AI Surveillance Murdoch University projects

REVASIA meeting, Phnom Penh, 17th June 2009

Professor Stan Fenwick Veterinary Public Health

Current and completed AI projects at Murdoch University

Epidemiology of HPAI in Bali, Indonesia Surveillance for HPAI in wild birds in Thailand Movement of birds through live bird markets in Bali and Lombok and their role in the epidemiology of avian influenza Development of a risk based surveillance system to demonstrate freedom from Highly Pathogenic Avian Influenza (HPAI) in Timor Leste Development of a marker vaccine for AI surveillance Development of a DNA vaccine for AI in chickens Epidemiology of HPAI and vaccination efficacy in Vietnam

Epidemiology of AI in Bali, Indonesia

Epidemiology of AI in Bali, Indonesia

(Tze Hoong Chua PhD)

1. Laboratory evaluation of five influenza antigen detection tests to estimate their diagnostic sensitivity 2. H5N1 infection in healthy village poultry in Bali 3. Risks for spread of viruses from live poultry markets in Bali 4. Phylogenetic relationship of H5N1 viruses circulating in the village study sites

Epidemiology of AI in Bali, Indonesia

1. Overall sensitivities of rapid antigen tests ranged from 36.3% to 51.4%. Could be used for rapid and preliminary flock investigations of H5N1 outbreaks in sick and dead birds but should not be used for surveillance testing of clinically healthy birds

2. Surveillance recovered H5N1 HPAI virus at a low isolation rate (0.09% in chickens and 0.13% in ducks) in apparently healthy village poultry.

Surveillance activities in villages in Bali

Epidemiology of AI in Bali, Indonesia

3. Isolates from the surveillance of apparently healthy birds shared close phylogenetic relationship with poultry viruses from outbreak cases (under subclade 2.1) and also contained the characteristic HPAI molecular pathotypes. 4. A case-control study of village household flocks was performed which identified risk factors that could influence the occurrence of H5N1 HPAI. Sale of poultry to collectors remained significantly associated with highly pathogenic poultry diseases (p0.1)

Surveillance for HPAI in wild birds in Thailand

(Jarunee Siengsenan PhD)

1. Existing data analysis from Thai Govt. surveillance 2. Serology and virology surveillance program for HPAI H5N1 in wild birds 3. Molecular epidemiology of virus isolates 4. Observational study of poultry/wild bird interactions on selected farms 5. Quantitative risk assessment

Study area selection based on district where infections were identified in open billed storks
Banglane District, Nakornpratom province

Bangsripa subdistrict Banglane subdistrict Klongnokkratong subdistrict Bangpasri subdistrict

Rice paddy fields

Villages and farms

Field sample collection Targeting multiple species/multiple trapping techniques working with Dept of Wildlife Samples including throat swabs, cloacal swabs, and serum

Laboratory tests Viral isolation: MDCK cell culture Haemagglutination test (HA) RT PCR: specific primers to H5, N1, and M genes Neutralization Test (NT) Gene sequencing Molecular analysis Sequence results were compared with other Thai sequences submitted to Genbank to generate phylogenetic trees. Softwares including BioEdit, Mega, and ExPasy were used to create trees.

 Total 421 healthy wild birds sampled(44 species) No concurrent poultry outbreaks Sero-prevalence in wild birds was 2.1% (8 out of 385 samples) Species tested positive by NT: Rock Pigeon (Columba livia) Asian Pied Starling (Gracupica contra) Spotted Dove (Streptopelia chinensis) Oriental Magpie Robin (Copsychus saularis) Blue-tailed bee-eater (Merops philippinus) Myna (Acridotheres spp.) Pond heron (Ardeola spp.) Species highlighted in red are common resident birds which have close contact with poultry on farms or in paddy fields

 Virus prevalence was 0.5% (2 out of 421 samples). Positive samples were from one Asian Pied Starling (Gracupica contra) and one White Vented Myna (Acidotheres grandis) both common resident species on farms
Asian pied starling White vented myna

Both viral samples clustered with other samples isolated in Thailand between 2004 and 2006 Clinically healthy resident wild bird species showed evidence of previous or current H5N1 infection with H5N1 viruses albeit at low prevalence. This data does not elucidate the direction of movement or source of virus transmission to the wild birds (domestic poultry to resident wild birds or vice versa; origin from migratory birds, domestic poultry or indirectly via human movements)

