KR101371355B1 - Oral fowl typhoid vaccine using a live adjuvant system - Google Patents

Abstract

The present invention relates to a vaccine for oral vaccination of oral livestock, and the oral live vaccine for preventing oral vaccination according to the present invention can minimize side effects due to invasive inoculation (subcutaneous and intramuscular inoculation), and cellular and mucosal immunity. The ability to continuously improve and maintain the ability to protect against outdoor strains is also excellent.

Description

Oral fowl typhoid vaccine using a live adjuvant system

The present invention relates to an oral attenuated live vaccine using a biotype immunopotentiator for preventing poultry typhoid infection in poultry.

Fowl typhoid is an acute or chronic bacterial infectious disease that causes sepsis in poultry. The cause is Salmonella Gallinarum, which can be easily infected at all ages, from chickens to young chicks to the chicken system, and has a high mortality rate. Poultry typhoid is mainly infected orally through feces, but can also be vertically transmitted through reproductive organs and eggs, making it difficult to eradicate it.

 The global outbreak with high mortality, along with Pullorum disease in the early 1900s, poses a serious threat to the poultry industry in each country, and in the 1950s in North America and in several European countries. It has successfully eradicated a strong defense program at the level, and in these countries, there have been few reports of occurrence since the 1980s. However, in Asia and South America, the continuous occurrence is causing huge economic losses to the poultry industry.

In Korea, an official occurrence was reported in 1992, and since 1994, it has been reported nationwide and has caused huge economic losses to date. The causative agent of poultry typhoid is an intracellular parasitic bacterium that proliferates even in macrophages, making it impossible to treat antibiotics. In other words, when antibiotics are administered to an individual infected with poultice, the level of antibiotics can be suppressed by the bacteriostatic action of antibiotics. However, the concentration of antibiotics in the blood decreases by stopping the use of antibiotics. In the beginning, since the bacteria are already present in the host cell, they start to proliferate again and the symptoms of the symptoms are caused. Antibiotic treatment of poultry typhoid is not only possible, but the use of indiscriminate antibiotics can lead to the emergence of resistant bacteria. Thus, the method for defense against poultice is not with antibiotics, but with vaccines.

Currently used poultry fever vaccines include bacteriophage vaccines and live vaccines, but because they are parasitic bacteria in cells, viable vaccines that simultaneously activate cellular and humoral immune responses have a high protective effect. The most prophylactic live vaccines against poultry typhoid are Salmonella gallinarum 9R (SG 9R), a live vaccine for poultry fever, which has been licensed for laying hens since 2001. Vac E is sold.

 Both SG 9R and SR2 vaccines are mutated strains of the LPS moiety that are part of the pathogenic factor of the causative organism, showing some degree of safety and defense. SG 9R and SR2 vaccines are not clear in their genetic properties or are likely to recover from pathogenicity and are known to have some side effects when applied to individuals, requiring the development of safer vaccine strains.

Above all, however, it has been pointed out that the vaccine developed for poultry fever prevents the protection against oral vaccination considerably.In fact, these two products also recommend subcutaneous or intramuscular vaccination. It is limited. In other words, the invasive inoculation method using a syringe (subcutaneous, intramuscular inoculation) not only causes physical stress of the applied animal and can cause side effects due to the inoculation, but is also disadvantageous in terms of economics because a professional should inoculate each individual. Do.

On the other hand, Salmonella Vac E probiotic vaccines, which have recently started to be marketed in some countries, recommend oral vaccination. However, this product was originally developed for the purpose of preventing Salmonella Enteritidis infection, and it is not well suited in the area of pandemic typhoid because of its low protection against the infection.

Despite the simplicity and economic advantages of oral vaccination, the defense against oral application of the existing poultry fever vaccine is not satisfactory. The main reason is that the orally inoculated vaccine is relatively poorly penetrated into the gut mucosa. This is because it does not cause enough. In order to solve this limitation of oral vaccination, a method of maximally increasing immunity using an appropriate adjuvant may be applied. The inventors have noted the heat-labile enterotoxin B subunit (LTB) of E. coli, among the many mucosal immune enhancers reported so far.

LTB is a transporter protein that is a component of heat-labile enterotoxin (LT) and lacks toxicity and, when orally inoculated with other antigens, mucosal membranes for antigens inoculated together due to the high immunogenicity of LTB And good adjuvant for causing systemic immune induction. LTB is usually applied by inoculation with the bacteriophage vaccine in the form of purified protein, but it has been mentioned above that the bacteriophage vaccine has no effect on the prevention of gamma typhoid. In addition, even if the purified protein LTB is injected with a live vaccine, the effect cannot be expected.

Accordingly, the present inventors attenuated the 'combined plasmid for continuous expression of LTB' produced in the present invention to the strain of agaposis, which has been attenuated by deleting two virulence genes recently, in order to develop an oral strain of live bacterium typhoid. Bio-type immunopotentiator strains were inserted into the cultivated strains at a certain ratio to prepare oral poultice fever vaccine composition, and when applied to laying hens, the present invention was completed by confirming sufficient immune response induction and effective defense ability by oral vaccination. It was.

An object of the present invention is to provide a safe poultry typhoid vaccine showing sufficient protective effect by oral vaccination.

The invention is lon And cpxR For Salmonella strains with gene deletion, Salmonella mutants further comprising an eltB (heat-labile enterotoxin B subunit) gene are provided. In one embodiment of the present invention, the Salmonella strain Salmonella It's Gallinarum .

