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USING THE IMMUNOSTIMULANTS AS A FRIENDLY ENVIRONMENT IN
CONTROLING THE VIRUS DISEASES ON THE SHRIMP AQUACULTURE IN
INDONESIA*1
1) Alexander Rantetondok
Department of Fisheries Faculty of Marine Science and Fisheries, Hasanuddin
University, Makassar Indonesia
Fax: 0411-586025; E-mail: alexander_ tondok@yahoo.com
ABCTRACT
Shrimp is the main exporting product from fishery sector of Indonesia. In 1991,
Indonesia is the second rank producer of shrimp next to Chinese by total production
more than 660.000 metric tons. Since 1993, however the production of shrimp have
been decreasing, dramatically. To control disease,especially bacterial disease,currently
has been used many kinds of antibiotic such as chloramfenicole,amphisiline,
gentamisine, neosine, tetrasicline, penicilline G. on others. Nevertheless ,in the reality
many of antibiotic are referred [as] generate strain new bacterium that resistant’s is not
effective in battling the disease. Nowadays, prevention and disease controling become
main priority in maintaining continuity of shrimp culture industry. Immunology of
shrimp is a key element in determining strategy for disease control at shrimp culture.
The usage of immunostimulants for example β-glucant and lipopolysaccharide (LPS)
can improve nonspesifik immune response because it can improve phagocytoses
activity from cellular defence, that have : effect on bactericidal activity. Using the
immunostimulants in the shrimp culture for control the disease is a friendly
environment because materials of the immunostimulants easy to reduse in the
environments.
Keywords:
Immunostimulants, enviroments, antibiotic, bactericidal
Foot Note: Field Study option: Aquaculture (Budidaya Perikanan)
Introduction
Shrimp is the main exporting product from fishery sector of Indonesia. In
1991, Indonesia is the second rank producer of shrimp next to Chinese by total
production more than 660.000 metric ton (Anonymus, 1991 in Prayitno,1997). This
succeeded is a short period because there is a failure of shrimp culture since 1989.
The biggest failure of shrimp culture was occurred in 1993,where the production of
shrimp downwardness till reaches 100.000 metric ton ( Anonymus,2002). According to
Dahuri (2001), production of shrimp Indonesia in 1998 only around 167.550 metric
ton, so the percentage of increase/year from 1994-1998 only 5.56%. In 2000
Indonesian shrimp productions was only 97.710 metric ton. (Anonymus,2002). In
comparison with production in 1991, there was a degradation production account for
14,80%. The main cause of shrimp failure culture was disease virus outbreaks and
conducting culture management that unfavorable and ecological potency degradation, as
a consequence decreasing of shrimp resistance to diseases. Loss that generated by
shrimp disease reaches 300 billion rupiahs per year for all ponds area in Indonesia
(Rukyani and Sunarto,1998). Hawever, grace on the encourage of fish farmers and the
applied of Shrimp Culture Health Management the production of shrimp by
brackish-water ponds have been increased from 242,500 ton in 2004 to 300,000 ton in
2005 (Putro, 2005).
In 2008 the production of acuaculture include shrimp increase up to 1690,000
tons, just the fourth rank after China with the total production of 32736,000 tons
( Dahuri, 2011).
Based on the observation on the shrimp export data from 2000 to 2004,(Table
1) both the export volume and value (price in US, dollar) have been fluctuated due to
fluctuation of the price ( export vulue tendency decrease ).
Tabel 1.
Indonesian Shrimp Export
Year
Volume (ton)
Value (Million US $)
2000
114,035.1
1003.26
2001
127,344.3
940.1
2002
122,050.0
840.35
2003
134,241.6
852.72
2004
127,846.3
823.96
(Source: Indonesian Statistic, 2004)
If we observe the shrimp export from Indonesia to US from January to August 2006 it is
indicated that the volume of shrimp export is smaller than of Thailand and China.
