WO2013015586A2 - Vaccine composition for preventing and treating colibacillosis and salmonellosis in poultry, containing a salmonella strain expressing virulence factors of pathogenic escherichia coli of poultry - Google Patents

Abstract

The present invention relates to a vaccine for poultry, containing an attenuated Salmonella strain that contains virulence factors of pathogenic Escherichia coli of poultry, and to a vaccine composition using same for preventing and treating colibacillosis and salmonellosis in poultry. The present invention further relates to a method for increasing immune reactions in poultry by inoculating the poultry with said vaccine so as to induce immune reactions in the poultry and thus to prevent colibacillosis and salmonellosis in a safe and effective manner.

Description

Vaccine composition for the prevention and treatment of pathogenic Escherichia coli and Salmonella bacterium in poultry containing Salmonella expressing pathogenic factors of poultry Escherichia coli

The present invention relates to vaccines and vaccine compositions for poultry comprising attenuated Salmonella strains expressing the pathogenic factors of Escherichia coli of poultry. More particularly, the present invention relates to vaccines and vaccine compositions for poultry that can effectively protect against coliform and salmonellosis of poultry and a method for increasing the immune response of poultry using the same.

Escherichia coli (Escherichia coli) is a bacterium that is commonly present in the intestines of warm-blooded animals. In most cases, Escherichia coli is a non-pathogenic Escherichia coli whose pathogenicity to humans and animals is not a problem. However, Escherichia coli is considered important in veterinary and public health. In poultry, pathogenic E. coli may die of acute pneumonia or subacutely cause pericarditis and airsacculitis. It is a respiratory infectious disease that is distinguished from an infectious disease caused by toxins of Escherichia coli that have settled in the intestine in pigs or humans, and leads to systemic diseases. Poultry is unlikely to have anatomical histological structure of the respiratory tract, which is about 60% thinner for gas exchange, making it more susceptible to respiratory infections caused by overcrowding. It is then settled in the lungs weak to air sacs and infections present only in poultry. Then, it enters the blood and enters the internal organs such as the heart, spleen, and liver. The condition of poultry coliform bacillus is completely distinguished from coliform bacteria such as pigs. Poultry coliformosis is very common worldwide, and morbidity rates are usually very high, around 50%, resulting in low growth resulting in economic losses. The mortality rate is usually 5% but sometimes up to about 20%, causing several viral and mycoplasma infections, including infectious tracheitis, and multiple infections, which are more severe. In Korea, avian coliosis has been noticed as the poultry industry has developed since the late 1970s, and its incidence and severity have increased rapidly in recent years.

   It is common for poultry coliosis to apply antibiotics initially, but a safe and effective prophylaxis is awaited, with the emergence of resistant bacteria and the addition of preventive antibiotics to feed. Existing Escherichia coli inactivation vaccines are known to be ineffective without serotypes. Therefore, it is necessary to develop effective and safe vaccines.

 The distinction between Escherichia coli and non-pathogenic Escherichia coli was classified into O: H serotypes by determining the type of O antigen and H antigen mainly after the fact that H (anti flagella) antigens were different from the past, and Escherichia coli isolated from poultry. It mainly belongs to O1, O2, O78, O8, O35 and so on. Recently, however, even when pathogenic Escherichia coli and serotypes coincide with each other, it is known to express pathogenicity only when expressing a pathogenic factor. As molecular biology techniques are common, knowledge about the presence of pathogenic and non-pathogenic Escherichia coli is rapidly increasing. It is increasing. For example, the pathogenic factors of poultry Escherichia coli include the adhesion factors adhesins (afa, papa, papG, f17, clp, eaeA genes), iron-related pathogenic factors iron-related (iutA genes, etc.), and temperature-dependent hemagglutinin (tsh). Genes), survivors in serum (iss genes), colicin V (cvi genes), endotoxins (eltB genes), which are also known for their immunopotentiation, are already known.

 It should be considered here that the pathogenic factors that contribute to the formation of E. coli are significantly different depending on the pathological characteristics of the animal and E. coli. As an example, in the case of porcine E. coliosis, the adhesion of intestinal membranes by an adhesion factor is very important for disease progression, so that antibody formation against adhesins defends the disease. However, in the case of poultry coliform bacillus, antibodies against adhesins alone cannot protect against coliform bacillus and there is a need for other factors (Avian Pathology. 2006. 35: 238-249.). Therefore, in order to develop a vaccine that can protect against poultry coliform disease, unique research is required based on the knowledge of the pathogenic factors of pathogenic poultry coliform, the anatomical physiology of poultry, and the characteristics of poultry coliform bacterium.

Salmonella Typhimurium (ST) is a Salmonella with a pathogenic serotype that causes paratyphoid in young poultry. ST, along with Salmonella Enteritidis, is a major cause of human infection, mainly through contaminated eggs, and has again grown in importance, especially in developed countries that have overcome Salmonella Typhi. In addition, ST is a parasitic bacterium in cells, particularly antigen presenting cells, which can effectively induce cellular and mucosal immunity, and thus is commonly used as a delivery system for foreign antigens. In another aspect, such cross-reactions can be used in vaccines because of their significant cross-reaction with serotypes that are particularly highly pathogenic in poultry such as Salmonella Gallinarum. Based on this knowledge, the present invention invents a vaccine that is intended to protect against both human infection of salmonella and infection in poultry.

