Vaccines Application in Indonesian Aquaculture New

Vaccines Application in Indonesian Aquaculture

I. Background

Fish is the largest creature in the waters and biodiversity which a high-protein food. Fresh fish contains 16-24% protein, 0.2 to 2.2% fat, vitamins, minerals and carbohydrates (Suseno, 2004). Aquaculture has developed because it has a good chance in the domestic and international market, such as the European Union, ASEAN, Hong Kong, China and Japan. One of the main obstacles aquaculture development, both in freshwater, brackish or sea ecosystems is a crop failure due to infectious and non-infectious disease. The problems of these diseases are very important because they can lead to decreased production, quality of fish and even death (Kabata, 1985).

In freshwater commodity, fish farming constraints due to Aeromonas infection began to arise since 1980 with a loss of about US$ 2 million. This is a pathogenic bacterium, spread very rapidly at high stocking density and can lead to death until reaches 90% of population (Kabata, 1985).

Another obstacle to the cultivation of freshwater is the presence of herpes disease in carp and koi. Koi Herpes Virus (KHV) is a new emerging disease known to cause gill and skin damage in koi and carp (Cyprinus carpio). The disease suspected to have been introduced into Indonesia through importation of koi from Hongkong. It is currently occurring in Indonesia since March 2002 starting in the area of Blitar in East Java. Since then it has been spreading rapidly throughout Java Island, Bali, southern part of Sumatra, East Kalimantan, and Central Sulawesi (Sunarto et al., 2005).

The history of KHV in Indonesia including the first 3-episodes of the outbreak was chronologically described by Sunarto et al. (2002). The first episode of mass mortalities of cultured koi (Cyprinus carpio) was recorded in March 2002 in Blitar, East Java. It occurred after heavy rains among new fishes introduced from Surabaya, the capital city of East Java. The fish were imported from China through Hongkong in December 2001 and January 2002. Blitar is well known as the centre for koi production in the country. The koi including the infected one were distributed over the country, with to Central Java, West Java and Jakarta as the main market (Sunarto et al., 2005).

The second disease outbreak occurred in cultured common carp (Cyprinus carpio) during the end of April 2002 in Subang regency, West Java. The gross signs of the diseased common carp were extremely similar with that observed previously in koi carp. Subang is one of the production centers of common carp in West Java. Since then the outbreaks spread to neighboring regencies mainly through fish movements (Sunarto et al., 2005).

The third episode of the outbreak occurred in end of May to early of June 2002 in cultured common carp in floating net cage at Cirata Reservoir, West Java. Weeks before the outbreak, farmers introduced common carp from Subang region due to the low price of fish (Sunarto et al., 2005).

The fourth episode of the outbreak occurred in February 2003. The outbreak affected cultured common carp in Lubuk Lingau regency, South Sumatera. The gross signs of the diseased common carp were extremely similar with that observed previously in koi and carp in Java islands. Common carp farms at Lubuk Lingau were infected with the disease coming from Cirata Reservoir, West Java through fish transfer by traders. The outbreak then spread to neighboring district and province including Bengkulu in the south and Jambi in the west (Sunarto et al., 2005).

The first report regarding the economic losses due to the outbreak was made by the head of the Association of Ornamental Fish Culture of Blitar regency, East Java. They reported that in Blitar alone, the outbreak destroyed high quality koi carp belong to 5,000 fish farmers with economic losses more than IDR 5 billions (US$ 0.5 millions) within the first 3 months period of the outbreak. As of July 2002, the NACA’s Task Force estimated that the loss revenue of the sector and the socio-economic impact to the rural farming communities was in the region of US$ 5 millions. As the outbreaks continued and spreading to new areas, the socio-economic impact due to the outbreaks was escalated. Directorate of Fish Health and Environment (DFHE) estimated that as of December 2002 and 2003, losses due to KHV were US$10 million and US$15 million, respectively (Sunarto et al., 2005).

Central Freshwater Aquaculture Development (Balai Besar Perikanan Budidaya Air Tawar, BBPBAT) monitoring results (2002-2005), showed that KHV virus attack all stages of common carps or koi. Based on data from the spread of KHV in Indonesia, almost all regions, there are strains of carp attack by KHV. However, there are not yet data about the percentage difference in mortality between strains of carp. The mortality percentage differences between strains of carps may be related to KHV tolerance (Mudjiutami, 2007). Results of monitoring and surveillance Directorate of Pests Fish Diseases, Directorate General of Aquaculture Ministry of Marine Fishery until 2009 showed the expansion of the spread of KHV in Indonesia that has got to Sumatra (kab. Tanah Datar, Lima Puluh Kota and Sawah Lunto), Jambi (Kab. Kerinci), NTB (West Lombok), South Kalimantan, East and West Kalimantan.

In brackish and marine water commodities, a major cause disease that affects fish farming and wild fish are Vibrio sp. This disease has been recognized since 1718 in Italy, with many epizootics documented throughout the 19th century. Types of bacterial disease found in fish farming include caudal fin ulcer and red mouth disease. The bacterial isolation and identification found some types of bacteria are closely associated with cases of suspected disease, namely Vibrio alginolyticus, V. algosus, V. anguillarum and V. fuscus. Vibriosis can be either acute or chronic depending on the type of Vibrio that attack, host condition, and environmental conditions. In general, this disease can cause death up to 100%.

In addition there is an also viral disease that causes the collapse of marine fish and shrimp cultivation, namely, Viral Nervous Necrosis (VNN) and White Spot Syndrome Virus (WSSV). VNN has spread in Japan, Korea, China, Southeast Asia, northern Australia, Austria, Iran, Israel, Greece, France, Norway, Canada and America. These diseases are extremely malignant, so rapidly spreading in various parts of earth. Thus, these pathogens were greatly feared by all countries that have fishing industry. These pathogens can cause mass mortality of farmed fish in a relatively short time (Chi, 2006 in Bimantara, 2009). Most

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of fishes being attacked by VNN in Asia, namely: Grouper, Epinephelus spp. in Southeast Asia and Japan, Asian seabass (Lates calcarifer Bloch.) in Southeast Asia, India and Australia. Seabass (Dicentrarchus labrax L.) in Northern Australia, Striped jack (Pseudocaranx dentex) in Japan, Red Drum (Sciaenops ocellatus) in Korea, Golden Grey mullet (Liza auratus) in Iran, and many others.

