CSIRO Livestock Industries

Objective 1 Reduce the risk of WSD in shrimp farms through the application of PCR-based detection tests and epidemiological probes.
A major experimental component of this project is a large longitudinal study of shrimp ponds in India. The aim is to obtain information on the quality of PCR screening results available to farmers and to use molecular epidemiological analysis to trace the sources of disease outbreaks in ponds. The longitudinal study was conducted at a NACA/MPEDA/ACIAR study area in the West Godavari District of Andhra Pradesh. The site comprises 27 farm clusters around 14 villages between Bhimavarum and the mouth of the Godavari River. Farmers participating in the study are members of "aquaclubs" formed as part of an ongoing extension program of best management practices. The farms were stocked in February - April 2005 with PCR-screened post-larvae (PLs) obtained from local hatcheries. Some farmers employed nursery ponds to improve survival rates during grow-out. Each nursery pond served to seed a number of grow-out ponds at the same location. A total of 27 nursery ponds were sampled on stocking; 19 of these were also sampled at harvest. A total of 457 grow-out ponds were sampled at the time of stocking. Samples were also collected during disease outbreaks from 14 ponds, emergency harvests from 52 ponds, and planned harvests from 277 ponds. Wild shrimp were collected from 23 ponds and crabs from 65 ponds during the period between stocking and harvest.

The shrimp samples were preserved in alcoholic fixative for subsequent analysis by PCR. Other samples collected from disease outbreaks and emergency harvest ponds were preserved in Davidson's fixative for histological analysis. All samples were transported to CIBA in Chennai and catalogued. Samples for PCR analysis were divided into two equivalent sets. Testing of samples in the first set will be conducted at CIBA and the College of Fisheries in Mangalore. The second set of samples will be transported to CSIRO in Australia. All histology samples were retained for preliminary analysis at CIBA. Laboratory analyses on all of these samples will be reported during the next period.

Objective 2 Reduce the risk of yellow head and other diseases in shrimp farms through the application of PCR-based detection tests and epidemiological probes.

Monodon slow growth syndrome (MSGS) is a newly emerging disease that has impacted severely on P. monodon production in Thailand with losses in 2004 estimated at ~40 million baht. Work at Centex Shrimp in Thailand has suggested that the disease is infectious. Examination of shrimp displaying signs of the disease has identified three infectious agents - a yellow head virus genotype, a new shrimp virus (Laem Singh virus - LSNV) and a microsporidium. It is not yet clear which, if any, of these agents is the primary cause of MSGS. LSNV has been partially sequenced and a PCR detection test developed. Using this test, the virus can be detected in healthy shrimp as well as those showing signs of slow growth. In June-July 2005, Dr Sitidilokratna of Centex Shrimp visited CSIRO to apply the PCR detection test to P. monodon samples from throughout the Indo-Pacific region. LSNV was detected in shrimp from Thailand, Malaysia and Indonesia but not in samples from Australia, India, Vietnam, Mozambique or Fiji.

Objective 3 Improve the effectiveness of PCR-based viral screening in hatcheries and service laboratories in India. Indonesia and other countries in the Asian region.
The project also aims to enhance technical capabilities in India and Indonesia through PCR training workshops and the inter-calibration of PCR testing performance between laboratories. The first PCR training workshop was held in Bogor, Indonesia in August 2005. The workshop was attended by 24 participants from laboratories throughout the Indonesian archipelago. A second PCR training workshop was held at CIBA in India in October 2005 with financial support from MPEDA. The workshop was attended by 25 participants from hatcheries and government, private and research laboratories in seven States, and 3 international participants (Sri Lanka, Bangaldesh and Myanmar) supported by ACIAR. Each workshop included practical sessions and an open Seminars which were attended by representatives from the research, government and industry sectors.

Mr Agus Sunarto and Mrs Isti Koesharyani from the Fish Health Research Laboratory, Jakarta, and Mrs Christina Handayani from the Center for Brackishwater Aquaculture Development, Japara, Indonesia visited AAHL in July 2005. These scientists assisted with preparation for the training workshop in Bogor and participated as trainers in the workshop. They were also instructed in the use of real-time PCR during the visit to AAHL. Dr Nursa Sittidilokratna of Centex Shrimp, Thailand visited AAHL in June-July 2005 to work with CSIRO staff on studies of LSV. Dr Sitidilokratna also advised on the implementation of training workshops and participated as a trainer.

This project aims to reduce risks of disease outbreaks in small-holder shrimp farms by a combination of strategies involving training of PCR laboratory technicians, the inter-calibration of PCR laboratories test performance and improved health management practices derived from a better understanding of sources of white spot disease on farms. A major aspect of the project is a large field study at a site in Andhra Pradesh, India. The study is the first in a series of investigations that aim to assess the quality of PCR screening available to small holder shrimp farmers and to identify the sources of disease outbreaks in shrimp ponds. The study involved a total of 457 ponds from 28 clusters in 15 villages. The farms were stocked in early 2005 with PCR-screened PLs obtained from local hatcheries. Samples of shrimp post-larvae (PLs) were collected from farmers at the time of stocking and from juvenile shrimp and wild crustaceans from the ponds during grow-out and at the time of disease outbreaks and planned or emergency harvest. Samples collected from the site were tested by laboratory staff in India and Australia by PCR to identify infected stock and assess the source of infection. Samples from disease outbreaks were also examined by histology to determine the cause of disease. The results indicate that very few of the PL batches used by the farmers in the study were positive by PCR tests conducted in India and Australia by the project team, suggesting that PCR screening conducted by local laboratories was relatively effective. However, PCR testing of samples collected from shrimp at planned or emergency harvest indicated there was a very high prevalence of WSSV infection in the study ponds with a high proportion of moderate-severe infections. There was also evidence that a small number of nursery ponds were the source of seed for a high proportion of disease outbreaks during grow-out. There was also evidence of clustering according to the village under study with 2 villages in particular being major foci of infection and disease. The data suggests that nursery ponds may be a weak link in the current disease management practice.

Work has also continued in the search for infectious agent associated with monodon slow growth syndrome (MSGS), a newly emerging disease that has impacted severely on P. monodon production in Thailand. At Centex Shrimp in Bangkok, a new shrimp virus (Laem Singh virus - LSNV) has been investigated. Although LSNV occurs commonly in P. monodon in Thailand, it is thought that progression of the infection in the optic nerve may be the cause of slow growth. To determine if LSNV also occurs in India, 205 samples collected in the field study in Andhra Pradesh were screened for the presence of the virus using PCR test developed at Centex Shrimp. Although no evidence of infection was detected, we have concerns about the stability of the LSNV RNA after prolonged storage in ethanol and new set of samples will be collected in early 2007.

To improve the reliability of PCR-based screening, the first of two inter-laboratory calibrations of WSSV PCR testing was conducted in India in June 2006. Forty-nine service laboratories from the government and private sectors and shrimp hatcheries received equivalent sets of randomly coded positive and negative samples comprising fixed shrimp tissues WSSV DNA for testing. Seventeen laboratories correctly identified all positive and negative samples and 3 laboratories failed to detect only one low positive sample. Six laboratories reported negative results for positive samples indicating problems with test sensitivity. Nine laboratories reported positive results for negative samples indicating problems with test contamination. Two laboratories reported incorrect results for both positive and negative samples. The results were collated returned to all participating laboratories is a form that allowed them to see the results of all laboratories but identify only themselves by code. A second inter-calibration is planned following the second PCR training workshop in early 2007 and it is expected that a national PCR laboratory accreditation program will commence in 2008. A similar inter-calibration commenced in Indonesia in December 2006.

The second in a series of 2 PCR training workshops has been completed in India. The second workshop was conducted in October 2006 for essentially the same group that attended the first workshop in 2005 including 23 from hatcheries and government, private and research laboratories in India and 3 participants from Sri Lanka, Bangaldesh and Myanmar. The second workshop provided more focussed training and assessment with each participant required to perform every step in the process at least once during the 4-day program and feedback was given on the basis of individual rather than group performance.

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.