In one embodiment of the present invention lon And It provides a mixed Salmonella mutant strain comprising a Salmonella mutant strain lacking the cpxR gene and the Salmonella mutant strain of claim 1, in one embodiment of the present invention Salmonella strain Salmonella Gallinarum , and in one embodiment of the invention the lon And The Salmonella mutant strain lacking the cpxR gene and the Salmonella mutant strain of claim 1 are in a ratio of 7: 3 to 9: 1.

In one embodiment of the present invention (a) eltB Contains the gene, lon And preparing a Salmonella strain lacking the cpxR gene; And (b) provides a method for producing a Salmonella mutant strain comprising the step of selecting the Salmonella strain of step (a), in one embodiment of the present invention Salmonella strain Salmonella It's Gallinarum .

In one embodiment of the present invention provides a vaccine composition for the prevention of poultry fever comprising the mutant strain of Salmonella, in one embodiment of the invention the vaccine composition for preventing poultry fever is for oral inoculation, of the present invention In one embodiment the Salmonella mutant strain is live bacteria.

In one embodiment of the present invention (a) provides a method for treating poultry typhoid comprising the step of orally inoculating the mixed Salmonella strain of claim 3 in birds, (b) Lon in one embodiment of the present invention And Salmonella mutants lacking the cpxR gene; Or lon and Salmonella mutants and lon lacking the cpxR gene And cpxR In Salmonella strains which have been deleted, the method further comprises orally inoculating birds with a mixed Salmonella strain comprising a Salmonella strain further comprising a heat-labile enterotoxin B subunit ( eltB ) gene. In an embodiment the lon And Salmonella mutants and lon lacking the cpxR gene And cpxR In Salmonella strains, the gene further comprising eltB (heat-labile enterotoxin B subunit) gene, and in one embodiment of the present invention, the step (b) is performed from 9 weeks after step (a) of claim 9 to Inoculated when 11 weeks have passed, in one embodiment of the present invention the Salmonella strain strain Salmonella Gallinarum , in an embodiment of the present invention the step is inoculated with ¹ Ⅹ ^{10 7} to 1 Ⅹ ^{10 9} cfu / 200 μl, the present invention In one embodiment of the step is inoculated with 1Ⅹ10 ⁵ to 1Ⅹ10 ⁸ cfu / 200μl, in one embodiment of the present invention the bird is at least one of chicken, duck, turkey, quail, waterfowl, pigeon or canary. .

In one embodiment of the present invention provides a feed additive for preventing poultry fever comprising the above strain of Salmonella. The Salmonella mutant according to an embodiment of the present invention are Salmonella Gallinarum , and in one embodiment of the present invention, the Salmonella mutant strain is characterized in that it is included in 1Ⅹ10 ⁷ to 1Ⅹ10 ⁹ cfu / 200μl.

The pJHL165 plasmid carries an aspartate semi-aldehyde dehydrogenase (asd) gene (hereinafter referred to as asd), which is essential for bacterial cell wall synthesis. Therefore, asd as a non-antibiotic marker instead of a selection marker using an antibiotic resistance gene by selecting a transformed cell when transforming a plasmid with an asd gene into a bacterium lacking the asd gene. Genes can be used. In other words, bacteria as host cells lacking the asd gene must use the asd gene for cell wall synthesis necessary for survival, and thus, live host bacteria after transformation must have a plasmid containing the asd gene.

In addition, the pJHL165 plasmid contains a multiple cloning site (MSC) (hereinafter referred to as MSC) capable of inserting foreign DNA and a β-lactamase signal sequence before the foreign DNA cloning site. ; blaSS) (hereafter blaSS) exists. β-lactamase is an enzyme encoded by an ampicillin resistance gene and has a characteristic of being secreted from gram-negative bacteria into the periplasmic space. Therefore, when the foreign gene is cloned upstream of blaSS, the foreign protein expressed by the foreign gene can be effectively secreted.

The pJHL165 plasmid is regulated by the Ptrc promoter and transcriptional termination of the cloned foreign protein is accomplished by the ρ-independent transcriptional termination sequence of 5ST1T2 (5S rRNA transcription terminator T1T2). In addition, the pJHL165 vector has a His-epitope gene located downstream of blaSS, and after transformation, an anti-his-tag antibody (anti-) is used to confirm the secretion of the foreign protein LTB protein by immunoblotting assay. His-tag antibody was used to make it easier to check the secretion of proteins without the use of antisera. PJHL165, a high copy number plasmid, has a replication origin of pBR ori so that the plasmid remains stable even after more than 50 generations in the presence of diaminopimellic acid (DAP). do.

DAP is an essential material used for bacterial cell wall synthesis. If cultured in a medium that lacks DAP, bacteria without the asd gene may not grow properly because of problems in cell wall synthesis, and the plasmid pJHL165 with the asd gene inserted therein. Bacteria with -LTB grow normally. On this principle, asd gene was used as a non-antibiotic marker for selecting transformed cells.

The vaccine provided by the present invention is for oral vaccination, which is safe, convenient and economical to inoculate.

