KR101411995B1 - Antibody composition of bovine colostrum for treating or preventing porcine wasting diseases - Google Patents

Abstract

The present invention comprises antibodies which are obtained by vaccinating the antigen of a pig epidemic diarrhea disease, the antigen of pig epidemic gastroenteritis, the antigen of a pig rotaviral infection, the antigen of a pig circovirus infection, the antigen of pig colibacillosis, and the antigen of pig salmonellosis to a milk cow. The present invention relates to an antibody composition for treating or preventing wasting diseases of a pig such as the pig epidemic diarrhea disease, pig epidemic gastroenteritis, pig rotaviral infection, pig circovirus infection, pig colibacillosis, and pig salmonellosis. The antibody composition according to the present invention is a high incidence antibody composition which treats and/or prevents the wasting diseases of pigs which cause death and growth delays.

Description

TECHNICAL FIELD The present invention relates to a cow's colostrum antibody composition for treating or preventing swine-

The present invention relates to a highly immunized dairy cow's colostrum antibody composition for treating or preventing a consuming disease that lowers the productivity of pigs, and more particularly, to an antibody composition capable of treating or preventing various kinds of pig diarrhea and respiratory diseases at the same time .

Productive damage to pig farms is severe due to the development of consumable diseases of pigs. It is estimated that a loss of KRW 690 billion will be incurred if the mortality rate of piglets due to consuming diseases reaches 18.5% in domestic pig farms, and KRW 1.1 trillion in damage if the mortality rate reaches 32.5%. In order to prevent such swine-wasting disease, it is necessary to stop the influx of the main pathogens to the pig farm first and thoroughly the pig breeding environment and hygiene management. In addition, administration of vaccines or immunizations is also a way of managing the immune system. Other pigs infected with the disease may be treated directly with antibiotics or through the treatment of symptoms. Immunogenesis is a type of therapeutic and preventive agent that has been used in humans and animals by producing immunity serum in the past. Recently, in Korea, highly immunogenic antibodies against human or animal pathogens have been developed in egg yolk, Have been developed and used. IgY preparations have the advantage of less production or side effects than conventional immunoserant agents and can easily and continuously obtain IgY antibody from immunized laying hens. However, there is a disadvantage that separate egg laying facility is necessary and continuous antigen inoculation and management are necessary . One method that can overcome these shortcomings and be applied to produce high-titer antibody preparations is to prepare immune formulations using colostrum produced by highly immunizing pathogens against cows. The colostrum secreted by the cow for a few days after delivery contains not only nutrients such as proteins, sugars, lipids, vitamins and minerals but also various growth factors and anti-microbial factors And has a bioactive component. Various growth factors promote the growth and development of newborns. The antimicrobial factors include immunoglobulin, lactoferrin, lysozyme and lactoperoxidase. In particular, colostrum ingested from the mother has a role of defense-related immunoglobulin for a few weeks after birth. The concentration of IgG, IgM, and IgA is about 100 times that of normal milk. Immunogens prepared by highly immunizing various pathogens in cows for the purpose of treating calf-related diseases, especially digestive diseases, have been developed and partially commercialized.

However, in Korea, there has not been developed a colostrum preparation for pigs using colostrum having high antibody titer and excellent therapeutic and preventive effects. Pigs are generally more dense than cows and are more susceptible to disease, so many preventive vaccines and treatments are used. In particular, if the level of immunity to disease in the sows is unbalanced and colostrum and lactation are inadequate or if normal mammary function is impaired, the piglets are likely to be infected with these diseases. In case of infectious diseases caused by proper immunodeficiency, not only direct damage such as infant mortality but also stagnation of growth and atrophy cause decrease in productivity of farms, resulting in greater economic damage.

There are various viruses and bacteria that cause gastrointestinal diseases, respiratory diseases and systemic diseases. In addition, disease occurrence is compounded by various pathogens rather than infection by single pathogens, which makes symptoms worse. The problem of disease can be solved by transferring the immune substance of a strong sow to the pathogen to piglets, but it is not easy to maintain such a balanced sow immunity by farm or pig group. Therefore, in order to enhance the immune system against these diseases, it is necessary to administer various immune preparations as described above. In particular, it is important to administer sufficient immunological substances that can tolerate diseases. Therefore, in the present invention, development of a preparation for enhancing the immunity of deficient pigs by using immuno-colostrum of a cow which is not applied in Korea is a main objective. Through the application of such a preparation, various diseases And to prevent damage from wasting disease.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention is directed to provide an effective antibody composition capable of simultaneously preventing pig consuming diseases.

The present invention aims to provide an antibody composition which is free from the immunological interference that can be induced by simultaneous inoculation and has a small side effect and thus has excellent therapeutic and preventive effects.

It also provides stable formulations that are less susceptible to various chemical and physical processes.

In order to solve the above technical problems, the present invention provides an antibody composition for treating and / or preventing swine exhaustion diseases. More preferably, the present invention is to provide an antibody composition for the treatment and / or prevention of swine-wasting disease using cow colostrum immunoglobulin.

Despite the existence of many other methods for antibody production, the inventors of the present invention have been studying methods for producing antibodies in the milk of livestock in order to increase the immunity while reducing the production cost by relatively high antibody production cost Thereby completing the present invention.

