(b) Description of the Related Art As farming techniques for shrimps became established, the farming of shrimps based mostly on Penaeus mondon has been actively conducted in various countries including Thailand, China, Mexico, Brazil, etc. However, since damage by shrimp infection symptoms was reported, it has become recognized as a serious problem in shrimp farming.

As shrimp infection symptoms, there are vibrio diseases of pathogenic bacteria and baculoviral infection symptoms of pathogenic viruses, etc. In particular, the shrimp white spot syndrome virus (WSSV) that occurred first in the Asian area has come to be known as the most lethal disease of shrimps so far, and a recent report has stated that this virus is spreading to Latin America as well as the United States.

Dr. Lightner (Arizona University, US) identified the white spot virus in

a shrimp aquafarm located on the Pacific coast of Central America in early <BR> <BR> 1999, and informed the Office Internationale des Epizooties (O. I. E. ) thereof.

This virus has caused serious damage to the shrimp farming industry in Asian countries for the past 6-7 years, and it is so lethal that it can kill more than 90% of shrimps in an aquafarm during an outbreak. In Korea, since 1993 in which damage due to this virus was first reported, it increased year by year until after 1996 when the damage reached its maximum, and it has been somewhat steady since. However, the white spot virus still functions as the most serious obstacle in shrimp farming.

White spots of 0. 5-2. 0 mm are observed in the shells of shrimps infected by the shrimp white spot virus. Such spots are due to the accumulation of calcium salts. They begin at the abdominal 5 and 6 shell segments and shell, and spread into the whole body thereby the shell and muscle to be easily separated owing to their loosened binding. The infected shrimps are tinged with pink or redbrown due to the diffusion of chromatophores, and almost 100% of them are killed within 3-10 days from the day the symptom is first observed.

As a control measure against shrimp infection symptoms, antibiotics are generally used against bacterial infection symptoms including vibrio disease. Recently however, due to a large dissemination of antibiotics, the appearance of resistant bacteria has become problematic, and in addition, the administration of antibiotics does not guarantee a sufficient control effect.

Also, there has not been any efficacious medicine developed for viral

infection symptoms, and accordingly, it can be said that there is no effective control measure.

In mammals such as domestic animals and humans, active immunity has been practiced for the control of viral infection symptoms. Active immunity refers to an immune function that is capable of defending against infection symptoms by virtue of corresponding antibodies against pathogens, which are produced by the self-immune ability stimulated by the administration of the pathogens such as inactivated or attenuated viruses, bacteria, etc. into species such as humans, domestic animals, etc. In the farming area, research on active immunity for the purpose of control of infection symptoms has been under active progress. In Japan (Patent Gazette No. 56-53286, Patent Gazette No. 56-53287, Patent Laid-Open No.

58-50026), active immunity for farmed fish has been practically used because of the research about vibrio diseases of the farmed fish.

In invertebrates such as shrimps, it has been known that no specific immune function using antibodies exists, but a control effect of vibrio disease infection by the proliferation activity acceleration function of shrimp hemocyte cells using the inactivated biomass of vibrio Penaeus (Japanese Patent Laid-Open No. 3-204820) has been disclosed. The immunity of invertebrates is assumed to be provided by an in vivo defense function by the proliferation of hemocytes, not by a specific immune function using antibodies, but no study about a detailed mechanism thereof has been conducted.

As a control measure against the infection symptoms of shrimps, passive immunity has been spotlighted. Passive immunity is generated by administering pathogens to domestic animals such as birds, not shrimps, to obtain the corresponding antibodies, which are then used for the control of shrimp infection symptoms. As known examples of the passive immunity development using yolk antibodies of laying hens, prevention of decayed teeth which occurs by the adhesion of Streptococcus mutans bacteria to <BR> <BR> teeth (J. Dent, Res. , 70,162-166, 1991), prevention of rotaviral diarrhea which occurs by the adhesion of rotavirus to epidermal cells in the intestine (Microbiol. Immunol., 34,617-629, 1990), and so on have been disclosed.

In the fish farming area, prevention of Pasteurella piscicida in yellow tail, prevention of vibriosis in flatfish, etc. are known examples of passive immunity, and the effectiveness of passive immunity using antibodies against the infection symptoms of fish has been disclosed (Japanese Patent Laid-Open No. 1-168246). However, the in vivo defense mechanism of invertebrates is significantly different from the immune mechanism of vertebrates, and there has been no concrete disclosure about the effectiveness of passive immunity for the control of infection symptoms of invertebrates such as shrimps.

SUMMARY OF THE INVENTION Therefore, it is an object of this invention to provide a control method against the shrimp white spot virus.

