KR20180062468A - Novel Lawsonia intracellularis and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel strain of Lawsonia intracellularis and use thereof. More specifically, the present invention relates to a strain of Lawsonia intracellularis, deposited with accession No. KCTC13152BP, a method for preparing the strain, a vaccine composition, containing the strain as an active ingredient, for preventing or treating proliferative enteritis, and a method for preventing or treating proliferative enteritis. According to the present invention, the strain of the present invention can be effectively used to prepare an attenuated vaccine which has safety, stability, immunogenicity, and high efficiency when the strain is administered to an object.

Description

Novel Lawsonia intracellularis strains and their uses {Novel Lawsonia intracellularis and Uses Thereof}

The present invention relates to a novel Rhodiola intracellularis strain and its use.

Porcine proliferative enteritis (PPE), known as ileitis, intestinal adenomatosis, hemorrhagic enteropathy, or necrotic enteritis, is a common complication of postpartum porcine pigs It is a naturally occurring disease that affects.

Proliferative pyelonephritis in pigs is a major problem in the pig farming industry in Korea and around the world. Numerous swine farms suffer from economic damage due to proliferative porcine swine, but it is difficult to isolate the causative organisms. to be. In the United Kingdom and the United States, Lawsonia intracellularis (L. intracellularis ) , which is a causative organism of proliferative ileitis, has been successfully isolated and purified in a laboratory. However, studies have been actively conducted on rosinia intracellularis, It is an absolute intracellular bacterium that can not be cultured by normal bacterial methods on cell culture media and requires epithelial cells to attach to grow. This prior culture method requires a lot of effort and is not suitable for large-scale cultivation.

In order to conduct research on prophylactic and therapeutic agents against proliferative ileitis, it is very urgent to secure domestic isolationism. However, to isolate Rosa intracellularis, 1) establishment of cell culture technology 2) 3) accurate diagnosis of proliferative ileitis; 4) securing of fresh specimens; 5) isolation and isolation of proliferative resistant pathogens, excluding other bacteria and viruses; and 6) And pathogenic reproduction experiments on proliferative enterococci.

Therefore, it is absolutely required to obtain Rosa intracellulare strains for vaccine development, diagnosis and immunological studies for prevention of proliferative ileitis occurring domestically.

The present inventor has made an effort to develop a novel vaccine against swine enteritis. As a result, the obtained strains were attenuated to prepare novel strains. The strains were used as a vaccine to confirm the excellent immunogenic effect, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a novel strain of Lawsonia intracellularis .

It is a further object of the present invention to provide a vaccine composition for the prophylaxis or treatment of proliferative enteritis.

It is still another object of the present invention to provide a method for preventing or treating proliferative enteritis.

Further, another object of the present invention relates to novel rosoni ah intra-cell-less (Lawsonia intracellularis ). < / RTI >

Hereinafter, the present invention will be described in more detail.

In accordance with one aspect of the present invention, the present invention relates to novel rosoni deposited as accession number KCTC13152BP ah intra-cell-less (Lawsonia intracellularis ).

According to a preferred embodiment of the present invention, the strain is selected from the group consisting of Lawsonia < RTI ID = 0.0 > intracellularis ) isolate is attenuated or inactivated by CV-1 cell infection and subculture, and has 16S rRNA base sequence as shown in SEQ ID NO: 1.

The term " Lawsonia " intracellularis , L. intracellularis ) "refers to the causative organism of proliferative ileitis and is used interchangeably with" proliferative ileitis "or" ileitis ".

The attenuation may be performed by methods known in the art. The attenuated strain may be attenuated by chemical treatment, for example, formalin treatment, or attenuated by ultraviolet irradiation or subculture.

As used herein, the term " attenuating "refers to having an extent of toxicity that is capable of inducing an immune response to a subject, but not so fatal to a subject as a vaccine. In this specification, attenuation is used interchangeably with "inactivation ". Such attenuation includes not only the ability of the strain to proliferate but also the ability to completely lose its proliferative capacity.

According to a preferred embodiment of the present invention, the attenuation is a continuous subculture of 5th to 200th generations, more preferably 80th to 120th generations, and most preferably 100th generations.

The new Lawsonia intracellularis strain deposited with the accession number KCTC13152BP according to the present invention is a novel strain having a different base sequence at a specific site as compared with a known strain. More specifically, a new Lawsonia intracellularis strain deposited with the known Accession No. KCTC13152BP of the present invention obtained through the above-mentioned continuous passage culture with the known Rosonia intracellularis (NC_008011.1) and its parent strain The differences in the sequence of the isolates of the inflorescence intracellularis were compared and analyzed.

