KR101378608B1 - A Novel Lactobacillus fermentum with Treating Activity for animal disease caused by viruses - Google Patents

Abstract

The present invention relates to a novel isolated Lactobacillus fermentum strain having a viral infection inhibitory activity, the novel isolated strain of the present invention is excellent in the prevention and treatment of one or more different types of viral infections, It can be widely used for the prevention and treatment of viral diseases.

Description

A novel isolated Lactobacillus fermentum with Treating Activity for animal disease caused by viruses}

The present invention relates to a novel isolated Lactobacillus fermentum strain having viral infection inhibitory activity.

Due to the recent surge in damage caused by livestock viral diseases, efforts have been made to suppress animal infections such as prevention and prevention of vaccinations, but there are no genetic variants of the virus and the incomplete efficacy of vaccines. I am struggling with my back. In particular, the respiratory system of animals in which gas exchange takes place is the most active site of innate immunity because it is easily exposed to external pathogens. There are many types of viruses that cause respiratory diseases, and the site of infection, host range, and clinical symptoms vary greatly depending on the virus. In addition, most viral respiratory diseases are characterized by a very fast propagation rate is difficult to prevent and treat a lot.

Influenza virus, a representative cause of respiratory diseases, is an RNA virus belonging to the family Orthomyxoviridae. The serotypes are classified into three types, type A, type B, and type C. Among them, type B and type C Has been identified only in humans, and type A has been identified in humans, horses, pigs, other mammals, and various types of poultry and wild birds (Webster RG et al., 1992. Microbiol Rev., 56 (1). ): 152-179).

In the case of influenza A, it is a problem when it spreads to a new host through genetic mutation, so minimizing the influenza epidemic in animals other than humans is attracting attention as an important means of preventing influenza pandemic in humans.

Currently, influenza countermeasures that can be used in animals are the only vaccination vaccines, but they are not able to prevent 100% of the virus infection and catch the mutation of the virus. Although influenza drugs have been developed, they are all expensive drugs developed for humans, and even these have limited effective serotypes and routes of administration, and side effects such as the appearance of resistant viruses, vomiting and dizziness (Ward P et. al., J. antimicrob . Chemother . , 55 (supp1), ppi5-i21, 2005).

Infectious bronchitis (IB) is a representative respiratory disease in the poultry industry worldwide. It is a representative acute respiratory infectious disease in chickens with very high propagation caused by the IB virus (IBV) of the family Coronaviridae. IBV does not have a high mortality rate during infection, but has a high morbidity (expressed as a percentage of the population per patient over a given period of time) and causes lower egg laying rates with coughing, runny nose, lower body weight, lower external and internal egg quality, as well as respiratory sequelae. This is causing enormous economic damage to the poultry industry worldwide by causing a wide variety of productivity declines, such as the presence of E. coli and chronic mortality (David cavanagh and Syed A. Naqi, 2003, infectious bronchitis , Disease of poultry 11 ^th edition : 101-119).

IBV is an RNA virus that has a characteristic that genes constituting virus particles are easily changed, similar to influenza virus. Therefore, a wide variety of IBV serotypes have been reported and reported worldwide, and it is estimated that there will be dozens of species including the mutated mutant virus in each serotype. In addition, these serotypes are difficult to use due to poor cross-reaction or cross-immunity (Jane KACook et al., Avian) . pathol . 28: 477-485, 1999, J. Ignjatovic and S. Sapats, Rev. sci . tech . Off . Int . Epiz ., 19 (2): 493-508, 2000).

Representative viral respiratory diseases in the hog industry around the world include Porcine reproductive and respiratory syndrome (PRRS) caused by the Porcine Genital Respiratory Syndrome Virus (PRRSV) of the Arteririridae family. Since the 1980s, the majority of pig-producing countries in North America, Europe and Asia have been causing fertility and respiratory diseases in pigs (KD Rossow, 1998, Vet). Pathol ., 35 (1): 1-20).

Currently, live and killed vaccines are used worldwide for the prevention of PRRS. However, live vaccines are effective in reducing the incidence of disease, the extent of clinical manifestations, the duration of virus release, etc., but have the disadvantage that they are effective only against homologous serotypes and that immunity does not last long. Zadok vaccines are not only delayed in induction of cellular immunity, but also have limited efficacy due to the inability to produce neutralizing antibodies required for virus removal (TG Kimman et al., 2009, Vaccine , 27: 3704-3718; W. Charerntantanakul, 2009). , Vet Immunol Immunopathol 129:.. . 1-13) provided with a countermeasure is urgently required. In addition, the continuous circulation of viruses in pig populations through subclinical infections, the continuous influx of susceptible breeding pig populations, and the emergence of mutant strains have made it difficult to prevent damage from these viruses (Michael PM et al., 2002, Viral). Immunol ., 15 (4): 533-47, 2002; Murtaugh MP et al. Virus Res . , 154 (1-2): 18-30, 2010).

On the other hand, probiotics (live probiotics) refers to a living microbial agent or component of a microorganism that acts on the normal bacterial flora in the intestinal tract and improves its balance to benefit the host. In particular, lactic acid bacteria is a generic term for bacteria that are characterized by producing a lot of lactic acid in the growth process, it is commonly used as a probiotic as a strain that exists in the gut of healthy animals. Generally known probiotics efficacy of lactic acid bacteria helps to suppress the growth of harmful microorganisms in the intestinal tract and balance the useful bacteria flora through sterilization of pathogenic bacteria, inducing changes in bacterial metabolism, and stimulating host immune function. will be.

Recently, attention has been paid to the pharmacological effects of the infection defense effect against pathogenic microorganisms by the reactivation of host immune function of lactic acid bacteria. In addition to the known diarrhea-induced gastrointestinal infectious diseases, there is a newly reported application for the prevention and treatment of respiratory and systemic viral infections (US Patent No. 7807185, US Patent No. 20040057965 and US Patent No. 7862808). However, most of the techniques limit subjects to human infectious diseases and are not intended to prevent or treat respiratory diseases in animals.

Cases have also been reported in some animals (Izumo, T. et al., 2010. Int . Immunopharmacol . 10: 1101-1106; Harata, G. et al., 2010. Lett . Appl. Microbiol . 50: 597-602; Korean Patent Laid-Open Publication No. 2005-0023475), these techniques include only limited efficacy against a single viral disease and do not exhibit antiviral combination efficacy against one or more viruses.

