KR20160009352A - Method for producing a composition for the treatment of diseases of dogs with immune antibody - Google Patents

Abstract

The present invention relates to a method for manufacturing a composition for treating diseases of dogs using an immune antibody by comprising the following steps: independently preparing antibodies against different infectious diseases infecting dogs; inoculating layers and milk cows with the manufactured antibodies for forming an immune system; collecting immune eggs from the layers and collecting immune foremilk from the milk cows; and extracting yolk of the collected immune eggs, mixing with the immune foremilk, and manufacturing into a powder.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a composition for treating dog disease,

The present invention relates to a method for producing a high-antigen antibody against dogs diarrhea and respiratory diseases by producing an antigen of each pathogen, inoculating the egg yolk into a laying hens and a pregnant cow to produce an immunoglobulin antibody and an immuno-colostrum, The product is manufactured after the addition of a good probiotic agent to improve the function, and the prevention of the diarrhea and respiratory disease by feeding to the young puppy, thereby reducing the mortality caused by the disease and minimizing the growth delay, thereby improving the productivity of the livestock farmer. To a process for the preparation of a composition for the treatment of diseases.

Specific antibodies (Specific IgG) obtained from sera of immunized animals have been widely used in diagnostic and pure science fields.

Another area where IgG can be used is indirect immunization (passive immunization). Indirect immunity refers to the production of a specific antibody against a pathogen in another animal and administration of the antibody to the subject animal for the prevention of disease. Several studies have shown that indirect immunity is effective in preventing infectious diseases.

IgG in egg yolk is called IgY (Immune globulin Yolk).

The colostrum secreted by the cow for a few days after the birth does not only contain nutrients such as proteins, sugars, lipids, vitamins and minerals but also contains various growth factors such as growth factors and anti-microbial factors It has an active ingredient. The antimicrobial factors include immunoglobulin, lactoferrin, lysozyme, and lactoperoxidase. In colostrum, immunoglobulin, which can be defended from the pathogen for several weeks after birth, plays a very important role in disease Do.

Recently, it has been used for the treatment or prevention of diseases by producing an immune antibody by using an animal in a specific infectious pathogen of a human body and by adding these antibodies to various foods.

In order to protect people or animals from various diseases, immune function is exerted by recovering after infection of one pathogen. In addition, for immunity without illness, it is usually made to obtain immunity by preparing and inoculating a specific pathogen with a preventive medicine, but it is also possible to inject an already prepared immunity substance into the body to obtain the effect on the disease.

In general, the use of vaccines and antibiotics in dog breeding can reduce the damage caused by the disease in dogs. However, young dogs are exposed to disease pathogens and are resistant to disease, In addition to being expected to suffer from damage, lack of maternal transit antibody is weak resistance against disease, making it difficult to defend.

Currently, the most common diseases in dogs in Korea are dog parvovirus, distemper virus, corona virus, canine adenovirus, and canine infectious tracheobronchitis (Kennel cough) And respiratory illnesses, combined with the high incidence of dog mortality during infection, new levels of prevention and treatment techniques may be required.

Korean Patent Laid-Open Publication No. 1020070050499 (May 15, 2007) discloses a disease or disorder caused by a pathogen, for example, infectious bronchitis caused by Bordetella bronchiseptica, (ICH) caused by canine adenovirus type 1 (CAV-1), respiratory disease caused by canine adenovirus type 2 (CAV-2), canine influenza Leptospira canricola, Leptospira canicola, Leptospira canricola, Leptospira canola, Leptospira cannabis, Leptospira cannabis, Leptospira cannabis, Leptospira cannabis, Leptospira canis, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, or Leptospira pomona, discloses a combination vaccine and a method for protecting a dog from leptospirosis caused by ira pomona,

Korean Patent Publication No. 1012438710000 (Mar. 03, 2013) discloses an open-label virus vaccine, a diagnostic agent and a method of using the same,

Korean Patent Registration No. 1001171420000 discloses a vaccine comprising an effective amount of a cell-associated CCV pemphrotic protein-containing wax to protect animals such as dogs from diseases caused by infection with canine corpuscular virus (CCV) The waxes produced from the culture are disclosed.