As a result, the 182329th nucleotide of the isolate of the parent strain of the cellulase, which is the parent strain, was compared based on the known Laosonia intracellularis (NC_008011.1). 339993 th nucleotide; 544127 th nucleotide; The 787441st nucleotide; And 1397904 nucleotides in the region corresponding to one or more of the positions, it was confirmed that the nucleotide sequence of the nucleotide sequence of Lawsonia < (R) > intracellularis ) strain is a novel strain different from the parent strain.

That is, the novel rosoni deposited as accession number KCTC13152BP of the invention ah intra-cell-less (Lawsonia intracellularis strains were obtained by isolating a strain of Lawsonia intracellularis from a subject afflicted with proliferative enteritis and culturing it continuously in CV-1 cells for up to 100 generations.

The strain has high immunogenicity and no pathogenicity. In addition, in one embodiment of the present invention, when the strain was orally administered to pigs and hemsters, immunization was induced.

In accordance with another aspect of the present invention, the present invention provides a method of inhibiting the growth of Rhodia intracellularis ( Lawsonia < RTI ID = 0.0 > intracellularis ) strains:

(a) Lawsonia < RTI ID = 0.0 > intracellularis ) positive testicular tissue;

(b) inoculating and infecting the separated cells of step (a) with CV-1 (ATCC CCL-70) cells; And

(c) infecting the infected cells of step (b) by subculturing the cells for 5 to 200 years.

The term "separator" is separated from neuropathic feelings once jangyeomgyun (L. intracellularis) Chapter benign tissue specimens from the Green Cross Veterinary Medicines Veterinary Institute, 2010 herein rosoni Oh intra-cellular leases (Lawsonia intracellularis ), and can be used interchangeably with "isolate" in the present invention.

More specifically, the method of the present invention comprises the steps of: obtaining a positive testicular tissue in a proliferative ileum and confirming the presence of a causative organism of proliferative ileitis by immunohistochemical staining and / or PCR; Infecting the CV-1 cells with the rosinia intracellularisseparation obtained from the intestinal tissue and then infecting the CV-1 cells with the inoculum containing the Rhodiola cellulase isolate under anaerobic conditions; The infected CV-1 cells were incubated for 2 to 5 hours and then replaced with medium supplemented with antibiotics; At 1-6 days after inoculation, infected cells were ruptured using 0.1% potassium chloride solution and a syringe, and the supernatant from which the nucleus was removed was inoculated into fresh CV-1 cells, By obtaining CV-I cells infected with rosonic intra-cellulase, new Lawsonia intracellularis intracellularis ).

That is, in the present invention, infected CV-1 cells were ruptured using a potassium chloride solution and a syringe, and the supernatant containing the Rosonia intracellularis strain in which the nucleus was removed was suspended in fresh CV-1 cells .

In the present invention, the inoculum may be a culture of rosio intracellularez obtained from an infected subject or a long intestine produced by scraping the mucosa from the ileum of a subject infected with rosio intracellulareis. In the case of preparing intestinal lysates, the portion of the ileum selected for cultivation should exhibit a severe lesion thickened throughout the intestine. Since the bacteria are inherently weak, the sample should preferably be stored at -70 C as soon as possible after autopsy. A member of the aminoglycoside group antibiotic, including antibiotics resistant to Rhodiola intracellularis, for example, vancomycin, amphotericin B or gentamicin and neomycin, is preferably added to the inoculum to inhibit contaminating bacteria Proliferates intra-cellulase. Regardless of whether the inoculum is a pure culture or intact, the inoculation of cultured cells can be carried out by a variety of techniques known in the art taught herein.

The bacteria and / or inoculated cultured cells are then cultured under a reduced O ₂ dissolved concentration. At a dissolved oxygen concentration of more than 10%, the proliferation of rosio intracellularis is suboptimal and ultimately stops proliferation at oxygen concentrations outside of this range. Preferably, the bacterial and / or inoculated cultured cells are cultured at a dissolved oxygen concentration ranging from 0.1% to 10%.

More preferably, the bacteria and / or cells are cultured at an oxygen concentration in the range of 0.1% to 8% and in the range of 5.0-7.0% The oxygen concentration is most preferred.

The proper concentration of carbon dioxide is also known as El. It is important for the proper propagation of intracellularis. At a carbon dioxide concentration ranging from 0% to 4%, it is non-optimal to proliferate and eventually the proliferation is stopped at a carbon dioxide concentration outside the range. Preferably, the carbon dioxide concentration is in the range of 6% to 10%, and the carbon dioxide concentration in the range of 7.5-9% is most preferred.

In addition, the cells are preferably cultured in a hydrogen concentration range of 73% to 96%. Nitrogen can be used in place of some or all of the hydrogen present. Most preferably, the cells are cultured in O ₂ , 8.8% CO ₂ in the range of 0.1 to 8.0%.

