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Scinzer Journal of Agricultural and Biological Sciences, Vol 1, Issue 1, (2015): 34-38

Disease Control Strategy of Motile Aeromond Septicemia in

Immunostimulatory Goldfish through Development of Marine
Algae Eucheuma Cotonii
Fitje Losung1, Antonius P. Rumengan1, Indriasari2, Remy E. P. Mangindaan1
1. Lecturer in the Faculty of Fisheries and Marine Sciences Sam Ratulangi University
2. Lecturer in the Faculty of Fisheries and Marine Sciences Khairun University
Corresponding author email :vera.losung @ yahoo.com
Abstract: Motile Aeromonand Septicemia disease (MAS) which attacks the carp is very harmful for fish farmers and requires
serious treatment. Immunostimulant need to be explored and developed to control the development and transmission of
pathogens in fish caused by the bacterium A. Hydrophila. Meanwhile Indonesian marine biota contained a lot of potential with
immunostimulatory activity. Extracts isolated from marine algae Eucheuma cotonii and tested its efficacy against pathogens
A.hydrophila in the carp, then effectively extracts tested time after administration of the fish in the field. Thus acquired the
right way and can be realistically applied by fish farmers. Carrageenan dose 10 mg / kg fish effectively used as
immunostimulant for carp and effective application are: intraperitoneal injection.
Keywords: Isolation; Immunostimulants; Pathogens; Algae, Aeromonas hydrophila
Introduction
The utilization of marine algae Eucheuma is still limited to the extraction carrageenan. The carrageenan content that
are very useful as a source of immunostimulatory compound has not been sought as optimal as possible. Carps are very
sensitive to the bacterium Aeromonas and can be observed instantly. Hence, the culture of carp require preventive actions to
provide protection against pathogens and parasites.
Both of these issues if it is correlated, it could be concluded that the use of red algae Eucheuma cottonii as a source of
immunostimulatory for carp could make a meaningful contribution and required precise handling and high costs.
The Aquaculture of carp continues to expand towards intensive fish farming . High density, feeding abundant cause
water quality to be conducive to the development of microbes, including fish pathogens such as Aeromonas hydrophila. When
an epidemic, acts like disinfestation, antibiotics, proved difficult to save the fish population alive. Therefore, prevention and
strengthening the immune system of fish is one of the best way out, in order to obtain extracts prepared to applied to the fish.
After applied carrageenan fish was confronted with pathogens.
Antibiotics can be used to kill harmful microorganisms, but excessive use will negatively affect the immune system
and balance of microorganisms that are important and often led to a more virulent strain of the pathogen
The red algae Eucheuma cottonii widely spread out in some mariculture area in North Sulawesi such as Nain,
Arakan, and Likupang . However, diversifying the utilization of seaweed and applied technology has not been a lot attention,
though this alga contains protein or glycoprotein superior and efficacious, such as agglutinin or lectin ( Mangindaan , 1997,
1998 , 1999) .
This type of algae known to contain a polysaccharide as the source of immunostimulatory compounds that are known
to stimulate the immune system of fish. Thus, it is very important to do further studies to address the various diseases of fish
consumption as a source of immunostimulatory. Immunostimulant is a substance that stimulate the immune system
(Baratawidjaya , 1991), or a compound that stimulates the defense mechanisms of the organism (Hou and Chen, 2005).
According to Fletcher (1982) in Rukyani et al (1997 ) , immunostimulatory mechanism is by increasing neutrophil oxidative
activity , increase activities of phagocytic cells such as macrophages and T lymphocytes or the workings of other cytotoxic
cells , and induces cytokine proteins (cytokines) such as interleukins , interferons , tumor necrosis factor , C - Reaktive protein
( CRP), complement and lysozyme . Immunostimulant is an alternative to the use of vaccines and antibiotics in fish disease
protection against attacks (Robertsoen et al , 1990) . Immunostimulatory polysaccharide types have been widely used in
improving the immune system of fish. Extraction of red algae (Rhodophyta) Eucheuma cotoni known to contain
polysaccharides (Trono, 1988).