No vaccines have yet been reported to effectively and safely prevent pathogenic E. coli and various Salmonellosis in poultry. Therefore, a multivalent vaccine that can effectively protect against infection with outdoor strains by effectively inducing an immune response to poultry using the pathogenic factors is very valuable.

It is an object of the present invention to provide a poultry vaccine comprising an attenuated Salmonella mutant strain incorporating a pathogenic factor of poultry pathogenic Escherichia coli which can induce an excellent immune response to poultry coliform bacillus and salmonella using one vaccine. .

It is another object of the present invention to provide a method that can effectively increase the immune response of poultry using the vaccine of the present invention as well as increase the immune response of chicks and eggs of vaccinated breeders.

In order to achieve the above object, the present invention is a pathogenic factor papA, papG, f17aA, f17aG, iron-regulated aerobactin receptor (iutA), CS31A surface antigen (clpG), afa8D (afimbrial adhesion), afa8E ( Provided is a poultry vaccine comprising an attenuated Salmonella mutant strain in which one gene selected from afimbrial adhesion) and intimin (eaeA) is introduced.

The present invention also provides a mixed poultry vaccine comprising one or more mutants or all nine mutants in the group consisting of the mutants.

In still another aspect of the present invention, there is provided a vaccine composition for preventing and treating pathogenic Escherichia coli and Salmonella bacterium of poultry comprising the vaccine.

In another aspect, the present invention provides a method for increasing the immune response of poultry, chicks, eggs characterized by inoculating the vaccine.

According to the present invention, by using a mixed poultry vaccine, it is possible to safely and effectively prevent and treat E. coli and Salmonella bacterium at once in poultry. In addition, the present invention can be usefully used to induce an immune response against E. coli and Salmonella bacterium in chicks or eggs of laying hens by inoculating the mixed poultry vaccine according to the present invention.

1 is an overview of the present invention.

2 is a vaccine strain production process.

Figure 3 is the concentration of IgG in the plasma for the papA, papG, iutA, CS31A antigen of Example 3 (A: 2 weeks; B: 3 weeks; C: values of each group measured at 4 weeks)

 Figure 4 is the concentration of small intestinal secretion IgA for papA, papG, iutA, CS31A antigen in Example 3 (A: 2 weeks; B: 3 weeks; C: values of each group measured at 4 weeks).

 Figure 5 shows the proliferation response index of peripheral lymphocytes stimulated with papA and iutA antigens measured at 3 weeks of age in Example 3.

 Figure 6 shows E. coli pathogenic factors papA, papG, iutA, CS31A antigen specific immune response measured at 3 weeks of age in Example 4 (4-1) (A: plasma IgG; B: small intestine secretion IgA; C: lymphocytes Proliferation response index)

Figure 7 is E. coli pathogenic factors papa and iutA antigen-specific immune response measured at 3 weeks of age in Example 4 (4-2). (A: plasma IgG; B: small intestine secreted IgA; C: lymphocyte proliferation index)

Figure 8 shows E. coli pathogenic antigen-specific plasma IgG response measured after vaccination in Example 5 (5-1) (A: papA; B: papG; C: iutA; D: CS31A)

Figure 9 is E. coli pathogenic antigen specific small intestinal secretion type IgA response measured after vaccination in Example 5 (5-1) (A: papA; B: papG; C: iutA; D: CS31A)

Figure 10 is E. coli pathogenic factor antigen-specific peripheral lymphocyte proliferation response index measured after vaccination in Example 5 (5-1) (A: papA; B: iutA.)

Figure 11 shows the E. coli pathogenic antigen-specific IgY concentration contained in the egg yolk obtained from the hens vaccinated in Example 5 (5-2) (A: papA; B: papG; C: iutA; D: CS31A)

12 shows E. coli pathogenic antigen specific IgA concentrations contained in egg whites obtained from hens vaccinated in Example 5 (5-2) (A: papA; B: papG; C: iutA; D: CS31A)

FIG. 13 shows E. coli pathogen antigen specific IgY concentrations in plasma of chicks obtained from fertilized eggs obtained from hens vaccinated in Example 5 (5-3) (A: papA; B: papG; C: iutA; D: CS31A )

14 shows E. coli pathogen antigen specific IgA concentrations in plasma of chicks obtained from fertilized eggs obtained from hens vaccinated in Example 5 (5-3). (A: papA; B: papG; C: iutA; D: CS31A)

Figure 15 shows E. coli pathogenic antigen-specific plasma IgG response measured in laying hens after vaccination in Example 6 (6-2) (A: papA; B: papG; C: iutA; D: CS31A)

Figure 16 shows E. coli pathogenic antigen specific small intestinal secretion type IgA response measured in laying hens after vaccination in Example 6 (6-2) (A: papA; B: papG; C: iutA; D: CS31A)

Figure 17 shows the E. coli pathogenic antigen antigen-specific peripheral lymphocyte proliferation response index measured in laying hens after vaccination in Example 6 (6-2) (A: papA; B: iutA)

