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 5 " ppt

Two scientists from the Regional Animal Health laboratory HCMC trained at AAHL in molecular techniques for the diagnosis of FMD and standardisation of the protocols and reagents to be us

CSIRO-AAHL AUSAID DAH-VN

CARD-072/04VIE

Ministry of Agriculture & Rural Development

062/05VIE 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 5: Progress Report

Date 31/12/2006

7 Implementation & Sustainability Issues 9

1 Institute Information

Project Name

Chi Minh City (RAHC-HCMC), South Vietnam

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

Completion date (revised)

Contact Officer(s)

In Australia: Team Leader

Supervisor Mammalian Virology

Organisation Australian Animal Health

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

Australia

In Australia: Administrative contact

Organisation Australian Animal Health

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

Australia

In Vietnam

Organisation Regional Animal Health Centre,

Ho Chi Minh City HCMC), South Vietnam

2 Project Abstract

The projects purpose is to determine why there are vaccine failures, and to investigate what serotypes of FMDV are circulating in Vietnam so that the correct vaccines can be used for these serotypes Regional laboratories are setup 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 and laboratory testing for both virus typing and sero-surveillance have begun 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 are established in provinces near the borders of Vietnam to study serotypes circulating in

Vietnam and to determine their origin The quality and collection of samples have increased giving more data on the FMD situation in Vietnam Molecular epidemiological studies of the FMDV isolates in these provinces will provide insights to the effectiveness of border control and the origin of FMDV circulating in Vietnam each year Virus isolation and molecular studies have started to be carried out on FMD samples from the field A diagnostic capacity for FMD would allow the early detection and identification of disease enabling better control of disease and help reduce loss of livestock and so productivity

3 Executive Summary

The main focus of this reporting period was to establish FMD diagnostics in the collaborating laboratories with a focus on the HCMC laboratory All consumables and reagents were supplied to the laboratories in Vietnam for the second year of the project AAHL staff

prepared the protocols necessary for the training of laboratory scientists Two scientists from the Regional Animal Health laboratory HCMC trained at AAHL in molecular techniques for the diagnosis of FMD and standardisation of the protocols and reagents to be used in the project for sequencing FMD isolates from Vietnam

The project carried out training at the HCMC and Hanoi laboratories in molecular

technologies for diagnosis of FMD and reviewed ELISA results for the first round of serveillance An important part of the laboratory training was Quality Assurance (QA) in the laboratory to ensure tests were running according to a standard protocol and with Internal Quality Assurance (IQC) to allow AAHL staff to audit the results from the laboratory and give the appropriate data to allow trouble shooting The QA records were reviewed during training visit to the laboratories

sero-The Regional Animal Health Centre in Ho Chi Minh City also continued to train the field veterinarians on how to collect samples and how to collect information necessary for the history of the animals on the villages in the project It was identified that data collected from the field was not suitable from the first round of sero-surveillance The funding was supplied for the second year of field studies

Avian Influenza (AI) continues to delay the sero-surveillance in the field as field staff deal with the AI outbreak

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

Implementation Approach and Strategy

The project approach is thought to be the most appropriate for developing an

understanding of FMD epidemiology in Vietnam The approach for technology transfer

is well established at AAHL and has been successfully applied in previous projects in Vietnam, Thailand and Indonesia The field studies and epidemiology and sero-

surveillance approaches have been 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 Progress to Date

5.1 Implementation Highlights

Main achievements for this reporting period were:

• Training of laboratory staff at AAHL Two representatives from the HCMC laboratory were trained in molecular techniques for the detection of FMD genome and the

standardisation of reagents and protocols needed to establish PCR in HCMC Training was carried out under a quality system emphasizing the importance of Quality

Assurance in the laboratory focusing on test maintenance, record keeping and data collection

• A consultant from AAHL completed training of HCMC-RAHC staff in molecular techniques and established the technology to be used to identify FMD isolates from the field in the HCMC laboratory Again Quality Assurance, record keeping and data collection was emphasized

• AAHL consultant reviewed 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,

• Further training of field veterinarians was carried out with feedback of the results from first rounds of testing and experiences from the first round of sample collection to increase the quality of samples and data collected