From January to September 2006 the volume of shrimp export from Indonesia to Japan
is smaller than Vietnam (Kompas, 6 Novem ber. 2006).
Acording to the Kompas News (N0v. 2006), the volume of shrimp export from
Thailand to US (Jan. to August 2006) was 120,872 ton; from China to US was 49,446
ton from Indonesia to US was 43,655 ton and from Vietnam to US was 22,954 ton.
From January .to September, 2006, the volume of shrimp export from Vietnam to Japan
was 32,364 ton; from Indonesia to Japan was 29,004 ton; from Thailand to Japan was
17,531 ton and from China to Japan was 10,663 ton. In 2006 the target of shrimp
production of Indonesia was 350,000 ton. To get this target the government of
Indonesia introduced the new species Vannamei (Litopenaeus vannamei) from Hawai
(US). This new species was cultured in both tradisional and semi intensive
brackish-water ponds to increase the shrimp production that decrease due to the diseases
outbreaks. Actually Litopenaeus vannamei is not resistance against the virus the same
as tiger shrimp P. monodon (endogenous species).
Recently, Myo Necrosis Virus has
been reported to cause mortalities L.vannamei reared in brackish-water ponds in
Situbondo (East Java) and others palce in Indonesia. Therefore, the research on the
genetical engineering to produce the brootstock that resistance to disease is needed,
especially to improve the genetic of the endogenous species tiger shrimp P. monodon.
To control disease,especially bacterial disease, currently has been used some
kinds of antibiotic such as chloramfenicole,amphisiline,gentamisine,
neosine,tetrasicline,penicilline G. Nevertheless ,in the reality many of antibiotic are
referred [as] generate strain new bacterium that resistant’s is not effective in battling the
disease. According to Rosily (1987), there are about 200 new strains from bacterium
Vibrio that resistant to more than two antibiotics. In consequence, it’s required to look
for new methods in effort for disease problem. One of way to control disease is by
increasing immune system of shrimp.
Nowadays, prevention and disease control become main priority in maintaining
continuity of shrimp culture industry. Immunology of shrimp is a key element in
determining strategy for disease control at shrimp culture. In consequence, direct
research at development come [from] (experiment) to evaluate and monitor
impenetrability level shrimp must be conducted (Bacheré, 2000).
According to Secombes (1994), the usage of immunostimulant for example
β-glucant and lipopolysaccaride (LPS) can improve nonspesifik immune response
because it can improve phagocytoses activity from cellular defence, that have : effect
on bactericidal activity.
Research on using β-glucant and lipopolysaccaride as a immunostimulant for
disease prevention in tiger shrimp (Penaeus monodon Fabricius) have been conducted
inat in door tank and out door (brackish-water ponds). The results of this research
indicated that both immunostimulants can enhance the immune response of shrimp to
prevent the outbreaks of disease especially the White Syndrome Baculovirus or White
Spot Syndrome Virus (Table 2) Rantetondok, 2002). This immunostimulant suggest as a
friendly environment due to the materials used will preven the disease and do not
destroy the enviromnts. The materials contens of the immunostimulants easy to reduce
so that the immunostimulants do not contaminate the shrimp . Nowadays, prevention
and disease control become main priority in maintaining continuity of shrimp culture
industry. Immunology of shrimp is a key element in determining strategy for disease
control at shrimp culture. In consequence, direct research at development come [from]
(experiment) to evaluate and monitor impenetrability level shrimp must be conducted
(Bacheré, 2000).
Tabel 2. The Avarage of Survival Rate (%) Post Larvae of Tiger Shrimp Using
Imunostimulant β-glucant and LPS after challenged test of White Spot
Baculovirus or White Spot Syndrome Virus during 96 hours
Treatments
Average
Duncan Range Test
Glucant 5 g/kg feed
9.670
A
LPS 30 g/kg feed
6.840
BA
LPS 20 g/kg feed
6.630
BA
Glucant 10 g/kg feed
6.170
BA
Glucant 15 g/kg feed
4.340
BA
Control
0.710
B
LPS 10 g/kg feed
0.710
B
Explanation: Treatment with average shown by Duncan test with the same character not
significantly difference .