The present invention relates to a method for the treatment of swine infectious gastroenteritis caused by Transmissible gastroenteritis virus (TGEV), swine diarrhea caused by Porcine epidemic diarrhea virus (PEDV), and Pocine rotavirus infection (PRV) Swine rotavirus infection, porcine circovirus type 2 (PCV2) induced porcine circovirus infection, porcine pathogenic Escherichia coli infection, porcine salmonellosis caused by infection of the genus Salmonella typhimurium , Swine pneumonia caused by Mycoplasma hyopneumoniae , Glasser's disease caused by infection of Haemophilus parasuis , swine feverulerosis caused by Pastuellar multocida , and streptococcus Streptococcus there is provided an antiviral composition for preventing swine fever disease using a colostrum immunoglobulin produced by inoculating cows with antigens of the above diseases in order to treat and / or prevent porcine digestive and respiratory diseases caused by swine streptococcal infection caused by swine .

As used herein, the term " consumable disease or disorder " is used broadly to broadly denote diseases that can cause physical exhaustion, such as digestive, respiratory, and fever, which cause diarrhea and the like.

According to embodiments of the present invention, the present invention encompasses an antibody obtained by inoculating a cow with a porcine epidemic diarrhea antigen, a porcine infectious gastroenteritis antigen, a porcine rotavirus infection antigen, a porcine circovirus infection antigen, a porcine colonic antigens antigen and a porcine salmonellosis antigen Which is characterized in that it is used for the treatment or prevention of swine-related diarrhea, swine infectious gastroenteritis, swine rotavirus infection, swine circovirus infection, porcine colicosis and swine-wasting disease of swine salmonellosis.

According to still another embodiment of the present invention, the composition is prepared by inoculating a cow with any one or more antigens selected from the group consisting of a swine pneumococcal pneumonia antigen, a Glucocorticoid antigen, a porcine sweat ulcer antigen and a porcine streptococcus infection antigen Wherein the swine wasting disease further comprises one or more diseases selected from the group consisting of swine pneumonia, Glazes disease, swine fever disease and swine streptococcal infection.

When an antigen inducing such diseases is administered in vivo, antibodies are generated in the serum by the stimulation of these antigens. When such an antibody is contacted with a specific antigen, it has a property corresponding to this specific antigen. do.

Antigens producing such immunoglobulins may interfere with the production of antibodies corresponding to the respective antigens when they are administered in vivo, thereby causing the problem of inhibiting antibody production.

The present invention intends to provide a composition in which a multi-species antibody is produced without such interference, and a produced multi-species antibody stably exists in the antibody composition.

The colostrum immunoglobulin antibody of the present invention can be obtained by injecting an antigen of the pig swine disease into a pregnant cow.

In the present invention, the term " injection " is performed by subcutaneous injection or intramuscular injection, preferably by intramuscular injection.

The term "injection" or "administration" in the present invention may vary depending on the age, sex, body weight and the like of the subject to be administered and may vary depending on administration route, disease severity, sex, Dose may vary.

The term " immunogen " or " antigenic substance " as used herein refers to a virus, a bacterial, a peptide, a polypeptide, a lactic acid bacterium expressing the polypeptide, a protein, a lactic acid bacterium expressing the protein, an oligonucleotide, a polynucleotide, a recombinant bacterium, , And the like. As a specific example, the antigenic substance may be in the form of an inactivated whole or partial cytostatic form, or an antigenic molecule obtained by conventional protein purification, genetic engineering techniques or chemical synthesis.

In general, when various kinds of antigens are injected into the body, there is a problem in that the antibody production rate is significantly reduced due to immunological interference or proper antibody production is not performed smoothly. In order to overcome this problem, the antigen is inactivated. However, if the antigen is inactivated, if there are too many different antigens, competition may occur in the immune response and immunity may be impaired. Also, even if an antibody is produced to prepare a preparation, stability may be a problem such that coagulation occurs due to high antibody concentration in a preparation containing the antibody.

Accordingly, the present invention researched cow colostrum immunogens that do not cause competition even when many kinds of antigens are inoculated at once.

The present invention is based on the surprising fact that inoculation of an antibody against 10 kinds of swine-wasting diseases at one time can effectively prevent or treat all 10 types of swine-related diseases, and can have a stable formulation form.

Particularly, the present invention can produce an optimal antigen for each pathogen and use it to inoculate a pregnant cow to produce a high-cost colostrum immune antibody, so that it is possible to treat and prevent various kinds of swine- It is a breakthrough technology.

Specifically, according to the embodiments of the present invention, the optimal antigens for each pathogen are inoculated into a pregnant cow, and the cow's colostrum is formulated and administered to young piglets to treat and / or treat pig consumable diseases such as diarrhea and respiratory diseases, Or prevention.

The strains used for the swine pathogens used to produce the colostrum immuno-antibodies of the present invention are shown in Table 1 below.

Disease name Strain Inoculation antigen Swine infectious gastroenteritis
(Transmissible gastroenteritis, TGE) Vaccine (Pyeongtaek Province) Virus-enriched antigen Swine diarrhea
(Porcine epidemic diarrhea, PED) The vaccine (SM98) Virus-enriched antigen Swine rotavirus infection
(Porcine rotavirus infection, RotaV) The separators (G4, G5, G9) virus
Concentrated antigen Porcine circovirus infection
(Porcine circovirus disease, PCV2) Recombinant baculovirus Recombinant antigen Porcine colicosis
( Escherichia coli infection, E. coli ) Standard week and separator Cell antigen Pig salmonellosis
( Salmonella typhimurium infection, S. typhimurium ) Separator Cell antigen Swine pneumonia
( Mycoplasma hyopneumoniae infection, M. hyopneumoniae ) Separator Cell antigen Glazier bottle
( Haemophilus parasuis infection, H. parasuis ) Separator Cell antigen Swine fever
( Pasteurella mutocida infection, P. mutocida ) A type Cell antigen Swine streptococcal infection
( Streptococcus suis infection, S. suis ) Separator Cell antigen

The antigens of the swine epidemic diarrhea are derived from vaccine strain SM98 strain.

