WO2011019134A2 - Vaccine composition including salmonella microorganism variants for preventing fowl typhoid - Google Patents

Abstract

The present invention relates to a vaccine composition including salmonella microorganism variants, the lon and cpxR genes of which are deleted from the DNA in the chromosomes thereof, for preventing fowl typhoid. The attenuated salmonella vaccine according to the present invention is a live vaccine rendering production and storage convenient and cost-effective. Being free of the risk of recurring pathogenicity, the vaccine is safe to administer to fowls. In addition, the protective strength of the vaccine against field strains is superior to that of conventional vaccines.

Description

Poultry typhoid vaccine composition comprising Salmonella mutant strains

It relates to an attenuated vaccine composition using Salmonella gallinarum mutant strains in which the functions of the lon gene and cpxR gene are impaired.

Poultry fever is a contagious disease that causes sepsis in poultry, caused by Salmonella Gallinarum, and is rapidly spreading and high in mortality not only in chicks but also in the chickens. It is a very important disease. Salmonella gallinarum, the causative agent of poultry fever, is characterized by the lack of adequate antibiotics and ineffective treatment. Therefore, it is most appropriate to prevent this disease by using an appropriate vaccine.

To date, live and dead vaccines have been used, including JOL966 from rough type I, a mutated O-specific chain of lipopolisaccharide (LPS) for laboratory passage. Vaccines also have some protective effects. However, due to the S-R variation (smooth-rough variation), the genetic characteristics of JOL966 are not clear, and most of all, there is a possibility that pathogenicity may be restored upon administration to a living body. The risk can be easily estimated by the fact that it is experimentally commonly used to recover pathogenicity by subcultured in artificial medium and administering the attenuated strain back to the living body.

On the other hand, lon is an ATP-dependent protease, which is a protease that regulates the host infection mechanism of Salmonella. lon has the effect of inhibiting the expression of invasive genes against SPI1 (salmonella pathogenicity islands), a group of genes involved in invasion of early mesenteric cells. Subsequently, Salmonella typhimurium (SPI2), which stimulates the type III secretion system and has a role in promoting SPI2, a gene involved in the stage of infection that leads to systemic infection and organ colonization through survival and proliferation in macrophages. Salmonella Typhimurium) has been reported in in vitro and in vivo mice (Takaya et al. Infection and Immunity, 2003).

In addition, CpxA / CpxR acts like autonomic nerves of bacteria. It is a two-component regulator system that is widely known in living organisms for detecting and responding to changes in the external environment. It is thought to be involved. In each role, CpxA is a sensor and cpxR is an activator. Phosphorylation (activation) of CpxR results in envelope stress response system, pilus assembly, type III secretion, motility, chemotaxis, adhesion, biofilm development It is involved in controlling a wide range of reactions. Deficient cpxA in Salmonella typhimurium impairs the ability to adhere to macrophages but lacks cpxR, and lack of cpxA or cpxR does not affect survival proliferation in macrophages. Infection in rats has been reported to have no effect on body distribution (Sue Humphreys et al. , Infection and Immunity, 2004). Thus, cpxR deficiency is expected to affect pathogenicity but not inhibit immune formation.

However, there have been no studies on live vaccines for the prevention of gamut typhus using Salmonella gallinarum mutant strains that have lon and cpxR genes deleted. Therefore, the present inventors confirmed that the vaccine composition including Salmonella mutant strain which artificially deleted two genes of lon and cpxR was inoculated to poultry, so that it was sufficiently attenuated and had sufficient immunity. The present invention has been completed.

It is an object of the present invention to provide a vaccine composition for preventing poultry typhoid including Salmonella mutant strains in which lon and cpxR genes are deleted.

Yet another object of the present invention is to provide a feed additive comprising the vaccine composition.

In order to achieve the above object, the present invention provides a vaccine composition for preventing poultry fever , including Salmonella mutant strains lon and cpxR gene is deleted from the chromosomal DNA of Salmonella.

The Salmonella bacteria include Salmonella typhimurium, Salmonella typhi, S. typhi, Salmonella paratyphi, Salmonella Sendai, Salmonella gallinarum, and S. gallinarum. It is preferably selected from the group consisting of Salmonella enteritidis.

The vaccine composition may be provided in powder form, for example, for oral administration in a livestock feed, or may be mixed with a suitable preservative for mixing with negative water or for intramuscular injection.

The vaccine composition of the present invention is preferably administered by oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous or nasal route.

