KR19990079333A - Oral immunization for yolk antibody for the prevention and treatment of swine pandemic diarrhea - Google Patents

Abstract

The present invention relates to an oral immunologic preparation that can minimize the damage caused by Porcine epidemic diarrhea virus (PEDV) infection, the immunological preparation according to the present invention of eggs laid by laying hens immunized by inoculating PEDV Anti-PEDV antibody is produced from egg yolk and formulated as an active ingredient, and thus the prepared immunologic agent can be used very safely because egg yolk antibody is extracted using a food additive. It shows an excellent protective ability, can be stored for a long time by the freeze-drying method, etc., there is an advantage that can be used by adjusting the concentration. By providing antibodies directly to swine pandemic diarrheal diseases, diseases caused by swine pandemic diarrheal virus can be prevented and treated.

Description

Oral Immunologicals Using Egg Yolk Antibodies for Prevention and Treatment of Swine Epidemic Diarrhea

The present invention relates to an oral immunologic agent capable of preventing and treating diarrheal diseases caused by infection with Porcine epidemic diarrhea virus ( PEDV), and more specifically, egg yolk of eggs laid by laying hens immunized with PEDV. The present invention relates to an immunologic agent prepared by extracting an antibody (anti-PEDV antibody) specific for the PEDV antigen from the compound.

In 1971, when Oldham reported an infectious disease (Oldham, J: Pig farming [Oct suppl], 72-73, 1972) characterized by acute diarrhea in growing pigs in the United Kingdom, the presence of a disease other than infectious gastroenteritis Gradually it became clear.

The major causes of swine viral diarrheal diseases include the current Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), and Porcine respiratory coronavirus (PRCV). PEDV and TGEV occur annually in Korea, causing enormous damage to the pig industry. In particular, PEDV causes diarrhea in pigs of all ages, including mammalian pigs and raised pigs, and shows a serious mortality rate of 50-100% in the case of mammalian pigs, and urgent measures are required.

Currently, vaccine prevention is the most widely used to prevent gastrointestinal diseases worldwide. To prevent gastrointestinal diseases using vaccines, vaccines are given to pregnant sows to form antibodies to sows and then to deliver. It is made by transferring the antibody to piglets through colostrum. As a method for preventing swine epidemic diarrheal disease, the present inventors have developed the swine epidemic diarrheal virus recently passaged over 90 generations in an animal cell Vero cell line. Attenuated swine pandemic diarrheal virus attenuated strain (KCTC 8641P) (Korean Patent Publication No. 96-23048 (July 18, 1996)) was inoculated into piglets to prevent the attachment and infection of PEDV to the gastric mucosa and small intestine of piglets. There is a way to suppress and protect against the swine epidemic diarrheal virus. However, the preventive medicine against PEDV is not easily used in the field due to poor domestic environment or lack of recognition of pig farmers, so it is still suffering from PEDV. Moreover, the method of transferring the antibody formed by inoculation of the virus to the new piglets through the colostrum of sows may cause a non-free state such as the antibody level of the sow during pregnancy, the timing of inoculation of the preventive drug, the specification and nutritional management level, and other E. coli or mastitis. Due to various conditions such as the presence of various diseases, colostrum secretion of sows and maternal function of piglets, there are many limitations in the prevention of diarrheal diseases caused by PEDV.

Accordingly, the present inventors have focused on developing more effective vaccines and antibodies that can eliminate the above-mentioned problems.

For example, studies have been reported to prevent and treat swine pandemic diarrheal disease by orally administering to a pig an antibody formed by inoculating a swine pandemic diarrheal virus, that is, an immunocondensed milk. However, this method also has the above-mentioned problems because it only inoculates PEDV into cows instead of pregnant sows and obtains antibodies to colostrum through colostrum.

Therefore, there is an urgent need to develop a new level of preventive and therapeutic agents that are more practical and easier to use outdoors than the passive immune system through colostrum.

In the late 80's and early 90's, however, several researchers began to study egg yolk antibodies using eggs laid by laying hens, which are readily accessible among birds and highly capable of producing antibodies. The purpose of this study is to predict the disease, that is, diagnose the disease. For the prevention and treatment of the disease, the chicken virus such as chicken infectious bursal disease virus and chicken rotavirus are used. Was performed.

Based on the above results, the present inventors formulated an antibody extracted from egg yolk produced by inoculation of swine epidemic diarrheal virus in laying hens to use egg yolk antibodies for the purpose of diagnosis and treatment as well as for the prevention and treatment of diseases. In the case of oral administration, it was found that the adhesion and infection of PEDV to the gastric mucosa and small intestine of pigs were suppressed, thereby completing the present invention.

