Management of CSF and FMD at the village level in Lao PDR

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.