The inoculated cells may be cultured in a binary gas incubator or other gas chamber containing appropriate hydrogen, oxygen and carbon dioxide concentrations and suspending the cells during culture. The chamber should include a means for maintaining the suspension of the cultured cells and a source for supplying and maintaining a gas monitor and a suitable gas concentration. The incubation temperature should be in the range of 30 캜 to 45 캜, and more preferably in the range of 36 캜 to 38 캜. Most preferably, the temperature is 37 < 0 > C. Essential equipment for cultivation and attenuation is readily available to those skilled in the art as taught herein.

By keeping inoculated cells in suspension during culture, each individual cell is exposed to a suitable mixture of growth medium and hydrogen, oxygen, and carbon dioxide to increase the cell and thereby the el. Intracellularis is maximally proliferated.

In accordance with another aspect of the present invention, the present invention provides a method of inhibiting the growth and / or proliferation of Rochea intracellularis ( Lawsonia < RTI ID = 0.0 > The present invention also provides a vaccine composition for preventing or treating proliferative enteritis comprising an intracellularis strain as an active ingredient.

The rosa intracellularis strain produced by the above-described method of the present invention can be used to produce an attenuated vaccine and a sterilized rosinia intracellularis vaccine. That is, the strains isolated and cultured by the method of the present invention can be sufficiently counted several times to select strains having weakened toxicity, or to weaken the toxicity by a chemical method, so that toxicity A vaccine composition is provided that secures the attenuated strain and uses a therapeutically effective amount of an attenuated vaccine as a vaccine.

In addition, the rosinia intracellulare toxin shippers of the present invention can be sterilized by heat treatment and used as a vaccine. The attenuated vaccine and the sterilized vaccine may contain pharmaceutically acceptable stabilizers, diluents, adjuvants, and the like.

The dosage of the composition is from 10 ⁵ to 10 ¹⁰ TCID ₅₀ , preferably from 10 ⁵ to 10 ⁷ TCID _{50 per} subject. The composition may be in the form of a solution, an injection, a tablet, and a capsule, but is not limited thereto.

In addition, the compositions of the present invention may additionally comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be selected from the group consisting of conventional excipients, disintegrants, binders and lubricants, and may be selected from the group consisting of microcrystalline cellulose, lactose, low-substituted hydroxy cellulose , Sodium starch glycolate and calcium monohydrogen phosphate anhydrous as a disintegrant; polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, and hydroxypropylcellulose as binding agents; magnesium stearate, silicon dioxide, talc and the like as a lubricant; You can choose to use it.

According to another aspect of the present invention, there is provided a method of preventing or treating proliferative enteritis comprising administering to a subject a vaccine composition as described above.

The subject is selected from the group consisting of pigs, humans, rabbits, moths, hamsters, foxes, horses, ostriches and emu, preferably selected from the group consisting of pigs, humans, rabbits, moths and hamsters, It is a pig.

Since the method of the present invention utilizes the vaccine composition described above, its description is omitted in order to avoid the undue complexity of the present disclosure in accordance with the repeating substrate.

According to another aspect of the present invention, the present invention provides a method of diagnosing proliferative enteritis comprising the steps of:

(a) preparing a sample of the object; And

(b) detecting a strain of Lawsonia intracellularis deposited in Accession No. KCTC13152BP in the sample.

According to a preferred embodiment of the present invention, the step of detecting the strain is to perform PCR amplification using a primer set to confirm the level of the PCR product.

If the level of the PCR product is higher than the normal level, it can be judged that it is infected with Lawsonia intracellularis.

Any of the methods known in the art can be used as long as PCR can be performed using known primer sets.

Any of the samples may be used as long as the sample is separated from the object and the detection of the strain of the intracellular cellulose of the present invention can be confirmed.

Since the method of the present invention is to detect the above-mentioned strains, common description thereof is omitted in order to avoid the excessive complexity of the present specification in accordance with repetitive description.

The novel Rhodiola intracellularis strain of the present invention is attenuated through submerged culture of 100 times and has excellent safety and stability and produces an immunized vaccine with high immunogenicity and high therapeutic efficiency when administered to a subject Can be effectively used.