Accordingly, an object of the present invention relates to an immunologic agent capable of preventing and treating swine epidemic diarrhea caused by Porcine epidemic diarrhea virus (PEDV).

Figure 1 is a result of comparing the titer of passive immune antibodies generated by inoculating a preventive drug against the swine epidemic diarrheal virus (PEDV) by enzyme immunoassay (ELISA).

Figure 2a is a result of comparing the time-dependent response of the passive immunized antibody produced by inoculating the epidemic of the swine epidemic diarrhea virus attenuated strain (KCTC 8641P) in the laying hens by the enzyme immunoassay (ELISA) .

Figure 2b is the immunity of the present invention formulated by inoculating the antigen of the epidemic diarrhea virus attenuated strain (KCTC 8641P) in the present invention into a laying hen, and then extracting the specific antibody from the egg yolk and egg yolk separated from the laying egg Results of enzyme immunoassay (ELISA) of porcine serum inoculated with preparations.

<Description of Drawing Symbols>

1. Negative yolk (1,000-fold dilution) 2. Positive yolk (1,000-fold dilution)

3. Negative pig serum (500-fold dilution) 4. Positive pig serum (500-fold dilution)

In order to achieve the above object, the immunological agent according to the present invention is characterized by extracting and formulating an anti-PEDV antibody from eggs laid by laying hens immunized with PEDV as an antigen.

Hereinafter, the present invention will be described in more detail.

Immunity against swine epidemic diarrhea according to the present invention by inhibiting the infection caused by PEDV can prevent the disease caused by this, and to remove the risk factors such as piglet dehydration, piglet death, etc., not inoculated by injection Because it is an oral preparation, anyone can use it easily. In addition, the immunologic agent of the present invention gives excellent immunity to newborn piglets with incomplete immune function. As can be seen in FIG. 1, the prophylactic drug requires an immunization period of about 2 weeks, but the immunologic agent of the present invention is immunogenic. Efficacy immediately after administration without period.

The immunological agent of the present invention is prepared by inoculating PEDV antigen into a laying hen and separating egg yolk of the immunized chicken egg. That is, after adding carrageenan, pectin, xanthan gum, etc. to the solution in which the separated egg yolk is diluted with phosphate buffer solution (PBS), the egg yolk soluble protein from which the yolk lipoprotein is removed by centrifugation is used. On the other hand, egg yolk antibody is preferably formulated as an immunologic agent, but after inoculation of the PEDV antigen into the laying hen, the egg yolk is separated from the immunized chicken egg, homogenized, sterilized under conditions of 63 ° C. for 3 minutes, and then the blowing temperature. Spray-dried at 145 ° C and blowing air temperature of 85 ° C to make a powder, and then mix and use in feed.

Although the kind is not specifically limited as a scattering system to be used, For example, it is preferable to use a sparing species, such as a white leg horn system, a Rhode island type, a high-line brown type, with high scattering rate. On the other hand, the method of immunizing PEDV in laying hens uses a general immunization method such as subcutaneous injection, intramuscular injection, intraperitoneal injection.

The dosage of the antigen is appropriately selected so that the desired antibody titer is obtained and the adverse effects on the laying hens are not adversely affected. In addition, additional immunity after initial immunization can be determined by the antibody titer of the antibody of interest.

The immunological agent of the present invention can be used for long-term storage by lyophilization, and the dosage can be freely adjusted according to the incidence of disease.

In addition, in order to maintain and improve the diarrheal disease prevention and treatment effect of the immunologic agent, appropriate means of use are required. For example, kelp meal, which can increase immunoprotein, may be added in an amount of 2 to 4%, and an enhancer for boosting the antibody, for example, to increase the antibody titer, for example, Korean Patent Publication No. 97- Immunopotentiators disclosed in No. 32884 [E. coli or purified protein of Staphylococcus aureus cowan I strain] ; And bodetella pertussis ATCC 9797 as an active ingredient, can be injected or added to feed.

Hereinafter, a method for preparing an immunologic agent will be described in detail.

(1) Preparation of swine pandemic diarrheal virus (PEDV) antigen

The PEDV strain used as an antigen can use the swine epidemic diarrhea virus attenuated strain (KCTC 8641P), field isolated PEDV strain, or PEDV strain officially identified in Korean Patent Application Publication No. 96-23048. have.

