KR100693077B1 - Biological composition for preventing and treating prdc - Google Patents

Abstract

A novel biological prophylactic and therapeutic composition isolated from domestic porcine respiratory disease complex(PRDC)-attacked grown pigs is provided to be able to protect pigs from the PRDC. The biological composition for preventing and treating PRDC comprises porcine circovirus type-2 and Actinobacillus pleuropneumoniae type 2 and type 5. The porcine circovirus type-2 is isolated from domestic PRDC-attacked grown pigs with a deposition number of KCCM10688P, and the Actinobacillus pleuropneumoniae type 2 and type 5 are isolated from domestic PRDC-attacked grown pigs with a respective deposition number of KCCM10686P and KCCM10687P. In the composition, the circovirus type-2 and Actinobacillus pleuropneumoniae type 2 and type 5 are inactivated by using formalin.

Description

Biological composition for preventing and treating PRDC}

The present invention relates to biological compositions and methods for the prevention and treatment of porcine respiratory disease complex (PRDC).

The disease that has been a problem recently in growing pigs (70 to 90 days of age) is a swine complex respiratory disease. This disease has been identified as being caused by a combination of viral and bacterial infections. The main symptoms are cough, mortality, decreased feed intake and mortality. In addition, it is a complex infection of viruses and bacteria, there is a disadvantage that can not be prevented and treated using antibiotics. The term PRDC will be used throughout this invention for this disease.

It is an object of the present invention to provide novel prophylactic and therapeutic biological compositions for protecting swine from swine complex respiratory disease (PRDC).

Another object of the present invention is to provide a method of protecting swine from swine complex respiratory disease.

The present invention provides a composition comprising swine circovirus type 2 and pleural pneumococcal types 2 and 5 inactivated by laboratory manipulation for use in vaccination.

Separation of the porcine circovirus type 2 and pleural pneumococcal type 2 and 5 can be carried out according to methods known to those skilled in the art. Specifically, it can separate according to the method of the following Example. Preferably, the pig circovirus type 2 is a strain separated from domestically grown PRDC pigs, and the pleural pneumococcal type 2 and 5 are characterized in that the host is separated from domestically grown PRDC pigs, more preferably. The swine circus virus type 2 is deposited using the deposit number (KCCM 10688P), and the pleural pneumococcal type 2 and 5 are deposited using the deposit number (KCCM 10686P) and deposit number (KCCM 10687P), respectively It is characterized by.

In addition, the pleural pneumococcal type 2 and 5 are sufficient to be mixed in a ratio of 1: 0.5-1.5, more preferably in a 1: 1 ratio.

The composition may be prepared according to standard techniques well known to those skilled in the pharmaceutical and veterinary arts. The content of such compositions can be determined by those skilled in the medical and veterinary arts, taking into account such factors as the age, sex, weight, race and condition of the particular subject and route of administration. The composition may be administered alone or in combination with other immunological antigens or vaccine compositions or therapeutic compositions or for subsequent administration.

The composition may include pharmaceutically acceptable carriers in addition to circovirus type-2 and pleural pneumococcal type 2 and 5 antigens. A pharmaceutically acceptable carrier herein refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient, is chemically inert and is not toxic to the subject to be administered. Various carriers are known in the art and include distilled, brine or mineral water.

In addition, the composition may include at least one adjuvant for enhancing immune response, which is generally used. Examples of adjuvants are mineral gels such as aluminum hydroxide, surfactants such as resorcithin, glycosides such as saponin or saponin derivatives, lantitidine, Avridine ™, carbopol, polyphosphazene, dimethyldioctadecylammonium bromide (DDA), Quill A, Ampigen, mineral oil, etc. are mentioned.

The compositions of the present invention may also include one or more pharmaceutically or veterinary acceptable preservatives. For example, it may include an EDTA salt, methiolate, and the like.

Examples of formulations of the compositions of the present invention include liquid preparations, such as suspensions for oral, nasal, anal, vaginal, intragastric administration, syrups, etc. and sterile suspensions or emulsions for subcutaneous, endothelial, intramuscular, or intravenous administration. do. It may also be a solid preparation of soluble components or particulates which is resuspended in a pharmaceutically acceptable diluent prior to use. Soluble components or particulates can be prepared according to techniques known to those skilled in the pharmaceutical and veterinary arts, such as coacervation, biacervation, coagulation, spray drying, bubble drying, precipitation, lyophilization and the like.

The composition may be an immunogenic active ingredient based on attenuated virus or a vaccine prepared with such active ingredient, wherein attenuation is carried out by conventional methods such as passage on cells, preferably on pig cells, in particular PK. Subculture in the same cell line as -15 cells. Such compositions typically comprise a carrier, excipient or diluent, adjuvant and lyophilized stabilizer, which are typically acceptable in pharmaceutical or veterinary terms. As adjuvant, immunoadjuvant can be used.