• The consultant from AAHL also reviewed the transfer of ELISA technology to each laboratory and the implementation of a Quality Assurance system into the laboratory The testing of sera in each laboratory was discussed with the Nth Vietnam sample collection needing to improve Also training of RAHC-HCMC staff in FMD serology using cell culture to carry out the Virus Neutralisation Test (VNT) was part of training

• Sampling for next round of sera collection completed for each province for

epidemiological studies and sero-surveillance The collection of tissue samples from outbreaks continued to increase giving valuable information on the serotypes

circulating in Vietnam

• ELISA technology in use at collaborating laboratories in HCMC, Hanoi and Can Tho, for sero-typing of FMD tissue samples from the field Sera from sero-surveillance tested at RAHC-HCMC and Hanoi laboratories

• Virus isolation in use at RAHC-HCMC Molecular technology and serology by VNT using cell culture has started to be used

• Supply of consumables and further reagents for testing of samples by FMD ELISA, molecular technology and cell culture

• Project meeting held at AAHL with Director of DAH, Vietnam and Director of

RAHO-HCMC to discuss project outcomes Training in QA and biosafety given Also held a meeting in Canberra with DAFF to discuss Australia’s preparedness for disease outbreaks Project achieving aims for the DAH in establishing framework for control of FMD

Report from Vietnam:

of sero-surveillance

Discuss improved data collection and records Expert from AAHL to complete molecular training and review FMD

diagnostics in collaborating laboratories

Chris Morrissy and Peter Durr

Chris Morrissy

Visit HCMC and Hanoi laboratories

-Virus Inactivation by BEI

to produce antigen for ELISA , practice on Blue Tongue virus and VSV

- Chris Morrissy

- 1 Participants from RAHC-HCMC

3 -13th

Nov – 8th

Dec., 2006

- Analysis of FMDV sequencing data

-Virus Inactivation by BEI

to produce antigen for ELISA , practice on Blue Tongue virus and VSV

- Chris Morrissy

- 1 participant from RAHC-HCMC

- Dr.Dong Manh Hoa ( Director of RAHC-HCMC, main Viet Nam partner)

2 Field activities

2.1.1 Visiting 8 provinces in the project (Middle and Southern ) , Oct.2006

• Transfer the equipments and documents ( form, questionnaire) for sample collection

• Transfer money for sample collection

• Feed back the results from second testing round and share the experience

on sample collection with the field veterinarian staff to increase the quality of collected samples

• Staff in charge : Dr Hoa, Dr Vu , Dr Ha (RAHC-HCMC) - Dr Quan , Dr.Quang (RAHC-Da Nang) – Dr Thanh , Dr Dung (RAHC-Can Tho)

2.1.2 Visiting 2 provinces in the project ( North ) ,Oct 2006

• Transfer the equipments and documents (form, questionnaire) for sample

collection

• Transfer money for sample collection

• Feed back the results from second testing round and share the experience

on sample collection with the field veterinarian staff to increase the quality of collected samples

• Staff in charge : Dr Cam , Dr Tung ( NVDC- Ha Noi)

2.2 Collect sera samples from 10 provinces for the third round of surveillance , 120 cattle

sera and 120 pig sera from each province

2.2.1 NVDC-Ha Noi : 02 provinces (Quang Ninh and Lang Son) , total 480 sera samples 2.2.2 RAHC-Da Nang : 02 provinces (Kontum and Quang Nam) , total 480 sera samples 2.2.3 RAHC-Can Tho : 02 provinces (An Giang, Kien Giang ), total 480 sera

2.2.4 RAHC-HCMC : 04 provinces (Binh Phuoc, Tay Ninh, Long An, Dong Thap), total

960 sera

2.3 Collect tissue samples for FMDV typing

2.3.1 NVDC-Ha Noi : 103 tissue samples from the North ,

2.3.2 RAHC-HCMC : 189 tissue samples from the South

3.1 National Vaterinary Diagnostic Centre- Ha Noi ( NVDC-Ha Noi):

3.1.1 Testing on tissue samples for FMDV typing: 103 samples, all of them are serotype O

3.1.2 Testing on sera for FMDV antibodies detection: 480 sera , test finished

3.2 Regional Animal Health Center Da Nang (RAHC-Da Nang)

3.2.1 Testing on sera for FMDV antibodies detection : 480 sera, test finished

3.2.2 Sending 480 sera to RAHC-HCMC for retest : 480 sera

3.3 Regional Animal Health Center Can Tho (RAHC-Can Tho)

3.3.1 Testing on sera for FMDV antibodies detection : 480 sera, test finished

3.2.2 Sending 480 sera to RAHC-HCMC for retest: 480 sera

3.4 Regional Animal Health Center HCMC(RAHC-HCMC)

3.4.1 Testing on tissue samples for FMDV typing: 189 samples, 01 of them is serotype Asia 1, 179 samples are serotype O and 09 samples are negative