18 is plasma cytokine concentration measured in laying hens after vaccination in Example 6 (6-2) (A: IFN-γ; B: IL-2; C: IL-6)

19 is a cytokine concentration in culture medium after stimulating lymphocytes collected from a laying hen after vaccination in Example 6 (6-2) with E. coli pathogenic factors (A: stimulation with papA, IFN-γ concentration; B: with iutA) IFN-γ concentration after stimulation; C: IL-2 concentration after stimulation with papA; D: IL-2 concentration after stimulation with iutA; E: IL-6 concentration after stimulation with papA; F: stimulation with iutA Post-IL-6 concentration)

The present invention is directed to the pathogenic factors papA, papG, f17aA, f17aG, iron-regulated aerobactin receptor (iutA), CS31A surface antigen (clpG), afa8D (afimbrial adhesion), afa8E (afimbrial adhesion), and intimin (eaeA). The present invention relates to a poultry vaccine comprising an attenuated Salmonella mutant strain incorporating one gene selected from the group.

Poultry refers to birds, such as chickens, pigeons, ducks, turkeys, quails, etc., which are useful for human life and cultivated by breeding wild birds. The present invention discloses an embodiment using chickens in poultry, but may include poultry which may be infected by E. coli and Salmonella bacterium without limitation thereto.

The present invention discloses a vaccine using Salmonella Typhimurium (ST) as a carrier, Salmonella typhimurium (hereinafter ST) as one of the preferred embodiments, but is not limited to this, Salmonella is Salmonella typhimurium (Salmonella typhimurium), Any one selected from the group consisting of Salmonella typhi, Salmonella paratyphi, Salmonella sendai, Salmonella gallinarium and Salmonella enteritidis It can be used as.

As a transporter, ST is a salmonella with a pathogenic serotype that causes paratyphoid in young poultry, while ST is a parasitic bacterium in cells, especially antigen presenting cells, which can effectively induce cellular and mucosal immunity. widely used as a delivery system). The lon and cpxR gene deletion type ST strains adopted in the present invention are very attenuated strains with low pathogenicity, and have excellent intestinal mucosa adhesion and antigen-presenting cell invasiveness after oral administration, thereby continuously providing immune stimulation to the host. In addition, the plasmid pBP244 with the asd gene used in the present invention has a signal peptide and SecA (ATPase), SecB (chaperone) and LepB (signal peptidase) for transporting pathogenic factor genes to the periplasmic region. Foreign antigen secretion efficiency is high.

The term "challenge infection" in the present specification is an act of promoting artificial infection by inoculating an outdoor strain with a vaccine animal vaccinated, and is commonly referred to as a challenge in English. The result may be an artificial infection (for example in the case of unvaccinated control animals), or the infection may not be achieved because the outdoor strain is excluded (for example, if the vaccine has a high potency), which is given by vaccination. It is the process of evaluating defenses.

The attenuated Salmonella strain comprising the pathogenic E. coli factor of the poultry of the present invention can be used independently as a vaccine that brings an effective prophylactic and therapeutic effect against E. coli and Salmonella. In addition, these attenuated strains may be mixed with each other at different ratios and used as vaccines and vaccine compositions, including but not limited to the pathogenic factors papA, papG, f17aA, f17aG, iron- of the pathogenic E. coli of the present invention. A combination of regulated aerobactin receptor (iutA), CS31A surface antigen (clpG), afa8D (afimbrial adhesion), afa8E (afimbrial adhesion) and intimin (eaeA) can be used as a vaccine.

The vaccine of the present invention is also directed to a poultry vaccine, characterized in that it further comprises an attenuated Salmonella mutant strain incorporating the eltB gene to increase the immune response.

The pathogenic Escherichia coli of the poultry of the present invention includes, but is not limited to, digestive disease, respiratory disease, arthritis, osteomyelitis, or urogenital disease.

In the vaccine composition, Salmonella mutant strains can be used as live or dead bacteria, and the dead bacteria can be inactivated by heating or formalin, although not limited thereto.

The method of inoculating the vaccine of the present invention can be inoculated by oral, intramuscular, intraperitoneal, intravenous, subcutaneous or nasal route. Preferably, the live vaccine can be orally inoculated at the first and second inoculations. More preferably, live vaccines are orally inoculated at the first and second inoculations, but live vaccines other than Salmonella mutant strains expressing and secreting the eltB gene at the second inoculation may be inoculated.

According to the present invention, when the breeder is vaccinated, the breeder can not only increase the immune response to E. coli and Salmonella, but also increase the immune response to the eggs laid by the breeder and the chicks hatching from them.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Preparation of Salmonella (Vaccine Strain ) with Poultry Escherichia Coli Pathogenic Factor Gene

