Sunday 27th September
Travel from Brisbane to Bangkok via Sydney, Qantas Flight Nos QF517, QF301
Overnight accommodation at the Amari Airport Hotel
Monday 28th September
Together with Professor Spradbrow, I visited the FAO headquarters, Bangkok where we met with Dr Denis Hoffmann, Regional Animal Health and Production Officer, Mr Mansour, Personnel Officer and Mr Xu Lingfeng, Country Project Manager.
I signed a formal offer of employment as an FAO consultant and received a Travel Authorization, equivalent to 80% of the Daily Subsistence Allowance ($US 780.00)
In the evening, we travelled with Dr Denis Hoffmann and Mrs Hoffmann from Yangon to Bangkok, That Airways Flight No TG305. We were met at the Yangon Airport by Dr Thet Swe, Director of the Livestock Breeding and Veterinary Department and driven to our accommodation at the Amanda Inn.
Tuesday 29th September
AM: Dr Denis Hoffmann, Professor Spradbrow and I were accompanied by Dr Thet Swe and Dr Tin Aung Myint to FAO headquarters in Yangon for a meeting with Mr Francis Rinville, the FAO representative in Myanmar. He stressed the need for our work with the small scale production and testing of Newcastle disease vaccine to be sustainable.
PM: At the Central Vaccine Laboratory, I met Dr Nyan Soe, the Assistant Director of Research and Biologics who manages the laboratory. I was introduced to my designated counterpart. Daw Mya Mya San, Research Officer and the four assistant researchers who would be attending the workshop. The materials from Australia and Bangkok were delivered. I inspected the laboratory and discussed preparations for the workshop. At the entrance to the laboratory, a well ventilated area containing desks and a white board was available for teaching and discussions. The virology laboratory containing a Laminar Flow unit, refrigerator and some bench space had been set aside for the practical work. One vial of Websters HR-V4 experimental vaccine was resuspended in diluent and used to inoculate eggs for antigen preparation.
Wednesday 30th September
AM: A formal breakfast meeting was held at 0800 by the Deputy Minister of the Ministry of Livestock and Fisheries. This was followed by the official opening which was addressed by the Deputy Minister, Mr Francis Rinville, FAO representative for Myanmar and Professor Spradbrow. Professor Spradbrow then commenced the workshops with a joint session attended by the participants of both the extension and the laboratory workshops. He covered the following topics:
· History of the Newcastle disease vaccine research projects sponsored by ACIAR
· Use of a seedlot system to produce Newcastle disease vaccine
· The need for ongoing co-operation between the extension and laboratory groups
· Outline of the educational objectives for both the groups
I addressed the group outlining my role assisting Professor Spradbrow with his Newcastle disease research and as the convenor of the laboratory workshops.
PM: I commenced the Laboratory workshop with a personal introduction and welcome to the participants of the workshop. I included further details of my research work with Newcastle disease vaccine and as convenor of three previous workshops held in Africa. Participants were asked to complete registration forms. Name tags, laboratory coats and laboratory manuals were distributed. I initiated discussion about the current production and testing of Newcastle disease vaccine in the Livestock Breeding and Veterinary Department. Participants from the Central Vaccine Laboratory and the Veterinary Assay Laboratory provided the following information. Two strains of Newcastle disease vaccine are produced in the Central Vaccine Laboratory. Komarov strain is stabilized with 3% skim milk, freeze dried and supplied in vials of 500 doses at a cost of 500 kyat per vial. F strain is supplied as a wet vaccine at a cost of 300 kyat per vial. It is stabilized by mixing three parts of allantoic fluid with one part of a solution containing 0.2% PVP and 10% lactose prior to storage at -20°C. It is further diluted with 6 ml of normal saline to give 300 doses administered by eye drop. Both vaccines cost one kyat per dose. Quality Control and Efficacy trials are carried out at the Veterinary Assay Laboratory. Quality Control involves titration of the vaccine and passage in eggs to check that the infectivity titre per dose is greater than or equal to 107 ELD50. They also carry out a Safety Test where 25 chickens are vaccinated and bled for serology two weeks post vaccination. Haemagglutination inhibition tests are used to determine antibody titres. These birds together with 10 unvaccinated controls are challenged with a field strain. The vaccine is approved for sale if 100% of the controls are killed by the field strain and 70% of the vaccinates survive the challenge.
Further discussions included:
· Quantifying the amount of lentogenic Newcastle disease virus in a vial of vaccine or in allantoic fluid harvested for vaccine, by ten-fold serial dilutions, inoculation of allantoic cavity often day old embryonated eggs, harvesting of allantoic fluid, HA test for presence of virus, calculation of infectivity titre using Reed Muench method.
