Báo cáo nghiên cứu khoa học " Development of an Improved Capability in support of National Bio-security for the Surveillance and Control of Foot & Mouth Disease in Cattle and Pigs - Milestone 3 " pptx

Virus isolation and molecular studies can now be carried out on FMD samples from the field and molecular epidemiological studies of the FMDV isolates in these provinces has provided insi

Ministry of Agriculture & Rural Development

Technical Report

Development of an Improved Capability in support

of National Bio-security for the Surveillance and Control of Foot & Mouth Disease in Cattle and Pigs

Milestone 3

National Reference Laboratory and Regional Laboratories operational and effective

By Chris Morrissy

Table of Contents

4 Introduction & Background _ 5

5 National Reference Laboratory and Regional Laboratories operational and effective 6

5.1 Implementation Highlights _ 6 5.2 Capacity Building _ 8 5.3 Publicity _ 8

7.2 Appendix 2 FMD Serotyping Results for Vietnam 2005 – 2009 _ 14

7.3 Appendix 3 Sequencing information from project 2005 – 2009 _ 15

7.4 Appendix 4 FMD serosurveilance results _ 24

1 Institute Information

Project Name

Vietnamese Institution Regional Animal Health Centre, Ho Chi Minh

City (RAHO - 6 ), South Vietnam

Vietnamese Project Team Leader Dr Dong Manh Hoa

Australian Organisation Australian Animal Health Laboratory

(AAHL), PMB 24, Geelong, 3213, Australia

Completion date (revised)

Reporting period

Contact Officer(s)

In Australia: Team Leader

Position: Diagnostic Virologist

Supervisor Mammalian Virology

Organisation Australian Animal Health

Laboratory (AAHL), PMB 24, Geelong, 3213,

Australia

Email: chris.morrissy@csiro.au

In Australia: Administrative contact

Name: Mr Chris Morrissy Telephone: +61 3 5227 5000

Position: Patents Contracts Officer Fax: +61 3 5227 5555

Organisation Australian Animal Health

Laboratory (AAHL), PMB

24, Geelong, 3213, Australia

Email: christopher.morrissy@csiro.au

In Vietnam

Organisation Regional Animal Health Centre,

Ho Chi Minh City (RAHO - 6 ), South Vietnam

Email: rahchcmc@hcm.vnn.vn

2 Project Abstract

The project’s purpose was twofold - to develop capacity for FMD (and other disease)

surveillance and diagnosis at both a laboratory and field level, and to investigate the

serotypes of FMDV circulating in Vietnam and the reason for vaccine failures Regional laboratories were set up with the reagents and methods to allow a diagnostic capability for FMDV diagnosis and serology Control strategies for understanding of FMD epidemiology have been implemented through veterinary and laboratory training workshops The project has highlighted the importance of having a laboratory network to identify what is happening

in the field and how to prevent and control disease outbreaks The pilot zones were

established in provinces near the borders of Vietnam to study serotypes circulating in

Vietnam and to determine their origin The number and quality of samples increased with each round of the project giving more data on the FMD situation in Vietnam Virus isolation and molecular studies can now be carried out on FMD samples from the field and molecular epidemiological studies of the FMDV isolates in these provinces has provided insights into the effectiveness of border control and origin of circulating FMDV Improved diagnostic capacity for FMD allows for the early detection and identification of disease enabling better control of disease and helps reduce loss of livestock and therefore increases productivity

3 Executive Summary

Over the life of the project there was improvement and advances in both activities in the field and laboratory The quality of both the diagnostic tests and the field data collected continued

to improve throughout the project

Throughout the project, consultants from AAHL worked with four diagnostic laboratories – RAHO – 6 - HCMC, NCVD - Hanoi, RAHO – 7 - Can Tho and RAHO – 4 - Da Nang - to improve FMD diagnostics and monitor the progress of the project The RAHO - 6 and NCVD laboratories now have virus isolation, virus neutralisation test, ELISA, PCR and sequencing techniques established for FMD diagnosis The project also worked with

NAVETCO research laboratory to establish the capability for FMD serology by ELISA RAHO - 6 is now well established in all these techniques and both RAHO - 6 and NCVD laboratories are also applying the new technologies to other disease problems in Vietnam eg RAHO – 6 have used virus isolation for Goat Pox, PRRS and CSF, NCVD used virus

isolation to identify a new isolate of PRRS in Vietnam The Can Tho and Da Nang

laboratories have the capability for FMD diagnosis for serotyping and post-vaccination serology by LP - ELISA