In this example all strains were cultured in Luria-Bertani (LB) broth. First, the genes of poultry pathogenic E. coli pathogenic factors were cloned from the pathogenic E. coli expressing each factor (Table 1) using the polymerase chain reaction (PCR) method. For example, after heat lysis of JOL439 (papA, papG), JOL132 (eaeA), JOL426 (f17aA, f17aG, clpG), JOL462 (afa8D, afa8E) and JOL718 (iutA), the separated DNA is shown in Table 2 as a template. Amplification was done using one primer. The base sequence including the base sequence of the primer mentioned in the present invention is disclosed in Genbank et al. A part of the gene amplified by the PCR method was stably cloned and inserted into the plasmid pBP244 to transform the attenuated Salmonella typhimurium (JOL912) with the plasmid by a known method. Attenuated Salmonella strains are preferred to use strains that are excellent in host invasiveness such as JOL912 and sufficiently attenuated. In addition, the produced strain was confirmed the secretion of foreign antigens by a known method, the preparation of the rabbit antiserum used was also carried out by a known method. Table 1 shows the field isolated strains used for vaccine strain production, the vaccine strains produced, and the field isolated Escherichia coli used for the purpose of challenge infection in this example in order to confirm the protective ability of the vaccine strains. Strains for challenge infection were well selected pathogenic strains and easy to observe clinical and pathological findings of artificial infection.

Table 1 Strains Used in the Examples

Strain		Explanation	How to get
Cloning production	JOL439	Escherichia coli wild-type isolate expressing papA and papG	Kyungpook National University (2162A)
	JOL132	Escherichia coli wild-type isolate expressing eaeA	Outdoor separation note
	JOL426	Escherichia coli wild type isolate expressing f17aA, f17aG, clpG	Outdoor separation note
	JOL462	Escherichia coli wild-type isolate expressing afa8D and afa8E	Outdoor separation note
	JOL718	Escherichia coli wild-type isolate expressing F18 fimbria	Outdoor separation note
	JOL908	Escherichia coli χ7213 with pBP244 (Asd expression plasmid)	Kim et al. J. Microbiol. Biotechnol. 2007, 17: 1316-1323. Reference
	JOL767	Escherichia coli χ7213 Δasd	Kim et al. J. Microbiol. Biotechnol. See 2007, 17: 1316-1323
	JOL912	Salmonella Thphimurium CK110 Δlon ΔcpxR Δasd	Korea Research Institute of Bioscience and Biotechnology Biological Resource Center KCTC11540BP
Vaccine strain	JOL924	JOL912 expressing papA of Escherichia coli
	JOL928	JOL912 expressing papG of Escherichia coli
	JOL930	JOL912 expressing iutA of Escherichia coli
	JOL954	JOL912 expressing clpG of Escherichia coli
	JOL953	JOL912 expressing eaeA of Escherichia coli
	JOL960	JOL912 Expressing f17aA of Escherichia coli
	JOL952	JOL912 Expressing f17aG of Escherichia coli
	JOL955	JOL912 expressing afa8D of Escherichia coli
	JOL956	JOL912 expressing afa8E of Escherichia coli
	JOL906	JOL912 expressing eltB of Escherichia coli
Challenge infecting strain	JOL718	ColV + Tsh + Iss + IuC + IutA + CS31A +	Outdoor separation note

TABLE 2 Primers mentioned in the examples

	direction	Primers (5 '→ 3')	Restrictionsites
papA	F	CCGC GGA TCC GCT CCA ACT ATT CCA CAG	BamHI
	R	CC CGC GTC GAC TTA CTG GTA ACT TAA ATT	SalI
papG	F	CCGC GGA TCC ATG AAA AAA TFF TTC CCA GCT TG	BamHI
	R	CC CGC AAG CTT TTA TGG CAA TAT CAT GAG CAG CG	HindⅢ
eaeA	F	CCG GGA TCC GAT GAA AAA CGG TCA GCC AGT	BamHI
	R	CCG AAG CTT TAT TTT AGC CGG GGT GGT TAT	HindⅢ
f17A	F	CCGC GAA TTC ATG TAT GAC GGT AAA ATT ACT	EcoRI
	R	CCGC AAG CTT TTA CTG ATA AGC GAT GGT GT	HindⅢ
f17G	F	CCG GAA TTC ATG GCA GTT TCA TTT ATT GGC AG	EcoRI
	R	CCG AAG CTT TTA CTG ATA GGA AAA TG	HindⅢ
clpG	F	CCG GAA TTC ATG TGG ACC ACT GGT GAT TTT AAT	EcoRI
	R	CCG AAG CTT TTA GTT ATA AGT TAC TGC CA	HindⅢ
afa8D	F	CCG GGA TCC GAT GGT TGA ACT GAG TCT T	BamHI
	R	CCG AAG CTT TTA TGA GCA TTC TCC GC	HindⅢ
afa8E	F	CCG GGA TCC GAT GCT AAC TTG CCA TGC TGT GA	BamHI
	R	CCG AAG CTT TCA GTC GGT CCA AGT AGT	HindⅢ
iutA	F	CGT GGA TCC CAA CAA ACC GAT GAT GAA ACG	BamHI
	R	CGG AAG CTT TCA GAA CAG CAC AGA GTA GTT CAG ACC	HindIII
eltB	F	CCGC GAA TTC GCT CCC CAG TCT ATT ACA G	EcoRI
	R	CCGC AAG CTT CTA GTT TTC CAT ACT GAT TG	HindIII

Example 2. Preparation of the vaccine

To prepare a live vaccine, attenuated Salmonella typhimurium in which the expression and secretion of each pathogenic factor was confirmed in Example 1 was inoculated in 5 ml LB Broth and incubated at 37 ° C. at a speed of 200 rpm for 16 hours. This culture was added to 15 ml 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 twice more in the same way, the final precipitate was suspended in a small amount of sterile PBS to determine the bacterial count by measuring the OD600 value. Attenuated Salmonella typhimurium expressing and secreting each pathogenic factor and attenuated Salmonella typhimurium expressing and secreting eltB are mixed to be 1 × 10 ⁷ cfu / 100 μl each to inoculate all strains with a single inoculation. The multivalent vaccine was prepared in a possible form.