1 is a schematic diagram showing a process for preparing a pJHL165-LTB plasmid according to an embodiment of the present invention.
Figure 2 is a photograph of Western blot results confirming the expression of LTB according to an embodiment of the present invention. Figure 3 is a graph showing the serum IgG antibody titer according to an embodiment of the present invention.
4 is a graph showing serum IgA antibody titers according to an embodiment of the present invention.
5 is a graph showing a stimulation index according to an embodiment of the present invention.
6 is a graph showing serum IgG antibody titers according to an embodiment of the present invention.
7 is a graph showing intestinal secretion type IgA antibody titers according to an embodiment of the present invention.
8 is a graph showing a stimulation index according to an embodiment of the present invention.

Hereinafter, the components and technical features of the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention and are not intended to limit the scope of the invention.

Example

Example  1. Biotype Immunostimulant strains ( JOL1229  Production of strains)

1-1. LTB  Construction of Expression Recombinant Plasmids

PJHL165 plasmid was used as an expression vector to prepare a recombinant plasmid to continuously express LTB used as an immunopotentiator (FIG. 1). First, the eltB gene, which is a gene expressing LTB used as an adjuvant, was amplified by PCR with the primers described in Table 2. The PCR amplification product and the expression vector pET28a plasmid were digested with Eco RI and Hin dIII, respectively, and then the DNA molecules digested using T4 DNA ligase were linked to each other, and the host bacterium E. coli BL21 (DE3). The pET28a-LTB vector containing the eltB gene was prepared by introducing into pLysS.

In order to clone the eltB gene into the pJHL165 plasmid to be used as the expression vector, the pET28a-LTB vector and the pJHL165 plasmid containing the eltB gene prepared as described above were digested with the corresponding restriction enzymes, and then ligated using a T4 DNA ligase to host. It was introduced into the bacterium E. coli χ 6212. Then, strains transformed with pJHL165-LTB inserted with LTB expression gene were selected in Luria-Bertiani (LB) agarrose plate without DAP (FIG. 1). ). Finally, in order to confirm that the pJHL165-LTB vector was inserted into the host cell, the plasmid was separated from E. coli χ 6212 and cut and confirmed by restriction enzymes corresponding to the LTB expression gene. Table 1 below describes the strains and plasmids used in the examples, and Table 2 below shows the primers used in the examples.

Strains and Plasmids Relative characteristics How to get it S. Gallinarum JOL394 Wild type, Nal ^R Invention JOL420 Wild type, highly virulent Invention JOL422 Wild type, used for antigen preparation Invention JOL966 9R strain Intervet JOL916 JOL394 ㅿ lon ㅿ cpxR KCTC 11539BP JOL967 JOL394 ㅿ lon ㅿ cpxR ㅿ asd Invention JOL1229 JOL967 containing pJHL165-LTB Invention
KCTC 12150BP E. coli χ 6212 F-λ-Φ80 △ lacZYA-argF) △ asdA4 BL21 (DE3) pLysS F-, ompT, hsdSB (rB- mB-, dcm, gal, λDE3), pLysS, Cmr Promega Plasmids pET28a IPTG-inducible expression vector; Kmr Novagen pET28a-LTB pET28a derivative containing eltB gene Invention pJHL165 Asd + vector, pBR ori, β-lactamase signal sequence-based periplasmic secretion plasmid, 6xHis Invention pJHL165-LTB pJHL165 derivative containing eltB Invention

primer Sequence (5′-3 ’) LTB-F CCGC GAA TTC GCT CCC CAG TCT ATT ACA G LTB-R CCGC AAG CTT CTA GTT TTC CAT ACT GAT TG

1-2. Immunostimulant strains ( JOL1229  Production of strains)

The pJHL165-LTB cloned with LTB was asd in the attenuated Salmonella mutant strain by deleting the toxic gene in the outdoor strain of Poultry typhoid bacteria by electroporation method. Salmonella Gallinarum ㅿ lon ㅿ cpxR ㅿ asd (hereinafter referred to as JOL967) which had the gene deleted were transformed to obtain a Salmonella gallinarum immune enhancer strain JOL1229.

Specifically, the JOL967 strain was incubated in a LB liquid medium containing DAP (50 μg / ml) until the mid-log phase and washed twice with sterile 10% glycerol containing distilled water. . Thereafter, the prepared JOL967 strain was added to a 0.2 cm cuvette and mixed with 0.1 ㎍ of plasmid. After applying an electric shock, the bacteria were recovered from the cuvette, and then placed in 1 ml of LB broth without DAP, followed by 37 for 1 hour. Incubated at? In order to select transformed Salmonella bacteria, colonies formed by smear plate method were selected in LB agar medium (gar) without DAP addition 100 μl cultured culture solution again. The selected Salmonella was incubated again in LB broth, the plasmids were separated, and digested with the same restriction enzymes as for cloning to confirm the presence of LTB. (Table 1)

1-3. Immunohistometry in Culture of Stimulators immunoblotting  by assay LTB  Confirmation of expression

LTB protein secretion from JOL1229 strain, an immunostimulant strain, was confirmed by immunoblotting assay. In order to prepare JOL1229 where the protein is secreted, the strain was incubated at 37 ° C. in an LB broth, centrifuged at 4000 rpm for 20 minutes, and the supernatant of the culture was passed through a 0.22 μm-pore-size filter. It was precipitated with 20% trichloroacetic acid (TCA, hereinafter TCA) to detect protein secretion.

The TCA precipitate was centrifuged and resuspended in cold phosphate buffered saline (PBS, PBS) and acetone. Protein samples secreted from JOL1229 were boiled for 5 minutes and then separated by SDS-PAGE. LTB expression was confirmed by Western blot analysis using an anti-his-tag antibody (anti-his-tag antibody) in the immunopoint analysis (Fig. 2).

