2000 Science
and Technology Award Thailand Toray
Science Foundation
Professor
Dr. Boonsirm Withyachumnarnkul was born in Bangkok on October 26,
1950. He obtained a B.Sc. degree in
Medical Science in 1969 and an M.D.
Degree in 1973 from Mahidol University,
Bangkok, Thailand. After an internship
in 1974, he became a faculty member
of the Department
of Anatomy, Faculty of Science,
Mahidol University, teaching gross
anatomy and neuroanatomy to medical
students. In 1975, he went to United
States of America to have his Ph.D.
studies at the Department of Anatomy,
Strong Memorial Hospital, University
of Rochester, Rochester, NY and finished
the degree in 1978. His research topic
was on pineal physiology. He then
went to pursue his post-doctoral training
at the Center for Medical Education,
Indiana University, at Gary, IN, working
on endocrinology of the hereditary
obese rats. In 1980, he returned to
Thailand and resumed teaching and
researches at the Department of Anatomy,
Faculty of Science, Mahidol University.
He received the King Anantamahidol
Award, as a recognition on a dedication
to medical teaching during 1989-1992,
and Inventer Award for teaching aids
from the National Research Council
in 1997. In 1999 he was appointed
Professor by His Majesty the King.
In 1993 following a few years in shrimp
research, Dr. Withyachumnarnkul was
invited by Charoen Pokephand Foods
Company Limited to be the company
consultant on shrimp researches. In
1997, he became the principal investigator
in the project, “Domestication
and Selective Breeding in Penaeus
monodon”, funded by the
Shrimp Culture Research and Development
Company Limited (SCRD) with the support
of the National Center for Genetic
Engineering and Biotechnology (BIOTEC),
under National Science and Technology
Development Agency (NSTDA).
Professor
Dr. Timothy William Flegel
Professor
Dr. Timothy William Flegel was
born in Canada on 18 February, 1943.
He obtained a B.Sc. degree in Science
(Zoology) in 1965 and and M.Sc. degree
in Botany (fungal physiology) in 1968
from the University of British Columbia,
Vancouver, Canada. He subsequently
taught Scientific English at Chulalongkorn
University and studied tropical fungi
at the Department of Microbiology,
Faculty of Science, Mahidol University
from 1969-1974. He completed his Ph.D
in Biology at Simon Fraser University,
Canada in 1977. In 1978, he returned
to Mahidol University. In 1986, he
was co-recipient of the Award for
Outstanding Achievement in Applied
Science from the NRC Thailand, for
research on soy sauce fermentation.
In 1987 he was appointed Spectial
Professor by His Majesty the King
and in 1988 he received the Thai Royal
Decoration, Commander Third Class,
Most Nober Order of the Crown. In
the same year, Dr. Flegel began collaborative
research on shrimp aquaculture with
Aquastar Company Ltd. that lasted
for almost 10 years. In 1991, he became
a Thai immigrant. In 1994, he was
awarded the Thai Royal Decoration,
Commander Third Class, Most Exhalted
Order of the White Elephant. During
this period, Dr. Flegel was instrumental
in founding the Shrimp Culture Research
and Development Company Limited (SCRD)
with the support of the National Center
for Genetic Engineering and Biotechnology
(BIOTEC), and in 1997, he was appointed
Director of the Shrimp Biotechnology
Program for BIOTEC. Dr. Flegel is
also editor for crustacean diseases
for the journal Diseases of Aquatic
Organisms, Inter-Research Science
Publisher, Luhe, Germany and, since
2000, a member of the Thai Academy
of Science and Technology Foundation.
He has published over 90 articles
and books since the beginning academic
career, 26 associated with shrimp.
In recognition of his contributions
to shrimp aquaculture, Dr. Flegel
was awarded the Ajinomoto Lecture
Award by Thai Society for Biotechnology
in 1997 and the Golden Shrimp Award
by the Surathani Shrimp Famer’s
Association in 1999.
Professor
Dr. Vichai Boonsaeng
Professor
Dr. Vichai Boonsaeng obtained
his B.Sc. degree in chemistry from
Mahidol University (formerly University
of Medical Sciences) in 1964. He pursued
further studies under a Colombo Plan
scholarship at the University of Otago,
New Zealand where he received a Ph.D.
degree in Biochemistry in 1975. He
was Chairman of the Department of
Biochemistry, Faculty of Science,
Mahidol University from 1992 to 1996
and in 1995 he was appointed Professor
of Biochemistry. He is married to
Dr. Paungtong Premprasit and has three
daughters. He is currently the Director
of Academic Research Division, the
Thailand Research Fund and a member
of Mahidol University Council. He
was awarded the Mahidol University
Prize for Excellence in Research in
1996, the National Research Council
of Thailand award for outstanding
research in 1996 and the National
Research Council of Thailand award
for Outstanding Researcher in 2000.