Example 3. Vaccine Effects in Broilers

3.1 Animals and Inoculation Conditions.

The 160-day-old Arbor Acres Plus-S broiler chicks were divided into three groups to measure the immune response (divided into groups 1A, 1B, 1C, and 1D according to the vaccination program), and 2 week-old challenge groups (2A, 2B). Group), 3 week old challenge test group (3A, 3B, 3C, 3D group), 4 week old challenge test group (4A, 4B, 4C, 4D group) (Table 3). Feeds without antibiotics were fed (Table 3).

In the first vaccination, 100 μl of PBS mixture containing 1 × 10 ⁷ cfu of each strain of JOL924, JOL928, JOL930, JOL954, JOL953, JOL960, JOL952, JOL955, JOL956, and JOL906 were used as vaccines. At the inoculation, 100 μl of PBS mixture containing all strains except JOL906 was administered to each individual. In addition, the field strain challenge was injected into the left thoracic sac directly by syringe with 100 μl of PBS turbidity containing 1 × 10 ⁷ cfu of JOL718 at 2, 3, or 4 weeks of age.

TABLE 3 Overview of Vaccine Validation Experiments in Broilers

Group	n	First inoculation a	Second dose b	Outline Description
1A	15	-c	-	Immunoassay with 5 euthanasias at 2, 3 and 4 weeks of age
1B	15	oral-	-	Immunoassay with 5 euthanasias at 2, 3 and 4 weeks of age
1C
	10	oral-	oral-	Immunization with 5 euthanasias at 3 and 4 weeks of age
1D
	10	oral-	muscle	Immunization with 5 euthanasias at 3 and 4 weeks of age
2A	10	-	-	Challenge 2 weeks old d
2B	10	oral-	-	Challenge 2 weeks old
3A	10	-	-	Challenge 3 weeks old
3B
	10	oral-	-	Challenge 3 weeks old
3C
	10	oral-	oral-	Challenge 3 weeks old
3D
	10	oral-	muscle	Challenge	3 weeks old
4A	10	-	-	Challenge 4 weeks old
4B
	10	oral-	-	Challenge 4 weeks old
4C
	10	oral-	oral-	Challenge 4 weeks old
4D
	10	oral-	muscle	Challenge	4 weeks old

a: Orally inoculated all at day 1 including JOL906 (eltB strain).

b: Inoculated by oral or intramuscular injection except JOL906 (eltB strain) at 2 weeks of age.

c: inoculated only with PBS

d: Field strain challenge was injected JOL718 into the air sacs to observe the protective effect.

3.2 Safety Check

Health status was checked daily after vaccination. In particular, the presence of diarrhea, appetite, and respiration caused by salmonella strains were observed. No symptoms were seen after vaccination.

3.3 Measurement of Immune Response

Immune responses to E. coli pathogenic molecule molecules by vaccination were measured at 2, 3 and 4 weeks of age (n = 5). Immediately before euthanasia, 1 ml of papa, papG, iutA, and clpG antigen-specific IgG concentrations contained in the plasma of blood collected from the jugular vein with a heparin-extractor, and a specific part of the small intestine (Meckel's diverticulum 10 cm in the middle) The same antigen-specific secreted IgA concentration contained in the washing solution washed with PBS was measured by ELISA. ELISA was performed using Chicken IgG or IgA ELISA Quantitation (Bethyl laboratories, TX, USA) (Matsuda et al., 2010. Vet. Res. 41:59). In addition, lymphocytes (peripheral lymphocytes) of blood collected by heparin-injector as described above were cultured in an RPMI-1640 medium containing papa or iutA for 72 hours and 40 degrees, and the presence or absence of lymphocyte proliferation reaction did not contain these antigens. Antigen-specific cellular immune responses were measured by comparison with responses in culture conditions (Rana & Kulshreshtha, 2006, Vet. Microbiol. 115: 156-162). The proliferation reaction was measured by the photometer using ViaLight® Plus (Lonza Rockland, ME, USA) (Matsuda et al., 2010. Vet. Res. 41:59).