In Korean Patent Registration No. 1012427570000 (Mar. 03, 2013), a recombinant protein comprising an antigenic determinant of canine coronavirus, a gene encoding the protein, an expression vector containing the gene, a host transformed with the expression vector A composition for preventing or treating a dog coronavirus infection comprising a cell, a vaccine containing the protein, and an egg yolk antibody produced using the protein, and a method for preventing or treating a dog coronavirus infection using the composition,

Korean Patent Registration No. 1005631970000 (Mar. 15, 2006) discloses VP2 recombinant proteins comprising an open-label virus neutralizing antigen determinant, a gene encoding the protein, an expression vector comprising the gene, and an expression vector It is understood that a composition for treating or preventing canine parvovirus infection comprising host cells, a vaccine containing the protein, and an antibody produced using the protein is disclosed.

1. Korean Patent Laid-Open Publication No. 1020070050499 2. Korean Patent Registration No. 101243871 3. Korean Patent Registration No. 100117142 4. Korean Patent Registration No. 101242757 5. Domestic Patent Registration No. 100563197

The main object of the present invention is to provide a composition capable of treating canine parvovirus, distemper virus, coronavirus, Canine adenovirus and dog infectious bronchitis simultaneously, which are digestive and respiratory diseases of dogs.

Another object of the present invention is to provide a process for preparing a composition as described below.

The present invention relates to a method for the prevention and treatment of canine parvovirus, distemper virus, coronavirus, Canine adenovirus and dog infectious bronchitis, which are major diseases of dogs, and to produce antigens of disease causative agents, , Immunostimulant was added to the immunogen, and the immunized preparation was periodically inoculated on the laying hens and dairy cows. In the laying hens, the immunized egg yolk with the immune antibody was obtained, and the colostrum collected within 30 hours was collected from the cow The immunostimulant was mixed with immune yolk sac, and the mixture was dried with powder using a spray dryer. Then, the active agent ( Bacillus subtills , Lactobacillus acidophilus , Enterococcus faecium ) is added to the animal to enhance the defense and resistance against the disease to treat the dog disease.

The present invention enhances the defense against dog disease so that it increases the resistance to disease without using antibiotics and is excellent in safety and stability to animal animals and is used for prevention and treatment of dog disease, It can greatly contribute to the increase of income and it can solve the problem about the residual antibiotic substance in the produced livestock products.

The present invention provides a method for preparing a vaccine, comprising: preparing antigens for different infectious diseases capable of infecting puppies, respectively;

Preparing the prepared antigens and the immunogen-forming antigen;

Inoculating the prepared immunostaining antigen into a laying hens and a cow;

Collecting immunologic eggs from the laying hens and collecting colostrum from a cow;

And collecting the egg yolk of the collected immunized egg to prepare a powder after mixing with an immunostimulated colostrum. The present invention also relates to a method for preparing a composition for treating a dog disease using an immunological antibody.

The present invention relates to a method for the prevention and treatment of canine parvovirus, distemper virus, coronavirus, Canine adenovirus and dog infectious bronchitis, which are major diseases of dogs, and to produce antigens of disease causative agents, , Immunoprecipitated by adding an immunopotentiating agent to the laying hens and dairy cows periodically, and in the laying hens egg yolk in which the immune antibody against the antigen was formed was obtained, and the colostrum within 30 hours was collected from the cow delivered The present invention also relates to a method for preparing a composition for treating a dog disease using an immunizing antibody.

The present invention is a substance that can be used for treatment of major causes of digestive and respiratory diseases that occur in young dogs, and diseases that cause great damage to dogs are as follows.

1. Canine parvovirus (CPV)

The dog parvovirus infection is a communicable disease that has been reported worldwide since 1978, and is a highly contagious and deadly digestive disease. Vulnerable, vomiting, and hemorrhagic diarrhea and dehydration are observed in infected dogs through mouth-infected diseases through the feces of the diarrhea, and it is severe after 5 days of oral infection and the susceptible puppy is 100% infected when exposed to virus .

2. Canine distemper virus (CDV)

Dog Distemper virus is a typical acute febrile viral disease. It has a 36 day incubation period and shows rhinitis, loss of appetite, starvation, bladder dryness, fever, severe respiratory symptoms (pneumonia), digestive disorders, neurological symptoms, The mortality rate is high, and when the neurological symptoms are shown, it is almost 100% deadly disease.

