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Generation of information and tools to support the management of the avian influenza crisis in poultry

Final Report Summary - FLUAID (Generation of information and tools to support the management of the avian influenza crisis in poultry)

The primary goal of the 'Generation of information and tools to support the management of the avian influenza crisis in poultry' (FLUAID) project was the joint development and application of novel technologies to combat avian influenza (AI) infections. This goal has been achieved through the interaction of leading European institutes along with the active collaboration of laboratories in Australia, Indonesia, Pakistan, South Africa, Egypt and Vietnam.

AI outbreaks have recently caused severe losses to the poultry industry, its stakeholders and, ultimately, to the EU taxpayer. In addition, the ongoing global H5N1 crisis is a serious concern for food security and human health. The increased relevance of AI in the fields of animal and human health has highlighted the lack of scientific information on several aspects of the disease, which has hampered the adequate management of some of the recent crises thus resulting in millions of dead animals and concern over loss of human lives and over management of the pandemic potential.

The individual objectives of the project, were:
- studies on the development of prototype vaccines
- development of companion diagnostic tests based on anti-N detection systems
- studies on antigenic drift and genetic variability of selected H7 isolates
- development and validation of antigenic penside diagnostic tests
- tropism, pathogenesis and transmission in different avian species.

The project was structured into the following work packages (WPs):
- WP1: Coordination
- WP2: Vaccination
- WP3: Diagnostics and novel technologies
- WP4: Tropism, pathogenesis and transmission in different avian species.

The Ruggedised advanced pathogen identification device (R.A.P.I.D) system would function well as a pen-side portable molecular device for the diagnosis of AI if used with the recommended protocols.

Key conclusions and observations as regards animal experiments were as follows:
1. Vertical transmission of H5 LPAIV in ducks does not occur.
2. An age related association with clinical disease and viral dissemination exists in Pekin ducks when infected with H5N1 of the Eurasian lineage.
3. LPAI infected quails shed high concentrations of virus via the respiratory apparatus even though they show no clinical signs. Infected quail can also transmit the virus to contact birds rapidly. This suggests that quail may act as a reservoir of infection play a role in virus transmission.
4. Vaccination of quail using standard vaccination protocols for chickens and turkeys does not provide clinical protection.
5. Quail farms should be carefully monitored and managed given their ability to mask infection and transmit to other avian species.
6. Native chickens develop substantial HI antibody titers directed against H5N1 upon vaccination with a heterologous H5N2 vaccine, and that this provides a level of protection that is generally sufficient to prevent a productive H5N1 infection.
7. Vaccination with a heterologous H7N1 vaccine protects 12-week-old turkeys against challenge with H7N7 HPAI virus and no transmission of the virus occurs between the vaccinated turkeys.
8. An H5N2 vaccine strain with a low level of genetic and antigenic homology with the H5N1 challenge virus is able to reduce transmission in ducks significantly.
9. Evaluation of different serological tests for the detection of antibodies against highly pathogenic avian influenza in experimentally infected ostriches revealed that validated ELISA tests can be considered suitable test for screening purposes and should be preferred to AGID. In addition, the HI test (under specific conditions) remains a suitable test to monitor the circulation of AI in ostriches.

Results indicate that certain avian isolates of IV, in particular the HP H5N1 can more efficiently infect and activate porcine DC. It is considered that the activation is due to the replicative form of the virus RNA (dsRNA) interacting with TLR3 and / or cytosolic helicases). The capacity of inactivated virus to activate the DC is likely to be transmitted via the remnants of the now inactivated RNA genome. There will be adequate RNA remaining to activate via perhaps TLR7, but certainly via cytosolic helicases. Only when the RNA of the virus is removed, as when using virosomes of H5N1 origin, there is no longer an activation of the DC. This would confirm that the activation was dependent on the viral RNA, but not necessarily functional RNA in terms of virus replication. However, replication of the virus would allow for an elaboration of the viral RNA present, and therefore the degree of DC activation and induction of pro-inflammatory cytokines.

Infection of duck breeders from two species (Muscovy and Mule) with a low pathogenic H5 influenza virus did not lead to infection of their progeny. Moreover, when inoculated early into embryonated duck eggs (Muscovy and Mule ones), this virus and four other LP H5 viruses did not permit embryos inoculated with high doses to survive and to hatch. Ducklings that hatched from inoculated eggs never showed the presence of virus RNA or antibodies to the challenge virus. These results therefore invalidate the hypothesis of vertical transmission of H5 LPAIV in ducks.

Findings reported suggest that Japanese quails are readily infected with LPAI H7 viruses and that they may represent a source of infection for other poultry species during outbreaks. Therefore, it is reasonable to postulate that, during the 2002-2004 Italian H7 epidemic, quails might have played a role as reservoir of infection.

The results of the study indicate that a two-dose vaccination programme commencing at four weeks old, based on an inactivated, conventional vaccine registered for chickens, is not successful in preventing clinical signs and mortality and in suppressing shedding of viable virus. All vaccine titres were below the value considered protective for animals (1 : 32).

The results demonstrate that a widely used H5N2 vaccine strain that has a HA1 protein homology of 84 % with the H5N1 challenge virus not only prevented severe morbidity and mortality but also significantly reduced virus excretion and transmission of H5N1 in ducks two weeks after vaccination.

The data generated indicate that vaccination with H7N1 in turkeys was highly efficacious, Turkeys vaccinated once showed almost no virus shedding after challenge with H7N7 virus, and no transmission of the virus to vaccinated contact turkeys was observed. When the birds were vaccinated twice, no virus shedding was demonstrated at all after challenge, and hence transmission could not be quantified. Unvaccinated turkeys however, showed symptoms of illness, shed virus and were fully able to transmit the virus to susceptible contact turkeys. These results indicate that vaccination of turkeys could be useful as additional control measure to control an epidemic of HPAI as it was able to induce clinical protection and reduce virus shedding and virus transmission.

The main results of this study indicate that native chickens develop substantial HI antibody titers directed against H5N1 upon vaccination with a heterologous H5N2 vaccine, and that this provides a level of protection that is generally sufficient to prevent a productive H5N1 infection. Moreover, the results provide a proof-of-principle that vaccination with a heterologous vaccine can reduce transmission levels of H5N1 influenza virus to the extent that no epidemics can occur. In addition, the unvaccinated native chickens were not resistant to infection, and showed signs of infection that are comparable to layer chickens infected with H5N1 virus.

It is concluded that the N7 iIFAT and the N7 NI assay are useful tests in detecting silent transmission, because the birds that are most significant in spreading virus will be detected. On the other hand, due to the lower specificity, the N7 iIFAT and the N7 NI are less suitable for declaring freedom of disease after vaccination.

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