Immunostimulants β-glucant can increase the survival rate and immune response
in tiger shrimp P. monodon cultured in fiber glass tanks (Rantetondok,2000). Research
on β-glucant and LPS as a immunostimulant for control disesae in tiger shrimp
indicated that these immunostimulants can increase the survival rate and the resistance
against White Syndrome Baculovirus or White Spot which one of a very dangerous
disease in brackish water ponds (tambak) in Indonesia (Rantetondok, 2002).
The research on the effect of two immunostumulants that is β-glucant and
lipopolysaccaride (LPS) on respiratory burst
conducted by the authors indicated that there was the strong relationship between
respiratory burst and survival rate of the tiger shrimp. Dose LPS 30 g/kg feed showed
the highest both in activity of respiratory burst and survival rate of the tiger shrimp
when fed in net enclosured during the juvenile growing (called “Pembantutan”)n
brackish-water ponds. Immune response can be seen in the increase of the respiratory
activity of cell of the shrimp (Rantetondok, et al, 2004).
The method of shrimp (invertebrate) to develop the immune-system called
ProPO System as shown in Fig. 1 (Modified by Alday-Zanz, 1965).
Hemocyte
Release the proPO when in contact with foreign material
ProPo System activated by:
Fungi
Bacteria
(β-3,glucans)
Gram negative
(LPS)
Gram positive
(Peptidoglycans)
Phenoloxidase
PHENOL
QUINONES
MELANIN
Fig. 1.Scheme
ProPo System (Alday-Zanz, 1995)
Immunostimulant Agents
One of the methods to encrease the non-spesific defence of animal in
aquaculture is to apply immunostimulants such as β-glucant and lipopolysaccaride
(LPS. This is the of one the best methods to prevent the disease in aquaculture
system (Scombes, 1994).
Nowdays some of antibiotics have been used to preven the bacterial disease such as
Furazolidone 12 ppm, Teramicyn 450 mg/kg body weight of shrimp added to feed, and
Furanace 1,3 ppm that effective as bactericidal especially to control Vibriosis (Taren,
1985). In the other hand however, the residual of these antibiotics have a side effect to
shrimp and humen due to the resintence of the phatogenic bacteria such as Vibrio to
Chloramphenicol, Penicilin G. others. The recidue of the these antibiotics can stay in
the body of shrimp 2 weeks after initial treatments, and these will be harmful for
humen being as a consumer whose dirctly withought waiting for 2 weeks afer initial of
treatments. These indicated that antibiotic can react as a theraupitic but it can be a lethal
for shrimp because at the dose over the tolerant limit it can be destructed the potensial
cell. Use of the antibiotic for long period an be resulted I in resistance of the
bacteria to some of the antibiotics. Therefore using of immunostimulats is a good choice
for control disease and it a friendly environments methods.
β-glucans are branched β-1,3 and β-1,6 linked polyglucoses and are major
structure polysaccharide in cell walls of most fungi yeast and mycelia fungi. High
organism have depeloped various recognition mechanisms for these conservative fungal
wall components. Β-gulcans are thuse able to stimulate non specific defence mechanism
in animal as well as palnts. It is well estabiblished that β-glucans in mammals increase
the resistance against microbial infections and stimulate anti-tumor mechanism. There is
now a growing body of evidence that β-glucans also function as a immunostimulator
in fish. They thus have a potential as a feed additives to counteract in immuosuppressive
states of fish and as vaccine adjuvants (Robertsen et al. 1994).
