detecting antibodies against H5 subtype of AI virus in ducks, the species that is considered to play a major role in the maintenance of HPAI H5N1 virus in Asia [21–23]. In both the experimental and field studies, the HI-HRBC test detected a greater number of sera as positive (titre ≥ 4 log2) than the HI-CRBC test, but overall there was a very good agreement between the HI tests. Previous studies have demonstrated that performing HI tests using horse RBCs could increase the detection of HI antibodies against avian influenza viruses in human sera [13, 14, 17–19]. Antibodies to the viral HA glycoprotein inhibit binding of avian-derived influenza viruses to their specific sialic acid receptor, which has a sialic acid, N-acetylneuraminic acid α-2,3-galactose (SAα2,3Gal) linkage . Horse RBCs express predominantly SAα2,3Gal linkages, whereas chicken RBCs express a mixture of SAα2,3Gal and SAα2,6Gal linkages , which accounts for the higher sensitivity of the avian influenza HI test using horse RBCs compared to chicken RBCs.
The experimental study showed that the HI-HRBC test was able to detect H5 antibodies at an earlier stage of serological response (8 dpi) than the HI-CRBC. At the later stages of infection, both HI-CRBC and HI-HRBC showed an equal dichotomised result. Better agreements were found between the two HI tests than between each HI test and the VN test, which might be due to differences in the binding specificities of antibodies detected in each of the tests. HI tests detect antibodies that inhibit viral hemagglutination, whereas the VN test detects antibodies that neutralize the virus and prevent replication in living cells ; not all antibody specificities that inhibit hemagglutination necessarily neutralize the virus, and conversely, not all virus neutralizing antibodies can inhibit hemagglutination caused by the virus .
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  or RIG-I pathways . 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  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.
A large disparity in the seroprevalence results between the two HI tests was demonstrated in the field sera. Overall, significantly (P < 0.001) more HI antibody-positive ducks were detected with HI-HRBC than HI-CRBC, but no sera that were positive in the HI-CRBC were negative in the HI-HRBC test. This indicates that the type of HI test used for assessing field sera has to be carefully chosen as the test performance will influence the reported measures of disease frequency, such as prevalence or incidence, for the population evaluated. In contrast a discrepancy between the proportions of positives identified with the HI tests was not evident in the experimental study. Compared to the experimental study, the H5N1-exposure status of ducks in the field study was unknown. Therefore, it is possible that some of the birds that tested negative in the HI-CRBC test were in the early response of H5N1 infection, which as the experimental study showed, is more likely to be detected with the HI-HRBC test than HI-CRBC.
The interpretation of the diagnostic test results should be conducted cautiously, particularly if it is performed to assess an agreement between tests using samples from different populations. The seroprevalence of ducks in the field was relatively low (8% or 2% using HI-HRBC and HI-CRBC, respectively) in contrast to the overall seroprevalence of 57-60% obtained in the experimental study. Factors related to the stage and severity of disease and the immune status vary within a population (and could be different in low- and high-prevalence populations) . Therefore the distribution of individuals sampled at different stages in the infection process will influence the prevalence estimated. On the other hand, it has been highlighted that the statistical relationship between kappa and prevalence (and bias) is multifaceted and complex [33, 44, 45]; two tests will have a higher kappa value if the prevalence is moderate (e.g. 50%) rather than very low or very high . Therefore the unadjusted kappa statistic was higher for the experimental study compared to the field study. In fact, it has been recommended by some statisticians to concentrate on populations with prevalence near 50% when comparing tests using the kappa statistic, rather than using adjusted measures such as the PABAK . However, in reality we often face situations with low or high prevalence values and this was also the case in our field study. Hence, we used the PABAK to evaluate the agreement of HI tests conducted with field samples and noted a similar agreement compared to tests conducted with samples collected in the experimental study.
The variety of individuals in terms of breed and age within the population are also important factors that might influence the ability of biological assays to detect infected and uninfected animals. The experimental study used homogenous birds, while in the field study ducks of genetically diverse breeds and of different ages were sampled. In addition, given the possibility of other sub-types of AI viruses circulating on duck farms, cross re-activity could have been another factor influencing the serological test results in the field study.
Adherence to recommend guidelines, such as the OIE Manual of diagnostic tests and vaccines for terrestrial animals , is essential to standardize diagnostic procedures in different laboratories. For example, OIE has recommended using RBCs from specific-pathogen or specific-antibody free chickens for HI tests to detect antibodies against AI viruses, including H5N1 HPAI  and we did so in both our experimental and field study. However, country-specific access to specific reagents might influence laboratory specific test variability. In this regard, our study might have had some limitations related to the use of different sources of H5N1 antigen and RBCs in the serological tests of sera between the two studies. Since we used the same H5N1 virus lineage (clade 2.1.1) for antigens, with 99.9% and 99.8% homologies detected in the hemagglutinin nucleotide and amino acid sequences among both viruses respectively (only one amino acid difference was found in a residue unrelated with either antigenic or glycosylation sites) , we assume that the variations in the HI results are likely to be related more to the exposure status and host-specific factors of birds (e.g. age, breed) than the types of antigen used.
It was beyond the scope and objective of this study to estimate the sensitivity and specificity of the HI tests, in particular in the absence of a gold standard for detecting antibodies against avian influenza viruses. Therefore, an assessment of HI test characteristics through future studies is recommended. Overall our finding support a previous study which reported that the use of horse RBCs increased performance of HI tests in measuring AI antibodies in other bird species . Thus, we suggest that the OIE should review these findings for an alternative serological method for the diagnosis and surveillance of HPAI in birds.