KR102467292B1 - Composition for respiratory disease treatment of animal by combined bacterial infection - Google Patents

Abstract

Actinobacillus pleuropneumoniae , which is the main bacterium of respiratory diseases, Pasteurella multocida and Mycoplasma hyopneumoniae Due to complex infection by bacteria including A therapeutic agent capable of effectively coping with respiratory diseases of pigs is disclosed. The present invention provides a composition for treating bacterial respiratory diseases in animals, comprising Marbofloxacin, Flunixin meglumin and Butaphosphan as active ingredients.

Description

Composition for respiratory disease treatment of animal by combined bacterial infection {Composition for respiratory disease treatment of animal by combined bacterial infection}

The present invention relates to a composition for treating respiratory diseases in animals, and more particularly, to a composition for treating respiratory diseases in animals caused by multiple infections of bacteria.

Pig respiratory disease (Swine Respiratory Disease, SRD) is a representative disease that causes mortality, growth retardation, weight gain reduction, and various economic losses such as increased costs for disease treatment. The causes of respiratory diseases in pigs are viruses such as porcine reproductive and respiratory syndrome (PRRS) virus and swine influenza virus (SIV) and Actinobacillus pleuropneumoniae , APP), Bordetella bronchiseptica ( Bordetella bronchiseptica ), Haemophilus parasuis ( Haemophilus parasuis ), Mycoplasma hyopneumoniae ( Mycoplasma hyopneumoniae ) It is caused by bacteria such as, and is secondary to Pasteurella of Pasteurella multoc ida, Streptococcus suis , Haemophilus parasuis , Actinobacillus suis, and Salmonella choleraesuis Symptoms are severe and last for a long time due to multiple infections.

In general, fever, respiratory symptoms (cough, runny nose, etc.), and anorexia appear when a bacterial respiratory disease occurs, and when acute high fever or fever persists, physiological changes such as reduced feed intake and depression are induced, and secondary respiratory viruses It is very important to promptly implement symptomatic treatment at an early stage because it is vulnerable to infection by

Marbofloxacin is a third-generation fluoroquinolone antibiotic that has a bactericidal action against gram-negative bacteria, some gram-positive bacteria, and Mycoplasma , including Pasteurella and Haemophilus. Marbofloxacin, like other third-generation fluoroquinolones, inhibits DNA-gyrase and topoisomerase IV to inhibit DNA replication and transcription, killing bacteria and killing Gram-positive bacteria. It exhibits a bactericidal action in a time-dependent manner, and causes a bactericidal action in a dose-dependent manner against Gram-negative bacteria. Marbofloxacin is a lipid-soluble organic acid that has a low binding to plasma proteins (less than 10%) and is distributed in high concentrations in tissues because of its good tissue permeability.

Flunixin Meglumine is a non-steroidal anti-inflammatory drug (NSAID) used in cattle and pigs. It inhibits cyclooxygenase (COX) involved in inflammatory reactions and reduces prostaglandin synthesis, resulting in anti-inflammatory, antipyretic and analgesic effects. indicates It is reported that an effective analgesic effect appears within 15 minutes when an appropriate amount of flunixin is administered, and the effect lasts up to 30 hours. Flunixin is mainly used for colic, musculoskeletal pain and ocular pain, and is used as an antipyretic to reduce the effects of endotoxemia. It is reported as

Butaphosphane (1-butylamino-1-methylethyl phosphonic acid) is a kind of organic phosphoric acid compound used as a phosphorus source in cattle, horses, pigs, sheep and goats, in liver carbohydrate metabolism and ATP synthesis. It is reported to improve energy metabolism by acting as a source of human phosphorus, stimulate feed intake, improve digestive function, improve immune function, improve liver and muscle function, improve homeostasis control function, and reduce stress.

These major drugs are prescribed in combination of one or two and are applied to various veterinary medicines, but the effect of the combination of the major drugs is unknown, and on the other hand, Actinobacillus flu, a major bacterium in respiratory diseases New moniae ( Actinobacillus pleuropneumoniae ), Pasteurella multocida ( Pasteurella multocida ) and Mycoplasma hyopneumoniae ( Mycoplasma hyopneumoniae ) A treatment that can effectively cope with the outbreak of respiratory diseases in pigs due to complex infection by bacteria is not presented.

Korean Patent Registration No. 1751773 discloses a multifunctional antibiotic composition for the treatment of respiratory diseases that can minimize lung damage caused by inflammatory reactions by using mabofloxacin, tylosin, and flunixin in combination, and as a pathogen causing respiratory diseases Actinobacillus pleuropneumoniae , Pasteurella multocida , Bordetella bronchiseptica , Haemophilus parasuis or Mycoplasma hyonyu Mycoplasma hyopneumoniae is mentioned.

In addition, Korean Patent Registration No. 2086462 discloses a combination antibiotic composition for veterinary treatment of respiratory diseases accompanied by E. coli or Salmonella infection, which contains cefquinom, lincomycin and butaphosphan as main components and chlorpheniramine maleate as a minor component, As pathogens causing respiratory diseases, Pasteurella multocida , Actinobacillus pleuropneumoniae or Streptococcus suis are mentioned.

However, referring to the specific test examples of these patents, it only shows a therapeutic effect on a single pathogen, and the main bacteria of respiratory diseases, Actinobacillus pleuropneumoniae , Pasteurella multocida multocida ) and Mycoplasma hyopneumoniae ( Mycoplasma hyopneumoniae ) There is no treatment that can effectively cope with the onset of respiratory diseases caused by complex infection by bacteria.

Therefore, the present invention is a major bacterium of respiratory diseases, Actinobacillus pleuropneumoniae ( Actinobacillus pleuropneumoniae ), Pasteurella multocida ( Pasteurella multocida ) and Mycoplasma hyopneumoniae ( Mycoplasma hyopneumoniae ) caused by bacteria including We would like to present a treatment that can effectively cope with respiratory diseases in pigs caused by complex infection.

In order to solve the above problems, the present invention provides a composition for treating bacterial respiratory diseases in animals containing Marbofloxacin, Flunixin meglumin and Butaphosphan as active ingredients .

In addition, the respiratory disease is caused by a complex infection of bacteria including Actinobacillus pleuropneumoniae , Pasteurella multocida and Mycoplasma hyopneumoniae It provides a composition for treating bacterial respiratory diseases of animals, characterized in that the disease.

In addition, the content of the active ingredient is 50 to 150 parts by weight of the marbofloxacin, 41.5 to 166 parts by weight of the flunixin meglumin, and 50 to 150 parts by weight of the butaphosphan. It provides a composition for treating bacterial respiratory diseases of animals.

In addition, the composition is formulated as an injection for pigs, and the dosage per prescription is 0.5 to 5 mg of Marbofloxacin, 0.2 to 3 mg of Flunixin and the butaphosphan per 1 kg of body weight. Butaphosphan) provides a composition for treating bacterial respiratory diseases in animals, characterized in that 0.5 to 5 mg.

The composition for treating bacterial respiratory diseases in animals according to the present invention contains marbofloxacin, flunixin meglumin and butaphosphan as active ingredients, and furthermore, the content ratio and By specializing the formulation and dosage, the main bacteria of respiratory diseases, Actinobacillus pleuropneumoniae , Pasteurella multocida , and Mycoplasma hyopneumoniae , including It can dramatically improve the treatment effect of respiratory diseases in pigs due to complex infection by bacteria.

1 is an electrophoresis photograph showing the results of confirming the presence or absence of pathogens by performing PCR to confirm the discharge of inoculated bacteria in an embodiment of the present invention;
Figure 2 is a graph showing the change in feed intake during the test period after inoculation of bacteria and administration of the test product in an embodiment of the present invention;
3 is a graph showing changes in body temperature after bacterial inoculation and test product administration in an embodiment of the present invention;
4 is a photograph showing respiratory clinical symptoms of pigs co-infected with Actinobacillus pleuropneumoniae , Pasteurella multocida and Mycoplasma hyopneumoniae in an embodiment of the present invention;
5 is a graph showing clinical symptom indicators according to the presence or absence of drug treatment after bacterial inoculation in an embodiment of the present invention;
6 is a graph showing the results of measuring the number of bacteria in the nasal cavity ( A. pleuropneumoniae ) according to the presence or absence of drug treatment after bacterial inoculation in an embodiment of the present invention;
7 is a graph showing the results of measuring the number of bacteria ( P. multocida ) in the nasal cavity according to the presence or absence of drug treatment after bacterial inoculation in an embodiment of the present invention;
8 is a photograph showing the lungs of an untreated group in an embodiment of the present invention;
9 is a photograph showing the lungs of a pathogen-infected group in an embodiment of the present invention;
10 is a photograph showing the lungs of the pathogen + maboxin-B strain test group in an embodiment of the present invention;
11 is a photograph showing the lungs of the pathogen + Mabomax test group in an embodiment of the present invention;
12 is a photograph showing lung tissue of an untreated group in an embodiment of the present invention;
13 is a photograph showing lung tissue of a pathogen-infected group in an embodiment of the present invention;
Figure 14 is a photograph showing the lung tissue of the pathogen + maboxin-B strain test group in an embodiment of the present invention;
15 is a photograph showing the lung tissue of the pathogen + Mabomax test group in an embodiment of the present invention;
16 is a graph showing changes in blood TNF-α content of each test group in Examples of the present invention;
17 is a graph showing changes in IL-1β content in the serum of each test group in Examples of the present invention;
18 is a graph showing changes in IL-6 content in the serum of each test group in Examples of the present invention;
19 is a graph showing changes in PGE ₂ content in the serum of each test group in Examples of the present invention;
20 is a graph showing changes in glucose content in the serum of each test group in Examples of the present invention;
21 is a graph showing changes in serum insulin content of each test group in Examples of the present invention;
22 is a graph showing changes in glucagon content in the serum of each test group in Examples of the present invention;
23 is a graph showing changes in total cholesterol content in the serum of each test group in Examples of the present invention;
24 is a graph showing changes in TG content in serum of each test group in Examples of the present invention;
25 is a graph showing changes in phosphorus content in the serum of each test group in Examples of the present invention;
26 is a graph showing changes in urea content in serum of each test group in Examples of the present invention;
27 is a graph showing the Calpain I activity measurement results of each test group in Examples of the present invention.

Hereinafter, the present invention will be described in detail through examples. Prior to this, the terms or words used in this specification and claims should not be construed as being limited to the usual or dictionary meaning, and the inventor appropriately uses the concept of the term in order to explain his/her invention in the best way. Based on the principle that it can be defined, it should be interpreted as meaning and concept consistent with the technical spirit of the present invention. Therefore, since the configurations of the embodiments described in this specification are merely the most preferred embodiments of the present invention and do not represent all of the technical spirit of the present invention, various equivalents and modifications that can replace them at the time of this application It should be understood that there may be

In a composition for treating bacterial respiratory diseases in animals, the present inventors have found that, in particular, by including Marbofloxacin, Flunixin meglumin and Butaphosphan as active ingredients, Actinobacillus fluro New moniae ( Actinobacillus pleuropneumoniae ), Pasteurella multocida ( Pasteurella multocida ) and Mycoplasma hyopneumoniae ( Mycoplasma hyopneumoniae ) Confirm that it shows a dramatic effect on the treatment of respiratory diseases caused by complex infections have led to the present invention.

Accordingly, the present invention provides a composition for treating bacterial respiratory diseases in animals, comprising marbofloxacin, flunixin meglumin and butaphosphan as active ingredients.

According to an application example of the present invention, the composition is active against bacteria including Actinobacillus pleuropneumoniae , Pasteurella multocida and Mycoplasma hyopneumoniae . It is particularly suitable for respiratory diseases caused by complex infections.

At this time, in the present invention, it was confirmed that a specific content of the active ingredient exists for maximizing the therapeutic effect of respiratory diseases caused by the bacterial complex infection, specifically, the marbofloxacin 50 to 150 parts by weight, 41.5 to 166 parts by weight of the flunixin meglumin (25 to 100 parts by weight as flunixin) and 50 to 150 parts by weight of the butaphosphan, and 80 to 120 parts by weight of marbofloxacin Part by weight, preferably 60 to 100 parts by weight of Flunixin meglumin and 80 to 120 parts by weight of Butaphosphan, more preferably 90 to 110 parts by weight of Marbofloxacin Part, 70 to 90 parts by weight of Flunixin meglumin and 90 to 110 parts by weight of Butaphosphan, more preferably 95 to 105 parts by weight of Marbofloxacin, the 77 to 87 parts by weight of Flunixin meglumin and 95 to 105 parts by weight of Butaphosphan, most preferably 98 to 102 parts by weight of Marbofloxacin, the flunixin meglu Min (Flunixin meglumin) 81 to 85 parts by weight and the butaphosphan (Butaphosphan) 98 to 102 parts by weight.

In addition, based on the content ratio as an excipient together with the active ingredient, 100 to 250 parts by weight of propylene glycol (solvent), 50 to 150 parts by weight of Gluconolacton, 30 to 130 parts by weight of pH HCl (hydrochloric acid) or NaOH (sodium hydroxide) 30 to 130 parts by weight so that the composition has a pH of 7.0 to 11.