In Indonesia, This pathogen attack fishes which reared in floating net cage and fish larvae are still kept in hatchery which spread across Indonesia. VNN be a major challenge in aquaculture industry because of the high levels frequency of disease occurrence that can lead mortality approaching 100% and wide distribution both in warm and cold waters as media of various kinds of fish live.

In an effort to control fish disease, still using antibiotics as a curative action. However, this is no longer permitted as the policy of "say no to antibiotics" and the demands of global markets of food safety, product quality, antibiotic free, and environmentally friendly. The European Union has some regulations related to residue limits of pharmacologically active substances in foodstuffs of animal origin, one of the most recent regulation is EC 470-2009. To follow up this regulation, the government issued a regulation of Minister of Marine Affairs and Fisheries Republic of Indonesia Number 02/men/2007 about monitoring of drug, chemical, biological, and contaminants residues in fish cultivation. Under those regulations, the National Residue Control Plan program was launched on 2010 in order to support quality assurance and food safety in fish cultivation to obtain residue-free fishery products. The realizations of this program are fish drug and chemical controlled with feed antibiotics free and monitoring residues implemented in fish cultivation.

An alternative disease control in aquaculture can be done with health care management by improving fish immune through vaccination and immunostimulation. These techniques are environmentally friendly and more efficient, because only by providing one-two times were able to increase the resilience of fish's body until the end of maintenance period. Some research of the vaccines used that have been done and showing good results. Based on research conducted by Prof. Kamiso on 2007, the use of vibriosis vaccines which added with immunostimulant showed survival rate reached 100%, compared with non-vaccinated groupers are only 12.5%. In some countries, like USA, Canada, Japan and Europe has produced several commercial vaccines. Vibrio vaccine use in Salmon, showing 100% Relative percent survival (RPS), while Aeromonas hydrophila vaccines showed 80-90% with 6-12 months protection. Recombinant protein vaccine use in Japan showed VNN infection resistance with 10-65% mortality rates and showed a high antibody titer up to 100 days. Based on Willoughby data on 1999, the vaccines use can increase fish production to reach 45,000 tons on 1997 in Norway.

II. Vaccine Research in Indonesia

2.1 Aeromonas hydrophila

A. hydrophila vaccine preparation needed to consider various aspects, such as heterogeneous antigens, relatively low immunity, and its application in field. There are two types of vaccine

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A. hydrophila, namely live vaccines or attenuated bacterial vaccine and death cells or only certain parts of attenuated (Ward, 1982). Research on A. hydrophila vaccine has been pioneered by The Bogor Research Institute for Freshwater Aquaculture (BRPBAT) since 1983. The pathology team pioneered and exploits fish immunology and uses it as a major breakthrough in prevention of fish diseases. In the early 1980s an epidemic of bacterial meningitis in a freshwater fish that attacks various species of fish in various sizes. Plague originated from West Java area and eventually spread to all parts of Indonesia with loss rate is estimated to reach hundreds of million rupiah. Results of epidemiological studies indicate that one type of pathogen that considered responsible for these cases is A. hydrophila. Since then the research and the study of biology, character and mechanism of the disease are done intensively to obtain a rational prevention technology, cheap, safe, efficient and effective.

The discovery of prevention techniques such as vaccines begins from the discovery of some evidence that in fish’s body has immunoglobulin-like which have ability to induce immunity that can be stimulated either through artificial or natural. Based on this evidence, several tests carried out on A. hydrophila isolates, about the potential immunogenic and virulence in vitro and in vivo to get one antigens candidate that have cross-react potential to other isolates.

Song et al. (1984) has managed to increase Japanese eel (Anguilla japonica) immune with A. hydrophila monovalent vaccine. The vaccine contents about 87% of fish. Mulia et al. (2004) has A. hydrophila vaccine test intramuscularly with various boosters. The results showed that A. hydrophila cell debris vaccine can increase level of relative protection, survival rate, and antibody titer.

2.2. Vibriosis

Vaccine is an active ingredient that is used to stimulate active immunity in animals and humans (Kuby, 1992). Vaccination for fish diseases control has been developed since the last two decades, but still a few of vaccines for fish diseases that are commercially available. The first commercial vibriosis vaccines are V. anguillarum and V. ordalii polyvalent vaccines for salmon and trout.

Factors to be considered in developing an effective vaccine are: 1. Increasing of an immune response does not mean that immunity has been achieved 2. Stimulation of memory cells is important to establish protection in future and this

depends on the pathogen incubation period.

In gram-negative pathogenic bacteria, outer membrane plays as an important role in infection and pathogenicity level to the hosts (Tsolis, 2002). Outer membrane layer is basically composed of protein, fat and sugar, which can easily be recognized as an element of foreign substances in host defense system. Among these components, membrane proteins have a complex role in many processes and cell physiology, which is very attractive as a candidate used to develop vaccines and diagnostic kits (Pizza et al., 2000; Qian et al., 1999). Protein outer membranes (POM) which have been identified include porin, siderophore and adhesin. Results showed that POM has proven to be a component of a potential vaccine for fish diseases control. This is because, according to Finlay & Falkow (1997) in Desrina et al.

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(2007) that many virulence factors and pathogenicity made its code so that POM in outer membrane is specific and strongly immunogenic. POM vaccines Research has been conducted by Desrina (2006), by giving 5μg of 73.43 kDa of POM vaccine by intraperitoneal and able to protect the tiger grouper seed with 50% RPS.

2.3. KHV

Recently, research and vaccine development continues to create new vaccines. DNA vaccines are a new generation of vaccines that obtained with encoding plasmid DNA that are immunogenic to the host. DNA vaccines can cause not only the humoral immune response, but also the cellular immune response, has the ability to protect against infection, can be purified in large quantities and because it is stable at room temperature or under high temperature, does not require refrigeration.