In addition, RAHO - 6 have learnt techniques in reagent production and have produced their own FMD antigen In developing such techniques they have also gained the ability to

trouble-shoot the ELISAs and problems with growth of FMDV Staff from RAHO - 6 and NCVD also visited AAHL during each year of the project for training and collaborative projects

In regards to field activities, there were significant improvements in the quality and number

of samples submitted to the laboratory for serosurveillance and serotyping for ELISA The latter allowed DAH to better understand and identify the circulating serotypes of FMD viruses in Vietnam in addition to gaining confidence in conducting large serosurveillance surveys There was also a significant improvement in the amount of data collected over each round of the project RAHO - 6 now has an epidemiological unit and this project has

assisted in developing the skills of the epidemiologists in serosurveillance, outbreak control and investigation of vaccine failure

Information from the serosurveillance in the project was reviewed to give an indication on the success of vaccination and the prevalence of FMD infection This data has been presented

at the OIE/SEAFMD regional meetings and to DAH

The project was invited to a number of regional meetings to present data from Vietnam on control of FMD As a result of these outputs of the project, Vietnam has been used as an example for others in the region to show the type of information that can be collected on serosurveillance and outbreak investigation The project coordinated activities with the AusAID capacity building project to allow both projects to achieve their objectives, eg combined PCR and sequence training with sequencing FMD isolates along with AI, PRRS and CSF isolates from Vietnam

Outbreaks of avian Influenza (AI) and PRRS had impact on both field and laboratory

activities of this project with DAH staff being overloaded with work

4 Introduction & Background

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 the veterinary laboratories in Vietnam and the training of DAH veterinarians in disease investigation and control This will strengthen the profile of DAH which will play a vital role in making Vietnam more economically competitive Improved animal health will lead to

an increase in rural productivity though increased animal production and indirectly in increased crop production Healthy animals will enable small farmers to be more

competitive in the local market Control of FMD and animal diseases in general will give poor farmers a more stable income stream and reduce their vulnerability to natural and economic problems 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 working model on which to base disease control This will directly 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

Specific Objectives for the Laboratories:

• Laboratory training manuals incorporating diagnostic techniques

• Functional tests at RAHO – 6 –HCMC and NCVD - Hanoi laboratories to allow

it to operate as national reference laboratories

• Functional ELISA tests at RAHO – 4 - Da Nang, RAHO – 7 - Can Tho &

NAVETCO laboratories to allow them to operate as regional laboratories

• Protocol manuals for submission and handling of samples and provision of feedback of results

• Laboratory Quality Control procedures documented and tested Internal Quality

control results documented and reviewed for each laboratory

Implementation Approach and Strategy

The approach for technology transfer is well established at AAHL and has been

successfully applied in previous projects in Vietnam, Thailand and Indonesia The

project approach used was also thought to be the most appropriate for developing an understanding of FMD epidemiology in Vietnam The field studies and serosurveillance approaches were designed and planned in conjunction with DAH to provide the

maximum necessary information to demonstrate the FMD situation in Vietnam and the effectiveness of FMD vaccines The diagnostic technologies that will be used in this approach are the standard diagnostic tests in use throughout the world to study FMD as directed by OIE

AAHL has a lot of experience with field surveys for prevalence of antibodies, as in the ACIAR projects in Laos and Thailand on FMD The Philippines is another example where OIE standard diagnostic tests are being used to control and eradicate FMD

5 National Reference Laboratories and Regional Laboratories

operational and effective

5.1 Implementation Highlights

The main achievements of the project were:

• The following FMD diagnostics were established in the collaborating laboratories:

™ RAHO - 6 and NCVD laboratories have established virus isolation, virus

neutralisation test, ELISA for antigen and antibody detection, PCR and

sequencing

™ RAHO – 4 and RAHO – 7 laboratories have the capability for FMD diagnosis for serotyping by ELISA (detection of antigen) from a FMD outbreak and serology by ELISA for post-vaccination surveillance

• A quality system was implemented in each laboratory for FMD diagnostics which included standardised methods, IQC and better record keeping

™ Standardised methods introduced for ELISAs, cell culture, PCR, sequencing and data collection ( eg ELISA methods attached to this email with this report )

™ Standard coversheets, result sheets and IQC record keeping forms ( eg ELISA forms appendix 1 )