Example  2. Oral Poultry fever  Preparation and Inoculation of Vaccine Compositions

In order to prepare oral live poultry typhoid vaccine, JOL1229 strain in which LLT expression was confirmed in Example 1 was inoculated into LB broth and incubated at 37 ° C. at a speed of 200 rpm for 16 hours. Was added to fresh LB broth at a rate of 1/100 (volume) and re-cultured for 3 to 5 hours under the same conditions. The culture was centrifuged at 4,000 rpm at 4 ° C to discard the supernatant and the precipitate was washed by resuspending in sterile PBS. After washing two more times in the same manner, and then suspended in sterile PBS again to measure the absorbance to check the number of bacteria.

Attenuated Salmonella gallinarum mutant strain (JOL916) was also prepared in this manner.

After measuring the absorbance of the prepared JOL916 strain and JOL1229 strain, respectively, in the case of oral inoculation was inoculated with 1 × 10 ⁸ cfu / 200μl bacteria.

Example  3. Oral Poultry fever  Evaluation of stability of live vaccine

To evaluate the stability of oral inoculation of live poultry fever vaccine composition, JOL916 strain and JOL1229 strain were mixed at a ratio of 80% and 20%, respectively, as shown in Table 3 below, and 1 × 10 ⁸ cfu / 200 μl was added to a 6-week-old brown laying hen. After oral inoculation, euthanasia was randomly selected three times a week for 5 weeks after inoculation, and pathological evaluation and microbiological examination of liver and spleen were performed. At the same time, the clinical symptoms of the vaccinated subjects were also observed. For the evaluation of pathological findings, the degree of enlargement and necrotic foci was observed for the liver and spleen, respectively, and scored from individual 0 to 3 (severe) by organ (Table 4).

group Inoculation head Vaccination (6 weeks old) Strain Name (Ratio) Inoculation route Capacity (cfu) A 15 JOL916 + JOL1229 (80% + 20%) Oral vaccination 1 × 0 ⁸ B 15 PBS Oral vaccination -

score liver spleen Hypertrophy Necrotic lesions Hypertrophy Necrotic lesions 0 Not enlarged No lesion Not enlarged No lesion One No hypertrophy but organization softens Less than 5 No hypertrophy but organization softens Less than 3 2 Enlarged to a degree that does not cover the proximal It is less than 20 more than five Enlarged to less than 1.5 times the diameter of a normal spleen It is less than ten more than three 3 Enlarged to fully cover the proximal 20 or more Enlarged to more than 1.5 times the diameter of a normal spleen more than 10

To confirm the presence of vaccine strains, the liver and spleens were ground in buffered peptone water (BPW, BPW) (Becton, Dickinson and Company, France), and the supernatant was brilliant green agar; BGA, hereinafter BGA) (Becton, Dickinson and Company, France) was inoculated in the plate medium to check for the appearance of colonies. If negative, enrichment cultures were incubated at 37 ° for 16 hours in BPW, and 1/100 of the Rappaport-Vassiliadis R10 broth (Becton, Dickinson and Company, France) was reinoculated at 42 ° C. After 48 hours of incubation, the presence of colonies of Salmonella was confirmed in BGA plate medium.

As shown in Table 5 below, no special clinical symptoms were observed including diarrhea, anorexia, and depression after vaccination. Internal organ pathology showed mild lesions (less than 1) in the liver and spleen up to 4 weeks after vaccination, and no lesions were observed at 5 weeks (Table 5).

group Gross pathologic ^a Week post vaccination long time One 2 3 4 5 A liver Hypertrophy 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Necrotic lesion 0.3 ± 0.6 0.7 ± 1.2 0.7 ± 0.6 0.3 ± 0.6 0.0 ± 0.0 spleen Hypertrophy 0.0 ± 0.0 0.3 ± 0.6 0.7 ± 0.6 0.3 ± 0.6 0.0 ± 0.0 Necrotic lesion 0.0 ± 0.0 0.7 ± 1.2 0.7 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 B liver Hypertrophy 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Necrotic lesion 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 spleen Hypertrophy 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Necrotic lesion 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

^a mean ± standard deviation

As shown in Table 5 above, microbiological examination confirmed that the vaccine strain was present in the liver and spleen up to 4 weeks after vaccination, and at 5 weeks no vaccine strain was isolated from all selected individuals. Therefore, the vaccine composition of the present invention is determined that the vaccine strain is removed from the body within 5 weeks after vaccination, and that the vaccine strain remains in the body for about 4-5 weeks without clinical symptoms and acts as a continuous immune inducing agent.

group long time Week post vaccination One 2 3 4 5 Bisexual ^a Colony can ^b positivity Colony count positivity Colony count positivity Colony count positivity Colony count A liver 3/3 ^* 1.73 ± 1.26 ^* 2/3 1.82 ± 1.58 1/3 0.62 ± 1.08 1/3 0.87 ± 1.50 0/3 0.00 ± 0.00 spleen 3/3 ^* 2.55 ± 0.41 ^* 3/3 ^* 3.54 ± 0.26 ^* 3/3 ^* 3.11 ± 0.68 ^* 1/3 1.21 ± 2.10 0/3 0.00 ± 0.00 B liver 0/3 0.00 ± 0.00 0/3 0.00 ± 0.00 0/3 0.00 ± 0.00 0/3 0.00 ± 0.00 0/3 0.00 ± 0.00 spleen 0/3 0.00 ± 0.00 0/3 0.00 ± 0.00 0/3 0.00 ± 0.00 0/3 0.00 ± 0.00 0/3 0.00 ± 0.00