In addition, the patented technology related to the antiviral use using lactic acid bacteria often emphasizes the efficacy of culture medium or cell extracts excluding lactic acid bacteria or exhibits the efficacy using complex strains (Korea Patent Registration 1104397, Korea Registered) Patent 0813637, US Patent Publication 20100034877, Republic of Korea Patent 0808910, Republic of Korea Patent Publication 2008-0081146.

Accordingly, the present inventors conducted a study for developing a next-generation antiviral agent that is safe and effective for inhibiting viral diseases in animals. As a result, the present invention was completed by confirming that the morphological, biochemical and genetic characteristics of Lactobacillus spp. Isolated from nature are novel and possess the effect of inhibiting infection and proliferation of one or more viruses.

It is an object of the present invention to provide a novel isolated Lactobacillus strain having the effect of inhibiting the development or symptomatic treatment of animal diseases due to viral infection.

Another object of the present invention is to provide a composition for preventing or treating virus infection in an animal, comprising the Lactobacillus strain as an active ingredient.

Still another object of the present invention is to provide a livestock feed additive or drinking water additive comprising the Lactobacillus strain as an active ingredient.

Still another object of the present invention is to provide a method for preventing or treating infectious diseases caused by viruses of animals using a composition comprising the Lactobacillus strain as an active ingredient.

In order to achieve the above object, in one aspect of the present invention, the present invention relates to a novel isolated Lactobacillus fermentum CJL-112 (Accession No. KCCM11184P) having a virus infection inhibitory activity.

Specifically, samples from healthy infants and fermented plants were taken, cultured in MRS solid medium and grouped according to colony observation to isolate strains of Lactobacillus sp .

One of the primary isolates, Lactobacillus strains showing the best anti-influenza effect was finally selected through mouse in vivo proliferation inhibition test against influenza virus, a representative respiratory virus in animals.

In order to identify the selected Lactobacillus strains, physiological, biochemical properties, sugar availability, and 16S rRNA gene sequencing were performed. As a result of physiological and biochemical characterization, the lactic acid bacteria were Gram-positive bacteria, did not form spores, were able to grow under both aerobic and anaerobic conditions, and did not have motility, and the shape of cells was bacilli. 16S rRNA gene sequence of the Lactobacillus strain is the same as SEQ ID NO: 1, Lactobacillus Permanent standard strain ( Lactobacillus) in molecular system taxonomic analysis fermentum IFO3956, GeneBank accession number NC_010610) showed the highest homology (99.9%). Therefore, the microorganism was identified as Lactobacillus fermentum ( Lactobacillus fermentum ), named as Lactobacillus fermentum CJL-112, and deposited on April 7, 2011 to the Korea Microorganism Conservation Center (Accession No. KCCM11184P).

Lactobacillus Permantum CJL-112 of the present invention is to freeze-dried by storage in -70 ℃ or suspended in sterilized 10% skim milk in a storage solution made by mixing a certain amount of glycerol components in water in order to preserve long-term stable desirable.

As another aspect of the present invention, the present invention relates to a composition for the prevention or treatment of infectious diseases caused by viruses of animals other than humans, comprising the Lactobacillus pertumtum CJL-112 as an active ingredient.

Veterinary compositions comprising the Lactobacillus fermentum CJL-112 of the present invention may further comprise suitable excipients and diluents according to conventional methods.

Excipients and diluents that may be included in the veterinary composition comprising the Lactobacillus fermentum CJL-112 of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber , Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, ste Aryl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methyl paraben, propyl paraben and mineral oil.

The veterinary composition comprising Lactobacillus permanent CJL-112 according to the present invention may further include a filler, an anticoagulant, a lubricant, a humectant, a spice, an emulsifier, a preservative, and the like. It can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to the formulations, the formulations being powders, granules, tablets, capsules, suspensions, emulsions, solutions, Syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile external preparations, and the like.

Veterinary composition according to the invention may vary depending on the age, sex, body weight of the animal, but the 10 ⁴ to 10 has the amount of ¹⁰ cfu / kg can be administered once a day to several times. In addition, the dose of Lactobacillus permanent CJL-112 may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, and age. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

Lactobacillus permanent CJL-112 of the present invention has a viral infection inhibitory activity, the virus is not particularly limited to this, may be an RNA virus or a DNA virus.

An RNA virus is a generic term of a virus having RNA as a gene, and has a (+) chain RNA which becomes an mRNA, a (-) chain RNA of a complementary chain, and a double stranded RNA as genes in a virus particle, and only one molecule of RNA And the like (coronavirus, paramyxovirus), a virus having two homologous RNA molecules (retrovirus), and a virus using eight different RNA molecules as genes (influenza virus). Usually, RNA synthetase (DNA synthetase in case of retrovirus) which has RNA as a template exists.

DNA viruses are classified as either circular DNA viruses or linear DNA viruses, depending on the shape of the gene. Examples of linear DNA viruses include parvoviruses with single stranded linear DNA in virus particles and adenoviruses, herpesviruses, and poxviruses with double stranded linear DNA. Most of them have a special repetitive sequence at the ends of the genome and show unique infection and proliferation patterns due to differences in genome structure and size. DNA viruses having circular DNA molecules as genomes are largely classified into two viral families. In other words, the genome of a papain virus (polyomavirus, SV40, papilloma virus, etc.) having a double-stranded closed-loop DNA molecule as a genome and a double-stranded cyclic DNA molecule including the outer strand portion (Hepadnavi) -ridae) virus (hepatitis B virus, Woodchuck hepatitis virus, etc.).

In the present invention, the virus that Lactobacillus permanent CJL-112 may have an infection inhibitory activity is not particularly limited, and may include all of the above RNA-type or DNA-type viruses, preferably orthomyxoviruses. (orthomyxovirus), paramyxovirus (paramyxovirus), coronavirus (coronavirus), aterivirus (arterivirus), herpesvirus (herpesvirus) or adenovirus (adenovirus).

In a specific embodiment of the present invention, the Lactobacillus permanent CJL-112 of the present invention is influenza A virus (H1N1 strain), low pathogenic avian influenza virus (H9N2 strain), infectious bronchitis virus (KM91 strain), porcine respiratory parasite syndrome virus (PRRS) By confirming that it has an infection inhibitory activity against viruses, LMY strains, etc., it was confirmed that it had antiviral complex efficacy against various viruses.