Dr. Vichai Boonsaeng has published
some 50 papers on the applicatiion
of DNA-based technology in the diagnosis
of infectious diseases of human and
animals, and especially for detection
of penaeid shrimp viruses. The methods
developed by his group for the detection
of these shrimp viruses (white shot
syndrome virus, yellow head virus
and hepatopancreatic virus) are now
used routinely in number of laboratories
in Thailand and abroad. For this achievement,
Dr. Vichai Boonsaeng was awarded the
Golden Shrimp Prize by the Surat-Thani
Shrimp Farmer's Association in 1997.
He has also introduced the technique
of DNA fingerprinting in Thailand
and this method is now in routin use
forensic laboratories throughtout
the country.
Achievements
of Research Team on Shrimp
Our Research Work on Shrimp 
Shrimp culture is a major Thai industry
and the country has been the world’s
leading exporter of farmed shrimp
for the past five years. In 2000,
the country exported cultured black
tiger shrimp (Penaeus monodon) worth
100,000 million baht (~2,325 million
US $) and the industry has been estimated
to support more than 1 million Thais.
There are several reasons for this
achievement; the major one is probably
that Thai shrimp culturists are mostly
small-scale farmers. With naturally-creative
minds and hard-work, they are always
willing to start anew, despite previous
crop failures that are unfortunately
still too common in shrimp culture.
Equally important is close co-operation
among Thai researchers in shrimp culture,
including those from both government
and private sectors. Contributions
from individual researchers have been
patched together to become a complete
picture, like putting together the
pieces of a giant jigsaw puzzle. The
activities include field studies and
laboratory studies using methods ranging
from traditional staining of tissue
sections to advanced biochemical techniques
like DNA-based technologies. The co-operation
involver various individuals from
different fields and organizations:
farmers, educators, researchers, and
business and marketing specialists.
This multidisciplinary approach is
also a key to our success.
The number of diseases affecting
cultivated penaeid shrimp has increased
steadily with expansion and intensification
of large-scale commercial cultivation.
In the 1990’s the most serious
losses occurred in Asia from the emergence
of new viral pathogens, like yellow
head virus (YHV) and white spot virus
(WSV).The coming of these pathogens
required in tensive research on their
basic biology, on host-pathogen interactions
and on the development of rapid and
sensitive detection methods. Through
our efforts and those of our post-graduate
students, other Thai colleagues, and
extension workers, our research on
these two viruses alone is estimated
to have prevented thousands of millions
of baht in lost shrimp production
to Thai shrimp farmers since 199.
It serves as an excellent example
of the benefit that was gained by
less than 10 million baht of Thai
Govermment investment in research.
In the coming years, more shrimp viral
pathogens will undoubtedly be found,
especially as cultivation expands
geographically and non-native species
are used for rearing. To lower risks
of disastrous viral outbreaks we recommend
continued investment in research and
more caution in the international
movement of living shrimp for aquaculture.
Together with researchers from the
Department of Fisheries, other universities
and the private sector, our group
from the Faculty of Science, Mahidol
University has studied white spot
disease and the yellow head disease
in cultured P. monodon, caused by
YHV and WSV infections, respectively.
These two diseases have caused more
than 15,000 million baht loss yearly
to Thai shrimp farmers. We have discovered
causative agents, modes of transmission
and prevention measures for the two
diseases. By studies in the laboratory
and the field, effective preventive
methods for shrimp culture have been
carefully planned and the private
sector has helped in applying the
researc results for full benefit on
the farm.
As consultants to Charoen Pokphand
Foods Co. Ltd., Aquastar Co. Ltd.
and other companies, we have been
able to recognize problems on shrimp
farms and to visualize more clearly
what technologies would be most appropriate
for famers. Through close association
with researchers from private companies,
the Department of Fisheries and other
universities, the direction of research
activities became more focused and
unnecesary duplication of activity
was avoided. The result has been a
continuous outflow of practical research
results to famers through company
contacts, extension training and publications
in scientific journals, magazines
and brochures.
Our first collaborative effort in
shrimp research was focused on the
causative agents of white spot and
yellow head diseases beginning in
1993. Our aim was to work on molecular
biology of the two viruses and to
develop sensitive techniques to detect
covert WSV and YHV infections in shrimp
by polymerase chain reaction (PCR)
technology.
PCR is rapidly becoming the most
important technique in the molecular
detection and diagnosis of viral diseases.
