KR20090065984A - Porcine disease of antigen manufacturing process and immune antibody production and composition for add electrolyte prevent disease - Google Patents

Abstract

A method for producing an antigen of porcine disease which is used in preventing and treating infectious disease, the main cause of porcine digestive tract disease, is provided to obtain a composition for preventing porcine diseases, shorten the use of antibiotics during raising pig and reduce the infection of piglet. A method for producing an antigen for immunity comprises: a step of mixing each infection medium of transmissible gastroenteritis virus of pigs, porcine epidemic diarrhea virus, rotarvirus, circovirus, escherichia coli and salmonellae with 30% of antigen and 70% of oil adjevant(Montnide ISA70); and a step of stirring with a homogenizer. A composition for preventing and treating transmissible gastroenteritis virus of pigs, porcine epidemic diarrhea virus, rotarvirus, circovirus, escherichia coli and salmonellae comprises powder containing immune antibody and electrolytic agent. A method for producing an immune yolk of powder type comprises: a step of inoculating the antigen of transmissible gastroenteritis virus of pigs, porcine epidemic diarrhea virus, rotarvirus, circovirus, escherichia coli and salmonellae into a hen; a step of collecting egg yolk which having immune antibody against the antigen; and a step of drying the moisture of egg yolk through spray drying and freeze-drying to powderize the yolk. The electrolytic agent contains 62.5g of sodium bicarbonate](NaHCO3), 37.5g of potassium chloride(KCl), 100g of glycine, 500g of glucose or dextrose, and 50g of antipyretic.

Description

Porcine disease of antigen manufacturing process and immune antibody production and composition for add electrolyte prevent disease}

The present invention relates to a method for producing an antigen of swine diseases, a composition for the production of immune yolk antibodies (IgY), and a control for swine diseases to which an electrolyte is added.

Specific IgG obtained from the sera of immunized animals has been used extensively in the diagnostic and pure science fields.

Another major field where IgG can be used is passive immunization. Indirect immunity refers to the production of specific antibodies against pathogens in other animals and the administration of these antibodies to the target animal to prevent disease. Indirect immunity has been shown to be effective in preventing infectious diseases through several studies.

IgG in egg yolk is called IgY (Yolk immunization).

Recently, a specific infectious pathogen of the human body is used to produce immune antibodies using animals, and these antibodies are added to various foods and used to treat or prevent diseases.

To protect against various diseases in humans or animals, immune function is exerted by the recovery of one pathogen after infection. In addition, to prevent immunity and to immunize with a specific pathogen to prevent immunity, it is possible to obtain immunity to obtain immunity by injecting an immune substance, which is usually made or externally, into the body.

In general, in pigs, the use of vaccines and antibiotics can reduce disease damage.However, due to population breeding, young piglets are exposed to pathogens and resistant to disease, resulting in a combination of many diseases that result in significant damage. Not only is it expected, especially in some young piglets, the lack of maternal transitional antibodies results in weak resistance to disease, making it difficult to defend against various diseases.

The diseases currently in question in Korea include swine infectious gastroenteritis virus (TGE), swine pancreatic diarrheal virus (PEDV), rotavirus (PRV), circovirus (PCV) and E. coli and Salmonella infections. Diarrhea is a strong gastrointestinal disease, and a combination of high mortality in infections may require new levels of prevention and treatment.

The present invention is to prepare each antigen of the disease causing agent that is causing great damage to the pigs of young age, and mix the oil adjuvant, and periodically inoculates the prepared immune agent into the laying hens, to obtain egg yolk with an immune antibody against the antigen in the laying hens The product manufactured by this product enhances the defense against diseases, thereby increasing the resistance to diseases without using antibiotics used for temporary treatment purposes, and contributes greatly to the income raising of pig farms by providing excellent safety and stability for the target animal. It is a good compositional material that can solve the problem of residual antibiotics on livestock products.

The present invention relates to a composition material for the prevention and treatment of swine infectious gastroenteritis virus, swine epidemic diarrheal virus, rotavirus, circovirus, E. coli, Salmonella infection.

More specifically, the present invention is a substance that can be used for the purpose of preventing and treating diarrheal infection, which is a major cause of digestive diseases that occur in young piglets at the same time, as a cause of the disease causing enormous damage to the pig industry. same.

