KR20090016236A - Bovine disease of antigen manufacturing process and immune antibody(igy, colostrum) production and composition for add electrolyte prevent diarrhea in calf - Google Patents

Abstract

A composition comprising an added electrolyte is provided to prevent bovine viral diarrhea, replace the antibiotics which have been used conventionally, and improve the safety and stability of use, thereby increasing income of the stockbreeding farmhouse. The method for producing an antigen of bovine disease comprises the steps of: inoculating the inactivated antigen of bovine virus diarrhea disease(NADL strain), rotarvirus(P44, 678 strain), coronavirus(BCV94 strain), Escherichia coli(K99 strain), Salmonella(S. enteritidis, S. typhimurum strain), and Pasteurella(P. multocida A, P. haemolytica strain) into the laying hen; collecting eggs in which the immune antibody against the antigen is formed from the vaccinated laying hen; obtaining the yolk from eggs and drying it; inoculating the inactivated antigen of bovine virus diarrhea disease(NADL strain), rotarvirus(P44, 678 strain), coronavirus(BCV94 strain), Escherichia coli(K99 strain), Salmonella(S. enteritidis, S. typhimurum strain), and Pasteurella(P. multocida A, P. haemolytica strain) into the milk cow; obtaining the first milk in which the immune antibody is formed and drying it; drying the obtained yolk and first milk; and obtaining the immune antibody of the yolk and first milk in a powder form.

Description

Bovine disease of antigen manufacturing process and immune antibody (IgY, colostrum) production and composition for add electrolyte prevent diarrhea in calf }

The present invention relates to a method for producing an antigen of bovine disease and the composition for the production of immune antibodies (egg yolk, colostrum) and control of calf diarrhea added with an electrolyte.

Humans or animals exert their immune function by recovering after infection with one pathogen to protect against various diseases. In addition, to prevent immunity, immunizations are made with specific pathogens for immunization, so getting immunity can be achieved by injecting an immune substance that is already made or externally into the body.

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

In general, the calf can reduce the damage caused by hygienic breeding and the use of vaccines. However, in the case of breeding the calves, they are exposed to various diseases. In particular, calf postpartum calves lack the maternal transitional antibodies, which makes them less resistant to disease.

Bovine diseases currently in question in Korea include bovine virus diarrheal disease (BVD), rotavirus (BRV), corona virus (BCV), and E. coli, Salmonella, Pasteurella infection, etc. These diseases are highly infectious digestive diseases As diarrhea is a symptom, and complex mortality is high in infections, new levels of prevention and treatment techniques are required.

The present invention is to prepare the antigens for each major cause of the disease that is causing great damage to the calf, and to mix the oil adjuvant, and periodically inoculates the prepared immune preparations in laying hens and cows, raising the immunity of the laying hens and cows to the antigen Products prepared by obtaining yolks and colostrums with immune antibodies when they are gone enhance their defense against diseases, thereby increasing their resistance to diseases without using antibiotics used for temporary treatment. It is excellent in safety and stability, which can contribute greatly to income raising of livestock farmers, and can solve the problem of residual antibiotics for livestock products.

The present invention relates to a composition for the prevention and treatment of bovine virus diarrhea, rotavirus, corona virus and E. coli, Salmonella, Pasteurella infection.

More specifically, the present invention is to include a method and composition of the composition as a composition that can be used for the purpose of preventing and treating infections for diarrhea, which is a major cause of livestock diseases.

Bovine virus diarrhea / Mucosal disease (BVD / MD) is a contagious disease of the mucous membranes of the digestive tract that causes ulcers, diarrhea and respiratory lesions. Young animals of 8 months of age are highly susceptible and usually develop in winter to spring. They are characterized by high fever, mucous, bloody diarrhea, leukopenia, decreased appetite, cough, shortness of breath, runny nose, and dehydration. It is a disease that affects all age groups, including death. Infection of cows in early pregnancy also causes abortion, surplus and stillbirth due to placenta, and newborn calves cause ataxia and weakness.

Bovine rotavirus (BRV) infection causes diarrhea when infected with newborn calves, and is one of the most damaging digestive diseases, mainly due to high mortality when infected with young calves less than one week of age. Falls.