Remark: test covers 3 serotype: serotype A, O and Asia 1

3.4.2 Testing on sera for FMDV antibodies detection: 1920 sera ( 960 sera in the region

and retest 960 sera from RAHC-Can Tho and Da Nang ) Test finished

3.4.3 Isolate FMDV by BHK cell line from 32 selected samples, 01 serotype Asia1 and 31

serotype O

3.4.4 Application of SNT protocol in testing phase :

Adapt 5 FMDV on BHK cell line to produce reference virus for using in SNT for

• Serotype O, PanAsia ( ME) topotype : 01

• Serotype O, South EastAsia ( SEA) topotype : 01

• Serotype O, Cathay topotype : 01

• Serotype A : 01

• Serotype Asia 1: 01

• Titrate these viruses

Setup SNT using the serum from vaccine trial on cattle as reference serum and to the vaccine which was selected for applying in FMD National Control program since

2006

3.4.5 RT-PCR application

Extract RNA from FMDV isolates

• Serotype O: 74 (52 from the South and 22 from the North)

• Serotype A: 9

• Serotype Asia 1: 4

Make cDNA from these RNA

Send cDNA and amplicons to AAHL for sequencing

The number of FMDV cDNA sequenced at AAHL:

Ngo Thanh Long

Note: Further detail on activities for reporting period in log frame

5.2 Smallholder Benefits

All pig and cattle and producers are potential beneficiaries Those that take up the advice and use vaccine according to recommendations will benefit financially through reduced losses due to death and disease in their cattle and pigs The benefits of a more profitable farming operation flow to all family members Farmers and district veterinarians will have improved knowledge and skill in disease prevention, knowledge on the selection of the right vaccine and improving the efficacy of vaccination

5.3 Capacity Building

Training and education of field veterinarians in disease prevention, disease investigation and sample collection has continued with lessons learnt from the first round of sero-surveillance and sample collection This training has already shown an impact with an increase in quality

of sample collection and number of samples collected and submitted to the laboratory The project has provided training and technology transfer of FMD diagnostics to each laboratory involved in the project Reagents and standard methods have been supplied to each

laboratory giving them the diagnostic capability for FMDV diagnosis and serology using ELISA technology, this technology is now being practised at each laboratory The RAHC-HCMC has virus isolation and cell culture techniques established and has begun to use molecular technology and serology by VNT using cell culture RAHC-HCMC now has capacity to carry out RT-PCR for FMD for detection of FMD antigen and for sequence analysis

5.4 Publicity

The CARD AusAID project have received publicity through the training programs and also through the achievements so far in the project in understanding FMD in Vietnam FMD is a

disease on importance in Vietnam and this has put our project into the lime light The project has been publicised through a press release in Australia and articles in news letters including the SEAFMD newsletter and on the internet The results from the project have been

presented at OIE/SEAFMD meetings during the project

to run other aspects of the farm

6.2 Gender and Social Issues

Application of new diagnostic tests will improve the ability of regional and provincial

diagnostic units to quickly and accurately assess FMD outbreaks, enabling rapid and

appropriate measures to be applied to control disease These benefits will begin during the project and continue to accrue with continued application The beneficiaries of this project will be both large and small farms and particularly smallholder farmers whose animals and incomes will be protected by better disease diagnosis, management and control Since

women at the village level are the primary animal handlers and managers, they will be major beneficiaries of the final outcome of better diagnosis and control of animal diseases