It is another object of the invention to provide a control method using

an antibody against the shrimp white spot virus.

It is another object of the invention to provide a yolk antibody against the shrimp white spot virus.

It is another object of the invention to provide a composition for control against shrimp infection symptoms comprising a yolk antibody against the shrimp white spot virus.

In order to solve such technical tasks, the present invention provides an egg discharged from animals that are immunized by the administration of the shrimp white spot virus or its proteins.

Also, the invention provides an antibody against the shrimp white spot virus, which is isolated from the egg.

Also, the invention provides an anti-shrimp white spot virus composition comprising the egg.

Also, the invention provides a control method against the shrimp white spot virus comprising (a) immunizing animals by administrating a shrimp white spot virus antigen thereinto; and (b) administering an antibody against the shrimp white spot virus which is obtained from the immunized animals, to shrimps.

DETAILED DESCRIPTION OF THE INVENTION The inventors produced antibodies against the shrimp white spot virus, and developed a composition for control against the shrimp white spot virus using them.

The antibodies of the invention have a specific binding activity to the

shrimp white spot virus. More preferably, the antibodies are proteins having a specific binding activity to the shrimp white spot virus, VP24, VP26, or VP18 proteins.

The antibodies of the present invention are produced by eggs discharged from animals that are immunized by the administration of antigens for the shrimp white spot virus. The animals are preferably birds, for example chickens, ducks, turkeys, etc. , and most preferably, laying hens.

The production of eggs comprising an antibody by injecting an antigen into animals to thereby immunize the animals can be easily carried out by a person having ordinary skill in the pertinent art.

For example, antigens can be injected into animals in such manners as intramuscular injection, subcutaneous injection, intravenous injection, intraperitoneal injection, or oral administration, and a preferred method is intramuscular injection. The injection amount of the antigen may be 10 ug to 1 mg in terms of the protein amount of the antigen, but more preferably, it is applied differently according to the state of animals or other conditions. In addition, the administration of antigens is carried out repeatedly until the amount of antibodies reaches its maximum, after the change in the amount of specific antibodies (antibody having a specific binding activity against a corresponding antigen) that appears in the yolk of an egg has been examined through methods such as an enzyme immunoassay, etc.

Antigens for the shrimp white spot virus are an inactivated shrimp white spot virus, an attenuated shrimp white spot virus, or shrimp white spot

virus proteins, and preferably one or more proteins selected from the group consisting of VP24 (SEQ iD NO: 1), VP26 (SEQ ID NO: 2), VP28 (SEQ ID NO: 3), and fragments thereof.

The antibodies of the present invention can be purified by conventional antibody isolation methods from the eggs. Also, the eggs themselves can be used as antibody sources by powdering and drying them, and whole egg powders, yolk powders, or aqueous yolk protein powders may be included as well.

Also, the invention provides an anti-shrimp white spot virus composition comprising said egg or yolk antibody. The composition can be used in an admixture of feeds or in water in which shrimps are raised.

The anti-shrimp white spot virus composition of the present invention comprises a specific antibody against the pathogen of shrimp infection symptoms, and it may further comprise whole egg powder, yolk powder, aqueous yolk protein powder, or egg antibody pure powder obtained from the eggs of immunized hens. A mixed amount of an egg or yolk antibody in the composition for control is not particularly limited, but it is preferably 0.01 wt. % to 10 wt. %, more preferably 0.1 wt. % to 1 wt. %, and most preferably it is adjusted suitably according to the titer of the yolk antibody.

Also, the composition for control can be formulated into conventional <BR> <BR> dosage forms such as powders, granules, pellets, etc. , and in the case of oral administration into shrimp larvae, formulation into particulate powders by a spray drying method, etc. is the most preferable.

The antibody against the shrimp white spot virus of the present invention can be produced in a large quantity with a low cost by using a recombinant protein derived from the isolated DNA of the protein known as the most pathogenic among virus proteins. Also, the yolk antibody has a specific control effect against the shrimp white spot virus, is harmless because it uses edible eggs, and has superior nutriment due to the enriched natural protein contained in the eggs.

Also, the invention provides a control method against the shrimp white spot virus comprising (a) immunizing animals by administrating shrimp white spot virus antigens thereinto; and (b) administering an antibody against the shrimp white spot virus, which is induced from the immunized animals, into shrimps. The antibody against the shrimp white spot virus can be purified from the immunized animals, and it is preferably a yolk antibody or an egg comprising the yolk antibody.

The following are preferred examples of the present invention for better understanding of the invention. However, the following examples are provided solely to understand the present invention more easily; the invention should not be construed to be limited thereto.