The vaccine composition of the present invention can be administered to poultry such as chickens, ducks, turkeys and quails, and wild birds such as waterfowl and pigeons, and ornamental birds such as canaries to prevent poultry fever.

The present invention further provides a feed additive comprising the vaccine composition.

Since the vaccine composition according to the present invention is a live vaccine and is easy to produce and store, it is economical. When administered to poultry, there is no fear of pathogenic return, and it is safer than the existing vaccine because it is superior to existing vaccines. , Safety, and defense are excellent.

1 is a schematic diagram showing a process of applying Salmonella gallinarum △ lon △ cpxR mutant strain (JOL916) as a chicken Salmonella vaccine.

Figure 2 is a weight curve showing the safety of JOL916 vaccination and defense against challenge. 916 (6) IM is the live vaccine vaccine group; -Control challenge without challenge challenge but inoculation; -/-Represents a healthy control with neither inoculation nor challenge.

Figure 3 is a diagram showing the cross-immunity to Salmonella gallinarum specific immune and Salmonella typhimurium induced after JOL916 vaccination (transition of plasma IgG). SG uses Salmonella gallinarum antigen; ST is Salmonella typhimurium Use of antigens; 916 (6) IM was inoculated with JOL916; CTRL represents the control.

Figure 4 is a diagram showing the cross-immunity to Salmonella gallinarum specific immunity and Salmonella typhimurium induced after JOL916 vaccination (transition of secretion type IgA in the digestive organs).

5 is a diagram showing the cross-immunity to Salmonella gallinarum specific immune and Salmonella typhimurium induced after JOL916 vaccination (lymphocyte stimulation response). SI represents a stimulation index.

Figure 6 shows the survival rate after challenge infection. 916 (6) IM was inoculated with JOL916; -Challenge challenge without vaccination but challenge challenge; -/-Indicates a group that was neither vaccinated nor challenged.

Figure 7 shows the diarrhea index after challenge infection.

8 shows the appetite index after challenge infection.

9 is a diagram showing the priming index after challenge infection.

Figure 10 shows the gross pathological findings in the internal organs after inoculation with a picture showing that JOL916 is now safer than the representative commercialized vaccine JOL966. 916 is the JOL916 inoculation group; 966 is the JOL966 inoculation group; CTRL represents a control.

11 shows plasma IgG following JOL916 and JOL966 vaccination. wpv is weeks post vaccination; 916 is the JOL916 inoculation group; 966 is the JOL966 inoculation group; CTRL represents the control.

12 shows the secreted IgA detected in the crop and ileum after JOL916 and JOL966 vaccination.

13 is a diagram showing the protection against outdoor strain challenge of JOL 916 in mortality compared with JOL 966. 916IM was inoculated with JOL916; 916PO was orally administered with JOL916; 966IM was inoculated with JOL966; CTRL represents the control.

14 is a graph showing the growth resistance of JOL916 against outdoor strain challenge as compared to JOL966.

15 is a diagram showing the defense against the challenge of outdoor strains of JOL916 compared to JOL966 as an appetite index.

FIG. 16 is a diagram showing the defense against outdoor strain challenge of JOL916 as a diarrhea index compared to JOL966.

17 is a diagram showing the defense against the challenge of outdoor strains of JOL916 compared to JOL966 as the priming index.

18 is a diagram showing the defense against outdoor strain challenge of JOL 916 in comparison with JOL 966 in gross pathological findings. 916IM is the JOL916 species inoculation group; 916PO is a JOL916 species oral group; 966IM represents the JOL966 primary muscle inoculation group.

19 is a diagram showing the protection against outdoor strain challenge of JOL916 compared to JOL966 as the number of isolated strains from the liver.

20 is a diagram showing the protection against outdoor strain challenge of JOL916 compared to JOL966 as the number of outdoor strains isolated from the spleen.

FIG. 21 shows pathological findings by dose indicative of safety of JOL916 vaccination in 1 week old chicks.

FIG. 22 shows body weight measurements showing safety of JOL916 vaccination in 1 week old chicks.

Figure 23 is Salmonella gallinarum specific cellular immunity induced after JOL916 vaccination in 1 week old laying chicks.

24 is a secreted IgA concentration in Salmonella gallinalum specific digestive organs induced after JOL916 vaccination in 1 week old hens.

25 is Salmonella gallinalum specific plasma IgG concentration induced after JOL916 vaccination in 1 week old hens.

Figure 26 is a clinical symptom showing the protection against outdoor strains of JOL916 vaccination in 1 week old chicks.