In addition, in order to maximize the therapeutic effect of respiratory diseases caused by the complex infection of the bacteria, it is preferable to formulate an injection. mg, preferably 0.2 to 3 mg of Flunixin and 0.5 to 5 mg of Butaphosphan, more preferably 1 to 3 mg of Marbofloxacin and 0.5 mg of Flunixin to 2 mg and the butaphosphan 1 to 3 mg, most preferably the marbofloxacin 1.5 to 2.5 mg, the flunixin 0.5 to 1.5 mg and the butaphosphan 1.5 to 2.5 mg.

When the composition for treatment according to the present invention is applied as an injection, as a pharmaceutically acceptable carrier, a pyrrolidone solvent, N,N-dimethylamide, N,N-dimethylformamide, DMSO (dimethyl sulfoxide), acetone, glycerol Glycerol formal, N-Methyl-2-Pyrrolidone, 2-Pyrrolidone, ethyl oleate, ethanol, isopropanol, 1,2-propanediol , glycerin, benzyl alcohol, dimethylisosorbit, triacetin, glycoether, propylene glycol, polyethylene glycol having an average molecular weight of about 200 to 600, triacetin, lidocaine, propylene glycol dicaprylate , Medium chain triglycerides, aluminum stearate, etc. can be used, and as antioxidants, ascorbic acid, sodium hydrogensulfite, sodium pyrosulfite, tocopherol, butylated hydroxyanasol, monothioglycerin etc. can be used, and as a preservative, methyl p-hydroxybenzoate (methylparaben), propyl p-hydroxybenzoate (propylparaben), thimerosal, benzalkonium chloride, phenol, cresol, paraoxymethylbenzoate It can be formulated using the like.

In addition, when administering the composition for treatment according to the present invention, in the case of an injection, it may be administered by a conventional intramuscular injection method.

Hereinafter, the present invention will be described in detail with reference to examples. In this example, the antibacterial and bactericidal effects of mabofloxacin on the causative agents of bacterial respiratory diseases in pigs were investigated, and Actinobacillus fluronumoniae ( Actinobacillus pleuropneumoniae ), Pasteurella multocida ( Pasteurella multocida ) and Mycoplasma high The efficacy and safety of the composition for treating respiratory diseases according to the present invention were evaluated by examining the clinical efficacy and safety in pigs for swine respiratory diseases caused by complex infections of Mycoplasma hyopneumoniae .

Materials and Methods

1. Manufacture of test products

Compositions according to Examples (Maboxin-B strain) and Comparative Examples (Mabomax 10% strain) were prepared with the following composition, formulation and properties, usage and dosage.

(1) Maboxin-B strain

1) Composition (in 1 mL of this drug): Marbofloxacin 100 mg, Flunixin meglumin 83 mg (50 mg as Flunixin), Butaphosphan 100 mg, as an appropriate amount of excipient Gluconolactone (KVP) 100 to 250 mg, monothioglycerin (KVP), sodium hydroxide (Sodium Hydroxide, KVP) 30 to 130 mg, propylene glycol (Propylene Glycol, KVP) 100 to 250 mg and an appropriate amount Water for Injection.

2) Formulation and Appearance: Injection, yellow or slightly yellow transparent liquid.

3) Usage and dosage: Swine. Intramuscular injection once a day for 3 to 5 days at the rate of 1 mL of this drug per 50 kg of body weight (2 mg of marbofloxacin, 1 mg of flunixin, and 2 mg of butaphosphan per 1 kg of body weight).

(2) Mabomax 10% share

1) Composition (in 1 mL of this product): Marbofloxacin 100 mg as an active ingredient, gluconolactone, monothioglycerol, disodium edetate, monoethanolamine, and water for injection as additives in appropriate amounts.

2) Formulation and Appearance: Injection, yellow or slightly yellow transparent liquid.

3) Usage and dosage: Swine. Intramuscular injection once a day for 3 to 5 days at the rate of 1 mL of this drug per 50 kg of body weight (2 marbofloxacin per 1 kg of body weight).

2. Clinical efficacy test

Animal experiments were conducted after obtaining approval through the review procedure of the Hoseo University Animal Experiment Ethics Committee (approval number: HSIACUC-19-138(1)).

(1) Experimental animals

1) Lineage, species and gender: Conventional Landrace pig, male (castrated pig)

2) Number of animals upon acquisition: 25 (45-50 kg)

3) Source: Hanabio Co., Ltd. (Suwon-si, Korea)

4) Animal acclimatization: After obtaining the animals, they were acclimatized in the animal room for one week.

5) Breeding environment: The animal was bred using the Hoseo University Safety Evaluation Center animal laboratory set to environmental conditions of temperature 22 ± 3 ° C, ventilation frequency of 10 to 15 times / hour, lighting cycle of 12 hours, and illumination of 150 to 300 Lux.

(2) Identification of the inoculated strain

In order to check whether the inoculated bacteria were excreted from the test animal, PCR was performed using the primers shown in Table 1 below to confirm the presence or absence of pathogens, and the results were shown in FIG. 1, and it was confirmed that each bacteria was not detected.

Bacterium Nucleotide sequences (5'→3') Target Band
(bp) APP F: ATA CGG TTA ATG GCG GTA ATG G
R: ACC TGA GTG CTC ACC AAC G 346 APP type 2 F: ACT ATG GCA ATC AGT CGA TTC AT
R: CCT AAT CGG AAA CGC CAT TCT G 500 PM F : ATC CGC TAT TTA CCC AGT GGR : GCT GTA AAC GAA CTC GCC AC 460 PM typeA F : TGC CAA AAT CGC AGT CAGR : TTG CCA TCA TTG TCA GTG 1044 Mycoplasma hyopneumoniae
(MH) F: ACTA GAT AGG AAA TGC TCT AGT
R: GTG GAC TAC CAG GGT ATC T 352

(3) Composition of test group and challenge inoculation

1) Composition of test group

The composition of the test group was configured as shown in Table 2 below.

test group treatment method Dusu untreated untreated control group 5 2-dose drug administration group 5-day injection into the muscle at a rate of 2 mL of test product per 50 kg of body weight 5 like
suction
energy
quality
ring pathogen infection group APP, PM, M. hyopneumoniae intranasal inoculation
- APP : 1 mL (5.06Х10 ⁸ CFU/mL)
- PM : 2 mL (5.81Х10 ⁹ CFU/mL)
- MH : 2 mL (5Х10 ⁸ CCU/mL) 5 Pathogen + Marboxin-B strain Intranasal inoculation of APP, PM, M. hyopneumoniae strains, confirmation of clinical symptoms, intramuscular injection at the rate of 1 mL of this drug per 50 kg of body weight for 5 days 5 Pathogen + Magic Antidote Intranasal inoculation of APP, PM, M. hyopneumoniae strains and confirmation of clinical symptoms, intramuscular injection for 5 days at a rate of 1 mL of a single drug (Mabomax 10% stock, Marbofloxacin 100 mg/mL) per 50 kg of body weight 5

2) Attack inoculation

To induce respiratory disease in pigs, 1 mL of APP (5.06×10 ⁸ CFU/mL), 2mL of PM (5.81×10 ⁹ CFU/mL, and 2mL of MH (5×10 ⁸ CCU/mL) were inoculated into the nasal cavity. All subjects The test substance was administered after the manifestation of clinical symptoms was confirmed.

(4) Clinical efficacy index

1) Weight measurement

Changes in daily weight gain were investigated by measuring body weight before induction of bacterial respiratory disease and before test product administration (Day 0) and on days 1, 3, 7, and 14 after administration. The daily weight gain was the value obtained by dividing the number of test days by the value obtained by subtracting the test product administration day (Day 0) from the body weight at the end of the experiment (Day 14).

2) Measurement of feed intake

Planned feeding was conducted to measure feed intake. That is, 2.5 to 3.5 kg of feed per head was supplied during the experiment period, considering the feed requirement per head per day. That is, the feed intake per head was measured by measuring the daily feed supply and remaining amount, and the feed was supplied by reflecting the feed intake per head the previous day.

3) Observation of clinical symptoms

Clinical symptoms were observed for 14 days from the start of test substance administration to the end of the experiment (refer to Table 3, clinical symptom indicators of respiratory diseases).

observation Score Appearance/behavioral abnormalities normal
(Movement is active and responds immediately to stimuli) 0 dullness
(Movements are slightly sluggish, responding to stimuli but slow) One Depression (movement is sluggish, responding to stimuli but very slowly) 2 transverse
(little movement, unresponsive to stimuli) 3 body temperature (rectal temperature) 38~39.5℃ (normal) 0 39.51~40.5℃ One > 40.5℃ 2 < 38 3 skin/hair normal 0 coarse hair One Cyanosis (nose, ears, legs) 2 Respiratory pattern or degree normal 0 increased effort, some abdominal breathing One Difficult breathing, abdominal breathing, panting 2 Dyspnea, open breathing 3 breathing rate
(Number of times/min) 18-40 0 41-60 One 61-80 2 81 to 100 3 >100 4 cough hits doesn't exist 0 1 to 10 One 11-30 2 31-50 3 > 51 4 cough pattern doesn't exist 0 dry cough One wet cough (phlegm) 2 frequent habitual cough 3 runny nose secretion doesn't exist 0 handful One usually 2 much 3 runny nose normal 0 clear runny nose One viscous grayish-white runny nose 2 mucopurulent 3 death Dead 20

4) Hematology analysis

Hematology and blood chemistry analysis was performed once before administration of the test product and on days 1, 4, 7, and 14 after administration of the test product.

For hematology analysis, blood was collected from the jugular vein, 1 mL of blood was put into a potassium EDTA bottle, mixed, and hematology was analyzed. WBC, RBC, hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean hemoglobin Mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet (PLT) were measured.

5) Measurement of the number of bacteria

After bacterial inoculation and drug administration, a sample was taken from the nasal cavity using a culture swab (Difco), cut and immersed in 1 mL of sterile physiological saline, and allowed to stand for 3 minutes. Then, after stirring for about 30 seconds, the mixture was diluted 10-fold up to 10-6. The stock solution and the 10-fold dilution were inoculated into chocolate agar and 5% sheep blood agar and incubated for 18 to 24 hours in an incubator at 37°C, 5% CO ₂ or 37°C, and then the number of bacterial colonies was counted. In the case of Mycoplasma hyopneumoniae , DNA was extracted from nasal exudate and PCR was performed to confirm the presence and presence of infection.

6) Autopsy and pathological findings

Pigs in all test groups were euthanized on the 14th day after test product administration at the end of the experiment, and gross findings were observed by necropsy (see Table 4, lung lesion score below), and tissue in which gross lesions were observed was collected and 10% After fixation in neutral formalin, H&E staining was performed to observe histopathological findings.

division score standard presence of pulmonary consolidation 0 no hardened area One Observation of 1 lesion of consolidation 2 Consolidation is observed in multiple areas of one lobe or consolidation is observed in one large area 3 Observation of consolidation across one lobe 4 Sclerosis develops over several lobes, abscess observed Presence of adhesions, pleurisy 0 No chest wall or interlobular adhesions and no pleurisy One Adhesions or pleurisy observed in less than 50% of the lung/pleural surface 2 Adhesions or pleurisy observed in >50% of the lung/pleural surface

(5) Antipyretic, analgesic, anti-inflammatory and metabolism promoting effect indicators

1) Analysis of serum cytokine content

Before challenge, before drug administration, and 1, 4, 7, and 14 days after drug administration, blood was collected from pig jugular veins, coagulated, centrifuged, and serum was collected and stored at -80 ° C.

- TNF-α content analysis

In order to analyze the effect of drugs on serum TNF-α secretion, the TNF-α content in serum was analyzed using an ELISA kit (Mybiosource). That is, 100 μL of serum was dispensed into each well to which Porcine TNF-α monoclonal antibody was attached, reacted at 37° C. for 90 minutes, and after the reaction was finished, washed twice with washing buffer. After washing, 100 μL of the previously prepared biotinylated Porcine TNF-α antibody solution was dispensed, reacted at 37° C. for 60 minutes, and washed three times. 100 μL of enzyme-conjugate liquid was dispensed, reacted at 37°C for 30 minutes, and washed 5 times with washing buffer. After dispensing 100 μL of the Color Reagent solution into each well, react at 37 ° C for about 30 minutes, stop the color reaction by adding 100 μL of Color Reagent C, and then measure the absorbance at 450 nm using an ELISA reader (Hidex sense). .

- Serum IL-1β content analysis

In order to analyze the effect of the drug on serum IL-1β, the serum content was analyzed using ELISA kit (Bioassay Technology Laboratory). That is, 40 μL of serum, 10 μL of anti-IL-1β antibody, and 50 μL of streptavidin-HRP were dispensed and mixed into wells to which Porcine IL-1β antibody was attached, and reacted at 37°C for 60 minutes. After the reaction, washing was completed. Washed 5 times with buffer. After adding 50 μL each of Substrates A and B, they were reacted at 37 ° C for 10 minutes. After stopping the color reaction by adding 50 μL of stop solution, the reaction was performed at 450 nm using an ELISA reader (Hidex sense). The absorbance was measured at.