7 Implementation & Sustainability Issues

7.1 Issues and Constraints

DAH continues to spend time controlling the AI outbreaks in Vietnam and this has made there workload high The collection of samples continues to be made more difficult by the need to train all veterinarians in each province in data collection and on how to collect samples, collection of blood and how to restrain cattle This increased training need has highlighted the need for further training of field veterinarians throughout Vietnam in disease investigation techniques with a focus on sample collection and data collection There is a need for further input in this area, large scale training of field veterinarians to ensure better knowledge and control of disease in Vietnam This could be achieved though better

collaboration of aid agencies in Vietnam especially those supplying training for AI Training

in data analysis by DAH staff is also an area that needs further input

7.2 Options

The government of Vietnam is looking at increasing the support to DAH and has increased funding for AI diagnosis and is looking to do the same for FMD diagnosis The increased funding for AI has seen laboratories updated with new equipment some of which will

improve all disease diagnosis which includes FMD

The money available to improve AI diagnosis can improve all disease diagnosis if this money

is used wisely, eg in training field veterinarians in AI diagnosis, the training should be

general to cover all diseases including FMD, CSF and other diseases of importance in

Vietnam Also in improving AI diagnosis in laboratories the training should emphasize how improvement can be applied to all areas of diagnosis, eg quality assurance in the laboratory

7.3 Sustainability

The DAH laboratories appear to be well supported by the central government The DAH laboratories are also receiving increased support from overseas funding agencies as part of the AI campaign to improve laboratory facilities

RAHC-HCMC is gaining in confidence as an institution This confidence is beginning to be passed on to other DAH laboratories The DAH scientific staff are quick to understand new information and are energetic in its implementation People have an obvious desire to do their jobs well This desire is obvious in staff in RAHC-HCMC and other laboratories The laboratories just need technical support to be encouraged to carry out the techniques that they need for diagnosis and research

The project is following a well tried model for successful technology transfer, of

demonstration and teaching of the technology in the donor facility followed by supply of reagents for implementation by the trainees at their own initiative in the recipient laboratory, subsequently backed up by consultancy visits to the recipient laboratory for support and fine tuning A useful indicator of likely success is the initiative and ability of the trainees to make

a useful attempt at implementation by themselves DAH and NAVETCO staff show a high level of involvement in this way The RAHC-HCMC now has established ELISA, cell culture and molecular technologies

The AusAID funded CSFV project, that linked DAH, NaVetCo and AAHL prior to this project, is proving sustainable NAVETCO and DAH not only maintains the diagnostic capability developed at that time, but on its own initiative and unassisted by AAHL is

conducting training courses and technology transfer to other labs in Vietnam

8 Next Critical Steps

In the next 6 months AAHL staff will review the project outputs and to examine the

information collected so far in the project and continue to provide support to the

establishment of the technologies transferred to the laboratories The next round of the surveillance for the collection of the samples will be implemented with any changes

sero-necessary In the next 6 months we will finalise the transfer of technologies for FMD

diagnosis and evaluate each laboratories capability to carry out FMD diagnosis RAHC will continue to use cell culture techniques to grow FMD isolates from the field The FMD isolates from the field will be send for further analysis of these isolates by PCR and sequencing Two trainees will visit AAHL to be trained in virus isolation and cell culture AAHL consultants to visit the laboratories to examine data from project and to review the techniques for FMD diagnosis

HCMC-9 Conclusion

The project activities are ahead of schedule with training, this will allow more time for the laboratories in the project to take up the technology The DAH laboratories are running the field trials and have provided further training to the field staff to improve sample and data collection from the field The sero-surveillance is in progress and is expected to be completed

on schedule in the south but the north are finding it difficult to collect samples from the field

to due to the AI workload The field veterinarians need further training in the collection of data and how to ensure the correct information is obtained from the farmer The staff at RAHC-HCMC will continue to deal directly with the veterinarians in the field to ensure the correct information is collected Training of the HCMCHCMC laboratory in molecular technology was completed, with a training course for 2 trainees from the laboratory held at AAHL RAHC-HCMC continues to carry out in country training where necessary to ensure problems are addressed with the transfer of technology to the laboratories in the project The establishment of cell culture has allowed the growth of FMD isolates from the field that could not be previously identified The growth of isolates has also allowed sending of higher quality samples to the world reference laboratory at Pirbright, UK, and allowing results to be returned in a quicker timeframe The introduction of molecular techniques at RAHC-HCMC has allowed sequecing results to be obtained in real time allowing DAH Vietnam to make informed decisions about serotypes of FMD circulating in Vietnam and the correct vaccine to use in each region to control FMD

This project continues to be important project for Vietnam with the importance of FMD increasing due to the large outbreaks that have occurred in Vietnam in 2006