Fish have non-specific and specific healthy immune system. Prior to successfully invade its host, the pathogen has to deal first with the physical and chemical body's defense barrier. Pathogens must penetrate the mucus and the mucus barrier in the outer parts of the body. Mucus has the ability to agglutinate antigen chemically. After that the pathogen must be able to pass through the skin or scales prior to the scales fish. After this section passes the pathogen has to deal with other non-specific defense system in the body (Tort et al., 2003).

KHV disease prevention using vaccines have been killed developed in BBPBAT Sukabumi on 2007 to 2009. Test results of the vaccine in field and laboratory conditions (floating net cage) gives the results of increased survival rate ranged from 59.5 to 63.3%. Unstable the immunogenic material gill phase as a source of virus is still a constraint that affects the success of conventional vaccination. Therefore a new research carried out using DNA vaccines.

DNA vaccine is a breakthrough experimental technique to protect the organism against disease by injecting pure DNA that contains no protein or peripheral material not expected to generate immunological responses. The vaccine is the result of genetic engineering in which the viral genes sequences that is immunogenic inserted into E. coli plasmid. Plasmids were then transformed and propagation in some E. coli bacteria. Plasmid isolation products from cultures of E. coli were then used as a vaccine (Santika and Sri Nuryati, 2009).

Challenge test at common carp which had been given anti-KHV DNA vaccine on laboratory scale, carried out by BBPBAT Sukabumi in cooperation with the Bogor Institute of Agriculture, shows the results that administration of anti-KHV DNA vaccine with 12.5% / 100 μl dose, was able to maintain the survival rate of common carp up to 72-96%, after 30 days of challenge test with active KHV (Santika and Sri Nuryati, 2009).

In addition a private company has issued a vaccine for the KHV disease, the first in Indonesia. KV3 branded vaccines already passed the field test and ready to use. Based on the results of the Fish Drug Commission Meeting on January 13, 2010, Fish and Environmental Health Directorate of the Directorate General of Aquaculture Ministry of Marine Affairs and

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Fisheries issued a letter number 429/DPB/PB430DA/1/2010. The letter states KV3 vaccine from a private company already graduated a field test.

According to Palar Batubara on 2010, a field test conducted over two years in floating net cage at Cirata and Jatiluhur Reservoir, West Java. "Tests conducted during the second cycle of carp rearing, the result is the survival level of harvested carp amounted to 95%," Palar said. Further explained, the company is the sole agent of KoVax Ltd. in marketing the KV3 vaccine for the Asian region including Indonesia. KoVax has a patent for KHV vaccines and diagnostic tools from the Hebrews University, Israel. The companies from the United States (U.S.), it has a major business to develop aquaculture vaccines, research, and manufacturing.

For while the product is sold to farmers in the form of seeds that have been vaccinated, this step is done to reduce the risk of failure of the vaccination process. Within one month ahead was to be distributed to the cultivators of vaccinated carp seed size 3-5 cm per seed (age one month). From the results expected if the farmer had seen and felt cultivation increased survival of carp seed that has been vaccinated it will generate confidence. In order to continue to strengthen the recognition of product quality KV3 vaccine, the KV3 vaccine is being sought in order to obtain SNI (Indonesian National Standard).

Center for Freshwater Aquaculture Development Sukabumi on 2009 has been test the efficacy of commercial vaccines KV3 for koi and common carp. This vaccine is a live attenuated vaccine (attenuated vaccine). This type of Vaccines has a weakness that potential for infection if the attenuation is not perfect done (Nuryati, 2010). Therefore Greedy (2008) in Nuryati (2010) did not recommend the use of this KV3 vaccine in fish. Greedy also stated that EU law does not permit the use of this vaccine. Attenuated vaccine does have limitations that can be enhanced by a DNA vaccine.

According to Alimuddin (2010), attenuated virus vaccines such as vaccines above has been available, however the price is still considered to be very expensive and from the testing that some indication of the virus is still virulent. DNA vaccine is an alternative effective and safe vaccine. Through research sponsored by the Department of Marine and Fisheries of West Java Province (year 2008) and Doctorate Grant research program by the SPS-IPB (year 2009), Sri Nuryati, S.Pi., M.Si which is a student of Veterinary Science Doctoral PS-SPS IPB, which at the same time teaching staff at the Aquaculture Department of Fisheries and Marine Science Faculty of Bogor Agricultural University has made a DNA vaccine for KHV. Results of reverse transcriptase PCR analysis showed that the gene contained by the vaccine expressed in the vaccinated fish which means that the vaccine is active. Furthermore, through laboratory-scale challenge test the vaccine can increase survival of common carp more than 95% for one month after vaccination. Non-vaccinated fish before the challenge test are death. This shows that DNA vaccines are very effective to cope with KHV virus attack.

2.1. 2.4. VNN

Betanodaviruses (Nodaviridae family) are agents which cause viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in various marine fish farming activities

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around the world. This disease occurs mainly during the period of hatchery fish larvae and the cultivation process is under way (Dennis et al., 2006)

VNN is a disease listed by Office International des Epizooties (OIE), which is marine fisheries production major problem in the world. Identification of virus causing VNN is obtained Nodaviridae family members to investigate the nucleic acid and structural proteins of the Pseudocaranx dentex virus larvae. There are two types of Nodaviridae family, namely Alphanodavirus and Betanodavirus, both types are highly malignant in infected fish. Betanodaviruses (Nodarideae family) is the agent causing VNN in marine fish cultivation. Betanodaviruses is a small virus, spherical, no capsid with a genome consisting of double helix. Nodaviruses is icosahedral viruses that are not wrapped with a genome consist of two single helix RNAs13. Piscine nodaviruses (betanodaviruses) infected more than 30 species marine fish larvae, especially during juvennil, and these infections commonly result in high mortality (Yukio, 2007).

Piscine nodaviruses can be classified into four genotypes based on nucleotide sequence of coat protein genes: SJNNV (striped jack nervous necrosis virus), RGNNV (redspotted grouper nervous necrosis virus), TPNNV (tiger puffer nervous necrosis virus), and BFNNV (barfin flounder nervous necrosis virus). Piscine nodavirus infections have been associated with high mortality rates in grouper cultivation in Taiwan, Singapore, Thailand, China, and Indonesia. Recently been documented the outbreaks of VNN between hatchery of the orange-spotted grouper and asian seabass in Philippines. By phylogenetic analysis, isolation from orange-spotted grouper and asian seabass have RGNNV genotype (unpublished data). Piscine nodavirus infection is a potential threat to cause damage to many species of fish aquaculture in the region. Viruses of this type are the agent that causes betanodavirus VNN, a devastating disease of marine fish farming industry worldwide (Chi, 2006,).

