Division 3 DIVA laboratory test for monitoring infection

As an alternative or in addition, testing for field exposure may be performed on the vaccinated birds itself by using DIVA laboratory tests. Several test systems have been developed in recent years that also allow the detection of field challenge of vaccinated birds. One method that has proved workable is the use of a vaccine containing a virus of the same haemagglutinin (H) subtype but a different neuraminidase (N) from the prevailing field virus. Antibodies to the N of the field virus act as natural markers of infection.

That system was used in Italy following the re-emergence of a LPAI H7N1 virus in 2000. In order to supplement direct control measures, a DIVA strategy was implemented using a vaccine containing H7N3 to combat an H7N1 field infection. Vaccinated and field exposed birds were differentiated using a serological test to detect specific anti-N1 antibodies. The same strategy was used to control LPAI caused by H7N3 in Italy in 2002 to 2003, in this case with an H7N1 vaccine and a serological test detecting antibodies against N3 specifically. In both cases vaccination with stamping out using this DIVA strategy resulted in eradication of the field virus.

Problems with that system arise if a field virus emerges that has the same N antigen as the existing field virus but are of another H subtype than H5 or H7 or if subtypes with the same N antigens are already circulating in the field. Particularly ducks are known to be carriers of more than one subtype. There was also a need to develop a suitable test that would allow the routine monitoring of flocks for anti-neuraminidase antibodies. In Italy an ‘ad hoc’ serological test based on an indirect fluorescent antibody assay, using as an antigen N proteins expressed by baculovirus recombinants was developed and used. That may have wider and easier application when an ELISA test is developed.

The use of vaccines containing only HA, such as recombinant vector vaccines, allows classical AGID tests or ELISA tests based on nucleoprotein, non-structural protein or matrix proteins to be used to detect infection in vaccinated birds.

For inactivated vaccines, a test that detects antibodies to the non-structural virus protein that are only produced during natural infection has been described. Such a system is yet to be validated in the field, but has the limitation that natural infection of a flock with any influenza virus, irrespective of subtype results in the production of antibodies directed against the non-structural protein.

The development of rapid and sensitive virus-detection methods, especially those that can be automated, such as real time RT-PCR, means that these could be used for simple widespread and regular testing of vaccinated birds for the presence of field virus. Agent detection, however, will be limited to a short window in the acute phase of the infection and cannot be used to conclude that a flock was not exposed to the virus in the past. This approach is most appropriate to the testing of vaccinated birds prior to movement to demonstrate freedom from active infection.

The number of samples to be tested by the systems of choice must enable the exclusion of a prevalence of AI virus infection in a flock of more than 15 % with confidence level of 95 %.