The term “infectious disease caused by a virus” of the present invention refers to a disease that is caused by a virus and is contagious. The disease is not particularly limited thereto, but is mainly a disease of an animal caused by an RNA virus or a DNA virus. It may mean. Preferably, the infectious diseases are poultry diseases such as avian influenza, Newcastle disease, chicken infectious bronchitis, avian pneumovirus infection, infectious laryngeal bronchitis; Swine influenza, swine respiratory syndrome, swine pandemic diarrhea, bullous stomatitis and the like; Diseases of dogs such as individual influenza, adenovirus infection, canpabovirus infection, canopies virus infection, canine measles, rabies; Diseases of cats such as feline calcivirus infection, feline herpesvirus infection, etc .; Equine influenza, equine encephalitis, equine arteritis virus infection, etc .; It may be a disease of the cow, such as Ozeski disease, bovine viral diarrhea, bovine infectious rhinitis.

As another aspect of the present invention, the present invention relates to a feed additive or drinking water additives for livestock useful for the prevention and improvement of infectious diseases caused by a virus comprising the Lactobacillus Pertumtum CJL-112 as an active ingredient.

The form of Lactobacillus permanent CJL-112 included in the additive is not particularly limited but may be in the form of a concentrate, powder or granules.

In addition to the above-mentioned active ingredients, the additive of the present invention is organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, and catechins (catechin). ), Alpha-tocopherol, rosemary extract (rosemary extract), vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like may further include any one or more than one.

In addition, the additives are amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, live microbial preparations, grains (milled or crushed wheat, oats, barley, corn and rice), vegetable proteins as auxiliary ingredients. Feed (flat, soy and sunflower), animal protein feed (blood, meat, bone meal and fish meal), sugar and dairy products (milk powder and whey powder), dry additives, lipids (animal fats and vegetable fats), nutritional supplements, digestive and It may be included in feeds with substances such as absorption enhancers, growth promoters, disease prevention agents, and the like.

As another aspect of the present invention, the present invention comprises the step of administering the composition for the prevention or treatment of Lactobacillus permanent CJL-112 or an infectious disease caused by the virus to an individual in need thereof, except human A method for treating an infectious disease caused by a virus in an animal.

The Lactobacillus permanent CJL-112, a composition comprising the same, and an infectious disease are the same as described above.

The method of administering the composition for the prevention or treatment of Lactobacillus permanent CJL-112 or the infectious disease caused by the virus is not particularly limited thereto, but is administered by oral or parenteral routes to an individual in need thereof. Can be selected, and can be preferably administered via a parenteral route.

First, when administered orally, it is preferable to administer the strain or composition to the feed mixed or administered in a separate oral dosage form other than the feed.

In addition, when administered via a parenteral route, it is possible to prevent loss due to metabolism, partial or total degradation of the drug, binding to food, degradation in the digestive tract or other causes, preferably subcutaneous, muscle, It can be administered by injection or infusion through intravenous, transdermal, dermal, nasal, arterial, abdominal, ocular, mucosal, skin or the like, more preferably in the manner of injection into the nasal or ocular. When administering through the parenteral route, it is preferable to administer in a dosage form suitable for the route of administration. For example, when administered through the nasal cavity, it is formulated in the form of a spray or inhalant, and when administered through the eye, it is formulated in the form of a spray or eye drop, and when administered through the mucous membrane or skin, It may be administered in the form of a patch or ointment.

The method for treating an infectious disease caused by the virus of the present invention can be applied to a plurality of animals including mammals, poultry and fish. More specifically, the method can be used in commercially important mammals such as pigs, cattle, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), furry animals (eg mink and fox), And zoo animals (eg monkeys and tailless monkeys), as well as pets (eg cats and dogs).

The novel isolated Lactobacillus bacteria of the present invention is excellent in the prevention and treatment of one or more different types of viral infections, and thus may be widely used for the prevention and treatment of viral diseases in animals.

1 is an electron micrograph of the Lactobacillus latement CJL-112 of the present invention.
Figure 2 is a schematic diagram showing the phylogenetic relationship with other bacteria based on the nucleotide sequence of the Lactobacillus Permanent CJL-112 of the present invention.
Figure 3 is an electrophoresis picture showing the SDS-PAGE results of Lactobacillus Permanent CJL-112 and Lactobacillus Permanent standard strain ATCC9338 according to an embodiment of the present invention. Protein patterns of both strains were observed with major proteins of about 145kDa, 80kDa, 70kDa, 51kDa and 48kDa.
Figure 4 is an electrophoresis picture showing the results of RAPD analysis after PCR using a primer after extracting and purifying the DNA of Lactobacillus Permanent CJL-112 and Lactobacillus Permanent standard ATCC9338 according to an embodiment of the present invention. (a) is a primer P2, (b) is an electrophoresis picture using the primer P3.
FIG. 5 shows the influenzavirus (A / NWS / 33 [H1N1]) challenge following administration of Lactobacillus permanent CJL-112 to BALB / c mice 6 times in total nasal cavity 10 days in accordance with an embodiment of the present invention. Changes in cytokine and IgA concentrations in mouse lungs were measured. (a) is IL-1β, (b) is IL-2, (c) is IFN-γ, (d) is IFN-α, (e) is IgA, (f) is IL-10, (g) is The concentration measurement result of TNF-α is shown. (*; p <0.05, **; p <0.01, hpc; hour of post challenge)

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Reference Example  1: Preparation of the virus

Reference Example  1-1: Influenza A Virus ( Influenza  A virus )

The A / NWS / 33 (H1N1) strain, which was a published virus, was purchased from the American Type Culture Collection, USA (ATCC), and used for storage for 48 hours in 10 days old SPF (Specific Pathogen Free) eggs for storage and storage at -80 ° C. .

Reference Example  1-2: Low pathogenic  Avian Influenza Virus ( low pathogenic avian  Influenza virus )

The A / Korean native chicken / Korea / K040110 / 2010 (H9N2) strain was used as the field strain isolated from domestic chickens for 48 hours in 10-day-old SPF eggs and stored at -80 ° C.

Reference Example  1-3: infectious bronchitis virus ( Infectious bronchitis virus )

The KM91 strain, a published virus, was distributed by the National Veterinary Research and Quarantine Service, and used for 48 hours in 10-day-old SPF eggs and stored at -80 ° C.