• Increased collaboration between the laboratories since the inception of the project

• Improvement in the quality and number of samples submitted to the laboratory for serotyping by ELISA ( summary of serotyping results appendix 2 ) This gave DAH a better understanding of the circulating serotypes of FMD viruses in Vietnam The improvement in sample collection also allowed for virus isolation from field samples that had not been possible prior to the project and in turn enabled sequence data to be obtained from FMD virus isolates

• Sequencing, genotyping and analysis of approximately 100 Vietnamese FMD isolates collected from 2006 onwards The isolates were sequenced and analysed at AAHL by a scientists from AAHL and RAHO – 6 The sequence data was then sent to WRL for confirmation and comparison to other FMD isolates This information was shared with SEAFMD in support of the regional project to control FMD

• Sequencing/Genotyping 2006/2008

Serotype O: 3 topotypes

Cathay (33%), ME-SA (PanAsia) (6%) & SEA (Myanmar 98) (41%)

Pigs: mainly Cathay & SEA

Cattle: mainly SEA

Serotype A: Thailand/Malaysia 97

Vaccine used for FMD Serotype A needed to change from A 22 to

A Malaysia 97 (also antigen for serology)

Serotype Asia 1: Jiangsu-China-2005 (isolated from the Nth & Centre)

and Myanmar 98 (isolated from the Centre)

Confirmed 2 sources of Asia 1 virus into Vietnam

• The analysis of FMD isolate sequence data from Vietnam was used to compare

sequences to FMD sequences from around the world and is shown in appendix 3

• Improvement in the quality and number of serum samples submitted to the laboratory for serology by ELISA This gave DAH a better understanding of the animals exposed

to FMD and the vaccination coverage in Vietnam

™ Two rounds of serosurvellance each year with first round collected prior to vaccination and the second round collected after vaccination

™ The data form serosurvellance showed vaccine coverage and number of animals exposed to FMD (eg summary table example attached to email)

™ Recommendations for improved serosurveillance can be found

• A number of sera samples were retested to compare antibody titres for samples from the provinces to allow identification by serology of the circulating isolate The results indicated that the combined use of the non-structural 3ABC ELISA and the structural LP-ELISA could be used to identify the serotype of FMD that had been circulating in the field (eg appendix 4 )

• Throughout the project AAHL consultants visited the laboratories to:

™ Establish FMD Elisas and standardised techniques

™ Validate an Elisa using antigen produced at RAHO – 6 using Vietnam isolates The production of FMD Elisa antigen allows the laboratories to be more self-sufficient and is the start of the laboratories capability to produce its own reagants

™ Establish and review cell culture and virus isolation for growth of FMD isolates from the field Cell culture is important to grow FMD virus to allow further analysis of FMD field isolates by PCR and sequencing Cell culture has also been used for serology (VNT) and for isolation of other disease agents, such as goat pox, CSF and PRRS

™ Review molecular techniques and establish a workflow for sample processing and testing to ensure quality results

™ Evaluate quality assurance records and data collection to ensure test records were being maintained and results were being interpreted correctly

™ Provided advice on field information required and produced data collection form

™ Analyse field and laboratory results to provide epidemiological input

™ Supply consumables and reagents for testing of samples by FMD Elisa,

molecular technology and cell culture

™ Train staff in biosafety techniques and assist with their implementation

™ Assess quality assurance procedures and provide input to quality assurance manuals

™ Assess accurately of tests by Proficiency Testing Samples supplied to

laboratories to test results produced were accurate

5.2 Capacity Building

The project has provided training and technology transfer of FMD diagnostics to each

laboratory involved in the project Reagents and standard methods were supplied to each laboratory giving them the diagnostic capability for FMDV diagnosis Serology and

serotyping (detection of antigen) by Elisa is now being practiced at all of the laboratories and RAHO – 6 and NCDV have also established virus isolation, cell culture, virus neutralisation for serology, molecular and sequencing techniques

Training and education of field veterinarians in sample and data collection showed an impact with an increase in quality and numbers of samples collected and submitted to the laboratory These skills will be vital in implementation of the National FMD Control Program