3. Canine coronavirus (CCV)

It is recognized as an important viral disease because it is mixed with dog parvovirus infectious disease. It is recognized as an important viral disease and it releases clinical symptoms such as vomiting, diarrhea and dehydration, stools, stamina, anorexia and bad stool. do.

4. Canine adenovirus (CAV)

Canine adenoviruses are of two types, type-1 and type-2.

Type-1 causes infectious hepatitis and causes high fever, nerve symptoms and serious eye damage,

Type-2 is a type of respiratory disease that is characterized by symptoms such as coughing, hoarseness, rhinitis and fever due to upper and lower respiratory tract infections. It is a combination infection with dog distemper virus and parainfluenza virus and canine infectious bronchitis infectious tracheobronchitis).

5. Canine infectious tracheobronchitis

Canine infectious tracheobronchitis (Kennel cough) is a highly contagious respiratory disease characterized by coughing and runny nose, and its causative agent is Bordetella It is a highly contagious respiratory disease caused by bronchiseptica . It causes secondary infection during distemper virus infection and induces bronchopneumonia. It shows severe symptoms especially in young puppy. It shows clinical symptoms such as runny nose, dry cough, and vomiting after 3 ~ 4 days after infection. The body temperature gradually increases above 39 ℃ and shows severe bronchopneumonia.

Accordingly, the present invention was made to develop a substance that can be used for the prevention and treatment of canine parvovirus, distemper virus, corona virus, Canine adenovirus, and dog infectious bronchitis, which are major diseases of dogs,

It is possible to effectively prevent and treat the infection of the above diseases, and the present invention has been made.

Hereinafter, the present invention will be described in detail with reference to the following examples.

Example 1. (Antigen production; canine parvovirus (CPV) culture)

The first step (virus inoculation culture)

A72 (Canine tumor cell origin) cells were suspended at a concentration of 200,000 per ml and cultured at 37 ° C for 2 to 3 days. When a single layer was formed at 50%, the culture medium for proliferation was removed, The virus was inoculated with 10 ^3.0 TCID ₅₀ per ml and incubated at 37 ° C for 4 days.

Second step (virus harvesting)

When the cytopathic effect (CPE) of the inoculated virus was more than 80%, the viral culture infectious fluid was collected, and the cells were frozen and thawed at -80 ° C for 2 times, centrifuged at 5,000 rpm for 20 minutes, Respectively. (Using supernatant as antigen)

Example 2 (Preparation of antigen; Canine distemper virus (CDV) culture)

The first step (virus inoculation culture)

10.0 ^4.0 TCID ₅₀ per ml of distemper virus was inoculated in a culture medium (see Table 1) in which 1 ml of a specific pathogen free egg (SPF) -developed egg of 10-day-old SPF next,

Second step (virus harvesting)

  When the cytopathic effect (CPE) of the inoculated virus was more than 80%, the viral culture infectious fluid was collected, and the cells were frozen and thawed at -80 ° C for 2 times, centrifuged at 5,000 rpm for 20 minutes, Respectively.

(Using supernatant as antigen)

Example 3 (Preparation of antigen; Canine coronavirus (CCV) culture)

The first step (virus inoculation culture)

A72 (Canine tumor cell origin) cells were suspended at a concentration of 200,000 per ml and cultured in a culture medium for proliferation (see Table 1) at 37 ° C to remove the culture medium for proliferation when a monolayer of the cultured cells was formed. the inoculated and incubated 3-5 days at 37 ℃ rotating the coronavirus of 10 ^4.0 TCID ₅₀ per ml, and then,

Second step (virus harvesting)

  When the cytopathic effect (CPE) of the inoculated virus was more than 80%, the cultured infectious broth was collected and centrifuged at 5,000 rpm for 20 minutes after repeating two freeze-thaw cycles at -80 to separate only the supernatant Respectively.

(Using supernatant as antigen)

Example 4 (Preparation of antigen; Canine adenovirus (CAV) culture)

The first step (virus inoculation and culture)

A72 (Canine tumor cell origin) cells were suspended in 200,000 cells per ml and cultured in a culture medium for proliferation (see Table 1) at 37 ° C to remove the culture medium for proliferation when a monolayer of cultured cells was formed. The canine adenovirus type-1 and type-2 of 10 ^4.0 TCID ₅₀ per ml were inoculated into the culture medium, respectively, and cultured at 37 ° C for 3 to 4 days.