The antigen of the porcine rotavirus infection is derived from any one or more isolates selected from the group consisting of isolates G4, G5 and G9, preferably the antigens are derived from both G4, G5 and G9 strains.

The antigen of the swine infectious gastroenteritis originates from the vaccine host Pyeongtaek.

The vaccine strain and the isolate may be inactivated with 2-bromoethylamine hydrobromide after concentration to produce an antigen, preferably 5 to 15 wt% of 0.1 M BEI to the total weight of concentrated pathogens, Preferably 7 to 12 wt.%, Most preferably 10 wt.%.

The vaccine strains and isolates of the pigs' harmful diarrhea, swine rotavirus infection, and swine infectious gastroenteritis antigen are inactivated after culturing the respective pathogens, preferably 1/3 of the original culture volume, preferably 1/5 It can be used by concentrating.

The antigen of the porcine circovirus infection is derived from porcine circovirus type 2. Preferably, the antigen of the porcine circovirus infection can utilize the recombinant capsid protein of the circovirus.

The recombinant capsid protein can be produced as an antigen by inactivation with bromoethylamide hydrobromide as well as the pigs' harmful diarrhea, porcine rotavirus infection and porcine infectious gastroenteritis antigen.

The porcine colic antigen can be obtained by culturing at least one strain of E. coli selected from the group consisting of K88, K99, 987P and F41 and at least one strain selected from the group consisting of LT, ST and Hly, And all of the above E. coli antigens can be obtained.

The pork stew par Relais increased antigen wave stew is Pasteurella Mule Toshi (Pasteurella multocida ) type A. < / RTI >

The pig salmonellosis antigen, epidemic pneumonia antigen, glomerular disease antigen and porcine streptococcus infection antigen can be obtained by isolates obtained by a general technique in the relevant technical field.

The porcine colonic antigens, porcine salmonellosis antigens, porcine epidemic pneumonia antigens, glabellar antigens, porcine parasitic strains, and porcine streptococcal infection antigens may be inactivated using formalin, preferably 0.3% formalin have.

The antigens may be used as an antigen for inoculation for production of colostrum immunoglobulin, and the inoculation antigen for inoculation of the milk of the present invention may be inoculated together with a veterinarily acceptable carrier. The term "veterinarily acceptable carrier " as used herein includes any and all solvents, excipients, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, do. Specifically, the inoculation antigen is Biostim. ^{TM .} Such as aluminum hydroxide gel, Iscoms, aluminum hydroxide gel, saponin, DEAE-dextran, miglyol and the like, preferably aluminum hydroxide gel.

The inoculating antigen may further comprise an immunostimulant, which may include ISA 25, ISA 51 and SWE (squalene-based oil-in-water emulsion) , Preferably ISA 25.

According to a preferred embodiment of the present invention, the inoculating antigen is inoculated 2 months before the cow is delivered.

According to an embodiment of the present invention, the colostrum secreted from the cow in which the antigen is inoculated is selected from the group consisting of swine infectious gastroenteritis, swine epidemic diarrhea, swine rotavirus infection, porcine circovirus infection, porcine colicosis, porcine salmonellosis, , Porcine pustulerosis, and porcine streptococcal infections.

The colostrum of the cow containing the antibodies can be dried and powdered, preferably spray-dried.

According to the present invention, it has been found that a composition comprising the colostrum immunoglobulin of the present invention can avoid aggregation between the various types of antibodies despite the high antibody concentration, and can preserve the activity of the antibody. The composition of the present invention may further include an excipient to prevent aggregation of the antibodies and improve the stability of the composition, in addition to the antibody separated from the cow's colostrum.

The excipient may include at least one selected from the group consisting of paclitooligosaccharide, glucose, glycine, KCl, ascorbic acid, sodium bicarbonate, and sorbitol.

The present invention also provides a method for producing a dairy cow colostrum immunoconjugate composition.

Specifically, the present invention relates to a method for the treatment and prophylaxis of pox diarrhea antigen, porcine infectious gastroenteritis antigen, porcine rotavirus infection antigen, porcine circovirus infection antigen, porcine colonic antigens, porcine salmonellosis antigen, swine pneumococcal antigen, An antigen, and a strain of swine-wasting disease consisting of a porcine streptococcal infection antigen;

Inactivating the strain to produce an inactivated mixed antigen;

Mixing the inactivated antigen and the excipient to prepare an inoculation antigen for production of colostrum immunoglobulin;

Inoculating the cow with the inoculating antigen; And

The method comprising the step of collecting a colostrum.

In a further embodiment, the present invention relates to a method for producing a dry colostrum by spray-drying cow colostrum comprising the immuno-antibody produced in said step; And mixing the dry colostrum and the medium to prepare a liquid colostrum immune antibody composition.

The medium contained in the liquid colostrum immuno composition may be water, that is, sterilized water, bacteriostatic water for injection (BWFI).

In connection with the step of inactivating the strain to produce an inactivated bacterial antigen, the meaning of fluorination as used herein has been broadly used, such as attenuation or decontamination. In particular, poisoning refers to artificially weakening the toxicity of living pathogens, which in turn means eliminating pathogen toxicity.

In the step of preparing the mixed antigen, a conventional method of mixing inactivated viruses or antigens can be applied, and a shaking machine (shaking machine) which rotates at 2000 rpm or less, preferably 1500 rpm or less, more preferably 1000 rpm ) For 30 hours by a method involving rotation and vibration.