Lyophilized stabilizers include carbohydrates such as sorbitol, mannitol, starch, sugar, dextran, glucose or proteins such as albumin, casein, or buffers such as alkali metal phosphates. Such vaccines preferably comprise 10 ³ to 10 ⁶ TCID ₅₀ .

In addition, the circovirus type 2 and the pleural pneumococcal type 2 and 5 is characterized in that inactivated. Inactivation of the virus can be carried out by methods known to those skilled in the art. That is, the antigen may be exposed to a chemical agent such as formaldehyde, paraformaldehyde, beta-propiolactone, ethyleneimine or derivatives thereof, or may be inactivated by heat or radiation.

In particular, the inactivation method using formalin is preferable. Inactivation with formalin (formaldehyde) has been proven to be reliable, certainty and stable in achieving inactivation compared to inactivation with other chemicals, thereby providing a stable product. This inactivation method is suitable for mass production, which saves manpower and production cost, and is efficient. It has been recognized worldwide for the Japanese encephalitis virus vaccine which has been used for decades.

Adjuvants can be added to inactivated antigens according to techniques known to those skilled in the art. Examples of adjuvants are mineral gels such as aluminum hydroxide, surfactants such as resorcithin, glycosides such as saponin or saponin derivatives, lantitidine, Avridine ™, carbopol, polyphosphazene, dimethyldioctadecylammonium bromide , Quill A, ampygen, mineral oil, and the like.

Inactivated vaccines of viruses preferably comprise 10 ⁵ to 10 ⁸ TCID ₅₀ .

Furthermore, the swine circovirus type 2 and pleural pneumococcal are preferably mixed in the range of 1: 0.5-1.5, and preferably mixed in a ratio of about 1: 1. This is because the ratio of virus to bacteria is preferably in the range of about 1: 1, and therefore, it is preferable to mix pigs circovirus type 2 and pleural pneumonia type 2 and type 5 in a ratio of 2: 1: 1 respectively.

In addition, the present invention can treat PRDC by administering the biological composition to pigs infected with PRDC. Conventional vaccines have been used to prevent diseases by administering vaccines to uninfected animals, but the reality is that PRDCs are already widespread in farms in Korea, and preventive vaccines alone do not work. Accordingly, the present invention provides a biological composition which already has an antibody of PRDC, that is, can be administered to pigs pre-infected with PRDC, and also provides a method of administering the composition.

Such compositions can be administered via a variety of veterinary acceptable routes. For example, it may be administered by oral administration in addition to feed or drinking water, instillation into the eye mucosa, intramuscular injection, intravenous injection, intradermal injection and the like. Depending on the route of administration, those skilled in the veterinary art can determine the dosage.

Hereinafter, a preferred embodiment of the present invention will be described in detail by way of example, but the present invention is not limited thereto.

Example 1

Vaccine manufacturing

1. Manufacture of pig circovirus vaccine

Vaccine was prepared using porcine circovirus type-2 (KCCM 10688P) using cells derived from porcine kidney called PK-15 in culture flasks. After culturing PK-15 cells in a 150 cm ² floor flask, 5 ml of porcine circovirus type 2 (10 ⁶ tissue culture infective dose per ml) was inoculated and DMEM containing 1.8% FBS (fetal bovine serum). 50 ml of Dulbecco's Modified Eagle medium was added and incubated in a carbon dioxide incubator at 37 ° C for 5 days.

  The culture flask cultured as above was put in a -75 ° C. cryogenic freezer, frozen for 3 hours, dissolved at room temperature (18 ° C.), filtered using a 0.8 μm syringe filter, and the filtrate was extracted. Formalin 500 μl was added to 40 ml of the filtrate to inactivate swine circovirus type-2. Thereafter, 8 ml of aluminum gel was added to prepare a vaccine.

2. Preparation of Pleural Pneumococcal Vaccine

Pleural pneumococci type 2 and type 5 are incubated first. Pleural pneumococcal type 2 (10 ⁵ colony forming units per ml) was inoculated in a culture tube containing 40 ml of brain heart infusion (BHI) containing NAD, and then enriched in an incubator at 37 degrees for 12 hours. After enrichment, 40 μl of culture medium was added with 500 μl of formalin to inactivate pleural pneumococcal type 2. The vaccine was then prepared by adding 8 ml of aluminum sections. In the same manner, a pleural pneumonia type 5 vaccine was also prepared.

  The final pleural pneumonia vaccine was prepared by mixing 1: 1 pleural pneumococcal type 2 vaccine and pleural pneumococcal type 5 vaccine prepared above.

.

3. Pig PRDC Vaccine Manufacturing

The vaccines prepared in 1 and 2 were mixed in a ratio of 1: 1 (pig circovirus type-2; 10 ⁷ tissue culture infective doses per ml; pleural pneumococcal types 2 and 5; 10 ⁷ colony forming units per ml) ) Was prepared.

Example 2

PRDC vaccination

Thirty five-day-old pigs were purchased and, by serometry, had already had swine circovirus type 2 antibodies (ie, infected with swine circovirus type 2) and antibody titers of pleural pneumonia were type 2 After confirming that both types and 5 are 1: 4 or less, randomly divided into three groups of 10 each.