Figure 1 shows the IHC results of the CV-1 cell monolayer (left) infected with Rosa intracellularis and the uninfected CV-1 cell monolayer (right).
FIGS. 2A and 2B show results of confirming that the isolated strains are strains of Rosa intracellularis.
FIG. 3 shows the results of intestinal gross lesions confirmed by the hamster for the virulence of the present invention.
Fig. 4 shows pathological examination results of the ileum in which the pathogenicity of the tobacco shippers was confirmed in hamsters.
Fig. 5 shows the gross lesion results of the ileum in which the pathogenicity of the present invention was confirmed in pigs.
Fig. 6 shows pathological examination results of the pancreas in which the pathogenicity of the present invention was confirmed in pigs.
FIG. 7 shows the results of confirming the protective effect of the tobacco shippers on the inoculation after the inoculation of the tobacco shippers into pigs.
FIG. 8 shows the average weight change of the test money for the inoculation after the inoculation of the vaccine shippers into pigs.
FIG. 9 shows the result of confirming the antibody response of the subject to the attacked inoculation after the inoculation of the vaccine shippers into pigs.
FIG. 10 shows the results of histological changes in the target organs of the present toxin shippers for the inoculation after inoculation of the vaccine shippers into pigs.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example  1. Escherichia coli Rosonia  Intracellularis ( Lawsonia intracellularis , L. intracellularis ) And culture

The present inventors isolated and cultivated Lawsonia intracellularis (L. intracellularis), which is a causative agent of pathogenesis, for the production of a novel Rhodiola intracellularis strain.

In 2010, bacteria were harvested from L. intracellularis positive intestinal tissue of pathological susceptible ginseng at the Institute of Green Cross Water Veterinary Medicine, and CV1 cells were inoculated to obtain isolates. Then, as shown in Fig. 1, The presence of the microorganism was confirmed by monoclonal antibody that specifically reacted with lees.

PCR was carried out by amplifying 16S rDNA using the primer shown in Table 1 below. As a result, it was confirmed that it was positive for Rhodiola intracellularis (Fig. 2A). As a result of the homology test with NCBI GenBank, 99% And thus it was confirmed that this strain was a novel strain not known as a isolate of a strain of Rosacea intracellularis (Fig. 2B).

Example  2. Cell culture

CV-1 (ATCC CCL-70) cells were used for infection with Rosa intracellularis. D-MEM supplemented with 1 to 10% of FBS (Fetal bovine serum), 0.1 to 5% of L-glutamine and Amphotericin B was used for the cell culture, Was carried out in a 37 ° C incubator supplied with 2 to 10% CO ₂ . Cells were subcultured by treatment with EDTA-Trypsin every 4 to 5 days and transferred to a new tissue culture flask at a concentration of 5 × 10 ⁵ cells per ml.

Example  3. Isolated Rosonia Intracellular  Cell infection

The diluted inoculum of Rosa intracellularis bacteria isolated in Example 1 was inoculated on the surface of the CV-1 cell monolayer, and the inoculated cell monolayer surface was rotated using centrifugal force of 1000 to 2,000 g. The inoculum flask was placed in an anaerobic culture container . Gas in anaerobic culture vessel were degassed to a pressure of 500mmHg and replaced with hydrogen gas for medical purity, again after re-degassed as in 500mmHg pressure of 5 to 10% of O _2, 5-10% CO ₂ mixed gas incubator For 3 hours at 37 DEG C, and then replaced with a medium supplemented with 50 to 300 ppm of antibiotics such as neomycin, gentamicin, vancomycin and the like in the same cell culture medium composition. At two and four days after inoculation, the same antibiotic supplemented medium was replaced with 5% FBS. On the 6th to 7th day after the inoculation, treatment for the passage was performed.

Example  4. Transfection of infected isolates

The cell lysate solution containing 0.1% KCl solution was used at a ratio of 1: 3 for separating strains. The culture supernatant of the tissue culture flasks that were one week old after the infection was collected. 0.1% KCl solution was applied to the surface of the cell monolayer in which the supernatant was removed and reacted for 10 minutes. At the end of the reaction, the 0.1% KCl solution was removed, and the cells on the bottom of the tissue culture flask were collected using a SPG solution containing 10% FBS and a cell scraper. The collected cells were physically ruptured 10 times through a 20 gauge emulsification needle. The nuclei generated in this process were removed by centrifugation at 100 g for 5 minutes, and the obtained supernatant was used to inoculate freshly prepared fresh CV-1 cells.

 CV-1 cells infected with Laosonia intracellularis were transfected by this method at 6-7 day intervals. When crossing the passage, one tissue culture flask was subcultured in a ratio of 1: 3 to make three tissue culture flasks in the next pass.

The strain isolated by the above method was attenuated by subculturing 100 or more strains, and the attenuated strain was transformed into Lawsonia intracellularis L1 / CV1 and deposited on Nov. 08, 2016 with the Center for Microbial Resources, Korea Research Institute of Bioscience and Biotechnology (Accession No .: KCTC 13152BP).

In addition, it is also possible to use a novel rosinia intracellularis (NC_008011.1) known in the art and a novel rosna intracellulareis ( Lawsonia) deposited with the accession number KCTC13152BP of the present invention obtained through the above- intracellularis ) and its parent strain, rosonic acid intracellularisse , were compared and analyzed.