Betanodaviruses are causatif agent of VNN in marine fish farming. Brain tissue of fish and other invertebrate animals have been tested with Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) with the aim to detect betanodavirus. Positive PCR test results obtained from the brain eight species of marine fish (shrimp fish Aeoliscus strigatus, Milkfish Chanos chanos, three spot damsel Dascyllus trimaculatus, Japanese anchovy Engraulis japonicus, Monocentris japonica pinecone fish, blue ribbon eel Rhinomuraena quaesita, look down on fish Selene vomer, yellow tang Zebrasoma flavesenes), a marine invertebrate species (spiny lobster Pamulirus versicolor), and two types freshwater fish (South American leaf fish and red piranhas Monocirrhus polyacanthus Pygocentrus nattereri). PCR detection level was 11/237 (4.64%). In subclinic, fish and invertebrate in aquariums infection by inoculum betanodaviruses.

VNN attacks among population in marine fish farming can occur with the transmission vertically or horizontally. In Korea, VNN attack symptoms were first reported to attack grouper (Epinephelus septemfasciatus). Red drum (Sciaenops ocellatus) mass mortality in hatchery associated with betanodavirus (Chi, 2006).

In the development of larvae, the first vacuolation need to be observed on spine, dorsal fin, swim bladder damage, then brain condition and within retina, marked on the spine are points

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to proliferation of VNN initial location (Nguyen et al., 1996). Naturally, young fish larvae which attacked by viruses can be detected in skin epithelial cells and intestinal epithelium, which simultaneously with Central Nervous System (CNS) nerve cells as early stages of infection or inflammation by VNN. Neurotropisme indications of VNN attack might gain access to CNS through peripheral nerves, for example through the automatic nervous on digestive organs, as well as through the sensor and is associated with motor nerve in skin epithelium (Dennis et al., 2006).

Grouper in grow-out phase can be infected by VNN with pernasal changes. VNN penetrate nasal epithelium via olfactory nerves and bladder, and attacked olfactory ears. Via intramuscular (IM), VNN through peripheral nervous system in muscular tissue edge, transported through the network Axon spine on spine. VNN can attack CNS via blood circulation as starting point injection (Dennis et al., 2006).

Common VNN clinical sign in several fish species namely swimming behavior erratic, and float to stomach caused by swim bladders swelling, melanosis and diminished appetite. Death (mortality) achieved a cumulative 34% and 56% for 10 weeks. VNN infected fish usually show neurological disorders condition associated with central nerves system and retina strong vacuolization (damage) (R. Thie'ry et al., 2006).

There are no other types of antibiotics and chemotherapy which can be used for viral diseases treatment. Prevention is more effective in controlling viral diseases. So we need a vaccine that can be used to boost immune system to be more resistant organisms from pathogenic viral infection attacks. For the procurement of vaccine virus is very helpful in suppressing the impact caused by the virus that causes a variety of viral diseases. To obtain the vaccine in suppressing malignancy VNN can be obtained from a variety of ways, including: recombinant vaccines, DNA vaccine, inactivated vaccine (chemical / biological) and immunization (immersion and injection).

2.5. WSSV

WSSV has a broad host range among decapod crustaceans (Lo et al., 1996; Flegel, 1997; and Alday-Sanz, 1998), and potentially lethal for most commercially cultivated penaeid shrimp species (OIE 2003). WSD causative Agent is white spot syndrome virus (WSSV) or white spot virus (WSV), is a double strand DNA virus (dsDNA) recently established by the ICTV to own a new genus Whispovirus, and family Nimaviridae. Virions have a large (80-120 x 250-380 nm), elliptical rods, and a trilaminar envelope (Wang, et al., 1995; Durand et al. 1997; Inouye et al., 1994, 1996; Kanchanaphum et al. 1998; van Hulten, et al. 2001).

Virions produced in nucleus of infected cells which hypertrophy without occlusion body production. In early reports, WSSV is described as non-occluded baculovirus, but analysis of WSSV DNA sequences has shown that is not related to baculoviruses (van Hulten et al. 2001; Yang et al. 2001). WSSV genome size has been reported for different isolates: 305.107 bp (GenBank Accession No. AF332093), 292.967 bp (GenBank Accession No. AF369029) and 307.287 bp (GenBank Accession No. AF440570) for viruses isolated from China, Thailand and China Taipei. The third sequence is almost the same isolates, with differences in size

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largely due to several small insertions and one large deletion (12 KBP). In accordance with the genome size of 300 kb, a total of 531 putative open reading frames (ORFs) were identified by sequence analysis, among which 181 ORFs likely to encode functional proteins. 181 Thirty-six ORFs were identified by screening and sequencing of a cDNA.

WSSV disease was founded and identified in Taiwan in 1992 which caused mass mortality of tiger shrimp (Penaeus monodon), kuruma shrimp (P. japonicus), yellow tail shrimp (P. penicillatus) and shrimp greasyback (Metapanaeus ensis) (Kasornchandra and Boonyaratpalin, 1996: Wang et al., 1997a; Kasornchandra et al., 1998 and Peng et al., 1998). The Nomenclature of this virus is very diverse, in Japan a virus that attacks P. Rod shapes called nuclear japonicas virus or RV-PJ (Inouye et al., 1994) or Penaeid Rod-shaped DNA virus (PRDV) also called Penaeid acute viremia (PAV) (Inouye et al., 1996). In Thailand it is known as viral diseases or systemic SEMBV Ectomesodermal or White Spot Syndrome Baculovirus Virus (WSSV) (Lightner, 1996). While in Taiwan, this virus is known as White Spot Disease (Mahardika et al., 2004). Lightner (1996) states that the WSSV caused by SEMBV viruses which classified as DNA (Dioxyribonucleic Acid) viruses and rod-shaped (Bacilliform). In morphology, size, cellular pathology and nucleic acids, WSSV (Pm NoBll-Type) are grouped on Non-occluded Baculovirus, subfamily Nudibaculoviridae and family Baculoviridae (Mahardika et al., 2004). The virus has the form of virions rod-shaped particles with a size of 305 x 127 30 11 nm, and in the nucleus, there is one that will join the nucleosome origin of replication (Wang et al., 1999).