5.3 Publicity

The CARD AusAID project received publicity in Vietnam, Australia and internationally through the training programs and also through the achievements in understanding FMD in Vietnam FMD is a disease of importance in Vietnam and the region and this put this project into the lime light The project has been publicised through a press releases in Australia and articles in newsletters including the SEAFMD newsletter and on the internet The results from the project have been presented at:

o OIE/SEAFMD meetings (eg presentation attached to email)

o EU FMD 2008 (eg presentation attached to email)

o WAVLD 2007, 2009

o Lower and Upper Mekong Working Group meetings in the region

With the laboratory and epidemiological capacity now available in the collaborating laboratories, particularly RAHO – 6, there is now the potential for a smaller, more focused study on vaccination failure This would be best limited to a smaller number of provinces in southern Vietnam, with a study protocol aimed specifically at investigating vaccination effectiveness

As the collaborating laboratories are now implementing a range of FMD diagnostic techniques it is considered that these abilities will be sustained and transferred to other laboratories

6 Conclusion

The project achieved its objectives by helping to improve the FMD and general diagnostic capacity of the network of veterinary laboratories The RAHO – 6 and Hanoi laboratories have provided and are continuing to provide support and training to other laboratories in the network

The project has highlighted the need for training of field veterinarians in the collection of

data and how to ensure the correct information is obtained from the farmer The staff at RAHO – 6 will continue to deal directly with the veterinarians in the field to ensure the correct information is collected in future surveys

The establishment of cell culture has allowed the growth of FMD isolates from the field that could not be previously identified at RAHO - 6 and at NCVD The increased FMD diagnostic capacity, combination of ELISA, PCR and cell culture for detection of FMD, has increased the number of FMD outbreaks identified in Vietnam The use of cell culture for growth of FMDV has resulted in the availability of higher quality samples for sequencing The

introduction of molecular techniques at RAHO – 6 has allowed sequencing results to be obtained quickly and has allowed informed decisions to be made about serotypes of FMD circulating in Vietnam and the correct vaccine to use in each region to control FMD AAHL, DAH and WRL have cooperated on analysis of the sequence data from Vietnam isolates Cell culture has also allowed the production of ELISA antigen which is a key reagent in FMD diagnostics RAHO – 6 is now producing own reagent which it has transferred to NCVD, RAHO – 4 and RAHO – 7

This project continues to be important for Vietnam with the repeated cyclical outbreaks of FMD The Vietnam Government and RAHO - 6 have invited AAHL to give advice on FMD control and have used the project as a model for the implementation of their control plan Despite the overwhelming requirement for control and surveillance of AI and more recently PRRS, the Vietnam Government is committed to the control of FMD This is evidenced by the currently active National FMD Control Program

The project highlighted the need to have coordination between the laboratory and the field staff in the control of FMD and in understanding the serosurveillance data post-vaccination and post-exposure to FMD from the laboratory results In the laboratory it is important to have the correct reagents to match the circulating field virus ( also highlighting the

importance of improved FMDV identification ) and in the field the field data to allow the interpretation of the laboratory results The whole veterinary network is important in the control of disease, there needs to be strong link between the field and laboratory to ensure both the actions in the field and the results in the laboratory are correct

This project set the standard for what was required for a laboratory network to function and the lessons learnt were used in handling HPAI and PRRS outbreaks in Vietnam and the projects and investigations that followed

Appendix 1

Standard coversheets, result sheets and IQC record keeping forms eg FMD 3ABC ELISA:

9.11 3ABC C-ELISA Test Coversheet

FMD 3ABC COMPETITION ELISA

SAN:

SAMPLE IDENTIFICATION:

TEST (3ABC C-ELISA): SUPERVISOR:

DATE PLATE COATED: OPERATOR:

DATE OF TEST: OPERATOR:

WASHING METHOD: INCUBATOR:

PLATE SHAKER: PIPETTERS:

NUMBER OF PLATES:

3ABC ANTIGEN DILUTION:

DETECTING ANTIBODY BATCH:

DETECTING ANTIBODY BATCH:

SERUM CONTROLS & SAMPLE DILUTIONS:

COATING BUFFER DATE: BLOCKING BUFFER DATE:

CONJUGATE BATCH: CONJUGATE DILUTION:

SUBSTRATE INCUBATION TIME:

Date Reported Signed

ELISA PLATE FORMAT

X

H

S8 S8

9.13 Quality Control Sheet

Date

Antigen

C++ C+ C- OD Max

Serotype Expected Expected Expected

Inhibition Inhibition Inhibition

80 – 100% 50 – 80% < 30% 0.8 – 1.5