- Serum IL-6 content analysis

In order to analyze the effect of the drug on serum IL-6, the serum content was analyzed using an ELISA kit (Bioassay Technology Laboratory). That is, 40 μL of serum, 10 μL of anti-IL-6 antibody, and 50 μL of streptavidin-HRP were dispensed and mixed into wells to which Porcine IL-6 antibody was attached, followed by reaction at 37°C for 60 minutes, and washing after the reaction was completed. Washed 5 times with buffer. After adding 50 μL each of Substrates A and B, they were reacted at 37 ° C for 10 minutes. After stopping the color reaction by adding 50 μL of stop solution, the reaction was performed at 450 nm using an ELISA reader (Hidex sense). The absorbance was measured at.

- Serum PGE ₂ content analysis

In order to analyze the PGE ₂ content in blood, the PGE ₂ content in serum was analyzed using an ELISA kit (Mybiosource). That is, after dispensing 100 μL of serum into wells to which Porcine PGE ₂ monoclonal antibody was attached, the mixture was reacted at 37° C. for 90 minutes, and after the reaction was completed, washed twice with washing buffer. Thereafter, 100 μL of biotinylated PGE ₂ antibody was dispensed and reacted at 37° C. for 60 minutes, and after the reaction was finished, washed three times with washing buffer. After washing, 100 μL of enzyme-conjugate liquid was dispensed, reacted at 37 ° C for 30 minutes, and washed 5 times with washing buffer. After adding and mixing 100 μL of color reagent liquid, a color reaction was induced at 37° C. for 15 to 30 minutes, and 100 μL of color reagent C was added to stop the color reaction. After stopping the color reaction, absorbance was measured at 450 nm using an ELISA reader (Hidex sense).

2) Metabolism accelerating effect index

- Blood chemistry analysis

Glucose (GLU), total cholesterol (T-CHO), triglyceride (TG), phosphorus (P), Blood urea nitrogen (BUN) concentration analysis was measured using a blood chemistry analyzer (COBAS C702), and phosphorus (P) was analyzed for inorganic phosphate (inogarnic phosphate). The blood urea concentration was analyzed by converting BUN.

- Analysis of glucagon content

In order to analyze the glucagon content in pig blood, the glucagon content in serum was analyzed using an ELISA kit (Mybiosource). That is, the wells to which the Porcine glucacon monoclonal antibody was attached were washed twice, and then 100 μL of serum was dispensed and reacted at 37° C. for 90 minutes. After the reaction was completed, the wells were washed twice with washing buffer. Thereafter, 100 μL of Biotin-labeled Antibody was dispensed and reacted at 37° C. for 60 minutes. After the reaction was completed, it was washed three times with washing buffer. After washing, 100 μL of HRP-Streptavidin Conjugate was dispensed and reacted for 30 minutes, then 100 μL of enzyme-conjugate liquid was dispensed and reacted at 37 ° C for 30 minutes. After the reaction was completed, the mixture was washed 5 times with washing buffer. After adding 90 μL of TMB Substrate to all wells, wrapping in foil, a color reaction was induced at 37 ° C for 10 to 20 minutes, and the color reaction was stopped by adding 50 μL of Stop solution. After stopping the color reaction, absorbance was measured at 450 nm using an ELISA reader (Hidex sense).

- Insulin content analysis

In order to analyze the insulin content in pig blood, the serum insulin content was analyzed using an ELISA kit (Millipore). That is, after dispensing 100 μL of serum into wells to which Porcine insulin antibody was attached, the mixture was reacted with shaking at room temperature for 150 minutes, and then washed 4 times with washing buffer. After washing, 100 μL of Detection Antibody was dispensed into each well, reacted while shaking at room temperature for 60 minutes, and washed 4 times with washing buffer. After washing, 100 μL of Streptavidin Solution was added to each well, shaken at room temperature for 45 minutes, and washed 4 times. After washing, 100 μL of TMB one-step substrate reagent was added to each well, wrapped in foil, and reacted while shaking at room temperature for 30 minutes. The color reaction was stopped by adding 50 μL of Stop solution to each well. After stopping the color reaction, absorbance was measured at 450 nm using an ELISA reader (Hidex sense).

- Measurement of Calpain I activity

In order to analyze Calpain I activity in pig muscle tissue, the content in muscle tissue was analyzed using an ELISA kit (Mybiosource). That is, muscle tissue collected after autopsy was washed with ice-cold PBS to remove blood present in the muscle, and 3 g of muscle was homogenized with 30 mL ice-cold and stored at -20 ° C for one day. Thereafter, thawing and freezing were repeated twice, centrifuged at 5,000 rpm for 5 minutes, and the supernatant was collected and stored at -20 ° C until the day of analysis. After melting the frozen sample, 100 μL was dispensed into each well and reacted at 37 ° C for 2 hours. After the reaction was finished, the contents were removed, and 100 μL of Detection Reagent A was dispensed and reacted at 37 ° C for 1 hour. After washing three times with Washing buffer, 100 μL of Detection Reagent B was added, reacted at 37°C for 1 hour, and washed 5 times with Washing buffer. Then, 90 μL of substrate solution was added, wrapped in foil, and reacted at 37 ° C for 20 minutes. After stopping the color reaction by adding 50 μL of stop solution, the absorbance was measured at 450 nm using an ELISA reader (Hidex sense).

statistical processing

Statistical processing was performed by F-test using the STATISTICA program. That is, one-way ANOVA analysis using LSD was performed, followed by post-hoc test by Duncan analysis.

Test result

1. Clinical efficacy test results

Actinobacillus pleuropneumoniae (APP), Pasteurella multocida (PM), and Mycoplasma hyopneumooniae (MH) were inoculated once intranasally to pigs in the test group, except for the untreated group, to induce respiratory disease in pigs, and the pathogen + Marboxin B injection group and the pathogen Pigs in the +Mabomax-administered group were intramuscularly injected with 1 mL of the test substance per 50 kg of body weight for 5 days, and changes in clinical symptoms were observed.

(1) Weight change

On the day of inoculation, the body weights of the untreated group, the pathogen-infected group, and the pathogen+drug group were 54.22±1.15 kg, 53.74±1.25 kg, 53.62±1.17 kg, and 53.78±1.89 kg, respectively. Two days after bacterial inoculation, the test product was intramuscularly injected for 5 days in the pathogen + Marboxin-B administration group and the pathogen + Marbomax administration group. The body weight of the Max-administered group was significantly lower than that of the untreated group.

At 14 days after drug administration, the average body weights of the untreated group, the pathogen-infected group, the pathogen+Maboxin-B main-administered group, and the pathogen+Mabomax-administered group were 72.38±1.08 kg, 58.92±2.60 kg, 66.92±3.26 kg, and 64.02±2 kg, respectively. 3.12 kg, and the total weight gain of each test group was 16.96±1.25 kg, 7.12±3.05 kg, 15.96±3.83 kg, and 12.44±1.94 kg. In addition, the daily weight gain of each test group was 1.21 ± 0.09 kg / day, 0.51 ± 0.22 kg / day, 1.14 ± 0.27 kg / day, and 0.89 ± 0.14 kg / day, respectively (see Table 5 below).

The total weight gain and daily weight gain of the pathogen + Marboxin-B main administration group and the pathogen + Mabomax administration group increased significantly compared to the pathogen infection group. , The total weight gain and daily weight gain of the pathogen + Marboxin-B week administration group showed a tendency to increase compared to the pathogen + Mabomax administration group (see Table 5 below).

test group Weight change (kg) Total weight gain (kg)
(D14-D0) daily weight gain
(kg/day) fungal inoculation
(D-2) Initiation of administration
(Day 0) end of experiment
(Day 14) untreated 54.22±1.15 55.42±1.02 72.38±1.08 16.96±1.25 1.21±0.09 pathogen infection group 53.74±1.25 51.80±0.68 ^*** 58.92±2.60 ^*** 7.12±3.05 ^*** 0.51±0.22 ^*** Pathogen + Marboxin-B strain 53.62±1.17 50.96 ± 1.43 ^*** 66.92±3.26 ^**,### 15.96±3.83 ^### 1.14±0.27 ^### pathogen
+Mabomax 53.78±1.89 51.58 ± 1.58 ^*** 64.02±3.12 ^***,## 12.44±1.94 ^*,## 0.89±0.14 ^*,##

^* p < 0.05, ^** p < 0.01, ^** p < 0.001 significantly different from normal group

^## p <0.01, ^### p <0.001 significantly different from challenge group

(2) Feed efficiency

For accurate measurement of feed intake, 3.0 to 4.0 kg of feed was supplied per head per day. During the experimental period, the total feed intake for each individual in the untreated group, the pathogen-infected group, the pathogen + Maboxin-B main administration group, and the pathogen + MavoMax administration group was 44.95 ± 0.67 kg, 24.57 ± 8.33 kg, 39.81 ± 4.92 kg, and 34.35 ± 34.35 ± 3 kg, respectively. 2.14 kg, and the feed efficiency of each group was 0.38±0.03, 0.28±0.04, 0.40±0.05, and 0.36±0.04, respectively. It was significantly higher than the pathogen infection group (see Table 6 and FIG. 2 below).

test group total weight gain
(kg/animal) total feed intake
(kg/animal) feed efficiency untreated 16.96±1.25 44.95±0.67 0.38±0.03 pathogen infection group 7.12±3.05 ^*** 24.57 ± 8.33 ^*** 0.28±0.04 ^** Pathogen + Marboxin-B strain 15.96±3.83 ^### 39.81±4.92 ^### 0.40±0.05 ^### Pathogen + Mabomax 12.44±1.94 ^*,## 34.35±2.14 ^**,## 0.36±0.04 ^##

^* p < 0.05, ^** p < 0.01, ^** p < 0.001 significantly different from normal group

^## p <0.01, ^### p <0.001 significantly different from challenge group

(3) Changes in body temperature

Actinobacillus pleuropneumoniae (APP), Pasteurella multocida (PM), and Mycoplasma hyopneumooniae (MH) were inoculated once intranasally to pigs in the test group, except for the untreated group, to induce respiratory disease in pigs, and the pathogen + Marboxin B injection group and the pathogen Pigs in the +Mabomax-administered group were intramuscularly injected with 1 mL of test substance per 50 kg of body weight for 5 days, and body temperature was measured twice a day (morning and afternoon) to observe changes in body temperature.

An increase in body temperature was observed in all test groups except for the untreated group until the second day (Day 0) after challenge inoculation (see FIG. 3).

In the case of the pathogen-infected group, obvious fever was observed in the initial stage of infection in most individuals, and then body temperature was maintained high until the end of the experiment in most individuals (see FIG. 3).

In the case of the group administered with the pathogen + maboxin-B, the body temperature decreased significantly in 4 animals on the 1st day after the test substance was administered, and the body temperature was significantly lower than the pathogen-infected group. , On the 4th day, all subjects showed normal body temperature, and a temporary increase in body temperature was observed in 1 or 2 subjects from the 5th day to the end of the experiment, but no significant difference was observed compared to the untreated group. In addition, the group administered with pathogen + maboxin-B showed a significantly lower body temperature than the pathogen-infected group on the 1st, 4th, 7th, 8th and 10th days after drug administration, and on the 4th and 10th days after drug administration The body temperature was significantly lower than that of the pathogen + Mabomax administration group (see FIG. 3).

In the case of the control drug pathogen + Mabomax administration group, the body temperature recovered to normal in 3 animals except 2 animals on the first day after drug administration, and continuously high body temperature was observed in 1 or 2 animals, and at some time point, the untreated group In comparison, the body temperature was significantly higher (see FIG. 3).

As a result of checking the change in body temperature of each test group after challenge inoculation and administration of the test substance, the pathogen + Mavosin-B injection group showed an antipyretic effect earlier than the pathogen + Mavomax administration group, and the antipyretic effect was continuously confirmed (Fig. see 3)

(4) Changes in hematology

Changes in the total number of white blood cells by bacterial inoculation and test product administration were observed.

The total number of leukocytes in the pathogen-infected group was 38.1±4.6×10 ⁹ /L on the second day after inoculation (Day 0), and showed a tendency to be consistently high until the end of the experiment (see Table 7 below).

In the case of the group administered with pathogen + maboxin-B, the total white blood cell count was 36.5±5.1×10 ⁹ /L on the 2nd day after inoculation, but the total white blood cell count was 28.4±4.6×10 ⁹ /L on the 4th day after the start of test product administration, indicating that the pathogen There was a significant decrease compared to the infected group, and then the total white blood count continued to decrease, and the total white blood cell count on the 14th day after intramuscular injection of the test product was 21.6±2.5×10 ⁹ /L, which was significantly lower than that of the pathogen infection group (Table 7 below). Reference).