The observation showed that shrimp cultivation in these areas has been attacked by WSSV on the stage of PL, prospective parent / size of consumption (subadult) and shrimp (adult). Sudha et al. (1998) states that the WSSV-infected shrimp will changes the experience in behavior such reduced swimming activity, swimming is not directed, and often swim on one side only. The shrimp also tend to schooling on the edge of the pond and swim to the surface. In the acute phase, there are white patches on the carapace with a diameter of 0.5-3.0 mm (Mahardika et al., 2004), and the first white spots appeared on cephalothorak, in fifth and sixth segments of the last abdominal and spread throughout his body cuticle (Wang et al., 1997a). In WSSV cases, white spots on the carapace has become the common sign (Wang et al., 1997b), but it become red for adult shrimp (Mahardika et al., 2004). Shrimps which infected by this disease will die in short period (Ministry of Maritime Affairs and Fisheries, 2004). In field observation (diagnosis), white spots or patches on shrimp carapace has not been found in samples collected. Sudha et al. (1998) states that if the shrimp were WSSV infected but have not found signs of white spots, categorized as type III (chronic), which has low tissue infection so that the white spots and redness of shrimp was not visible. Sudha et al. (1998) also said that death will occurred in 15-28 days. The attack target organs which can be use as attack indicator of such as gill cells, hepatopancreas and intestine. Hepatopancreas, intestine and gills cells which attacked by WSSV disease characterized by damaged core hipertopi (eosinophilic hypertrophy) and cell inclusion bodies.

Histopathologic specific changes infectious WSSV was the finding of pathological changes in cells. Pathological changes such as abnormalities caused the development of existing and varion buildup inside cells, which caused by viral infection. These changes are called Inclusion Body (Alifudin et. al., 2003). Inclusion Body observed a swelling of cell nucleus

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(hyperthropi cell nucleus) which are eosinophilic (reddish color) and move to the edge of cell nucleus. Inclusion Body can also be basofilik (colored blue) where the cell nucleus is very large in threshold to be broken (karyolisis) and it ended with cell lysis where cells nucleus out of the cell.

Changes in salinity did not affect WSSV virus. This is known in several studies that use freshwater crayfish Cherax quadricarinatus (Djauhari, 2005), Procambarus clarkii (Jha, 2006; Wang et al.; 1998) and Pacifastus leniusculus (Jiravanichpaisal et al., 2001) can be WSSV infected by oral, immersion and injection. On research, it have known that the WSSV virus against freshwater crayfish Pacifastus leniusculus result in total mortality on 10 days post infection (Jiravanichpaisal et al., 2001) while Procambarus clarkii reaches 81% within 18 days after consuming a diet of shrimp (Penaeus monodon ) which positive WSSV (Wang et al., 1998). WSSV virus is able to spread through cannibalism among shrimp or through contaminated water (Chang et al., 1996).

According to Mahardika et al. (2004), WSSV virus damages the stomach, gills, epithelial cells, subkutikula, lymphoid organ, antennal gland and haemocyte. Then through histopathologically observed, the cell degeneration occurs in the form of enlargement on the various tissues such as the mesodermal and ectodermal layer of skin, connective tissue, lymphoid organs, glands and antenal haematopoetic, gill and nervous tissue (Wang et al., 1997a).

Based on these, the researchers conducted various researches in controlling this disease. The research team Faculty of Veterinary Medicine, Bogor Agricultural University (IPB), for example, develop a study to provide passive immunization with Y-immunoglobulin (Yolk immunoglobulin) in white shrimp. This research conducted by Sri Murtini, Retno D Soejoedono, Okti N Putri and Heru from Faculty of Veterinary Medicine at IPB is applying specific antibodies against WSSV7 on Y-yolk immunoglobulins (Ig-Y), which is formulated in shrimp feed to control WSSV disease.

Sri Murtini explains utilization Ig-Y research performed on a variety of bacterial diseases, both on terrestrial and marine animals. But a few scientists who examined disease caused by viruses in fish and shrimp. This research, beginning with the Ig-Y produced in chickens. "You do this by chickens vaccinating with WSSV virus with a certain dose." Three weeks pasca vaccination, researchers found anti WSSV antibody in chicken’s serum.

Subsequently, one week after the fourth vaccination, the researchers re-discovered anti-WSSV antibody in chicken yolk egg. Anti-WSSV antibody in yolk egg was tested by researchers from AGPT IPB.They are also testing chicken’s serum after vaccination. Chicken yolk egg which tested is proved contain Ig-Y is able to function as antibodies against WSSV virus. Ig-Y is imunoglobulin or antibody proteins which will work to minimize the WSSV virus attacks and make this virus can not infect the host white shrimp, by making eggs containing anti-Ig-Y WSSV for shrimp feed.

Sri and her colleagues tested the Y-lg shrimp feed with a certain dose of infected white shrimp in ponds. The dose varied, ranging from 5 to 20 percent. This feed test results proved

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quite satisfactory. In ponds, which were given Ig-Y 20%, for example, WSSV-infected shrimp condition began to improve after three days feeding this nutritious. While shrimp fed with Ig-Y content of 5 and 10% getting better condition on the fifth day. Different results seen in virus-infected shrimp and are not fed with Ig-Y. Shrimp like this die in a week.

The death shrimp then were tested by PCR to detect WSSV virus. It goal was to find out the causes of death, whether WSSV infection or due to other causes. From the PCR results concluded that the death of white shrimp which fed with anti-WSSV not find the virus, known as white spot ini. Based on this study, the research team concluded that the yolk egg containing Ig-Y that can be formulated in shrimp and capable of providing passive immunity of white shrimp WSSV virus-infected.