This disease, called swine transmissible gastroenteritis virus (TGEV), is a diarrheal disease caused by the corona virus. It is mainly characterized by causing diarrhea, and severe diarrhea and rapid dehydration at the same time, the weight is reduced, most of the pigs within 7 days of age are dead, pigs suddenly develop foster diarrhea, the incidence is close to 100% The mortality rate is low.

② Porcine epidemic diarrhea virus (PEDV) infectious disease causes diarrhea mainly in piglets of young age, and mortality rate in Korea is nearly 100%. It also causes diarrhea in cultivated pigs and almost no mortality. Most of the symptoms are caused by vomiting, loss of appetite, lack of appetite, and recovery.

③ Porcine rotavirus infection (PRV) infection is characterized by causing severe foster diarrhea in piglets 1-4 weeks of age, most recovers, but causes mortality of 10-15%. The initial symptoms are dehydration after severe diarrhea such as depression, loss of appetite, avoiding exercise and vomiting.

④ Porcine circovirus (PCV) is reported for the first time in Korea in 1998. It is a disease that occurs mainly in weaning pigs and growing pigs due to the spread of disease nationwide. Weight loss, atrophy, dyspnea, pneumonia, diarrhea and neurological symptoms , Anemia and jaundice (liver damage), and immune function is lowered. In case of mixed infection, the disease helps to cause high mortality and atrophy systemic congestion syndrome. Also, it is easy to infect diarrhea or respiratory diseases. As a result, the disease spreads throughout the farm and reduces the therapeutic effect of antibiotics.

⑤ Escherichia coli ( Escherichia coli ) occurs mainly in young piglets 1 to 4 days of age, a lot of foster diarrhea and death in severe cases.

Weaning piglets are characterized by gray-white diarrhea. Infected piglets are repelled, atrophy, and in severe cases, dehydration and sepsis are accompanied by mortality.

⑥ Salmonella (Salmonellosis) increase is characterized by acute and chronic enterocolitis diarrhea on mortality is high acute sepsis and yiyujadon the grower an epidemic caused by infection with Salmonella spp as chief complaint, and often occurs even symptoms of pneumonia and meningitis.

  Sepsis is common in young piglets and suddenly dies, has high body temperature, is vigorous, and has pneumonia and neurological symptoms. Some pigs are infested with uncontrollable infections, and there are many difficulties in the prevention of pesticides.

Accordingly, the present inventors have studied to develop a substance for the purpose of preventing and treating pig infectious gastroenteritis virus, swine epidemic diarrhea virus, rotavirus, circovirus, E. coli and Salmonella infection, which are major diseases of pigs,

  It has been confirmed that the present invention can be effectively prevented and treated for the infection of the above diseases.

The main object of the present invention is to provide a composition capable of preventing and treating swine infectious gastroenteritis virus, swine epidemic diarrheal virus, rotavirus, circovirus, E. coli and Salmonella infection, which are the main diseases of swine.

Another object of the present invention is to provide a method for producing a composition as follows.

Another object of the present invention to provide a feed containing the following composition materials.

The present invention provides a composition material capable of simultaneously preventing and treating infections for swine infectious gastroenteritis virus, swine epidemic diarrheal virus, rotavirus, circovirus, Escherichia coli and Salmonella infection, which are major diseases of pigs,

As manufacturing a feed containing the composition substance,

Compositions capable of preventing and treating swine infectious gastroenteritis virus, swine pancreatic diarrheal virus, rotavirus, circovirus, Escherichia coli and Salmonella infectious disease, as a substance that can replace the use of antibiotics, will greatly contribute to the income increase of pig farmers. There is a number.

Hereinafter, the present invention will be described in detail by way of Examples.

1. Antigen Production of Major Disease Causes

end. pig Infectious gastroenteritis  virus( TGEV Culture

When the ST (Swine testicle) or SK (Swine kidney) cells were cultured in a cell growth medium (Supplementary Note 1) at 37 ° C, when the monolayer of cells formed 50%, the growth medium was removed and the TGE virus (175 L strain) contained. The cultured infection solution is harvested and used as an antigen when the denatured culture solution (Supplementary Note 2) is added and cultured at 37 ° C. for 2-3 days while the cytopathic effect caused by the inoculation virus is about 80% or more.