^a number of positive samples after enrichment

^b mean ± standard deviation log ₁₀ cfu / g

^* Statistically significant difference compared to the control group ( P ≤ 0.05)

Example  4. Oral Poultry fever  Probiotic vaccine composition and SG  Comparison of Immune Responses of 9R Strains

In order to confirm the optimal composition ratio for the effective application of the oral live poultice virus vaccine composition and at the same time to compare the immune response with the existing commercial live poultice virus vaccine SG 9R strain (hereinafter referred to as JOL966 strain), as shown in Table 7, Different mix ratios of JOL916 and JOL1229 strains were orally inoculated 1 × 10 ⁸ cfu / 200 μl into 6-week-old brown laying chicks, and the JOL 966 strain was inoculated intramuscularly at a dose of 2 × 10 ⁷ cfu / 200 μl. In order to confirm the induction of the immune response of the vaccine, ELISA was performed to measure the concentration of plasma IgG and intestinal sIgA and to evaluate the peripheral lymphocytes proliferation assay (LPA). Sexual and cellular immune responses were measured.

group Inoculation head Vaccination (6 weeks old) Challenge Infection (3 weeks after vaccination) Strain Name (Ratio) Inoculation route Capacity (cfu) Strain name Inoculation route Capacity (cfu) A 10 JOL916 + JOL1229 (50% + 50%) Oral vaccination 1 × 10 ⁸ JOL420 Oral vaccination 1 x 10 ⁶ B 10 JOL916 + JOL1229 (80% + 20%) Oral vaccination 1 × 10 ⁸ JOL420 Oral vaccination 1 x 10 ⁶ C 10 JOL916 + JOL1229 (90% + 10%) Oral vaccination 1 × 10 ⁸ JOL420 Oral vaccination 1 x 10 ⁶ D 10 JOL966 Inoculation 2 x 10 ⁷ JOL420 Oral vaccination 1 x 10 ⁶ E 10 PBS Oral vaccination - JOL420 Oral vaccination 1 x 10 ⁶

Samples for ELISA measurements were collected at random from 5 chicks per group at 1 week, 2 weeks and 3 weeks after vaccination, and plasma was isolated from peripheral blood which inhibited coagulation by adding heparin. Meanwhile, the digestive tract washing solution was orally administered with a washing solution (48.5 mM PEG [polyethylene glycol 3350], 40 mM Na2SO4, 20 mM NaHCO3, 10 mM KCl, 25 mM NaCl) orally by 12 ml / kgBW (body weight), and 30 minutes later pilocarpine) was intramuscularly injected at a dose of 13 mg / kgBW (20 mg / kgBW for BW <300 g). Each chick was placed in a clean container and 5 ml of STI / EDTA solution (0.1 mg soybean trypsin inhibitor in 50 mM EDTA, pH 8.0) was mixed with the effluent well. After 30 minutes, the effluent was recovered and centrifuged (13,000 rpm, 2 min, 4 ° C.) to obtain a clear supernatant. The supernatant was 100 mM PMSF (phenylmethylsulfonyl fluoride), 1% sodium azide and 5% BSA. Each of 10 ml of bovine serum albumin was added and then stored at -20 ° C.

Plasma-specific IgG and intestinal secretion type IgA were subjected to indirect ELISA using an outer membrane protein (OMP). The OMP antigen was adsorbed onto an ELISA plate (flat-bottom 96 well high binding ELISA plate, Greiner, Germany) to perform ELISA using a chicken IgG (or IgA) ELISA quantification kit (Bethyl, Tx, USA). Specifically, to determine the optimal concentration of antigen and sample, cross titration test is performed to determine antigen coating, blocking, primary antibody reaction, secondary antibody reaction, HRP-substrate staining according to the determined concentration. (substrate colorization), the OD value was measured at 492 nm after the reaction was stopped.

LPA was performed three weeks after vaccination to measure cellular immune responses (Barta et al., 1992, Avian Dis). In order to obtain peripheral lymphocyte samples, 1 ml of heparinized blood was centrifuged at 40 rpm (<10 rcf) for 15 minutes at room temperature to separate mononuclear leukocytes, and antibiotics and fetal bovine serum (FBS) were added. After washing and resuspending with one RPMI-1640 complete medium, and confirming the living cell number by trypane blue exclusion test, the cell number was adjusted to 1 × 10 ⁵ cfu / mL. 100 μl of cell suspension per well and 50 μl of 0.1 μg / ml soluble antigen (control wells RPMI-1640) were placed in a 96-well cell culture plate at 40 ° C. in the presence of 5% CO _2. Time incubation (Rana et al, 2006). Incubation ViaLight ^ⓡ Plus Assay Kit (Lonza, USA), the value of the well stimulation by measuring the degree of luminescence in aqueous antigen with and stimulates the antigen instead of the ratio of the value of the control wells were added only RPMI-1640 Index (stimulation index; Obtained by SI) and compared by group.

The water-soluble antigen used in LPA was prepared using a precipitate obtained by centrifugation of 200 ml of JOL422 strain culture. The procedure of freeze-thaw was repeated three times by washing twice with PBS and then resuspending in 40 ml of PBS. The supernatant obtained by centrifuging the solution for 20 minutes at 4,250 × g, 4 ° C. after sonication was further centrifuged at 30,000 × g, 4 ° C. for 20 minutes to recover the supernatant to determine the concentration.