In bovine coronavirus (BCV) infections, the flow rate decreases in severe cases, resulting in decreased feed efficiency and reduced beef gain. In young calves, the incidence is high and it is also a one-dimensional body of the pandemic pneumonia. The disease mainly occurs in the small intestine and colon, especially severe vigorous atrophy, fusion, and dropping of villi are observed, yellow watery diarrhea and severe dehydration due to malabsorption, weakness and lethargy. Is an important disease in calves so young that it is called neonatal calf diarrhea.

E. coli (Escherichia coli ) is caused by intestinal infection. In calf, diarrheal disease, atrophy and mortality are the main causes, and in calf and wean calves, the stress is caused by intensive group management.

Salmonella typhimurium and Salmonella enteritids are the causative agents of common infectious diseases that cause food poisoning in humans without host specificity. Is acute or chronic gastrointestinal infectious disease caused by Salmonella infection, mainly in calves, fever, enteritis and sepsis, and encephalitis, arthritis, lactic acid and mastitis.

Pasteurella pneumonia ( bovine) pneumonic pasteurellosis ) A disease caused by various stress factors such as long-distance transport, reasons, segregation , climate change, feed change, deterioration of specification conditions, and viral and bacterial infections, among which Pasteurella A bacterium called Hemolyticer ( pasteurella haemolytica ) is known to be the most important cause.

Therefore, the present inventors have studied to develop a substance capable of preventing and treating infectious diseases of bovine virus diarrhea, rota virus, corona virus, Escherichia coli and Salmonella and Pasteurella, which are the main diseases of livestock, It was confirmed that the present invention can be prevented and treated.

It is a main object of the present invention to provide a composition capable of preventing and treating infectious diseases of bovine virus diarrhea, rotavirus, corona virus, Escherichia coli and Salmonella and Pasteurella which are the main diseases of livestock.

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

Still another object of the present invention is to provide a feed containing the following composition.

The present invention provides a feed comprising a composition, a preparation method thereof, and an electrical composition, which can simultaneously and simultaneously prevent infection of bovine virus diarrhea, rota virus, corona virus, Escherichia coli, Salmonella, Pasteurella infection, which are major diseases of cattle. In doing

The virus diarrheal disease, rota virus, corona virus, E. coli, Salmonella, Pasteurella infectious disease prevention and treatment composition of the present invention can replace the antibiotics used in the past while ensuring safety and stability, It can greatly contribute to income growth.

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

1. Antigen Production Method for Major Disease Agents

end. small Virus diarrheal disease ( BVD Culture

MDBK (Bovine kidney) cells were cultured in a cell proliferation medium (Supplementary Note 1) at 37 ° C, and when the monolayer of cells formed 50%, the culture medium for proliferation was removed, and a virus (NADL strain) was added to the cell maintenance medium (Supplementary Note 2). Inoculate and incubate at 37 ℃ for 2 ~ 4 days when the cell degeneration effect by inoculation virus is about 80% or more, harvest it and use it as antigen.

I. small Rota  virus( BRV Culture

When MA104 (Green monkey kidney) cells were cultured in a cell proliferation medium (Supplementary Note 1) at 37 ° C., when a monolayer of cells was formed, the culture medium for proliferation was removed, and then the virus (P44, 678 weeks) were diluted and inoculated for 1 hour, and then sensitized for 1 hour, followed by adding a cell maintenance culture medium (Supplementary Note 2) with 0.5 µg of crystallized trypsin added to α-MEM containing no FBS. When the cell denaturation effect by the inoculated virus occurs over 80% during time incubation, it is harvested and used as an antigen.

All. Bovine coronavirus ( BCV Culture

MDBK (Bovine kidney) cells were cultured in a cell proliferation medium (Supplementary Note 1) at 37 ° C, and when a monolayer of cells was formed, the culture medium for proliferation was removed, and then a virus (BCV 94 strain) was inoculated into the cell maintenance medium (Supplementary Note 2). Incubate at 37 ℃ for 4 ~ 5 days when the cell degeneration effect by inoculation virus is about 80% or more and use it as an antigen.

la. Escherichia coli ( E. coli Culture

The strain (k99 strain) was transplanted into Sheep blood agar medium and cultured at 37 ° C. for 16-20 hours, and a typical single colony of E. coli was incubated in Brain heart infusion broth for 16-18 hours, followed by inactivation with 0.3% formalin. It is then concentrated and used as an antigen.