I. pig Pandemic diarrhea  virus( PEDV Culture

After culturing Vero (Green monkey kidney) cells in a cell growth medium at 37 ° C and forming a monolayer of cells, the culture medium for proliferation was removed and then inoculated with PED virus (SM98P strain) in a medium containing 10 µg / ml of crystallized trypsin. After sensitization at 37 ° C for 1 hour, a maintenance medium containing 0.5 µg of crystallized trypsin per ml was added to α-MEM containing no FBS. When about 80% or more occurs, the cultured infection solution is harvested and used as an antigen.

All. Porcine Rotavirus ( PRV Culture

After culturing MA104 (Green monkey kidney) cells at 37 ° C in a cell proliferation medium, when the monolayer of cells was formed, the culture medium for proliferation was removed and Rotavirus (10, A1 strain) was added to the medium to which 10 µg of crystallized trypsin was added. Were incubated at 37 ° C. for 1 hour, and then added to the culture medium containing 0.5 μg of crystallized trypsin per ml to α-MEM without FBS, followed by incubation at 37 ° C. for 24 to 48 hours. When cytopathic effect occurs about 80% or more, each culture infection solution is harvested and used as an antigen.

la. pig Circovirus ( PCV -2 type) culture

Hi-5 cells derived from insect cells were cultured at 27 ° C. in cell culture medium (Supplementary Note 3). When monolayers of cells were formed, the culture medium for proliferation was removed and 1 ml of the recombinant virus (PCV-2) was added to Grace's medium. Diluted in 50ml, inoculated 3ml each of circovirus into 175cm2 flask, sensitized at 27 ℃ for 1 hour, and then cultured for 5-7 days by adding culture solution (Supplementary Note 3). When more than% occurs, the cultured infectious solution and infected cells are harvested and centrifuged at 6,000 rpm for 20 minutes, and the infected cells are resuspended with an appropriate amount of PBS (Supplementary Note 4) and sonicated, and centrifuged at 8,000 rpm for 20 minutes. The supernatant is collected and inactivated before use as an antigen.

hemp. Escherichia coli ( E. coli Culture

Strains (k88ab, k88ac, k99, 987P, F41 strains) were transplanted to Sheep blood agar medium, respectively, and cultured at 37 ° C. for 16-20 hours each, and a typical single colony of E. coli was selected for 16-18 hours at Brain heart infusion broth. After incubation, 0.3% formalin is added to the culture infection solution, inactivated, and concentrated to use as antigen.

bar. Salmonella Salmonella Bacterial culture

The strain ( S. typhimurum strain) was transplanted into Sheep blood agar medium, incubated for 16-20 hours at 37 ° C, and a typical single colony of Salmonella was incubated for 15-18 hours in brain heart infusion broth, and then cultured in the infected solution. 0.3% formalin is added to inactivate the culture infection solution, and then concentrated and used as an antigen.

[Bookkeeping 1. Cell growth culture composition (1,000ml)]

α-MEM... … … … … … … … … … … … … … 930 ml

Fetal bovine serum (FBS)…. … … … … … 50 ml

7.5% NaHCO ₃ … … … … … … … … … … … … 20 ml

Penicillin G-Na... … … … … … … 100unit / ml

Streptomycin sulfate… … … … … 100 µg / ml

Fungizone… … … … … … … … … … 0.025 µg / ml

[Supplementary Supplement 2. Cell Maintenance Culture Ingredient Composition (1,000ml)]

α-MEM... … … … … … … … … … … … … … 950 ml

Fetal bovine serum (FBS)…. … … … … … 30 ml

7.5% NaHCO ₃ … … … … … … … … … … … … 20 ml

Penicillin G-Na... … … … … … … 100unit / ml

Streptomycin sulfate… … … … … 100 µg / ml

Fungizone… … … … … … … … … … 0.025 µg / ml

[Appendix 3. Hi-5 Cell Culture Composition (1,000ml)]

Grace's medium… … … … … … … … … … 900 ml

Fetal bovine serum (FBS)…. … … … … 100 ml

Penicillin G-Na... … … … … … … 100unit / ml

Streptomycin sulfate… … … … … 100 µg / ml

Fungizone… … … … … … … … … … 0.025 µg / ml

2. Production of Antigen for Immunogenesis

30% antigen and 70% of the oil adjuvant (Montnide ISA70) mixed with the infectious broths of swine infectious gastroenteritis virus, swine pandemic diarrheal virus, rotavirus, circovirus, Escherichia coli and Salmonella were respectively emulsified, and then a homogenizer was applied. Prepare an antigen for use immunization.