The content of the antibody composition according to the present invention can be determined by those skilled in the medical and veterinary arts, taking into consideration such factors as the age, sex, body weight, species and condition of the subject to be administered and route of administration. The antibody compositions may be administered alone or in conjunction with other immunological antigens or vaccine compositions or therapeutic compositions, or subsequent administration.

In particular, the appropriate dosage of the compositions of the present invention will depend, for example, on the particular antigen, host, mode of administration, and nature and severity of the condition to be treated, against the swine-wasting disease to be utilized. Dosage frequency in prophylactic use is usually administered once to three times a day, more usually twice a day, usually once a day, once a day, once a day, or once a week .

The compositions of the present invention are suitable to be administered to a pig either over a series of treatments or once in a pig and can be administered to the pig at any time after diagnosis and the agent may be administered to a subject as previously described May be administered with other drugs or therapies useful for the treatment or as a single therapeutic agent.

The antibody preparation of the present invention is preferably orally administered.

The antibody preparations according to the present invention can treat and / or prevent porcine exhaustion infections that frequently occur in pigs and cause mortality and delayed growth.

Furthermore, the present invention can prevent swine-consuming diseases at the same time, thereby contributing to reduction of cost of swine farms and improvement of productivity.

In particular, the antibody preparation of the present invention can stably produce various kinds of antibodies without inducing immunological interference between the inoculated antigens, thereby effectively preventing pig consuming diseases.

In addition, a higher-valency antibody that is superior to the existing antibody preparation can be obtained.

Brief Description of the Drawings Fig. 1 is a diagram showing constituents and strains of an antigen for inoculation of a dairy cow of the present invention. Fig.
FIG. 2 is a graph showing the results of measuring the antibody titer of each disease using colostrum of a cow in which pig disease pathogen antigens (10 species) were repeatedly inoculated and unvaccinated cows in the present invention.
FIG. 3 is a graph showing the results of investigating the effect of a colostrum-immunizing antibody preparation administered to pigs suffering from severe diarrhea and respiratory diseases of pigs.
Figure 4 shows the results of Western blot analysis of recombinant capsid protein of porcine circovirus.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

The antibody preparation for the prevention of swine exhaustion disease of the present invention was prepared using colostrum obtained after inoculation of an antigen for inoculation of a dairy cow as follows into a pregnant cow.

The pig viruses (TGEV, PEDV, RotaV) were inactivated after being cultured in a suitable cell line for the representative vaccine strain or the outdoor strain virus.

Bacteria such as E. coli , S. typhimurium , M. hyopneumoniae , H. parasuis , P. mutocida , and S. suis were inactivated after culture and centrifuged. In the case of PCV2, Protein) was expressed using a bequiro virus expression system, and then the culture broth and cells were disrupted and inactivated.

The antigens were mixed and diluted at an appropriate concentration, and the mixture was emulsified with an aluminum hydroxide gel and an oil adjuvant (ISA25) as an excipient. The animals were highly immunized by inoculating the pregnant cows three times at three week intervals from two months before delivery.

The final colostrum immunoassay was prepared from the immunized dairy cows by mixing with the excipient after powder drying, and the colostrum was prepared by enzyme immunoassay (ELISA) The activity of the immune antibody contained was measured.

The immuno-antibodies showed high antibody titer, indicating that each antibody to the disease-causing antigens had a P value of at least 3.5 (phosphorylated serum) / N (negative serum).

To evaluate the efficacy of the colostrum immunoassay, four pigs with severe digestive and respiratory diseases were treated with appropriate dosing and dosing according to the age of the pig and the stage of feeding, . As a result of the administration of this product to pigs with severe diarrhea symptoms due to the combined infection of viruses and bacteria in piglets, the survival rate and the clinical symptom relief effect of diarrhea were confirmed, and the inhibitory effect of symptoms on bacterial diarrhea was also observed. The effect of reducing the symptoms was also confirmed when applied to farms with severe respiratory symptoms.

Example 1: Cultivation of major disease causative agents

1. Pig infectious gastroenteritis (TGE) virus culture

Swine testes (ST) cells were cultured in α-Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum (FBS) and antibiotics, and the cell density was about 70-80% monolayer Was inoculated with 1 MOI (inoculated amount / number of cells) of TGE virus vaccine strain (Pyeongtaek Province (KVCC-VR0000190)) after washing three times with PBS. The inoculated cells were shaken slowly at 37 ° C for 60 min. After the virus was adsorbed, the inoculum was discarded and α-MEM supplemented with trypsin (10 ug / ml) and antibiotics was added. When the cell denaturation effect (CPE) of the inoculated virus was about 80%, the cells were frozen and thawed three times at -70 ° C and room temperature, and then the recovered culture supernatant was stored at -70 ° C.

2. Culture of PED epidemic diarrhea (PED) virus

African monkey kidney Vero cells were cultured in D-MEM supplemented with 5% FBS and antibiotics, washed three times with PBS at a cell density of about 70-80% monolayer, and then cultured in PED virus vaccine strain (SM98 strain KVCC-VR1300037)) was inoculated with 1 MOI. The inoculated cells were shaken slowly at 37 ° C for 60 min. After the virus was adsorbed, the inoculum was discarded and D-MEM supplemented with trypsin (10 ug / ml) and antibiotics was added. When the cell denaturation effect (CPE) of the inoculated virus was about 80%, the cells were frozen and thawed three times at -70 ° C and room temperature, and then the recovered culture supernatant was stored at -70 ° C.