In the pathogen + Mabomax administration group, the total white blood cell count was 38.4±7.5×10 ⁹ /L on the 2nd day after inoculation, but the total white blood cell count decreased to 32.3±4.7×10 ⁹ /L on the 4th day after the start of test product administration. The blood count continued to decrease, and the total white blood cell count on day 14 was 24.8±3.0×10 ⁹ /L, which tended to be lower than that of the pathogen-infected group (see Table 7 below).

Parameters Group Day-2 Day 0 Day 1 Day 4 Day 7 Day 14 WBC
(×10 ⁹ /L) untreated 18.0±1.5 18.9±1.2 18.7±1.8 18.8±2.4 17.9±2.3 19.4±2.0 pathogen infection group 18.6±1.7 38.1 ± 4.6 ^*** 37.5 ± 6.7 ^*** 36.3 ± 6.7 ^*** 34.9 ± 4.1 ^*** 30.6±6.1 ^*** Pathogen + Marboxin B strain 18.8±1.9 36.6±5.1 ^*** 32.3 ± 2.4 ^*** 28.4 ± 4.6 ^{**, #} 25.6 ± 2.9 ^{**, ###} 21.6±2.5 ^## Pathogen + Mabomax 19.1±2.5 38.4 ± 7.5 ^*** 35.2±4.7 ^*** 32.3 ± 4.7 ^*** 28.3 ± 3.3 ^{***, ##} 24.8±3.0 ^*,# RBC
(×10 ¹² /L) untreated 6.0±0.5 6.4±0.1 6.0±0.2 5.9±0.3 6.1±0.2 6.2±0.4 pathogen infection group 6.3±0.3 6.4±0.7 5.9±0.7 6.0±0.2 6.2±0.5 5.6±0.9 Pathogen + Marboxin B strain 6.2±0.1 6.2±0.5 5.9±0.3 5.9±0.5 5.8±1.1 6.2±0.2 Pathogen + Mabomax 6.0±0.6 6.3±0.4 6.0±0.2 6.0±0.3 6.8±0.6 6.0±0.6 HGB
(g/dL) untreated 11.2±1.4 11.4±1.0 11.3±0.6 11.8±0.6 11.8±0.8 12.3±1.4 pathogen infection group 11.7±0.9 11.4±1.4 11.3±0.9 11.3±0.4 11.3±0.7 11.5±0.7 Pathogen + Marboxin B strain 11.2±0.7 11.5±0.6 11.4±0.7 11.2±0.9 11.3±1.8 11.7±0.9 Pathogen + Mabomax 11.3±0.9 11.6±1.4 11.0±0.9 11.5±0.8 12.3±1.3 11.5±1.0 HCT
(%) untreated 37.1±3.7 39.0±2.0 36.3±1.1 37.1±1.9 38.4±2.1 39.9±3.6 pathogen infection group 38.3±2.3 38.2±3.1 36.0±3.3 36.4±0.9 36.6±3.5 35.7±4.1 Pathogen + Marboxin B strain 36.8±2.3 37.9±3.6 35.6±1.5 37.2±3.1 36.6±6.0 38.1±2.6 Pathogen + Mabomax 36.7±3.4 39.3±3.3 35.3±1.5 37.5±1.4 40.7±5.4 37.5±3.9 MCV
(fL) untreated 61.9±3.0 61.5±2.6 60.1±3.1 63.2±2.5 62.5±2.3 64.5±2.9 pathogen infection group 60.6±2.3 62.0±4.1 61.4±2.9 61.0±3.2 59.1±2.0 64.0±5.8 Pathogen + Marboxin B strain 59.6±3.0 61.5±6.5 60.6±1.8 62.7±1.5 63.5±5.4 62.0±3.8 Pathogen + Mabomax 61.5±2.8 62.7±3.2 59.2±2.8 62.3±2.3 60.1±4.9 63.3±6.7 MCH
(Pg) untreated 18.6±1.8 17.9±1.5 18.7±1.2 20.0±1.5 19.1±1.3 19.8±1.6 pathogen infection group 18.4±1.4 18.5±1.9 19.2±1.1 18.8±1.2 18.2±0.7 20.9±4.4 Pathogen + Marboxin B strain 18.1±1.2 18.6±2.1 19.3±0.9 18.8±1.4 17.5±5.1 19.0±1.4 Pathogen + Mabomax 18.9±2.2 18.5±1.6 18.3±1.0 19.1±1.4 18.2±1.1 19.4±1.7 MCHC
(g/dL) untreated 30.1±2.3 29.2±1.8 31.1±0.9 31.8±2.0 30.7±1.3 30.8±1.4 pathogen infection group 30.5±2.2 29.8±1.6 31.4±0.8 30.9±1.3 30.9±1.5 32.4±3.9 Pathogen + Marboxin B strain 30.5±1.9 30.4±2.4 31.9±0.9 30.1±2.2 30.8±1.1 30.8±0.9 Pathogen + Mabomax 30.7±2.5 29.6±1.3 31.1±2.8 30.7±1.3 30.4±1.2 30.8±0.7 PLT
(×10 ⁹ /L) untreated 383.2±58.9 335.0±68.7 366.2±43.0 338.2±31.5 329.0±31.2 308.0±60.8 pathogen infection group 378.2±66.4 306.8±54.0 386.4±63.2 334.6±37.9 317.4±52.9 330.0±82.9 Pathogen + Marboxin B strain 349.6±64.7 351.6±46.2 359.6±74.4 328.6±62.3 330.6±37.6 312.0±55.9 Pathogen + Mabomax 381.4±67.7 324.0±46.8 358.2±52.6 342.4±30.2 348.6±75.5 347.2±89.6

^* p <0.05, ^** p <0.01, ^*** p <0.001: significantly different from normal group

^# p <0.05, ^## p <0.01, ^### p <0.001: significantly different from challenge group

(5) Clinical symptoms

The clinical index was calculated according to Table 7 while observing clinical symptoms of all test subjects at least once a day from the 1st day after bacterial inoculation to the 14th day after administration of the test substance. That is, on Day-2 before drug administration, APP, PM, and MH were intranasally inoculated into the pathogen-infected group, the pathogen + Maboxin-B strain, and the pathogen + Mabomax test group excluding the untreated group to induce pneumonia, and then pneumonia was induced. The test substance was administered on Day 0 when clinical symptoms were confirmed.

In the case of the challenge group, there are some differences in the severity of clinical symptoms by time from the first day after inoculation (Day-1) to the end of the experiment, but cough, clear or mucus runny nose, rough hair, body temperature rise, and respiratory rate in all animals Clinical symptoms such as increase were continuously observed (see FIGS. 4 and 5).

In the pathogen + Maboxin-B injection group, the test substance (Maboxin-B injection) was intramuscularly injected with 1 mL of this drug per 50 kg of body weight for 5 days. A decrease in respiratory rate, a decrease in nasal mucus secretion, and clear runny nose were observed, and after the 5th day, a temporary increase in body temperature, a small amount of clear runny nose, and an increase in respiratory rate were intermittently observed in some individuals, and the reduction effect of clinical symptoms was superior to that of Marbomax. (see FIGS. 4 and 5).

In the pathogen + Mabomax administration group, as a result of intramuscular injection of 1 mL of this drug per 50 kg of body weight for 5 days, the test substance (Mabomax 10% stock) was injected intramuscularly for 5 days. A decrease in the amount of mucus secretion and clear runny nose was observed, and after 6 to 7 days, a temporary increase in body temperature, a small amount of clear runny nose, coughing, and an increase in respiratory rate were intermittently observed in some individuals (see FIGS. 4 and 5).

(6) Measurement of the number of bacteria in the nasal cavity

Actinobacillus pleuropneumoniae , Pasteurella multocida , and Mycoplasma hyopneumoniae were intranasally inoculated once, and 1 mL of the test product per 50 kg of body weight was injected intramuscularly five times, and nasal exudate was swabbed for each individual to examine the number of bacteria present in the nasal cavity.

Actinobacillus pleuropneumoniae in the nasal cavity of the pathogen-infected group was detected in most subjects until the end of the experiment, and 1.17±2.35 Log ₁₀ CFU/mL was detected at the end of the experiment. In the pathogen + maboxin-B injection group, the intranasal Actinobacillus pleuropneumoniae was 4.95±0.61 Log ₁₀ CFU/mL before drug administration, but on the 1st day after administration of maboxin-B injection, it was 4.02±0.69 Log ₁₀ CFU/mL in the pathogen-infected group. From the 4th day, bacteria were detected in only one individual, and from the 7th day, bacteria were not detected in all individuals. In the pathogen + Mabomax administration group, there was a significant decrease compared to the pathogen infection group from the 3rd day (2.06 ± 1.99 Log ₁₀ CFU / mL) after the start of Mabomax administration, and bacteria were not detected in all subjects from the 8th day (Table 8 and see Figure 6).

Day pathogen infection group
(Log ₁₀ CFU/mL) Pathogen + Marboxin-B strain
(Log ₁₀ CFU/mL) Pathogen + Mabomax
(Log ₁₀ CFU/mL) Day-2
(attack inoculation) - - - Day-1 4.45±0.57 4.84±0.39 4.78±0.26 approximately
water
two
female Day 0 4.56±0.32 4.95±0.61 4.83±0.36 Day 1 4.81±0.35 4.02±0.69 ^# 4.11±0.50 Day 2 4.67±0.25 2.29±2.14 ^# 2.85±1.70 Day 3 4.53±0.22 1.84±1.76 ^# 2.06±1.99 ^# Day 4 3.36±1.96 0.68±1.52 ^# 1.28±1.83 Day 5 3.57±2.02 0.60±1.34 ^# 0.66±1.47 ^# Day 6 4.12±0.37 0.51±1.14 ^### 0.58±1.31 ^### Day 7 3.30±1.92 0 0.45±1.00 ^## Day 8 3.47±1.98 0 Day 9 3.15±1.86 0 Day 10 2.88±2.63 0 Day 11 2.47±2.26 0 Day 12 2.34±2.17 0 Day 13 1.36±1.93 0 Day 14 1.71±2.35 0

^# p <0.05, ^## p <0.01, ^### p <0.001: significantly different from challenge group

Pasteurella multocida in the pathogen-infected group was detected at 3.10±2.83 Log ₁₀ CFU/mL at the end of the experiment. However, 1.50 ± 2.09 Log ₁₀ CFU/mL and 2.19 ± 2.06 Log ₁₀ CFU/mL of Pasteurella multocida bacteria were detected on the 3rd day after the start of drug administration in the group administered with the pathogen + Maboxin-B and the group administered with the pathogen + Mabomax, but the pathogen infection There was a significant decrease compared to the group, and thereafter, Pasteurella multocida bacteria continuously decreased in the nasal exudate, and the bacteria were not detected in all subjects from the 7th day after the start of drug administration (see Table 9 and FIG. 7 below).

Day pathogen infection group
(Log ₁₀ CFU/mL) Pathogen + Marboxin-B strain
(Log ₁₀ CFU/mL) Pathogen + Mabomax
(Log ₁₀ CFU/mL) Day-2
(attack inoculation) - - - Day-1 4.82±0.32 5.60±0.99 5.15±0.20 approximately
water
two
female Day 0 5.14±0.64 5.96±1.19 5.99±0.72 Day 1 5.55±0.35 4.72±1.59 4.60±0.40 Day 2 4.67±0.67 2.68±2.53 3.41±1.96 Day 3 5.22±0.83 1.50±2.09 ^## 2.19±2.06 ^# Day 4 4.20±2.41 1.25±1.81 1.39±1.93 Day 5 3.11±2.93 0.70±1.58 0.66±1.48 Day 6 4.06±2.29 0.41±0.91 ^## 0.58±1.30 ^## Day 7 4.30±2.60 0 0 Day 8 4.04±2.31 0 0 Day 9 3.39±3.13 0 0 Day 10 4.03±2.35 0 0 Day 11 2.86±2.75 0 0 Day 12 3.15±2.88 0 0 Day 13 3.09±2.83 0 0 Day 14 3.10±2.83 0 0

^# p <0.05, ^## p <0.01: significantly different from challenge group

As a result of confirming the presence of Mycoplasma hyopneumoniae in the nasal cavity, in the case of the pathogen-infected group, M. hyopneumoniae was continuously confirmed in the nasal cavity of 3 to 4 animals until the end of the experiment, whereas in the pathogen + Marboxin-B main administration group, after the start of drug administration M. hyopneumoniae was detected in 3 to 4 animals until the 2nd day, 2 animals were detected on the 3rd to 4th day, and 1 animal was detected on the 5th to 6th day, but M. hyopneumoniae was not detected from the 7th day onwards. In the pathogen + Mabomax administration group, M. hyopneumoniae was detected in 3 to 4 animals until the 2nd day after the start of drug administration, then in 2 animals on the 3rd to 5th day, and in 1 animal on the 6th to 7th day, but after the 8th day, M. hyopneumoniae was not detected (see Table 10 below).