It is a biochemical method to allow
for the amplification of very specific
sequences of genetic material from
pathogens of interest, so that small
numbers of pathogens in a test sample
can be detected; a single, individual
pathogen can be detected when a double
PCR of “nested PCR” test
is used. This level of sensitivity
was previously impossible and it is
necessary when testing animals that
carry low levels of patogens and show
no signs of disease. To develop PCR
assays for YHV and WSV, special chemical
reagents called “primers”
were needed and preparing them was
a difficult and exacting task requiring
advanced skills in microbiology and
genetic engineering. We first had
to multiply, purify and characterize
the pathogens before we could extract
and analyze their genetic material
to develop primers and test them.
During this process, we discovered
that the genetic material of WSV was
DNA while that of YHV was RNA. The
DNA was good for direct PCR but the
RNA of YHV made our task of developing
a PCR test more difficult. An extra
step was required in what is called
an RT-PCR amplification process where
the viral RNA is first converted to
DNA.
Our original PCR method for WSV
detection was quickly adopted in Thailand
for the screening of broodstock and
postlarval shrimp and it helped to
stop the spread of the virus and restore
production to former levels. We subsequently
developed a nested PCR technique to
improve test sensitivity in a single
tube assay to reduce the chance of
contamination. We also incorporated
chemicals to yield a quantitative
assay of the pathogen and an asurace
for integrity of the test. Similar
techniques were used to develop RT-PCR
assays for YHV with the same degree
of sensitivity as for WSV. Together,
we used these methods to prove that
WSV and YHV could be transmitted vertically
and horizontally from shrimp to shrimp
and from carrier species to shrimp.
We proved, for example, that several
crab species could transmit these
viruses to cultivated shrimp. In addition,
we are using these techniques to study
genetic variation in the viruses,
in order to develop epidemiological
diagnostic kits. These will be useful
to trace the source of outbreaks in
broodstock and carriers and help in
developing better disease prevention
programs. Also, by studying the genes
encoding viral proteins, we hope to
produce cheap immunodiagnostic reagents
that can be used in pond-side tests
and for research on the possibility
of improving shrimp defense against
these diseases.
In addition to YHV and WSV, we have
also studied Hepatopancreatic parvovirus
(HPV) and monodon baculovirus (MBV).
As with YHV and WSV, we have developed
or adapted PCR-based assays for these
pathogens. We are currently using
the reagents to study pathogen biology
and epidemiology. Although HPV and
WBV generally do not kill host shrimp,
we have discovered that they are associated
with retarded growth that is estimated
to cause many millions of baht in
lost production to Thai shrimp farmers
every year. We are assisting in the
implementation of effective prevention
programs for these viruses in order
to further improve the efficiency
of shrimp production, to make Thai
shrimp more competitive on the international
market and to increase profitability
for the shrimp industry. By cooperating
with other scientists in Thailand,
Australia, Sweden and the Americas
we are gaining a better understanding
of the relationships and the international
spread of shrimp viruses. This will
help in providing better, more rational
disease control, with the maximum
degree of freedom in the international
trade of farmed shrimp.
In comparison to viruses, bacteria
constitute a much less serious threat
to shrimp farmers because most can
be controlled by appropriate pond
management techniques and because
proper chemotherapy is possible in
the event of management failure However,
even for traditional pathogens like Vibrio harveyi and V.
parahaemolyticus (the most serious
bacterial pathogens for shrimp in
Thailand), we still need to understand
why some strains are lethal and others
are not. In cooperation with the Department
of Fisheries, our group was the first
to discover that many strains of V. harveyi pose no threat to
shrimp until they acquire the ability
to produce deadly toxins from viral
partners. These partners are not shrimp
viruses but viruses of the bacteria
themselves. This is an exciting new
area of research that may lead to
new methods of bacterial disease control.
By studying the bacteria and their
viral partners we will come to understand
the nature of the toxins, their mode
of production and their mode of action
in shrimp. The knowledge will allow
us to develop practical intervention
measures that will protect shrimp
by preventing toxin production or
blocking its deadly action. We believe
that natural products from marine
algae and friendly bacteria in shrimp
ponds may be useful in this regard.
Such biological controls for bacterial
diseases would obviate the need for
antibiotic or chemical use in shrimp
cultivation and reduce concerns for
negative environmental impacts that
result from the practice.