Fungi Mitake (Grifora frondosa) found ii Japan is branched and β-1,6 linked
polyglucoses and it have a potential as immunostimulator to increase non-spesific
defence mechanisms in humen (Ganjar, 1997). Futhermore it was decided that β-glucans
can stimulate the immune system of the body such as macrophages, natural cell killer, T
cell killer (cytotoxic), T cell helper, limfokine, interleukin-1, and interleukin-2 which
can inbihibit the growth of cancer and virus. According to Tahir (1996) β-glucans have a
important prospect as a immunostimulator. Tiger shrimp which is treated with β-glucans
at concentration of 0.5-1 mg/ml (immersion treatmen) following the challenger test by
bacteria V. vulnivicus at consentration of 5 x 104 CFU/ml for 12 hours indicated that
there was an increasing in phenoloxidase activity of hemocyte 1 month after the initial
treatments.
P. javonicus, orally treatment by β-1,3 glucans (extracted from
Schizophyllum commune) showed the increasing of fagocytic activity of hemocyte
defence mechanisms against bacteria Vibrio sp (JICA, 1994). Trout immersed by
chitosan or glucan for 30 minutes or injection for 1-3 h days before challenger test by
bacteria Aeromonas salmonicida indicated the good defence mechanisms of this spesies.
Injection administration (treatment) is better than the inmersion (Anderson,and Siwicki,
1994). β-glucans are major structural polysaccharides in cell walls of most
yeast,filamentous fungi and mushrooms are also secreted as a slime or mucilage by
some fungi (Rosenberger, 1976 in Robersten, 1994). From an evolutionary point of
view, these structure probably represent one of the most ancient micronial cell wall
components. Consequently, higher organism may have develop various recognism
mechanism for these conserved fungal wall polysaccharide early in the evolution. This
may be the reason why β-glucans have been found to stimulate or activate the
non-spesipic defence mechanism in a wide range of higher organisms,plants (Darvill
and Albersheim, 1984 in Robersten et al., 1994). Tiger shrimp treated by Chitin in
different dose can increase the chitinase activity in the ingestion canal of the shrimp.
Chitinase activity increase accordingly to the increasing of chitinase percentage in feed
(Fox, 1993). Extract of abalone which are a glycoprotein component can increasing the
fagositic activity and chemiluminesence of kidney leukocitye when intraperitonial
injection to the rainbow trout at the dose of 20 mg/kg feed. This encreasing accur 5
days afre treatments and following bt the increasing of defence mechanism against V.
anguillarium attact (Secombes, 1994). Lipopolysaccharide (LPS) are O-antigen and
endotoxin in Gram-negative bacteria can increase the fagositic activity of leucocite in
sea bream Pagrus major and limp macrophages in plate fish Pleuronectes platessa
when in jection at dose of 500 μg for 5 days and showed the increasing of higher
migration compare to the control (MacArthur et al., 1985). Futhermore, LPS also can
stimulate directly the fagositic in culture medium.
Bacterium cell, Vibrio sp killefd by 0.5% formaldehide and added in feed
encapsulated can increase the survival rate in tiger shrimp larvae. It is assumed that the
bacterium cell are a substance whuch can increase the defense mechanisms in shrimp
larvae against the attact of bacteria in water (Itami, et al., 1991). LPS extracted from
Cytophaga bacteria, and injection to turbotfish, Scophthalmus maximus than challenger
tested by the same bacteria 21 days after initial injection can decrease the mortality
0-5 % lower than control of 90-100 % mortality. Fish vaccinated showed the higher
haemoglutination titer compare to the control ( Al-Harbi and Austin, 1992). The result
of the challenger test by the Vibrio spp against the tiger shrimp poslarvae (PL 20)
treated for 96 hours by LPS extracted from E. coli showed the tendency of the more
dereasing the mortality the more increasing the dose of LPS and the duration of
treatment(Tahir,1996). They thus have a potential as feed additives to increase the
phagositosis of bacteria so that it can increase defence mechanism against the
micropatogen in the brackish-water ponds.
