Báo cáo Nghiên cứu khoa học Research and capacity building for the control of FMD in Vietnam

Collaboration for Agriculture and Rural Development (CARD) Program 82 RESEARCH AND CAPACITY BUILDING FOR THE CONTROL OF FMD IN VIETNAM Project title: Development of an Improved Capability in support of National Biosecurity for the Surveillance and Control of Foot & Mouth Disease in Cattle and Pigs Code of CARD project: 072/04VIE Author(s): Mr Chris Morrissy1 , Dr. Dong Manh Hoa2 Project Implementing organisations: 1 Australian Animal Health Laboratory (AAHL) 2 Regional Animal Health Office No.6 , Ho Chi Minh City, Vietnam EXECUTIVE SUMMARY The aims of this project were to develop capacity for FMD diagnosis, surveillance and control at both a laboratory and field level within the wider animal health network in Vietnam. Improved diagnostic capacity will facilitate early detection and identification of FMD enabling better disease control. Specifically, successful capacity development at regional laboratories will enable quality assured laboratory capability for FMDV diagnosis and serology which provide a greater understanding of FMD epidemiology and facilitate the coordination and implementation of effective vaccination control strategies via the wider animal health network in Vietnam On completion, this project has realised all laboratory-based objectives at a national level. However, due to the lack of epidemiology support and the diversion of Department of Animal Health (DAH) resources to deal with outbreaks of AI and PRRSV, field-dependent objectives were only achieved to a varying extent throughout Vietnam as a whole. Nevertheless all project objectives were fully realised and with great effect in southern Vietnam. Despite considerable achievements on a regional basis, this project clearly demonstrates the consequences of not having a fully integrated FMD diagnostic and surveillance network at a national level. The success achieved by RAHO-6 will act as an impetus to other regional offices in relation to the requirement for closer integration and clear, effective, two- way communication between laboratory and field-based personnel. During the course of the project there was a clear improvement and advances in both laboratory and field-based activities for the diagnosis and control of FMD. The AAHL Scientific Coordinator has mentored and liaised extensively with four diagnostic laboratories; RAHO-6 [HCMC]; National Centre for Veterinary Diagnostics (NCVD) [Hanoi]; Regional Animal Health Office – 7 (RAHO-7) [Can Tho]; Regional Animal Health Office – 4 (RAHO-4) [Da Nang] and one research laboratory, National Veterinary Company (NAVETCO) [HCMC]. As a result of successful capacity development and technology transfer from AAHL the RAHO-6 and NCVD laboratories now possess comprehensive, quality assured capabilities to diagnose FMD and perform sero-surveillance. In addition, both laboratories are capable of performing FMD virus isolation, virus neutralisation, ELISAs, PCR and sequencing/genotyping for the characterisation of FMD field isolates. All FMD laboratory diagnostic capabilities have been subject to internal quality assurance following on-site appraisal by the AAHL Scientific Coordinator. This has resulted in the recognition of both RAHO-6 and NCVD as FMD Reference Laboratories in Vietnam. In addition, quality assured FMD diagnostic capability in the form of the AAHL FMD Ag ELISA [for the detection of virus] and both the AAHL FMD C-ELISA and LP-ELISA [for post-vaccination surveillance] have been successfully established at RAHO-4, RAHO-7 and NAVETCO. Chriss Morrissy, Dong Manh Hoa 83 Following the establishment of comprehensive FMD diagnostic, virus isolation and serotyping capability at RAHO-6, tests have been implemented with both zeal and determination and of particular significance, with a long-term strategic vision. For example, the isolation and propagation of FMD serotypes in cell culture has been used by RAHO-6 to produce their own FMD antigens for in-house ELISA use and are distributing this antigen for use in other regional laboratories in Vietnam. In addition, ongoing sero-surveillance, vaccine efficacy monitoring and the serotyping of FMD field isolates has facilitated the evidence-based selection of the most appropriate FMD vaccine serotype composition and the identification of disease incursions from adjacent countries. Central to the achievement of project objectives in regions such as southern Vietnam was the close integration of laboratory and field-based activities. Overall, there was a significant improvement in the amount of data and quality of field specimens submitted to the laboratories over each surveillance round. During the course of this project, this closer integration, collaboration and communication between RAHO-6 laboratory/office and field personnel in southern Vietnam has facilitated the acquisition of the necessary high quality field data and clinical specimens. This highly effective two- way integration has resulted in an