Example  One: Lactobacillus Permanent CJL Isolation of -112 Strain

Example  1-1: Sample acquisition and Candidate strain  detach

Samples obtained from fecal and fermented plants of Korean infants were diluted in stages and incubated in MRS solid medium (Difco, USA) for 24 hours under anaerobic conditions at 37 ° C. Strains isolated from each sample were grouped according to colony observation to isolate strains. Selected colonies were purely separated by culturing in a new medium over 3 times, and the pure cultured cells were stored in a medium to which 20% glycerol was added and stored at below 70 ° C.

Example  1-2: Anti-influenza  Selection of strains with excellent efficacy

Six-week-old SPF BALB / c mice (Orient, Korea) were divided into 10 groups per group, and were divided into a test group to which lactic acid bacteria were administered, a positive control group to which no lactic acid bacteria were administered, and a negative control group to which virus was not challenged.

Nine pure test specimens were incubated in MRS liquid medium (Difco, USA) for 24 hours at 37 ° C for 24 hours, and then the cultures were centrifuged and washed with sterile distilled water. The concentration was> 10 ¹⁰ cfu / ml. It was suspended in sterile distilled water and used for the test.

Administration of the prepared lactic acid bacteria was largely divided into oral and nasal administration. In the case of oral administration, lactobacillus suspension was administered using oral sonde at 300 μl daily, starting from 21 days before challenge and 14 days after challenge. In the case of nasal administration, a 90 μl dose of lactic acid bacteria suspension was administered to the nasal cavity after ketamine anesthesia (100 mg / kg) for 6 times at intervals of 3 to 4 days for 21 days prior to challenge with influenza virus.

For influenza virus challenge, influenza A / NWS / 33 (N1H1) virus fluid with a titer of 10 ^4.0 TCID ₅₀ / ml was administered to the nasal cavity at 90 μl dose per animal after ketamine anesthesia (100 mg / kg). .

The body weight, feed and drinking intake, and mortality were observed during the test period, and the clinical symptoms and the suppression of mortality expression were measured after influenza virus challenge against lactobacillus nasal administration. Retests were performed when mice used in negative controls without influenza A / NWS / 33 (H1N1) virus challenge did not survive more than 80%.

The oral and nasal administration results are shown in Table 1 and Table 2, respectively.

Changes in Clinical Symptoms after Influenza Inoculation after Oral Lactobacillus Administration in Mice group
Dose strain ^A Average weight (± SD) (kg) Clinical Symptom ^B (MTO) % Mortality (MDT) PI ₁₄ Before vaccination After inoculation  Test group CJL-A 20.3 ± 0.7 14.8 ± 4.4 10/10 (3) 7/10 (8.9) 1.7 CJL-B 20.1 ± 1.5 15.1 ± 1.3 10/10 (3) 8/10 (8.4) 1.8 CJL-C 20.9 ± 1.0 15.4 ± 0.4 10/10 (3) 9/10 (8.1) 1.9 CJL-D 21.1 ± 0.6 16.4 ± 1.1 10/10 (3) 7/10 (9.4) 1.7 CJL-E 20.9 ± 1.0 15.4 ± 0.4 10/10 (3) 8/10 (8.0) 1.8 CJL-F 20.9 ± 0.7 0.0 ± 0.0 10/10 (3) 10/10 (7.5) 2.0 CJL-G 20.9 ± 1.0 18.2 ± 0.0 10/10 (3) 9/10 (7.8) 1.9 CJL-H 20.1 ± 1.4 0.0 ± 0.0 10/10 (3) 10/10 (8.0) 2.0 CJL-I 20.2 ± 0.8 0.0 ± 0.0 10/10 (3) 10/10 (8.6) 2.0 Attack
Control group - 20.4 ± 0.8 0.0 ± 0.0 10/10 (3) 10/10 (7.8) 2.0 voice
Control group - 20.0 ± 1.0 20.8 ± 0.7 0/10 0/10 0

^A) Test Lactobacillus is administered orally every day from 21 days before challenge to 14 days after challenge with influenza virus (A / NWS / 33 [H1N1]) at a dose of 300 μl per animal.

^B) Clinical Symptoms / Head of Vaccination

ND; not determined

Mean time of onset clinical signs (MTO); Mean clinical presentation time

Mean deathh time (MDT); Average lethal time

Pathogenicity index ₁₄ (PI ₁₄ ); Pathogenicity index, average score per mouse observed daily over a period of 14 days, normal: 0, sick (fever, eyelids, hairy abnormalities): 1, death: 2

As shown in Table 1, seven candidate strains (CJL-A, CJL-B, CJL-C, and CJL-) showed delayed mean mortality or decreased pathogenicity indices compared to the challenge group through oral administration of lactic acid bacteria. D, CJL-E, CJL-H, CJL-I) were selected first.

Changes in Clinical Symptoms Following Influenza Inoculation After Lactic Acid Bacteria Administration in Mice group
Dose strain ^A Average weight (± SD) (kg) Clinical Symptom ^B (MTO) % Mortality (MDT) PI ₁₄ Before vaccination After inoculation  Test group CJL-A 19.9 ± 1.8 18.2 ± 2.4 10/10 (3) 40 (9.5) 1.4 CJL-B 19.9 ± 1.8 15.9 ± 2.1 10/10 (3) 50 (10.2) 1.5 CJL-C 20.7 ± 1.5 16.8 ± 3.7 10/10 (3) 80 (9.0) 1.8 CJL-D 19.6 ± 1.3 16.3 ± 1.9 10/10 (3) 40 (9.8) 1.4 CJL-E 19.9 ± 1.0 15.6 ± 2.3 10/10 (3) 0 (ND) 0 CJL-H 20.4 ± 0.8 16.8 ± 2.3 10/10 (3) 100 (8.3) 1.3 CJL-I 20.4 ± 0.8 18.9 ± 1.9 10/10 (3) 50 (10.2) 1.5 Attack
Control group - 20.2 ± 0.7 0.0 ± 0.0 10/10 (3) 10/10 (8.0) 2.0 voice
Control group - 20.3 ± 0.7 21.1 ± 0.9 0/10
(ND) 0/10
(ND) 0

^{A) The} test lactic acid bacteria were administered at a dose of 90 μl per bird in total 6 times at intervals of 3-4 days for 21 days prior to challenge with influenza virus (A / NWS / 33 [H1N1]).