   As a result, the specific plasma IgG concentrations of the measured papA, papG, iutA, and CS31A antigens were improved two to three times from the vaccinated group (group B) to the non-vaccinated group (group A) at 2 weeks of age (FIG. 3). . High values were maintained up to 3 weeks of age even when no second dose was given. It was confirmed that the high antibody titer was maintained by 4 weeks of age by the second inoculation, but the antibody titer was higher than the case of intramuscular inoculation in the case of oral inoculation (group C). Thus, by orally inoculating the attenuated Salmonella vaccine strain of the present invention at 1 day and 2 weeks of age was confirmed that high systemic immunity to the pathogenic factors of Escherichia coli is maintained at least from 2 weeks to 4 weeks old. Observation of secretory immunity against the same antigen showed a similar pattern of elevated plasma IgG concentration. In particular, it was determined that mucosal immunity could not be effectively maintained due to a decrease in secretion type IgA value at 4 weeks of age (FIG. 4). However, oral vaccination was found to effectively induce mucosal immunity against pathogenic Escherichia coli. As a result of peripheral lymphocyte proliferation by papA and iutA at 3 weeks of age, strong oral immunization was induced twice, indicating that strong cellular immunity was induced (FIG. 5). As a result of the above immunoassay, it was found that the antigen-specific immunity is formed very effectively by orally inoculating the vaccine of the present invention at 1 and 2 weeks of age.

3.4 Defense Rating

 Under the same vaccination conditions, 2, 3, and 4 weeks of age were injected into the air sac to compare the protection against the vaccine program. After challenge with an outdoor strain, the mortality was checked daily for one week, and pathological findings were observed in the case of mortality. One week after challenge, all surviving animals were euthanized, and gross lesions of the bladder, pericardium, and liver were evaluated. . In addition, at least one lesion was identified and counted according to the group. Here, the air sac injection of the outdoor strain is a very powerful artificial challenge, which is much stronger than the infection occurring in nature, and is used only for the verification of vaccine defense ability.

The results are summarized in Table 4. In the challenge of 2 weeks of age, the unvaccinated group (group 2A) showed 100% morbidity and mortality, and the pathologic score was 9 points. In contrast, only 60% of the vaccinated patients (group 2B) and 40% died. Pathological findings also showed significant differences. The non-vaccinated group showed 100% morbidity and mortality in the 3-week-old challenge test, but there was a statistical difference of 50% mortality and 60% morbidity in the vaccinated group (Group 3B). The 3C group, which was given two more oral vaccinations, had only 20% morbidity and no animals died. The 3C group showed very good defensiveness in the pathologic score. On the other hand, when the second inoculation was administered by intramuscular injection (3D group) was less protected than the 3B group on the basis of morbidity rate, and showed the same morbidity rate as the unvaccinated group. However, mortality remained at 20%, lower than the vaccinated group. In the challenge of 4 weeks of age, the unoccupied mortality rate remained at 40%, suggesting subclinical infection as the age increased. Chicken E. coli has a low mortality rate even if the morbidity is high depending on age. The morbidity rate of the 4A group was 100% and the pathologic score was not low, but mortality was not high. In comparison, the 4B and 4D groups showed some degree of defense, and the 4C group that received two oral doses showed the best protection. The results of these defense tests were in good agreement with those of the immunoassay test above.

   In the above results, it was confirmed that by orally inoculating the vaccine of the present invention at 1 day and 2 weeks of age, it is possible to effectively protect against E. coli.

Table 4 Vaccine Defense by Vaccine Program in Broilers

Group	n	vaccine		Outdoor Strain Challenge Defense
		1st inoculation	2nd inoculation	Mortality (%)	% Affected	Pathologic score (mean ± SE)
2A	10	-	-	10 (100%)	10 (100%)	9.0 ± 0.00
2B	10	oral-	-	4 (40%) **	6 (60%) *	3.8 ± 4.16 *
3A	10	-	-	10 (100%)	10 (100%)	9.0 ± 0.00
3B	10	oral-	-	5 (50%) *	6 (60%) *	5.4 ± 4.32
3C	10	oral-	oral-	0 (0%) **	2 (20%) **	1.2 ± 1.92 **
3D	10	oral-	muscle	2 (20%) **	10 (100%)	5.8 ± 2.64
4A	10	-	-	4 (40%)	10 (100%)	6.4 ± 2.32
4B	10	oral-	-	4 (40%)	6 (60%) *	4.6 ± 3.68
4C	10	oral-	oral-	2 (20%)	2 (20%) **	1.8 ± 2.88 *
4D	10	oral-	muscle	2 (20%)	6 (60%) *	5.6 ± 2.50

Mortality and morbidity were examined by Chi-square test to see if there was statistical difference between the inoculated group and the non-pointed group. Mann-Whitney U test was performed to investigate the statistical difference between the inoculated and non-vaccinated groups. * Indicates P <0.05, ** indicates difference with a probability of P <0.01.

Example 4. Investigate the use of immune enhancing strains in broilers.

4.1 Strength and weakness investigation of defenses with and without immune enhancing strains.

A 75-day-old Arbor Acres Plus-S broiler chick was divided into three groups, and the vaccine composition was not vaccinated (group A), the vaccine strain containing no eltB gene expression strain (group B), and the vaccine composition containing eltB gene expression strain. The inoculation group (group C) was divided (n = 25). That is, group B was orally inoculated 100 μl of PBS suspension containing 1 × 10 ⁷ cfu of all strains of JOL924, JOL928, JOL930, JOL954, JOL953, JOL960, JOL952, JOL955, JOL956 at 1 day of age. Was inoculated orally with 100 μl of PBS suspension containing 1 × 10 ⁷ cfu of all strains, including JOL906 (Table 5).