Movement of birds through live bird markets in Bali and Lombok and the role of markets in the epidemiology of AI
(Johanna Johnson PhD)

1. Cross-sectional studies to determine trends in quantity, sources, destinations and management of chickens and ducks at selected markets in Bali and Lombok 2. Detection of HPAI H5N1 virus in selected markets in Bali and Lombok 3. Qualitative assessment of risk associated with chicken and duck movements 4. Social network analysis of market movements

Questionnaires for Collectors, Vendors and Customers developed and applied at selected markets in Bali and Lombok

Cross-sectional studies of live bird markets in Bali and Lombok

1. Live birds in the supply chain come from various sources in the island such as villages, and sub-district traditional markets 2. Birds from backyard farmers are collected by village level collectors then brought to sub-district and district level live bird markets. From these markets the birds are purchased by customers for different purposes such as consumption (households, and restaurants), offerings in ceremonies and religious festivals, and for replacement stock for farmers

Cross-sectional studies of live bird markets in Bali and Lombok

3. This study found that the numbers of birds at markets tends to increase during certain times and months of the year such as Galungan and Kuningan in Bali, and Fasting and Hari Raya months in Lombok . 4. Vendors, Collectors, and Customers all want and tend to buy healthy birds 5. Transportation and management of birds involve poor biosecurity practices. Collectors and vendors do not separate birds according to bird species and sources. Birds are mixed in cages during transportation and at markets. Birds are also moved from one market to another with mobile vendors and collectors

? HPAI infection in duckling at a poultry market and close contact with child

Development of a risk based surveillance system to demonstrate freedom from HPAI in Timor Leste
(Acacio Amaral PhD)

1. To demonstrate the absence of Highly pathogenic Avian Influenza (HPAI) in Timor Leste. 2. To define the potential pathways for the introduction of AI into East Timor and carry out a qualitative and quantitative analysis of the risks .

Development of a risk based surveillance system to demonstrate freedom from HPAI in Timor Leste 1. Planned, targeted surveillance strategies for virus detection 2. Development of questionnaires for farmers, livestock officers, quarantine and other related stakeholders 3. Identification of possible risk pathways for HPAI introduction from infected countries into the country

Development of a risk based surveillance system to demonstrate freedom from HPAI in Timor Leste 4. Identification of risk factors for disease transmission to domestic poultry from wild birds 5. Collection of samples from targeted high risk areas at the border of Timor Leste and Indonesia

(Trevor Ellis, Cassie James, Stan Fenwick, DIVA Solutions)

Development of a marker vaccine for AI surveillance

1. 2. 3. 4. 5. 6.

Tetanus toxoid (TT) evaluated as an exogenous marker for poultry vaccination No evidence of natural antibodies to TT in poultry ELISA developed to detect Ab in chickens, competitive ELISA for detection in ducks Long-lasting Ab response post TT vaccination in chickens and ducks No interference with immunity when TT and AI vaccines given together, evaluated via challenge trial with H5N1 Trial batch of TT/H5N2 vaccine formulated by Intervet and tested in a field trial in Hong Kong, detection of TT Ab allowed rapid detection of vaccinated birds

Benefits of an AI marker vaccine

1. TT is a highly suitable marker for AI vaccination in poultry allowing simple and effective monitoring of AI vaccination status via cheap, rapid ELISA testing

2. TT is a good candidate as it is cost-effective, easy to manufacture, and is a commercially available vaccine for food animals and humans 3. The versatility and robustness of the exogenous TT marker means that it can be formulated with any vaccine type (inactivated, reverse genetics) or with any poultry vaccine (AI, ND)

Benefits of an AI marker vaccine

4. TT strategy can improve control of poultry movements from farms to markets based on serological evidence of successful vaccination using approved vaccines as birds would need to show positive antibody responses to the TT marker with concordant HA antibody levels that were adequate for protection 5. Other benefits include evaluation of vaccination methodology and efficacy, evidence of vaccination coverage - market and slaughterhouse surveillance

Epidemiology of HPAI and vaccination coverage in Vietnam

(Stephanie Desvaux PhD)

1. To estimate vaccination effectiveness (qualitative and quantitative evaluation) within chicken and duck farms in a selected area of northern Vietnam 2. To study the serological response profile of vaccinated birds and the duration of immunity under field conditions 3. To estimate the backyard poultry population dynamics and vaccination coverage in selected households in the study area