Second step (virus harvesting)

When the cytopathic effect (CPE) of the inoculated virus was more than 80%, the cultured infectious broth was collected and centrifuged at 5,000 rpm for 20 minutes by repeating two freeze-thaw cycles at -80, (Using the supernatant as an antigen).

Example 5 (Preparation of antigen; Bordetella bronchiseptica culture)

The first step (seed culture)

The lyophilized seeds ( Bordetella bronchiseptica ) was inoculated into 10 ml of Tryptic soy broth medium and cultured at 37 ° C for 12 to 15 hours.

The second step (culture)

  Inoculate 10 ml of seedlings in 1,000 ml of Tryptic soy broth medium and incubate shaking at 37 ° C and 150 rpm for 18 hours.

Third step (collection and treatment of cells)

The cells were inoculated with 0.3% formalin by inoculating the infected culture with shaking at 150 rpm for 18 hours at 37 ° C. The cells were collected by centrifugation at 8,000 rpm for 30 minutes, washed once with PBS (phosphate buffered saline, pH 7.2) And suspended in PBS.

(Optical density (OD) was measured at 410 nm wavelength using a spectrophotometer and then used as an antigen)

Example 6 (Preparation of antigen; Dermonecrotoxin (DNT) toxoid culture)

Dermonecrotoxin (DNT) toxoid was isolated from Bordetella bronchiseptica ) was cultured in Tryptic soy broth at 37 ° C for 18 hours, and then centrifuged at 8,000 rpm for 30 minutes to collect the bacteria. The cells were washed twice with PBS and suspended in an appropriate amount of PBS. The cells were disrupted using Sonication, rpm for 30 minutes.

Centrifuged supernatant was assayed for protein concentration using the BCA protein assay reagent kit (Pierce Co.) and the crude DNT antigen concentration was then used at 500 μg / ml.

[Table 1] Composition composition for cytokine production (1,000)]

 Alpha MEM ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ

 Fetal bovine serum (FBS) · · · · · · · · · · · · · · · · · 50 ml

7.5% NaHCO ₃ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ 20ml

Penicillin G-Na ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ

Streptomycin sulfate ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ

Fungizone · · · · · · · · · · · · · · · · · · · 0.025 μg / ml

[Table 2] Composition composition for cell-holding culture (1,000 ml)

Alpha MEM ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ

Fetal bovine serum (FBS) · · · · · · · · · · · · · · · · · · · 30 ml

7.5% NaHCO ₃ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ 20ml

Penicillin G-Na ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ

Streptomycin sulfate ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ

Fungizone · · · · · · · · · · · · · · · · · · · 0.025 μg / ml

[Table 3. Phosphate buffered saline (1,000 ml)]

Sodium chloride ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ

Potassium chloride ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ

KH ₂ PO ₄ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ ㆍ

Na ₂ HPO _{4 - - - - - - - - - - - - - - - - - - - - - - -}

Example 7 (Preparation of antigen for immunogenesis: for inoculation of laying hens)

(7 ml ), distemper virus (5 ml ), coronavirus (7 ml ) and Canine (5 ml ), which were concentrated five times in the supernatants cultured in Examples 1 to 5, To the antigen of adenovirus (5 ml ), 6 ml of Bordetella Bacterial cultures of bronchiseptica infection and toxin were mixed with 30 ml of antigen , and oil adjuvant (Montnide ISA70) (70 ml) was mixed. The mixture was emulsified at 1,000 rpm for 30 minutes and then an immunogen was prepared using a homogenizer.

Example 8 (Preparation of antigen for immunogenesis: for inoculation of milk)

20 ml of canine parvovirus, 10 ml of distemper virus, 20 ml of corona virus, 10 ml of canine adenovirus, which were cultured in Examples 1 to 5, and Bordetella 5 ml of oil adjuvant (Montnide ISA25) and 15 ml of aluminum hydroxide gel were added to 20 ml of bronchiseptica antigen and emulsified at 1,000 rpm for 30 minutes to prepare an immunogen.