Day pathogen infection group
(number of positive animals/number of total animals) Pathogen + Marboxin-B strain
(number of positive animals/number of total animals) Pathogen + Mabomax
(number of positive animals/number of total animals) Day-2
(attack inoculation) 0/5 0/5 0/5 Day-1 3/5 4/5 2/5 approximately
water
two
female Day 0 4/5 4/5 4/5 Day 1 4/5 4/5 3/5 Day 2 3/5 3/5 3/5 Day 3 3/5 2/5 2/5 Day 4 4/5 2/5 2/5 Day 5 3/5 1/5 2/5 Day 6 3/5 1/5 1/5 Day 7 3/5 0/5 1/5 Day 8 4/5 0/5 0/5 Day 9 3/5 0/5 0/5 Day 10 4/5 0/5 0/5 Day 11 4/5 0/5 0/5 Day 12 3/5 0/5 0/5 Day 13 3/5 0/5 0/5 Day 14 4/5 0/5 0/5

(7) Autopsy findings

1) Gross pathological findings

No specific findings were observed in the lungs of the untreated group (see FIG. 8).

In the pathogen-infected group, adhesions on the chest wall of the left and right lungs, focal or extensive purulent nodules, and purplish-red sclerosis were observed in some lung lobes (see FIG. 9).

In the group administered with pathogen + maboxin-B, chest wall adhesions and extensive purulent nodules of the right anterior lobe and mesenchymal lobe were observed in one animal, and slight reddish hardening (flesh change) of the left anterior lobe and mesenchymal lobe were observed, but in the other four animals Slight purplish red sclerosis (flesh change) and focal purulent nodules (1 to 2 sites) were observed in some lung lobes, but no specific lesion was observed overall (see FIG. 10).

In the pathogen + Mabomax administration group, adhesions to the chest wall of the right middle lobe, purulent nodules, and sclerosis (flesh change) of the left anterior lobe were observed in 2 animals, and mild sclerosis and localized purulent nodules (1 to 2) were observed in the remaining 3 animals. site) was observed, but no specific lesion was observed overall (see FIG. 11).

2) Histopathological findings

After autopsy, the lung lesion sites and lung lymph nodes of the untreated group, the pathogen-infected group, the pathogen + Marboxin-B strain, and the pathogen + Marbomax test group were collected, fixed in 10% neutral formalin, and stained and observed.

As a result of histological examination of the lungs of the untreated group, no specific findings other than weak alveolar wall thickening were observed (see FIG. 12).

As a result of histological examination of the lungs of the pathogen-infected group, severe inflammatory cells (mainly polymorphonuclear leukocytes and macrophages) and fibrin infiltration, alveolar wall and lobular interstitial thickening, and abscess formation were observed in the bronchial and alveolar cavities in 4 animals. In , bronchial lymphoid tissue hypertrophy and local alveolar wall thickening were observed (see FIG. 13 ).

As a result of histological examination of the lungs of the group administered with pathogen + maboxin-B, inflammatory cells (mainly polymorphonuclear leukocytes and macrophages) and fibrin infiltration were observed in the lumen of the bronchi and alveoli in one lung, and bronchial pneumonia was observed in three lungs. Bronchial lymphoid tissue hypertrophy and localized alveolar wall thickening were observed in the lungs of one animal, and no specific findings were observed in the other animal (see FIG. 14).

As a result of histological examination of the lungs of the group administered with pathogen + Mabokmax, inflammatory cells (mainly polymorphonuclear leukocytes and macrophages) and fibrin infiltration were observed in the lumen of the bronchi and alveoli in 2 lungs, and bronchial pneumonia was observed in 3 lungs. Bronchial lymphoid tissue hypertrophy and focal alveolar wall thickening were observed (see Fig. 15).

(8) Measurement of the number of bacteria in lung tissue

After autopsy, lung lesion tissues of the challenge inoculation group and the efficacy test group were collected and the number of bacteria present per 1 g was investigated.

In the pathogen-infected group, APP and PM bacteria were detected at (8.29±10.55)×10 ⁴ CFU/g and (2.14±2.06)×10 ⁶ CFU/g, respectively, whereas in the pathogen+maboxin-B-administered group, 2.86 in one animal. ×10 ³ CFU/g and 1.33×10 ⁴ CFU/g were detected, and in the pathogen + Mabomax administration group, 3.92×10 ³ CFU/g of APP bacteria was detected in one lung, and PM bacteria were detected in two lungs. 5.66×10 ⁴ CFU/g and 2.67×10 ⁵ CFU/g bacteria were respectively detected in (see Table 11 below).

In addition, in the case of the pathogen-infected group, Mycoplasma hyopneumoniae was detected in the lungs of 4 animals, whereas Mycoplasma hyopneumoniae was not detected in the lungs of the pathogen + Marboxin-B injection group and the pathogen + Mabomax administration group (see Table 11 below).

test group object number Lung lesion index APP PM MH pathogen infection group 3 3 0 2.08×10 ⁴ - 11 5 0 1.53×10 + 13 5 1.83×10 ⁴ 8.30×10 ⁵ + 14 6 2.06×10 ⁵ 3.07×10 ⁶ + 16 6 1.90×10 ⁵ 5.22×10 ⁶ + Mean±SD 5.0±1.2 (8.29±10.55)×10 ⁴ (2.14±2.06)×10 ⁶ 4 Pathogen + Marboxin-B strain 5 0 0 0 - 12 4 2.86×10 ³ 1.33×10 ⁴ - 15 0 0 0 - 18 One 0 0 - 20 One 0 0 - Mean±SD 1.8±1.6 (5.72.±12.79)×10 ² (2.67±5.96)×10 ³ 0 Pathogen + Mabomax 6 0 0 0 - 7 4 3.92×10 ³ 5.66×10 ⁴ - 8 2 0 0 - 10 4 0 2.67×10 - 19 2 0 0 - Mean±SD 2.4±1.7 (7.84±17.53)×10 ² (6.48±11.6)×10 ⁴ 0

2. Anti-pyretic, analgesic, anti-inflammatory and metabolic promoting effects

(1) Changes in serum cytokine concentration

1) Changes in TNF-α content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge vaccination, on the day of drug administration, and on the 1st, 4th, 7th, and 14th days after starting drug administration for each test group Blood was collected to confirm the content of TNF-α in serum.

After challenge inoculation, TNF-α in the blood of the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen Mavomax-administered group were 273.7±41.9 pg/mL, 287.4±25.2 pg/mL, and 265.4±33.1 pg/mL, respectively, without treatment. Significantly increased compared to the group (see Table 12 (unit: pg / mL) and Figure 16).

The concentration of TNF-α in the blood of the pathogen + maboxin-B injection group on the 1st and 4th days after the start of drug administration was 236.4±32.1 pg/mL and 239.4±31.6 pg/mL, respectively, compared to the pathogen-infected group (Day 1: 295.1±20.0 pg/mL, Day 4: 289.9±15.0 pg/mL) and the pathogen + Mabomax administration group (Day 1: 273.9±30.9 pg/mL, Day 4: 276.9±39.5 pg/mL), while significantly lower than those without treatment There was no significant difference from the group (Day 1: 209.6±17.8 pg/mL, Day 4: 212.7±11.1 pg/mL). The concentration of TNF-α in the blood of the pathogen-infected group and the pathogen+Mabomax-administered group was significantly higher than that of the untreated group (see Table 12 and FIG. 16 below).

On the 7th day after the start of drug administration, the concentrations of TNF-α in the blood of the pathogen + Marboxin-B main administration group and the pathogen + Mabomax administration group were 216.9±24.2 pg/mL and 226.9±24.8 pg/mL, respectively, and the pathogen-infected group (258.4±25.0 It was significantly lower than pg/mL) (see Table 12 and FIG. 16 below).

Even on the 14th day after the start of drug administration, the TNF-α in the blood of the pathogen + Marboxin-B main administration group and the pathogen + Mabomax administration group were 195.1 ± 22.2 pg/mL and 206.7 ± 23.4 pg/mL, respectively, in the pathogen-infected group (244.8 ± 31.9 pg). /mL) was significantly lower than that of There was no significant difference in the concentration of TNF-α in the blood between the pathogen + Maboxin-B injection group and the pathogen + Mabomax administration group compared to the untreated group (Day 7: 210.3 ± 10.9 Day 14: 199.2 ± 22.7). It was significantly higher than the group (see Table 12 and FIG. 16 below).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 205.7±12.2 187.5±22.6 212.3±25.9 202.0±28.6 Day 0 (start of drug administration) 215.4±27.8 273.7±41.9 ^* 287.4±25.2 ^** 265.4±33.1 ^* Day1 209.6±17.8 295.1 ± 20.0 ^*** 236.4±32.1 ^##,a 273.9 ± 30.9 ^** Day 4 (end of drug administration) 212.7±11.1 289.9 ± 15.0 ^*** 239.4±31.6 ^#,a 276.9 ± 39.5 ^** Day 7 210.3±10.9 258.4±25.0 ^** 216.9±24.2 ^# 226.9±24.8 ^# Day 14 199.2±22.7 244.8 ± 31.9 ^* 195.1±22.2 ^# 206.7±23.4 ^#

^* p <0.05, ^** p <0.01, ^*** p <0.001: significantly different from normal group

^# p <0.05, ^## p <0.01: significantly different from challenge group

^a p <0.05: significantly different from Marbomax group

2) Changes in IL-1β content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected and the content of IL-1β in serum was confirmed.

After challenge inoculation, the plasma IL-1β of the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen Mavomax-administered group were 42.019±10.982 ng/L, 44.810±7.746 ng/L, and 36.898±4.830 ng/L, respectively. The concentration of IL-1β in the blood of the infected group and the group administered with pathogen + maboxin-B was significantly higher than that of the untreated group (see Table 13 (unit: ng/L) and FIG. 17).

The concentrations of IL-1β in the blood of the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen+Mabomax treatment group on day 1 after drug administration were 42.019±10.982 ng/L, 44.810±7.746 ng/L, and 36.898±4.830, respectively. ng/L, and the pathogen infection group was significantly higher than the untreated group (30.232±2.556 ng/L) (see Table 13 and FIG. 17 below).

On the 4th day after the start of drug administration, the concentration of IL-1β in the blood of the pathogen+Maboxin-B main administration group was 31.173±3.408 ng/L, respectively, in the pathogen-infected group (43.962±15.062 ng/L) and the pathogen+Mabomax-administered group (36.274±5.717 ng/L), whereas the concentration of IL-1β in the blood of the pathogen-infected group and the pathogen + Mabomax administration group was significantly higher than that of the untreated group (28.592±3.554 ng/L) (see Table 13 and FIG. 17 below). ).

On the 7th day after the initiation of drug administration, the concentration of IL-1β in the blood of the pathogen + Marboxin-B main administration group and the pathogen + Mabomax administration group was 32.005 ± 4.654 ng / L and 38.263 ± 4.218 ng / L, respectively, compared to the pathogen-infected group (50.125 ± 13.164 ng / L), and the pathogen infection group was significantly higher than the untreated group (30.712 ± 1.403 ng / L) (see Table 13 and FIG. 17 below).

On the 14th day after the start of drug administration, the concentration of IL-1β in the blood of the main group administered with pathogen + Marboxin-B was 29.764 ± 2.669 ng/L, which was compared to the group infected with pathogen (41.244 ± 4.403 ng/L) and the group administered with pathogen + Mavomax (35.173 ± 4.767 ng). /L) was significantly lower than The pathogen-infected group was significantly higher than the untreated group (29.457 ± 3.674 ng/L), and significantly lower than the pathogen-infected group from the 4th day to the 14th day after the start of drug administration, and on the 4th and 14th days, pathogen + Mabomax It was significantly lower than that of the administration group (see Table 13 and FIG. 17 below).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 28.241±3.965 27.776±2.404 27.608±3.564 29.638±1.945 Day 0 (start of drug administration) 29.843±1.629 42.019±10.982 ^* 44.810 ± 7.746 ^** 36.898±4.830 Day1 30.232±2.556 43.962±15.062 ^* 35.882±4.520 36.274±5.717 Day 4 (end of drug administration) 28.592±3.554 52.263±15.854 ^** 31.173±3.408 ^{#, a} 45.239±12.107 ^* Day 7 30.712±1.403 50.125 ± 13.164 ^** 32.005±4.654 ^## 38.263±4.218 ^# Day 14 29.457±3.674 41.244±4.403 ^*** 29.764±2.669 ^{##, a} 35.173±4.767 ^*,#

^* p <0.05, ^** p <0.01, ^*** p <0.001: significantly different from normal group

^# p <0.05, ^## p <0.01: significantly different from challenge group

^a p <0.05: significantly different from Marbomax group

3) Changes in IL-6 content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected and the IL-6 content in serum was confirmed.

After challenge inoculation, IL-6 in the blood of the pathogen-infected group, the pathogen + Maboxin-B injection group, and the pathogen Mavomax-administered group were 150.527±23.411 ng/L, 146.016±40.757 ng/L, and 139.950±35.131 ng/L, respectively, untreated. It showed a higher concentration than the group (see Table 14 (unit: ng/L) and FIG. 18 below).

