Emergency Assistance for Control of Avian Influenza in Indonesia
OSRO/RAS/401/JPN
Technical Report
by
J. Garcia-Garcia
Vaccine Production Specialist
7- 18, June 2004
Food and Agriculture Organization of the United Nations
Rome
1
TABLE OF CONTENTS
Objectives of the mission
3
Background
3
Main findings and conclusions
4
1. Avian Influenza vaccination Programme
4
2. Local vaccine production capabilities
5
3. Vaccine Development
3.1. Master Seed
3.2. SPF embryos for vaccine production
3.3. Antigen content of the vaccine
3.4. Inactivation procedure
3.5. Oil emulsion vaccines
3.6. Vaccine efficacy studies
3.7. Vaccination efficacy at farm level
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5
8
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9
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9
11
4. Vaccination using heterologous vaccine
11
5. Vaccine needs for Indonesia
11
6. Personal suggestions
12
Summary of conclusions and recommendations
12
Complementary information
13
Annex 1. Terms of Reference
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2
Objectives of the mission
This mission to Indonesia was carried out from June 7 to 14, 2004 under the
FAOTCP/INS/3001 “Emergency Assistance for Control of Avian Influenza in
Indonesia ‘ launched by FAO in response to Avian Influenza (AI) outbreak in Asia.
The main objective of the mission was to provide technical assistance to the
Ministry of Agriculture for the establishment of a programme to control the outbreak
of Avian Influenza (AI), based on the use of inactivated oil-emulsion vaccine. A
further objective was to advise on appropriate vaccines and to assess short,
medium and long term needs. Also relevant is the national technical capabilities for
vaccine production, including capacity for vaccine production and testing and
expected quality of locally produced vaccine.
The Terms of Reference of the mission are attached in annex 1.
Background
The Indonesian poultry industry in Indonesia has been infected with Avian
Influenza virus, H5N1 subtype, likely since August 2003. However, infection was
not clearly characterized at that time because of shortfalls in diagnostic capability;
avian influenza was officially confirmed and reported in February 2004.
Once the disease had been detected, AI vaccines were introduced, mainly from
China, without Government authorization, and others imported through regulatory
channels. The Minister of Agriculture decided to initiate a widespread vaccination
program to reduce the economic impact on the poultry industry, particularly small
producers. At this time, the Government plans to provide highly efficacious AI
vaccine, free of charge, to all small farmers and owners of backyard chickens.
The present situation in Indonesia is characterized by a lack of sound on-farm
biosecurity measures; no programme of vaccination for broilers, and little or no
meaningful control of the movement and sale of poultry and poultry products (eggs
and meat). The presence of live bird markets, which incorporate slaughterhouses,
selling poultry for human consumption, and live bird markets selling ornamental
species, including indigenous and fighting cocks, further complicates the
management of AI. Chickens and ducks in live bird markets can be important
reservoirs of infection.
3
Main findings and conclusions
1. Avian Influenza vaccination Programme
One of the most important elements is a regular monitoring or surveillance program
to determine the extent of infection. This could be achieved by taking samples from
broiler farms located in the main poultry production regions in Indonesia. Broiler
producers are not currently using vaccine. Information obtained from regular
surveillance in the broiler production sector would be useful in making decisions on
the location of infection, priorities for vaccine use and, potentially regionalization.
Any vaccination programme must be supported by the application of minimum
biosecurity measures at farms and associated premises, such as bird and egg
transport facilities. Cleaning and disinfections of trucks and cages used in the
transport of chickens and eggs before leaving distribution centers (markets) is
strongly recommended. Cleaning and disinfections of premises and equipment at
markets - both for poultry and ornamental species - is also important.
Additional inspection points in the roads and highways should be established to
control the movement of the commercial birds and their products to prevent the
infection in free areas.
In order to evaluate vaccine performance, Government Veterinary Services must
have basic information on patterns of use. This would be facilitated by introducing
a requirement for Government authorization of vaccine purchase and use.
Indonesian vaccine production laboratories should also be required to inform the
Ministry of Agriculture of the amount of vaccine produced and sold and the
companies/individuals purchasing the product.