FIG. 27 shows the results of anatomical pathology findings showing protection against outdoor strains of JOL916 vaccination in 1 week old chicks.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in more detail based on an Example. However, the following Examples are for illustrating the present invention, and the scope of the present invention is not limited by the following Examples.

EXAMPLE

Example 1. Preparation of Salmonella gallinarum mutant strain

Salmonella gallinarum outdoor strain JOL394 (Table 1)lon The 5 'and 3' ends of the gene were amplified by PCR using lon-F-XbaI and lon-R-XhoI, lon-F-XhoI and lon-R-XbaI as primers, respectively.^®Cloned to -T vector (PromegaCo., Wi, USA) (Table 2). Subsequently, digestion was performed with restriction enzyme XhoI and ligation to obtain a gene sequence in which the lon gene was deleted. This sequence was cleaved with XbaI and cloned into a suicide vector pMEG375 (by Dr. Curtiss R. III. Dozoisetal., 2003) to obtain pBP294. In the same manner, a suicide vector pBP210 having a sequence deleted with the cpxR gene was obtained. Deletion of the full length lon gene and full length cpxR gene was confirmed from the sequences of pBP294 and pBP210.E.coliχ7213 is transformed with pBP294 and pBP210, respectively.E.colis was incubated with LB culture medium to which 50ng / ml of DL-α, ε-diaminopimelic acid (DL-α, ε-Diaminopimellicacid, DAP, Sigma) was added. E. coli with pBP294 was introduced into JOL394 in LB-DAP agar by conjugation to delete the lon gene in Salmonella gallinalum JOL394. Since JOL394 was unable to supply the π-protein, pBP294 failed to replicate and did not persist and naturally disappeared when it was not introduced into the chromosome. At the same time, resistant bacteria were selected from LB agar to which 50 µg / ml ampicillin was added using only those bacteria to which pBP294 was introduced showed antibiotic resistance. These selected colonies were incubated in LB liquid medium without antibiotics at 600 nm to have an absorbance of 0.5 to 1.0, and then diluted appropriately and selected in a medium containing sucrose in LB. the DNA of pBP294 is a sucrose-sensitive genesacBBecause of the presence of a host that continues to retain this gene (with a plasmid), only the host with the missing gene (plasmid) is viable. Selected colonies were again cultured by tooth-picking in LB / sucrose agar and LB / ampicillin agar, and colonies that grow in LB / sucrose agar and do not grow in LB / ampicillin agar were selected as mutated strains, S. Gallinarum Δlon (JOL914). The selected strains were identified for mutation through PCR. to the nextS. ΔcpxR was introduced into Gallinarum △ lon strains using the same conjugation and selection method.S. Gallinarum ΔlonΔcpxR (vaccine strain) was obtained.

Table 1 Strains and Plasmids

Strain	Relevant characteristics	Source or reference
Salmonella Galinarum
JOL394	Wild type, Nal R	Quarantine Salmonella Galinarum No. 169
JOL916	JOL394 ㅿ lon ㅿ cpxR	Accession Number KCTC 11539-BP
E. coli
χ7213	Thi-1 thr-1 leuB6 fhuA21 lacY1 glnV44.asdA4 recA1 RP4 2-Tc :: Mu [λpir]; Km R	Edwards et al. (1998)
Plasmid
pMEG375	sacRB mobRP4 oriR6K. Cm R , Ap R	Dozois et al. (2003)

Nal: nalidixic acid, Cm: Chloramphenicol, Ap: ampicillin

TABLE 2 Primers Used in the Examples

primer	Sequence (5'-3 ')	SEQ ID NO: a
lon-F-XbaI	T / CT AGA CAG GAG TTC TTA CAG GTA GA	502616-502635
lon-R-XhoI	C / TC GAG TCA ATG CGT TCA GAA CGC TC	503386-503405
lon-F-XhoI	CTC GAG GGA ATG CAG GTT GTA ACC GC	505705-505724
lon-R-XbaI	TCT AGA CCA CAC TCC GCT GTA GGT GA	506475-506495
cpxR-F-XbaI	TCT AGA CAT CAT CTG CGG GTT GCA GC	3514975-3514994
cpxR-R-XhoI	CTC GAG CTC GGT CAT CAT CAA CTA AC	3515995-3516014
cpxR-F-XhoI	CTC GAG GTG GTC GCG GCT ATC TGA TG	3516645-3516664
cpxR-R-XbaI	TCT AGA GAT AAT TTA CCG TTA ACG AC	3517665-3517684

^a Sequence number of gene NC_011274 as the accession number of GenBank

Example 2. Cultivation and inoculation of Salmonella gallinarum mutant strain (JOL916 strain)