3. Swine Rotavirus ( RotaV ) Culture

To isolate RotaV from pigs, R. monkey kidney (TF104) cells were cultured in a roll tube to about 70-80% monolayer with 5% FBS and α-MEM supplemented with antibiotics. The supernatant was filtered through a 0.45-μm filter, and typsin (10 ug / ml) was added to the supernatant. The supernatant was filtered through a 0.45-μm filter, Lt; 0 > C for 60 minutes to make an inoculum. The inoculated solution was added to the cells washed three times with PBS, and the virus was adsorbed while slowly shaking at 37 ° C for 60 minutes. The inoculum was discarded and washed once with PBS. After immunization with trypsin (2.5 ug / ml) and α-MEM supplemented with antibiotics, cytotoxic effect (CPE) was observed inoculated. Fluorescent antibody test confirmed the presence of RotaV. The confirmed RotaV was subjected to G typing using the VP7 gene coding for viral glycoprotein, and the finally identified RotaV was purified by plaque assay and stored at -70 ° C. In the present invention, G4, G5, and G9 RotaV, which are the most frequently occurring in domestic pig farms, were isolated and mass-cultured according to the above method.

Detection and characterization of pig rotavirus in domestic pig farm G type Number of farms Separator Homology (%) nucleotide amino acid G2 2 5 81.3-85.5 86.1 to 89.8 G3 One 2 - - G4 7 18 83.2-95.6 89.0-96.8 G5 11 29 82.5 to 99.8 89.0-100 G9 6 16 92.5 to 99.7 94.3 to 99.6 G11 One 6 - -

4. Pig swine virus ( PCV2 ) Production and culture of recombinant virus

Since PCV2 is difficult to isolate and proliferate, capsid protein, a major defense antibody production protein, was constructed using baculovirus expression system. In other words, the ORF2 gene coding for the capsid protein in the gene for outdoor isolation was cloned and inserted into baculovirus transfer vector pFastBacHT (Invitrogen), transformed into Bacmid (DH 10BAc) E. coli containing baculovirus DNA, and the recombinant baculovirus DNA Were transfected into insect cells (Sf9). After the purified recombinant baculovirus was purified by a plaque assay, cell culture was performed to confirm the expression of the recombinant capsid protein by ELISA and western blot. As a result, a recombinant capsid protein of about 28 kDa was identified as shown in FIG. The final confirmed recombinant baculovirus was inoculated into Sf9 cells at 10 MOI. After 3 days, the infected cells were collected, frozen at -70 ° C and 3 times at room temperature, and stored at -20 ° C after ultrasonication.

5. Porcine E. coli ( E. coli ) Culture

In order to cultivate pathogenic E. coli in pigs, LT, ST, and Hly isolates were cultured according to islet model according to the four major species (K88, K99, 987P and F41) and toxin-producing strains. The strain was inoculated into a cottonseed blood culture medium, cultured at 37 ° C for 20 hours, and then a typical single shot was firstly cultured in a BHI (Brain heart infusion) liquid medium. In this culture, BHI liquid medium was inoculated with 2% of the primary culture solution, cultured at 37 ° C for 20 hours with shaking at 100 rpm, and then 0.3% formalin was added thereto to inactivate the cells and centrifuged to collect the cells.

6. Pig Salmonella ( S. typhimurium ) Culture

In order to cultivate S. typhimurium isolated from pigs, seeds were inoculated into a cotton blood culture medium, cultured at 37 ° C for 20 hours, and a typical single colony was cultured in a BHI (Brain heart infusion) liquid culture medium. In this culture, BHI liquid medium was inoculated with 2% of the primary culture solution, cultured at 37 ° C for 20 hours with shaking at 100 rpm, and then 0.3% formalin was added thereto to inactivate the cells and centrifuged to collect the cells.

7. Swine pneumococcus ( M. hyopneuminiae ) Culture

A M. hyopneuminiae bacteria isolated from pig lungs infected with porcine epidemic pneumonia, yeast extract (0.05%), Lactalbumin hydrolysate (0.0125%), BHI broth (0.5%), 10X HBSS (0.3%), 0.2% Phenol (0.137% ) Were added to Mycoplasma broth for 5-7 days. Then, the cells were inactivated by addition of 0.3% formalin and centrifuged at 7,000 rpm for 50 minutes.

8. Pig Pasteurela ( P. mutocida ) Culture

In order to cultivate P. mutocida (A type) isolated from pigs, inoculated on a cotton blood culture medium and incubated for 20 hours at 37 ° C., a typical single colony was first cultured in a BHI (Brain heart infusion) liquid medium. This culture was prepared by inoculating 2% BHI liquid medium with 2% inoculum and incubating at 37 ° C with shaking at 200 rpm for 20 hours. The cells were inactivated by adding 0.3% formalin and centrifuged to collect cells.

9. Glucocorticoid ( H. parasuis ) Culture

After incubation at 37 ° C for 20 hours, a typical single colony was inoculated with β-NAD (40 ug / ml) to cultivate H. parasuis isolated from pigs. Primary culture was added to the added BHI (Brain heart infusion) liquid medium. The culture was performed by inoculating the BHI liquid medium supplemented with β-NAD (40 ug / ml) with 2% of the primary culture solution and incubating at 37 ° C. with shaking at 100 rpm for 20 hours. The cells were inactivated by adding 0.3% formalin and centrifuged Cells were collected.

10. Porcine streptococcus ( S. suis ) Culture

In order to cultivate S. suis isolated from pigs, seeds were inoculated into a cotton blood culture medium, cultured at 37 ° C for 20 hours, and a typical single elucidation was performed in a BHI (Brain heart infusion) liquid culture medium. This culture was prepared by inoculating 2% BHI liquid medium with 2% inoculum and incubating at 37 ° C with shaking at 200 rpm for 20 hours. The cells were inactivated by adding 0.3% formalin and centrifuged to collect cells.