As shown in Figures 3 and 4, serum IgG antibody titers in both vaccination groups (groups A, B, C, D) began to increase from 2 weeks after vaccination to 3 weeks after vaccination It was measured at a high level. Intestinal secretory IgA antibody titers increased first after 1 week of vaccination in Group A, and then maintained until 2 weeks after vaccination in Group A, and intestinal secreted IgA antibody titers from 2 weeks after vaccination in Group B. Began to increase and continued to increase until 3 weeks after vaccination. In the control group, both serum IgG and gut secreted IgA antibody titers showed no significant change from before vaccination to 4 weeks after vaccination.

In addition, as shown in Figure 5, peripheral lymphocyte differentiation measurement showing a cellular immune response also showed that the stimulation index (SI) value increased more than 2 times compared to the control group in all vaccinated groups, in particular In group B, the SI was about 3.1, which was the highest among the vaccine groups. Taken together, the results of measuring the immune responses showed that group B (vaccinated group with JOL916 and JOL1229 in a ratio of 80:20) was higher than other vaccinated groups in humoral and cellular immune responses. Confirmed.

Example  5. Oral Poultry fever  Probiotic vaccine composition and SG  Comparison of Defenses of 9R Strains

In order to confirm the protective effect of oral poultry typhoid live vaccine against the protective effect of the existing commercial poultry typhoid live vaccine strain JOL966 strain, 1 × 10 ⁶ cfu of JOL420 (National Veterinary Research and Quarantine Service) was used as shown in Table 7 above. Survival was observed for 2 weeks by oral inoculation, and all recovered subjects were euthanized 2 weeks after field strain inoculation, and then assessed by visual examination of liver and spleen gross lesions. In gross pathological evaluation, the liver and spleen were classified into two items, namely, enlargement and necrotic foci, respectively, and the higher the lesion, the higher the score (Table 4).

As shown in Table 8 below, the vaccinated control group (Group E) died 80% after challenge but the vaccinated groups (Groups A, B, C, D) were 20%, 10% and 10%, respectively. , 0% mortality, and compared with the non-vaccinated group (Group E) (Chi-square test), but in group A, B, C, D group, there was statistical difference between groups. Not shown.

group Inoculation head Vaccination
Strain Name (Ratio) Days post challenge Mortality (%) One 2 3 4 5 6 7 8 9 10 11 12 13 14 A 10 JOL916 + JOL1229 (50% + 50%) 0 0 0 0 0 0 One 0 One 0 0 0 0 0 20 * B 10 JOL916 + JOL1229 (80% + 20%) 0 0 0 0 0 0 0 0 0 One 0 0 0 0 10 * C 10 JOL916 + JOL1229 (90% + 10%) 0 0 0 0 0 0 0 0 0 0 One 0 0 0 10 * D 10 SG 9R 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0* E 10 PBS 0 0 0 0 0 One One 0 2 2 0 One One 0 80

Post hoc examinations conducted two weeks after challenge were scored and examined as a result of gross lesions. As a result, as shown in Table 9, the unvaccinated control group showed a serious pathology of 2 or more, but the vaccinated groups Most of the scores were observed to be less than 1 or in and around 1, confirming the protective effect of the vaccine through the state of internal organs of each individual. Therefore, although the oral inoculation of live vaccines generally has a relatively low absorption efficiency in the individual of the vaccine compared to intramuscular or subcutaneous inoculation, the protective effect of the oral live poultry fever vaccine containing JOL1229 is known to be used for intramuscular vaccination. Compared with the protective effect of the live vaccine, it was judged not to decrease.

group Vaccination
Strain Name (Ratio) Gross lesion score (mean ± standard deviation) liver spleen Hypertrophy Necrotic lesions Hypertrophy Necrotic lesions A JOL916 + JOL1229 (50% + 50%) 0.7 ± 1.2 0.7 ± 1.2 1.3 ± 1.3 0.8 ± 1.2 B JOL916 + JOL1229 (80% + 20%) 0.5 ± 1.0 0.6 ± 1.2 0.6 ± 1.1 0.8 ± 1.2 C JOL916 + JOL1229 (90% + 10%) 0.3 ± 0.9 0.5 ± 1.1 0.6 ± 1.1 0.7 ± 1.1 D SG 9R 0.3 ± 0.5 0.9 ± 0.7 0.7 ± 0.7 1.1 ± 1.0 E PBS 2.2 ± 1.3 2.3 ± 1.2 2.2 ± 1.3 2.3 ± 1.1

Example  6. Oral Poultry fever  Probiotic vaccine composition and SG  Comparison of Immune Responses in the 1st-2nd Vaccination Program of 9R Strains

For comparison of the immune response between the live oral live poultry fever vaccine and the existing commercial live poultry fever vaccine strain in the first-second vaccination program, which is the vaccination method used in the actual laying hen farm. As described above, the first vaccine was inoculated at 6 weeks of age, the second vaccine was inoculated at 16 weeks of age, and the corresponding vaccine strains were inoculated to the laying hens.