Lysozyme
Lysozyme is one of the indicators to decide the effectifness of the
immunostimulants or immune response of the organism.
According to Ellis (1990), lysozime has anti bacterium activity that caused lisis
and can also personate opsonim. Specific substrat was found on lysozime is β-(1-4)
“linked” with N-acetyl-D-Glikosamin, N-acetyl-asam muramil peptidoglucant that
found at some wall cell of bacterium and if it was digested will cause plasma fall to
pieceses caused by osmosis pressure. When that's lysozime at haemomocyte shrimp will
have the character of anti bacterium that mean that immune system of shrimp has been
activated. Thus giving β-glucant and LPS that functioned as [the] goals from
recognition of protein form (Pattern Recognition Protein) can generate power of body
defence shrimp pass by improvement lysozime.
Besides direct influence as anti bacterium, lysozime is also reported can
improve phagocytosis.
Lysozyme found in serum or mucus of fish especially the tissue wich enreach in
leucocyte such as kidney, gaster, and limp (Engstad, et al,1992).
Tiger shrimp treated by β-glucant and LPS indicated the increasing of the
lysozyme activity which can be seen in the high survival rate of the juvinel after
perform the challenger test by Vibrio harveyi bacteria. (Fig. 2) (Rantetondok dkk, 2003)
LPS 20
Figure 2. The average of lysozyme activity by the treatments
Note : LPS 30 g/kg feed cannot be determinated
The Methods of Immunostimulants Applied to Shrimp
β-glucant : Immunostimulant was given to shrimp through mix with crumble
feed with 3 doses each : 5 gr/kg feed, 10gr/kg feed and 15gr/kg feed . Feed amount that
given is 10% from shrimp biomass weight per day and decreasing according to age of
shrimp, at the end of research only 5% from shrimp biomass weight. Feed with
immunostimulant was given only at first day once a week, and the observation of the
effect immunostimulant was conducted at week 2, 4, 6, 8th. Shrimp sampling was
conducted every week to weight shrimp biomass that will be a reference to count
amount of given feed.
Lipopolysaccharide (LPS) : was given to shrimp through mix LPS with crumble feed
with 3 doses each 10gr/kg feed, 20gr/kg feed and 30gr/kg feed. Immunostimulant
giving method through shrimp feed is precisely the same with at B-glucant.
Shrimp that has been stimulated its immune system by using both types of
immunostimulant was challenged infected by pathogen bacterium Vibrio harveyi with
dose 5 x 104 CFUs/ml through inverted for 96 hour. (Sung et al,1998).
Challenger test was conducted at the end of culture period of larvae phase at week
2, 4, 6, 8th, from each set of experiment unit to calculate survival rate. The method to
calculate survival rate was by undertake shrimp sampling account for 10 shrimps of
each container of research basin. Hereinafter, shrimp was put into 21 units- 1 litre glass
based on replicate and type of its immunostimulant. Shrimp prior to put into glass was
given disinfectant KMnO4 4 ppms for 1 hour. The water that will be used to challenge
test was sterilized with ultra violet ray. Hereinafter each unit of experiment glass was
infected by V.harveyi bacterium with dose and time such as those which have been
mentioned above.
The methods of the immunostimulants applied to shrimp is useful to improve
the oral administration because this is one of the best methos beside the others methods
such injection,immersion, spraying and others. Nevertheless it is needed to improve the
administration of immunostimulant applied to shrimp eg. using the microencapsulated
feed for shrimp larvae and the ability of the shrimp to eat the such feed in culture
medium ( brackish-water ponds or net enclosures for culture the post larvae). The
develop of research on the applied of immunostimulants in the field is needed
inconneting to pruduce the seed which specipic patogen resistant. On the other hands
the research on the geneticall engineering to produce the seed which resistance to
disease is also needed so that the production of the shrimp can be increased in
Indonesia.
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