Scinzer Journal of Agricultural and Biological Sciences, Vol 1, Issue 1, (2015): 34-38

Polysaccharides have the ability to bring certain biological information for structural changes, including the formation
of stimulating T cells and able to induce forms of different effector cells such as T cells, NK cells ( natural killer ) and
macrophages ( Anonimous , 2001). This opinion was also expressed by Okuda et al (1972); Hamuro et al (1978) and Azuma
(1997 ) in Yano et al (1991) on the ability of the polysaccharide to the nonspecific immune response as the activation of
macrophages , NK cells , T - lymphocytes and interferon production as well as activities related to the activity of the
alternative complement work .
This research aimed to achieve techniques / strategies to prevent MAS disease development on many carp fish that
cause many loses to the fish farmers
Methods
The study was designed in a scheme which includes:
Collection of algae.
Algae were collected from several places in Manado bay waters then were taken to the Marine Biological Laboratory
Chemical Materials FPIK Unsrat rinsed with fresh water, then weighed in a dry aired. Algae were weighed again to determine
the algae ratio.
Extraction
Satari carrageenan extraction method (1998) were applied in; algal flour was trnsfereed in glass beaker and then
added water and 2 % NaOH solution. Furthermore, it was boiled for 4 hours at a temperature of 90 C. After that, added
Celite which had been dissolved and re- heated for 30 minutes , filtered with Whatman paper, then added a solution of 10 %
NaCl . After it heated for 15 minutes, It was stirring frequently. Furthermore , 95 % ethanol was added and kept stirring
slowly. Polysaccharide clot attached to the agitator are separated, weighed , and then dried in an oven for 6 hours at a
temperature of 75 C. Subsequently weighed again to determine the weight percentage of carrageenan algal flour.
Test Preparation and Fish Containers
Container used aquarium measures 60 cm x 40 cm x 40 cm . In each container filled 5 fish 40-50 grams of test size
and aerated . fish acclimatized for 14 days . During acclimatization lasts fish fed commercial pellets as much as 5 % of body
weight per day to the frequency of feeding 2 times a day .
Test Challenge
Each group is given a goldfish route carrageenan injection , intraperitoneal during the week with 5 different doses .
Furthermore inoculated the whole fish then was challenged with pathogenic bacteria Aeromonas hydrophila bacteria with
density 0 ( control ), 1x108; 5x107; 1x107; 5x106 and 1x106 cells / ml for 3 days . Each treatment had 3 replications. Each
level alive escaped was compared with controls that received only saline.
Testing phagocytosis index
Phagocytosis index calculation is intended to measure the level of greed phagocytic cells (neutrophils) after the test
fish treated.
Counting rate of gluttony can describe how active the fish phagocytic cells against attacks of disease or other disorder
of the body's defense mechanism.
Data collected in the determination of phagocytosis index ( IP ) were : the number of neutrophils and the number of
yeast cells that expressed in the formula terfagosit of le Morvan et al (1977 ) .
IP = ( % N -consuming yeast cells ) x ( Z eaten )
Description: IP = phagocytic index
N = neutrophils
Z = yeast
The target of this research were as follows :
Fishes tested in the field did not die after 60 days of extract
Socialization results can be well understood and carried out by the fish farmers
Results And Discussion
Algae
Eucheuma cotonii algae have been collected from Manado bay waters , washed with sea water . After it was brought
to the Marine Biological Laboratory Chemical Materials FPIK Unsrat , in rinse with fresh water, and then weighed in a dry