The plasma IL-6 concentrations of the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen+Mabomax treatment group on day 1 after drug administration were 150.527±23.411 ng/L, 146.016±40.757 ng/L, and 139.950±35.131, respectively. ng/L, and was significantly higher than that of the untreated group (89.450±27.326 ng/L) (see Table 14 and FIG. 18 below).

On the 4th and 7th days after the start of drug administration, the plasma IL-6 concentrations of the pathogen-infected group were 190.655±48.492 ng/L and 184.884±49.719 ng/L, respectively, in the untreated group (Day 4: 95.334±18.576 ng/L, Day 4). 7: 108.395 ± 22.860 ng/L), while pathogen + Marboxin-B main administration group (Day 4: 139.875 ± 44.106 ng/L, Day 7: 135.757 ± 23.007 ng/L) and pathogen + Marbomax There was no significant difference in blood IL-6 concentration of the administration group (Day 4: 163.355 ± 78.383 ng/L, Day 7: 151.142 ± 37.012 ng/L) from that of the untreated group (see Table 14 and FIG. 18 below).

On the 14th day after the initiation of drug administration, the plasma IL-6 concentrations of the pathogen + Marboxin-B main administration group and the pathogen + Mabomax administration group were 103.951 ± 27.697 ng / L and 118.356 ± 23.730 ng / L, respectively. /L) was significantly lower than There was no significant difference between the pathogen infection group, the pathogen + Maboxin-B main administration group, and the pathogen + Mabomax administration group compared to the untreated group (29.457 ± 3.674 ng / L) (see Table 14 and FIG. 18 below).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 86.608±14.883 84.710±13.819 85.502±12.3885 85.192±19.437 Day 0 (start of drug administration) 88.972±18.695 144.145±32.755 151.570±57.907 161.523±78.904 Day1 89.450±27.326 150.527±23.411 ^* 146.016±40.757 ^* 139.950 ± 35.131 ^* Day 4 (end of drug administration) 95.334±18.576 190.655±48.492 ^* 139.875±44.106 163.355±78.383 Day 7 108.395±22.860 184.884±49.719 ^** 135.757±23.007 151.142±37.012 Day 14 92.072±6.977 159.394±30.690 103.951±27.697 ^## 118.356±23.730 ^#

* p < 0.05, ** p < 0.01, significantly different from normal group

^# p < 0.05, significantly different from FMDV vaccine group

4) Changes in PGE2 content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected and the content of PGE ₂ in serum was confirmed.

After challenge inoculation, the blood PGE2 concentrations of the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen Mavomax-administered group were 277.6±33.0 pg/mL, 285.5±23.8 pg/mL, and 269.9±52.8 pg/mL, respectively, in the untreated group. Significantly increased compared to (see Table 15 (unit: pg / mL) and Figure 19).

On the 1st day after the start of drug administration, the blood PGE2 concentration of the pathogen + Marboxin-B main administration group was 246.3 ± 27.5 pg/mL, which was significantly lower than that of the pathogen-infected group (326.1 ± 52.9 pg/mL), and the pathogen-infected group and the pathogen Mabomax The administration group (284.0±25.9 pg/mL) was significantly higher than the untreated group (215.3±25.0 pg/mL) (see Table 15 and FIG. 19 below).

On the 4th and 7th days after the start of drug administration, the concentrations of PGE2 in the blood of the pathogen + maboxin-B main administration group were 234.1±37.6 pg/mL and 196.4±11.4 pg/mL, respectively, compared to the pathogen-infected group (Day 4: 292.8± 27.5 pg/mL). mL, Day 7: 270.4±34.3 pg/mL) and the pathogen + Mabomax administration group (Day 4: 282.7±31.8 pg/mL, Day 7: 233.3±33.2 pg/mL). The concentration of PGE2 in the blood on the 4th and 7th days of the pathogen-infected group was significantly higher than that of the untreated group. There was no difference, and it was significantly lower than the pathogen infection group (see Table 15 and FIG. 19 below).

On the 14th day after the start of drug administration, the blood PGE2 concentrations of the pathogen + Marboxin-B main administration group and the pathogen + Mabomax administration group were 181.1 ± 21.3 pg/mL and 210.4 ± 29.5 pg/mL, respectively, and the pathogen infection group (265.8 ± 23.7 pg/mL) mL), and the pathogen-infected group was significantly higher than the untreated group (192.8±21.9 pg/mL) (see Table 15 and FIG. 19 below).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 234.9±34.2 211.0±32.1 222.3±30.1 208.3±33.2 Day 0 (start of drug administration) 215.5±15.5 277.6 ± 33.0 ^* 285.5±23.8 ^** 269.9±52.8 ^* Day1 215.3±25.0 326.1 ± 52.9 ^*** 246.3±27.5 ^## 284.0±25.9 ^** Day 4 (end of drug administration) 200.5±27.0 292.8 ± 27.5 ^*** 234.1±37.6 ^#,a 282.7 ± 31.8 ^** Day 7 208.2±16.8 270.4±34.3 ^** 196.4±11.4 ^###,a 233.3±33.2 ^# Day 14 192.8±21.9 265.8 ± 23.7 ^*** 181.1±21.3 ^### 210.4±29.5 ^##

^* p <0.05, ^** p <0.01, ^*** p <0.001: significantly different from control group

^# p <0.05, ^## p <0.01: significantly different from challenge group

^a p <0.05: significantly different from Marbomax 10% inj. group

(2) Metabolism stimulating effect

1) Changes in glucose content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected and the glucose content in serum was confirmed.

After challenge inoculation, the blood glucose concentrations of the pathogen-infected group, the pathogen + Marboxin-B administration group, and the pathogen + Mavomax administration group were 36.0±10.1 mg/dL, 38.0±12.8 mg/dL, and 32.6±10.5 mg/dL, respectively, without treatment. It was significantly decreased compared to the group (see Table 16 (unit: mg/dL) and FIG. 20 below).

On day 1 after the start of drug administration, the blood glucose concentrations of the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen+Mabomax treatment group were 52.8±9.8 mg/dL, 71.8±12.7 mg/dL, and 59.0±13.9 mg/dL, respectively. Although significantly lower than that of the untreated group (98.4±11.4 mg/dL), the group administered with the pathogen + maboxin-B was significantly higher than the group infected with the pathogen (see Table 16 and FIG. 20 below).

On the 4th day after the start of drug administration, the pathogen-infected group (75.2±7.1 mg/dL) and the pathogen-infected group (79.6±10.5 mg/dL), excluding the pathogen+Maboxin B injection group (84.0±6.5 mg/dL) Blood glucose concentration was significantly lower than that of the untreated group (91.0±4.5 mg/dL) (see Table 16 and FIG. 20 below).

On the 7th and 14th days after the start of drug administration, the blood glucose concentrations of the pathogen + maboxin B injection group were 86.8±10.3 mg/dL and 83.2±9.3 mg/dL, respectively, compared to the pathogen-infected group (Day 7: 68.4±8.4 mg/dL). , Day 14: 54.8±15.2 mg/dL) and pathogen + Mabomax administration group (Day 7: 74.4±6.3 mg/dL, Day 14: 68.2±9.9 mg/dL), and was significantly higher than the pathogen infection group and pathogen + The blood glucose concentration of the Mabomax administration group was significantly lower than that of the untreated group (Day 7: 96.4±1.8 mg/dL, Day 14: 89.4±9.1 mg/dL) (see Table 16 and FIG. 20 below).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 83.8±7.4 82.6±8.5 84.8±6.2 82.4±6.4 Day 0 (start of drug administration) 84.8±8.9 36.0±10.1 ^*** 38.0±12.8 ^*** 32.6±10.5 ^*** Day1 98.4±11.4 52.8 ± 9.8 ^*** 71.8 ± 12.7 ^{**, #} 59.0 ± 13.9 ^*** Day 4 (end of drug administration) 91.0±4.5 75.2±7.1 ^** 84.0±6.5 79.6±10.5 ^* Day 7 96.4±1.8 68.4 ± 8.4 ^*** 86.8±10.3 ^##,a 74.4 ± 6.3 ^*** Day 14 89.4±9.1 54.8 ± 15.2 ^*** 83.2±9.3 ^##,a 68.2±9.9 ^*

^* p <0.05, ^** p <0.01, ^*** p <0.001: significantly different from normal group

^# p <0.05, ^## p <0.01: significantly different from challenge group

^a p <0.05: significantly different from Marbomax group

2) Changes in insulin content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected and the insulin content in serum was confirmed.

After challenge inoculation, the plasma insulin concentrations of the pathogen-infected group, the pathogen + Marboxin-B administration group, and the pathogen + Mavomax administration group were 5.264±1.191 μIU/mL, 5.128±0.472 μIU/mL, and 5.839±1.223 μIU/mL, respectively, without treatment. It showed a higher tendency compared to the group (4.739 ± 0.643 μIU / mL) (see Table 17 (unit: μIU / mL) and FIG. 21).

On the 1st and 4th days after the start of drug administration, the plasma insulin concentrations of the pathogen+maboxin-B injection group were 5.364±0.227 μIU/mL and 5.217±0.453 μIU/mL, respectively, compared to the pathogen-infected group (Day 1: 6.753±1.647 μIU/mL). mL, Day 4: 5.937 ± 2.079 μIU / mL) and the pathogen + Mabomax administration group (Day 1: 6.317 ± 2.453 μIU / mL, Day 4: 6.069 ± 1.336 μIU / mL) lower (see Table 17 and FIG. 21 below) .

On the 7th day after the start of drug administration, the blood insulin concentration of the pathogen + Maboxin B injection group was 5.013 ± 0.426 μIU/mL, and the pathogen infection group (6.472 ± 1.256 μIU/mL) and the pathogen + Mavomax treatment group (6.258 ± 0.856 μIU/mL) ), and the pathogen infection group and the pathogen + Mabomax administration group were significantly higher than the untreated group (4.646 ± 0.248 μIU / mL) (see Table 17 and FIG. 21 below).

At 14 days after the start of drug administration, the blood insulin concentrations of the untreated group, the pathogen-infected group, the pathogen + Maboxin-B injection group, and the pathogen + MavoMax administration group were 5.294±1.038 μIU/mL, 6.229±1.107 μIU/mL, 4.866± Significance between test groups was not observed at 0.745 μIU/mL and 6.086±1.264 μIU/mL (see Table 17 and FIG. 21 below).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 4.970±0.742 4.717±0.455 4.866±0.572 4.988±0.472 Day 0 (start of drug administration) 4.739±0.643 5.264±1.191 5.128±0.472 5.839±1.223 Day1 5.527±0.708 6.753±1.647 5.364±0.227 6.317±2.453 Day 4 (end of drug administration) 4.644±0.395 5.937±2.079 5.217±0.453 6.069±1.336 Day 7 4.646±0.248 6.472±1.256 ^** 5.013±0.426 ^#,a 6.258±0.856 ^** Day 14 5.294±1.038 6.229±1.107 4.866±0.745 6.086±1.264

^** p <0.01: significantly different from control group

^# p < 0.05: significantly different from challenge group

^a p <0.05: significantly different from Marbomax group

3) Changes in glucagon content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected to confirm the content of glucagon in serum.

After challenge inoculation, the blood glucagon concentrations of the untreated group, the pathogen-infected group, the pathogen + Maboxin-B injection group, and the pathogen + MavoMax treatment group were 1352.7±255.7 pg/mL, 1396.3±420.5 pg/mL, and 1272.5±108.9 pg/mL, respectively. No difference was observed between each test group in terms of mL (see Table 18 (unit: pg/mL) and FIG. 22 below).

On day 1 after the start of drug administration, the blood glucagon concentrations of the untreated group, the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen+MABOMAX treatment group were 1357.9±288.8 pg/mL, 1532.5±426.0 pg/mL, and 1327.3±, respectively. They were 187.5 pg/mL and 1394.1±257.3 pg/mL, and the blood glucagon concentrations of each test group on the 4th day after the start of drug administration were 1291.3±222.6 pg/mL, 1473.5±424.6 pg/mL, 1314.8±221.7 pg/mL and 1352.9 ±245.4 pg/mL, no significant difference was observed between each test group (see Table 18 and FIG. 22 below).

