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  blue diamond KOREA-US AQUACULTURE -> diseases->Vaccine development
Panthogenic agents Vaccine development

Fish vaccine development in Korea 

♦ Introduction

Infectious disease such as Edwardsiellosis, Streptococcosis and Vibriosis poses the biggest single threat to aquaculture in Korea.

Vaccines have become one of the major approaches to combat fish diseases in recent years and have made a major contribution to improvements of fish health in aquaculture.

Developing fish vaccine could potentially save aquaculture producers money worldwide by preventing this disease. Reduction of disease outbreak has a flow on effect to decreased antibiotic usage, aiding in the retention of the state's "clean green" image. Other advantages are reducing antibiotic use to control this bacterium in culture fish, making a safer, more environmentally friendly consumer product.

Many people research on the development of effective vaccine, immunostimulant and adjuvant in fish. The introduction of a new generation of both oil- and non-oil adjuvant has greatly improved the efficacy of bacterial vaccines and has resulted in an impressive reduction in mortalities.

The vaccination strategies for the control of viral and bacterial diseases in aquaculture are being studied. Recent studies have investigated the mechanisms of induced resistance of DNA vaccines, the use of cytokines as adjuvant and the potency of novel fish antimicrobial peptides. Theses include the use of live, genetically attenuated bacterial pathogens and DNA vaccines such as recombinant iridovirus.

♦ General Conditions for Use

ο Water temperature should not fall below 8during vaccination or for two weeks post vaccination.

ο The fish must not be stressed unduly. Vaccination must not be combined with grading, transport or other stressing procedures.

ο Fish to be vaccinated should not weigh less than 10 grams.

♦ Reports on fish vaccine in Korea

⟩ Vibrio anguillarum

Vibriosis has caused severe economic losses in the fish farming industry worldwide. Vibrio anguillarum is a gram-negative, comma-shaped rod bacterium classified to the family Vibrionaceae. It is a halophilic bacterium which causes vibriosis or hemorrhagic septicemia in wild marine fish, cultured-marine fish, freshwater fish, and other aquatic animals in Korea.

# Immunological Effect of Experimental Vibrio anguillarum Vaccine and its Safety in Fish
D. Y. Cho, Y. G. Kim, Y. G. Jang, W. Huh and C. H. Kweon The Korean Journal of Laboratory Animal Science, 17(4), 291-296, 2001

Vibrio anguillarum bacterin was experimentally tested for the safety and efficacy in fishes. When the fish were immersed with experimental vaccine or intraperitoneally inoculated, the immune responses were detected within 2 weeks after second vaccination with the highest titer at 8 weeks post vaccination. In addition, the duration of immunity were detected more than 6 months after vaccination. In order to investigate the protective efficacy, the survival rate of fishes were evaluated after challenge exposure from first week throughout 7 weeks post vaccination. Although 100% survival was evident from the group of intraperitoneal vaccination, however, the low survival rate(60%) were detected from the immersed fishes. Nevertheless, the higher survival rate than control (30%) were detected from the immersed fishes, confirming the presence of protective immunity. The present result indicate thatVibrio anguillarum vaccine can be safely applied as an effective prophylatic method to prevent the economic losses from clinical vibriosis.

⟩ Edwardsiella tarda

Edwardsiellosis is a generalized septicaemia, which is often associated with poor water quality and stress. Disease signs may include small cutaneous lesions that can develop into necrotic abscesses, distended abdomen and swellen anus due to the accumulation of ascitic fluid, pigment loss, enlarged kidney, and abscesses on internal organs.

# Characteristics and antigenicity of Edwardsiella tarda isolated from Tilapia (Oreochromis niloticus)
Myung-Joo Oh and She-kyu Chun J. Fish Pathol., Vol.2, No. 2, 83-90, 1989

In March 1987, a bacterial disease occurredamong cultured tilapia(Oreochromia niloticus), in the aquarium of fish pathology laboratory of National Fisheries University of Pusan. The diseased fish showed abdominal inflammation of ascites.

The causative organisms isolated from the kidney were Gram negative rods. On SS agar colonies developed within 48 hours at 25°C.

On the basis of the morphological and biochemical characteristics. The organisms were identified as Edwardsiella tarda.

They were sensitive to chloramphenicol, ampicillin and oxytetracycline. The pathogenicity was proved positive by intraperitoneal injection.

Three kinds of antigen were prepared with E. tarda for the immune response in tilapia : i.e., by inactivating the strain with 0.4% formalin for 24hrs at 25, ultrasonicating it 3 times with 5 watts for 30 seconds each time and filtrating it through milipore filter. The formalin killed antigen showed good efficacy at the injection concentration of over 107 cells per fish of 50g. All the immunized tilapia showed only a little agglutination titer to the three antigens. The highest survival rate was recorded in challenged tilapia immunized with formalin killed cells.