Salmonella mutant strain (JOL916) was suspended in Tryprose Soybean Broth (TSB) with 20% glycerol and stocked at -80 degrees. The stock was inoculated in TSA (Tryptose Soybean Agar) solid medium, incubated at 37 ° C for 16 hours, inoculated again in TSA, and cultured under the same conditions three times in total to obtain an S-type colony. One colony was inoculated into TSB and cultured for 16 hours in a shaking incubator (200 rpm, 37 ° C) was added to TSB at a ratio of 1/100 (volume), and the concentration was 10 ⁹ cfu (colony forming unit) in the incubator. Incubate until about / ml. The culture solution was centrifuged at 4,000 rpm at 4 ° C., washed three times under the same conditions with PBS (Phosphate buffered saline), and the OD ₆₀₀ values were measured. The concentration was appropriately diluted or concentrated to inoculate the live vaccine. For the production of vaccinated vaccines, the cells were resuspended in 1% formalin-added PBS at an appropriate concentration and inactivated at 4 ° C. for 24 hours.

Inoculation with live or dead bacteria is possible, but inoculation with live bacteria is superior in defense. The route of administration may be intramuscular or subcutaneous injection and oral administration. In this case, 2 × 10 ⁶ cfu was inoculated into the muscle to observe the presence of side effects, immunity, and defense against outdoor strains.

In this experiment, the bacterial count count was performed after colony count method (overnight incubation, 37 ℃) in TSA, the relationship between the OD ₆₀₀ value is as follows. However, C is the number of bacteria (unit: × 10 ⁹ cfu) by the colony measurement method, OD ₆₀₀ is OD ₆₀₀ value.

C = 0.8687 × OD ₆₀₀ - 0.0417

Example 3 Side Effect Observation Experiment of Salmonella Gallinarum Mutant

In order to observe the side effects of the Salmonella gallinarum mutant strain (hereinafter referred to as JOL916 strain) obtained by Example 2, 2 × 10 ⁶ cfu / 100 μl was inoculated intramuscularly (IM) into 5 week old laying chicks. Before and after the inoculation, the body weight was measured by 3-4 days when the side effects started to occur, 6-7 days when the recovery started, and 10 days after the recovery, and the presence or absence of side effects caused by the inoculation was examined (Table 3). .

TABLE 3

group	Inoculation head	Vaccination	Challenge	25 days after vaccination
A
	8	JOL916, IM, 2 × 10 6 cfu	JOL422, 2 × 10 6 cfu, IM
B	8	-
C	8	-	-

IM: intra-muscular inoculation; cfu: colony forming unit

In addition, health status was monitored mainly on fecal changes, appetite loss and restlessness after vaccination. As shown in the middle curve in FIG. 2, it is reasonable to think that a slight weight loss on the 3rd day after vaccination looks the same in all groups including the non-vaccinated group as a transport stress and not the effect of vaccination. Do. There were no clinical symptoms, including diarrhea, anorexia, and depression after vaccination.

Example 4 실험 Specific Immunity and Cross-Immune Induction Confirmation Test of Live Vaccine of JOL916 Strain

In order to confirm specific immunity and cross-immune induction of the live vaccine of JOL916 strain, cross-response to Salmonella gallinarum specific immunity and Salmonella typhimurium was measured after vaccination in the same chick. The results are shown in Figures 3-5. To this end, the concentrations of plasma IgG (plasma IgG) and intestinal secretion type IgA (intestinal S-IgA) were measured by ELISA using LPS extracted from Salmonella gallinarum and Salmonella typhimurium. Peripheral lymphocytes proliferation assay was performed using soluble antigen.

LPS was isolated from outdoor strains JOL420 (Salmonella gallinarum outdoor strain) and JOL389 (Salmonella typhimurium outdoor strain) using LPS extraction kit (Intron Biotechnology, Korea) and these antigens were isolated from a flat bottom 96 well high binding ELISA plate (flat). Adsorbed onto a bottom 96 well high binding ELISA plate, Greiner, Germany, the chicken IgG (or IgA) ELISA quantification kit (quantitation kit, Bethyl, Tx, USA) was used following the instructions. Samples are given in Example 3 The samples were taken from the same chick, plasma was isolated from heparin-added peripheral blood and digestive tract lavage was obtained by the following method (Berthelot-Herault et al., Vet Immunol Immunopathol, 2003). That is, the washing liquid (48.5 mM PEG [polyethylene glycol 3350], 40mM Na₂SO₄, 20mM NaHCO₃, 30 mM NaCl) orally administered 12 ml / kgBW (body weight) 30 minutes after pilocarpine (pilocarpine) at a dose of 13mg / kgBW (20mg / kgBW for BW <300 g) Inject muscle. Each chick is 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) is mixed with the discharge. After 30 minutes, the effluent is recovered and 10 μl of 100 mM PMSF (phenylmethylsulfonyl fluoride) is added per 1 ml, followed by centrifugation (13,000 rpm, 2 min, 4 ° C.) to obtain a clear supernatant. 100 mM PMSF, 1% sodium azide, and 5% BSA (bovine serum albumin) were added to the supernatant, respectively, 10 μl per ml and stored at 4 ° C. to be used as samples for the next day ELISA.