However good our control of bacterial
diseases may become, we will still
need rapid, sensitive and specific
detection methods that can be used
to monitor cultivated shrimp and to
help in disease diagnosis. To this
end, we have developed a PCR test
for V. parahaemolyticus,
similar to the tests we have developed
for shrimp viruses. Like the viral
assays, the test can be carried out
using a minute quantity of shrimp
blood, drawn without harm to the shrimp
and giving results within a few hours,
without the need for bacterial isolation
and purification. A similar test is
being developed for V. harveyi,
and our target is to develop a multiplex
test that can be carried out for simultaneous
detection of the four major Vibrio pathogens of shrimp found in Thailand.
Application of these tests on a routine
basis would provide advance warning
of disease outbreaks and allow for
timely intervention to prevent loss.
Control of all shrimp diseases requires
a level of understanding that we still
lack, not only for the pathogens but
also for the shrimp host. Admirable
progress has been made elsewhere with
respect to crayfish cellular defence
mechanisms. These are known to be
based on pattern recognition proteins
targeted against bacterial and fungal
cell wall components. In Thailand,
we are in the early stages of similar
work on P. monodon in cooperation
with leading crayfish scientists in
Sweden. In contrast to bacteria, our
knowledge of the shrimp response to
viral pathogens is almost totally
lacking and this is an exciting new
area of work in which we are now involved.
Our group was the first to demonstrate
that shrimp infected with YHV and
WSV show high levels of programmed
cell death (apoptosis). The molecular
mechanisms behind this phenomenon
open up an exciting new area of research
on shrimp-viral interaction. Our investigations
at the molecular level for both the
host shrimp an its pathogens aim to
rationally assess and improve the
benefits of proposed therapies and
preventative measures for disease.
This work is going hand in hand with
a concerted effort to develop certified
domesticated shrimp stocks and secure,
but affordable, cultivation systems.
During the application of PCR in
the field, in cooperation with scientists
at private companies such as Charoen
Pokphand our test procedures were
modified so that they could be practically
applied with less error. The work
could not have been accomplished without
contributions from research facilities
on the farm. Application of the research
results led to the production of virus-free
postlarvae from hatcheries and to
the introduction of closed system
culture, once we knew that natural
crustacean species were important
virus carriers. In closed system culture,
virus-carriers are prevented from
entering shrimp ponds, and water exchange
is minimal; whenever necessary, using
water from a virus-and carrier-free
reservoir. Closed-system shrimp culture
has gained popularity and been proven
to reduce the risk of crop failure.
In closed systems, water treatment
becomes a major concern, as waste
products from the shrimp, dead plankton,
left-over feed and other sources pollute
the water. Water in the shrimp ponds
needs to be cleaned, but not by water
exchange as that could introduce viruses
into the system. With help from company
researchers and an Israeli expert,
bioremediation has been developed.
The principle of this method is to
provide water with an optimal carbon-to-nitrogen
ration, to help the growth of beneficial
bacteria. These bacteria, in turn,
help reduce the nitrogen waste.
Since aquaculture still depends
on nature, even the strictest prevention
measures against viruses or carriers
cannot guarantee 100% success. To
further strengthen shrimp health,
we have worked to develop and introduce
probiotics and vaccines to farmers.
Although the products still need further
development, initial success has been
observed and we are involved in continuous
research on the mechanisms of prevention
and on improvements in efficacy.
In addition to research on shrimp
diseases, we have also played a role
in the domestication and selective
breeding of P. monodon broodstock
for the Thai consortium, the Shrimp
Culture and Research and Development
Company (SCRD). This on-going project
has passed to the fourth generation
of domesticated stock, and the feasibility
of commercial production has been
demonstrated. Concurrently, research
activities in this area include molecular
genetics of P. monodon, with
the help from Dr. Sirawut Klinbungar
from the National Science and Technology
Development Agency (NSTDA) and Dr.
Anchalee Tassanakajorn at Chulalongkorn
University. These concerted activities
demonstrate that research at any level,
with the right application, can benefit
society and can really be “research
for development”. Mahidol University
and BIOTEC recognize the importance
of the shrimp industry to Thailand
and the valuable contribution the
can be made to its overall development
by research in biotechnology and molecular
biology. To this end, the two have
joined efforts in establishment of
a new center of excellence in Shrimp
Biotechnology and Molecular Biology
to be located at the Faculty of Science.
Starting in the first quarter of 2001
our group activities will be conducted
out of this center. Our aim is to
create a national focus for cooperative
research and training in this specialty
area as an element in the overall
national program for development of
a sustainable and profitable shrimp
industry that will have minimal negative
impact on the environment. We believe
that this goal can be reached only
by good teamwork and not by a single
scientist or by individual scientists
working in isolation. A look at our
publication list and the wide scope
of out collaboration both within Thailand
and abroad bears witness to this belief.
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