# Studies on Immuno-responses of Eel, Anguilla japonica-Serological Studies on Edwardsiella tarda
Soo-Il Park J. Fish Pathol., Vol.2, No. 2, 83-90, 1989

Edwardsiella tarda, the causative organism of Edwardsiellosis, was isolated from the water and the eel, Anguila japonica of the recirculating eel culture ponds in 1988. E. tarda was always isolated from the water of eel culture ponds in the number of log 2.30-log4.78 cfu/&13206; without distinguishable seasonal characteristics.

The counts of isolated E. tarda were changed with the health condition of the eel. i. e., a number of E. tarda were isolated from all checked organs, intestine contents, liver, spleen and kidney of the diseased eel, however, it was hardly isolated from spleen and kidney of the healthy eel.

The isolates were classified according to o-agglutinating test into three serotypes (Ⅰ,Ⅱ,Ⅲ). There were no distinct differences in the composition of the serotypes between intestine contents od eel and pond water samples (Ⅰ, 52.4-53.3%;Ⅱ, 20.0-23.8%; Ⅲ, 4.8-6.7%). SerotypeⅠ was ascendant in the spleen (66.7%) and the kidney (73.7%) samples. By experimental challenge, most of serotypeⅠproved highly virulent to eel in comparison with the other serotypes tested.

# The effects of adjuvant and vaccine against Edwardsiellosis in tilapia, Oreochromis niloticus
Joo-Seok Lee and Soo-Il Park Journal of Fish Pathology, Vol.5, No.1, pp.19-27, 1992

A formalized bacterin(FKC) of Edwardsiella tarda, conjugated with/without Freund's complete adjuvant(FCA), Freund's incomplete adjuvant(FIA) and potassium sulfate(PAS), was administered by intraperitoneal injection to tilapia respectively. The tilapia were reared under indoor recirculating filter system. There were agglutinin titer increment in the tilapia which were administered not only FKC bacterin but also adjuvant vaccines. But the producing time of the highest level of antibody in the immunized tilapia was different among vaccine preparation, I, e. FKC+FCA group and PAS+FKC group are between the 2nd and the 4th week, and FKC alone group is the 2nd week. In the challenge experiment with 2.5107 CFU/mol or 2.5108CFU/mol of E. tardaT1123 to the tilapia immunized with vaccines, RPS above sixty was recorded both challenge dose in the tilapia which were 3 weeks after immunization with FKC+FCA, PAS+FKC and FKC alone, and the former dose 8 week after immunization with FCA+FKC and PAS+FKC. There were some resistance enhancement against E. tarda in the tilapia which were injectedwith adjuvant alone than that of control. As the results, the FCA or PAS adjuvant vaccine is effective to sustain the defensible period against edwardsiellosis

# Immune response of eel against fish pathogen E. tarda
Soo-Il Park, Yoon -Jeong Choi and Joo-Seok Lee
Journal of Fish Pathology, Vol.6, No.1, pp.11-20, 1993

To study the immune responses of the japanese eel. Anguillajaponica, fish were injected intraperitoneally with several types of Ewardsiella tarda antigen. i e., FKC(formalin killed cells), HKC(heat killed cells) or LPS(lipopolysaccharide). and the changes of immunocytes numbers. phagocytosis and agglutination titre in the peripheral blood of the fish were investigated.

The number of lymphocytes in the peripheral blood of eels were decreased until 6 hours after injection, and then were turn to normal levels after 24hours of injection. However, the level were slightly increased and were remained after 24 hours.

The number of neutrophils of FKC. HKC or LPS injected fish were the highest at 12 hours after injection and were decreased slowly after that.

Three weeks after the injections, the agglutination of antibody titre of all immunized groups were reached at 128 and were remained this level thereafter. However 6weeks after the injections, that in HKC injected fish were dropped the level up to 4.

Fish were injected with LPS and the blood from the fish were bled after 12 hours. Then the blood were incubated with E. tarda. Six hours after incubation, the phagocytic index was reached the highest level. 28.3. One week after the LPS injection, the blood were again bled and incubated with FKC and HKC, treted as same LPS injected fish as above, were 18.8 and 10.7, respectively. The phagocytic index of the conrol fish was 1.2, The antibacterial activities of normal antiserum against E. tarda were shown for both FKC and LPS injected fish, but not for HKC injected fish. The RPS(relative percentage of survival) of HKC, FKC and LPS injected fish in the challenge test were 10%, 20% and 30%, respectively. These results suggest that the effect of protection of the eel which were injected with antigen were varied with the method of preparation of the antigen.