Significant progress was made in this reporting period. With respect to the setting-up of the project in Myanmar, a project vehicle was purchased in Australia and shipped to Myanmar in November 2003. The vehicle will allow project activities to be carried out more effectively, in particular for visits by project staff to field sites and for collection of samples from chickens in the project survey. A project office was established in the buildings of the Livestock Breeding and Veterinary Department (LBVD) at Insein, Yangon. Office equipment and computers were purchased and an internet connection to the project office was set up. This was a major achievement and allows the project to have its own internet account and email address. The email address aciar [at] mail4u [dot] com [dot] mm
Progress relating to each of the project objectives is outlined below:

Objective 1 To enhance the production, quality and distribution of thermostable Newcastle disease vaccine in Myanmar Items of equipment for the viral vaccine production laboratory were purchased and transported to Myanmar. Data loggers were also obtained and taken to Myanmar. Myanmar staff were provided with basic instructions on the use of the data loggers and associated software. The data loggers will be used from March 2004 when the quarterly vaccine distribution program commences.

Objective 2 To develop appropriate extension materials to enhance sustainable usage of Newcastle disease vaccines and to improve the capacity of government staff in extension methodologies In November 2003, Dr Henning conducted a training session on collection of questionnaire data from village households. This is one facet of extension techniques. Training and development of extension methodologies will be extended prior to and during the vaccination intervention stage of the survey. The extension materials related to Newcastle disease control that are available in Myanmar have been collected by project staff and are currently being reviewed.

Objective 3 To improve the capacity of government staff to diagnose the major diseases of poultry In December 2003, a one-week workshop in diagnostic pathology was conducted by Dr Ian Wilkie and Professor Allan Frost. This course concentrated on the methodology of rational diagnosis with emphasis on gross pathology and microbiology. Practical classes utilised chickens as the demonstration animal, however, the principles taught are applicable to all species.

Objective 4 To determine the major constraints on the production of village poultry in Myanmar, and to specifically evaluate the impact of Newcastle disease and the effect of vaccination The major activity of the project is to conduct a survey to identify constraints on poultry production in villages in Myanmar and to determine the impact of vaccination against Newcastle disease. Following discussions among the Australian project personnel, including Dr Henning (epidemiologist), Dr Rutherford (agricultural economist), Dr Wilkie (pathologist), Dr Meers and Prof Spradbrow (virologists) and Dr Pym (poultry production specialist), two questionnaires were designed for this survey. One will be used for an initial interview with village householders (baseline study) and the other will be used for monthly data collection. The baseline questionnaire was tested in a pilot trial in 4 selected households. Based on results, several questions were modified, the questionnaire was shortened and other changes made to simplify the interview process.
Following consultation with Myanmar project staff, the survey strategy was finalised. The survey will be divided into two stages: a collection of background data (over a 6-month period) and an intervention study with Newcastle disease vaccination (over a 12-month period). The villages to be involved in the survey were selected and lists of households prepared. The sample size for the survey was calculated, based on a two stage sampling frame. A total number of 307 households will be sampled, from 10 villages in two separate townships (one north and one south of Yangon).
An information session was held in November 2003, attended by all of the field veterinarians, district and township veterinary officers and staff from the LBVD who will be involved in the survey. The questionnaires were explained, and a practical exercise was held to instruct on methods for collection of blood samples from chickens. The survey commenced in November 2003.

Objective 5 To genetically characterise field isolates of Newcastle disease virus from Myanmar and to confirm the efficacy of I-2 vaccine against these isolates Equipment for the molecular studies was purchased and transported to Myanmar. Discussions were held regarding the timing and format of a training workshop on molecular techniques. This will be held in February 2004 and will involve 3-5 trainees.

Significant progress was made during this reporting period. A baseline study to identify major constraints to village chicken production was completed in May 2004 and was followed in June 2004 by a 12-month intervention study. Results of the baseline study were presented at one international poultry conference and at a number of other international meetings. Necessary laboratory equipment was purchased for diagnosis of poultry diseases and Livestock Breeding and Veterinary Department (LBVD) staff members were trained in molecular techniques, serological diagnosis of poultry viruses and database work. Extension pamphlets on the intervention strategies and I-2 vaccination were produced. Specific progress relating to each of the project objectives is outlined below.

Objective 1

Items of laboratory equipment were purchased for the vaccine production laboratory and diagnostic laboratory. Participants from LBVD attended an ACIAR-funded refresher course in Newcastle disease vaccine production and quality control in Laos and were trained in problem solving during vaccine production. Two refrigerators for both veterinary township offices and chilly bins for every enumerator participating in the study were purchased for vaccine storage and distribution in the project villages. Data loggers were used to monitor the exposure of vaccine virus to environmental temperatures.

Objective 2
An extension pamphlet describing the management changes and different causes of mortality in village chicken based on the data collected in the baseline study was developed and has been distributed to households where management changes are being incorporated. A folded extension pamphlet describing the use of I-2 vaccine to protect birds from Newcastle disease was developed. LBVD staff members were trained in database management and database work as the foundation for further epidemiological analysis.

Objective 3
A training course on the use of molecular techniques to identify agents causing poultry diseases was conducted in February 2004 in Myanmar. Laboratory equipment for molecular techniques, including a PCR machine (thermocycler) and electrophoresis equipment was also supplied. An ACIAR-funded training workshop on the serological diagnosis of H5N1 avian influenza was held in Laos in June 2004 with 2 participants from Myanmar.

Objective 4

A baseline study to identify major constraints to village chicken production was conducted until May 2004. A total of 307 households were sampled, from 10 villages in two separate townships (one north and one south of Yangon) in Yangon Division. The outcomes of this survey led to the treatments currently applied in the intervention study. Results of the baseline study were presented internationally and in Myanmar and were incorporated in the extension material developed. In addition a Rapid Rural Appraisal was conducted in another region (Mandalay Division) to monitor regional differences. Currently two journal papers are being drafted on this work and will be submitted to international journals.
The ongoing intervention study, which commenced in June 2004, involves several treatment groups of partial and total flock vaccination against Newcastle disease, placebo vaccination and improved chick rearing management and various combinations of those two treatments over a 12-month period. A total of 160 households in 12 villages are involved in this study.

Objective 5

Laboratory personnel were trained in the genetic characterization of virus isolates using reverse transcriptase polymerase chain reaction (PCR). In addition, laboratory equipment for conducting molecular techniques was supplied. Field isolates of Newcastle disease virus have been collected for future genetic characterization. PCR products have already been generated from ten field viruses isolated in 2004.

Significant progress was made during this reporting period. A 12-month intervention study assessing different strategies to improve village chicken production was completed in July 2005. The results of the study were presented at one international poultry conference in Africa and at a number of other meetings. Necessary laboratory equipment was purchased for diagnosis of poultry diseases and Livestock Breeding and Veterinary Department (LBVD) staff members were trained in vaccine production, molecular techniques and database work. Three Myanmar project staff members visited The University of Queensland in April 2005 and were further trained in molecular techniques and veterinary epidemiology. An external project review was conducted in May 2005 and an extension of the project was recommended by the reviewers. Extensions until 30 June 2006 were granted, to allow further work to be conducted including extension work, data analysis and training in epidemiology. Specific progress relating to each of the original project objectives is outlined below.

Objective 1

Items of laboratory equipment were purchased for the vaccine production laboratory and diagnostic laboratory. A vaccine production course was held by Australian project personal in June 2005 in Pyin Oo Lwin (Mandalay Division) and included participants from the central and regional vaccine production laboratories in Myanmar.

Objective 2

After data collection was completed in July 2005, data analysis commenced and will be further conducted in the extension period of the project. Extension materials based on the outcomes of the intervention study will be prepared in the extension period of the project.

Objective 3

Further training of a Myanmar scientist in molecular techniques was conducted in April 2005 at The University of Queensland. Another scientist was further trained in the field of veterinary epidemiology during this visit. This improved the capacity of the diagnostic laboratory and the epidemiology unit at LBVD to investigate and diagnose poultry diseases.

Objective 4

The intervention study, which commenced in June 2004, involved several treatment groups of partial and total flock vaccination against Newcastle disease, placebo vaccination and improved chick rearing management and various combinations of those two treatments over a 12-month period. A total of 160 households in 12 villages were involved in this study. This study was completed in July 2005. Preliminary data analysis showed that improved chick rearing led to a significant reduction in chick mortality during the first 6 weeks of age. There was a significant increase in number of birds sold after a period of 6 months in the group with management changes, resulting in a significantly larger income for these farmers and an increase in the number of households consuming home produced chicken meat. Mortality due to disease was reduced in groups using Newcastle disease vaccination, but was offset by deaths due to other causes. Further data analysis will be conducted in the extension period.