III.Field Applications

3.1 Aeromonas hydrophila vaccine

Aeromonas hydrophila vaccine use in field applications has yet to have any data, but the community has a lot to use this vaccine. From the research that has been done by BRPBAT Bogor, then the production of A. hydrophila vaccines which is sold freely by BRPBAT Bogor. Efficacy test of Aeromonas hydrophila vaccines test currently only on laboratory-scale and will test in the field on 2011. Efficacy laboratory-scale testing of 100 tilapia vaccinations with Aeromonas hydrophila vaccines by immersion results showed 90-100% survival rate after challenge test, while for the non-vaccinated fish only reach 40%. Moreover the antibody titers of vaccinated fish reach 211 HAU/25μl which means an increase in immunity against Aeromonas hydrophila.

3.2 Vibriosis vaccine application

3.2.1. Application in Central Sea Water Aquaculture Development (BBPBL) Lampung

Vaccination Program in BBPBL Lampung has been running on grouper culture, both at the parent and the seed. Unfortunately, in the implementation of vaccination has not been supported by the existence of a good recording system, so the vaccination result can not be described statistically. Vaccination is applied polyvalent vibrio vaccine. In fish seed, vaccine is applied in tiger grouper while for broodstock, is applied to giant grouper. Vaccination applied in BBPBL Lampung depending on fish condition report, if any fish which showed illness symptoms and otherwise ill based on laboratory test results, the vaccination carried out on other fish are estimated to remain healthy. Vaccination is applied using vibrio vaccine polyvalent with densities 107 cells / ml

Vibrio vaccination on tiger grouper had been done in January-March 2007. The seed-sized fish in this vaccination are 6 cm long, with 120 fishes, which 40 fishes were injected intraperitoneally, 40 others are immersion and the last 40 vaccinated orally. Fish vaccinated by injection using a dose of 0.1 ml/fish with bacterial density 107 cells/ml. Fish vaccinated by immersion using diluted vaccine until the bacterial density into107 cells/ml, and then fish in

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the soak for 15 minutes. Orally vaccinated fish was done by the fish fed with vaccine 3-5% per day for 10-15 consecutive days. Booster performed 7 days after the first vaccine.

Parameter measuring the success of vaccination carried out with calculation of survival rate, relative level of protection (Relative Percent Survival/RPS) and Mean Time to Death (MTD). Here are the results of the calculation parameters:

Route of vaccination

Survival rate (%)

RPS (%)

MTD (days)

Non-vaccinated 0 0 6

Injection 100 100 ~

Immersion 42.5 42.5 9

Oral 72.5 72.5 14

From data obtained, it was obvious that after vaccination against disease, the fish resistances become much stronger were shown in increased survival rate, relative percent survival and Mean Time to Death reaches 100%. These data also indicates the use of immersion as a way of vaccination is less effective because it raises stress on fish. Thus, for vaccine application, the fish condition must be really comfortable and healthy.

Besides the parameters calculation, symptoms that occur after vaccination should be continuously monitored, it is aimed at monitoring fish condition during maintenance. Here are symptoms which appear:

Day Non-vaccinated

Injection Immersion Oral

1-3 Active, high Appetite

Lethargy, loss of appetite, inactivity swim

Lethargy, loss of appetite, the fish in the primary vessel

Somewhat active, gather in the primary vessel


Began to eat, began to swim actively

Weakness, appetite began to increase

Active, Appetite increased


Active, high Appetite Active, high Appetite Active, high Appetite

Booster


Do not swim actively. Decreased appetite, gather

Do not swim actively. There is no appetite, gather

Decreased appetite, do not swim actively


Active swimming, high appetite

Active swim, appetite began there






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The symptoms which appear indicate that the fish in healthy condition, the decline in conditions such as fatigue and decreased appetite indicate side effects of vaccines in fish. To determine the effectiveness of the vaccine then performed challenge test. The symptoms were observed also as follows:

Day External symptoms during the challenge test

1-2 The fish swam off, lethargic and always quiet, low appetite, and excessive mucus

3-5 Fish started to move actively, appetite had begun to exist, the mucus begins to decline, has begun to respond

6-8 Fish have started to swim on the surface with a swirling motion (whirling), increased appetite

9-21 Fish are active swimmers, gather, lenders are normal, and high appetite

Vaccination to parent fish was applied to giant grouper. Vaccination being done if there are grouper giant which showed any marks the emergence symptoms in reddish spots on fish abdomen. Usually these symptoms will begin to re-emerge about 2-3 months after vaccination. Fish vaccinated using a vibrio vaccine with dose of 1ml/fish via injection intraperitoneal. The number of breeders which routinely vaccinated every three months is about 17 fish. The results of vaccination showed reduced levels of disease symptoms giant grouper incidence. But the recording of this vaccination does not exist.

3.2.2. Applications in Situbondo

Brackish Water Aquaculture Development Centre (Balai Besar Perikanan Budidaya Air Payau, BBPBAP) Situbondo has successfully created vibrio polyvalent vaccine to prevent vibriosis disease on 2008 to 2009. Vaccines are made in isolation from the tiger grouper, adult and larvae of Humpback grouper. The bacterial isolates obtained from 29 isolates of bacteria, but after Koch's postulates test, only obtained 14 isolates which have pathogenic properties. Four of the most virulent isolates then being pathogenicity test and obtained the LD 50. These four isolates are identified similar to V. alginolyticus, V. trachuri, V. fluvialis, and V. Pelagius with an average similarity level of 80%. Then these four isolates being test antibody titers and cross reaction to search for vaccine candidates. From these tests is known that TKL-2 isolate, in which were identified similar to V. alginolyticus, giving high antibody titer against all vibrio isolates tested so that these serve as vaccine candidates.

In the following year i.e. 2010, the vaccine is applied to tiger grouper cultivation floating net cage in BBPBAP Situbondo. Vaccination applied to tiger grouper with 10 cm total length which kept on floating nets size of 1×1×1.5 m with a density of 50 individuals per plot.

Acclimatization of these fish is less than a week before vaccination. After the acclimatization process, tiger grouper were vaccinated with vibrio polyvalent vaccine (107 cells/ml) which was developed by BBPBAP Situbondo with dose 0.1 ml per fish via injection intraperitoneally using automatic injector. After one week, booster being done with the same steps as in the initial injection.

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From the vaccination applied, obtained data of antibody titer values, length and average weight of fish, and survival rate.

