WO2004004764A1 - Composition against white spot disease - Google Patents

Abstract

A composition against white spot disease which contains an antibody obtained from an egg of a hen having been immunized with at least one member selected from among the whole virion of shrimp white spot virus, the envelope thereof, a protein constituting the envelope and an immunogenic fragment thereof; and a method of preventing or treating white spot disease which comprises administering the above antibody to crustaceans.

Description

 Anti-white spot disease composition

Technical field

 The present invention relates to a composition which is effective for the prevention and treatment of white spot disease virus (white spot syndrome virus) infection of crustaceans such as shrimp and rikiji, ie, white spot disease. For more information, see Crustacean white spot disease.

Has excellent preventive and therapeutic effects on white spot disease virus infection

The present invention relates to a composition which is extremely effective in protecting against infection from the environment, reducing death and loss associated with infection with the virus, or treating the virus. Background art

 White spot disease is currently untreatable. The main preventive measure for white spot disease is hygiene. If white spot disease virus is detected in a shrimp pond, incinerate all shrimp and disinfect the pond water. However, it is difficult to prevent transmission of the virus due to the large number of carriers of the virus. Also, unlike mammals and various fish, shrimp and other crustaceans have no immune cells, so vaccine development is not possible. Disclosure of the invention

 White spot disease is an acute viral disease found in various types of shrimp. Rinji and other crustaceans are known to be carriers of the virus. Shrimp are infected at all stages of development, spread very quickly, and often have a mortality rate of 100% within a week. Shrimp and other crustaceans cannot produce antibodies to the virus because they lack immune cells. Therefore, by providing an exogenous specific antibody, the pathogenic virus can be neutralized outside and inside the body, and infection can be prevented.

The present inventors have conducted intensive studies to solve the above-mentioned problems and found that All virions and envelopes of white spot syndrome virus, and chicken eggs obtained by immunizing chickens with at least one selected from the constituent proteins of the envelope and immunogenic fragments thereof. The present inventors have found that administration of an antibody to shrimp or other crustaceans can neutralize the virus and eliminate its pathogenicity, thereby completing the present invention.

 That is, the present invention includes the following inventions.

 (1) Chicken immunized with at least one member selected from the whole region of the white spot syndrome virus and the envelope, and the proteins constituting the envelope and the immunogenic fragments thereof. An anti-white spot syndrome composition comprising an antibody obtained from an egg.

 (2) The composition according to the above (1), which is used for prevention or treatment of white spot disease.

 (3) A crustacean feed containing the composition according to (1) or (2).

 (4) A method for preventing or treating white spot disease, which comprises administering the antibody according to (1) to a crustacean.

 Hereinafter, the present invention will be described in detail.

 In the present invention, as the antigen, at least one selected from all virions and envelopes of the shrimp white spot virus, and the constituent proteins of the envelope and immunogenic fragments thereof is used.

 The white spot disease virus used as an antigen is not particularly limited as long as it is an available virus strain, and examples thereof include the WSSV strain described in J. Virol. 75: 11811-11820 (2001). In addition, these viruses can be used in the form of shrimp cells or shrimp and other crustaceans.

 The envelope of the present virus can be obtained, for example, by the method described in Virol. 266: 227-236 (2000) in which fractionation is performed by ultracentrifugation after Nonidet-P40 treatment. Examples of the protein constituting the envelope include a 28 kD protein (VP 28) described in Virol. 285: 228-233 (2001), and a protein described in J. Gen. Virol. 83: 257-265 (2002). The 19 kD protein (VP19) can be used.

The immunogenic fragment comprises one or more epitopes, There is no particular limitation as long as it can be used as.