every decreasing incidence of FMD outbreaks from many outbreaks in 2006, approximately 100 outbreaks in 2007 to none in 2008, 3 outbreaks in 2009 and only one isolated outbreak being detected in the 2010 reporting period in southern. This significant achievement has been noted by DAH, attests to the effectiveness of the project implementation approach, and highlights the successful RAHO-6 “lab-to-field” and “field-to-lab” integration as an exemplar to other regional laboratories / offices. Sero-surveillance and epidemiological studies have enabled the acquisition of important data in relation to vaccine coverage, the serotyping and genotyping of FMD field isolates and the prevalence of FMD infection in Vietnam. This data has enabled driven science-based changes in vaccine recommendations to be made with great effect in the field. This project has not only been of benefit to Vietnam but has also lead to a greater knowledge and understanding of circulating FMDV genotypes and the benefits of sero-surveillance for the whole region. In acknowledgement of the significance of this work, project participants have been invited to a number of regional [SEAFMD LabNet 2010; SEAFMD LMWG 2008] and international meetings [EU-FMD; OIE Subcommittee 2010] to present Vietnam’s highly effective implementation strategy for the control of FMD. Of particular significance, it should be noted that as a result of this project Vietnam is recognised internationally as model example to other counties in the region in relation to the successful implementation of FMD diagnostic tests, sero-surveillance, outbreak investigations and disease control. 1. Introduction Objectives of the project: 1. To establish an effective laboratory network for the diagnosis and control of FMD by the provision of resources and training of staff in required methods and quality assurance. 2. To provide accurate data to explain failure of vaccination to control FMDV and to develop new effective vaccine application strategies. Completing these objectives will improve the diagnostic capability of veterinary laboratories in Vietnam and achieve training of DAH veterinarians in disease investigation and control. This will strengthen both the role and the profile of DAH which will play a vital role in making Vietnam more economically competitive. In addition, improved animal health will lead to an increase in rural productivity though increased animal production. Healthy animals will enable small farmers to be more competitive in the local market and the control of FMD will reduce poor farmers’ vulnerability to FMD outbreaks and result in a more stable income stream. Establishing a diagnostic network which extends from the North to South Vietnam, from the laboratory to the farm level, reinforced by training and education, will give Vietnam a more integrated animal health network and greatly facilitate disease control. This will directly CARD Project 072/04 – Control FMD 84 increase the competitiveness and productivity of the national agricultural system which includes the major areas of concern including the Mekong Delta and the Central Coast. 2. Research contents and methods 2.1 Research Contents Four DAH laboratories, including RAHO – 6 in South Vietnam, RAHO – 7 in Can Tho in the Mekong Delta, RAHC – 4 in Da Nang in central Vietnam, NVDC in Hanoi in North Vietnam and NAVETCO, in HCMC in South Vietnam participated in the project. RAHC – 6 was the lead Vietnamese organisation and was responsible for the implementation of the project in Vietnam. Development and implementation of control strategies are dependent on an understanding of FMD epidemiology and requires laboratory testing for virus typing, genotyping and serosurveillance. Serosurveillance will be carried out using ELISA in each laboratory. Two ELISAs were used at each laboratory to test for FMD antibody, the Liquid phase (LP) ELISA to test for structural antibodies to FMDV and the 3ABC NS ELISA to test for non-structural (NS) antibodies to FMDV. The LP ELISA detects antibodies to FMDV from vaccination and infection, while the NS ELISA detects antibodies from FMDV infection only. These two ELISA were used to estimate the prevalence of infection, serotypes present in Vietnam and the efficacy of FMD vaccine. Approximately 3000 serum samples were collected each year for testing. ELISA technology was transferred to each laboratory though in-country training and training at AAHL. ELISAs were setup under Quality Assurance (QA) with training in record keeping, Internal Quality Control (IQC) and participation in PT. In RAHO – 6 the Virus Neutralisation Test (VNT) was established as the confirmatory test for serology and used in conjunction with the ELISA tests. For serotyping FMDV from disease outbreaks in the field each laboratory used the serotyping antigen capture ELISA for serotypes O, A and Asia 1. RAHO – 6 and NCVD also has the capability for virus isolation and PCR & genotyping. PCR products and cDNA from field isolates produced at RAHO – 6 and NCVD were