On the 7th and 14th days after the start of drug administration, the concentrations of Glugacon in the blood of the group treated with the pathogen + maboxin B were 1308.6±271.5 pg/mL and 1274.5±175.5 pg/mL, respectively, and the untreated group (Day 7: 1185.7±293.0 pg /mL, Day 14: 1164.6 ± 273.1 pg/mL, pathogen infection group (Day 7: 1565.6 ± 318.5 pg/mL, Day 14: 1645.8 ± 312.0 pg/mL) and pathogen + Mabomax administration group (Day 7: 1459.2 ± 357.8 pg/mL, Day 14: 1443.5±420.7 pg/mL), but no significant difference was observed, but the concentration of glucagon in the blood of the pathogen + Marboxin-B injection group showed a tendency to be lower than that of the pathogen infection group and the pathogen + Mabomax administration group. (see Table 18 and FIG. 22 below).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 1397.4±215.8 1407.1±316.7 1397.3±210.3 1432.9±223.7 Day 0 (start of drug administration) 1352.7±255.7 1396.3±420.5 1272.5±108.9 1376.9±274.6 Day1 1357.9±288.8 1532.5±426.0 1327.3±187.5 1394.1±257.3 Day 4 (end of drug administration) 1291.3±222.6 1473.5±424.6 1314.8±221.7 1352.9±245.4 Day 7 1185.7±293.0 1565.6±318.5 1308.6±271.5 1459.2±357.8 Day 14 1164.6±273.1 1645.8±312.0 1274.5±175.5 1443.5±420.7

4) Changes in total cholesterol content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected to determine the total cholesterol content in serum.

After challenge vaccination, the blood total cholesterol levels of the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen+Mavomax treatment group were 62.6±9.9 mg/dL, 61.8±16.3 mg/dL, and 58.0±10.6 mg/dL, respectively. It was significantly decreased compared to the treatment group (92.2 ± 6.3 mg / dL) (see Table 19 (unit: mg / dL) and Figure 23).

On day 1 after the start of drug administration, the blood total cholesterol concentrations of the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen+Marvomax treatment group were 50.8±5.8 mg/dL, 64.6±7.4 mg/dL, and 55.4±8.6 mg/dL, respectively. dL was significantly lower than that of the untreated group, and the group administered with the pathogen + maboxin-B was significantly higher than the group infected with the pathogen (see Table 19 and FIG. 23 below).

On the 4th day after the start of drug administration, the blood total cholesterol concentration of the pathogen + Marboxin-B injection group and the pathogen + Marbomax administration group was 75.2 ± 4.9 mg/dL, and the pathogen infection group (54.4 ± 4.9 mg/dL) and the pathogen + Marbomax treatment group. It was significantly higher than that of the administration group (67.0±7.8 mg/dL). However, the total cholesterol concentration in the blood of the pathogen-infected group, the pathogen + Marboxin-B administration group, and the pathogen + Mabomax administration group was significantly lower than that of the untreated group (94.2 ± 6.3 mg / dL) (see Table 19 and FIG. 23 below). .

On the 7th day after the start of drug administration, the blood total cholesterol concentrations of the pathogen + Marboxin B injection group and the pathogen + Mabomax administration group were 80.8 ± 5.4 mg / dL and 75.6 ± 7.2 mg / dL, respectively, and the pathogen infection group (60.2 ± 9.1 mg / dL) ) and significantly lower than the untreated group (90.6±5.7 mg/dL). In addition, the pathogen infection group was significantly lower than the untreated group (see Table 19 and FIG. 23 below).

On the 14th day after the start of drug administration, the blood total cholesterol concentrations of the pathogen-infected group, the pathogen+Maboxin injection B injection group, and the pathogen+Mavomax treatment group were 71.8±10.9mg/dL, 83.6±7.2 mg/dL, and 81.0±8.0 mg/dL, respectively. dL, and the total cholesterol in the blood of the pathogen-infected group was significantly lower than that of the untreated group (92.0±13.9 mg/dL) (see Table 19 and FIG. 23 below).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 1397.4±215.8 1407.1±316.7 1397.3±210.3 1432.9±223.7 Day 0 (start of drug administration) 1352.7±255.7 1396.3±420.5 1272.5±108.9 1376.9±274.6 Day1 1357.9±288.8 1532.5±426.0 1327.3±187.5 1394.1±257.3 Day 4 (end of drug administration) 1291.3±222.6 1473.5±424.6 1314.8±221.7 1352.9±245.4 Day 7 1185.7±293.0 1565.6±318.5 1308.6±271.5 1459.2±357.8 Day 14 1164.6±273.1 1645.8±312.0 1274.5±175.5 1443.5±420.7

^* p <0.05, ^** p <0.01, ^*** p <0.001: significantly different from normal group

^# p <0.05, ^## p <0.01, ^### p <0.001: significantly different from challenge group

^a p <0.05: significantly different from Marbomax group

5) Changes in TG content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected and the TG content in serum was confirmed.

After challenge inoculation, the blood TG concentrations of the pathogen-infected group, the pathogen + Marboxin-B administration group, and the pathogen + Mavomax administration group were 33.6±9.2 mg/dL, 29.1±10.1 mg/dL, and 30.8±10.4 mg/dL, respectively, without treatment. It was significantly decreased compared to the group (46.3±3.7 mg/dL) (see Table 20 (unit: mg/dL) and FIG. 24).

The blood TG concentration of the pathogen-infected group showed a higher tendency from the 1st to the 14th day after the start of drug administration than the untreated group, the pathogen+Marboxin-B injection group, and the pathogen+Marboxin-B injection group, but the pathogen+Marboxin-B injection group The blood TG concentration of the administration group and the pathogen + Mabomax administration group was lower than that of the untreated group on the 1st day after the start of drug administration, but was similar to that of the untreated group from the 4th day to the 14th day after the start of drug administration (see Table 20 and FIG. 24 below) ).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 49.8±4.1 51.4±10.8 47.8±7.5 46.8±7.6 Day 0 (start of drug administration) 46.4±3.7 33.6±9.2 ^* 29.1±10.1 ^* 30.8±10.4 ^* Day1 45.4±8.3 52.2±16.9 36.4±12.6 37.7±7.3 Day 4 (end of drug administration) 46.0±8.9 52.0±10.6 45.8±4.0 49.0±15.4 Day 7 49.8±11.4 56.2±19.3 48.0±15.2 53.8±13.1 Day 14 52.0±5.1 63.4±12.6. 51.2±6.5 52.2±13.5

^* p <0.05: significantly different from normal group

6) Changes in phosphorus content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected and the phosphorus content in serum was confirmed.

No significant change was observed in the blood phosphorus content of the pathogen-infected group, the pathogen + Maboxin-B injection group, and the pathogen + MavoMax administration group according to challenge vaccination and drug administration. Significant differences were observed when compared to the untreated group. did not (see Table 21 (unit: mg/dL) and FIG. 25 below).

On the 1st, 7th and 14th days after the initiation of drug administration, except for the 4th day after initiation of drug administration, the blood phosphorus content of the untreated group, the pathogen-infected group, the pathogen + Marboxin-B injection group, and the pathogen + Mavomax administration group was significant. No difference was observed. However, on the 4th day after the start of drug administration, the blood phosphorus concentration of the pathogen + Marboxin-B injection group was 10.8 ± 0.8 mg/dL, and the pathogen infection group (9.2 ± 0.9 mg/dL) and the pathogen + Mavomax administration group (9.3 ± 0.7 mg) / dL) (see Table 21 and FIG. 25 below).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 10.5±0.8 10.3±0.6 10.4±0.8 10.0±0.9 Day 0 (start of drug administration) 11.3±1.1 10.8±1.4 11.3±0.9 11.5±0.9 Day1 11.1±1.4 10.5±1.4 10.9±0.9 9.8±0.8 Day 4 (end of drug administration) 10.4±1.5 9.2±0.9 10.8±0.8 ^{#, a} 9.3±0.7 Day 7 10.5±0.8 9.7±1.9 10.9±1.5 10.5±0.9 Day 14 12.0±1.6 11.0±1.0 12.5±1.0 12.3±1.6

^# p < 0.05: significantly different from challenge group

^a p <0.05: significantly different from Marbomax group

7) Changes in urea content in serum

Untreated group, pathogen-infected group, pathogen+Maboxin-B injection group, pathogen+Mabomax treatment group Before challenge inoculation, on the day of drug administration, and on the 1st, 4th, 7th and 14th days after starting drug administration for each test group Blood was collected and the urea content in serum was confirmed.

After challenge inoculation, the blood urea concentrations of the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen+Mavomax treatment group were 25.81±2.62 mg/dL, 22.43±2.54 mg/dL, and 26.75±5.99 mg/dL, respectively, without treatment. It was significantly increased compared to the group (16.78 ± 3.07 mg / dL) (see Table 22 (unit: mg / dL) and Figure 26).

On the 1st day after the start of drug administration, the blood urea content of the pathogen+Maboxin-B injection group was 17.42±1.58 mg/dL, which was found in the pathogen-infected group (21.40±2.33 mg/dL) and the pathogen+Mabomax-administered group (22.47±2.27 mg/dL). ), while the concentration of blood urea in the pathogen-infected group and the pathogen + Mabomax administration group was significantly higher than that of the untreated group (16.61 ± 2.15 mg / dL) (see Table 22 and FIG. 26 below).

On the 4th, 7th and 14th days after the start of drug administration, no significant difference was observed in the blood urea content of the untreated group, the pathogen-infected group, the pathogen + Maboxin-B injection group, and the pathogen + Mavomax administration group (Table below). 22 and Figure 26).

Day untreated pathogen infection group Pathogen + Marboxin-B strain Pathogen + Mabomax Day-2
(attack inoculation) 17.89±3.13 18.96±2.83 17.08±2.38 19.05±2.95 Day 0 (start of drug administration) 16.78±3.07 25.81±2.62 ^** 22.43±2.54 ^* 26.75 ± 5.99 ^** Day1 16.61±2.15 21.40±2.33 ^** 17.42±1.58 ^{#, a} 22.47 ± 2.27 ^** Day 4 (end of drug administration) 16.05±1.57 19.82±3.75 17.93±3.19 19.13±3.30 Day 7 18.15±3.89 21.10±3.13 17.16±3.26 20.12±4.60 Day 14 18.92±1.95 20.76±4.74 18.10±2.46 19.65±4.47

^* p < 0.05, ^** p < 0.01: significantly different from normal group

^# p < 0.05: significantly different from challenge group

^a p <0.05: significantly different from Marbomax group

8) Measurement of Calpain I activity

Intramuscular Calpain I activity was measured in the untreated group, the pathogen-infected group, the pathogen + Marboxin-B injection group, and the pathogen + Mavomax administration group.

The intramuscular concentrations of Calpain I in the untreated group, the pathogen-infected group, the pathogen+Maboxin-B injection group, and the pathogen+Mavomax treatment group were 35.6±4.1 ng/mL, 34.6±3.4 ng/mL, and 35.2±4.2 ng/mL, respectively. mL and 35.1±6.4 ng/mL, no significant differences were observed between each test group (see FIG. 27).

The present invention is an antibiotic, anti-inflammatory and metabolism promoter Actinobacillus pleuropneumonae in order to confirm the therapeutic effect of 'Marboxin-B strain' containing mabofloxacin, flunixin and butaphosphan as active ingredients for swine respiratory disease (SRD), The clinical efficacy of 'Marboxin-B strain' was evaluated by inducing respiratory diseases in pigs through combined infection with Pasteurella multocida and Mycoplasma hyopneumoniae , and antipyretic, anti-inflammatory and metabolic stimulating effects were also evaluated.

Marbofloxacin is a 3rd generation fluoroquinolone broad-spectrum antibiotic that is used to treat severe infections such as respiratory diseases, digestive diseases, and sepsis caused by Gram-negative bacteria. It is also used for urinary tract infections and skin infections caused by Gram-negative bacteria or some Gram-positive bacteria. have. In addition, mabofloxacin has low affinity for plasma proteins, high tissue permeability, is distributed in various organs, has high bioavailability when injected intramuscularly into pigs, and accumulates in the cytoplasm of leukocytes, neutrophils and macrophages, so it is effective in treating Mycoplasma. do.

After inoculating A. pleuropneumoniae , P. multocida , and M. hyopneumoniae , the causative bacteria of respiratory diseases, into the nasal cavity of pigs once to induce respiratory diseases, intramuscular injection of 1 mL of maboxin-B strain per 50 kg of body weight for 5 days Results , From day 1 after mavosin-B administration, body temperature decrease, cough and respiratory rate decrease, nasal mucus secretion decrease, and clear runny nose were observed in most subjects, and after 5 to 6 days, some subjects temporarily increased body temperature and a small amount of clear Only runny nose and increased respiratory rate were intermittently observed, but clinical symptoms were significantly suppressed until the end of the experiment, and the effect of improving feed efficiency and weight gain was also remarkable. In addition, A. pleuropneumoniae , P. multocida , and M. hyopneumoniae bacteria were not detected in the nasal exudate of the pathogen + maboxin-B injection group from the 7th day after the start of administration. Purulent nodules and purplish red sclerosis did not appear or were weak, and the bacteria removal effect in the lung tissue was remarkable.

On the other hand, in the group administered with mabofloxacin alone, 10% mabomax, the pathogen + mabomax administration group, most of the subjects showed a decrease in body temperature, a decrease in cough and respiratory rate, a decrease in mucus secretion, and clear runny nose on the 2nd to 4th day after the test substance was administered. It was observed, and after 6 to 7 days, a temporary increase in body temperature, a small amount of clear runny nose, coughing, and an increase in respiratory rate were intermittently observed in some individuals, and the effect of improving feed efficiency and weight gain was confirmed. In the pathogen + Mabomax administration group, bacteria were not detected from 7 to 8 days after the start of drug administration, and even in the lungs, chest wall adhesions, purulent nodules, purple sclerosis, etc., observed in the pathogen-infected group, did not appear or were weak. , The effect of removing bacteria in the lung tissue was also remarkable, but overall, the effect of improving clinical symptoms was weak compared to the group administered with Maboxin-B.