· Use of aseptic technique in laboratory work to minimize bacterial contamination
Thursday 1st October
AM: Professor Spradbrow conducted a joint session for participants of both the laboratory and the extension workshops. The following topics were addressed:
· Quality control of a wet vaccine produced by regional laboratories
· Standardization of Haemagglutination Inhibition (HI) tests between laboratories in Myanmar. Importance of preparing and distributing standard positive and negative sera to all the laboratories.
· Economic benefits of vaccinating village chickens
· Conducting pilot trials of vaccines in village chickens
· Protection offered by maternal antibodies against Newcastle disease
The participants of the laboratory workshop returned to the Central Vaccine Laboratory where I asked the Dr Nyan Soe, the manager of the laboratory to contribute to a discussion about Laboratory Safety, disposal of sharps and biological wastes. Laboratory rules appropriate for the Newcastle disease vaccine workshop were developed. The participants were divided into four working groups of four per group. Ten day old embryonated eggs were candled and marked by each of the groups in preparation for inoculation.
PM: The techniques of ten-fold serial dilution and egg inoculation were demonstrated. A freeze dried vial of the I2 seed vaccine was opened and suspended in diluent. To prepare I2 working seed, an Assistant Researcher experienced with the practice of egg inoculation was selected to inoculate seventy eggs with the I2 seed virus. One of the working groups titrated this suspension of the I2 seed and inoculated eggs. The remaining three groups were given suspensions of Websters HR-V4 experimental vaccine to titrate.
Friday 3rd October
AM: The joint session of participants of both workshops was held at the Central Vaccine Laboratory. The techniques used to collect blood from chickens by wingbleeding, vaccination by eye drop and wing tagging were demonstrated. Blood was collected into an anticoagulant in order to be used for the preparation of 10% red blood cells. Participants from the extension workshop were shown where and how the Newcastle disease vaccine is produced at the Central Vaccine Laboratory.
Participants of the laboratory workshop were taken to the government owned Number 1 breeding farm. Arrangements had been made with the Assistant Manager for birds to be made available for wing bleeding. The participants each bled a minimum of three chickens and readily mastered the demonstrated technique which minimizes the trauma experienced by the bird.
PM: Participants assisted with the preparation of 10% red blood cells used in the Haemagglutination (HA) and Haemagglutination Inhibition (HI) tests. Eggs inoculated the previous day were candled and dead eggs were discarded. The serum collected in the morning had been kept at 37°C to encourage separation of the serum from the clot. The participants transferred the serum to microfuge tubes. A centrifuge for these tubes was not available. The tubes were left at 4°C for any red blood cells to settle overnight.
Saturday 3rd October
AM: Serum samples prepared the previous day were transferred to microfuge tubes, numbered and stored at -20°C. Participants were impressed with the visible lack of haemolysed red blood cells in the samples. Allantoic fluid from the eggs inoculated with Websters HR-V4 four days earlier was harvested. The titration and testing of this allantoic fluid by the HA test and subsequent dilution to 4 HA units for use as the haemagglutinin in HI tests was demonstrated. The positive and negative sera brought from Australia were tested using this diluted allantoic fluid. Unfortunately the negative serum tested positive. I concluded these samples had been incorrectly labelled in Australia prior to my departure.
PM: Accompanied by Dr Tin Aung Myint and Daw Mya Mya San, the Australian FAO consultants were taken to the Bogyoke Aung San Market. In the evening we accepted an invitation to visit the residence of the Australian Ambassador to Myanmar.
Sunday 4th October
The workshop was not held. Accompanied by Dr Thet Swe, Dr Tin Aung Myint, Daw Mya Mya San and my Australian colleagues Professor Spradbrow and Dr Denis Hoffmann, the day was spent visiting the Shwedagon Pagoda, Zoological Gardens, Natural History Museum, Aquarium and the new port and industrial zone at Thilawa. We inspected the holding area for goats under quarantine which is being constructed at Thilawa under Dr Thet Swe's supervision.
Monday 5th October
AM: There was further discussion and practice of the calculation of infectivity titres, (EID50) using the Reed Muench method. In response to a request from participants, some time was devoted to the use of logarithm tables to calculate dilution factors involved in preparation of wet vaccine from allantoic fluid.