The first group of pigs were vaccinated, and the vaccine developed at 4 and 7 weeks of age was inoculated into 1 ml of muscle regardless of body weight. Challenge was inoculated. Pigs in the second group were challenged with pig circovirus type 2 and pleural pneumococci type 2 and 5 only at 9 weeks of age without the vaccine developed as a positive control. Aggressive vaccination was performed by dropping 4 ml of the culture medium containing 10 ⁵ TCID ₅₀ of swine circovirus type 2 and pleural pneumococcal type 2 and type 5 into the pig's nose.

Pigs in the third group did not receive the vaccine developed as a negative control, nor did they inoculate the swine circovirus type 2 and pleural pneumococci type 2 and 5 with challenge. From 4 weeks of age, pigs in group 1 and 2 were tested for clinical symptoms in 3 groups over 5 weeks once weekly after challenge with pig circovirus type 2 and pleural pneumococcal type 2 and 5.

After the final 5 weeks of vaccination (when the pigs were 12 weeks old), all live pigs were euthanized and the lesions were examined. This experiment was performed three times independently.

Example 3

Mortality Survey

 In the first group of vaccinated pigs, no significant depression was observed after 9 weeks of inoculation with swine circovirus type 2 and pleural pneumococcal type 2 and 5, and no pigs died until the end of the experiment.

  In the positive control pigs of the second group, the pigs were vaccinated at 9 weeks of age after the pig circovirus type 2 and pleural pneumococcal type 2 and type 5, and the pigs were depressed from 5 hours of inoculation, and 2 heads died at 12 hours of inoculation. Three heads died 24 hours after the inoculation, and there were no deaths, but respiratory diseases such as persistent cough and dyspnea were observed until the end of the experiment.

  No specific symptoms were observed in the third group of negative control pigs during the experiment.

2. Lung lesion investigation

The lung lesion index was measured by the Ministry of Agriculture and Forestry Notice No. 1996-58. The final lung lesion index was calculated by measuring the index of determination of left and right lung lesions using a lattice measuring instrument of 1 cm in width and length.

  Pulmonary lesion index 0 was observed in 6 heads, lung lesion index 1 in 3 heads, and lung lesion index 2 in 1 head of the group 1 vaccinated pigs (mean lung lesion index; 0.5).

  In the positive control pigs of the second group, lung lesion index 3 was observed in two of the five lived to the end of the study, lung lesion index 4 in one, and lung lesion index 5 in two (mean lung lesion index; 4.0). .

  The lung lesion index could not be observed in the third group of negative control pigs.

3. Experimental Results

In the first group of vaccinated pigs, no pigs died at the time of challenge vaccination, but in the second group pigs, five pigs died, and in the lung lesion index of pigs that survived after the end of the experiment. The mean pulmonary lesion index was 0.5 in the group, whereas the mean pulmonary lesion index was found to be 4.0 in the second group, which was not vaccinated. It was confirmed that the respiratory disease caused by the causative agent has a prophylactic effect.

As described above, according to the present invention, it was confirmed that there is an effect of preventing respiratory diseases caused by porcine circoviruses and pleural pneumonia.

While the invention has been described in connection with specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made within the scope of the invention without departing from the spirit of the invention supported by the following claims.

Claims (7)

It comprises swine circovirus type 2 and pleural pneumococcal type 2 and type 5, wherein the pig circovirus type 2 is a state isolated from the swine development pigs of swine complex respiratory disease, using the deposit of KCCM 10688P deposited. And, the pleural pneumococcal type 2 and type 5 are isolated from the swine complex respiratory disease development growth pigs in Korea using the deposit number KMCCM 10686P and KMCCM 10687P, respectively, biological composition for the prevention and treatment of swine complex respiratory disease. [Claim 2] The biological composition for preventing and treating swine complex respiratory disease according to claim 1, wherein the circovirus type 2 and pleural pneumococci type 2 and 5 are inactivated. The biological composition of claim 2, wherein the inactivation is made of formalin. The method of claim 1, wherein the mixing ratio of the pig circovirus type 2 and pleural pneumococcus is mixed at a ratio of 1: 0.5 to 1: 1.5 (V / V), wherein the type 2 and 5 of the pleural pneumonia are The mixing ratio is 1: 0.5 to 1: 1.5 (V / V) of the biological composition for the prevention and treatment of swine complex respiratory disease, characterized in that the mixing The method of claim 4, wherein the mixing ratio of swine circovirus type 2 and pleural pneumococcal type 2 and type 5 is mixed at a ratio of 2: 1: 1 (V / V). Biological Compositions A method for protecting pigs from swine complex respiratory diseases comprising administering to a pig in need thereof an effective amount of a composition according to claims 1 to 5 The method of claim 6, wherein the composition is administered orally, nasal, anal, intramuscular, intradermal, intravenous or subcutaneous.