It is also recommended that the Government introduce controls over the movement
to slaughter of vaccinated chickens, based on prior testing and negative serology
of sentinel chickens. Flocks that show evidence of continued virus circulation,
despite the use of vaccine, should be sent to designated slaughterhouses,
authorized by the Government, for processing infected birds. Infected birds must
not be sent to live bird markets.
Serological testing of sentinel chickens, based on 35 samples per farm (or other
recommendation of epidemiologists familiar with the local situation), can be used to
determine the performance of an AI vaccination programme. Results of such
testing must be available prior to marketing of birds.
Observation of disease or mortality in sentinel birds should be investigated with the
assistance of the diagnostic laboratory to determine the cause. If AI infection is
established, the total population in the farm should be depopulated.
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For a proper evaluation of the performance of a vaccination campaign, it will be
necessary to consider the performance of vaccinated flocks in specified numbers
of farms, in specified regions, during a specified time period, as well as background
information on flock health, intercurrent disease and use of other vaccines.
Control of AI in Indonesia will require the establishment of measures and controls
relating to vaccine production and use, movement controls, routine surveillance
and reporting.
2. Local vaccine production capabilities
Two private laboratories (VAKSINDO and MEDION) and one official laboratory
(PUSVETMA) have been selected to produce vaccine locally. These laboratories
vary greatly in terms of production capacity, availability of trained people, minimum
standards of facilities, implementation and capacity to conduct quality control and
quality assurance.
The following observations can be made with regard to the production capabilities
of the three laboratories:
 VAKSINDO has the maximum capability for vaccine production. They have
more experience, better conditions and availability to external expertise.
 MEDION has a lower level of technology and should make investments to
improve bio-containment, modernize equipment and improve Good
Manufacturing Practice (GMP). For example, they should tighten measures
relating to access to the facility, change wood for cement, paint with epoxy
and have sanitary curves in the laboratory. It was of the consultant
impression that they have the most experienced personnel in vaccine
production of the three visited laboratories.
 PUSVETMA does not currently have the capability to produce good quality
vaccine. The facilities and equipment are too old and have very poor
sanitary conditions. Significant investment would be needed. However, the
personnel appear to be highly motivated and could be trained in commercial
private laboratories.
3. Vaccine Development
3.1. Master Seed
There are at least two strains used in local vaccine production, one isolate was
produced at the Drug and Pharmaceutical Assay Laboratory, Bogor province. This
strain was originally characterized as Newcastle Disease (ND) virus, before it was
redefined as AI virus.
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This strain was submitted for vaccine production at the Government Vaccine
Laboratory (PUSVETMA), Surabaya City. Reportedly, the virus did not grow in
chicken embryos. The laboratory then selected two isolates obtained from different
locations it the district of Surabaya. They grew those virus isolates and mixed them
to develop their own Master Seed, to produce the vaccine made early this year. At
PUSVETMA they have lyophilized their own Master Seed and keep it at
refrigeration temperature.
Private laboratories indicated that they are using the strain received from Bogor
laboratory. However, it is not possible to ascertain which virus is in use and it is
necessary to characterize the virus used by each vaccine producer.
The isolates being used for local vaccine preparation are highly pathogenic virus
H5N1, isolated in Indonesia from different times and places. Ideally, the virus
should offer the same immunogenicity, even if there are minor genetic differences.
Personnel at the Bogor Laboratory indicated that they perform 10 serial passages
in chicken embryos to reduce the pathogenicity of the isolated virus. In the
absence of scientific information to confirm this procedure, it must be assumed that
the master seed used in the vaccine is highly pathogenic.
Comments and Recommendations
- It is necessary to characterize the virus isolates that have been used (and any
others of interest) to identify the most appropriate candidate for vaccine
preparation, based on pathogenicity in susceptible chickens, by IV injection. This
step should be done in a Government laboratory under biosecure conditions.
- Vaccine efficacy studies that require challenge with characterized highly
pathogenic virus should also be conducted by government authorized personnel.