Monoclonal Vero (African green monkey kidney) cells were inoculated with swine influenza diarrhea virus attenuated strain (KCTC 8641P) and tryptose Phosphate Broth (TPB) and yeast extract were added at a concentration of 0.1-10%. Then, α-MEM medium to which trypsin was added at 1 to 5% was added, and cultured at 37 ° C. for 4 to 7 days.

The cultured PEDV was treated with polyethylene glycol (MW 6000-8000) by the method reported by Chu et al. (Am. J. Vet. Res. 25: 103-107, 1985), and concentrated and harvested by centrifugation. The laying hens are dissolved in buffer (10 mM Tris-Cl, 100 mM NaCl, 1 mM EDTA, pH 7.0-8.0) and emulsified and suspended together with auxiliaries such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and commercialized oil. Used as antigen for immunization.

(2) Immunity of laying hens

The antigen prepared in (1) was intramuscularly injected into a white leghorn system at 0.2 to 1 ml per animal. Immunization was repeated three times in total for three to four weeks, and then immunization was performed once every four to six weeks to maintain the antibody titer. After injection, blood and eggs were collected at each cycle, and antibody titers in serum and egg yolk were examined by enzyme immunosorbent adsorption (ELISA) (see FIGS. 2A and 2B).

That is, after separating the yolk from the egg collecting stand for 30 minutes for each cycle in the yolk and, λ- carrageenan solution at room temperature by mixing (0.1~0.5%, V / V) , 1000 × g, for 10 minutes Centrifugation yielded a yolk soluble protein fraction containing a specific antibody. The obtained yolk soluble protein fraction sample solution and blood were diluted 1,000 to 2,000 times with physiological phosphate buffer solution (hereinafter referred to as PBS) at pH 7.4 to prepare a sample solution. The antigen solution was prepared by diluting PEDV used for immunization with PBS 300 times (absorbance measurement at a wavelength of 405 nm). Antibody titer was determined by ELISA reader after color development, and the results are shown in Table 1.

Blood and Yolk Antibodies Change after Inoculation of Swine Pandemic Diarrhea Virus process week serum Egg yolk Primary vaccination 0 0.231 0.133 2 1.103 0.236 2nd vaccination 4 0.782 1.242 6 2.531 1.307 8 1.121 Tertiary vaccination 10 1.229 12 1.287 14 1.524 1.269

As can be seen from Table 1, the antibody increased from 2 weeks after the first inoculation, showing the highest antibody distribution in egg yolk after 6 weeks (2 weeks after the second inoculation).

(3) Preparation of Immunologicals

Egg yolk was isolated from the eggs produced from the laying hens immunized with PEDV as in (2), homogenized with a homomixer with the same amount of PBS, and then mixed with aqueous solution of γ-carrageenan (0.1-0.5%). The solution was allowed to stand at room temperature for 30 minutes and then precipitated yolk lipoprotein by centrifugation (10000 × g, 10 minutes), and then filtered using a filter paper (Whatman, No. 2 Paper filter). The filtrate was lyophilized to obtain an egg yolk soluble protein powder (hereinafter referred to as "anti PEDV immunologic agent") containing a specific antibody against PEDV.

(4) Survival rate after administration of immunologic agent

In order to test the prevention and treatment effect of the immunologic preparation prepared in the above (3), 4ml of anti-PEDV immunosuppressive drug was administered to the 2-4 day old piglets causing diarrhea in PED-producing farms once a day for 3 days to investigate the mortality. The results are shown in Table 2. The infection was determined by identifying and identifying pathogenic E. coli in diarrhea of piglets with PEDV.

Survival Rate after Administration of Preventive Agents Action Diarrhea Treatment head Mortality (survival rate) Clinical Effect Oral administration 2 to 4 days of age 128 heads 19 (85%) Dehydration Prevention and Diarrhea Relief Nothing 〃 125 heads 87 (30%)

As can be seen in Table 2, the survival rate of piglets administered with the immunologic agent of the present invention was 85%, significantly higher than 30% of the control group, and diarrhea and dehydration symptoms were also alleviated.

PEDV immunologic agent of the present invention is safe in vivo and by administering it can reduce diarrhea, dehydration and death caused by PEDV infection, thereby improving the productivity of pigs.

Claims (1)

After inoculating the porcine epidemic diarrhea virus (PEDV) antigen into the laying hen, the egg yolk isolated from the egg of the immunized chicken is diluted with phosphate buffer solution, and the egg yolk dilution solution is added with carrageenan, pectin or xanthan gum and centrifuged to give egg yolk. An immunological agent against swine epidemic diarrheal disease, which contains an egg yolk soluble protein from which lipoproteins have been removed.