< Example  2> Colostrum antibody  Production inoculation antigen production

Virus antigens (10 ⁷ TCID ₅₀ / ml or more) such as TGEV, PEDV, and RotaV were concentrated to 1/5 of the original culture volume using a Pellicon filter (5K), and 0.1 M BEI (Bromoethylamide hydrobromide) The virus was inactivated by adding 10%. In addition, PCV2 capsid protein (supernatant of cell supernatant) prepared using baculovirus expression system was inactivated by the same method and diluted and suspended to OD value of 0.5 by Spectrophotometer (410 nm). E. coli , S. typhimurium , M. hyopneuminiae , H. parasuis, P. mutocida , S. suis Were inactivated by the spectrophotometer (410 nm) to an OD value of 1.0. Each of the antigens was mixed to make a uniform suspension. Aluminum hydroxide gel and an oil adjuvant (ISA25) were added at 10% and 5% of the total suspension and emulsified in a homogenizer at 1,000 rpm for 30 minutes.

< Example  3> Colostrum antibody  Production antigen vaccination

The cow colostrum immunogen production antigen prepared in 2 above was inoculated into 2 ml of the buttock muscle per 3 times every 3 weeks from 2 months before delivery. For the control colostrum production, PBS was inoculated in place of the antigen in the same manner.

< Example  4> Colostrum Collection and Immune Antibody Production

The colostrum within 30 hours of cow cows inoculated with the antigen for producing the colostrum immunizing antibody 3 was collected in a sterilized container and spray-dried. Collected colostrum was powder dried using a spray drier. For the piglets, colostrum powder was supplemented with 2 mg of paracooligosaccharide (2%), glucose (5%), glycine (1%), KCl (0.1%), sodium bicarbonate (1%), and Sobitol (0.2%) were added to the powder. The dry preparation was mixed with water and diluted with water to prepare a preparation for oral administration to the piglets. The pigs for the weaning pigs and breeding pigs were prepared in the same manner as the piglets for pigs and the dose was adjusted according to the body weight.

< Example  5> Collect Colostrum Potency  Measure

The colostrum (including the control group) produced in Example 4 was used to measure the immune antibody titer for each pathogen. E. coli and S. typhimurium were subjected to coagulation and enzyme immunoassay (ELISA) using cell antigens, and ELISA was performed for other pathogens. The ELISA antigens were centrifuged at 25,000 rpm for 3 hours, and the virus pellet was suspended in PBS and subjected to further deep ultrasound sonication. After culturing, each microorganism was subjected to ultrasonic treatment to purify OMP Respectively. ELISA antigen concentration for TGE, PED, RV and PCVD viruses was adjusted to 200ng / ml and other bacterial pathogens were coated at 300ng / ml. The concentration of the aggregation reaction antigen was adjusted to a OD value of 1.5 at a wavelength of 410 nm using a spectrophotometer, and then used as an antigen for flocculation. 1 g of the spray-dried colostrum powder was diluted with 10 ml of distilled water, sonicated, and centrifuged. The supernatant was diluted 10-fold in each of the coated plates and dispensed. After 1 hour of reaction, the supernatant was washed and then washed with anti-bovine IgG Was added at a concentration of 20 ng / ml. After 1 hour of reaction, the cells were washed and the TMB substrate was dispensed. After about 20 minutes, the stop solution was added thereto, and the absorbance at 450 nm was measured. The positive / negative value (P / N value) of each sample was calculated by inversely multiplying the dilution factor showing an OD value twice or more than the OD value of the negative sample.

Table 4 below shows the results of measuring the antibody titer of colostrum of cows vaccinated with a pathogen antigen and unvaccinated cows to each disease according to the present invention. The antioxidant activity of the TGE and PED viruses was 5,120 to 10,240 times, the RV virus was 10,240 times, and the PCV2 was 2,560 to 5,120 times. . E. coli The ELISA of the standard and outdoors isolates was measured from 5,120 to 10,240 times that of the untreated 20 times and the agglutination titers ranged from 640 to 2,560 times that of the untreated 20 times. The activity of S. typhimurium was measured from 5,120 to 10,240 times and the activity of the coagulation reaction from 320 to 1,280 times. For the other bacterial diseases, the inoculated right antibody titer was 5,120 to 10,240 times.

Ant colony Antibody titers of cows and unvaccinated cows Disease name Antibody titer Non-inoculated colostrum Inoculation colostrum 1 Inoculation colostrum 2 Inoculation colostrum 3 ELISA Cohesion ELISA Cohesion ELISA Cohesion ELISA Cohesion TGE 10 5,120 10,240 10,240 PED 10 5,120 10,240 10,240 RotaV 10 10,240 10,240 10,240 PCV2 10 2,560 5,120 2,560 E. coli (Standard Note) 20 20 5,120 640 10,240 1,280 10,240 640 E. coli (Separator) 20 20 5,120 640 10,240 2,560 10,240 640 S. typhimurium 10 10 5,120 320 10,240 1,280 10,240 640 M. hyopneuminiae 10 5,120 10,240 5,120 H. parasuis 10 5,120 5,120 5,120 P. multocida 10 5,120 10,240 5,120 S. suis 40 5,120 10,240 5,120

< Example  5> Colostrum antibody preparation  Efficacy test using

One. Colostrum antibody preparation  Used Piglet  Diarrhea treatment and prevention effect (A farm)