sequenced at AAHL and used for genotyping. RAHO – 6 and NCVD were trained in the techniques and the software used to obtain sequence data from FMD field isolates. It was estimated that approximately 60 samples will be received per year. Budget estimates were based on previous CSF CARD and DVE ACIAR projects and the number of samples to be tested, to calculate the budget for consumables, reagents and time necessary for training. Training and implementation of the project was based on training in Vietnam and AAHL which allow continual reinforcement of the training at each laboratory. Setting up the technology under QA system means the laboratories results can be constantly reviewed through IQC and PT. The constant contact between laboratories and to the field staff and AAHL means there is a strong collaborative approach in this project. Figure 1 Location of Provinces & Laboratories in Project Geography of the project 1. LANG SON 2. QUANG NINH 3. QUANG NAM 4. KONTUM 5. BINH PHUOC 6. TAY NINH 7. LONG AN 8. DONG THAP 9. AN GIANG 10. KIEN GIANG CAN THO DA NANG HA NOI (NCVD) HO CHI MINH CITY¯ CARD Participating Provinces CARD Participating Laboratories 0 75 150 225 300 Kilometers Chriss Morrissy, Dong Manh Hoa 85 The project was carried out in 6 provinces in the lower Mekong Delta which border with Cambodia, 2 provinces in central Vietnam that border with Laos and 2 provinces in North Vietnam that border with China (see Figure 1). One district was selected from each province and two communes were be selected from each district (1 commune with 100% vaccination and 1 other chosen at random from district), thus in total, the project covered 20 communes. These communes were pilot zones in each region and detailed register and history of the animals in these zones were established. Workshops to train field veterinarians in collection of samples and in maintaining records of the history of disease and vaccination in each zone were carried out early in the project. The initial workshop was held at RAHO – 6 by AAHL personnel with the assistance of RAHO - 6 staff for South Vietnam. The workshop in the centre of Vietnam was carried by RAHO - 6 using the materials produced for the first workshop and with the support of AAHL staff and the final workshop in the North was carried out solely by RAHO – 6 staff. 2.2 Research Methods Field studies:  120 cattle & 120 pig sera were to be collected each round from each District (Total 2400 each round) to look at effectiveness of vaccination and exposure to FMD virus, twice a year. The sera was used to measure FMDV antibody titre to serotype O, A & Asia 1 by LP ELISA after vaccination in order to determine the efficacy of the vaccination program. The sera were also tested for antibody to 3ABC antigen to assess the prevalence of infection. The 3ABC positive sera were tested further by LP ELISA to determine if antibody titre to FMDV serotypes (O, A and Asia1) could be used to determine the FMD serotypes circulating in the commune.  Routine disease investigation samples (tissues) from the pilot zones as well as the whole of Vietnam were tested by serotyping ELISA to determine FMD serotype and then virus isolation was carried to isolate FMDV. RT-PCR was also used to detect FMD, RT-PCR and virus isolation will be carried out primarily at RAHO – 6 and NCVD  Field samples and virus isolates were genotyped after production of cDNA and PCR products at RAHO – 6 and NCVD and sequencing at AAHL. Geno-typing was confirm by the World Reference Laboratory (WRL) at Pirbright in the UK. Laboratory Training Vietnam & Australia: AAHL staff carried out training both in Vietnam and at AAHL for scientists from the participating laboratories. AAHL staff maintained frequent contact with the laboratories to review the data from the laboratories and provide support and direction. The project also supplied equipment and reagents to the Vietnamese laboratories for the diagnostic tests. Nucleotide sequencing of the FMDV isolates collected during the project will be carried out at AAHL. The data collected over the 3 years of the project gave an invaluable perspective of the FMDV situation in Vietnam and the effectiveness of the FMD vaccines used to control FMD. A workshop on the diagnostic technologies for detection of FMD was be held at RAHO - 6 led by personnel from AAHL for staff from the laboratories participating in the project as well as provincial laboratories. The workshop focused on ELISAs and transferring these assays to each of the participating regional laboratory along with the quality assurance needed to maintain these tests in their laboratories. The workshop also provided knowledge and training to other laboratories not directly involved in the project. CARD Project 072/04 – Control FMD 86 3. Research results and discussions 3.1 Successful capacity development for the diagnosis, surveillance and control of FMD in Vietnam. Specifically, the following quality assured FMD diagnostics were established in the collaborating laboratories  RAHO6 and NCVD laboratories have established comprehensive cell culture, virus isolation, virus neutralisation test, ELISA, PCR, sequencing/genotyping capability  It should be noted that both RAHO-6 and NCVD have achieved FMD Reference Laboratory status in Vietnam  RAHO4, RAHO7 and NAVETCO laboratories also have the capability for FMD diagnosis and serology by ELISA for post-vaccination surveillance  All laboratories in the project are able to utilise the AAHL LP-ELISA, C-ELISA and 3ABC ELISA to establish the sero- prevalence of FMD in herds and vaccine coverage post-vaccination  All laboratories have implemented an in- house Quality Assurance system to monitor the accuracy of test results. 