The studies must be done under biosecure conditions, with established disposal
procedures for all materials, including birds, used in these studies. At present
private laboratories are conducting these studies. Official veterinarians
appropriately trained in biosecurity and vaccine evaluation, should conduct, or
supervise, pathogenicity and challenge testing to measure vaccine efficacy. Proper
management and interpretation of these studies should be done by an expert to
support decision-making by the Indonesian Ministry of Agriculture.
- Molecular characterization can be accomplished at an avian influenza reference
laboratory such as Weybridge (UK) or the South-Eastern Poultry Research
Laboratory of USDA- ARS in USA. The latter laboratory has been contacted and
USDA import permits obtained and submitted, by fax, to the office of the General
Director of Animal Health of Indonesia.
- Apart from molecular characterization of the isolates it is necessary to
demonstrate that the virus selected for vaccine purposes is free of other virus
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contaminants and other infectious agents, before any isolated virus can be defined
as a Master Seed. Limiting dilution techniques, in three fold dilutions would be a
required first step.
- It is recommended that the Asia-Pacific Region establish, as soon as possible,
their own BSL3 or BSL4 level reference laboratory that can conduct necessary
studies.
- Low Pathogenic H5N1 virus must be used for vaccine purposes because:

The HP AI virus H5N1 has been associated with human infection. During
virus replication in the chicken embryo, for vaccine manufacturing purposes,
it is possible that viral mutations may arise, with the possible result that the
virus could gain a capability to infect humans. Recent studies have shown
isolates of H5N1 obtained in China are more pathogenic for mice than
previous HP isolates.

HP virus readily kills embryos and the resulting lysis of embryo tissues can
result in vaccine contamination by blood and other substances.
Furthermore, antigen quantity in chicken embryos inoculated with HPAI is
lower than LPAI. Fluids should only be collected from live embryos
inoculated with LP virus.

HP virus presents a risk to spread within or outside the laboratory during
transport and disposal of used embryos and other contaminated materials (I
was informed that the embryos used for production are transported in plastic
bags, which are readily pierced, with subsequent contamination of the
environment). Accidental spillage of low pathogenic virus can also occur but
the epidemiological consequences are much less serious.

Failures in the inactivation procedure may result in the spread of HP virus
during vaccination programs. For that, proper inactivation must be
confirmed, by testing fluids in embryos at least during two passages. An
additional test should be conducted by vaccinating chickens with double
dose (1 ml) and housing them with unvaccinated chickens. The
unvaccinated chickens should be seronegative 21 and 28 days after being
placed in contact with vaccinated chickens.
- Indonesia has many AI virus isolates (more than 60) stored at the Tropical
Disease Center laboratory in Surabaya City. It is likely that among them, a low
pathogenic virus exists that would be suitable for use as a Master Seed.
- Another strategy may be considered for the isolation of LP virus. This consists of
a field study in which AI free chickens are placed in a serological positive farm and
sampled (blood and tracheal and cloacal swabs) every 7 days. Swabs are kept at -
7
70° C in the laboratory. HI tests are carried out on the serum of the newly
introduced chickens. When serum samples react as positive, test swabs taken 14
and 21 days before serum samples became positive, by inoculation into embryo
chickens in an attempt to isolate the virus. The sentinel birds described above may
not show clinical signs of disease despite being seropositive, as these birds may
be infected with LP virus.
Swabs should be prepared with polyester fiber instead of cotton. Appropriate
transport media containing antibiotics should be used. More detailed information
can be found in the WHO diagnostic manual1. The allantoic fluid collected from the
chicken embryos should be diluted 1:10 in PBS and inoculated intravenously into
SPF chickens. These chickens should not show clinical signs in the 10 - 12 days
following inoculation. The resulting isolate should be tested for virus titre in
embryos and immunogenic studies in chickens before it can be considered as a
Master seed candidate
Preparation of vaccine from scratch, using local sources, may take at least one
year, assuming the availability of appropriate facilities, personnel and equipment.
3.2. SPF embryos for vaccine production
SPF embryos must be used in vaccine production to ensure that vaccines are not
contaminated with other infectious microorganisms for poultry.