Objective 5

Further training of a visiting scientist in molecular techniques was conducted at The University of Queensland in April 2005. Arrangements were made to transport to The University of Queensland, amplified DNA products from approximately 10 PCR-positive samples. These PCR products will undergo genetic sequencing during the extension period of the project.

During the reporting period two project extensions were provided to the project (extension I: 01/05/2005-30/06/2006; extension II: 01/07/2006-30/06/2007). The focus of both extension periods was the dissemination of the study results to the village chicken-owning community and the training of veterinary staff. Extension materials on village chicken health issues were developed (booklets, flip charts, calendars, pamphlets) and distributed among the farming community. Farmer workshops were organised to train and discuss village chicken health issues. Village personnel who had significant association with poultry issues were identified to conduct these workshops in the future. These key people will also distribute the equipment for improved chick-management and inform the Livestock Breeding and Veterinary Department (LBVD) about any constraints and health problems in raising chicken in the villages. A supply of chick starter feed, bamboo coops and creep feeders to the villages was established. The extension work was expanded into 31 villages in the Yangon Division and promoted in two other states and divisions (Mandalay Division and Shan State). The extension work was evaluated very positively by an external extension specialist who also recommended conducting the extension work over several more years.

Data from the field studies were analysed. Results of the studies were published internationally in journals and presented at international conferences. This was the first time that village chicken information from Myanmar was published internationally. Field isolates of Newcastle disease virus were transported the University of Queensland for sequencing. Phylogenetic analysis showed that these velogenic Myanmar isolates form a separate cluster within genotype VII viruses.

Training in Veterinary Epidemiology was provided to LBVD staff from Yangon and from the regional LBVD centres and also to field veterinarians. In addition a training workshop on project planning, extension skills and disease awareness was conducted.

The overall objective of the project was to be achieved through 5 different activity areas. The progress in each of these areas is as follows:

1. Development, evaluation and implementation of a simple, rapid diagnostic test for CSF.

This aspect of the project work is to be conducted principally by Mr James Conlan who has enrolled as Masters Degree student at Melbourne University through the School of Veterinary Science. Mr Conlan started his program at the CSIRO Australian Animal Health Laboratory in Geelong, organised planning meetings with his co-supervisors (project leader and Prof Colin Wilks) and returned to Laos to implement the plan developed. Some of the reagents required to develop the test were procured from AAHL or produced by the student while in Geelong, and in addition the Lao DLF project leader (Dr Syseng Khounsy) serially immunised rabbits with the local CSF vaccine strain to produce a hyperimmune serum suitable for application to the test format. The proposed format for the test (a bead binding assay using coloured beads) was not successfully replicated when a new batch of monoclonal antibody was produced and so far the reason for this change has not been determined. However the reagents have been adapted to a immuno-magnetic bead ELISA for detection of CSF antigen and preliminary results indicate that this could be a suitable test system for a small laboratory. Potential advantages are that it will be a faster and possibly more sensitive test than the current antigen detection ELISA used in the central laboratory. Dr Wilks and Gleeson planned to visit the project in late July to assess the laboratory work first hand and to plan the next phase of this research.

2. Establishment & validation of a system to apply locally produced CSF vaccine in Lao villages.

There were two key aspects of this work undertaken. The first was to assess the potency of vaccine produced by the DLF Vaccine Institute. The prevailing anecdotal evidence is that the vaccine produced by DLF does not protect against disease in the field, One pen trial carried out in the previous project indicated that the vaccine was potent in improved breed pigs, but DLF monitoring of the response to vaccine in commercial piggeries suggested that the vaccine was not potent. So it was a high priority to have an 'in-vivo' assessment of any batches to be used in the project areas. The methodology involved acquiring native breed pigs from local sources, immunising the pigs according to the recommended protocol and then assessing immune responses. In the course of the year 3 batches of vaccine were assessed because according to the laboratory assessments all batches failed. Assessments were carried out using a commercially available ELISA that measures antibodies to the protective antigen and the antibody test established by the previous project. In order to check the reliability of the ELISA results Mr Conlan set out to establish the virus neutralisation test. This involved transport of cell cultures from AAHL, purchase of media, calibration of the carbon dioxide incubator and production and titration of virus stocks. The second aspect was to investigate the reason(s) for the lack of potency of the vaccine. The project purchased temperature loggers for monitoring vaccine storage in the field. When the temperature cycle to the main storage freezer at the Vaccine Institute was monitored, a rise and fall of temperature was detected. Further investigation indicated that the company employed to refurbish the freezer had incorporated an 8 hour freeze-defrost cycle that is not suitable for vaccine storage. The recommendation from the vaccine plant is that the vaccine is stored at -20oC but this is not practical for field offices in Lao PDR. An experiment has been planned to investigate the storage of vaccine at 4oC - 8oC, the temperature of a domestic refrigerator. This will require 'in-vivo' potency assessments by pig immunisation after various storage intervals.

3. Evaluation of the impact of the CSF vaccine program in the village pig production system.

This aspect of the project is behind schedule because the vaccine batches have failed to meet potency test requirements. In December 2003 Dr Ross Cutler accompanied the project leader to evaluate the village production systems in the current project area in Borikhamxay and in the study area in Xieng Khouang for the proposed collaboration with the Centro Internationale Agricultura Tropicale (CIAT) Forages and Livestock Systems Project (FLSP). His mission report was submitted to ACIAR, DLF and CIAT. Additional villages had been recruited to the program in Borikhamxay where a student from the Danish Veterinary College participated in the recruitment process, but her report to the Faculty has not been completed. A significant proposal to the project from the December mission was that in the new participating villages the main point of contact should be a woman involved with pig raising. Meetings were held with FLSP officers in Xieng Khouang to outline the activities likely under the project, and an overall methodology for the vaccination program was established with Dr Syseng. Impacts of the vaccination program will be measured using the methodology developed in the previous project.

4. Monitoring the epidemiology of FMD and CSF in Lao PDR.

Dr Syseng received 5 days of intensive training in the Epi-Info database program at a training course conducted at AAHL in October 2003. The AAHL project leader and Mr Conlan also participated in the course. In December 2003 field specimens from 32 outbreaks of FMD were submitted to the WQorld Reference Laboratory for FMD with the cooperation of the Department of Livestock Development Thailand and the OIE SEAFMD Regional Coordination Unit, Bangkok. The project counterpart had not received definitive results at the end of the reporting period, and further follow up was required with the World Reference Laboratory and the SEAFMD RCU. In Dr Syseng had carried out an investigation of an outbreak of FMD in North-west Lao near the border with Thailand. This outbreak was confirmed as type A, the first occurrence of type A in the history of project activity in Lao. The sample was sent for submission to WRL but at the end of the reporting period no result is available. The investigation of the outbreak appeared to indicate that the disease was carried by fomites from Thailand to the outbreak village. The field investigation indicated that the outbreak had not spread to neighbouring villages.

5. Communication of project findings to extension staff and animal health and production scientists in national, regional and international networks.
The activity is not yet applicable but it is anticipated that before the next report, Mr Conlan would have had an opportunity to report on his work at a regional meeting. One possible venue would be the Society for Tropical Veterinary Medicine international meeting in Hanoi in June 2005.

The overall objective of the project was to be achieved through 5 different activity areas. The progress in each of these areas is as follows:

1. Development, evaluation and implementation of a simple, rapid diagnostic test for CSF.

The test development has progressed well and Mr Conlan has prepared his data for submission for a Master of Science degree at Melbourne University. The test format was modified to a read-out using ELISA generated colour development has been termed an immuno-magnetic bead (IMB) ELISA. The binding conditions of the reagents and the conditions for the performance of the IMB test have been standardised. Limited validation using the diagnostic ELISA used for antigen detection as the gold standard has established the proof of principle of the IMB test format. Mr Conlan has undertaken operator variability studies with the project staff at the National Animal Health Centre that show the IMB ELISA can be successfully transferred to the central laboratory environment in Lao PDR. Mr Conlan will submit his thesis for examination in February 2006.