Peripheral lymphocyte differentiation measurements are described in Barta et al. (Avian Dis, 1992) .Refer to the research results, and centrifuged 0.8 ml of heparinized blood at 40 rpm (<10 rcf) at room temperature for 10 minutes to recover mononuclear leukocytes with plasma and added antibiotics and fetal bovine serum. After washing and resuspending with a RPMI1640 complete medium, the cell number was adjusted to 1 × 10 ⁵ cfu / ml after confirmation of the number of living cells by trypane blue exclusion test. 100 μl of this suspension per well and 50 μl of 0.1 μg / ml aqueous antigen solution (Rana et al, 2006) in a 96-well plate (control well for RPMI) and 40 ° C. at 40 ° C. in the presence of 5% CO _2. Time incubation. After incubation, the cells were treated with a ViaPlus assay kit (Lonza, USA), and the ratio of the values of wells stimulated with specific antigens and those of control wells with RPMI instead of antigens were measured. ) And compared by group.

As shown in Figures 3 to 4 it can be seen that the mutant strain induces immunity to Salmonella typhimurium at the same time as the specific immunity to Salmonella gallinarum.

Example 5 실험 JOL916 strain confirms the defense of the live vaccine

In order to confirm the defense ability of the live vaccine of JOL916 strain, Salmonella gallinarum outdoor strain JOL422 was inoculated (2x10 ⁶ cfu) by muscle inoculation (challenge infection) as compared to the defense ability.

Here, the control group for the vaccinated group was compared with the non-vaccinated group (-/ challenge) and the healthy control group without vaccination or challenge (-/-).

The number of deaths was observed every day, and the weight was measured immediately before challenge, 3-4 days, 6-7 days, and 10 times, and the average value was calculated for each group. Clinical symptoms were fecal, appetite, and restlessness. Fecal status is Salmonella gallinarum Symptoms of abnormal feces during infection were evaluated as 0 points (normal), 1 point (yellow mixed with normal stools), 2 points (yellow-green), and 3 points (green contents only), and scored by individual and averaged by group. Was calculated and presented as a fecal score. In addition, appetite decline and restlessness were evaluated from 0 to 3 points. The results are shown in FIGS. 7 to 9.

Vaccination group showed a survival rate of 100% with a healthy control group not vaccinated neither vaccine nor outdoor strain after challenge challenge (Fig. 6). In addition, the vaccinated group showed acute and severe clinical symptoms after challenge, and all died, but the JOL 916 vaccinated group showed mild clinical symptoms with slight weight loss up to 6 days, and none were inoculated. We can see that we returned to the middle curve parallel to the group. As such, 2 × 10 ⁶ cfu of JOL916 muscle was inoculated to obtain excellent defense without side effects.

Example 6 Comparison of Safety and Immune Responses of JOL916 and Conventional Vaccines

As shown in the following [Table 4], in order to compare the safety and immune response of JOL916 strain and JOL966 strain, 2 × 10 ⁷ cfu, which is the standard inoculation of JOL966, was inoculated by intramuscular injection, which is generally performed in Korea.

Table 4

group	Inoculation head	Inoculation
		Bell	Route of administration	Urine (cfu)
A	20	JOL916	IM		2 × 10 7
B	20	JOL966	IM		2 × 10 7
C	12	-	-

IM: intra-muscular, cfu: colony forming unit

In order to compare the apparent side effects in healthy chicks, five mice were euthanized each week to observe gross pathological findings. In the liver and spleen, lesions such as enlargement, white spots, and soft (liver) were evaluated, and they were classified according to the individual organ lesions by 0 points (normal), 1 point, 2 points, and 3 points (very severe). As shown in FIG. 10, JOL916 was determined to be more safe than JOL966.