hemp. Salmonella Salmonella Bacterial culture

Strains ( S. enteritidis , S. typhimurum ) were transplanted to Sheep blood agar medium, and cultured at 37 ° C for 16-20 hours, and the typical single colonies of Salmonella were incubated for 15-18 hours in Brain heart infusion broth. After 0.3% formalin was added, the cultured infection solution was inactivated and concentrated to use as an antigen.

bar. Pasteurella Pasteurella Bacterial culture

Strains ( P. multocida A, P. haemolytica ) were transplanted to Sheep blood agar medium, and cultured at 37 ° C for 16-20 hours, and the typical single colonies of Pasteurella were selected for 16-20 hours in Brain heart infusion broth. After incubation with each other, 0.3% formalin was added to inactivate and concentrated to use as 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

2. Production of Antigen for Immunogenesis

end. Antigen for laying hens

Bovine virus diarrhea, Rota virus, corona virus and E. coli, Salmonella, Pasteurella antigen (30%), respectively, mixed to make an emulsion, and then mix the oil adjuvant (Montnide ISA70) at a rate of 70% and Homogenizer Emulsify and homogenize and save in 2 ~ 7 ℃ cold room.

I. Antigen for cow inoculation

Bovine virus diarrhea, Rota virus, Corona virus and E. coli, Salmonella, Pasteurellae antigen (85%) were mixed to make an emulsion, and then 5% Oil adjuvant (Montnide ISA25) and 10% aluminum hydroxide gel were added. Emulsify for 30 minutes at rpm, homogenize and store in 2 ~ 7 ℃ cold room.

3. Antigen inoculation method

end. Laying hen

(1) Inoculation time: 1st vaccination-20 ~ 21 weeks old after birth, 2nd vaccination-3 weeks after 1st vaccination, additional vaccination every 3 months

(2) Inoculation site: breast or leg muscle

(3) Inoculation amount: 1.0 ml / water

I. milk cow

(1) Inoculation time: 6 weeks before the first vaccination, 3 weeks before the second vaccination

(2) Inoculation site: muscle

(3) Inoculation amount: 3.0 ml / horse

4. Powder Drying of Immune Antibodies

end. Spray Drying Method

Immunity-forming material (50 ~ 90% of egg yolk, 10 ~ 50% of colostrum) is mixed well and moisture is dried by spray method in the form of disk and nozzle using a spray dryer. Drying to powder.

I. Freeze drying method

After immunization material (50 ~ 90% of egg yolk, 10 ~ 50% of colostrum) is well mixed and rapidly frozen to -40 ℃, the freeze dryer's freezer temperature is -80 ℃ and vacuum A process of drying the powder with a moisture content of less than 10%.

5. Egg yolk  Immune Antibody Extraction Manufacturing Process

In order to extract the immune antibodies in egg yolks, the collected eggs are washed and disinfected. Then, only egg yolks are separated and mixed, and then mixed in a ratio of ionized water (pH 4.0) 5 and egg yolk emulsion 1 containing immune antibodies in a cold room (4 ° C.). After the preservation, the supernatant was collected and centrifuged at 8,000 rpm for 5 minutes to prepare a mixture of the electrolyte solution at 4: 1 ratio.

The electrolyte solution that maximizes the preventive treatment effect of the disease is a mixture of 12.5 g of sodium bicarbonate, 7.5 g of Potassium chloride, 20 g of Glycine, 100 g of Glucose or Dextrose, and 10 g of antipyretic agent, and adds egg yolk extract and electrolyte solution. Manufacturing process.

6. Paste (Paste) manufacturing process

Add 60 ~ 80% of the powdered immune antibody (eg yolk, colostrum) at the rate of 20 ~ 40% of the electrolyte solution, and homogenize it into a slightly thick and smooth dough using a kneader, and then fructo-oligosaccharides A process of preparing a paste by adding at least 30%.

The electrolyte solution that maximizes the preventive effect of disease is mixed with a solution of 12.5 g of sodium bicarbonate, 7.5 g of Potassium chloride, 20 g of Glycine, 100 g of Glucose or Dextrose, and 10 g of antipyretic agent in 1 liter of distilled water. Manufacturing process.