As a result, the 182329th nucleotide of the parent strain, Rosonia intracellularisse (LI), which is the parent strain, was compared based on the known Rosonia intracellularis (NC_008011.1); 339993 th nucleotide; 544127 th nucleotide; The 787441st nucleotide; And 1397904 nucleotides in the region corresponding to one or more of the positions, it was confirmed that the nucleotide sequence of the nucleotide sequence of Lawsonia < (R) > intracellularis ) strain (LI-100) is a novel strain different from the parent strain.

The results of the above sequence analysis are shown in Table 2 below.

Known Laosia intracellularis location Reference sequence Altered sequence LI LI-100 Genotype sequence Genotype sequence NC_008011.1 182329 CA CAA CA CAA NC_008011.1 339993 C T C T NC_008011.1 544127 C T C T NC_008011.1 787441 C A C A NC_008011.1 1397904 T G T G

Example  5. In animals Poisonous  Pathogenicity confirmation

The present inventors confirmed the pathogenic disappearance by inoculating the susceptible shippers prepared in Example 4 with the target animals and susceptible hamsters and pigs by inoculation.

As shown in Table 3, 8 piglets negative for the 3-week-old rhona-intracellularis antibody and 10 for 3 weeks of the Syrian hamster were each inoculated into a 100-pass culture broth ( L. intracellularis , accession number: KCTC13152BP ) In group A and a group B in L. intracellularis cultured in 5th line.

For each test vaccine, 2 ml of 3-week-old piglets and 3 ml of 3-week-old hamsters were orally administered, and 2 pigs and 2 hamsters were used as a control. After 21 days of diarrhea, anorexia and mortality were observed, pigs and hamsters were examined for autopsy and gross lesions and histopathological findings were observed.

5-1. Assessment of Clinical Symptoms

The present inventors inoculated the toxin shippers prepared in Example 4 and observed clinical symptoms thereof.

As a result, as shown in the following Table 4, the clinical symptoms (safety investigation) in pigs were as follows: vaccine group A using the isolate (accession number: KCTC13152BP) cultured in the 100 passages as the antigen showed dyspnea, vomiting, Diarrhea, etc., whereas vaccine group B, which used the isolates cultured in the 5th line as an antigen, showed some symptoms such as anorexia and diarrhea. In addition, the negative control group survived without symptoms such as dyspnea, vomiting, anorexia and diarrhea.

As shown in the following Table 5, the clinical symptoms (safety study) in the hamster were as follows. The vaccine group A using the isolate (accession number: KCTC13152BP) cultured in the 100 passages as the antigen showed dyspnea, vomiting, anorexia, diarrhea , While vaccine group B, which used the isolates cultured in the 5th passage as an antigen, showed some symptoms such as anorexia and diarrhea. In addition, the negative control group survived without symptoms such as dyspnea, vomiting, anorexia and diarrhea.

5-2. Evaluation of gross lesions

The present inventors inoculated the toxin shippers prepared in Example 4 and observed gross lesions therefrom.

As a result, as shown in Fig. 3, in the case of a gross lesion in a hamster, a typical lesion of a porcine proliferative ileus was not observed in a vaccine group A using an isolate (accession number: KCTC13152BP) Vaccine group B, which used 5 isolates as an antigen, had thickened thickened small intestine and wrinkled mucosa and congestion. It was shorter than that of small intestine of control group. In addition, no negative lesion of the proliferative pancreatitis was observed in the negative control group.

In addition, as shown in Fig. 5, in the case of a gross lesion in a pig, a typical lesion of a porcine proliferative ileum was not observed in a vaccine group A using an isolate (accession number: KCTC13152BP) Vaccine group B, which used culture broth as an antigen, had thickened thickened small intestine and wrinkled mucosa and congestion. In addition, no negative lesion of the proliferative pancreatitis was observed in the negative control group.

5-3. Histopathological evaluation

The present inventors inoculated the toxin shippers prepared in Example 4 and observed the histopathological state thereof.

As a result, as shown in Fig. 4, the histopathological findings in the hamster were that the vaccine group A using the isolate cultured in the 100th passage (accession number: KCTC13152BP) as an antigen was normal small intestine and no enteritis , And Crypt epithelial cell proliferation and inflammatory cell infiltration were observed mainly in the mucosal layer of the vaccine group B using the isolates cultured in the five major passages and the cytoplasmic part of the negative ectopic epithelium and macrophages, Were found in the cytoplasm. In addition, the negative control group was not identified as a normal small intestine.

In addition, as shown in Fig. 6, the histopathological findings in pigs showed that the vaccine group A using the isolates cultured in the 100th passage (accession number: KCTC13152BP) as an antigen was normal small intestine, Vaccine group B, which used cultured isolates as an antigen, showed a decrease in small villus length and proliferation of negative echo epithelium, localized abscesses with inflammatory cells such as neutrophils in the negative lumen, The cytoplasm of negative and epithelial cells and the cytoplasm of infiltrated macrophages were identified. In addition, the negative control group was not identified as a normal small intestine.