PM: Eggs were chilled prior to harvesting. Each group harvested allantoic fluid from the eggs they had inoculated four days earlier. This was tested for the presence of virus by the HA test. The Reed Muench method was employed to calculate the infectivity titre of the original viral suspension from these results. Each group also practiced harvesting allantoic fluid from eggs which had tested HA positive. This allantoic fluid was labelled and stored at 4°C. The allantoic fluid from the eggs inoculated with the I2 seed virus was harvested, tested for the presence of virus and centrifuged. A four ml aliquot was set aside for further testing and the remainder of the supernatent was given to Dr Nyen Soe for storage at -40°C. This will constitute the working seed for the I2 vaccine in Myanmar.
U Muang Maung Nyunt, Director General of the Livestock Breeding and Veterinary Department, Dr Aung Kyaw Lynn, Deputy Director General of the Livestock Breeding and Veterinary Department and Dr Hla Myint, Advisor to the Director General, all visited the laboratory to see the harvesting of the I2 working seed.
Tuesday 6th October
AM: Each group tabled their HA results and calculated infectivity titres on the whiteboard. A discussion was generated around the interpretation of these results. Each group titrated and tested antigen for use in the HI tests.
PM: The serum samples collected on Friday were thawed and distributed for testing by the HI test. Each group tested at least sixteen samples. Results were recorded on the microtitre recording sheets.
In the evening U Maung Maung Nyunt, Director General of Livestock Breeding and Veterinary Department, and the Lt. Col Khin Maung Aye, Managing Director of Livestock, Feedstuff and Milk Products Enterprise, hosted a dinner in honour of the visiting FAO consultants.
Wednesday 7th October
AM: Results of the HI tests carried out the previous day were tabled and discussed. The Geometric Mean Titre (GMT) was calculated to be 26.7. This indicates the birds have a protective antibody titre. They had been vaccinated at fourteen days old with the F strain vaccine made at the Central Vaccine Laboratory and vaccinated again at ten weeks old with Komorov vaccine imported from Singapore. This second vaccination had been carried out a few days prior to our bleeding the chickens. The manager indicated their regime for control of Newcastle disease would include vaccination with Komorov every five months. I asked that she be informed of the results of the HI tests.
Dr Win Hlaing from the Central Diagnostic Laboratory provided serum samples from a commercial chicken farm. These samples had already been tested for HI antibodies at the Central Diagnostic Laboratory. They were numbered and distributed to the working groups for retesting using the procedure for the HI test being practiced in the workshop. The positive antiserum used in the Assay Laboratory for their HI tests was also provided for testing. The serum samples collected from chickens at the number one breeding farm were thawed and pooled. Each group repeated HI tests on this pooled serum with good agreement of the result of a titre of 28.
The results of the two HI tests of the serum from the commercial chicken farm, firstly at the diagnostic laboratory and the tests just completed at the workshop were tabled. Geometric Mean Titres(GMT) were calculated and compared. For the tests of the serum samples carried out at the Diagnostic Laboratory, a GMT of 23.1 was calculated. This was compared with a calculated GMT of 25.2 for the same samples tested at the workshop. I stressed that this did not prove that the results obtained at either of the labs were wrong. Rather I used these results to demonstrate the need for standardization of HI tests between laboratories.
PM: A discussion was carried out about the application of vaccine to feed stuffs. Participants planned and carried out the application of enough vaccine to cooked white rice to vaccinate ten village chickens. The vaccine used was allantoic fluid containing Websters HR-V4 vaccine harvested on Monday and stored at 4°C. The vaccine coated cooked white rice was fed to chickens owned by an employee who lived nearby. Ten grams of the vaccine coated cooked white rice was removed prior to distribution to the chickens. It was mixed with 10 ml of antibiotic solution and stored at 4°C for 30 minutes. The amount of virus recovered from the rice was assayed by titration in eggs. The aliquot of the I2 working seed that had been kept at 4°C was also titrated in eggs to establish the infectivity titre. Allantoic fluid harvested Monday was centrifuged and 1.5 ml aliquots distributed into microfuge tubes and stored at -40°C. This is suitable for distribution to the laboratories for use as the haemagglutinin in HI tests. Aliquots of the pooled serum were also prepared. This serum had an HI titre of 28. It could be used to standardize HI tests in different laboratories until the official departmental standard positive and negative serum is prepared.
Thursday 8th October
AM: Visits were arranged to three of the central laboratories at Insein who had staff participating in the workshop.