3. Antigen inoculation method

end. Inoculation time: 1st vaccination-19 ~ 21 weeks of age,

               3 weeks after the 2nd vaccination and 1st vaccination

I. Inoculation site: Forefoot or leg muscles.

All. Inoculation amount: 1.0 ml / water.

4. Powder Drying Process of Immune Antibodies

end. Spray Drying Method

Process of drying the moisture of egg yolk with immunity by spray method of disk and nozzle type by using spray dryer.

I. Freeze drying method

Rapidly freezing immunized egg yolk to -40 ℃ and drying the moisture of immune substance under freezer dryer's freezer temperature at -80 ℃ in a vacuum. .

All. Immune Yolk Antibodies  And Electrolyte  Furtherance

The mixing ratio of the powdered immune yolk antibody and electrolyte agent to effectively prevent and treat the disease infection of young piglets is 60 ~ 80% of the powdered yolk powder containing immune antibody and 20 ~ 40% of the electrolyte agent. Process of mixing composition in proportion.

5. Immune Antibody Extraction Process in Egg Yolk

In order to extract the antibody in the yolk with the antibody, the collected eggs are washed and sterilized, and only the yolk is separated and mixed, followed by mixing in a ratio of ionized water (pH 4.0) 5 and the yolk emulsion 1 containing the immuno-antibody. The supernatant was collected and stored at 4 ° C.), followed by centrifugation at 8,000 rpm for 5 minutes to mix the electrolyte preparation solution in a ratio of 4: 1.

6. Paste manufacturing process

Add 60-80% of the powdered immune yolk antibody at the rate of 20-40% of the electrolyte preparation, homogenize it into a slightly thick and smooth dough using a dough machine, and then add more than 30% of fructo-oligosaccharides. The process of manufacturing the paste.

7. Manufacturing process of electrolyte

The composition content of the electrolyte formulation is

62.5 g of sodium bicarbonate (NaHCO ₃ ),

  Potassium chloride (KCl) 37.5 g,

  100 g of glycine,

  500 g of Glucose or Dextrose,

  50 g of antipyretic agents, respectively.

8. Antibodies to Antigen Test

After three weeks of inoculation of pig infectious gastroenteritis virus, swine pandemic diarrheal virus, rotavirus, circovirus, Escherichia coli and Salmonella infection antigens into the laying hens, the eggs of immunized laying hens were obtained three weeks later. As measured by Enzyme-linked immunosorbent assay (ELISA),

The antibody titer of swine infectious gastroenteritis virus was 1,280 times or more, the swine epidemic diarrheal virus 1,280-2,560 times and the circo virus 1,280 times, and the rotavirus antibody value of 1,280 times or more, respectively.

Escherichia coli showed 2,560-fold and Salmonella infection showed 2,560-fold or higher antibody titer, respectively.

9. Efficacy test on piglets

end. In powder  Test

Efficacy test of the composition with electrolytes added to the immune yolk antibody was performed by selecting 40 pigs at 7 days of age, and 20 animals were fed three times per day for 5 days by mixing 0.5% (500 g per 100 kg of feed) with the composition. Mild diarrhea (4/20), dehydration (3/20) and shortness of breath (3/20) showed mild clinical symptoms and showed a faster recovery than the control group.

Twenty non-administered controls also showed complex clinical symptoms, including typical diarrhea (7/20), dehydration (6/20), and dyspnea (5/20) (Table 1).

[Table 1. Efficacy test for powder preparation]

 division  Piglets Clinical observation Watery diarrhea dehydration Dyspnea throw up Cardiac symptoms (depression) Test group 20 4/20 3/20 3/20 1/20 0/20 Control 10 7/20 6/20 5/20 1/20 1/20

I. Extracts  Test

The efficacy test of the composition of the yolk yolk extract with the addition of the electrolyte solution was selected. 40 day-old piglets were selected at 1 day of age, and 20 animals were fed twice with 5 ml of the composition and observed for 10 days. Mild diarrhea (3/20), dehydration (2/20), and shortness of breath (3/20) showed mild clinical symptoms and showed faster recovery than the control group.