VAKSINDO advised that they are producing their own SPF chickens and embryos,
but the number they reported did not match with the production they claimed.
MEDION advised that they import SPF eggs to produce vaccine. It is important to
confirm that this is the case.
PUSVETMA does not have access to SPF eggs and requires support from FAO or
the Government for their provision as well as additional support for equipment and
materials required for vaccine production.
3.3. Antigen content of the vaccine
Antigenic mass is important to provide appropriate immunogenicity in a vaccine
and is a function of virus content and amount of allantoic fluid in the vaccine
formulation.
AI vaccine should contain 12 - 16% of allantoic fluid. Information obtained from the
two private laboratories indicates they are using less than 10% in the vaccines they
are producing. This makes the vaccine less expensive but provides a lower quality
(less immunogenic) vaccine which in the long run can be more costly.
1
http://www.who.int/csr/resources/publications/influenza/en/whocdscsrncs20025.pdf
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Vaccines that are obtained through illegal or unofficial channels are likely to have a
lower low antigenic mass and therefore provide poor protection.
It is important to determine the titre of the working seed virus in harvested fluid. It is
recommended to use 109 ELD50 /ml. The virus titre in the production fluid would be
close to 108.5 to 109 ELD50 /ml with a minimum of 32 hemagglutinating units (HAU)
for vaccine production.
The virus titre will depend on the virus isolate used for vaccine production (i.e. the
Master Seed selected). It may be necessary to modify production parameters, such
as the dilution of the working seed, temperature for incubation, time to harvesting,
handling of harvested fluid and amount of the fluid in the vaccine, in order to
establish the best results. It is necessary to conduct chicken challenge trials to
determine the best conditions and proper formulation.
The use f combined vaccines containing both Avian Influenza and Newcastle
Disease (ND) viruses could be considered in the future. In such case the content of
ND virus must be higher than that of AI virus.
3.4. Inactivation procedure
Vaccine fluids must be inactivated as soon as they are obtained. Maintaining them
without inactivation allows for bacterial growth. The use of beta-propiolactone to
inactivate avian influenza virus should be considered. This method requires
considerable experience in order to obtain reliable results. Both private and official
laboratories advised that they are aware of the procedure, but they have more
experience with formaldehyde inactivation. The use of formaldehyde is acceptable
and may be preferable until such time as training and supplies can be obtained to
utilize beta-propiolactone in an effective and repeatable manner.
3.5. Oil emulsion vaccines
Oil-emulsion vaccines are preferable to aluminum hydroxide-based products.
However, oil based products will produce a more extensive reaction at the injection
site. While Indonesian producers currently prefer to administer the vaccine by intramuscular injection, it is likely that this will be done by subcutaneous injection once
the vaccination programme is established in birds of 10-14 days of age.
Subcutaneous injection will reduce the extent of the inflammatory response.
Vaccine producers should ensure that the vaccine is not too viscous, to ensure
easy application. More viscous vaccines are harder to administer and can lead to
failures in vaccination technique.
3.6. Vaccine efficacy studies
Vaccine efficacy should be measured using serological studies that must be
conducted for each vaccine batch before the lot is approved. Experimental
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challenge studies in isolation units may be also considered to evaluate vaccine
efficiency to prevent clinical signs in susceptible chickens.
Serological studies should be conducted in farm that is free of AI, located in an AIfree area and with good biosecurity measures. Every batch should be tested and
all experimental formulations and experimental procedures tested in groups of 25
chickens per trial. Those activities could be conducted by personnel of
PUSVETMA.
The field and experimental protocol for each trial is as follows:
 For basic and experimental vaccine studies; the serological reaction of
vaccinated chickens would be tested after vaccination. Birds should be
sampled at day 0 of the study and every 7 days up to 42 days.
 For field studies with commercial poultry and vaccine batches, a serological
study should be conducted at 21 and 28 days after vaccination.
 Evaluation of the serological response will be the guide to achieve high
efficacious vaccine.
 It is not necessary to conduct challenge studies for each vaccine batch prior
to approval but, it is necessary to do so for vaccine licensing procedures
and for yearly renewal of the license.