To compare how long each vaccine strain survives in chicks, the liver and spleen are pulverized in BPW (Buffered peptone water, Becton, Dickinson and Company, France) and the supernatant is bred in BGA (Brilliant Green Agar, Becton, Dickinson and Company, Inoculation in France) was carried out to determine the number of colonies.If negative, the incubation culture was performed at 37 ° C. for 16 hours in BPW, and then Rappaport-Vassiliadis R10 broth, Becton, Dickinson and Company, France. ) Was inoculated with 1/100 volume, and after 48 hours of incubation at 42 degrees, the presence of Salmonella was confirmed in BGA (Table 5).

Table 5

Long-term / group		Post-Vaccination
		One
	2	3	4
liver	916	1/5	2/5	0/5	0/5
	966	3/5	2/5	2/5	0/5
	CTRL	0/3	0/3	0/3	0/3
spleen	916	5/5	1/5	0/5	1/5
	966	4/5	3/5	4/5	1/5
	CTRL	0/3	0/3	0/3	0/3

As shown in Table 5, JOL916 is believed to be alive faster than JOL966. JOL916 may also remain slightly in the spleen at 4 weeks, but emphasizes that it does not in itself represent pathogenic manifestations. In other words, if there is no problem in the state of health, weight ratio, and pathology, it is reasonable to consider that it is an immunogenic source.

In addition, plasma IgG measurement was measured by ELISA in the same manner as in Example 3 by separating the outer membrane protein (outer membrane protein) fraction from the outdoor strain of Salmonella gallinarum (Fig. 11). Intestinal S-IgA measurement was performed by removing 10 cm of the crop and ileum, washing with PBS containing the same additive as in Example 3, centrifuging, and measuring the result by ELISA. .

Example 7.Comparison of Protective Capacity of JOL916 Strain and Existing Vaccine

JOL 916 live vaccine and JOL 966, the most commonly used vaccine, were inoculated into 5-week-old laying chicks and challenged with outdoor strains after 25 days and tested for protection (Table 6).

Table 6

group	Inoculation head	Vaccination			Challenge
		Bell	Route of administration	Capacity (cfu)
A	15	JOL916	IM		2 × 10 7	JOL4222 × 10 6 cfuIM25 dpv
B
	15	JOL916	PO		2 × 10 9
C	15	JOL966	IM		2 × 10 7
D	15	-

IM: intra-muscular

PO: Per Os (by mouth)

cfu: colony count unit

dpv: days post vaccination

The mortality and clinical symptoms were assessed and compared after challenge. Mortality reached 100% in the vaccinated group and 10% in the JOL966 vaccinated group by day 12 (FIG. 13). The growth curve is a curve excluding the dead individuals in the JOL966 group, but the JOL916 inoculation group shows the best growth rate (FIG. 14). In the JOL966 vaccinated group, the weight gain was reduced shortly after challenge but gradually recovered. The control group was stopped due to death. In addition, the clinical symptoms were severely worsened in the control group and died. The JOL 966 inoculated group recovered with clinical symptoms such as weak depression in up to 5 animals after challenge. The JOL 916 inoculated group was weaker than the JOL 966 inoculated group. Only one symptom was shown (FIGS. 15-17).

Comparison of the gross pathological findings of internal organs after euthanasia at 12 days after inoculation with outdoor strains showed that vaccination by intramuscular injection of JOL916 was superior to JOL966 (existing vaccine). As a result of counting the number of outdoor strains invaded and propagated in the internal organs, the muscle injection of JOL916 was also well protected against the outdoor strains (FIGS. 18 to 20).

Example 8 Pathogenicity Investigation Study of JOL 916 Strain in Young Chick

JOL 916 prepared as in Example 2, 10 ⁶ cfu / 100 μl, 10 ⁷ cfu / 100 μl, 10 ⁸ cfu / 100 μl, 10 ⁹ cfu / 100 μl, 10 ¹⁰ cfu / 100 μl 1 week old chick (Brown Nick Oral administration to 20 rats at each concentration, and for comparison, 10 ⁵ cfu / 100 μl, 10 ⁶ cfu / 100 μl, 10 ⁷ cfu / 100 μl, 10 ⁸ cfu / 100 μl, 10 ⁹ cfu 20 animals were orally administered in the same manner. The mortality was checked for 3 weeks to calculate the 50% lethal dose (LD ₅₀ ). As shown in Table 7, the half lethal dose (LD ₅₀ ) by outdoor strain JOL394 was 3.0 X 10 ⁷ in the one-week-old laying chicks, whereas the half lethal dose by JOL916 was> 2.8 X 10 ¹⁰ , which was significantly attenuated in young chicks. It was confirmed (Table 7).