In other words, typical lesions of porcine proliferative pancreatitis in the pigs inoculated with antigen B (5 genera) were mainly evident in the small intestine, and the intestine was thickly thickened, and the corneal mucosa and red blood cell findings were confirmed. On the other hand, the autopsy findings of pigs inoculated with antigen A (100 units, accession no.: KCTC13152BP) showed no such pathological changes.

In addition, in the hamster group inoculated with antigen B (5 units), it was confirmed that the hamster group inoculated with antigen A (100 units) was significantly hypertrophied and congested compared with the intestinal wall of the hamster group. ) In the inoculation group was significantly shorter than that of the hamsters in the inoculation group.

Therefore, it was confirmed that the isolate (accession number: KCTC13152BP) cultured in the passage number 100 in the CV-1 cells according to the present invention was attenuated and usable as a vaccine.

Example  6. Identification of immunogenicity and defense effects on the target animal

The present inventors administered orally to piglets (3 weeks old or older), which is a target animal, according to the usage and dosage as the test vaccine of the present invention, as described in Example 5 above, as an effective veterinarian (100 strains cultured strain, KCTC13152BP) , It was confirmed whether or not an effective level of antibody titer was formed and the ability to defend against an attack.

The vaccine was orally administered to 3 out of 7 3 week old pigs susceptible to Lawsonia intracellularis by 2 ml each, and the remaining 4 vaccines were used as non-vaccine control.

6-1. Identify the immunogenicity of the vaccine

To confirm the immunogenicity of the vaccine of the present invention, the present inventors administered vaccine group before vaccination and after 1 week, 2 weeks, 3 weeks (attacking inoculation), 4 weeks, 5 weeks, All of the control samples were collected and the serum was separated and immobilized (56 ° C, 30 minutes), and antibody titers were measured by IFA (Indirect fluorescent antibody) test method.

The IFA test method is as follows: The cells after the 24 hours at a concentration of in each well of a 96 well tissue culture plates 5 X 10 ³ 10 ⁵ culture Lawsonia intracellularis . This tissue culture plate was incubated at 37 ° C in anaerobic condition (8.8% CO ₂ , 8% O ₂ ) for 5 days, then the supernatant was removed and a solution of acetone and methanol was diluted to 50:50, . Serum was separated from the collected blood, diluted serially with dilution serum of the first 1:30 concentration, applied to the infected cell plate, and reacted at 37 ° C for 30 minutes. Then, the cells were washed 5 times with PBS for 30 minutes and then reacted with anti-swine IgG FITC conjugate at 37 ° C for 30 minutes and observed under a fluorescence microscope. When the bacterial antigens emitting fluorescence were observed at a magnification of 200 to 400 times, it was judged to be positive.

As a result, as shown in Table 6, vaccine group immunized with vaccine group showed a steady increase from the 21st day after inoculation until the end of the experiment. In addition, the control pigs were all negative and it was confirmed that the test vaccine of the present invention had a satisfactory level of immunogenicity.

6-2. Confirmation of defense effect against attack inoculation

In order to confirm the protective effect of the vaccine of the present invention, the present inventors used porcine, which was disclosed for testing by an antibody, as a pig for protective effect test. In other words, vaccination three weeks later, in order to determine the protective efficacy against ileitis causative organism (L. intracellularis), L. intracellularis culture broth with a pathogenic vaccine group 3 and control group 2, two two (10 ^7.7 TCID ₅₀ / dose ) Were administered orally. Two of the remaining control groups were untreated and examined for clinical symptoms such as diarrhea, anorexia and mortality for 3 weeks. In addition, we checked periodically whether or not the inoculated bacteria were fecalized through the feces for 21 days after the attack. After the observation period, pigs were autopsied for clinical pathologic findings and L. intracellularis was detected from the specimens such as the spleen. The growth rate of the test money during the defensive effect test period was also confirmed.

As a result, as shown in Table 7 below, when the vaccine was administered to the piglets to which the vaccine was administered to confirm the defense efficacy, the vaccinated group survived without clinical symptoms of ileitis, In the control group, clinical symptoms such as diarrhea and anorexia were shown.

As shown in the following Table 8 and Fig. 7, when the naked eye lesion of the test pig at the time of autopsy was checked, no intestinal lesion specific to the ileum was observed in the vaccinated pig group, but in the positive control group, The test vaccine of the present invention has a satisfactory defense effect.

Furthermore, as shown in Table 9 below, bacterial excretion through the feces was observed, which was detected from the 7th day after inoculation in the feces of the vaccine group pig, and continued until the 35th day. On the other hand, no cells were observed in the feces of negative control pigs during the same experimental period.