^B) Clinical Symptoms / Head of Vaccination

ND; not determined

Mean time of onset clinical signs (MTO); Mean clinical presentation time

Mean deathh time (MDT); Average lethal time

Pathogenicity index ₁₄ (PI ₁₄ ); Pathogenicity index, average score per mouse observed daily over a period of 14 days, normal: 0, sick (fever, eyelids, hairy abnormalities): 1, death: 2

As shown in Table 2, three candidate strains (CJL-A, CJL-D, and CJL-E) that significantly reduced mortality were identified through nasal administration of lactic acid bacteria among the seven selected lactic acid bacteria. Among them, Lactobacillus spp. CJL-E, which was shown to completely inhibit mortality due to influenza infection, was selected.

Example  1-3: Selection strain  Identification, Physiology and Biochemical Characterization

Pure isolated final selection strain CJL-E was incubated for 18-24 hours in 37 ° C conditions in MRS liquid medium (Difco, USA).

After incubation, the physiological characteristics, sugar availability, and 16S rRNA gene sequencing of the final selected strain CJL-E were performed.

The strain is a Gram-positive bacterium, without forming a spore, capable of growing under both aerobic and anaerobic conditions, and lacking motility. 1 is an electron micrograph of the CJL-E of the present invention.

In addition, the nucleotide sequence of 16S rRNA gene was analyzed for lactic acid bacteria identification and classification. The base sequence of the 16S rRNA gene of the selected strain identified through the analysis was described in the sequence list (SEQ ID NO: 1) (FIG. 2). Figure 2 is a schematic diagram showing the phylogenetic relationship with other bacteria based on the nucleotide sequence of CJL-E according to an embodiment of the present invention.

As shown in Figure 2, 16S rRNA sequence analysis of the final selection strain showed the highest homology (99.9%) with Lactobacillus fermentum standard strain (Lactobacillus fermentum IFO3956, GeneBank accession number NC_010610) Lactobacillus fermentum ) And was named Lactobacillus permanent CJL-112, and was deposited with the Korea Microorganism Conservation Center on April 7, 2011 (Accession No. KCCM11184P).

Specific biochemical properties and sugar availability of Lactobacillus permanent CJL-112 were confirmed using API 50 CHL (Biomerius Co., Ltd.), and the results of analysis were shown in Table 3 below.

Sugar Availability of Lactobacillus Permanent CJL-112 characteristic result characteristic result characteristic result Glycerol - Mannitol - d-raffinose + Erythritol - Sorbitol - Starch - D-arabinose - α-methyl-D-mannoside - Glycogen - L-arabinose + α-methyl-D-glucoside - Xylitol - Ribose + N-acetyl-glucosamine - b-Gentibiose - D-xylose + Amygdalin - d-turanose - L-xylose - Arbutin - d-lyxose - Adonitol - Esculin - d-tagatose - β-methyl-D-xylose - Salicin - d-fucose - Galactose - Cellobiose - l-fucose - Glucose + Maltose + d-arabitol - Fructose + Lactose - l-arabitol - Mannose - Melbiose + Gluconate + Sorbose - Sucrose + 2-Keto-gluconate - Rhamnose - Trehalose - 5-Keto-gluconate + Dulcitol - Inulin - Inositol - Melezitose -

+: Positive,-: negative

As shown in Table 3 above, Lactobacillus permanent CJL-112 is L-arabinose, ribose, D-xylose, glucose, fructose, maltose, melbiose, sucrose, d-rapinose, gluconate and 5- It was confirmed that the keto-gluconate can be metabolized.

Example  2: Lactobacillus Permanent CJL Cell Wall Protein Pattern Analysis of -112 Strain

The cell wall protein pattern analysis of Lactobacillus fermentum CJL-112 strain was performed by Angelis et al. (Angelis et al., 2001. Appl . Environ . Microbiol . 67: 2011-2020). For comparison, Lactobacillus standard strain ATCC9338 was also used in the analysis. Lactobacillus species incubated for 24 hours using MRS liquid medium were washed twice with 0.05M Tris-HCl (pH 7.5) solution containing 0.1M CaCl2. The cells were suspended in the same solution to prepare 1 ml of OD 10.0 (A ₆₀₀ ) solution, followed by centrifugation at 8000 xg for 5 minutes. In order to extract proteins from the cells, the soaked cells were suspended in 1 ml of extract solution (0.01M EDTA, 0.01M NaCl, 2% SDS), and allowed to stand at room temperature for 60 minutes and reacted at 100 ° C for 5 minutes. After the reaction, the supernatant was recovered by centrifugation for 10 minutes at 11600 xg and 4 ° C. 15 µl of the recovered supernatant and 3 µl of 5X SDS sample solution were mixed and then boiled for 5 minutes. This was followed by the development of cell wall proteins of Lactobacillus permentum species on 4-12% NuPAGE Bis-Tris (Invitrogen) gels. Gels were stained at room temperature for 1 hour using Coomassie blue staining solution (FIG. 3). Figure 3 is an electrophoresis picture showing the SDS-PAGE results of CJL-112 and Lactobacillus Permanent standard stock ATCC9338 according to an embodiment of the present invention. As shown in Figure 3, the protein pattern of CJL-112 was observed as the main protein of about 145 kDa, 80kDa, 70kDa, 51kDa and 48kDa. This is the same as the protein pattern of the standard strain, it was confirmed that CJL-112 is a kind of Lactobacillus percentage.

Example  3: Lactobacillus Permanent CJL Of -112 strain RAPD  analysis

Randomly amplified polymerhic DNA (RAPD) analysis of Lactobacillus permanent CJL-112 used the method of Angelis et al. (Angelis, MD et al., 2001. Appl . Environ . Microbiol . 67: 2011-2020). The primers used for the test were P2 (5'-ATGTAACGCC-3 ', SEQ ID NO: 2) and P3 (5'-CTGCGGCAT-3', SEQ ID NO: 3), and PCR conditions were 94 ° C 1 after 4 min treatment at 94 ° C. The reaction for 45 minutes at 35 ° C. for 1 minute and 72 ° C. for 2 minutes was repeated 45 times, and the reaction was completed after 5 minutes at 72 ° C. The amplified PCR product was confirmed by electrophoresis on 1.5% agarose gel, stained with EtBr and irradiated with UV (FIG. 4). 4 (a) shows the results of RAPD analysis after PCR using primer P2 after extracting and purifying the DNA of Lactobacillus permanent CJL-112 and Lactobacillus permanent standard ATCC9338 according to an embodiment of the present invention. Figure 4 (b) shows the extraction and purification of the DNA of the Lactobacillus Permanum CJL-112 and Lactobacillus Permanent standard strain ATCC9338 according to an embodiment of the present invention and then PCR using primer P3 RAPD An electrophoretic photograph showing the results of the analysis.