Example 1: Production of Shrimp White Spot Virus Antigen (1) Isolation of Shrimp White Spot Virus DNA The tissues of killed shrimps were mixed with PBS and crushed into fine pieces, and then they were centrifuged for 5 min. to thereby obtain precipitates. The precipitates were mixed with 2.5 mL of a lysis buffer, to

which 0.2 ml of SDS (20 %) and 1 mL of proteinase K were then added, and after 1 ml of NaCI was added thereto, they were completely mixed. 10 ml of 99.9% EtOH was added to the mixed solution to thereby recover DNA, which was then added to 920, of a suspended buffer (genomic DNA extraction kit, Bid 01) to suspend it. To the suspended solution were added 25, xi of RNase MIX, 50 of a cell lysis buffer, and 12 rye of a proteinase mixture, which were then sufficiently mixed. After the mixed solution was cultured at 56 °C for 2 hours, it was sequentially mixed with 250 fli of a saltout mixture and EtOH and then dried. The dried products were dissolved in 10 mM Tris (pH 8.0) to thereby recover DNA.

(2) PCR Amplification of VP24, VP26, VP28 Genes To examine the type of virus and expression after cells were transfected with a viral gene, an RT-PCR was performed.

A DNA oligomer derived from the nucleotide sequence of a corresponding gene was used as a primer in the RT-PCR. After mRNA was extracted, cDNA was synthesized using oligo-dT and reverse transcriptase (Moloney Murine leukemia virus), and a PCR was performed again using this cDNA as a template. The amplified PCR products were identified on 1.0 % agarose gel, they were cloned in a pGEMT-easy vector, and then their nucleotide sequence was analyzed.

SEQ ID NO: 1 is the nucleotide sequence of Vp24 of the shrimp white spot virus, SEQ ID NO : 2 is the nucleotide sequence of Vp26, SEQ ID NO: 3

is the nucleotide sequence of Vp28, and they are identical to the known nucleotide sequences.

(3) Subcloning of VP24, VP26, and VP28 Protein Genes All the genes of the shrimp white spot virus isolated throughout the world have been cloned and stored in GenBank. Hence, using the gene specific primers obtained from GenBank, VP24, VP26 and VP28 genes were subcloned into the expression vector for bacteria, pET-28b (+), which was then transformed into BL21.

(4) Mass Production of VP24, VP26, and VP28 Proteins The transformants were cultured in a large quantity and then centrifuged to thereby obtain shrimp white spot virus proteins, VP24, VP26, and VP28 antigens.

After the transformants were spread onto an LB (Luria-Bertani) media and incubated at 37 °C for 6-12 hours, they were transferred into 5 L of liquid media containing kanamycin and then cultured while shaking at 90 rpm for 6 hours. After 2 ml of 1 M IPTG were added to the culture media, which was then cultured again for 2 hours under the same conditions, it was centrifuged (4,000 rpm, 15 min, 4 °C) to thereby obtain a precipitate which was then lysed in a cell lysis buffer (20 mM Tris-HCI, pH 7.4). The lysate was then centrifuged at 3,000 rpm at 4 °C for 15 min. to eliminate the precipitated E. coli biomass and to collect only the supernatant portion containing proteins, which was then ultracentrifuged (13,500 rpm, 4 °C, 30 min). In order to raise the purity of the obtained precipitates, ultracentrifugation was repeated three

times under the same conditions using a washing buffer to thereby obtain the final products containing VP24, VP26, and VP28 proteins.

(5) Preparation of Composite Protein Antigen A composite protein antigen for a vaccine was prepared by blending the recombinant proteins VP24, VP26, and VP28 in the same ratio.

Example 2: Production of Yolk Antibody against the Shrimp White Spot Virus Leg muscles of 21-week-old laying hens (Hy-Line Brown) that had begun to lay eggs were injected first with 0.3 mL (protein amount: 100 , ug/hen) of an emulsified composition wherein the shrimp white spot virus composite protein antigen complex and Freund's adjuvant (Sigma Chemical Co. ) were mixed in a 1: 1 ratio. Thereafter, a booster injection was carried out a total of three times at two week intervals for 6 weeks until sufficient antibodies were formed.

The change in the titer of white spot virus antibodies transferred into the yolk of eggs was determined, and immunized eggs having a high antibody titer were collected and then broken out. The broken yolk solution was homogenized, sterilized at 63 °C for 30 min. , and spray-dried to produce yolk antibody powders against the shrimp white spot virus.