7. Content of electrolyte preparation and immune antibody

Compositions that can prevent and treat the major diseases bovine virus diarrhea, rota virus, corona virus and E. coli, Salmonella, Pasteurella infection,

A composition comprising an electrolyte preparation mixture consisting of sodium bicarbonate, Potassium chloride, Glycine, Glucose (Dextrose), antipyretic, and immunized yolk antibodies against colitis, rotavirus, corona virus and E. coli, Salmonella, Pasteurella infection and colostrum When the calf is fed, it is confirmed that the disease can be effectively prevented or treated,

In addition, the mixing ratio of the immune antibody (egg yolk, colostrum) and the electrolyte preparation in powder form and the composition of the electrolyte preparation to maximize the prophylactic effect of these diseases is 62.5g of sodium bicarbonate, 37.5g of Potassium chloride, 100g of Glycine, Glucose Alternatively, 500 g of Dextrose and 50 g of antipyretic agents are mixed and mixed at a ratio of 60 to 80% in powder form (egg yolk and colostrum) containing immune antibodies and 20 to 40% of electrolyte.

8. Antibodies to Antigen Test

end. Egg yolk  Antibodies

The antibody titers contained in egg yolk were measured from chicken eggs immunized three weeks after bovine virus diarrheal disease, rota virus, corona virus and E. coli, Salmonella, Pasteurella infectious antigen secondary to laying hens.

Antibody value for bovine virus diarrheal disease was 640 ~ 1,280 times, Rota virus showed 640 ~ 2,560 times antibody value, and corona virus antibody value 640 ~ 1,280 times, respectively.

Escherichia coli showed 1,280 ~ 5,120 times, Salmonella 1,280 ~ 2,560 times, and Pasteurella showed 640 ~ 2,560 times antibody titer, respectively.

I. Antibodies to Colostrum

Bovine virus diarrheal disease, rota virus, corona virus and E. coli, Salmonella, Pasteurellae antigens were inoculated every three weeks, and then measured the antibody value in colostrum after delivery,

The antibody value for bovine virus diarrheal disease was 2,560 ~ 10,240 times, and the rotavirus showed 5,120 ~ 20,480 times the antibody value, and the corona virus also showed the antibody value 5,120 ~ 20,480 times.

E. coli 10,240 ~ 40,960 times, Salmonella 5,120 ~ 20,480 times, Pasteurella showed 5,120 ~ 40,960 times the antibody value.

9. Efficacy test

end. Test for Immune Antibody Powder

To determine whether it is possible to increase the effect of preventing and treating infections against bovine disease antigens in calves raised on farms, the composition of electrolytes added to yolk antibodies containing colostrum was tested. As a result of daily supplementation, it could be effectively prevented and treated, and showed clinical symptoms such as diarrhea (2/30), dehydration (1/30) and dyspnea (2/30). The calf showed a faster recovery than the control.

In the non-administrated control group, clinical symptoms such as watery diarrhea (8/30), dehydration (6/30), and dyspnea (8/30) were severely observed. Marie was able to confirm the death (Table 1).

[Table 1. Efficacy test on the immune antibody powder formulation]

division calf Salary Clinical observation for one month after delivery Watery diarrhea dehydration Dyspnea Atrophy and mortality survival Test group 30 10g two days 2/30 1/30 2/30 0/30 30/30 Control 30 Non-payment 8/30 6/30 8/30 * 2/30 28/30

* Mortality: Enteritis and Sepsis Complex Infection

I. Yolk extract preparation  Test

In order to verify that the composition of the yolk yolk extract with the electrolyte solution can be added to the calf, it is possible to increase the prevention and treatment of infections against the antigens of small disease. Three days of feeding resulted in mild clinical symptoms such as diarrhea (1/10) and dyspnea (2/10).

Ten non-administrated controls also showed clinical symptoms such as conventional diarrhea (4/10), dehydration (2/10) and dyspnea (3/10).