As shown in FIG. 4, the RAPD patterns of the Lactobacillus permanent CJL-112 using primers P2 and P3 were all different from those of the Lactobacillus permanent standard strain ATCC9338. This confirmed that CJL-112 is a novel Lactobacillus permanent strain that is distinguished from the standard strain ATCC9338.

Example  4: Lactobacillus Permanent CJL Inhibition of Chicken Infectious Bronchitis Virus Infection and Growth by -112 Administration

One hundred day-old SPF chicks were divided into three groups (oral administration, eye drop administration, and non-administration challenge control group) with 30 animals per group.

Lactobacillus permanent CJL-112 was used as a test target bacterium. For the preparation of test subjects, MRS liquid medium was prepared, incubated at 37 ° C. for 24 hours, the culture solution was centrifuged, washed with sterile distilled water, and suspended in an appropriate amount of sterile distilled water for use in the test.

From the second day of breeding, the test subject bacterium Lactobacillus permanent CJL-112 was prevented daily according to the group administration route. In the case of the oral administration group, test bacteria at a concentration of 2 × 10 ^9.0 cfu / ml were orally administered at a dose of 500 μl per animal. For the eye drop group, test bacteria at a concentration of 3x10 ^10.0 cfu / ml Topical administration was conducted at a 30 μl dose per horse.

On day 7, 14, and 21 days after the prophylactic administration, 10 animals of each group were randomly selected and challenged with 10 ^5.0 EID ₅₀ of infectious bronchitis virus KM per strain, and isolated and bred. Five days after each challenge, the animals were euthanized and the organs and kidneys were collected and inoculated into the egg. Urinary fluids were collected from egg cultures cultured for 3 days and dot-immunoblotting assay was used to confirm the proliferation of challenged virus in chicken organs (Song, CS et al., 1998, Avian). Dis . 42 (1): 92-100) (Table 4).

Preventing Chicken Infectious Bronchitis in Lactobacillus Permanent CJL-112 Administration in SPF Chickens CJL-112
Route of administration ^A CJL-112
Duration of administration ^B Virus positive sorghum / Avail sorghum ^C Agency kidney Control group Administered group Control group Administered group oral- 1 week 10/10 8/10 5/10 6/10 2 weeks 10/10 10/10 9/10 4/10 ^* 3 weeks 10/10 10/10 8/10 5/10 Eye drops 1 week 10/10 7/10 5/10 4/10 2 weeks 10/10 10/10 9/10 4/10 ^* 3 weeks 10/10 10/10 8/10 4/10

^A daily oral or instillation of CJL-112 at a dose of 10 ^9.0 cfu / bird to a 2-day-old SPF chick

^B On day 7, 14, and 21 days after commencement of ^C CJL-112, 10 chickens were challenged with 10 ^5.0 EID ₅₀ chicken infectious bronchitis virus per head.

^C Five days after challenge, the protective effect was calculated by re-isolation of challenge virus in chicken organs and kidneys.

* p <0.05, by Fisher exact t-test, compared with non-administered challenge control

As shown in Table 4, the re-separation rate of infectious bronchitis virus in the respiratory tract and kidneys was reduced by oral and instillation of CJL-112, compared to the challenge control group, especially when the CJL-112 was administered for 2 weeks. Regardless, the virus re-separation rate P was less than 0.05, indicating a significant decrease.

As a result, it was confirmed that Lactobacillus permanent CJL-112 exhibited excellent antiviral efficacy against not only influenza virus but also chicken infectious bronchitis virus.

Example  5: Lactobacillus Permanent CJL By -112 administration H9N2 brother Low pathogenic  Avian influenza infection and propagation suppression effect

Sixty three-week-old SPF chicks were divided into two groups (administration group, non-administration challenge group), 30 animals per group, and isolated.

Lactobacillus permanent CJL-112 was used as a test target bacterium. For the preparation of test subjects, MRS liquid medium was prepared, incubated at 37 ° C. for 24 hours, the culture solution was centrifuged, washed with sterile distilled water, and suspended in an appropriate amount of sterile distilled water for use in the test.

Low pathogenic avian influenza (A / Korean native chicken / Korea / K040110 / 2010 [H9N2]) Lactobacillus fermentum CJL-112 was administered daily nasal from 1 week before challenge to 14 days after challenge. For the test, 3 × ^{10 9} cfu / ml lactobacillus permanent CJL-112 was administered by nasal route at a dose of 500 μl per animal, and 100 μl of chicken at a concentration of 10 ^7.0 EID ₅₀ / ml per animal for influenza virus challenge. Inoculated into the nasal cavity.

During the test period, 30 chickens per group were reared in two 0.4 m² steel grid cages (Cage 1 = 10 challenge chickens, 10 direct contact chickens, 2 cages 10 indirect contact chickens), and between two cages. The distance was set to maintain a distance of about 40 cm to allow direct or air propagation of the inoculated virus.

The symptoms of clinical symptoms were observed daily for 14 days after influenza virus challenge, and oral and total excretory swabs were performed in the contact and airborne groups at 3, 5, 7 and 9 days of challenge and real-time influenza virus genes were exchanged. Detected using time RT-PCR. In addition, the presence of influenza specific antibodies was evaluated by collecting blood 14 days after challenge and performing hemagglutination inhibition.

The results are shown in Tables 5 and 6.

Inhibitory Effect of H9N2 Avian Influenza Infection on Lactobacillus Percentum CJL-112 Administration in SPF Chickens division group Sorghum Seroconversion ^A Morbidity ^B CJL-112
Administered group Attack group 10 10/10 7/10 Contact infection group 10 10/10 6/10 Airborne infection group 10 5/10 0/10 CJL-112
Non-administrated control Attack group 10 10/10 7/10 Contact infection group 10 10/10 7/10 Airborne infection group 10 8/10 3/10

^A Hemagglutination-inhibition assay (HI) was performed on sera collected 14 days after challenge with antigen A / Korean native chicken / Korea / K040110 / 2010 (H9N2). Results refer to HI test positive count / total test count

^B Clinical symptom manifestation / total test count

As shown in Table 5, the clinical symptom observed in the airborne group was 80% seroconversion rate and 30% morbidity rate in the CJL-112 non-administered control group (number of patients in a given period, as percentage of population). ). On the other hand, in the case of administration of CJL-112, despite the 50% seroconversion rate, no clinical symptom expression system was observed.