Plasma IgG, secreted IgA, and cellular immunity were examined by euthanizing five randomly selected rats from each group at 3 weeks of age. As shown in FIG. 6, plasma IgG response and cellular immunity were induced. In the group containing the immune enhancing strains (group C) showed a significantly stronger response. However, the secreted type IgA response showing mucosal immune response was stronger in the vaccinated group than in the non-vaccinated group, regardless of the presence of immune enhancing strains.

All animals were injected with PBS suspension containing 1 × 10 ⁷ cfu of field strain JOL718 at 3 weeks of age in the same manner as in Example 3 to compare the protection against the vaccine. Mortality, morbidity, and gross lesion score were performed in the same manner as in Example 3. In addition, the average weight gain for 7 days immediately before the field strain inoculation, and 6, 24, 48 hours after the field strain was inoculated in the peripheral blood was also tested. For the latter test, 50 μl of jugular vein blood was collected from 5 groups at each time, and applied to Eosin-Methylene blue (EMB) agar (Difco) and incubated at 37 ° C for 24 hours.

   As a result, the vaccine-vaccinated group (Group A) had a mortality rate of 60% and morbidity rate of 100%, and inoculated strains were isolated at a high rate up to 48 hours in peripheral blood. The gross lesion score was close to perfect score, and the weight gain for 7 days was 46.0 g. On the other hand, vaccination except for immune-enhancing strains significantly improved mortality rate of 25%, morbidity rate 45%. Bacterial isolates from gross lesions, weight gain, and peripheral blood also showed protective effects. However, vaccinations containing immune enhancing strains showed stronger defenses. These results are consistent with the measurement of immune response.

Table 5 Summary of Immune Enhancing Strain Usage Test 1 and Protective Effects

group	n	Vaccination	Observations after challenge with outdoor strain
		Inoculation contents	% Mortality	% Morbidity	Gross lesion average + SE	Weight gain (g)	Bacterial separation
							6h	24h	48h
A	20	PBS	60	100	8.9 + 0.4	46.0	5/5	4/5	5/5
B	20	Except JOL906	25 *	45 **	5.4 + 2.1	56.8	5/5	2/5	1/5 **
C	20	With JOL906	10 **	30 **	3.5 + 1.9 *	63.4 *	5/5	1/5	0/5 **

4.2 Strength and weakness investigation of defense by the content of immune-enhancing strains

The 75-day-old Arbor Acres Plus-S broiler chicks were divided into three groups, vaccinated group (Group A), and vaccine composition inoculated group (group B) containing eltB gene expression strains in the same amount as other strains, eltB. Gene expression strains were divided into vaccine composition inoculation groups (group C) containing 10 fold of other strains (n = 25). That is, group B was orally inoculated 100 μl of PBS suspension containing 1 × 10 ⁷ cfu of all strains of JOL924, JOL928, JOL930, JOL954, JOL953, JOL960, JOL952, JOL955, JOL956, and JOL906 at 1 day of age. group C, the cell suspension 100 μl PBS containing JOL906 other strains of 1 × 10 ⁷ and JOL906 by a 1 x 10 cfu ⁸ cfu were inoculated orally (Table 6).

At 5 weeks of age, five randomly selected rats from each group were euthanized and plasma IgG, secreted IgA, and cellular immunity were examined in the same manner as in Example 3. Plasma IgG and secreted IgA responses as shown in FIG. And vaccination group containing the non-vaccinated group (Group A) and the immune enhancer 10-fold in the group containing only 1 × 10 ⁷ cfu of the same content as other strains in inducing cellular immunity (group B). Significantly stronger than the C group). On the other hand, group C showed similar immune response pattern as the non-vaccinated group.

All animals were injected with PBS suspension containing 1 × 10 ⁷ cfu of field strain JOL718 at 3 weeks of age in the same manner as in Example 3 to compare the protection against the vaccine. Mortality, morbidity, gross lesion score was performed in the same manner as in Example 3. In addition, the average weight gain for 7 days immediately before the field strain inoculation was obtained and compared.

   As a result, the vaccinated group (Group A) had a mortality rate of 40% and morbidity rate of 100% with a gross lesion score of 4.4 and a 7-day weight gain of 72.0g (Table 6). Meanwhile, in group B, mortality rate was 20% and morbidity rate was 40%, and gross lesion score and weight gain were statistically different. However, the vaccinated group (group C) containing 10-fold immune enhancing strains showed similar mortality, gross lesion score, and weight gain as the non-vaccinated group. These results are in agreement with the results of the immune response measurements.

In the above results, it can be seen that the immune-enhancing strain JOL906 can give the animal excellent protection by orally inoculating 100 μl of the vaccine composition containing the same 1 × 10 ⁷ cfu as other monarchs.

Table 6 Summary of Immune Enhancing Strain Usage Test 1 and Protective Effects

group	n	Vaccination	Observations after challenge with outdoor strain
		Inoculation contents	Mortality (%)	% Morbidity	Gross lesion average + SE	Weight gain (g)
A	20	PBS	40	100	4.4 + 2.8	72.0
B	20	1 x 10 7 cfu for every monarch	20	40 **	0.6 + 1.0 **	93.4 *
C	20	JOL906 is 1 x 10 8 cfu	40	70	4.3 + 1.2	73.4

Example 5. Increased immune response in chicks through breeder vaccination.