Further, as shown in Table 10 below, the germination in the intestinal tissues was confirmed, and the intestinal tissues were not detected in the field of the vaccine group but in the field of the positive control group.

As shown in FIG. 8, when the average weight change of the test money after the inoculation of the attack was confirmed, the decrease rate of the growth rate was confirmed in the positive control group and the growth rate of the vaccine group was higher than that of the positive control group.

Example  7. Confirmation of the protective effects against the target animal

The inventors of the present invention found that the test vaccine of the present invention [green p100 high-valency (10 ^9.3 TCID ₅₀ / dose) and green p100 low-valency (10 ^6.0 TCID ₅₀ / dose); 1 dose = 1 ml] and Boehringer vaccine (10 ^6.0 TCID ₅₀ / dose), which is a commercially available vaccine, are vaccinated against piglets (3 weeks old or more) Fecal PCR and serum (ELISA) tests were performed. In addition, the pigs whose observation period was over were autopsied and their clinical pathologic findings were confirmed. The experimental conditions are shown in Table 11 below.

Defensive effect test Group Green
(p100, high band) Green
(p100, low band) Boehringer Vaccine Cont. challenge Neg. Population Three Three Three Three Two Object number 6B-3
6B-6
6R-9 6R-1
6B-7
6B-9 6R-4
6R-5
1-1 G2 enemy
G2 sulfur
G2 no Vaccination 2ml / two, Oral 2ml / two, Oral 2ml / two, Oral - - Attack inoculation 3 weeks after inoculation - PHE (P5) 2 ml (10 ^7.7 TCID ₅₀ / dose) - Autopsy 6, 10 - autopsy

PHE (Proliferative Hemorrhagic Enteropathy)

7-1. Feces PCR  Check the result

The feces of each experimental group were analyzed by PCR.

As a result, as shown in Table 12 below, all of the vaccine groups (high-frequency group, low-frequency group and Boehringer vaccine group) in the fecal PCR were fecalized from the 35th day after the attack in comparison with the non-vaccine control group It was confirmed that the amount of microbes discharged was small.

p100, high station Boehringer Vaccine p100, low band challenge cont. Object number 6B-3 6B-6 6R-9 6R-4 6R-5 1-1 6B-7 6B-9 6R-1 G2 enemy sulfur radish Date of collection One 2 3 4 5 6 7 8 9 10 11 12 11/10
(7DPI) - - - - - - - - - - - - 11/18
(15 DPI) - - - - - - - - - 11/24
(21DPI) - - - - - - - - - 12/1
(28 DPI) ± ± - + About + About - + About - + About + About + About + About 12/8
(35 DPI) ++ - - + About + About - + About + +++ ++ + - 12/15
(42DPI) + + ++ +++ ++ + 12/22
(49DPI) + + About +++ + +++ +++ 1/14
(72 DPI) - sudden death + - - -

p100 < / RTI > high band refers to the vaccinated group (10 ^9.3 TCID ₅₀ / dose) of the present invention.

The p100 low band indicates the vaccination group of the present invention (10 ^6.0 TCID ₅₀ / dose).

The Boehringer vaccine refers to the comparator vaccine (low-cost, 10 ^6.0 TCID ₅₀ / dose).

challenge cont. refers to a non-vaccine control group.

7-2. Serum (ELISA) test results confirmed

Serum of each experimental group was analyzed by ELISA.

As a result, as shown in Table 13, it was confirmed that ELISA antibodies showed immunoreactivity to proliferative enterococci, and that one of the high-frequency vaccine group and one of Boehringer's vaccine group were positive at 3 weeks after the vaccination, On the 7th day after the inoculation, all of the high - frequency vaccine groups were positive, but the Boehringer and low - vaccine groups and the control group began to be positive from the 2nd week to all the pigs were positive at the third week.

p100, high station Boehringer Vaccine p100, low band challenge cont. Object number 6B-3 6B-6 6R-9 6R-4 6R-5 1-1 6B-7 6B-9 6R-1 G2 enemy sulfur radish Date of collection One 2 3 4 5 6 7 8 9 10 11 12 11/3
(0DPI) - - - - - - - - - 11/17
(14DPI) - - - - - - - - - 11/25
(22DPI) - - + - + - - - - - - - 12/1
(28 DPI) - ± + - - - - - - - - - 12/8
(35 DPI) + ± - - ± - - + + + - - 12/17
(44DPI) + + - + - + + + + + + + 12/22
(49DPI) + + + - + + +

7-3. Clinical pathology results confirmed

Clinical pathologic results of each experimental group were analyzed.

As a result, as shown in Table 14, all of the vaccinated groups showed significantly lessened pathological findings on the gross lesions and histopathological examination of the ileum after the inoculation than the non-vaccinated control group.