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Scinzer Journal of Agricultural and Biological Sciences, Vol 1, Issue 1, (2015): 34-38

aired. Algae we do now weighed again to determine ratio algae. Of wet algae collected and dried obtained dry weight of algae
( 9.53 % ) . In the extraction of carrageenan obtained 45 %.
Carrageenan number different than other locations. Differences in location determines the amount of supply of nutrients for the
algae needs . In a previous study obtained carrageenan content of algal origin E.cotonii Arakan waters higher than the same
type of algal origin island of Nain waters.
Aeromonas hyrophylla
Hydrophylla Aeromonas obtained from Quarantine Fish North Sulawesi , TSA cultured in media for 48 hours and
then injected into a few fish to increase the ferocity of bacteria . This is repeated up to 4 times . Bacteria in the can , next to the
fish tested at a later stage and used for the manufacture of FKV (Formalin Killed Vaccine).
The test animals Carps ( Cyprinus carpio ) on average 50 g / carrageenan injected with 0 , 2 , 5 , 10 20 mg / kg of fish
every 2 days during the week. On day 10 fish were injected ( challenge test ) to test bacteria for 6 days and observed fish
behavior : death , reddish fins , and the inside of the fish bleeding . In the test fish pathogenic bacteria Aeromonas hydrophyla
injection with bacterial density of 1x107 cells / ml (corresponding previous testing, the lowest concentration that can kill fish
more than 50 %). External symptoms of fish disease motile Aeromonand Septichemia (MAS) include body color becomes
dark, rough skin and bleeding arise, the ability to swim down and often gasping for air at the water surface due to damaged
gills making it hard to breathe ( Afrianto and Liviawaty, 1992). Internal signs include redness and bleeding in the peritoneum
(the lining of the abdomen), and most of the visceral organs (Austin and Austin , 1993). The symptoms are consistent with
those observed in fish after injected bacterial pathogen A. hydrophyla. Attacked fish Aeromonas hydrophila generally showed
clinical signs of the presence of small wounds on the surface of the body that causes the calendar scales, especially on the gills
and bleeding anus , ulcers, anemia, organ damage, especially the kidneys and liver.
The survival of fish that were subjected to carrageenan shown in Figure 1 and the survival of fish that were given the
vaccine appears in Figure 2 .
Of the two images appear that carrageenan administration was superior in enhancing the immune response of fish.
Carrageenan treatment with doses of 20 and 10 mg / kg bw was not significantly different , thus the treatment of 10 mg / kg bw
have been effective , ie 80 % of fish may protect against pathogen attack .
This finding is supported with the results of the study (Sakai, 1999) which states that the immunostimulatory
(carrageenan) increase resistance to infectious diseases is not due to increased specific immune responses but by increasing the
non-specific defense mechanisms. Given fish immunostimulants usually show an increase in phagocytic activity. Increased
genetic killed on macrophages is critical of the fish that were immunostimulatory ( Kajita et al, 1990) . glucan Biological
Defense Modifier is potentially activate the immune system.
Phagocytic cells is important in the body's defense system against infectious microorganisms fish. Phagocytosis index
calculation is intended to measure the level of greed phagocytic cells (neutrophils) after the test fish treated and describes how
active the fish phagocytic cells encounter other disease or disorder of the body's defense mechanism. The influence of
carrageenan to phagocytosis by carp pronephros phagocytes obtained 20.2 4.4 ( Figure 3 ) . From the results obtained it turns
out resistance goldfish given karaginanan activate phagocytic cells are non - specific.

Figure 1. Live Survival of Carp treated with carrageenan in various doses

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Scinzer Journal of Agricultural and Biological Sciences, Vol 1, Issue 1, (2015): 34-38

Figure 2. Live survival of Carp treated with formalin killed vaccine in various doses

Figure 3. Fagositosis Pronephros Carp against Aeromonas hydrophila.

Conclusions And Recommendations
Conclusion
This research has successfully isolated carrageenan compound from algae Eucheuma cotonii to be used for
immunostimulatory materials for carp.
Carrageenan dose 10 mg / kg fish effectively used as an immunostimulant for carp.
Effective applications is intraperitoneal injection.
Recomendation
The result need to be applied in the field (fish ponds) for it can be socialized to fish farmers
Training needs to be done in the carrageenan extraction and injection techniques.
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