2. Establishment & validation of a system to apply locally produced CSF vaccine in Lao villages.

In the previous period there was concern about commencing the vaccination program in the villages until the reliability of the vaccine was established. The established antibody detection ELISA indicated that in many vaccinated pigs there was no humoral antibody response. Some of Mr Conlan's activity was redirected to the evaluation of the vaccine by use of the virus neutralisation test that showed that the vaccine was stimulating an antibody response in some pigs. A decision was made to boost the initial vaccination in villagers with a second vaccine a month later.

The current recommended temperature for the storage of CSF vaccine produced at the National Vaccine Production Plant is -20oC. Most provincial animal health offices do not have proper equipment to hold the vaccine at this temperature. Because commercially available live CSF vaccine can be stored for up to 12 months at -20oC, a trial was carried out to compare the potency of the vaccine (as measure by antibody response) after longitudinal storage of the vaccine at 4oC and at -20oC. This assessment indicated that the vaccine is not stable at 4oC, but maintains potency for up to 4 months at -20oC as specified by the manufacturer. Professor Wilks undertook a limited assessment of the vaccine production facility and reported his concerns about the production process.

The local project leader organised and delivered the vaccination program in an additional 8 village in Borikhamxay province and in FLSP villages in 2 districts in Xiengkhouang province.

3. Evaluation of the impact of the CSF vaccine program in the village pig production system.

As indicated above this program was delayed because of the constraints on the evaluation of the vaccine. Sera have been collected from a sample of pigs vaccinated in the village program but the testing is not yet complete. Production data has been collected from the project villages during the vaccination trial.

The project will undertake an evaluation of the villagers' attitude to the impact of the vaccination program as implemented by the project, to evaluate and attempt to provide some information to influence policy about CSF vaccine production. At present it is likely that the routine use of locally produced vaccine in the villages will not prevent disease outbreaks because of the relative instability of the vaccine and the difficulty of maintaining the proper storage conditions out in the provincial centres.

4. Monitoring the epidemiology of FMD and CSF in Lao PDR.

The project team from the Animal Health Centre and the provincial livestock and fisheries office have continued to collect the monthly production data from the villages enrolled in the project. There have been no outbreaks of CSF reported from the project villages. The project has been collaborating with the SEAFMD campaign by undertaking FMD serological surveys in three provinces. Limited results indicate that FMD is not endemic in Savannakhet, Xiengkhouang and Houaphan. There was one type O outbreak reported in the year, and a follow up investigation will be undertaken by the project to determine the origin.

5. Communication of project findings to extension staff and animal health and production scientists in national, regional and international networks.

The project has communicated the findings of the FMD studies and activities undertaken project to the Annual meeting of the SEAFMD program. A workshop planned for late June for communication of project findings to DLF was cancelled because Mr Conlan was unavailable due to illness.

The overall objective of the project was to be achieved through 5 different activity areas. The progress in each of these areas is as follows:

1. Development, evaluation and implementation of a simple, rapid diagnostic test for CSF.

Mr Conlan has submitted his thesis entitled "Improved diagnostic and management of Classical Swine Fever in Laos for a Master of Science degree at Melbourne University. He also gave a presentation summarizing the results of his work at AAHL. During this year his work concentrated on the further development of the IMB ELISA into a portable format suitable for use in provincial laboratories. That included experimental work to test the possibility to link the mab directly to the conjugate to shorten the turn around time of the assay. Results indicated that the saving of time did not justify the costs and this approach was abandoned. Another achievement was the production of reference samples to be used in an external quality assurance program and the training of national laboratory staff and selected provincial staff in the proper use of the test including troubleshooting exercises in Vientiane and a provincial lab. Laboratory consumables, e.g. conjugate stabilizer, beads and equipment, e.g. 4 microcentrifuges, pipette were purchased to support these activities. Further assessment of the IMB ELISA in relation to the AC ELISA is ongoing, preliminary results indicate the new rapid format performed at room temperature is equivalent to the IMB-ELISA described in Mr Conlan's MSc thesis. Training of National staff was carried out in Vientiane in early October and training of 8 laboratory staff from 4 provinces was carried out on 26 and 27 October.

2. Establishment & validation of a system to apply locally produced CSF vaccine in Lao villages.

Further experiments to assess the efficacy of the locally produced lapinised C-strain vaccine have been carried out. The vaccine was stored at 4 C for 4 and 8 weeks to test the effect of storage temperature on immunogenicity. A further experiment examines the effectiveness of the CSF vaccine at the village level when the vaccine has not encountered any temperature fluctuations i.e. storage at only -20 C. Eighty-nine sera have been tested at NANC (by ELISA) and AAHL (by NPLA and ELISA). Results are currently being analysed by Tess Vitesnik. A final forty-six sera are currently being tested at NANC and have been sent to AAHL, with results to be available by the end of October. Pigs and temperature loggers were purchased for these experiments.

3. Evaluation of the impact of the CSF vaccine program in the village pig production system.

The impact and success of the village vaccination program is most clearly demonstrated by the dramatic decrease in mortalities associated with CSF outbreaks that have occurred in villages in which the project is working. Three outbreaks of CSF have occurred in 2 of the 16 villages in this report period; the first in Houana in September 2005 and in Houana and Phontong villages in May 2006. From the production and health survey data collected in these villages, in total, 6 young pigs died during the 3 outbreak periods. These mortalities did not exceed the average monthly mortality for young pigs in either village. Before the vaccination programs began during this project, CSF associated mortalities were far greater. During a CSF outbreak in April & May of 2003 in Houana village, greater than 60 young pigs died in a single month, exceeding the average monthly mortality number for the village by almost 40 times. The impact of these decreases can not be underestimated, e.g. the loss of 60 pigs is equivalent to
500-1000 $US depending on the weight and the average income of a rural family is in the range of 200$US./year

Surveys assessing farmer views of the CSF vaccine and their perception of the vaccine's efficacy have been carried out in five villages in Bolikhamxay province. Results are being compiled and analysed by Tess Vitesnik.

4. Monitoring the epidemiology of FMD and CSF in Lao PDR.

The project has been collaborating with the SEAFMD and FAO-ADB campaigns for the control of transboundary animal diseases in the Upper Mekong countries. This involved the undertaking of FMD and CSF serological surveys in 5 provinces in the north, Oudomxay, Luang Prabang, Houaphan, Xayabouly and Phongsaly. Serological surveillance for FMD was carried out using a NS-protein ELISA, approximately 2% of cattle and buffalo in Luang Prabang and Xayabouly provinces were positive, no positive samples were detected in the other three provinces. CSF surveillance resulted in findings similar to those found in the previous ACIAR project AS1/1994/038, greater than 20% of pigs surveyed were positive for antibodies to CSF virus.

5. Communication of project findings to extension staff and animal health and production scientists in national, regional and international networks.

An outbreak of FMD was recently reported in northern Vietnam close to the border with Lao. Urgent action was taken to provide valuable information to Lao farmers in the neighbouring provinces of Phongsaly, Luang Prabang and Houaphan. In collaboration with SEAFMD and FAO-ADB, public awareness campaigns were conducted in these provinces. Radio broadcasts and posters were used to disseminate appropriate information.

An international final workshop with all main stakeholders, project participants and invited participants from neighbouring countries is scheduled for 20-21 November in Vientiane. The workshop is followed by a two 2-day training courses 23-24 November 2006. One training course will focus on the use of the IMB ELISA and other laboratory techniques for CSF diagnosis and the other training course will focus on pig health, production and housing. The later course is a interdisciplinary event which involves staff from Forage and Livestock Systems, CIAT. It is planned to have a CSF resource booklet in both Lao and English language published.

The project was externally reviewed in November 2006 and a review committee recommended that the project be extended for a further two years. The aim of the extension is to undertake additional activity considered necessary to provide the research base on which to plan and implement CSF control.

The specific objectives that will achieve this are:
1. Undertake experiments to further validate the IMB-ELISA and develop a system to introduce the test into the mainstream of CSF diagnosis nationally and regionally
2. Develop and implement a program for the adoption of CSF vaccination as a control strategy in village production systems,
3. Develop and distribute an education package for the control of CSF at the village level to complement Objectives 1 and 2.
4. To maintain a diagnostic capability for FMD by providing FMD reagents

Project implementation
Due to administrative circumstances the project started in May 2007.