[Table 2. Efficacy test for yolk extract preparation]

division calf Salary Clinical observation for one month after delivery Watery diarrhea dehydration Dyspnea Atrophy and mortality survival Test group 10 30 ml 3 days 1/10 0/10 2/10 0/10 10/10 Control 10 Non-payment 4/10 2/10 3/10 0/10 10/10

All. In paste  Test

In order to confirm the effect of preventing and treating the infection of the calf's bovine disease, the paste preparation prepared by adding the electrolyte and adding fructo oligosaccharide to the immunized powder-type immune antibodies (eg yolk and colostrum) using kneading machine Twenty animals were fed 10g per head for three days before and after colostrum feeding for 10 days, and showed clinical symptoms such as foster diarrhea (2/10) and dyspnea (2/10). Showed the condition.

On the other hand, in the non-administrative control group, typical symptoms of diarrhea (4/10), dehydration (2/10), dyspnea (3/10), and slight atrophy (1/10) were found. )

Table 3. Efficacy test for paste preparation

division calf Salary Clinical observation for one month after delivery Watery diarrhea dehydration Dyspnea Atrophy and mortality survival Test group 10 10g three days 2/10 0/10 2/10 0/10 10/10 Control 5 Non-payment 4/10 2/10 3/10 1/10 10/10

As described above, the present inventors focus on a method using a yolk antibody specific for the above diseases in order to develop a substance capable of preventing and treating bovine virus diarrhea, rota virus, corona virus, E. coli, Salmonella, Pasteurella infection. In the course of various studies in accordance with the present invention, the composition of the addition of an electrolyte agent to egg yolk powder containing colostrum, the composition of the electrolyte addition to the specific antibody extract of egg yolk, and the preparation of a paste preparation to which fructo oligosaccharides were added The preventive and therapeutic effects on the disease could be achieved and the formal effects were also confirmed.

Claims (7)

Bovine virus diarrheal disease (NADL strain), rotavirus (P44 strain 678), coronavirus (BCV94 strain), Escherichia coli (k99 strain), Salmonella ( S. enteritidis , S. typhimurum strain), Pasteurella ( P. multocida A) , P. haemolytica Note) Immunization fungal strains, antigens, which are inoculated into laying hens and cows of diarrhea, rotavirus, coronavirus, E. coli, Salmonella, Pasteurella infection And A composition for the prevention and treatment of bovine virus diarrhea, rota virus, corona virus, Escherichia coli, Salmonella, Pasteurella infection characterized by the composition and content of the electrolyte agent. Bovine virus diarrheal disease (NADL strain), rotavirus (P44 strain 678), coronavirus (BCV94 strain), Escherichia coli (k99 strain), Salmonella ( S. enteritidis , S. typhimurum strain), Pasteurella ( P. multocida A) , Inoculate the inactivated antigen of P. haemolytica into the laying hen, egg from which the immune antibody against the antigen is formed from the inoculated laying hen, and then obtain egg yolk from it and dry it; Bovine virus diarrheal disease (NADL strain), rotavirus (P44 strain 678), coronavirus (BCV94 strain), Escherichia coli (k99 strain), Salmonella ( S. enteritidis , S. typhimurum strain), Pasteurella ( P. multocida A) , Inoculation of cows with inactivated antigens of P. haemolytica ) to obtain colostrum with immune antibodies and drying them; Drying the obtained yolk and colostrum, and obtaining the immune antibodies of the yolk and colostrum in powder form. The method of claim 2, A process of mixing the powdered colostrum and egg yolk in an electrolyte formulation, including the composition and content of the electrolyte formulation in powdered yolk and colostrum containing the immune antibody by inoculating the antigen into the laying hens and cows. Egg yolk and colostrum, which is an immune antibody, comprising a composition for the prevention and treatment of viral diarrheal disease, rotavirus, corona virus, Escherichia coli, Salmonella, Pasteurella infection, characterized in that the spray dried powder and lyophilized powder. Feed containing composition for the prevention and treatment of bovine virus diarrheal disease, rotavirus, corona virus, Escherichia coli, Salmonella, Pasteurella infection characterized by the process of extracting immune antibody in egg yolk and composition and mixing ratio of electrolyte solution . The process of preparing fructo oligosaccharides in the form of powdered immune substances (egg yolk, colostrum) and electrolyte preparations and paste preparations prevents diarrhea caused by bovine virus diarrheal disease, rotavirus, corona virus, Escherichia coli, Salmonella and Pasteurella infection. And a composition for treatment. A feed comprising a composition for the prevention and treatment of bovine virus diarrhea, rota virus, corona virus, E. coli, Salmonella, Pasteurella infection.