 It has been reported that antibodies can recognize an amino acid sequence consisting of three amino acids (F. Hudecz et al., J. Immunol. Methods, 147: 201-210 (1992)). As the minimum unit, a peptide having 3 or more amino acid residues can be considered, preferably 5 or more amino acid residues, more preferably 10 or more amino acid residues, or a protein constituting the envelope or an immunogen thereof. Functional fragments can be synthesized by peptide synthesis methods such as the liquid phase method and the solid phase method. Further, an automatic peptide synthesizer may be used. IV, Tokyo Kagaku Dojin, 1979, Izumiya et al., "Basics and Experiments in Peptide Synthesis", Maruzen, 1985, Edited by The Biochemical Society of Japan, "Chemistry for Protein Chemistry 2", Tokyo Chemistry According to the method described in Doujin, 1989 Can be synthesized.

 Furthermore, the constituent proteins of the envelope or the immunogenic fragments thereof may be obtained from DNA or RNA having a corresponding nucleotide sequence by genetic engineering techniques (for example, “Sequence Chemistry Experimental Course 1 Genetic Research Method I” edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin, 1996, Ed., The Biochemical Society of Japan, edited by The Biochemical Chemistry Lecture 1, Gene Research Method II, Tokyo Kagaku Dojin, 1996, Japan Biochemical Society, Ed. Method III ”, Tokyo Kagaku Dojin, 1987).

When the immunogenic fragment is a low molecular substance, a fragment obtained by binding a carrier to the fragment is usually used. Examples of the carrier include keyhole limpet hemocyanin (KLH), sea urchin serum albumin (BSA), human serum albumin (HSA), chicken serum albumin, poly-L-lysine, polyalanyl lysine, dipalmityl lysine, Tetanus toxoid or polysaccharide can be used. Glutaraldehyde method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide method, maleimidobenzoyl-N-hydroxysuccinimide ester method, bisdiazobenzidine A known method such as the N-succimidyl-13- (2-pyridyldithio) propionic acid method can be used. In addition, the immunogen can be obtained by adsorbing the immunogenic fragment to a carrier such as nitrocellulose particles, polyvinylpyrrolidone, and ribosome. The chicken used for the preparation of the antibody is not limited, as long as it is a laying hen.

Examples of the immunization method include subcutaneous injection, intramuscular injection, and oral administration of an immunogen (the protein constituting the envelope or an immunogenic fragment thereof). Inoculated immunogenic amount of ^{1 0 3 ~ 1 0 9 TC} ID 5. (TCID _5Q : 50% infectious dose of cultured cells), or 0.05 to 2 mg as various proteins or peptides derived from this virus. A higher antibody titer can be obtained by boosting 3 to 10 weeks after the initial immunization. Two weeks after the booster, antibodies specifically reacting to the white spot virus are produced not only in chicken serum but also in egg yolk. Chicken immunized in this way usually maintains a high antibody titer for about 3 months. Even if the antibody titer is decreasing, a high titer can be maintained by boosting with the same procedure. The antibody activity in the serum and egg yolk is measured by a fluorescent antibody method, ELISA or virus neutralization reaction, and expressed as an antibody titer.

 The immunogen is preferably mixed with an adjuvant and injected by immunization. As the adjuvant, known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, and B. pertussis adjuvant can be used.

 The antibody of the egg obtained as described above can be used as a solution as whole egg or yolk, or dried and powdered with a spray dryer or the like, or defatted from whole egg or yolk and used as an egg. A water-soluble protein may be used, and the antibody may be further purified or roughly purified.

 The composition of the invention is applied to crustaceans, especially shrimp. The dose of the antibody is generally 0.01% to 10% of the diet, but is preferably adjusted appropriately according to the type of crustacean, the stage of development, the degree of virus infection, and the like.

 The anti-white spot disease composition of the present invention comprises a solution having a concentration of each active ingredient of 0.001% to 10%, and further has a powder, granule, tablet, or paste form in which the concentration of each active ingredient in the feed is increased. The salary ranges from 0.001% to 10%. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the test results of Example 4. FIG. 2 shows the test results of Example 5. This description includes part or all of the contents as disclosed in the description and Z or drawings of Japanese Patent Application No. 2002-199033, which is a priority document of the present application. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described specifically with reference to examples, but these examples do not limit the scope of the present invention in any way.