Table antibody titer results

Treatment BO B1 B2 B3

Control 2 6 2 5 2 5 2 4

Of vaccinated fish 2 6 2 7 2 8 2 9

BO: Bleeding before vaccination BO: Bleeding a week after vaccination

B1: Bleeding a week after vaccination BO: Bleeding the end of observation

The antibody titers results showed that the vaccine tended to increase the antibody. Fish length and weight data also showed an increase in body weight of vaccinated fish at 21.69%. These indicate that fish in a more healthy condition so that its appetite are also improving. At the end of observation the average survival rate of vaccinated fish were reach 68.165%, while non-vaccinated fish about 50%. This is in accordance with the opinion of Kamiso (1990) and Kamiso et al. (2005) that vaccine can increase fish resistance, especially a high level of survival live. Survival rate obtained indicates that resistance of vaccinated fish higher than non-vaccinated fish against V. alginolyticus during maintenance. This shows that the V.alginolyticus polyvalent vaccine can provide good protection.

BBPBAP Situbondo actually has two vaccines types, namely streptococcal and vibrio vaccines. In field application, the streptococcal vaccine has not been done while vibrio vaccine has been applied to the broodstock and seed grouper. Vaccines use report in grouper showed that the vaccinated parent has diseases incidence which tend to be lower than prior to vaccination. Recently BBAP Situbondo have been vaccinating with vibrios as many as 20.000 grouper belonging to private and the surrounding communities, so that the recording of data as a result of vaccination continued to be monitored.

Here are the procedure adopted in BBPBAP Situbondo vaccination both on breeding and seed grouper:

A. Parent fish vaccination procedures:

1. Broodstock selection Broodstock which to be vaccinated must be in good health, should not be attacked by disease and health status examination should be performed prior to fish vaccination. Fish condition must be physically clean and fish also should be kept in free of pest water and fish diseases.

2. Acclimatization and adaptation This process aims to minimize the stress level of fish to be vaccinated. Stress can decrease health conditions so that vaccination of stress fish would cause fish illness because of the immune system weakened to receive the vaccine and this vaccine can actually cause fish

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sick. In this process if there are parasites, then fish should be cleaned and cleared of parasites attack.

3. Time and method of vaccination Vaccination of parent carried out about two weeks before spawning, in order to produce seeds that have been brought immunity from its mother so as not susceptible to disease and can be specific pathogenic free. Vaccination can be done with injection, soak, or oral (via feed). Vaccination is usually done via intramuscular injection.

4. Maintenance Fish that have been vaccinated are kept in free of pests water and fish diseases in which both environmental conditions and water quality of the feed should be kept.

5. Breeding

B. Fish vaccination procedure

1. Seed Selection Seeds to be vaccinated must be in good health, should not be attacked by disease and health status examination should be performed prior to the vaccination of fish. Fish condition must be physically clean and fish also should be kept in free of pests water and fish diseases.

2. Acclimatization and adaptation This process aims to minimize the stress level of fish to be vaccinated. Stress can decrease health conditions so that vaccination of stress fish would cause fish illness because of the immune system weakened to receive the vaccine and this vaccine can actually cause fish sick. In this process if there are parasites, then fish should be cleaned and cleared of parasites attack.

3. Time and method of vaccination Vaccination of seed fish is done about 15-20 days after spawning. Vaccination can be done with injection, soak, or oral (via feed). For delivery by injection can be done through intraperitoneal, intramuscular and intra-venous. Seed vaccination is usually carried out through intraperitoneal between two fin fish

4. Booster Repetition of the vaccine or booster aims to strengthen the fish immune system. Seed booster is done about a week (7 days) after the first vaccination.

5. Maintenance Fish that have been vaccinated are kept in free of pest’s water and fish diseases which both environmental conditions and water quality of the feed should be kept for 3-7 days. Then these fish are ready to sell or exaggerated.

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3.2.3.Application in Batam

Institute for Marine Aquaculture Batam has applied polyvalent Vibrio vaccine in February until June 2010. The vaccine used was polyvalent Vibrio vaccine with densities 107 cells/fish. The vaccination method in laboratory scale tests were orally through feed. Vaccination is done every day for the last 3 (three) days of vaccination via feed. Preparation vaccines in food is done by spraying the 1000 ml vaccine into 2000 g of feed pellets which had been sprinkled with grains 6 white egg as binder vaccine. Feed plus vaccine was given during three consecutive days to test fish, while as a control given the same diet without the vaccine. Maintenance begins on day 4 to day 14, which will be a booster with the same treatment. Two weeks after the booster antibody titers were observed.

To test the vaccine efficacy, challenge test carried out by intramuscular with a dose of 10 6-07

CFU/ml and subsequently maintained in the aquarium with good condition. During the challenge test fish mortality rates and time of death occurrence will be observed, the other observation is that the symptoms occur.

Asian seabass of the seed with length and weight and different density conditions were vaccinated with vibrio polyvalent vaccine used best dose from laboratory scale test results through the feed. The booster vaccination conducted after one week with the same method and dose of vaccination. Both vaccination and booster conducted over three consecutive days via feed, which the feed were given chicken white egg as a binder of feed and vaccine. One week after booster, these fish moved in floating nets mesh size of 3×3×3 m3 in density 1000 fish/net for further observation during five months (150 days of research).

The observation during five months activities are as follows:

A. Fish Weight

No. Sampling Date Observations Fish Weight (grams)

Test fish Control

1 March 15 180 182

2 April 16 230 223

3 May 16 300 287

4 June 16 410 395

B. Fish Length

No. Sampling Date Observations of Fish Length (cm)

Test fish Control

1 March 15 22 23

2 April 16 31 30

3 May 16 39 38

4 June 16 47 45

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C. Blood Analysis No. Carcass analysis Fish samples

Test fish Control

1 Haemoglobin (g / dl) 6.2 5.9

2 Leucocytes (cells / μ liter) 501.290 487.490

3 Hematocrit (/ μ liter) 32.3 27.9

4 Thrombocyte (/ μ liter) 289.240 233.450

5 Erythrocytes (million / μ liter) 1.89 1.62

D. Fluctuations in Total Bacteria General (TBU) and Total Bacteria Vibrio (TBV) during the observation.

E. Survival rate

Treatment Survival rate (%)

Non-vaccinated 83

Vaccinations 92

Based on the above data, it is known that vaccination gives better results than non-vaccinated fish, which is fish survival rate at the end of test is 92% and 83% for control, average fish’s length and weight in final sequentially for the test is 35.1 cm and 410 g, 32.8 cm and 395 g in the control fish. Observations of vaccinated fish blood, showed improve the immune response based on increasing the number of leucocytes concentration and haematocrite.