 Challenge studies can be done in SPF or AI-free commercial birds.
 Experimental challenge studies with highly pathogenic virus should be
undertaken at 21 and 28 days after vaccination; to measure vaccine efficacy
based on prevention of morbidity and mortality.
 Challenge studies must be conducted under appropriate biosecurity
measures to prevent accidental virus release. The challenge facility should
be distant from the experimental farm and should utilize “HEPA” air filters
and positive pressure. It may be possible to use VAKSINDO or MEDION
facilities or the facility at Bogor laboratory.
 Personnel working in the experimental farm should never visit any
diagnostic laboratories, vaccine production facilities or the experimental
challenge facility, to assure the farm will stay free of AI
For these studies, syringes, needles, serum collectors (drinking straws are very
useful and cheap) and material for HI test, protective clothing and footwear must
be obtained for exclusive use on the farm. The AI free commercial experimental
farm must have a shower facility and an appropriate means of disposing of birds,
including safe transportation of birds to slaughterhouse.
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3.7. Vaccination efficacy at farm level
It is recommended that a technical document be prepared for use in training
vaccine crews working on farms. Important subjects include: how to administer the
vaccine properly, biosecurity measures, the effects of AI and the result of
inappropriate vaccination.
In order to avoid spread of virus, vaccination crews must change protective
equipment, clean and disinfect shoes, take a shower and have a break from
vaccination (of at least one day) in between farms. Use of oral antiseptic after
brushing teeth is recommended.
4. Vaccination using heterologous vaccine
It is recommended that Indonesia use heterologous LP H5 that does not represent
a human health risk. For local production of heterologous vaccine, it is important to
ensure proper inactivation of virus to avoid the inadvertent introduction of an
additional strain of circulating virus.
Antibodies due to vaccination can be differentiated from those due to infection with
field virus by the use of the Neuroaminidase and Neuroaminidase Inhibition (NI)
Assay. The general procedure can be found at the following Internet address in the
manual for diagnostic and surveillance published on-line from WHO1.
It is recommended that a Department of Animal Health laboratory hold the Master
seed and provide the Working Seed to the laboratories that will produce the
vaccine. This will ensure official control of strains used in vaccine production. Only
one Working Seed from one Master Seed should be approved for vaccine
production in the country.
Master Seed should be stored at –70°C stabilized with sucrose and bovine serum
albumin and should be tested once in a year to certified viability and titre.
5. Vaccine needs for Indonesia
To control AI outbreaks it will be necessary to intensify the vaccination scheme
throughout the country. Based on epidemiological data, the use of vaccine in free
regions with low risk of infection should be limited. Better vaccination coverage in
broilers is recommended in regions at high risk of infection – which will require
increased supplies of vaccine. As local vaccine production seems to be
insufficient, it is likely that vaccines would be illegally introduced without
Government approval. Such vaccines should be tested and the results presented
to the producers to convince them that this is a dangerous practice. The
Government of Indonesia needs to produce inexpensive, high quality vaccine and
1
http://www.who.int/csr/resources/publications/influenza/en/whocdscsrncs20025.pdf
11
the working relationship between private laboratories and Government will be
important to achieve this result.
6. Personal suggestions
Government personnel working at PUSVETMA laboratories may be in the best
position to conduct studies to isolate and characterize a low pathogenic strain that
could be used as the Master Seed. They could also conduct vaccine efficacy tests,
serological and challenge studies for all AI vaccines (both locally produced and
imported or smuggled). Personnel of this laboratory should work with molecular
biologists at the Tropical Disease Center to gradually acquire the capability to do
more sophisticated analyses.
The Government should invest in a better vaccine production facility with
appropriate equipment and trained people, in order to be competitive with private
producers based on the quality of their products.
7. Summary of conclusions and recommendations
 Consider the use of a heterologous H5 AI LP virus for local vaccine
production, but whether homologous or heterologous LP virus should be
used.
 Virus characterization is necessary to assure purity of the virus and the
absence of adventitious infectious agents in the vaccine candidate.
