Vaccination of domestic poultry with H5 vaccines has been implemented in several countries to limit the spread of H5N1 AI virus. Many studies have shown that vaccination of poultry with antigenically related vaccines protected birds from morbidity, mortality and virus excretion [
28‐
32]. However, insufficient efficacy of the vaccines was reported after the infection with antigenic drift variants [
16,
18,
19]. A recent study has also shown that H5N1 clade 7.2 viruses have not been eradicated from poultry in China through intensive vaccination [
33]. In the contrast to chickens, less is known about HPAI H5N1 infection in vaccinated turkeys. In this manuscript, we describe a vaccinal break in a meat-turkey flock vaccinated twice with two different inactivated H5 vaccines. Clinical signs and sudden onset of mortality were reported few weeks after the last vaccination. Interestingly, 29% mortality within 10 days was observed and surviving turkeys showed a transient drop in feed consumption and body weight. In the available literature no data are available on the efficacy of Re-5 to protect turkeys against challenge with Egyptian H5N1-like viruses. Re-5 was efficient to protect chickens from mortality after experimental infection with early H5N1 viruses in clade 2.2.1 and 2.2.1.1, although virus shedding was significantly higher than in birds vaccinated with a local vaccine [
34]. In an experiment, 20 to 100% mortality during a 10 days observation period were described in turkeys vaccinated with either H5N1 or H5N2 vaccines after challenge with Egyptian HPAIV H5N1 of clade 2.2.1.1 [
35].
In this study, data about the levels of anti-H5 antibodies in serum samples from the infected turkey flock are unfortunately missing. However, antigenic characterisation indicated antigenic drift particularly against serum samples from clade 2.2.1.1 in poultry in Egypt and to lesser extent against serum samples from genetically related 2.2.1.2 virus (Table
2). Also, TK16 showed remarkable antigenic drift against the recent H5N8 2.3.4.4 viruses, descendent clade from the vaccine strain. Unlike 2.2.1.1 viruses which circulated in Egypt from 2007 to 2014, viruses in clade 2.2.1.2 showed less, if any, antigenic variation, and vaccination of chickens was efficient even with highly diverse H5 vaccines derived from the Egyptian viruses [
34,
36]. However, TK16 showed a substantial genetic diversity at the protein level when compared with VacDK06 (clade 2.3.4) especially in HA and NA. In HA TK16 possessed up to 28 amino acids different from VacDK06. In addition to the 2.2.1.2 clade-specific signature (residues D43N, S120D, ∆129S I151T, R162K, G272S, R325K) which enabled efficient replication of the Egyptian 2.2.1.2 viruses in human cells and mice [
24,
37,
38], several mutations in the immunogenic epitopes (L71I, I83A, R140K, S141P, D155N, A156T, V174I, P181S, T263A) [
39] mostly in the HA1 head domain (Fig.
3) were observed in the new 2.2.1.2a subclade including TK2016. Some of these mutations are shared with viruses in clade 2.2.1.1 (e.g. L71I, R140K, S141P and A156T). These enabled the latter viruses to escape from the humoral immune response induced by H5 vaccines [
40] and resulted in vaccination failure in chickens [
34]. In the NA, unlike 2.2.1.1 clade viruses which contain only a single amino acid change compared to the viruses in 2006 and few mutations compared to the vaccine strains [
41], clade 2.2.1.2 viruses including TK16 possess several substitutions in the NA. Residue A20, I29, I34, S44, A46, S48, S50, T52, K53, A56, and K58 are located in the stalk region [
42] while the others are positioned at the head domain of the NA monomer (Fig.
3). Some amino acid changes are in the NA immunogenic epitope A (362, 366 equivalent to N2 numbering 385, 389), epitope B (180 equivalent to N2 199) or epitope C (residue 319 equivalent to 339 N2 numbering) [
43]. Meanwhile, 378 (equivalent to 402 N2 numbering) is located close to immunogenic epitope A, 204 (N2 numbering 223) close to epitope B and between two framework and functional residues of the sialidase, and 326 (equivalent to 349 N2 numbering) is close to epitope C [
43]. Together, continuous circulation of clade 2.2.1.2 viruses in vaccinated poultry resulted in the emergence of another antigenic variant sub-clade distinct from the human viruses in clade 2.2.1.2 resembling the situation in 2006 when the parental clade 2.2.1 diverged into two distinct clades: 2.2.1.1 in poultry in 2007–2014 and 2.2.1.2 in non-vaccinated poultry and humans in 2008–2016.
The source of infection in this flock is not yet clear. There was no infection in adjacent layers-flock premises according to the official reports. Since the turkey flock was vaccinated against NDV several days before starting the increased mortality the vaccination crew may be responsible for the introduction of the virus. This remains speculative, however. Moreover, phylogenetic analysis indicated a very close relationship of TK16 with recent viruses from Israel and Gaza in 2015. Since the flock was kept in an open system house, transmission by feral birds (e.g. pigeons, sparrows or egrets) or migratory birds (e.g. waterfowl) can not be excluded. Legal poultry trade between Egypt and both regions is not known and smuggling of poultry or feed is unlikely.