3.2 The project has both driven and inspired an increase in collaboration between the Vietnamese laboratories and helped nurture a more integrated animal health laboratory network  This will be essential not only for the control of FMD and has since been applied to other animal diseases such as resent PRRSV outbreaks in Vietnam. 3.3 Improvement in the quality and number of samples submitted to the laboratory for serotyping by ELISA  The closer integration of laboratory and field-based personnel, e.g. as achieved by RAHO-6, combined with improved diagnostic capacity and capability throughout Vietnam has led to the serotyping of a greater number of clinical specimens at both RAHO-6 and NCVD.  In addition, the significant improvement in sample collection and specimen submission has allowed virus isolation from field samples. This was not been possible prior to the initiation of this project and of particular significance has enabled genotyping of FMDV field isolates.  This has enabled DAH to achieve a greater understanding of the serotypes of FMD viruses circulating in Vietnam and provided essential baseline data in relation to the future control of FMD throughout Vietnam. 3.4 Genotyping and analysis of approx. 100 Vietnamese FMD field isolates collected from 2006 onwards  Genotyping data was detailed in Appendix 1 of the earlier Milestone 3 report.  Sequence data was sent to WRL for additional confirmation and comparison to other FMD isolates.  This information was shared with SEAFMD as part of the regional project to control FMD.  Genotyping has elucidated the reason underlying vaccine failures in the field and has successfully established capability in Vietnam to determine the source of a FMD outbreak and to make evidence-based decisions in relation to whether the current vaccine serotype compositions are appropriate.  Genotyping, combined with the ability to carry out VNT for determining antigenic variation of field isolates, has enabled Vietnamese laboratories to investigate future vaccine failures  Of particular significance, this has already resulted in the elucidation of vaccine failures in the field as a result of inappropriate vaccine composition usage Chriss Morrissy, Dong Manh Hoa 87 as detailed in the earlier Milestone 8 Report 3.5 Standard forms and protocols for collection of field data and trained field veterinarians in how to carry out disease investigation for control of disease and vaccine failure 3.6 A model approach for a program to control FMD with a strong laboratory and field component 3.7 Throughout the project AAHL consultants have mentored and liaised extensively with collaborating laboratories to achieve the following:  Established a comprehensive portfolio of internally and externally quality assured FMD diagnostic techniques at the newly established FMD Reference Laboratories at RAHO-6 and NCVD and related quality assured FMD ELISAs at all participating laboratories  Appraised quality assurance records and data collection under in-house QA systems to ensure test records were being maintained and results were interpreted correctly  Established and appraised cell culture and virus isolation capability for growth of FMD isolates from the field which has enabled further genotyping of FMD field isolates  Validated an in-house ELISA using FMD antigen produced at RAHO-6 using Vietnam isolates. The production and supply of this antigen to other laboratories facilitates future sustainability  Established and appraised molecular techniques to ensure best practice workflow for FMD diagnostic PCR under local conditions  Provided advice on the design and format of sample submission, data collection forms and outbreak investigation surveys to facilitate the acquisition of high quality field data and specimen submissions  Analysed and interpreted field and laboratory  Supplied consumables, reference IQC reagents and EQA PT reagents to confirm successful establishment of quality assured FMD diagnostic tests  On-going appraisal of laboratory quality assurance procedures and advice in relation to individual laboratory quality assurance manuals and SOPs 4. Conclusions and recommendation This project has realised objectives in relation to FMD diagnosis, surveillance and control via successful capacity development and subsequent implementation of quality assured FMD diagnostics