Use of M2e ELISAs for longitudinal surveillance of commercial poultry in Indonesia vaccinated against highly pathogenic avian influenza

- Application of DIVA test is important for an effective control of H5N1 in poultry.
- An experimental M2e ELISA was evaluated for DIVA in vaccinated commercial layers.
- M2e antibodies were not detected in surveyed flocks indicating lack of H5N1 challenge.
- Finding of M2e ELISA was supported by other convectional but more laborious tests.
- The M2e ELISA could be a useful monitoring tool to ascertain freedom from H5N1 in vaccinated poultry.

In countries where highly pathogenic avian influenza virus (HPAIV) H5N1 is endemic and controlled by vaccination, post-vaccination serological monitoring is essential to differentiate vaccinated poultry from those that are infected. The objectives of this study were to validate two experimental ELISAs that detect antibodies raised against the M2e protein of avian influenza virus that can be used for DIVA purposes. Results from the sM2e and tM2e ELISAs were compared with other conventional tests for the detection of H5N1influenza virus (virus isolation and RT-PCR) using samples collected from 16 commercial flocks in Indonesia. These comprised vaccinated layers aged between 18 and 68 weeks old that were sampled at ten-weekly intervals. A small number of sera were positive in sM2e and tM2e ELISA, 14 (0.6%) and 17 (0.7%) respectively, with low OD420 (0.1-0.3), but only 4 sera were positive in both tests. At the flock level, the incidence of M2e positive sera was low (4%), well below previously established minimum of 40% for an HPAIV H5N1-infected flock. Conventional M and H5 gene RT-PCRs indicated that none of 16 flocks were infected at any time during the study. No virus was isolated from any of the 480 pooled swab samples, except from one, for which the combined data analysis suggest to be the result of a laboratory cross-contamination. Clinical disease, mortalities or reduction in production performance, indicative of field H5N1 challenge, were not observed either in any of the flocks. Birds from two surveyed flocks, challenged in the laboratory with an Indonesian HPAIV H5N1 developed M2e antibodies in 50% and 55% of surviving birds with OD420 in the range of 0.35-1.47 in tM2e ELISA, confirming the validity of the criteria established for use of M2e ELISA for DIVA purposes. Overall these results showed that the tM2e ELISA could be a useful monitoring tool to ascertain freedom from H5N1 infections in vaccinated commercial poultry.