 (Example 1)

12-week-old hens, emulsifying and suspension 0. 5 m 1 and Freund's complete adjuvant 0. 5 m 1 containing white spot disease virus 10 ⁸ TCID ₅₀ cultured in E-bi-infected cells, the left and right pectoralis Was injected 0.5 ml each for the first immunization. Similarly, a second immunization was performed 6 weeks later. Two weeks after the second immunization, the neutralizing antibody titer of the anti-white spot disease virus antibody in the serum of this chicken was 20000-fold. The neutralizing antibody titer of the eggs laid by the chicken was 20000-fold. This antibody titer lasted for about 3 months.

 (Example 2)

 The purified white spot disease virus was treated with Nonidet-P40 and centrifuged. The envelope remaining in the soluble fraction was recovered from the viral virion. A 12-week-old hen was immunized using 0.1 mg of this envelope as an antigen. Similarly, a second immunization was performed 6 weeks later. Two weeks after the second immunization, the neutralizing antibody titer of the anti-White spot disease virus antibody in the serum of this chicken was 16000-fold. The eggs laid by the chicken had a neutralizing antibody titer of 16,000-fold. This antibody titer lasted for about 3 months.

 (Example 3)

The 28 kD protein of the white spot virus envelope (VP 28) (Virol. 285: 228-233 (2001)) was purified from the envelope and used as a chicken immunogen. Was 8000 times. Also, the envelope 19 kD protein (VP 19) (J. Gen. Virol. 83: 257-265 (2002)) When produced and immunized by the method, the obtained titer of neutralizing antibody for chicken eggs was 8000-fold. (Example 4)

Black tiger (Penaeus monodon) shrimp (1 g body weight), 1% by weight of whole egg powder (neutralizing antibody titer: 12,000 units Zg) containing anti-white spot disease virus antibody was added to the diet for 40 days. Bred. White spot disease virus 10 ⁹ TC ID ₅ . Was experimentally infected in each shrimp tank, and no abnormalities were observed in the treatment group. However, in the control shrimp group that did not receive the anti-white spot disease virus antibody powder, the disease began to develop on day 10 after virus infection and died 100% on day 32 (Fig. 1).

 (Example 5)

10% (10) of each shrimp group (100 / group) were injected with 10 ⁸ TC I Dso / m 1 virus suspension 10H1 and infected experimentally. Was paid. The control shrimp group became ill on day 6 after virus infection and died 100% on day 25. In the group treated with the antibody powder, 10 virus-infected shrimp died on day 12. The remaining 90 animals did not show any abnormalities until day 40 (Figure 2).

 In Examples 4 and 5 above, when the anti-white spot disease virus chicken egg antibody was continuously fed to shrimp after being mixed with the feed, there was no onset when experimentally infected with white spot disease virus. . In addition, as a result of treating the shrimp group inoculated with the virus with the antibody, the mortality rate was significantly reduced as compared with the control group. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial potential

 Since the anti-white spot disease composition of the present invention acts specifically on the white spot disease virus, it exhibits an extremely effective preventive effect against white spot disease of epipis and other crustaceans. It is extremely effective in stopping the transmission of this disease in shrimp populations previously infected with the virus.

Claims

 1 · Obtained from eggs of chickens immunized with the whole period and envelope of white spot syndrome virus (shrimp) and at least one selected from the envelope's constituent proteins and immunogenic fragments thereof. An anti-white spot syndrome composition comprising the antibody obtained.  2. The composition according to claim 1, which is used for prevention or treatment of white spot disease.  3. A crustacean feed containing the composition according to claim 1.  4. A feed for shellfish containing the composition according to claim 2.  5. A white box, wherein the antibody according to claim 1 is administered to a crustacean. A method for preventing or treating spot disease.