Example 9 (Immunization-forming antigen inoculation method: Layers)

The immunizing preparation for laying hens produced according to the above Example 7 was inoculated first to the muscle of the forefoot in 0.8 ml per marrow on a 20-week-old laying hens, and the second inoculation was inoculated three weeks after the first inoculation. 3 months.

Example 10 (Immunogenesis antigen vaccination method; cow)

The immunized preparation for cow immunization produced according to Example 8 was inoculated into the gluteal muscles in 2.0 ml of the marriage three times at intervals of three weeks, two months before delivery of the pregnant cow.

Example 11 (Collection of immunized eggs)

After 2 weeks from the third inoculation of the immunizing preparation according to Example 9, the eggs with the immunized antibodies were collected hygienically, 150 ppm of sodium hypochlorite was sprayed on the eggs to clean them, and then the moisture of the egg surface was completely After drying, the dried egg was harvested and only the egg yolk in which the immunized antibody was formed was collected and used to prepare powder using a spray dryer.

Example 12 (Colostrum Collection with Immunity)

About 10 liters of colostrum within 30 hours was collected from the mother after inoculation of the immunizing preparation according to Example 10 and used in the sterilized container to prepare powder using a spray dryer.

Example 13 (Drying step of powder of an immunizing antibody)

Using 1 liter of immunostain oil mixed with 1 liter of immune yolk solution which is immune to the pathogen by the spray method of the disk and nozzle using a spray dryer, the moisture is dried and the water content is less than 10% Dried to powder.

Example 14

(5g), glycine (1g), potassium chloride (0.2g) in egg yolk dried powder (85g) containing immunoglobulin antibody and immunosoluble antibody against canine parvovirus, distemper virus, coronavirus, canine adenovirus and dog infectious bronchitis, ( Bacillus sp.) At a ratio of 100: 3 by weight of the culture medium to the culture medium containing Ascorbic acid (0.1 g), Sodium bicarbonate (1 g), Sorbitol (0.2 g) and Fructooligosaccharide subtills (1 × 10 ¹⁰ CFU), Lactobacillus acidophilus (1 × 10 ¹⁰ CFU), Enterococcus In faecium (1 × ¹⁰ 10 CFU) was prepared in the product by the addition of any one selected.

Experimental Example 1 (Antibody test for antigen)

Eight pregnant cows were selected in order to measure antibody titers against immunized egg yolk solution and immunized colostrum collected according to Examples 11 and 12, and 5 cows were used as a test group and 3 cows as a control group.

In the test group

In accordance with Example 9, the laying hens were inoculated with the antigen for immunity-forming and the five cows were inoculated with the antigen for immunity-forming according to Example 10.

In accordance with Example 12, 5 cows of the test group were delivered, and then the immunized colostrum was collected, and the immunostaining solution collected in accordance with Example 11 was collected. Five liters of immunized colostrum was mixed with 5 liters of immunized egg yolk, Were prepared according to Example 13, respectively.

In the control group, the egg yolk of the laying hens not inoculated with the antigen was collected, and the three colostrum of the cow were respectively collected, and 5 liters of egg yolk was mixed with 5 liters of the colostrum to prepare three experimental animals according to the Example 13.

Antibody tests against 8 test samples (5 test groups, 3 control groups) prepared according to Example 13 were each measured by an enzyme-linked immunosorbent assay (ELISA), and the following test results were obtained .

Test material The antibody CPV CDV CCV CAV Bordetella
bronchiseptica Test group EUT 1 640 512 1,280 128 1,280 EUT 2 1,280 128 2,560 512 2,560 EUT 3 1,280 512 2,560 256 1,280 EUT 4 2,560 512 1,280 256 2,560 EUT 5 1,280 128 1,280 128 2,560 Control group EUT 1 10 10 10 10 20 EUT 2 10 10 10 10 20 EUT 3 10 10 10 10 20

Experimental Example 2 (Efficacy Test on Immunized Agents on Outdoor Farms)

end. Field test (A farm)

  We evaluated the efficacy of the product after diarrhea on a farm of 90 dogs of mother dogs after feeding the product to a severe dog. Diarrhea and autopsy of the dead puppy on this farm showed that parvovirus, coronavirus and canine adenovirus were present, and distemper virus and dog infectious bronchitis were complex.