 The Government should hold the Master Seed and if possible the working
seed, previously tested to ensure that it is immunogenic and protective for
susceptible chickens
 Establish continuous surveillance to identify epidemiological scenarios for
vaccine use
 Government should establish the minimum requirements for vaccine
production based on antigenic mass content.
 SPF embryos should be used in vaccine production
 The Government will require proper facilities for vaccine production,
including use of biosecurity measures, better than private laboratories.
 Vaccine development from scratch, using local strains, will take at least one
year to produce a good quality vaccine; Improvements in vaccine quality will
take more time.
 All AI viruses isolated in the country should be submitted to a government
laboratory for pathogenicity testing and management. This work should be
done only by expert government personnel. Poultry producers and other
diagnostic or pharmaceutical laboratories should not conduct any
pathogenecity or challenge study.
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8. Complementary information
It is important to remember that the protective immune response to AI does not rely
only on antibody formation. Rather, protection is a function of the cellular response,
mainly by cytotoxic cells. This concept is not well understood by veterinary
practitioners or poultry producers, who have years of experience with vaccination
against Newcastle disease. Thus, they expect the humoral response to guarantee
against AI infection. If the vaccine induces a good serological response 21 days
after vaccination, users will have confidence in vaccines.
AI vaccines do not prevent infection in all vaccinated chickens. However, the
majority of properly vaccinated chickens will not become infected. If they get
infected, they excrete lower levels of the virus into the environment than
unvaccinated infected chickens. In this situation, maintenance of flock immunity to
AI is important to minimize the effect of HP virus and ensure the disease is not
allowed to propagate and spread from flock to flock.
The vaccination scheme must be extensive and repeated in order to achieve good
long term results.
Maintenance of infection reservoirs will increase the amount of circulating virus and
increase the likelihood of AI spreading.
Movement controls should be implemented – and would have added benefit in
controlling other infectious diseases.
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Terms of Reference
International Consultant: Avian influenza vaccine expert
Under the general supervision of the Emergency Operations Service (TCEO) and the local
FAO Representative, the technical supervision of the Chief, Animal Health Service
(AGAH), FAO headquarters, in collaboration with the Regional Animal Health and
Production Officer, RAP, and in close collaboration with the National Project Coordinator
and other consultants, the avian influenza vaccine expert will undertake the following
activities:
 In collaboration with the veterinary epidemiologist and the Director of Animal
Health review the current AI vaccine-based control programme in Indonesia;
 Based on the existing information on the outbreaks of the disease and information
on the characterized strains that have been isolated, advise on the use of appropriate
avian influenza vaccines to be used in Indonesia;
 Assess the short, medium and long term needs for use of AI vaccines in Indonesia;
 Evaluate the domestic vaccine production capacity to provide sufficient quantities
of AI vaccines required for use in Indonesia by visiting local manufacturing
facilities;
 Design a field study plan to evaluate the response to vaccination and compare
results produced by different types of vaccines used;
 If appropriate, provide advice on standard operating procedures, quality control and
assurance in order to produce standardized, safe and highly efficacious vaccine for
Indonesia;
 Advise the Government of Indonesia in the deployment of AI vaccines linked to
their control programmes;
 Prepare a technical report (in English) after each mission for submission to TCEO
and the Regional Animal Health and Production Officer in RAP;
 Carry out any related tasks as directed by the FAO Representative.
Duty Station:
capital with local travel as required.
Duration:
two working weeks.
Qualifications: The International Vaccine Expert will be a microbiologist or a veterinarian
graduated from a recognized university with postgraduate degree in vaccine development
and production. He/she will have at least five years of relevant field experience and be an
internationally recognized expert in avian influenza vaccine manufacture, quality control
and deployment.
He/she will have level C proficiency in English.
Security: Consultant must be aware of security phase of country of assignment and
understand the implications for his/her own security. As soon as s/he arrives at the duty
station, through the FAO Representation or directly s/he must contact the designated UN
security officer to be briefed on all the recommended security.
Vaccinations: Consultant must ensure that he/she has received any necessary medical
vaccinations/ medical care before departing from home address.
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