At 3 weeks, the animals were examined by gross euthanasia after euthanasia. Lesions of the individual organs with 0 (normal), 1, 2, and 3 (very severe) lesions were evaluated in the liver and spleen by assessing lesions such as enlargement, white spots, and softening of the liver. Classification was evaluated by. As a result, the pathogenicity was significantly reduced compared to the outdoor strain, and especially when inoculated with 2.0 X 10 ⁶ cfu, pathological findings were not observed at all visually (FIG. 21).

TABLE 7

Strain	Relevant genotype of strain	LD 50 (CFU)
JOL394	Parental wild type, lon +, cpxR +	3.0 X 10 7
JOL916	△ lon, △ cpxR	> 2.8 X 10 10

Example 9. Confirmation of growth inhibition of JOL916 strain in young chicks

Sixteen week old Brown Nick chicks were orally vaccinated 2 × 10 ⁷ cfu / 100. Clinical symptoms were observed every day before and after inoculation, and the clinical symptoms were evaluated by scoring points ranging from 0 (health) to 3 (severe) by depression, appetite loss, and diarrhea. No clinical symptoms were observed in the vaccinated group.

In addition, growth inhibition was examined by weighing before and after 10 days of inoculation. As a result of analyzing the weight gain by multiple comparison test of the general linear model using SPSS ver 10 (P = 0.646), there was no side effect due to vaccination (Fig. 22).

Example 10. Defense Immunoassay Test of JOL916 Strain in Young Chicks

Eighty-five week old Brown Nick chicks were divided into three groups of 18, orally vaccinated 1 × 10 ⁷ cfu / 100 ul to the JOL394 and JOL916 vaccinated groups. Clinical symptoms and mortality were observed daily before and after inoculation. 55.6% of the deaths occurred in the JOL 394 inoculation group, but there were no deaths due to vaccination in the JOL 916 inoculation group (Table 8).

Table 8

Strain	Volume	Route of administration	Number	Our company	% Mortality	95% CI
JOL394	1 x 10 7	oral-	18	10	55.6	33-79
JOL916	1 x 10 7	oral-	18	0	0	0
Control	PBS	oral-	18	0	0	0

CI: Confidence interval

Outer membrane protein (hereinafter referred to as OMP) and soluble antigen were prepared in JOL422 to measure antigen-specific immune responses. OMP was obtained by Modified lysozyme-osmotic shock method. Centrifuge (7000 g, 10 min, 4C) 150 ml of culture to obtain pellets. Lysozyme was added (400 μl of 4 mg / ml) by resuspension in 800 μl of 100 mM Tris-HCl (500 mM sucrose, 0.5 mM EDTA, pH8.6). Immediately, 3.2 ml of 50 mM Tris-HCl (250 mM sucrose, 0.25 mM EDTA, 2.5 mM MgCl 2, pH8.6) was added and incubated for 15 min on ice. This was centrifuged (7000 g, 6 min) to obtain pellets. The pellet is resuspended in 4 ml of 20 mM Tris-HCl (pH8.6), sonicated and centrifuged (7000 x g, 6 min, 4C) to obtain supernatant. The supernatant was centrifuged (132,000 g, 4C, 1 hr) using an ultrafast centrifuge to obtain pellets. Resuspend the pellet in 4 ml of 20 mM Tris-HCl (pH8.6) containing 1% Sarkosyl, incubate for 30 min on ice and centrifuge again with a high-speed centrifuge (132,000 g, 4C, 1). hr) to obtain a supernatant. Aqueous antigens were prepared in pellets by centrifugation of 200 ml of culture. Freeze-thaw was repeated three times by washing twice with PBS and 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.

The feed for measuring immune response was collected as follows. Peripheral lymphocytes and plasma were centrifuged with 0.8 ml of heparinized blood at 40 rpm (<10 rcf) at room temperature for 10 minutes to recover mononuclear leukocytes with plasma and added RPMI1640 complete media with antibiotics and fetal bovine serum. After washing, resuspending and confirming the number of living cells by trypane blue exclusion test (trypane blue exclusion test) was adjusted to 1 × 10 ⁵ cfu / ㎖. Plasma was obtained by centrifugation at 1500 rpm for 5 minutes and stored in a freezer (-20 ° C). The small intestine wash was excised to a specific length of the small intestine 10 cm in length and washed with 1 ml of PBS to centrifuge the wash solution (13,000 rpm, 2 min, 4 ℃) to obtain a clear supernatant. 10 μl of 100 mM PMSF, 1% Sodium azide, and 5% BSA (bovine serum albumin) were added to the supernatant, respectively, and stored at 4 ° C. to be used as samples of the next day ELISA.