1. Veterinary Assay Laboratory. We met with U Maung Maung, Assistant Director and Dr Wah Wah Han, Research Officer. Both had attended the extension workshop. We discussed their current HI testing procedure and the need for standard positive and negative controls. Bacterial contamination of the Newcastle disease vaccines prepared at the central vaccine laboratory are tested by inoculation of Maconkey and nutrient agars, nutrient, thioglycollate and mycoplasma broths. We were shown the sections of the laboratory where the quality control and efficacy testing is carried out. This included a very well maintained area for the housing of animals in wire cages. Interest was expressed by U Maung Maung and Dr Wah Wah Han in conducting trials to compare the Komorov and F strains of Newcastle disease vaccine currently produced at the Central Vaccine Laboratory with commercial HR-V4 and I2 vaccine. We discussed the methodology of such trials and the need for improved facilities for housing experimental chickens.
2. Central Diagnostic Laboratory. The group were met by U That Mang, Deputy Director, Daw TinAye Kyi, Assistant Director and epidemiologist and Daw Ah Mar, Research Officer in Virology and Serology. Chicken serum is routinely tested for Newcastle disease antibodies by the HI test. We were shown the materials currently used to carry out this testing.
3. Veterinary Medicine Plant. Daw Nan Yin Yin Myint, the manager of the plant was a participant in the laboratory workshop. This is a large commercial plant involved in the production, sale and distribution of imported vitamins, antibiotics, essential amino acids and water soluble feed supplements. The plant is not involved in the sale or distribution of vaccines.
PM: The formal closing ceremony was held at the headquarters of the Livestock Breeding and Veterinary Department and presided over by Dr Aung Kyaw Lynn, the Deputy Director General. All the participants were handed a Certificate of Attendance. At the Central Vaccine Laboratory, I closed the nine days of collaborative laboratory work by inviting Professor Spradbrow to join the participants and myself for a general discussion. Dr Hla Myint also contributed.
Topics covered included:
· Frequency of vaccination
· Duration of immunity
· Dilution of I2 working seed prior to inoculation in eggs
· Vaccine efficacy trials - comparison between vaccines, application of vaccine to feed, vaccination of village chickens
· Possibility of a Newcastle disease vaccine newsletter
· Theoretical aspects of growth of Newcastle disease virus in embryonated eggs
· Suggested experiments to optimize production of vaccine
Participants were asked to anonymously complete an evaluation form recording their response to various aspects of the workshop. Laboratory daybooks were distributed to the participants. Copies of references brought from Australia were left with Dr Nyan Soe at the Central Vaccine Laboratory for participants to access.
Friday 9th October to Sunday 11th October
At my own expense and accompanied by Mrs Meredeth Hoffmann, wife of Dr Denis Hoffmann, Regional Animal Health and Production Officer, FAO, Bangkok, I travelled by air to Bagan where I stayed two nights and visited the historical sites close to Bagan. In order for me to extend my stay in Myanmar and travel beyond Yangon, official approval was required. This was arranged by U Maung Maung, Director General of the Livestock Breeding and Veterinary Department and supported by Lt. Col. Khin Maung Aye, Managing Director, Livestock, Feedstuff and Milk Products Enterprise, and Dr Than Hla, Deputy Director, Animal Health and Development Division, Livestock Breeding and Veterinary Department. Their support is gratefully acknowledged. They also arranged for two of their staff members. Daw Cho Cho Thein and Daw Yin Yin Sann from the Training Section to travel from Yangon to Bagan. Together with U Shwe Soe, Veterinary Officer of the Livestock Breeding and Veterinary Department at Nyang Oo township, they met us on our arrival at the Nyang Oo Airport. They drove us to our hotel, escorted us to visit many pagodas and arranged an excursion to Mt Poppa. Their company and guidance during the two day visit was greatly appreciated.
On returning to Yangon on Sunday 11th October, we were met at the airport by Dr Tin Aung Myint, taken to the Amanda Inn and accompanied for further sightseeing and shopping in Yangon.
Monday 12th October
AM: Dr Nyen Soe visited at the Amanda Inn. He brought the results for the titration of the L working seed and the recovery of the HR-V4 virus applied to cooked white rice. The titration confirmed high titre of 109 8 EID50 of virus in the I2 working seed. The working seed can now be thawed and aliquots prepared for storage at -40°C. We agreed one of the , aliquots should be thawed and titrated to further check the infectivity titre of the aliquots of working seed. We had final discussions about the workshop, the proposal for a newsletter to keep people informed about progress with the vaccine and distribution of the surplus materials brought from Australia. Dr Hla Myint also telephoned to discuss the results of the titration of the I2, working seed.
Mrs Hoffmann and I were driven to the Yangon Airport and caught the That Airways Flight No TG 3 04 to Bangkok.
PM: At 2050,1 left Bangkok on That Airways Flight No TG 983 for Brisbane via Sydney.
Tuesday 13th October
AM: I arrived at Brisbane International Airport at 1120.