Experiments necessary to further develop and validate the IMB antigen ELISA at AAHL
At this stage the IMB Ag ELISA uses spleen tissue samples and can be used only for samples from dead pigs. Control activities will be enhanced if the test can be used to detect infected pigs shortly after infection with CSF virus in blood samples of living pigs. Information about the diagnostic window of such a test will help to diagnose CSF outbreaks at earlier stages of infection and more timely quarantine actions. This will help reduce the spread of a CSF outbreak. A blood test will also allow livestock health officials to identify and monitor chronically infected pigs.
For that purpose it is necessary to conduct experiments at AAHL to investigate the potential to detect viral antigen in blood samples rather than spleen tissue samples. The next step is to further assess the sensitivity of the IMB Ag ELISA against suitable gold standards, such as virus isolation and RT PCR from experimentally infected pigs. The antigenic characteristics of the virus used in the experimental infection available at AAHL. The field viruses will have an impact on the comparability of the results.
Experiments to better assess the specificity of the test are being carried out at AAHL, e.g. samples from experimentally infected pigs with other Pestiviruses such as BVD and BD and also samples from CSF viruses used for vaccine strains are used to better characterize the specificity of the IMB Ag ELISA.
The outcome will allow an assessment as to how useful the test is for the early detection of CSF outbreaks and to better understand the importance of chronically infected pigs.

Experiment to determine vaccination schedule of piglets against CSF
To further determine the best timing for the vaccination of piglets against CSF approximately 320 piglet sera were sent to AAHL for testing with the NPLA and if possible also with the CEDI test. Blood samples were taken in weekly intervals from 0-3 months of age. Piglets were born from 2 sows at NAHC (16 piglets) and 3 sows from Bolikhamxay province (18 piglets). Sows were vaccinated against CSF prior to mating. The outcome of this experiment will help to determine the most suitable vaccination schedule for piglets. Samples are being tested at AAHL at the moment.

Production and distribution of test reagents for CSF
The normal shelf life of an IMB Ag ELISA kit is approximately 2 months, the main limitation being the stability of the working stock of conjugate. Colour development in positive samples decreases, conversely, colour intensity in negative samples increases. This has caused problems in the past because samples may not be available for testing in such a short period and the use of old conjugate may lead to the reporting of false negative and false positive results. Commercially available kits normally have a minimum of 12 month shelf life which is much better under the conditions in which these kits are used. Production of crucial test reagents such as coated IMBs, mabs, lyophilised conjugate is carried out at AAHL. AAHL is carrying out experiments to determine whether the conjugation of the HRP to the mab and lyophilisation of conjugate is a viable solution. If successful this will be an important step to increase the shelf life of the kit.
Once produced, sufficient quality controlled reagents these will be sent to NAHC. NAHC will be responsible to do further quality control and include lyophilized internal controls, SOP and eqa sheet. The test reagents then can be sent out as a test kit to other provincial or regional labs. It was suggested that AAHL can be the contact address for other interested laboratories, e.g. from Thailand, China, Cambodia, Vietnam, Myanmar and Indonesia.
It was suggested to use the SCAHLS validation template to formally assess the validation of the IMB ELISA. That would be useful as step to more formally certify and register the IMB ELISA.

Adaptation and preliminary validation of IMB-ELISA into a blocking format for the sensitive detection of antibodies to CSF virus
These experiments are carried out at NAHC. Jamie Conlan is in the process of adapting the IMB ELISA to a 96 well plate format and the production of reference sera from 5 vaccinated pigs on filter paper and as normal serum has been carried out. The next step is to further develop and test an inhibition ELISA. Once these experiments are completed successfully the use of the test to monitor CSF vaccination programs and for routine serological surveillance will be determined.

Conduct participatory problem diagnosis in upland villages to identify incentives to incorporate CSF vaccination in the village production system.
This activity addresses the question what are the drivers to incorporate vaccination into a production system in Lao highlands. NAHC staff is involved in a survey design and to conduct a survey. Jamie Conlan has contacted John Edwards to further discuss the role of a Lao PhD student in this research. It was also suggested to approach Nancy Burgeois from VSF in Vientiane.

ACIAR proceedings and CSF resource booklet
The proceedings represent a history of the activities and results of the project starting in 2003 and a snapshot of the diagnosis, epidemiology and control of CSF and FMD in Southeast Asia including countries such as Thailand, China, Cambodia, Vietnam and Myanmar. A preliminary version of the proceedings (> 20 papers, > 40 international authors > 200 pages) was submitted to ACIAR's publications manager and comments and changes were received. One contribution is still outstanding and once received the proceedings will be submitted to ACIAR for publication.
The resource booklet is a useful and easy to understand document (22 pages) which addresses the major constrains and possible solutions to control CSF at the village level. The next steps will be to include graphics and photos and then translate it into Lao. It was suggested to do a back translation from Lao into English to better detect any translation errors. Another quality control step is to distribute it to field staff and to other experienced interdisciplinary colleagues for further comments.

Maintain diagnostic capability at NAHC for FMD
NAHC holds reagents for the diagnosis of FMD serotype (O, A and Asia) specific ab and ag ELISA and FMD specific NSP ELISA. NAHC periodically undertakes quality control testing to assure that reagents are still performing within upper and lower limits. CSIRO/AAHL will assist in QA/QC and troubleshooting activities and can supply necessary reagents.

Jamie Conlan visited AAHL in June 2007 to train laboratory staff from the Mammalian laboratory in the production of IMB ELISA reagents, test performance, troubleshooting and results interpretation.
Axel Colling visited the project in August to assist in the implementation and coordination of project activities, especially to progress a number of documents such as the ACIAR proceedings and a CSF resource booklet.

Classical swine fever
The IMB CSF Ag ELISA has proved to be reliable for the diagnosis of CSF antigen in tissue samples and continues to be used at the diagnostic laboratory in Vientiane capital. Transfer of technology to provincial laboratories was successful and proficiency test rounds were performed.
Experiments regarding sensitivity and specificity of the IMB CSF Ag ELISA were carried out at AAHL. The diagnostic window and the suitability of different tissues incl. swabs in comparison with the standard antigen detection ELISA were assessed using samples from pigs that were experimentally infected with CSF virus. Positive IMB ELISA results were obtained as early as 5 days post infection. To further assess the specificity samples from other Pestiviruses, e.g. Bovine Viral Diarrhoea BVD (7 strains) and Border Disease Virus (BD) (2 strains) were tested and results compared with the standard antigen detection ELISA. The specificity of the IMB ELISA was found to be good as it did not cross-react with BVD and BD samples.
The use of blood samples in the IMB-ELISA and the adaption of this test into an antibody detection assay proved to be difficult. It is not likely that these problems can be solved within the remaining time of the project.
Problem diagnosis of CSF vaccination in upland villages and identification of incentives to incorporate CSF vaccination in the village production systems has not progressed because no suitable staff could be identified given the budgetary constrains of the project.

Another major output was to present and promote the knowledge generated through laboratory experiments and field studies through workshops, symposia and scientific publications. A poster entitled "Application of Immunomagnetic Bead Technology for Improved Diagnosis of Classical Swine Fever in a Low Technology Setting" was presented at the World Association of Veterinary Laboratory Diagnosticians, WAVLD 11-14 November 2007 in Melbourne by Conlan et al. and a paper entitled "Development and evaluation of a rapid immunomagnetic bead assay for the detection of classical swine fever virus antigen", Trop Anim Health Prod. is in press.

ACIAR proceedings No. 129 "Management of classical swine fever and FMD in the Lao PDR" were submitted for publication. They include 20 papers from more than 40 authors. Six papers are concerned with pig production and extension, 6 papers are country reports from China, Myanmar, Cambodia, Vietnam, Thailand and Lao PDR about CSF and FMD diagnosis and epidemiology and 8 papers are related to diagnosis and vaccination of CSF and Foot-and-Mouth Disease

The identification and assessment of major risk factors, e.g. pigmeat traders and trading practices such as "sell when sick", pig populations greater than 150, being in Bolikhan district etc has lead to recommendations and advice for vaccination and quarantine procedures. Results have been presented at a regional trans-boundary animal disease meeting in Bangkok in January 2008 and formed the basis for the CSF resource booklet which is being translated into Lao.
Approximately 300 pig serum samples from a maternal antibody transfer CSF experiment conducted in 2003 were sent to AAHL to determine the persistence of maternal antibodies against CSF virus. Results indicate that maternal antibodies may impact on vaccine uptake for more than 10 weeks. These findings were presented in a paper from Khounsy et al. "Recommended vaccine programs for village based pig production systems in Lao PDR" in ACIAR proceedings and in the CSF resource booklet. Another important observation is the low sensitivity of the Cedi ELISA and the CTB-ELISA to detect maternally derived CSF antibodies in comparison with the NPLA. These tests don't seem to be suitable for this purpose and a paper is intended to be published.