At the observation of fish final length reach 47 cm, its means that during the 150 days of maintenance there is increase gain 0.247 cm length/head/day. It is better when compared with the control fish with final length 46 cm which means daily increasing rate was 0.233 cm/head/day. Likewise fish weight increasing rate. The average weight at the end of maintenance period on the test fish is 410 grams, which means daily weight rate gain was

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Grafik Fluktuasi TBU dan TBV selama masa pengamatan

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Total Bakteri Vibrio

Grafik Fluktuasi TBU dan TBV selama masa pengamatan

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2.47 g/head/day. This compares favorably with the daily weight growth rate of control fish were 2.37 g/head/day. While the survival rate for vaccinated fish about 92% better than control fish was 83%.

From proximate analyze and blood profiles in vaccinated and control fish, it is known that the vaccinated fish have better nutritional content compared with control fish and especially the content of leucocytes and haematocrite blood profile showed concentrations increase in vaccinated fish leucocytes contain about 501.290 (j / μ liter) and haematocrite was 32.3 (j / μ liter). While the control fish leucocytes about 487.490 (j / μ liter) and haematocrite 27.9 (j / μ liter). This means that the immune system that you have on vaccinated fish was better than the control fish. This can happen because vaccination is one effort to deal with animals (including fish) diseases by giving vaccine into the animal's body in order to have resistance from disease attack.

Based on common bacteria and Vibrio total number, weekly fluctuations in water are very high. TBU range from 20 to 4.25 x 103 and Total Bacteria Vibrio 20 to 6.8 x 102 is a sufficient condition alarming occurrence of both primary and secondary infection in asian seabass cultivation. But vaccination showed effectiveness in providing resistance to disease attack. This is evidenced by relatively high survival rate of 92% versus 83% with control fish.

3.3 KHV Vaccine

According to Alimuddin (2010), attenuated virus vaccines such as vaccines above has been available, however the price is still considered to be very expensive and from the testing that some indication of the virus is still virulent. DNA vaccine is an alternative effective and safe vaccine. Through research sponsored by the Department of Marine and Fisheries of West Java Province (year 2008) and Doctorate Grant research program by the SPS-IPB (year 2009), Sri Nuryati, S.Pi., M.Si which is a student of Veterinary Science Doctoral PS-SPS IPB, which at the same time teaching staff at the Aquaculture Department of Fisheries and Marine Science Faculty of Bogor Agricultural University has made a DNA vaccine for KHV. Results of reverse transcriptase PCR analysis showed that the gene contained by the vaccine expressed in the vaccinated fish which means that the vaccine is active. Furthermore, through laboratory-scale challenge test the vaccine can increase survival of common carp more than 95% for one month after vaccination. Non-vaccinated fish that are before the challenge test are all done to death. This shows that DNA vaccines are very effective to cope with KHV virus attack.

Administration vaccine through injection used at the first step of this research. In subsequent studies will be developed a simple method to mass applied in the field, such as through live food and feed. Because of the ability of DNA vaccine protection large enough, the vaccination method development for applications in the field need to be implemented. This research is expected to contribute to prevent KHV infection in fish farming that have occurred in Indonesia since 2002, which until now the problem is not getting the correct solution (Alimuddin, 2010).

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Although the application via intramuscular injection is a method that can be considered in vaccination, but the development of applications with other methods should be developed for example by immersion or by mixing with a feed with consideration for environmental security.

3.4 VNN Vaccine

Research was conducted by Gondol Research Institute for Mariculture to know the effect of different doses and duration of immersion about humpback grouper juvenile (Cromileptus altivelis) immune against VNN infection. There are two stages of the experiment, first stage, 360 juvenile challenged with VNN during six hours immersion. Then the fish were reared for two weeks, and observed clinical symptoms and mortality. In second stage, with the same amount of fish given 15 mUL inactive vaccine by soaking treatment A (one hour), B (2 hours), C (3 hours), and D (0 hours / control). After 10, 20, and 30 days post vaccination 10 juvenile each challenged with VNN for 6 hours. Further observations should be made as in first stage. The results of VNN challenge in first stage show the treatment of 15 ml/L gives survival rate amounted to 86.67% after 25 days post-vaccination. The result of second stage is on immersion treatment for 3 hours gives survival rate after challenge about 86.67% after 20 days post-vaccination. Vaccination dose of 15 ml/L with 3-hour soaking can improve the humpback juvenile immunity against VNN infection.

VNN vaccine applications in grouper seed after 30 days VNN challenged test can reduce mortality also increase survival rate and titer of humpback grouper seed. Vaccine dose was obtained by humpback grouper seed treatment with injection of 0.2 ml vaccine per fish intraperitoneally.

3.5 WSSV vaccine

WSSV vaccine was applicated by the The Research Institute for Brackish Water Aquaculture (Balai Riset Perikanan Budidaya Air Payau, BRPBAP) Maros which consists of several stages: (1) Production of WSSV vaccine, (2) WSSV LC50 Test, (3) Vaccines application by immersion method, and (4) WSSV challenge test. Parameters observed included the highest survival rate of giant tiger prawn (Penaeus monodon) post-larvae in treatments. But apparently there were no significant differences for all treatments. Another report on WSSV vaccine application is not yet known.

IV. Conclusion

Vaccines application has many advantages, especially to increase the fish's immune system against various bacterial and viral diseases. Various researches and its application in Indonesia showed some vaccines such as Aeromonas hydrophilla, Vibriosis, KHV, and VNN have been able to clinically test in preventing disease caused by the target organism. Further research and monitoring needed for vaccination technology develop included new vaccines produce and the administered methods in order to obtain an effective vaccine, efficient, and economical and applicable.

References

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