Specifically, group A was orally inoculated at a dose of 1 × 10 ⁸ cfu / 200 μl by mixing JOL916 and JOL1229 strains at a ratio of 80% to 20% in both the first and second inoculations, and group B was 1 In the primary inoculation, JOL916 and JOL1229 strains were mixed at a ratio of 80% to 20%, and orally inoculated at a dose of 1 × 10 ⁸ cfu / 200 μl. In the second inoculation, only 1 × 10 ⁸ cfu / Inoculated orally at a dose of 200 μl. Group C was inoculated intramuscularly with a dose of 2 × 10 ⁷ cfu / 200 μl of JOL966 strain in both primary and secondary vaccinations. In order to confirm the induction of the immune response of the vaccine, humoral and cellular immune responses were measured by measuring concentrations of plasma IgG and intestinal sIgA by indirect ELISA and LPA. Is the same as the immune response measurement method in Example 4.

Samples for the ELISA measurement were randomly selected from five groups, collected once before vaccination and every 2 weeks after the first vaccination, and a total of eight samples of blood and intestinal lavage were collected and stored. Blood samples were taken twice a week after the third vaccination and three weeks after the second vaccination.

Number of loop immunizations Primary vaccination (6 weeks old) 2nd vaccination (16 weeks old) Challenge infection
(4 weeks after vaccination) Strain Name (Ratio) Inoculation route Capacity (cfu) Strain name Inoculation route Capacity (cfu) Strain name Inoculation route Capacity (cfu) 10 JOL916 + JOL1229 (80% + 20%) Oral vaccination 1 × 10 ⁸ JOL916 + JOL1229 (80% + 20%) Oral vaccination 1 x 10 ⁶ JOL420 Oral vaccination 1 x 10 ⁶ 10 JOL916 + JOL1229 (80% + 20%) Oral vaccination 1 × 10 ⁸ JOL916 Oral vaccination 1 x 10 ⁶ JOL420 Oral vaccination 1 x 10 ⁶ 10 JOL966 Inoculation 2 x 10 ⁷ JOL966 Inoculation 2 x 10 ⁷ JOL420 Oral vaccination 1 x 10 ⁶ 10 PBS Oral vaccination - PBS Oral vaccination - JOL420 Oral vaccination 1 x 10 ⁶

As a result, as shown in Figure 6, in all vaccinated groups (groups A, B, C) serum IgG antibody titers maintained a high level after the first vaccination. In group A and group B, oral vaccine groups, the titer of plasma IgG antibody did not increase significantly at 1 week after the first vaccination, but significantly increased at 3 weeks after the 1st vaccination. High levels were maintained before and continued after the second vaccination. In group C with intramuscular SG 9R, the titer of plasma IgG antibodies increased at 1 week after the first vaccination, compared to the other vaccine groups, and from 3 weeks after the first vaccination, as with other vaccine groups. The titer remained high.

In addition, as shown in FIG. 7, intestinal secretory IgA antibody titers increased up to 5 weeks after the first vaccination in the group A and group B (P <0.05), and then decreased after 1 vaccination. It increased again from week 1 and maintained at 3 weeks later (P <0.05). In group C, the titer of intestinal secreted IgA antibody did not increase at all after the second vaccination as well as 5 weeks after the first vaccination.

Serum IgG and gut secreted IgA antibody titers, both unvaccinated controls, did not show any significant changes before vaccination, or until 3 weeks after the second vaccination.

As a result of measurement of peripheral lymphocyte differentiation showing cellular immune response, as shown in FIG. 8, stimulation index (SI, hereinafter SI) in all vaccinated groups 3 weeks after the first vaccination compared to the control group. The value was increased more than 2 times, and at 3 weeks after the 2nd vaccination, the SI value was increased by about 3 times compared to the control group. In particular, in the second vaccination, the group B, except for the JOL1299 strain, had an SI value of 3.3 times higher than that of the control group, showing the highest average value among the vaccine groups.

Taken together, the results of measuring the immune response showed that the plasma IgG concentration showing the systemic immune response can be obtained from the commercial live bacteria of the group (Group A and Group B) inoculated using the oral gamma typhoid vaccine composition. Compared with the vaccinated group (Group C), the levels were similarly high, and the gut secreted IgA concentrations related to mucosal immunity showed better response in the oral poultice virus vaccine group. In the cellular immune response, the groups vaccinated with the oral strains of live bacterial vaccine (Groups A and B) had a similar or better response than the group vaccinated with commercial strains of commercial strains of bacterial strains (Group C). In addition, the mixed inoculation of the immune booster strains were performed only at the first time, and it was confirmed that it was advantageous to exclude the second inoculation.

Example  7. Oral Poultry fever  Probiotic vaccine composition and SG  Comparison of Defenses in the 1st-2nd Vaccination Program of 9R Strains

To compare the protective effect between oral and live commercial strains of live strains of live poultice fever in the 1st-2nd vaccination program, 4 weeks after the 2nd vaccination as shown in Table 10. JOL420 strain, oral, was inoculated 1 × 10 ⁶ cfu orally for 2 weeks, and all recovered subjects were euthanized 2 weeks after inoculation with outdoor strains, and then gross lesions of liver and spleen were scored as a post test. Evaluated. Post test was evaluated in the same manner as the post test method in Example 5.