Peripheral lymphocytes proliferation assay was performed to measure cellular immune response (Barta et al., 1992, Avian Dis). 100 μl of cell suspension per well and 50 μl of 0.1 μg / ml aqueous antigen solution (Rana et al, 2006) in a 96-well plate (control wells RPMI-1640) at 40 ° C. in the presence of 5% CO ₂ . Incubated for 40 hours. After incubation, the cells were treated with the ViaPlus assay kit (Lonza, USA) to measure the luminescence, and the ratio of the values of wells stimulated with specific antigens and those of control wells with RPMI-1640 instead of antigens were measured using the Stimulation index (SI). It was calculated as and compared by group.

Specific IgG in plasma and IgA in small intestine wash were subjected to indirect ELISA using OMP as antigen. Antigen was adsorbed onto a flat-bottom 96 well high binding ELISA plate (Greiner, Germany) to use a chicken IgG (or IgA) ELISA quantification kit (Bethyl, Tx, USA). To determine the optimal concentration of the side, antigen and sample, cross titration tests were performed to determine antigen coating, blocking, primary antibody reaction, secondary antibody reaction, and HRP-substrate staining according to the determined concentration. substrate colorization) and determine the OD value at 492 nm after stopping the reaction.

As a result of the immune response measurement results, the cellular immunity by JOL916 showed an excellent response similar to the response by JOL394 (Fig. 23). IgA response in the gut was weaker than the reaction by JOL394 but showed a specific reaction (FIG. 24). In addition, the plasma IgG concentration showing the systemic immune response showed an excellent response in the JOL916 inoculation group (FIG. 25).

Example 11 Protective Activity Confirmation of JOL916 Strain in Young Chick

Twenty one-week-old Brown Nick chicks were divided into two groups of 10 JOL 916 and control groups, and the vaccination group was orally vaccinated 2 × 10 ⁷ cfu / 100 ul. Clinical symptoms and mortality were observed daily before and after inoculation, but no clinical symptoms and mortality were observed. Two and a half weeks after vaccination, high pathogenic outdoor strain Salmonella gallinarum JOL422 was orally inoculated 2 × 10 ⁶ cfu / 100 ul for 10 days to observe clinical symptoms and mortality. Clinical symptoms were evaluated by scoring points ranging from 0 (health) to 3 (severe) by category of depression, loss of appetite, and diarrhea. As shown in Table 9 and FIG. 26, the control group showed severe symptoms and 80% died, and the vaccinated group showed very weak symptoms at 5 and 6 days and recovered and did not die.

On the 10th day after the inoculation of the outdoor strain, all the surviving animals were euthanized to evaluate the gross findings. Hypertrophy and white spots were observed in the liver and spleen, and scored from 0 (normal) to 3 (severe) for evaluation. As shown in FIG. 8, the control group showed severe denaturation in all of the surviving animals, but the vaccination group showed a good condition, and thus the protective effect of the vaccine was confirmed.

Table 9

group	Days post challenge										death rate(%)
	One	2	3	4	5	6	7	8	9	10
Control	0	0	0	0	0	4	One	2	One	0	80
Vaccination group	0	0	0	0	0	0	0	0	0	0	0

The present invention is industrially available as a vaccine composition for preventing poultry typhoid including salmonella mutant strains in which Ion and cpxR genes are deleted, or as a feed additive comprising the vaccine composition.

Claims (7)

1. Poultry typhoid vaccine composition comprising a Salmonella mutant strain deleted lon and cpxR gene in the chromosomal DNA of Salmonella.
2. The method of claim 1,

   The Salmonella bacteria vaccine composition, characterized in that selected from the group consisting of Salmonella typhimurium, Salmonella typhi, Salmonella paratypy, Salmonella Sendai, Salmonella gallinarum and Salmonella enteritidis.
3. The method of claim 1,

   Vaccine composition, characterized in that administered by oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous or nasal route.
4. The method of claim 1,

   A vaccine composition, characterized in that for administering to the algae the vaccine composition to prevent poultice.
5. The method of claim 4, wherein

   The bird is a chicken, duck, turkey, quail, waterfowl, pigeon, canary vaccine composition, characterized in that.
6. The method of claim 4, wherein

   The bird is a vaccine composition, characterized in that the chick 6 weeks, 4 weeks, 2 weeks or less than 1 week old.
7. Feed additive comprising the vaccine composition of claim 1.

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