FMD
NAHC holds reagents for the diagnosis of FMD serotype (O, A and Asia) specific antibody and antigen ELISAs and also for FMD specific non-structural protein (NSP) ELISA. The laboratory periodically undertakes quality control testing to assure that reagents are still performing within limits and AAHL has assisted in QA/QC and troubleshooting activities including proficiency testing.
NAHC sent over 300 bovine serum samples from FMD outbreaks to AAHL. These samples were tested at AAHL to further validate the sensitivity of the 3 abc FMD ELISA.

Staff changes and travel
During this final year Syseng Khounsy left as project leader and was replaced by Phout Inthavong. Jamie Conlan left the project in December 2007. The departure of two key project staff had an impact on the delivery of some outputs.
Jamie Conlan visited AAHL in June 2007 to train laboratory staff in the production of IMB reagents, test performance, results interpretation and troubleshooting. Consumables, e.g. dropper and wash bottles, magnets and magnetic beads were purchased to produce a number of test kits which can be used in Lao and other labs in the region, e.g. Indonesia, Vietnam and China.
Axel Colling visited the project in August 2007 to assist in the implementation and coordination of project activities, especially to progress a number of documents such as the ACIAR proceedings and a CSF resource booklet.

ASSESSMENT OF THE RISK OF INTRODUCTION OF FMD IN INDONESIA AND IMPROVEMENT OF EARLY DETECTION
Questionnaires have already been administered by Dr Pebi Suseno to Quarantine officers and members of the Animal Health Division to identify potential routes of introducing FMD into Indonesia. This includes both identification of geographical regions and evaluation of the likelihood of infection from legal and illegal sources of animal and animal product movement. This information together with livestock numbers has been used to identify the 10 provinces with the highest risk of having an introduction of FMD. Five villages from each province will be selected for strategic (targeted) sampling for FMD. Two workshops with Quarantine Officers and members of the DGLS were held in 2006/07 to discuss risks of FMD, the consequences of introduction and to identify deficiencies in knowledge and awareness of the disease by field staff and farmers. A presentation to the annual conference of the Indonesian Veterinary Association was made on the risks of FMD. A deficiency in current material suitable for farmers on the dangers and signs of FMD was identified and existing material will be updated during 2007/08.
SURVEILLANCE PROJECT- ALOR ISLAND
Important background information collected:
To date the Australian research team has made three trips to Alor and Kupang. There was a delay in commencing this component of the project because of workloads by staff in Kupang and Alor. During the last 10 months essential background information has been collected in relation to the role and function of the Department of Agriculture on Alor and the importance of livestock to farmers. It has become evident that although there are strong procedures in place to provide animal health services- the lack of resources, and interestingly farmer perceptions about the importance of livestock health, make providing adequate services difficult. A two-day workshop was organised on CSF and diseases of pigs in Alor and animal health workers were involved in a training session on collection of blood samples from chickens and pigs.
It is apparent that there are difficulties not only associated with the ability of remote villages to effectively contact the Agriculture Department but also with the ability of the Department to respond promptly and provide the necessary resources for a disease problem. There is also reluctance for many farmers to notify the Department of instances of disease and death in their livestock.
There has been a vaccination programme for CSF in place on Alor since 2002. The aim of this program is to vaccinate every pig on Alor yearly, in the hope of achieving eradication in the future. Unfortunately, some farmers continue to refuse the vaccination of their pigs.
Although there is no denying the importance of animals such as pigs in the culture of the people of Alor - it would seem that the farmer's main concern is the mere existence of the pig regardless of body condition and/or health. The pig functions as an asset and is traded for money to pay for such things as a child's education. It is also slaughtered for religious ceremonies, special events and parties. An issue highlighted by one of the volunteers from a farmer's NGO group we visited during one of our visits was that there is no market system in place in Alor. Farmers therefore are not motivated to produce the largest, healthiest pig for market. Animals are thus mainly only kept for private consumption or as a long term asset. Crops on the other hand provide daily returns and are probably the mainstay of the farmer's income, and as a consequence receive the most time and effort. This could have implications for the effectiveness of syndromic surveillance given that the farmers seem to pay little attention to the health of their animals and spend the majority of their day tending to their gardens and crops.
Survey
Thirty seven villages have been selected and demographic data collected. Questionnaires and instructions for the cross-sectional, cohort and socioeconomic studies have been designed. The questionnaire for the socioeconomic study has been pre-trialled and refined and the questionnaire for the cross-sectional study is currently being trialled. It is hoped that the socioeconomic and cross-sectional study will get underway shortly. The logistics associated with the cohort study and vaccine trial is currently being worked on; this aspect of the project will commence subsequent to the onset of the cross-sectional study.

In light of the background information collected, incorporated into the questionnaires, are targeted questions pertaining to identifying reasons for why farmers often fail to contact the Agricultural Department in the evident of disease or death in their livestock, and also why some often object to the routine vaccination of their pigs against CSF. Once this information has been collected it is hoped that we will be able to develop incentives aimed at improving this situation.

A framework for a national surveillance system for foot and mouth disease (FMD) in Indonesia was established at a workshop organised as part of this project and attended by staff from Government, University and Research institutions. Technical guidelines have been drafted for this surveillance. As part of the improved surveillance system a field investigation of a suspect case of FMD was investigated and samples collected and tested by the national FMD laboratory (PUSVETMA). All samples were test-negative and the results highlight the success of having a national integrated surveillance system.
Another important outcome of the work has been in relation to incentives. Villagers were asked what type of incentives would encourage them to report animal diseases and a high percentage stated that more information about diseases was necessary. They also indicated that free vaccinations and free vitamin injections would also encourage them to report to the government officials about diseases. An outcome of this research was the need to explore the decision making processes of villagers. An understanding of how decisions are made about animal disease identification and reporting will be important towards the development of a surveillance program on Alor Island and will form the basis of the upcoming years work.
A pre-trial of the questionnaires for the cross-sectional study, prospective cohort study and vaccination trial for classical swine fever (CSF) was conducted in July 2007. Active surveillance (sampling) for CSF has subsequently commenced. The cross sectional study is complete and in total 690 samples were collected. The cohort study is on-going and to date 529 samples have been collected. A field based vaccination trial of 4 commercially available CSF vaccines has also been instigated and to date 1526 samples have been collected. A valuable component of the cohort and vaccination studies has been collecting data on changes in animal numbers and animal movements over time. Knowledge of movement of animals is essential to understand disease dispersal and to design potential methods of disease control. Questionnaires have been administered to farmers involved in these studies and the results of these will aid in further understanding farmers actions and help in designing effective surveillance systems for remote areas.

The AYAD student, Michael Bragg, was located on Alor Island for 5 months in 2007 and during this time, studied 16 villages across Alor Island. This allowed a better understanding of the importance of livestock in villages on Alor and enabled Michael to successfully complete his Honours degree at the University of Western Sydney with second class honours. This analysis provided valuable insights into the importance of livestock to farmers and the day to day functioning of villages in rural Alor. Although pigs are culturally important to most villagers, it was found that they had little concern for disease in their pigs and were more concerned about their family and crops. Pigs were less important for day to day consumption in comparison to crops, as pigs were mainly eaten at cultural events.