The proinflammatory cytokines TNF-α, IL-1β and IL-6 are induced by pathogen infection and are involved in the immediate immune response and the acquired immune response of Th1 and Th2 cells. TNF-α is a representative cytokine that increases the activity of the immune system and is produced in macrophages and promotes the production of factors necessary for an inflammatory response. IL-1β, along with TNF-α, is involved in inducing an acute or chronic inflammatory response, causing a local inflammatory response, and acting to remove neutrophils and phagocytes by moving them to the inflammatory site and activating them. Although IL-6 has a weaker ability to induce inflammation than TNF-α and IL-1β, it has the ability to deepen inflammation and activates lymphocytes to increase antibody production. COX, an inflammation-inducing factor, is an enzyme that converts arachidonic acid to PGE2 and is classified into COX-1 and COX-2. The mechanism of action of many anti-inflammatory drugs is the inhibition of prostaglandin synthesis, which is caused by the inhibition of COX-2 production and activity.

Flunixin, an active ingredient of Marboxin-B strain, inhibits cyclooxygenase (COX) involved in inflammatory reactions and reduces prostaglandin synthesis, thereby exhibiting anti-inflammatory, antipyretic and analgesic effects. Therefore, in order to confirm the effect of flunixin on antipyretic and anti-inflammatory effects, changes in concentrations of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and fever factor PGE2 were confirmed.

After inoculation with Actinobacillus pleuropneumoniae , Pasteurella multocida , and Mycoplasma hyopneumoniae , TNF-α, IL-1β, and IL-6 in the serum of the pathogen-infected group, the pathogen + Marboxin-B injection group, and the pathogen + Mavomax administration group except for the untreated group were challenged. increased. However, on the 1st day after the initiation of drug administration of the flunixin-containing maboxin-B injection, the blood TNF-α of the pathogen + maboxin-B injection group was 236.4±32.1 pg/mL, compared to the pathogen-infected group (295.1±20.0 pg/mL). and pathogen + Mabomax (273.9 ± 30.9 pg/mL), and PGE2 concentration (246.3 ± 27.5 pg/mL) was also significantly lower than that of the pathogen infection group (326.1 ± 52.9 pg/mL). TNF-α (239.4±31.6 pg/m), IL-1β (31.173±3.408 ng/L) and PGE2 (234.1±37.6 pg/mL) in the blood of the pathogen+maboxin-B injection group on the 4th day after drug administration Concentrations of the pathogen-infected group (TNF-α: 289.9±15.0 pg/mL, IL-1β: 52.263±15.854 ng/L, PGE2: 292.8±27.5 pg/mL) and the pathogen+Mabomax-administered group (TNF-α: 276.9± 39.5 pg/mL, IL-1β: 45.239±12.107 ng/L, PGE2: 282.7±31.8 pg/mL). On the 7th day after the start of drug administration, blood TNF-α (216.9±24.2 pg/mL), IL-1β (32.005±4.654 ng/L), and PGE2 (196.4±11.4 pg/mL) in the pathogen-infected group (TNF-α: 258.4±25.0 pg/mL, IL-1β: 50.125±13.164 ng/L ng/L, PGE2: 270.4±34.3 pg/mL), and in particular, PGE2 was significantly lower in the pathogen + Mabomax group (233.3±33.2 pg). /mL) was significantly lower than Even on the 14th day after the start of drug administration, TNF-α (195.1±22.2 pg/mL), IL-1β (29.764±2.669 ng/L), and IL-6 (103.951± 27.697 ng/L) in the blood of the group administered with pathogen + maboxin-B L) and PGE2 (181.1±21.3 pg/mL) in the pathogen-infected group (TNF-α: 244.8±31.9 pg/mL, IL-1 β: 41.244±4.403 ng/L, IL-6: 159.394 ±30.690 ng/L , PGE2: 265.8±23.7 pg/mL), and the concentration of IL-β was significantly lower than that of the pathogen+Mabomax-administered group (35.173±4.767 ng/L).

In other words, flunixin, an active ingredient of Marboxin-B strain, effectively reduces pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and PGE2, a fever mediator, so that the pathogen + Marboxin-B treated group A decrease in body temperature occurred from the first day after the start of drug administration, and a continuous increase in body temperature was observed in 2 to 3 animals in the pathogen-infected group and the pathogen + Mavomax administration group, whereas only a temporary increase in body temperature was observed in the pathogen + Marboxin-B injection group. Observed. In addition, by reducing the excessive immune response, an increase in feed intake was induced faster than the pathogen-infected group and the pathogen + Mabomax administration group, and it was found that clinical symptoms were alleviated.

In addition, to confirm the metabolic promoting effect of maboxin-B strain, glucose, insulin, glucagon, total cholesterol (T-CHO), triglyceride (TG), phosphorus (P) and urea contents Changes were confirmed.

After challenge vaccination, the blood glucose concentrations of the pathogen-infected group, the pathogen+Maboxin-B main administration group, and the pathogen+Mavomax administration group were 36.0±10.1 mg/dL, 38.0±12.8 mg/dL, and 32.6±10.5 mg/dL, respectively, without treatment. It was significantly decreased compared to the group (84.8±8.9 mg/L). However, in the case of the pathogen + Maboxin-B main administration group, the blood glucose concentration began to increase from the 1st day after the start of drug administration, and the blood glucose concentration was continuously higher until the 14th day than the pathogen infection group or the pathogen + Mabomax administration group. That is, the blood glucose concentrations on the 1st, 4th, 7th, and 14th days after the start of drug administration in the pathogen + marboxin-B main administration group were 71.8±12.7 mg/dL, 84.0±6.5 mg/dL, and 86.8±10.3 mg, respectively. /dL and 83.2±9.3 mg/dL in the pathogen-infected group (Day 1: 52.8±9.8 mg/dL, Day 4: 75.2±7.1 mg/dL, Day 7: 68.4±8.4 mg/dL, Day 14: 54.8±15.2 mg/dL) and pathogen + Mabomax administration group (Day 1: 59.0±13.9 mg/dL, Day 4: 79.6±10.5 mg/dL, Day 7: 74.4±6.3 mg/dL, Day 14: 68.2±9.9 mg/dL ) tended to be higher than blood glucose.

As a result of confirming changes in insulin and glucagon, which are hormones related to blood glucose, after challenge vaccination, the insulin concentration of the pathogen-infected group, the pathogen + Marboxin-B injection group, and the pathogen + Mavomax administration group tended to be higher than that of the untreated group. , The blood insulin concentration of the pathogen-infected group and the pathogen-administered group, except for the pathogen + Marboxin-B strain, maintained a high concentration until the end of the experiment. /mL) and the insulin concentration of the pathogen + Mavomax administration group (6.258 ± 0.856 μIU / mL) were significantly different from the pathogen + Marboxin-B injection group (5.013 ± 0.426 μIU / mL) and the untreated group (4.646 ± 0.248 μIU / mL). was significantly higher than that of However, in the case of glucagon, which is involved in glucose synthesis in the liver, no significant difference was observed between each test group.

It has been reported that the low blood glucose content after challenge inoculation is due to an increase in glucose consumption in order to produce factors for immune cells to defend against and eliminate pathogens caused by pathogen infection. In other words, it has been reported that glucose use and energy consumption increase by about 50% when an infection occurs, and to compensate for this, gluconeogenesis in the liver and glucose use in insulin-sensitive tissues (skeletal muscle, adipose tissue) are reduced to increase blood sugar levels. It has been reported that my glucose content can be increased.

In the present invention, in the group administered with pathogen + maboxin-B, blood glucose increased due to an increase in feed intake and promotion of metabolism, but no significant changes were observed in insulin and glucagon compared to the untreated group. However, the reason why the insulin content of the pathogen-infected group and the pathogen + Mavomax administration group tended to be higher than that of the pathogen + Marboxin-B main administration group is to supply energy (glucose) necessary for immune cells while the immune response continues to appear. It became.

In addition, as a result of confirming changes in the content of total cholesterol and triglyceride related to lipid metabolism in the present invention, the total cholesterol content decreased in all test groups except for the untreated group by challenge inoculation, but the TG content No significant changes were observed. In the group administered with the main pathogen + Marboxin-B, the content of total cholesterol increased compared to the group infected with the pathogen or the group administered with the pathogen + Mabomax, which was determined to be due to an increase in feed intake and promotion of lipid metabolism. In addition, according to several studies, it has been reported that total cholesterol in blood decreases and TG increases during pathogen infection or inflammatory reaction. In the present invention, total cholesterol in the blood was reduced in the early stage of infection, and no increase in TG was observed.

Phosphorus (P) is an essential element as the second most abundant mineral in the body of animals after calcium and is essential for the maintenance and development of skeletal tissue, maintenance of acid-base balance and osmotic pressure, energy use and conversion, protein synthesis, cell differentiation and growth, appetite control, It is a key regulator of metabolism including cell signaling and nutrient metabolism. Butaphosphan, which is contained in Marboxin-B strain, is known to provide phosphorus necessary for gluconeogenesis in the liver as an organic phosphorus.

As a result of checking the phosphorus content in blood in the present invention, no significant difference was observed between each test group at most time points, but the phosphorus content of the pathogen + maboxin-B strain on the 4th day after the start of drug administration was the same as that of the pathogen-infected group and the pathogen + A significant increase was observed compared to the Mabomax administration group.

In addition, as a result of confirming the urea content, which is a biomarker of protein degradation, the urea content increased in the pathogen-infected group, the pathogen + Marboxin-B injection group, and the pathogen + Mavomax administration group except for no treatment after challenge. This is related to the increase in TNF-α and IL-6 by pathogen infection, and it seems that protein degradation is increased to supply amino acids necessary for protein synthesis in the acute phase. These results were similar to reports of hypoproteinemia in pigs injected intramuscularly with LPS (Daiwen et al., 2008, Johnson, 2012, Webel et al., 1997). However, in the group administered with the main pathogen + Marboxin-B, the blood urea concentration significantly decreased on the 1st day after the start of drug administration compared to the group infected with the pathogen and the group administered with the pathogen + Mabomax. Proteolysis was judged to be reduced.

Calpain 1 (μ-calpain) is a Ca2+ dependent, non-lysosomal cysteine protease, and is mainly expressed in porcine skeletal muscle. Calpain induces the degradation of IkB, an endogenous inhibitor of NF-kB that induces an inflammatory response, and mediates the signaling of TNF-α, IL-1, and COX-2, which are proinflammatory factors, to be involved in the inflammatory response. In the present invention, the fact that there was no difference in the content of Calpain 1 in the muscle of each test group was determined that there was no sensitive change depending on the disease state because the muscle contained a large amount of Calpain 1.

In conclusion, Marbofloxacin-B, which contains marbofloxacin, flunixin, and butaphosphan as active ingredients, marbofloxacin effectively removes germs when a disease occurs due to pathogen infection, and flunixin also reduces excessive inflammation caused by pathogen infection. Not only does it suppress the reaction to reduce fever, pain relief, improve vitality, alleviate clinical symptoms and increase feed intake, butaphosphan properly provides phosphorus necessary for the body and promotes sugar metabolism, effectively reducing hypoglycemia caused by pathogen infection. It was confirmed to be helpful in improving weight gain by blocking it, and it was evaluated as a safe drug in pigs and a rapid and excellent effect of alleviating symptoms and improving symptoms compared to marbofloxacin alone for respiratory diseases.

Preferred embodiments of the present invention described above have been disclosed to solve the technical problems, and those skilled in the art will be able to make various modifications, changes, additions, etc. within the spirit and scope of the present invention. , such modifications and changes should be regarded as belonging to the scope of the following claims.

Claims (4)

delete delete delete A composition for the treatment of swine respiratory disease (SRD) comprising marbofloxacin, flunixin meglumin and butaphosphan as active ingredients,
The swine respiratory disease (Swine Respiratory Disease, SRD) is Actinobacillus pleuropneumoniae ( Actinobacillus pleuropneumoniae ), Pasteurella multocida ( Pasteurella multocida ) and Mycoplasma hyopneumoniae ( Mycoplasma hyopneumoniae ) Bacteria including It is a disease caused by complex infection of
The content of the active ingredient is 98 to 102 parts by weight of Marbofloxacin, 81 to 85 parts by weight of Flunixin meglumin and 98 to 102 parts by weight of Butaphosphan,
The composition is formulated as an injection for pigs, and the dosage per prescription is 1.5 to 2.5 mg of Marbofloxacin, 0.72 to 1.3 mg of Flunixin, and 0.72 to 1.3 mg of Butaphosphan per 1 kg of body weight. ) A composition for treating bacterial respiratory diseases in animals, characterized in that 1.5 to 2.5 mg.

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