As shown in Table 11 below, 70% of the non-vaccinated controls (group E) died after challenge, and all of the vaccinated groups (groups A, B, and C) had a mortality rate of 10% (P < 0.01).

group Inoculation head Vaccination Days post challenge Mortality (%) 1st inoculation strain 2nd inoculation strain One 2 3 4 5 6 7 8 9 10 11 12 13 14 A 10 JOL916
+ JOL1229 JOL916
+ JOL1229 0 0 0 0 0 0 0 0 0 0 0 0 One 0 10 B 10 JOL916
+ JOL1229 JOL916 0 0 0 0 0 0 0 0 0 0 0 0 0 One 10 D 10 SG 9R SG 9R 0 0 0 0 0 0 0 0 0 0 0 One 0 0 10 E 10 PBS PBS 0 0 0 0 0 2 One One 2 0 0 One 0 0 70

In addition, as shown in Table 12, in the post hoc examination performed 2 weeks after challenge infection, as a result of scoring gross lesions, the control group showed severe pathological findings of 2 or more in all, and the average of the scores of all the vaccinated groups Less than 1 or identified as 1. Therefore, even in the first-second vaccination experiments, the protective effect of the live oral poultice virus vaccine did not decrease compared to the protective effect of the conventional commercial live vaccine for live poultice.

group Vaccination Gross lesion score (mean ± standard deviation) liver spleen 1st inoculation strain 2nd inoculation strain Hypertrophy Necrotic lesions Hypertrophy Necrotic lesions A JOL916 + JOL1229 JOL916 + JOL1229 0.7 ± 1.1 0.4 ± 1.0 0.4 ± 1.0 0.7 ± 0.9 B JOL916 + JOL1229 JOL916 0.6 ± 1.0 0.8 ± 1.1 0.7 ± 1.2 0.5 ± 1.0 C SG 9R SG 9R 0.6 ± 1.1 0.6 ± 1.1 0.7 ± 1.1 1.0 ± 1.2 D PBS PBS 2.1 ± 1.4 2.1 ± 1.4 2.1 ± 1.4 2.3 ± 1.3

Comparing the comparison of the immune response of Example 6 and the comparison of the protective effect against the outdoor strains of Example 7, the oral gamma typhoid live vaccine vaccination group in the first-second vaccination program that can be applied in the actual laying hen farm Group A with JOL1229 strain and group B with JOL1229 strain at the first inoculation were similar in both the first and second inoculations, and these orally inoculated vaccine groups (groups A and B) were intramuscularly inoculated. The results were similarly comparable when compared to a commercial Poultry Typhoid Vaccine Group C.

Korea Biotechnology Research Institute KCTC12150BP 20120306

Claims (21)

delete delete mixing Salmonella mutants of the lon gene and cpxR the Salmonella mutants and lon and cpxR gene deletion and containing the deletion mutant Salmonella further comprises a eltB (heat-labile enterotoxin B subunit ) genes.
The method of claim 3, wherein
The Salmonella strain Salmonella Mixed Salmonella strains, characterized in that Gallinarum .
The method of claim 3, wherein
The lon and cpxR gene is a Salmonella mutants and lon and cpxR gene deletion is deletion and Salmonella mutant strain further comprises a eltB (heat-labile enterotoxin B subunit ) gene is 7: characterized in that the ratio of 1: 3 to 9 Mixed Salmonella mutants.
delete delete Poultry typhoid vaccine composition comprising a strain of Salmonella strains of claim 3.
The method of claim 8,
Poultry fever vaccine composition for preventing poultry fever is characterized in that for oral inoculation.
The method of claim 9,
Poultry typhoid vaccine composition, characterized in that the Salmonella mutant strain is a live bacteria.
(A) method of treating poultry fever comprising the step of orally inoculating the mixed Salmonella strain of claim 3 to the bird.
12. The method of claim 11,
(b)Lon And cpxR Salmonella mutant strains in which the gene is deleted; or
lon And cpxR Salmonella mutant strains having a gene deleted andlon And cpxR In Salmonella strains with gene deletion,eltB(Heat-labile enterotoxin B subunit) Poultry fever treatment method comprising the step of orally inoculating a bird with a mixed Salmonella strain comprising a Salmonella strain further comprising a gene.
13. The method of claim 12,
Lon above And Salmonella mutants and lon lacking the cpxR gene And cpxR The Salmonella strain having the gene deleted, wherein the strain of Salmonella further comprising a heat-labile enterotoxin B subunit ( eltB ) gene is 7: 3 to 9: 1 ratio.
13. The method of claim 12,
Wherein (b) step is inoculated when 9 to 11 weeks after step (a), characterized in that the inoculation.
12. The method of claim 11,
Salmonella strain giving the Salmonella A method for treating poulticeous , characterized in that it is Gallinarum .
12. The method of claim 11,
The step is a method for treating poultry fever, characterized in that inoculation with 1Ⅹ10 ⁷ to 1Ⅹ10 ⁹ cfu / 200μl.
13. The method of claim 12,
The step is a method for treating poultry fever, characterized in that inoculation with 1 × 10 ⁵ to 1 × 10 ⁸ cfu / 200 μl.
13. The method according to claim 11 or 12,
Said bird is at least one of chicken, duck, turkey, quail, waterfowl, pigeon or canary.
Feed additive for preventing poultry fever, including the Salmonella mutant strain of claim 3.
20. The method of claim 19,
The Salmonella mutant is Salmonella Feed additive for the prevention of poultry fever , characterized in that the Gallinarum .
20. The method of claim 19,
The Salmonella mutant strains feed additive for the prevention of poultry fever, characterized in that it comprises 1 × 10 ⁷ to 1 × 10 ⁹ cfu / 200 μl.

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