Guidelines for a surveillance program for FMD has been established and incorporated into a contingency plan in the event FMD is detected in Indonesia. A quantitative risk assessment was conducted identifying high risk areas for targeted sampling in Indonesia and the INDOVET PLAN for FMD was updated and results presented to the 29th World Veterinary Congress in Vancouver, Canada in July 2008.
The cohort study for Classical Swine Fever (CSF) in Alor is on-going and is due to be completed in October 2009. Of the 300 pigs initially involved in the study, 103 remain and 954 blood samples have been collected over five sampling periods.
The vaccine trial for CSF is due for completion in September 2009. Of the 300 pigs initially involved and vaccinated with one of four vaccines or a control, 101 remain and 1758 blood samples have been collected from eight sampling periods.
Our research has determined that syndromic surveillance cannot be effectively implemented on Alor given the current situation. For syndromic surveillance to be effective, farmers must first recognise signs of disease and then report this to an animal health authority. Our research identified a multitude of factors associated with farmers failing to report disease including a lack of: awareness of disease signs, incentives to report and access to animal health officials. On Alor farmers spend most of their time tending to their crops, which are located up to five kilometres from the households where livestock are kept. As a result, farmers spend very little time feeding, observing or caring for their pigs, even though they have significant cultural importance. The low priority placed on pigs together with a severe lack of education relating to pig health, husbandry and nutrition, culminates in the farmer's inability to recognise signs of disease. If farmers do recognise disease it is often difficult for them to access an official to report to, and there is no incentive to do so. Only two villages have an animal health worker (AHW) in permanent residence; farmers in other villages may have to travel a day or more to reach one. Because travel is costly and diverts time from income-earning activities, farmers are unwilling to spend time travelling to report diseases, especially because they receive no perceivable benefit from doing so.
There is no conventional livestock market system in Alor nor is there an abattoir for pigs. Pigs are sold and traded between friends, family and neighbours and consequently the movements of animals go unrecorded and uncontrolled. This combined with the failure of farmers to report livestock diseases, means that disease surveillance currently relies on active monitoring by authorities. However, due to monetary and personnel constraints this system is ineffective and expensive. Consequently, for surveillance to be improved it must be acknowledged that the farmer is an important source of information and an effort must be made to improve their knowledge and reporting levels.
By addressing the key factors affecting disease reporting, the situation may be improved to a point where syndromic surveillance is possible on Alor. However we are of the opinion that syndromic surveillance could work in other regions with more intensive livestock systems. In Alor we identified two main incentives that could be used to encourage farmers to report diseases. Firstly, farmers have a strong desire to improve their knowledge on livestock issues. By offering training to farmers on livestock health, husbandry and nutrition the farmers' ability to detect and prevent disease/poor performance will be improved and they may be more motivated to report disease. We are conducting research to determine information delivery techniques appropriate for Alor. Secondly, farmers have a strong desire to have healthy pigs. Most farmers interviewed stated that they liked their pigs receiving injections because it made them healthy. Hence, offering timely and effective treatments to sick pigs could also motivate farmers to report disease. However, access to treatments and vets/animal health workers remains an issue. In one village with a permanent AHW all nine farmers interviewed told the AHW when they had a sick pig. In another village, five of seven farmers travelled five kilometres to a neighbouring village to report livestock diseases to the AHW. Providing access to an AHW in every village or cluster of villages could greatly improve disease reporting rates, however this is expensive and alternatives are being investigated.
The improved plan for the surveillance of FMD in Indonesia is of significant benefit to Australia and helps maintain confidence in Indonesia's status of freedom from FMD. If all of Australia's immediate northern neighbours (Timor Leste, Indonesia and Papua New Guinea) are free from FMD it reduces the risk of disease incursion into the northern region of Australia. Knowledge of the Indonesian situation for CSF, which has been exotic to Australia for over 40 years, is also of benefit in developing appropriate surveillance measures in the northern part of Australia.

Objective 1. To understand the epidemiology of highly pathogenic avian influenza (HPAI) H5N1 virus (e.g. seroprevalence, duration of virus shedding) in small holder duck production systems in Indonesia and Vietnam)
This objective will be achieved through field investigations. The field sites are located in Java in Indonesia and in the southern provinces of Vietnam to coincide with the largest duck populations and where there has been evidence of HPAI infection. A survey strategy for a longitudinal study that provides statistical confidence and considers the cost of sampling and the resources available was developed together with staff of the research organisations in Indonesia and Vietnam. An appropriate study design has been chosen and the sample sizes were estimated. Sampling frames were developed and study units from these sampling frames selected. In both countries 16 villages are involved in the study. A total of 80 duck-owning households in Vietnam and 96 duck-owning households in Indonesia were selected and farmers will be interviewed at two-monthly intervals over a period of 12 months. In addition blood samples and cloacal swabs will be collected from ducks and chickens and tested for haemagglutination inhibition antibody titres and for H5N1 viral RNA by real-time PCR.
Collaborating staff and sample collectors were trained in data collection, which commenced in Indonesia in March 2007 and in Vietnam in May 2007.
In addition in Vietnam a case-control study was conducted to identify risk factors associated with the HPAI outbreaks in the Mekong Delta from December 2006 to January 2007. A questionnaire was developed to investigate these outbreaks in five affected provinces and to collect information about potential risk factors for the emergence and spread of HPAI. In total 23 case farms were selected, along with 46 respective control farms. The questionnaire data was collected in February/ March 2007. The data obtained are currently being analysed.

Objectives 2&3. To understand the role of ducks as maintenance hosts and amplifiers of H5N1 virus during and after infection, and to understand the pathogenesis of HPAI H5N1 infection in ducks
The first activity under these objectives is the isolation of HPAI H5N1 viruses in Indonesia and Vietnam from samples collected in the longitudinal studies. Discussions have taken place, reagents purchased and personnel selected to undertake this activity in each of the research institutions in Indonesia and Vietnam.

Significant progress was made during this reporting period. The longitudinal studies conducted in Indonesia and Vietnam progressed well. In Indonesia, serological data from four bi-monthly samplings from March 2007 to September 2007 indicated that the seroprevalence of HPAI infection in unvaccinated ducks and in-contact chickens was relatively low, ranging from 0 to 6% in ducks and 0 to 10% in chickens between districts and samplings. This suggests that birds were highly susceptible to infection. Although the seroprevalence differed between districts, there was no clear temporal pattern apparent over this sampling period. No birds were vaccinated against H5N1 in Indonesia. Positive H5 PCR results were obtained from 28 ducks and 5 chickens during the same sampling period indicating that virus was circulating among these small-holder flocks. This was further reflected in a number of disease outbreaks that occurred in the study villages. H5 virus was isolated from 43 ducks and 52 chickens that died during these outbreaks.
In Vietnam, most of the birds in the longitudinal study were reported to be vaccinated. Although serological results from two samplings in May and July 2007 indicated that only 60% of vaccinated ducks and 39% of vaccinated chickens had protective titres, no mortality due to HPAI was reported in any of the study villages suggesting that the flock level of protection was adequate. When the antibody response to vaccination over time was analysed, it appeared that chickens responded later than ducks. Chickens had lower antibody titres than ducks at both under 3 weeks and over 15 weeks post vaccination. The sentinel unvaccinated birds in Viet Nam showed higher seroprevalences than the equivalent samples from birds in Indonesia, ranging from 2-26% in ducks and 3-21% in chickens between provinces and samplings. These results from Viet Nam suggest that despite not achieving protective levels of immunity in all birds, vaccination against HPAI can decrease the likelihood of disease outbreaks and reduce mortality in the presence of active exposure to the virus. Analysis of the case-control study conducted to identify farm-level risk factors associated with the HPAI outbreaks that occurred from December 2006 to January 2007 in the Mekong Delta region was continued during this period. In a preliminary analysis it appeared that vaccination status, other poultry species kept on the farm and the sharing of scavenging areas could be potential risk factors. A descriptive pilot study of nomadic duck farms was also conducted to describe this management system in detail and to evaluate the feasibility of conducting a longitudinal study on nomadic duck flocks.
Using H5N1 virus isolates from Indonesia and Viet Nam, isolated in 2006 and 2004 respectively, domestic Pekin ducks were infected by the mucosal route (oral, nasal, ocular). Viral shedding patterns were determined by virus isolation and titration of oral and cloacal swabs. Virus was shed from both the oral and cloacal routes; it was first detected 24 hours after challenge and continued to day 5 after challenge. Virus shedding was detected in all but two birds challenged with the Vietnamese strain, and in 10 of the 15 challenged with the Indonesian strain. The Vietnamese strain caused severe morbidity with fever and depression, whereas the Indonesian strain caused only transient fever. Both viruses had a predilection for a similar range of tissue types, but the quantity of tissue antigen and tissue virus titres appeared to be considerably higher with the Vietnamese strain.
John Allwright Fellowships were awarded to four candidates aligned to the project and they will commence their postgraduate studies in Semester 2, 2008 or in Semester 1, 2009. One project team member from Indonesia received training in molecular diagnostics at the Australian Animal Health Laboratory, Geelong. The project has been represented at a number of scientific meetings during this reporting period and two papers in conference proceedings have been published or accepted. In addition four journal articles are in final stages of preparation. Project meetings were held in Brisbane on 7 January 2008 and in Yogyakarta on 27 March 2008, to discuss progress and present results of the project.