Inhibition of Viral In Vitro Emission During H9N2 Avian Influenza Horizontal Infection by Lactobacillus Percentum CJL-112 Administration in SPF Chickens division group Virus re-isolation results ^A 3 dpc 5 dpc 7 dpc 9 dpc Mouth Total excretion steel Mouth Total excretion steel Mouth Total excretion steel Mouth Total excretion steel CJL-112
Administered group Attack group 10/10
(4.2 ± 0.2) 4/10
(2.3 ± 0.9) 9/10
(4.0 ± 0.5) 7/10
(4.0 ± 1.0) 7/10
(2.2 ± 0.5 ^* ) 8/10
(2.7 ± 1.0) 8/10
(2.1 ± 0.5) 6/10
(1.6 ± 0.7) contact
Infection group 9/10
(4.9 ± 0.7) 2/10
(1.2 ± 0.8) 10/10
(5.9 ± 0.2) 6/10
(2.4 ± 0.8) 10/10
(5.8 ± 0.2) 9/10
(4.9 ± 0.8) 10/10
(4.0 ± 0.3 ^*** ) 9/10
(5.1 ± 0.7) Air propagation
Infection group 0/10
(0) 0/10
(0) 5/10
(1.6 ± 0.6) 0/10
(0) 3/10
(0.9 ± 0.5) 0/10 ^{+ + +}
(0) 3/10
(1.1 ± 0.6) 0/10
(0) CJL-112
Non-administrated control Attack group 8/10
(3.4 ± 0.6) 7/10
(2.3 ± 0.6) 6/10
(2.3 ± 0.7) 7/10
(2.5 ± 0.7) 10/10
(3.5 ± 0.2) 10/10
(4.3 ± 0.5) 8/10
(2.9 ± 0.5) 9/10
(3.4 ± 0.6) contact
Infection group 9/10
(4.0 ± 0.6) 3/10
(0.9 ± 0.4) 10/10
(5.9 ± 0.2) 4/10
(1.7 ± 0.8) 9/10
(5.7 ± 0.6) 5/10
(2.5 ± 0.9) 10/10
(5.6 ± 0.3) 9/10
(6.2 ± 0.8) Air propagation
Infection group 0/10
(0) 0/10
(0) 3/10
(1.1 ± 0.6) 2/10
(0.6 ± 0.4) 5/10
(1.5 ± 0.5) 8/10
(4.3 ± 0.9) 5/10
(2.6 ± 0.9) 0/10
(0)

dpc, day post-challenge

^A challenge virus re-isolation / total test count (mean virus titer logEID ₅₀ / ml ± standard error)

^* p <0.05 by two-tailed Student's t- test compared with non-administered challenge group

^*** p <0.001 by two-tailed Student's t- test, compared with non-administered contact control group

^＋＋＋ p <0.001 by Fisher's exact test, compared with non-administered airborne control group

As shown in Table 6, as a result of virus reseparation, virus titers in the oral swabs of the challenge group and the contact group treated with CJL-112 at 7 and 9 days after challenge were significantly decreased compared to the control group (respectively, respectively). p <0.05 and p <0.001). Moreover, 7 days after challenge, total excretory virus re-separation rate of airborne infectious system in control group reached 80% (4.3 ± 0.9log EID ₅₀ / ml), while challenge virus in CJL-112 treated group It was not detected at all (p <0.001).

Through these results, it was confirmed that Lactobacillus permanent CJL-112 of the present invention showed excellent antiviral efficacy against H9N2 type low pathogenic avian influenza virus as well as human influenza virus.

Example  6: Lactobacillus Permanent CJL Respiratory Genital Syndrome with -112 Administration Porcine reproductive and respiratory syndrome , PRRS Inhibition and transmission effect

Eighteen piglets of three weeks old, PRRS virus negative, were divided into three test groups (four dosing groups, four non-administered challenge control groups, and two non-administered negative control groups).

Lactobacillus permanent CJL-112 was used as a test target bacterium. For the preparation of test subjects, MRS liquid medium was prepared, incubated at 37 ° C. for 24 hours, and the culture solution was centrifuged, washed with sterile distilled water, and suspended in an appropriate amount of physiological saline.

PRRS Virus (LMY week) 2 ml of Lactobacillus permanent CJL-112 (1 × 10 ¹⁰ CFU / ml) was daily sprayed through the nasal cavity in the piglets administered daily from 7 days before challenge to 21 days after challenge. The administration method was a method of spray administration using a spray gun, a total of 2 ml of the strain solution 1 ml in one nasal cavity at a distance of about 1 cm from the nasal cavity of the piglets. The same amount of physiological saline was administered to piglets of the non-administered challenge control group and the negative control group.

For virus challenge, four randomly selected piglets were anesthetized and stabilized in the administration group and the non-administration group, and then challenged and inoculated by dropping the virus solution in the nasal cavity. A total of 2 ml of 1 ml in one nasal cavity is inoculated, and the total amount of virus inoculated in each piglet has a viral titer of ⁵ × 10 ⁵ TCID ₅₀ . Four piglets remaining in the administration group and the non-administration group were isolated from the inoculated piglets for 24 hours, and then recombined for contact infection.

The clinical symptoms were observed for 21 days after challenge, and specimens (blood, pulmonary lavage fluid, nasal and oral swabs, lung tissue, etc.) were collected according to the planned schedule. Lung lavage fluid was collected at weekly intervals during the test period. ELISA and real-time RT-PCR were used to detect the concentration of IgA and RNA of challenge virus, and calculate the titer.