In order to evaluate the efficacy of the product prepared according to Example 14, 180 dogs of 1 day old dog were selected and 110 dogs of the test group were fed into the oral cavity for 3 days by dissolving 1 g of the product twice per day in the test group, For 14 days and examined the viability and clinical observations of the dog.

As shown in the table below, survival rate was 89.0% and survival rate was 37.1% in all 70 control groups, surviving 98 days after 14 days in a total of 110 test groups. (Table 5). In addition,

Clinical observations in the test group showed mild clinical symptoms such as vomiting (8/110), dyspnea (7/110), runny nose and cough (15/110) and nausea (5/110) Compared with that of the control group.

In the untreated control group, complex clinical symptoms such as vomiting (45/70), dyspnea (44/70), runny nose and cough (59/70), and ankylosis (18/70)

Survival rate after 1-day-old puppy feeding division Number of test marijuana Days observed Feed amount (days) Survival Marissa Survival rate (%) Test group 110 14 days 1g / 3 days 98 89.0 Control group 70 14 days No treatment 26 37.1

Clinical observations after feeding to 1-day-old puppies division observe
Days Clinical observation (symptom manifestation / number of testes) throw up Dyspnea Runny nose, cough A star formation Test group 14 days 8/110 7/110 15/110 5/110 Control group 14 days 45/70 44/70 59/70 18/70

I. Field test (B farm)

  We evaluated the efficacy of diarrhea after feeding the products to the farms which were damaged due to the diarrhea caused by diarrhea in our 60 dogs. Diarrhea and autopsy of parasitized young dogs on this farm showed that parvovirus, Canine adenovirus and coronavirus were dominant, and distemper virus and dog infectious bronchitis were complex infections.

In order to evaluate the efficacy of the product manufactured according to Example 14, 110 dogs of 1 day old dog were selected. In 70 dogs of the test group, 1 g of the product was dissolved twice per day for 3 days, and 40 dogs For 14 days and examined the viability and clinical observations of the dog.

As shown in the table below, the survival rate was 84.2% after surviving for 14 days in 70 test groups, and survival rate was 35.0% in 14 control groups in total, (See Table 7). In addition,

Clinical observations in the test group showed mild clinical symptoms such as vomiting (9/70), dyspnea (11/70), runny nose and cough (13/70), and nausea (9/70)

In the untreated control group, complex clinical symptoms such as vomiting (22/40), dyspnea (24/40), runny nose and cough (24/40), and ankylosis (12/40)

Survival rate after 1-day-old puppy feeding division exam
Marie Days observed Feed amount (days) Survival Marissa Survival rate (%) Test group 70 14 days 1g / 2 days 59 84.2 Control group 40 14 days No treatment 14 35.0

Clinical observations after feeding to 1-day-old puppies division Days observed Clinical observation (symptom manifestation / number of testes) throw up Dyspnea Runny nose, cough A star formation Test group 14 days 9/70 11/70 13/70 9/70 Control group 14 days 22/40 24/40 24/40 12/40

As described above, in order to develop a substance capable of simultaneously treating canine parvovirus, distemper virus, coronavirus, Canine adenovirus, and dog infectious bronchitis,

While conducting various studies focusing on the method of using immuno-colostrum mixed with specific immunoglobulin antibody against gastric diseases,

The product was prepared by adding probiotics such as probiotics, glucose, and fructooligosaccharides to the dried powder mixed with the immunoglobulin antibody and the immuno-colostrum, and it was possible to improve the anti-infectivity by manual immunization through feeding to the dog. And the efficacy of the treatment was confirmed.

In addition, the dogs with symptomatic symptoms in the test group showed rapid recovery. Also, by preventing disease infections and growth retardation in growth period and treating diseases without using antibiotics, disease management and environmentally friendly breeding It is expected that it will contribute to the improvement of public health and reduce the death of dogs and puppies, and at the same time, it will greatly contribute to the income increase of farmers who raise dogs and dogs.