Significant progress was made during this reporting period. The longitudinal studies conducted in Indonesia and Vietnam were completed in early 2009.
In Indonesia, serological data show that the bird-level seroprevalence of HPAI infection in the sampled ducks and in-contact chickens was relatively low, ranging from 1 to 4% in ducks and 0 to 2% in chickens. This suggests that the birds are highly susceptible to infection. However, about 20% of the duck flocks and 2% of the in-contact chicken flocks had at least one seropositive bird, indicating that these flocks had been exposed to HPAI virus. A total of 96 households were visited seven times over the period of one year in Indonesia. In about 16% of flock-visits ducks were H5 antibody positive while chickens at the same sampling were negative, indicating that ducks had been exposed to H5 and might have been carriers of the virus. H5-positive PCR results were obtained from about 3% of the flocks monitored in Indonesia. During HPAI outbreak periods nearly 12% of flocks had healthy birds from which H5 virus was isolated. This indicated that virus shedding was increased during outbreak periods. Outbreaks were common with nearly half of the monitored flocks experiencing at least one outbreak over the 12-month study period. Mortality during the outbreaks was high among chickens.
In Vietnam, most of the birds in the longitudinal study of 80 household-flocks were reported to be vaccinated. Serological results indicated that only in half of the vaccinated duck and chicken flocks, 50% or more of birds per flock had protective levels of antibody. However, no mortality due to HPAI was reported in any of the study villages suggesting that the flock level of protection was adequate. When the response to vaccination over time was analysed, it appeared that only 50% of vaccinated birds had protective titres 4 weeks post vaccination. The sentinel unvaccinated birds in Viet Nam showed higher H5 bird-level seroprevalences than the equivalent birds in Indonesia, ranging from 21-29% in ducks and 13-20% in chickens. About 49% of the duck flocks and 26% of the chicken flocks were probably exposed to HPAI indicated by protective H5 titres found in at least one unvaccinated bird per flock. Flock-level virus prevalence in Viet Nam was low with only 0.2% of flocks having birds that shed H5 virus over the study period. These results from Viet Nam suggest that despite not achieving protective levels of immunity in all birds, vaccination against HPAI can decrease the likelihood of disease outbreaks and reduce mortality in the presence of active exposure to the virus.
Longitudinal studies on the nomadic duck management system were conducted over a period of six months in both countries. In these studies the HPAI field virus antibody prevalence and incidence in moving duck flocks will be determined, the patterns of movement will be described and risk factors associated with HPAI infection will be identified. Data from these studies are currently being compiled into databases.
Using H5N1 virus isolates from Indonesia and Viet Nam, isolated in 2006 and 2004 respectively, domestic Pekin ducks were infected by the mucosal route (oral, nasal, ocular). Both isolates had a predilection for muscle tissues and brain tissue, with little evidence of infection of epithelial surfaces. However, virus was also detected in feather shafts, which may have implications for virus transmission and could become an additional sampling source. Chickens infected with both of these virus isolates succumbed one day post infection. There was a predilection for blood vessel endothelium and lymphoid tissues. No strain differences in pathogenicity or tissue tropism were observed, but larger amounts of virus were shed from chickens infected with the Indonesian isolate compared to the Vietnamese isolate.
An informal project review was conducted during this reporting period and the project was considered to be producing excellent results in a strong collaborative environment between investigators and researchers from Indonesia, Vietnam and Australia. A financial extension until 30 September 2010 was granted. In this extension period the moving duck study will be completed and additional field studies on bio-security issues relating to moving duck flock management will be conducted. This will include surveys of people who provide transport for moving duck flocks, of rice paddy owners who provide scavenging areas and of hatcheries, which provide ducklings and purchase fertilized eggs from the moving duck flocks.
The project has been represented at a number of scientific meetings during this reporting period, three conference papers have been presented and three additional conference papers have been accepted. In addition two journal articles are published or in press and two additional papers are in the final drafting stage.

Highly pathogenic avian influenza (HPAI) caused by an H5N1 virus continues to be a serious concern to both human and animal health. The key aim of the research project is to understand the role that domestic ducks play in the transmission and maintenance of HPAI H5N1 virus in Indonesia and Vietnam. This knowledge will allow better management and containment of outbreaks of HPAI, which will ultimately reduce the risks to human health and lead to more informed decisions on control of the disease in poultry.
This collaborative project involves researchers at two Australian institutions (University of Queensland, Australian Animal Health Laboratory), two Indonesian institutions (the Disease Investigation Centre at Wates, Yogyakarta, and the Research Institute for Veterinary Science-Balitvet- in Bogor), and two Vietnamese institutions (National Institute of Veterinary Research in Hanoi, and Department of Animal Health in HCMC). The project activities can be broadly divided into field and experimental components, with the former being conducted at UQ, Wates and HCMC and the latter conducted at AAHL, Balitvet and NIVR.
Longitudinal studies on the nomadic duck management system were conducted over a period of six months in both countries. Forty-eight moving duck flocks in four provinces of the Mekong Delta of Viet Nam and 54 moving duck flocks in six districts of Central Java in Indonesia were monitored at monthly intervals for a period of 6 months. The HPAI field virus antibody prevalence and incidence in moving duck flocks was determined, the patterns of movement was described and risk factors associated with HPAI infection in moving duck flocks identified. Preliminary analysis indicated that the overall crude bird-level HPAI H5 antibody prevalence (HI titre16) was about 15.5% in Viet Nam and 5.3% in Indonesia. External risk factors related to moving duck management of monitored study flocks were explored through interviews of people associated with moving duck production system. These include transporters of moving duck flocks, rice paddy owners who provide their rice fields for scavenging, and hatcheries which provide ducklings and/or purchase eggs from moving duck flocks. Databases for these biosecurity surveys are currently being created.
In related studies on the infectivity of poultry viruses to ducks, two H5N1 virus isolates (subclades 2.1.1 and 2.1.3) from the initial longitudinal field study on 'stationary' duck flocks in Indonesia were each inoculated into groups of 14 chickens and 14 ducks. Both virus isolates were highly pathogenic in chickens, but not able to cause apparent disease in ducks. In chickens, both of the virus isolates replicated in most body tissues and were shed orally and cloacally at high titres; conversely, their replication in ducks was limited and they were shed intermittently from the oral route only, and at very low concentrations. No evidence of long-term infection or sustained virus shedding in ducks was observed.
The nucleotide sequences of haemagglutinin (HA) and neuraminidase (NA) genes of virus isolates from the field study in Indonesia were analysed. Of 83 isolates analysed, 79 belonged to HA subclade 2.1.3, one belonged to subclade 2.1.1 and three belonged to the undefined Indonesian 2.1 subclade. A subset of 19 isolates analysed by antigenic mapping were antigenically similar, even those from distinct genetic subclades, indicating that there have been no significant mutations in the HA epitopes of these H5N1 isolates.
The capacity-building activities of the project continue, with two project members from Indonesia and Vietnam undertaking an intensive 3-week training course in veterinary epidemiology and data analysis in Brisbane. One project member is enrolled in a masters program in Veterinary Epidemiology at the Gadja Mada University in Yogyakarta, supported in part by the project. Three John Allwright Fellowship holders from Indonesia are aligned with the project and in addition to their PhD or MPhil research studies, have undertaken additional training in bioinformatics or epidemiology over the past year. In both Indonesia and Vietnam, field and government veterinarians were actively trained in questionnaire design, interviewing techniques and sample collection in the field. Scientific impacts are also a feature of the project, with much interest from government and non-government authorities in both Indonesia and Vietnam. Other researchers in the HPAI field frequently seek advice from the project team regarding study design, and project members have been invited to present their findings at a range of national and international meetings and conferences. Ten conference papers and four journal articles were presented or published during this reporting period.
The project is now in the final stages of a 1-year extension. Final project meetings will be held in June 2010 in Yogyakarta and HCMC to review results of the project and to formulate recommendations based on these findings that can be used for policy decisions in both countries.