The efficacy of colostrum-immunized antibody was evaluated in an integrated breeding farm in which the severity of diarrhea due to 650 pig sows was severe. Diarrhea and autopsy biopsy of the piglets of this farm showed that PEDV was superimposed and RotaV and pathogenic E. coli were complex infected. In order to confirm the effect of colostrum immunosuppressive drug on the 1 day old newborn piglets, 2 ml of colostrum preparation was directly administered for 2 days to 1 day old piglets at 1 day of age for each day of labor, and in the untreated group, Were administered in the same manner and the number of pigs survived for one week. As shown in Table 5, the survival rate was 94.1% in a total of 576 dogs, 543 dogs survived after 1 week, and survival rate was found to be 17.5% in 86 dogs of 492 dogs. Therefore, as a result of the colostrum feeding test on the farm where the PEDV, RotaV and pathogenic E. coli were infected in the newborn piglet interval, the piglets survival rate was improved about 76% for one week. The effect was analyzed to be very.

Survival rate after colostrum immunizing antibody administration to newborn piglet (1 day old) of pig infected diarrhea, pig rotavirus and pathogenic Escherichia coli complex infected farm division Head One week survival number Survival rate (%) Treatment group ^{1 )} 576 542 94.1 Untreated group ^{2 )} 492 86 17.5

 1) 2 ml of colostrum preparation twice a day for 2 days orally

2) Oral administration of 2 ml of saline twice daily for 2 days

In addition, colostrum preparation was prepared in liquid form for one week old pigs on the same farm so that they could be consumed on an average of 20 ml per one day. The survival rate was measured for 2 days once a day and for up to 21 to 28 days. As shown in Table 6, the survival rate was 88.8% in a total of 986 groups, and the survival rate was 36.5% in a total of 855 groups. Therefore, as a result of the colostrum administration test on the farm in which the PEDV, RotaV and pathogenic E. coli were infected in the poultry section, the survival rate of the pigs was about 52% for the reason, so that this product was used for the treatment and prevention of diarrhea The effect was analyzed to be very good.

Survival rate after the administration of colostrum immunoassay for the piglets (1 week old) in pig infected diarrhea, pig rotavirus, and pathogenic Escherichia coli combined farm division Head Survival head Survival rate (%) Treatment group ^{1 )} 986 876 88.8 Untreated group ^{2 )} 855 312 36.5

 1) Oral administration of colostrum preparation 20ml twice a day for 2 days

2) Oral administration of saline 20ml twice a day for 2 days

2. Colostrum antibody preparation  Used Piglet  Diarrhea treatment and prevention effect (B farm)

The efficacy of colostrum immunotherapy was evaluated in an integrated breeding farm which suffered from the growth congestion of weanling pigs and weaned pigs due to diarrhea of 350 size sows and weaned pigs. As a result of diarrhea feces and autopsy biopsy of the piglets of this farm, RotaV and pathogenic E. coli were complex infection. First, the colostrum preparation was prepared in liquid form for 10 days old piglets so that the average amount of colostrum immunizing preparation could be taken 20 ml per one day. The colostrum preparation was supplied for 2 days once a day, and survival rate was measured until 24 days. As shown in Table 7 below, the survival rate was 91.8% in 382 patients in the total of 416 patients, and survival rate was 56.8% in 196 patients in 345 patients. Therefore, as a result of the colostrum administration test on the farm where the RotaV and the pathogenic E. coli were infected in the section of the mammalian pigs, the survival rate of the pigs was increased by about 35% for the reason, so that this agent is effective for the treatment and prevention of diarrhea It was analyzed to be very.

Survival rate after the administration of colostrum immunizing antibody against the piglets (10 days old) of pig rotavirus and pathogenic Escherichia coli complex infected farm division Head Survival head Survival rate (%) Treatment group ^{1 )} 416 382 91.8 Untreated group ^{2 )} 345 196 56.8

 1) Oral administration of colostrum preparation 20ml twice a day for 2 days

2) Oral administration of saline 20ml twice a day for 2 days

In addition, the suppository formulations were prepared in liquid form so that the average intake of 30 ml per one 4-week-old pigs of the same farm was fed for 2 days once a day, and the presence of diarrhea symptoms was examined 10 days after administration. As shown in Table 6, in the total of 315 dogs, the incidence of diarrhea was 19% and the incidence of symptoms was 6%. In the total of 230 dogs, 189 dogs continued to have diarrhea and 82.2% . Therefore, as a result of the colostrum administration test on the farm in which RotaV and pathogenic E. coli were infected in the weedy pig section, the symptoms of diarrhea were reduced by 76.2%, so that this formulation was effective in the treatment and prevention of diarrhea Respectively.

The incidence of clinical symptoms after the administration of colostrum immunoassay for 4 weeks of weaning pig infested with pig rotavirus and pathogenic Escherichia coli division Head Number of diarrhea ^{3 )} Expression rate (%) Treatment group ^{1 )} 315 19 6.0 Untreated group ^{2 )} 230 189 82.2

 1) Oral administration of colostrum preparation 20ml twice a day for 2 days

 2) Oral administration of saline 20ml twice a day for 2 days

3) Individuals with diarrhea symptoms after 10 days of administration

3. Colostrum antibody preparation  Used Upbringing  Diarrhea treatment and prevention effect (C farm)

Pathogenic E. coli And S. typhimurium The efficacy of the colostrum immunoassay was evaluated in an integrated breeding farm with diarrhea caused by infection. In other words, the colostrum preparation was prepared as a liquid form so that the average amount of 40 ml of the colostrum immuno antibody preparation per 10 weeks of breeding pigs could be ingested. The colostrum preparation was supplied for 2 days once a day, and the presence of diarrhea symptom was examined 10 days after the administration. As shown in Table 9 below, the symptom occurrence rate was 8.3% in a total of 180 dogs with 15 diarrhea patients, and 72 dogs in the non-treated dogs with 175 dogs showed diarrhea symptoms with a symptom occurrence rate of 41.1% . Therefore, pathogenic E. coli And S. typhimurium , the results showed that diarrhea was reduced by about 32.8%, indicating that this formulation is effective in the treatment and prevention of diarrhea in breeding stock.