Therefore, it was confirmed that the bacterial discharge amount through feces, the gross lesion of the ileum and histopathological pathologic findings were relatively alleviated as compared with the vaccine in which the high-frequency vaccine of the present invention was commonly used and the low-frequency vaccine of the present invention.

Example  8. Confirmation of defense effect after attack

The present inventors used the test vaccine (GCVP; 10 ^6.0 TCID ₅₀ / dose) of the present invention and 2 ml of a commercially available conventional vaccine (BI; 10 ^6.0 TCID ₅₀ / dose) ), 3 weeks after inoculation, the bacterial release (shedding) period, antibody response and histopathologic changes were observed when an inoculation (oral administration of 2 ml of pathogenic L. intracellularis culture broth (10 ^7.7 TCID ₅₀ / dose) Further confirmed.

8-1. Identify when shedding

The duration of bacterial release by each experimental group was analyzed.

As a result, as shown in Table 15, the vaccine group quickly and efficiently discharged the pathogens competitively out of the conventional vaccine control group.

Group GCVP BI Control group Shedding duration 56 dpi = 70 dpi = 70 dpi

8-2. Confirm antibody results

Antibody response was analyzed by each experimental group.

As a result, as shown in FIG. 9, it showed a periodic serologic (ELISA) positive rate of proliferative acute gastroenteritis after an inoculation after vaccination, and there was no difference in the serological immune response between the vaccine of the present invention and the Boehringer vaccine .

8-3. Check pathology results

The pathological changes in the target organs of each experimental group were analyzed.

As a result, as shown in Fig. 10, the pathological histopathological changes of the small intestine (including the ileum) were quantified by performing an attack inoculation on animals of all test groups as pathogenic causative bacteria at 3 weeks after the vaccination. Compared with the non-vaccinated group, the vaccine group showed almost the same pathological changes as the conventional vaccine group, the Boehringer vaccine group, which means that even when the present invention is a low-dose vaccine, do.

Therefore, it can be confirmed that the vaccine containing the novel Rosa intracellularis strain according to the present invention exhibits an effective and safe vaccine efficacy as compared with the conventional vaccine control.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Biotechnology Research Institute KCTC13152BP 20161108

<110> GREEN CORSS VETERINARY PORDUCTS CO., LTD <120> Novel Lawsonia intracellularis and Uses Thereof <130> GCVP1-15p <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 633 <212> RNA <213> Lawsonia intracellularis 16S rRNA <400> 1 cttgagacgg ggataacggc tggaaacggt cgctaatacc ggataaccat tatggtaaaa 60 agttgtagtg caaagggtag cctctgtatg taagctatcg cttgaagatg agtccgcgtc 120 ccattagcta gttggtgagg taaatgctca ccaaggcgat gatgggtagc cgatctaaga 180 ggataatcgg ccacactgga actggaacac ggtccagact cctacgggag gcagcagtgg 240 ggaatattgc gcaatgggcg aaagcctgac gcagcgacgc cgcgtgaggg atgaaggtct 300 ttggatcgta aacctctgtc aggggggaag aaatggatgg gctctaatag ggtctatttt 360 tgacggtacc cccagaggaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga 420 gggtgcaagc gttaatcgga attactgggc gtaaagcgcg cgtaggtggt tatataagtc 480 aggggtgaaa gcccaccgct taacggtgga acagcctttg atactgtata acttgagtat 540 cggagagggt ggcggaattc caggtgtagg ggtgaaatcc gtagatatct ggaggaacac 600 cgtggcgaa ggcggccacc tggacgataa ctg 633

Claims (7)

Lawsonia intracellularis strain deposited with accession number KCTC13152BP. The strain of claim 1, wherein the strain is attenuated or inactivated. 3. The strain according to claim 2, wherein the attenuation is by 5 to 200 generations by continuous subculture. The strain according to claim 1, wherein the strain has the 16S rRNA base sequence set forth in SEQ ID NO: 1. Deposited as accession number KCTC13152BP, comprising the steps rosoni ah intra-cell-less (Lawsonia intracellularis ) strain:
(a) Lawsonia &lt; RTI ID = 0.0 &gt; intracellularis ) positive testicular tissue;
(b) inoculating and infecting the separated cells of step (a) with CV-1 (ATCC CCL-70) cells; And
(c) infecting the infected cells of step (b) by subculturing the cells for 5 to 200 years. Wherein the first to fourth deposited by any one of the accession No. of KCTC13152BP wherein rosoni ah intra-cell-less (Lawsonia The present invention relates to a vaccine composition for preventing or treating proliferative enteritis. A method for preventing or treating proliferative enteritis, comprising administering the vaccine composition of claim 6 to a subject.

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