Overview Objectives

Project work is based around the following objectives:
to assess the breadth and duration of immunity induced by various inactivated AI vaccines
to characterise AI isolates from vaccinated poultry, quails and pigeons, at genetic and antigenic levels
to develop reagents for ELISA-based ‘DIVA’ testing (differentiation of infected from vaccinated animals) to differentiate between wild infection and vaccinated animals
to provide specialised training and capacity building for Indonesian scientists
to develop sustainable protocols for investigating apparent vaccine failures for HPAI
to develop a simple state-transition model of HPAI infection.

Project Background and Objectives

In Indonesia outbreaks of highly pathogenic avian influenza (HPAI) in poultry were first notified in August 2003. Initial cases were not contained and HPAI strain H5N1 has since spread throughout the country, with 30 out of 33 provinces now affected. Mortalities due to H5N1 have occurred in layers, broilers, native chickens, quails, pigeons, aquatic, wild and cage birds. Total losses in October 2005 were estimated to be more than 9 million chickens.
Human infections with H5N1 also occurred in Indonesia, with a disturbing 72 confirmed cases (55 of which were fatal - a 76% mortality rate). The source of H5N1 infections for the majority of human cases has been linked to direct contact with poultry.
For this reason, the Indonesian Government has opted for vaccination as a control measure in the hope it will reduce the incidence of H5N1 infection in all poultry and thus allay the threat to human health. But the efficacy of most of vaccines for control of HPAI in Indonesian situations is unclear. And vaccination is to be undertaken in the context of an infection that has become endemic and where a large proportion of poultry are kept under low biosecurity. This project stems from an Indonesian initiative, and addresses some of the issues identified as priorities in the Government of Indonesia Strategic Plan for Control of HPAI.

Progress Reports (Year 1, 2, 3 etc)

In Indonesia outbreaks of highly pathogenic avian influenza (HPAI) in poultry were first reported in August 2003. Initial cases were not contained and HPAI strain H5N1 has since spread throughout the country, with 31 out of 33 provinces now affected. Mortalities due to H5N1 have occurred in layers, broilers, native chickens, quails, pigeons, aquatic, wild and cage birds. Human infections with H5N1 also continue to occur in Indonesia, with the disturbingly high rate of deaths. Indonesian Government has opted for vaccination as a control measure in order to reduce the incidence of H5N1 infection and thereby the threat to human health. Vaccination has been undertaken in the context of an infection that has become endemic, where a large proportion of poultry are kept under low biosecurity and the unique nature of the H5N1, which has a number of properties not previously encountered in AI. Therefore vaccination strategies developed in countries with good resources, highly structured poultry industries, geographical compactness, and where infections occurred with less aggressive AI viruses, might need to be adjusted.

The strategies used in this project aim to identify the most efficacious vaccination strategy to reduce the incidence of HPAI in poultry. Specifically the project aims are to: (i) Assess the breadth and duration of immunity induced by various inactivated AI vaccines; (ii) characterise AI isolates from poultry vaccinated with selected vaccines (iii) develop reagents to differentiate field infections in vaccinated poultry (DIVA); (iv) develop protocols for investigating apparent vaccine failures for HPAI and (v) develop a simple state-transition model of HPAI infection.

The project is a collaboration between Indonesian Research Center for Veterinary Science (Bbalitvet), Bogor, Indonesia and three organisations in Australia: the University of Melbourne School of Veterinary Science (UoM), CSIRO Australian Animal Health Laboratory (AAHL) and AusVet Pty/Ltd.

The project commenced in late September 2007 when the majority of legal agreements were signed and project development process completed. This includes exchange of material of transfer agreements (MTA) related to the transfer and use of Indonesian HPAI strains in Australia. Initially it was ensured that standardized procedure and reagents are in place enabling comparison of results with other studies conducted internationally and in Indonesia. Baseline collection of data on antibody status in selected commercial flocks has commenced, as has the laboratory trail to establish levels and duration of antibody responses in controlled conditions. It was attempted to source reagents for various DIVA tests that have been published in international literature, but none were available. In the view of changing HPAI situation in Indonesia, several approaches for development of DIVA test have been considered and generation of several different reagents has commenced. Protocols and documentation on how to approach and undertake investigations of vaccination failures have been generated. A simple state transition model was developed and tested using standard parameters. This model appears functionally sound and shows promise as the basis of future modelling scenarios if suitable parameters for the Indonesian situation can be developed.