The incidence of clinical symptoms after administration of colostrum immunoassay for growth donors (10 weeks old) of pathogenic Escherichia coli and salmonella complex infected farm division Head Number of diarrhea ^{3 )} Expression rate (%) Treatment group ^{1 )} 180 15 8.3 Untreated group ^{2 )} 175 72 41.1

 1) Oral preparation for 2 days twice a day in 40ml of colostrum preparation

 2) Oral administration of saline solution 40ml twice a day for 2 days

3) Individuals with diarrhea symptoms after 10 days of administration

4. Colostrum antibody preparation  Used Piglet  Respiratory treatment and preventive effect (D farm)

The efficacy of colostrum immunotherapy was evaluated in an integrated breeding farm with respiratory symptoms such as cough and abdominal respiration in weaned piglets. In other words, in order to confirm the suppressive effect of the colostrum immunizing agent on the 1 st day old newborn piglets, 2 ml of the colostrum preparation was directly orally administered to the 1 day old piglets twice a day for 2 days, The physiological saline was administered by the same method and the presence of respiratory symptoms in pigs (26 days old) was examined. As shown in Table 10 below, a total of 580 animals were found to have respiratory symptom manifestation rate of 2.1% in 12 animals, and in a total of 410 non-treated animals, 72 animals continued to have cough and abdominal respiratory symptoms And 17.6% respectively. Therefore, as a result of the colostrum administration test on the farm where the respiratory viruses and bacteria were infected in the piglets section, respiratory symptoms were reduced by about 15.5%, indicating that this product was effective in the treatment and prevention of respiratory diseases of piglets .

The incidence of clinical symptoms after the administration of the colostrum antibody preparation to the newborn piglets (1 day old) of the farms with respiratory disease in the weaned piglets division Head Respiratory symptom manifestation ^{3 )} Expression rate (%) Treatment group ^{1 )} 580 12 2.1 Untreated group ^{2 )} 410 72 17.6

1) 2 ml of colostrum preparation twice a day for 2 days orally

2) Oral administration of 2 ml of saline twice daily for 2 days

3) Weaning (26 days) Individuals with respiratory symptoms

Claims (17)

A cow colostrum antibody obtained by inoculating a cow with a porcine circulating diarrhea antigen, a porcine infectious gastrointestinal antigen, a porcine rotavirus infection antigen, a porcine circovirus infection antigen, a porcine colonic antigens antigen and a porcine salmonellosis antigen.
An antiviral composition for the treatment or prevention of swine diarrhea, swine infectious gastroenteritis, swine rotavirus infection, swine circovirus infection, swine colicosis and swine-wasting disease of swine salmonellosis. The composition according to claim 1, wherein the composition comprises a cow colostrum antibody obtained by inoculating a cow with one or more antigens selected from the group consisting of a swine pneumococcal antigen, a Glucocorticoid antigen, a porcine pustular enteritis antigen and a porcine streptococcal infection antigen Further,
Wherein the swine-wasting disease further comprises any one or more diseases selected from the group consisting of swine pneumonia, Glazes disease, swine fever disease, and swine streptococcal infection. delete 3. The antibody composition according to claim 1 or 2, wherein the cow colostrum immunoglobulin antibody has a P value (P value> 3.5) / N (negative serum value) of each antibody against the disease. The antibody composition of claim 1, wherein the antigen of swine epidemic diarrhea is derived from vaccine strain SM98 strain. The antibody composition according to claim 1, wherein the antigen of the porcine rotavirus infection is derived from any one or more isolates selected from the group consisting of isolates G4, G5 and G9. The antibody composition according to claim 1, wherein the antigen of the swine infectious gastroenteritis is derived from a vaccine strain Pyeongtaek. 8. The antibody composition according to any one of claims 5 to 7, wherein the vaccine strain or isolate is inactivated with bromoethylamide hydrobromide after concentration to produce an antigen. The antibody composition according to claim 1, wherein the antigen of the porcine circovirus infection is derived from porcine circovirus type 2. 10. The antibody composition according to claim 9, wherein the antigen of the porcine circovirus infection uses a capsid protein of a circovirus. 11. The antibody composition of claim 10, wherein the capsid protein is inactivated with bromoethylamide hydrobromide to produce an antigen. 3. The antibody composition according to claim 2, wherein the porcine parasitolaginous antigen is derived from Pasteurella multocida type A. 13. The antibody composition of claim 12, wherein the antigen is a bacterium inactivated with formalin. The antibody composition according to claim 1 or 2, wherein the cow colostrum immunoglobulin antibody is produced by administering an antigen and an excipient to a cow. 15. The antibody composition of claim 14, wherein the excipient is a mixture of an aluminum hydroxide gel and an immunostimulant. 3. The pharmaceutical composition according to claim 1 or 2, wherein the antibody composition further comprises one or more excipients selected from the group consisting of paclitooligosaccharide, glucose, glycine, KCl, ascorbic acid, sodium bicarbonate and Sobitol Lt; / RTI &gt; antibody composition. 3. The antibody composition according to claim 1 or 2, wherein the swine-wasting disease comprises swine diarrhea and respiratory disease.

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