Example 3: Antibody Separation from Yolk After the yolk solution collected in Example 2 above was filtered, 5 mi of yolk solution was mixed with 5 mL of distilled water and further mixed with 20 ml of lambda carrageenan, and then it was placed at room temperature

for 30 min. This mixture was centrifuged under the conditions of 3,000 xg, 15 min. , and 20 °C to collect only the supernatant. After 19 % sodium sulfate was added to the above supernatant and completely dissolved, the resultant was placed at room temperature for 40 min. and centrifuged at 10,000 xg for 15 min. to thereby obtain a precipitate, which was then dissolved in a phosphate buffer to obtain purified immunoglobulin (IgY).

Example 4: Determination of Titer of Shrimp White Spot Virus Antibody To examine antigen-antibody response of the immunoglobulin obtained from Example 3 above against the shrimp white spot virus, the titer of the antibody was determined using the ELISA method.

Recombinant proteins VP24, VP26, and VP28 were diluted to a concentration of 0. 5, ag/mL and coated onto a 96-well plate. The total antibodies were first diluted to a concentration of 100 ug/mL and then continuously diluted twice and put into each well to allow them to be reacted.

After the end point was determined by color development by goat anti-chicken IgY-AP conjugate, titer per mg of antibody was expressed. As a result of this experiment, the antibody titers of the yolk solutions produced in Example 3 above were all about 20,480 for each protein.

Example 5: Preparation of Composition for Control and Control Effect In this example, the control effect of immune yolk antibody against the shrimp white spot virus was investigated.

Shrimp larvae were divided into four groups, each containing 500 larvae. As a control, a group was used that was treated with assorted feeds

that are normally administered in shrimp aquafarms. After the yolk antibody was added to the normal assorted feeds identical to those used in the control group in concentrations of 0.5 %, 1.0 %, and 2.0 %, respectively, the feeds and water were mixed in a 1: 1 ratio, well kneaded, and then powdered again by lyophilization. The groups treated with the thus-obtained feeds were designated as experiment groups 1 to 3, respectively.

For infection with the shrimp white spot virus, 3 g of shrimps that were infected with the white spot virus, killed, and stored in a frozen condition at -80 °C were sterilized and homogenized in 30 in of saline buffer solution <BR> <BR> and then centrifuged (12, 000xg, 30 min. ) to thereby obtain supernatants, which were then filtered using a millipore filter of 0.45 ßm.

Virus infection was carried out by administering the filtrates into each test water tub in an amount of 10 m. C. The survival rate of shrimp larvae was determined by examining the number of killed shrimp larvae in each group for 15 days.

The survival rates of shrimp larvae in control and experiment groups are as shown in Table 1.

Table 1 Control Experiment Experiment Experiment Group Group 1 Group 2 Group 3 Yolk Antibody Content (%) 0 0. 5 1. 0 2. 0 Shrimp Survival Rate (%) 56 72 88

The control group that was not treated with yolk antibodies showed a 0% survival rate, whereas the experiment groups showed an increasing survival rate in relation to the administration concentration of yolk antibodies.

The survival rates of the comparative groups comprising yolk antibodies showed a statistically significant difference (P<0.01), and hence, the reliability of the experiment results was supported.

Example 6: Treatment Effect of Immune Yolk Antibody Against the Shrimp White Spot Virus The treatment effect of the immune yolk antibody against the shrimp white spot virus was examined using Malaysian Penaeus orientais in Namsamdo, Damgang, Guangdongsheng, China. Under the circumstances of the farming having already proceeded for 80 days, 15 days after the attack of white spot virus, 300 thousand shrimps were reduced to 150 thousand and white spots occurred throughout the head and body of the shrimps. At that point, as a result of administration of the immune yolk antibody powders against the shrimp white spot virus that was added to the previous feeds at a concentration of 2 %, on the first day of administration 500 shrimps were attacked, on the second day of administration 9 shrimps were attacked, and on the third day of administration 60 shrimps were attacked, and from the third day evening, the attacked shrimps began to ingest the feeds. On the fourth day of continuous administration 60 shrimps were attacked, on the fifth day of administration 20 shrimps were attacked, on the sixth day of administration 6 shrimps were attacked, and on the seventh day of

administration 30 shrimps were attacked. From the eighth day of administration, the number of dead shrimps decreased as time went on, and on the ninth day of administration, no more newly dead shrimps were observed.

From the experiment in which the immune yolk antibody against the shrimp white spot virus was tested in an actual aquafarm, it can be seen that it had a treatment effect on shrimps that had already been attacked by the virus.

As described above, the yolk antibody against the shrimp white spot virus of the present invention showed excellent control effects when applied to shrimps. Also, as the yolk antibody was produced from eggs that are used as food, it is safe without toxicity and persistency in a shrimp body.