Where H5N1 is endemic, as is now the case in Indonesia, vaccine failures and disease in vaccinated birds must be expected. To further the investigation of vaccine efficacy and vaccine failures (Objectives 1 and 3) we aim to evaluate effectiveness of vaccination in commercial Sector 3 and develop methodologies, which Indonesian veterinarian authorities can apply to apparent vaccine failures. A comprehensive epidemiological study has commenced using the protocol developed in year 1. Sixty farms in three districts with highest concentration of poultry in the province of West Java were visited and data collected on flock health, vaccination and management. Analysis showed variable vaccination practices and importantly that disease outbreaks due to H5N1 are un-common. This finding has significant implications and supports the current vaccination policies. To confirm this observation samples were collected from selected farms for analysis of immunity and H5N1 status. A database, accessible to staff in Indonesia and Australia, has been developed for entry and analysis of epidemiological data. A bio-economic model (Objective 6) of Indonesian poultry flocks was developed and will be used to evaluate financial impacts of HPIA infection and prevention in surveyed flocks.
Characterisation of H5N1 isolates from vaccinated commercial poultry have progressed (Objective 4). Surveillance in village poultry by FAO has shown that H5N1 is already changing significantly in Indonesia. We attempted to characterise H5N1 isolates in Sector 3 where vaccination is comprehensive and biosecurity level is variable. Samples were collected from 20 multi-age farms vaccinated with four vaccines. A molecular test indicated that a large proportion of farms were positive for H5N1. This finding was unexpected and contradicted the survey data from Sector 3. Attempts to recover AI from these samples were unsuccessful. Further sampling will be carried out to elucidate this finding. Six H5N1 isolates collected by Bbalitvet between 2003 and 2007 were fully sequenced to obtain background data for comparison with isolates from vaccinated flocks. Sequence analysis showed that, in addition to variations in haemmagglutinin gene, variations also occurred in two other genes that play a role in AI infectivity.
Control of H5N1 in poultry is achieved using H5N2 inactivated vaccines. This strategy enables detection of H5N1 infections in vaccinated poultry using tests known as DIVA (differentiation of infected from vaccinated animals). Several approaches were undertaken to generate cheap, reliable and robust DIVA tests suitable for application on a large scale (Objective 2). Three H5N1 proteins, neuraminidase (N1), NS1 and M2 were evaluated as DIVA antigens. The N1 was purified and used in an immunoassay with a range of reference and field sera. Results showed that N1-based test could differentiate between vaccinated and non-vaccinated chickens however it could not be used for DIVA because of cross-reactive antibodies. This work was undertaken in Bbalitvet and a paper describing this work has been submitted for publication. The NS1 and M2 proteins are present in infected but not in vaccinated chickens, and are therefore good candidates for DIVA strategy. Both proteins are however difficult to obtain in sufficient quantities, and cheaply. We evaluated different in vitro expression systems to generate NS1 and M2 in large quantities and sufficient test specificity. Comparison of five expressed proteins identified one M2 construct that was able to differentiate infected and vaccinated chicks, with minimal non-specific reactions. Further modifications to this M2 construct are being made to produce a robust and simple DIVA test for routine diagnostic applications in Indonesia.
Information from this project contributes to showing that currently used vaccines are controlling H5N1 efficiently and that Sector 3 does not contribute to maintenance of H5N1 in the environment; therefore no need to curtail or re-structure this sector. Application of the DIVA test will support vaccination as an effective strategy potentially resulting in eradication, the goal of the currently implemented strategy.
During the period a John Allwright Fellow, commenced postgraduate study leading to Master of Veterinary Science. One project team member received training in Australia. Workshops on sampling for epidemiological survey were held with staff from two Disease Investigation Centres in West Java. One paper was accepted for publication. Three team members attended the 7th International Symposium on Avian Influenza, at the University of Georgia, Athens, USA. The Annual Project Meeting was held in Bbalitvet, Bogor, in March 2009.
The presence of H5N1 influenza virus in South East Asia, including Indonesia, is a potential source for transmission to Australia. Incursion and spread of H5N1 in Australia would represent a significant threat to public health and also to the profitability and sustainability of the local poultry industry. Australian Government is vitally interested in upgrading the readiness and strengthening the technical capability to respond effectively to H5N1 incursion. This project contributes to the national capacity building in several areas including disease monitoring and better understanding of the epidemiology of disease, capabilities that will help in formulating better response to the disease. Research links with Indonesia offer unique opportunity to broaden the knowledge and skill base in dealing with a quickly changing viral pathogen, experience applicable to other zoonotic agents and pathogens of veterinary importance in Australia.

Project ID
AH/2006/050
Project Country
Commissioned Organisation
University of Melbourne, Australia
Project Leader
Dr Jagodina Ignjatovic
Email
jagodai@unimelb.edu.au
Phone
(03) 9731 226536
Fax
(03) 9731 2366
Collaborating Institutions
CSIRO Livestock Industries, Australia
Directorate General of Livestock Services, Indonesia
Research Institute for Veterinary Science, Indonesia
AusVet Animal Health Services, Australia
Project Budget
$1,021,840.00
Start Date
01/03/2007
Finish Date
28/02/2010
Extension Start Date
01/03/2010
Extension Finish Date
30/09/2010
ACIAR Research Program Manager
Dr Doug Gray
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