Claims (16)

A method for preparing a composition for treating a dog disease using an immunizing antibody,
Preparing antigens for different infectious diseases, each capable of infecting a puppy;
Preparing the prepared antigens and the immunogen-forming antigen;
Inoculating the prepared immunostaining antigen into a laying hens and a cow;
Collecting immune eggs from the laying hens and collecting colostrum from a cow;
And collecting egg yolk of the collected immunized egg and mixing with immunostaining colloid to prepare a powder. The method for preparing a composition for treating a dog disease using an immunological antibody according to claim 1,
The use according to claim 1, wherein the antigen of the dog disease is any one selected from the group consisting of canine parvovirus antigen, distemper virus antigen, coronavirus antigen, Canine adenovirus antigen and dog infectious bronchitis. For preparing a composition
The method according to claim 1, wherein preparing antigens for different infectious diseases capable of infecting the puppy, respectively,
A72 (Canine tumor cell origin) cells were suspended at a concentration of 200,000 per ml and cultured at 37 ° C for 2 to 3 days. When a single layer was formed at 50%, the culture medium for proliferation was removed, The virus was inoculated with 10 ^3.0 TCID ₅₀ per ml and incubated at 37 ° C for 4 days.
When the cytopathic effect (CPE) of the inoculated virus was more than 80%, the virus culture infectious fluid was collected, and the cells were frozen and thawed twice at -80 ° C., followed by centrifugation at 5,000 rpm for 20 minutes. In isolation
A method for preparing a composition for treating a dog disease using an immunizing antibody, which comprises preparing an antigen for a different infectious disease that can infect a dog.
The method according to claim 1, wherein preparing antigens for different infectious diseases capable of infecting the puppy, respectively,
10.0 ^4.0 TCID ₅₀ per ml of distemper virus was inoculated in a culture medium (see Table 1) in which 1 ml of a specific pathogen free (SPF) developing egg of 10-day-old SPF was incubated at 37 ° C for 5 days next,
When the cytopathic effect (CPE) of the inoculated virus was more than 80%, the virus culture infectious fluid was collected, and the cells were frozen and thawed twice at -80 ° C., followed by centrifugation at 5,000 rpm for 20 minutes. ≪ / RTI > wherein the antibody is administered to a mammal in need thereof.
The method according to claim 1, wherein preparing antigens for different infectious diseases capable of infecting the puppy, respectively,
A72 (Canine tumor cell origin) cells were suspended at a concentration of 200,000 per ml and cultured in a culture medium for proliferation (see Table 1) at 37 ° C to remove the culture medium for proliferation when a monolayer of the cultured cells was formed. the inoculated and incubated 3-5 days at 37 ℃ rotating the coronavirus of 10 ^4.0 TCID ₅₀ per ml, and then,
When the cytopathic effect (CPE) of the inoculated virus was about 80% or more, the cultured infectious agent was collected and centrifuged at 5,000 rpm for 20 minutes at a temperature of -80 ° C for 2 hours. ≪ / RTI > wherein the antibody is administered to a mammal in need thereof.
The method according to claim 1, wherein preparing antigens for different infectious diseases capable of infecting the puppy, respectively,
A72 (Canine tumor cell origin) cells were suspended in 200,000 cells per ml and cultured in a culture medium for proliferation (see Table 1) at 37 ° C to remove the culture medium for proliferation when a monolayer of cultured cells was formed. The canine adenovirus type-1 and type-2 of 10 ^4.0 TCID ₅₀ per ml were inoculated into the culture medium, respectively, and cultured at 37 ° C for 3 to 4 days.
When the cytopathic effect (CPE) caused by the inoculated virus occurred at about 80% or more, the cultured infectious broth was each inseminated, followed by two freeze-thaw cycles at -80, followed by concentration and centrifugation at 5,000 rpm for 20 minutes And separating only the supernatant from the supernatant. ≪ RTI ID = 0.0 > 11. < / RTI >
The method according to claim 1, wherein preparing antigens for different infectious diseases capable of infecting the puppy, respectively,
The lyophilized seeds ( Bordetella bronchiseptica ) was inoculated in 10 ml of Tryptic soy broth medium and cultured at 37 ° C for 12 to 15 hours,
Tryptic soy broth medium was inoculated with 10 ml of seedlings and inoculated with 0.3% formalin by inoculating the culture broth with shaking at 37 ° C and 150 rpm for 18 hours. The cells were then collected by centrifugation at 8,000 rpm for 30 minutes, saline, pH 7.2), and suspended in PBS. The method for preparing a composition for treating a dog disease using an immunological antibody according to claim 1,