Twenty non-administered controls also showed complex clinical symptoms, including typical diarrhea (7/20), dehydration (3/20), and dyspnea (5/20) (Table 2).

Table 2. Efficacy test for extract preparations

 division  Piglets Clinical observation Watery diarrhea dehydration Dyspnea throw up Cardiac symptoms (depression) Test group 20 3/20 2/20 3/20 0/20 1/20 Control 20 7/20 3/20 5/20 1/20 1/20

All. Test for Paste Formulations

To test the efficacy of paste formulations containing electrolytes and fructo oligosaccharides in the form of immunized yolk antibodies with immunity, 39 piglets at 1 day of age were selected. After 10 days of observation, the patient showed clinical symptoms such as diarrhea (4/19), dehydration (2/19) and dyspnea (2/19), and recovered faster than the control group.

Twenty non-administered controls also showed complex clinical symptoms, including typical diarrhea (6/20), dehydration (4/20), and dyspnea (5/20) (Table 3).

Table 3. Test for Paste Preparation

 division  Piglets Clinical observation Watery diarrhea dehydration Dyspnea throw up Cardiac symptoms (depression) Test group 19 4/19 2/19 2/19 0/19 1/19 Control 20 6/20 4/20 5/20 1/20 0/20

As described above, the present inventors have developed a substance capable of preventing and treating swine infectious gastroenteritis virus, swine epidemic diarrheal virus, rotavirus, circovirus, E. coli and Salmonella infection,

While conducting various studies focusing on the use of immune yolk antibodies specific for the above diseases,

A composition in which an electrolyte agent is added to an immune yolk antibody powder,

A composition in which an electrolyte is added to a specific antibody extract of egg yolk in which an immune antibody is formed,

The preparation of the paste prepared by adding the electrolyte preparation and the placto oligosaccharide to the immune yolk antibody powder can confirm the prophylactic and therapeutic effects in the efficacy test for the above diseases, and the piglets showing clinical symptoms in the test group showed a rapid recovery state. It was also confirmed that there is a formal effect.

Claims (8)

Swine infectious gastroenteritis virus (175L strain), swine pandemic diarrheal virus (SM98P strain), rotavirus (10, A1 strain), circovirus (PCV-2 type), coliform bacteremia (k88ab, k88ac, k99, 987P, F41 strain), Fungal solo of Salmonella ( S. typhimurum ) infection and A method for producing antigens for immunization of swine infectious gastroenteritis virus, swine epidemic diarrheal virus, rotavirus, circovirus, Escherichia coli and Salmonella inoculated in laying hens. The method of claim 1, A process for inoculating the laying hens with antigens of swine infectious gastroenteritis virus, swine pandemic diarrheal virus, rota virus, circovirus, Escherichia coli, Salmonella infection, and obtaining egg yolk of an egg with an immune antibody against the antigen from the inoculated laying hens. The method of claim 2, A step of drying the egg yolk of the egg, which has an immune antibody, by spray drying and freeze drying the water of the egg yolk into a powder. A process of preparing an electrolyte formulation and inoculating an antigen into a laying hen to mix and rate the composition with an immune yolk antibody having an antibody. The composition characterized by the mixing ratio of the immune yolk antibody in the form of a powder containing an immune antibody and an electrolyte preparation is used for the prevention and treatment of infectious gastroenteritis virus, swine epidemic diarrheal virus, rota virus, circovirus, E. coli and Salmonella infection in pigs. Formulations comprising the composition material. The composition characterized by the process of extracting the immune antibodies contained in egg yolk and the composition and mixing ratio of the electrolyte solution is used for the prevention and treatment of infectious gastroenteritis virus, swine epidemic diarrheal virus, rota virus, circovirus, Escherichia coli, Salmonella infection Formulations comprising a compositional substance as. Processes and paste preparations prepared by adding fructo oligosaccharides to powdery immune yolk antibodies and electrolyte preparations for the prevention and treatment of infectious gastroenteritis virus, swine epidemic diarrheal virus, rotavirus, circovirus, Escherichia coli, Salmonella infection Formulations comprising the composition material. A feed containing infectious gastroenteritis virus, swine epidemic diarrheal virus, rotavirus, circovirus, E. coli, and Salmonella infectious immune yolk antibody and electrolyte, comprising a composition for the prevention and treatment of diseases.