The test results are shown in Tables 7-9.

division group PRRSV antigen positive rate in blood after challenge v ^A (detected virus titer) ^B 0 days 2 days 4 days 5 days 7 days 10 days 14 days 21 days CJL-112 administration group Attack group 0/4
(0) 3/4 3/4
(2.54) 4/4 1/4
(3.62) 4/4 3/4
(4.44) 4/4
(4.96) Contact infection group 0/4
(0) 0/4 0/4 3/4 0/4
(0) 0/4 2/3
(4.10) 2/2
(3.72) CJL-112
Non-administrated control Attack group 0/4
(0) 1/4 1/4
(3.67) 2/4 3/4
(3.42) 2/4 4/4
(4.06) 1/3
(3.94) Contact infection group 0/4
(0) 1/4 2/4
(0) 1/4 0/4
(0) 1/3 3/3
(4.60) 2/2
(4.82) Negative control group 0/2
(0) 0/2 0/2
(0) 0/2 0/2
(0) 0/2 0/2
(0) 0/2

^A positive two / test animal two

^B log ₁₀ TCID ₅₀

As shown in Table 7, the detection time of virus in the blood of the contact infected piglets administered Lactobacillus permanent CJL-112 tended to be delayed by 4 days compared to the non-administered challenge control group.

division group Weekly gain rate after challenge 1 week 2 weeks 3 weeks 4 weeks CJL-112 administration group Attack group 0.90 0.84 1.16 1.95 ^* Contact infection group 1.15 1.58 0.90 2.55 CJL-112
Non-administrated control Attack group 0.75 1.30 0.83 0.36 Contact infection group 0.70 1.78 1.33 2.25 Negative control group 0.80 1.56 2.51 5.08

^* p <0.05 by two-tailed Student's t- test compared with non-administered challenge group

In addition, as shown in Table 8, a significant reduction in the weight gain rate of the non-administered group was confirmed during PRRS virus challenge, whereas the increase rate of the piglets in the challenge group treated with Lactobacillus fermentum CJL-112 was 3-4 weeks after infection. It was confirmed that the increase significantly in the period.

division group PRRSV-specific IgA Concentration ^{A in} Lung Wash 0 days 4 days 7 days 14 days 21st CJL-112 administration group Attack group 0.055 -0.120 -0.018 -0.038 1.033 ^* Contact infection group -0.007 -0.095 -0.100 -0.027 0.478 CJL-112
Non-administrated control Attack group -0.148 -0.192 -0.162 0.001 0.487 Contact infection group -0.140 -0.210 -0.083 0.015 0.207 Negative control group -0.153 -0.130 -0.230 -0.163 0.057

^* p <0.05 by two-tailed Student's t- test compared with non-administered challenge group

^A Sample-to-positive ratio (S / P ratio), (test average OD-negative control average OD) / (positive control average OD-negative control average OD)

Finally, as shown in Table 9, the PRRSV-specific IgA antibody titer in the pulmonary lavage fluid of the challenge group administered Lactobacillus pertumtum CJL-112 was higher than that of the non-administered control group.

Through these results, it was confirmed that the administration of Lactobacillus permanent CJL-112 had an effect on the formation of pig PRRS virus-specific antibody and delayed virus proliferation, and inhibited weight loss due to viral infection.

Example  7: Lactobacillus Permanent CJL Effect of -112 Administration on Lung Immune Response in Mouse Influenza Infection

33 three-week-old BALB / c mice were bred into 16 lactobacillus permanent CJL-112 administration groups and 17 PBS (phosphate buffered saline) administration groups. CJL-112 prepared MRS liquid medium and incubated for 24 hours at 37 ℃, centrifuged to wash the sterilized distilled water and then suspended in sterile distilled water at a concentration of 3X10 ⁹ cfu / ㎖ was used for the test.

A total of 6 ketamine anesthesia (100 mg / kg) was administered intranasally, followed by a 90 μl dose of CJL-112 suspension intranasally and 10 μl of PBS in the control group for 10 days prior to challenge with influenza virus. Administered nasal.

Ketamine anesthesia (100㎎ / kg) after administration to the titer 10 ^4.0 TCID ₅₀ / ㎖ of influenza A / NWS / 33 (N1H1) 90㎕ grains per the virus solution into the nasal dose for the mouse to transfer a lethal dose of influenza virus attack inoculation After that, they were kept in isolation with the non-vaccinated group.

After 0, 6, 24, and 48 hours of inoculation, 4 mice were euthanized in each group, and the lungs were aseptically collected and then emulsified. However, four mice in the dosing group and five mice in the non-administration group were randomly selected and used to measure the cytokine and IgA concentrations in each lung emulsion prior to the challenge.

The measured cytokine and IgA concentrations are shown in FIG. 5.

As a result, the concentrations of IL-1β (a), IL-2 (b), IFN-γ (c), IFN-α (d), and Ig A (e) in the CJL-112 treated group were significantly higher than those in the non-administered group. The concentrations of IL-10 (f) and TNF-α (g) decreased significantly.

Thus, administration of Lactobacillus permanent CJL-112 induces increased IL-1β, IFN-γ and Ig A secretion in the lungs of the mouse, activating the early immune response in the body, and especially significantly secreting IL-2 secretion after influenza virus infection. Increasingly, it was found that they induce Th1 type cellular immune response. In addition, it was confirmed that the secretion of IFN-γ, which plays an important role in killing the virus, contributes to the elimination of influenza virus and mouse survival by inducing rapid and high levels.

Korea Microorganism Conservation Center (overseas) KCCM11184P 20110407

Attach an electronic file to a sequence list

Claims (11)

Lactobacillus fermentum CJL-112 with accession number KCCM11184P having inhibitory activity against influenza A virus, low pathogenic avian influenza virus, infectious bronchitis virus, or swine respiratory syndrome.
Avian influenza, chicken infectious bronchitis, swine influenza, porcine respiratory syndrome, dog influenza, and equine influenza, comprising the Lactobacillus fermentum CJL-112 of claim 1 as an active ingredient. Composition for the prevention or treatment of infectious diseases caused by viruses.
delete An antiviral composition for a virus selected from the group consisting of orthomyxovirus, coronavirus, and aterivirus, comprising the Lactobacillus permanent CJL-112 of claim 1 as an active ingredient.
delete Livestock feed additives comprising the Lactobacillus fermentum CJL-112 as an active ingredient.
Avian influenza, chicken infectious bronchitis, swine influenza, swine respiratory syndrome, canine influenza, and horses, comprising administering to the subject in need thereof the Lactobacillus permanent CJL-112 or the composition of claim 2. A method for preventing or treating an infectious disease caused by a virus in an animal other than a human, which is selected from the group consisting of influenza.
Drinking water additive comprising the Lactobacillus Permanent CJL-112 of claim 1 as an active ingredient. 8. The method of claim 7, wherein said administration is carried out via an oral or parenteral route.
The method of claim 9, wherein the parenteral route is nasal or ocular.
The method of claim 9, wherein the method via the parenteral route is administered to the animal via spray vaccination.

Images