at both a regional and a national level in Vietnam. Specifically, both the RAHO-6 and NCVD laboratories have attained FMD Reference Laboratory status and possess comprehensive quality assured FMD diagnostic capabilities and the ability to isolate, serotype and genotype FMDV field isolates. On a national level, RAHO-4, RAHO-7 and NAVETCO also have the capability to perform ELISAs for the detection of FMD antigen and antibodies, further consolidating FMD diagnostic capabilities within the wider animal health network in Vietnam. This project has significantly aided DAH gain a greater understanding and appreciation of the logistic requirements and field skills that will be required for the effective control of FMD in Vietnam. In addition, this project has also generated important baseline data in relation to vaccination coverage in the field and the prevalence of FMD serotypes in circulation in Vietnam. In relation to FMD surveillance, resultant capabilities for serology by ELISA has enabled post-vaccination surveillance and has facilitated the investigation of the efficacy of vaccine coverage and ongoing surveillance which has demonstrated continual improvement in relation to FMD vaccine coverage in the field. The cost of FMD vaccine is expensive and is the main cost in a FMD control campaign for the Vietnam government, this project has highlighted the need for post vaccination seroserveillance supported by QA laboratory to ensure money being spend on vaccination is being delivered correctly and giving protection. Of particular significance, in CARD Project 072/04 – Control FMD 88 relation to FMD control, the subsequent serotyping and genotyping of FMDV field isolates has enabled evidence-based assessment of the prevalence and risks associated with individual FMD serotypes in specific regions. This has resulted in highly informed science- based recommendations being made to Department of Animal Health (DAH), field veterinarians and farmers with regard to the selection of the most appropriate FMD vaccine serotype formulations to be used in the field. The effectiveness of this approach was clearly evident from the fact that in southern Vietnam the incidence of FMD outbreaks has fallen continuously during the course of the project with only one isolated outbreak being reported in the 2009-2010 reporting period. Throughout the course of this project both the quality of field data and the integrity of specimen submissions showed continuous improvement. However it was widely recognised at an early stage that the critical component for FMD control was the requirement for closer integration, support and communication between laboratory and field personnel. In areas where this critical issue was addressed, the effectiveness of the implementation strategy adopted was readily apparent. For example, the implementation strategy at RAHO-6 where successful capability development and quality assurance in the laboratory was the driving force behind more effective training and education of field veterinarians realised project objectives and resulted in effective FMD management in the field. Specifically, the two-way communication, support and collaboration between the RAHO-6 laboratory / office and field veterinarians facilitated highly effective disease diagnosis, surveillance and control in southern Vietnam. The RAHO-6 epidemiology section have been highly proactive in engaging and training provincial veterinarians and field staff and it is considered likely that this highly successful approach will exhibit similar utility when implemented elsewhere in Vietnam once sufficient resources and training of field personnel is accomplished. From an Australian perspective, this project has been highly successful, enabling AAHL personnel to gain hands-on experience in dealing with FMD diagnostics, outbreak control and achieved further confirmation of the effectiveness of the AAHL FMD diagnostic tests and staff capabilities in relation to FMD preparedness in Australia. Project objectives were achieved throughout Vietnam and with considerable success in southern Vietnam. However, throughout the implementation of this project one of the key lessons learnt was in relation to the importance of complete and accurate field information and timely laboratory submission of appropriate clinical specimens. Whereas laboratory scientists were accustomed to accurate recording of results, in the field the recording of information such as comprehensive vaccination records and related data was not common place. Through the implementation of a series of data collection and field surveillance forms, data collection, management and laboratory submissions have improved dramatically throughout the course of this project. Nevertheless, it was clearly evident that continued training and support of field veterinarians and the greater integration and coordination of “lab-to-field” and “field-to-lab” activities will be critical for the successful implementation of FMD control measures at a national level.