The method according to claim 1, wherein preparing antigens for different infectious diseases capable of infecting the puppy, respectively,
Dermonecrotoxin (DNT) toxoid was isolated from Bordetella bronchiseptica ) was cultured in Tryptic soy broth at 37 ° C for 18 hours, and then centrifuged at 8,000 rpm for 30 minutes to collect the bacteria. The cells were washed twice with PBS and suspended in an appropriate amount of PBS. The cells were disrupted using Sonication, rpm, 30 minutes
The centrifuged supernatant was measured for protein concentration using the BCA protein assay reagent kit (Pierce Co) and then the crude DNT antigen concentration was used at 500 μg / ml. The composition for the treatment of dog disease using immuno-antibodies .
2. The method of claim 1, further comprising the steps of: preparing the prepared antigen and an immunogen;
To 30 ml of antigen mixed with 6 ml of cultured Bordetella bronchiseptica infectious disease and toxin in antigens of dog parvovirus (7 ml), distemper virus (5 ml), coronavirus (7 ml) and canine adenovirus (5 ml), oil adjuvant (Montnide ISA70) , Emulsifying the mixture at 1,000 rpm for 30 minutes, and then preparing an immunogen by using a homogenizer
A method for the preparation of a composition for treating a dog disease using an immunological antibody.
The method according to claim 1, further comprising producing the prepared antigen for immunization and antigen formation; is canine parvovirus 20ml, 10ml distemper virus, corona virus 20ml, 10ml Canine adenovirus Bordetella 5 ml of an oil adjuvant (Montnide ISA25) and 15 ml of aluminum hydroxide gel are added to 20 ml of bronchiseptica antigen and emulsified at 1,000 rpm for 30 minutes to prepare an immunizing preparation for inoculation of a dog. .
The method according to claim 1, wherein the step of inoculating the prepared immunogenicity antigens to the laying hens and cows is carried out by adding 5 ml of an oil adjuvant (Montnide ISA25) to 20 ml of dog bovetella bronchiseptica antigen in 20 ml of dog parvovirus, 10 ml of distemper virus, 20 ml of coronavirus, 10 ml of canine adenovirus , And 15 ml of aluminum hydroxide gel are added and emulsified at 1,000 rpm for 30 minutes to prepare an immunizing preparation for inoculation of a dairy cattle.
The method according to claim 1, wherein the step of inoculating the prepared immunogen antigens to the laying hens and cows comprises: a step of inoculating the egg yolk immunizing preparation into a 20-wk-old laying hens by 0.8 in each case, Wherein the vaccine is inoculated three weeks after the inoculation and then inoculated three months after the inoculation. The method for preparing a composition for treating a dog disease using the immunological antibody according to claim 1,
The method according to claim 1, wherein the step of inoculating the prepared immunogen antigens to the laying hens and dairy cows is carried out by administering an immunizing preparation for dairy cows immunizing a pregnant cow at a rate of 2.0 ml / Lt; RTI ID = 0.0 > immunological < / RTI >
The method according to claim 1, wherein the step of collecting the immunized eggs from the laying hens and collecting the colostrum from the cows comprises collecting the immunized preparation hygienically from the second week after the third inoculation with the immunized antibody, and then adding 150 ppm of sodium hypochlorite to the eggs Sprayed and cleaned. Then, the water on the surface of the egg is completely dried in a drying chamber. Then, the dried egg is harvested and only the egg yolk in which the immunized antibody is formed is collected and prepared as a powder using a spray dryer. A method for the preparation of a composition for treating a dog disease using an immunological antibody.
The method according to claim 1, wherein the step of collecting the immunized eggs from the laying hens and collecting the colostrum from the cow comprises collecting about 10 liters of the colostrum within 30 hours from the mother after inoculation of the immunizing preparation into the cow, Wherein the antibody is produced by using a spray dryer. The method for producing a composition for treating a dog disease according to claim 1,
The method according to claim 1, wherein the egg yolk of the collected immunized egg is mixed with an immunostable colostrum to prepare a powder. The step of spraying the egg yolk with the immune colostrum is performed using a spray dryer, And 1 liter of an immunized colostrum is mixed with 1 liter of an egg yolk solution and the water is dried to prepare a powder having a moisture content of less than 10%, thereby producing a composition for treating a dog disease using the immunological antibody.