# Effects of Immersion Vaccination in different Concentration of Edwardsiellosis Vaccine on Live Flounder, Paralichthys olivaceus
M. G. Kwon and J. D. Bang Journal of Fish Pathology, Vol.17, No.3, pp.171 177, 2004

This study was performed to investigate the effects of prolonged immersion vaccination in a dilute vaccine on the farmed olive flounder, Paralichtyhs olivaceus. Juvenile olive flounders were immersed in the several diluted (100, 10-1, 10-2 and 10-3) commercial edwardsiellosis bacterins (5mg/l) fro 2min (short immersion, SI) and 24 h (prolonged immersion, PI). After immunization, fish were analyzed to investigate on non-specific (serum lysozyme and bactericida activity), specific immune responses (antibody agglutinin titer) and resistances against Edwardsiellosis tarda infection. The PI of the commercial vaccine diluted with 10-1 and 102showed better results over the untreated control in terms of serum lysozyme, bactericidal activities and resistance against E. tarda infection. These results suggest that the prolonged treatments of the diluted vacine might be anew approach improving the vaccination potential.

# Effect of extracellular products (ECPs) of Edwardsiella tarda on olive flounder, Paralichthys olivaceus
Deok-Chan Lee†, Yi-Cheong Kim, Jin-Woo Kim and Soo-Il Park.. Journal of Fish Pathology, Vol.18, No.3, pp.215-225, 2005

The effect of extracellular products (ECPs) prepared from highly virulent Edwardsiella tarda PoKF-000623 on the physiological and the immunological function in the olive flounder, Paralichthys olivaceuswas evaluated. The virulence of ECPs on the fish, 5.6 g of average body weight was detected 0.714 ㎍ g fish-1 as 96 h-LD50 by intraperitonial injection. After intramuscularly injected with 0, 4 and 40 ㎍ECPs fish-1 to fish average sized 59.1 g of body weight, the fish were measured the glucose and total protein oncentration in sera, and immune activity of kidney macrophage. The fish injected with 40 ㎍protein fish-1 showed a significant increase in the glucose and total protein concentration in sera. But, the fish were changed in cellular immune response as lowed chemiluminescence response and bactericidal activity of head kidney macrophage. These results were suggested that ECPs of E. tarda could be effect on the physiological and the immunological factors,especially in the function of kidney macrophage concerning to edwardsiellosis infection.

⟩ Streptococcus iniae

Systemic infection with Streptococcus iniae represents a serious fish health and economic problem threatening aquaculture of several commercially important species. Signs of the disease in fish include erratic swimming, whirling motion at the surface of the water, darkening of the skin, blindness, popeyes, and small lesions on the body, fins, and anus.

Antibiotic treatments currently used to control the disease in fish suppress streptococcus disease signs, but doesn't completely eliminate the bacterium from treated fish. Due to the poor understanding of immune response of olive flounder to these pathogens, there is no effective vaccine against streptococcosis. A variety of methods were studied to develop vaccine against streptococcosis, such as formalin-killed bacterin, formalin-killed bacterin combined with E. tarda or nodavirus.

Shin (2004) reported that the flounder immunized S. iniae formalin-killed bacterin combined with its ECPs was able to prevent effectively from challenging virulent S. iniae isolate. Moreover, bacterins of S. iniae isolates were able to cross-protect against L. garvieae infection.

⟩ Viral systemic necrosis of carp virus(VSNCV)

# Immune responses to the vaccines of viral systemic necrosis of carp virus(VSNCV) of common carp, Cyprinus carpio L.
Mi-Young Cho, Myung-Joo Oh, Sung-Ju Jung, Sang-Gyu Sohn, Jin-Woo Kim, Soo-Il Park Journal of Fish Pathology, Vol.16, No.3, pp.175 - 181

VSNC is a viral disease causing significant economic losses in cultured carp Ciprinus carpioL. in Korea. Carps were immunized with prepared vaccines against VSNCV and examined specific and nonspecific immune responses. Carps were injected by 0.2㎖of formalin-killed vaccine(FKV), heat-killed vaccine(HKV) or D-MEM, respectively and dealt with boost with same way two weeks later. The lysozyme activity of serum and chemiluminescent reponses of head-kidney leucocytes showed increased responses during 2~7days post-first injection(pfi) and post-boost(pb) in the vaccinated fish, and then decreased to the level of control. As measured by ELISA, vaccinated groups showed a significant increase in VSNCV-specific serum antibodies between 1 week pfi and 6weeks pb with a peak at 2 weeks pb. Results of the virus challenge showed that the fish vaccinated with FKV have induced protective immunity, while HKV injection hardly provided protection.

Field trials of formalin-killed vaccine manufactured by Green cross Co., Ltd Field trials of formalin-killed Streptococcus <em>iniae</em> vaccine in fish farms.
The Edwardsiella tarda formalin-killed vaccine
manufactured by Green cross Co., Ltd
Field trials of formalin-killed Streptococcus iniae vaccine in fish farms.