5.1 Vaccination and Measurement of Immune Response in Breeders.

Forty female 15-week-old Brown Nicks were divided into two groups (n = 20) to be vaccinated (group A) and vaccinated (group B). All were allowed to eat free foods containing no water and no antibiotics. Group B was vaccinated by oral administration of 200 μl PBS suspension containing 1 × 10 ⁸ cfu at 15 and 18 weeks of age (Table 7). No adverse reactions occurred after inoculation.

Blood sampling and intestinal lavage were performed to measure plasma IgG response and secretory IgA response in five rats each week until vaccination and vaccination. Intestinal lavage was performed by known methods using pilocarpine (Porter & Holt, Avian Dis. 1992, 36: 529-536). In addition, plasma sampling and ELISA were performed in the same manner as in Example 3, and cellular immunoassay was performed in the same manner as in Example 3. As a result, it was confirmed that specific plasma IgG response to E. coli pathogenic factors began to appear after 2 weeks of the first inoculation, and significantly stronger after the second inoculation (Fig. 8). On the other hand, secretory type IgA response measured in the small intestine showed a strong value two weeks after the first inoculation, but did not become stronger by the second inoculation showed a statistically elevated value (Fig. 9). Peripheral lymphocyte proliferation also showed significant response to E. coli pathogenic factors after 2 weeks of the first inoculation and maintained high values even after the second inoculation (FIG. 10). The vaccine was inoculated at 15 and 18 weeks of age to confirm that strong immunity was maintained until spawning.

5.2. Changes in Transition Antibody Concentrations in Eggs from Vaccination Breeders

Laying hens started laying eggs at 18 weeks of age. From 18 weeks of age to 36 weeks of age at the end of the experiment, four to five eggs were randomly selected from each week to determine the concentration of specific transfecting antibodies against E. coli pathogens contained in yolks and whites. In order to extract immunoglobulins (mainly IgY) from egg yolks stored at 4 degrees, a known chloroform method was performed (Polson, Immunol. Invest. 1990, 19: 253-258). In addition, known PEG-based Ig isolation was performed to extract immunoglobulins (primarily IgA) from the whites (Polson et al. Immunol. Invest. 1985, 14: 323-327). Antigen-specific IgY and IgA concentrations contained in the extracted samples were determined by the same ELISA method as in Example 3.

As a result, it was confirmed that the concentration of IgY increased until 4 weeks after spawning and decreased after 6 weeks, but the statistically high value was maintained after 18 weeks (FIG. 11). The IgA concentration showed the highest value immediately after the start of laying and gradually decreased, and after 8 weeks, the concentration of IgA was similar to the amount of IgA contained in the eggs laid in the vaccinated group (FIG. 12).

5.3. Measurement of Transient Antibodies in Chicks of Vaccine Breeding

Next, the laying hens were artificially fertilized three weeks after the start of laying. Fertilized eggs were stored at 20 degrees and incubated using the incubator, and blood was collected at 3, 7, 14-day-old chicks from each broiler group, and the concentration of E. coli pathogen-specific IgG and IgA in plasma was measured by ELISA. Measured.

As a result, plasma IgG concentration was the highest at 3 days of age, but gradually decreased (FIG. 13), and IgA concentration was the highest at 7 days of age and similar to chicks of the non-vaccinated group at 3 and 14 days of age (FIG. 14).

5.4. E. coli protection test in chicks vaccinated

In addition, 30 chickens were randomly selected from each group and sprayed with PBS suspension containing 1 x 10 ⁸ cfu of outdoor strain in a confined space at 3 days of age, and then confirmed daily mortality for 10 days thereafter. An anatomical examination was performed to confirm the recognition. On day 10, all animals were euthanized and euthanized identically. Pathological evaluation and morbidity evaluation in the anatomical examination was carried out in the same manner as in Example 3. As a result, 13.3% of vaccinated chicks died and 26.6% of them were ill and the lesion score was 1.7 + 3.2. However, chicks of the vaccinated group had no mortality and only 6.6% morbidity. In addition, the lesion score was only very low 0.2 + 0.8. From the above results, it was confirmed that considerable protective force was given to the transition antibody by vaccination.

TABLE 7 Protective effect of chicks by vaccination against breeders in laying hens

Army (mother)	Vaccinations (on mother laying hens)			Military (cub)	Defensive Effect of Outdoor Strains (In Young Chicks)
	n	Primary inoculation (15 weeks old)	Second inoculation (18 weeks of age)		n	Challenge	Waste water (%)	Yi Hwansoo (%)	Lesion Score (Average + SE)
A	20	PBS	PBS	I	30	Spray spray at 3 days of age	4 (13.3)	8 (26.6)	1.7 + 3.2
B	20	Oral vaccination	Oral vaccination	II	30		0 (0.0)	2 (6.6) *	0.2 + 0.8 **

5.5. Investigation of Egg Contamination by Vaccine

 In order to investigate the presence of egg contamination due to vaccine strains, eggs were divided into five groups and stored at 4 degrees per week, and then the vaccine strain (mutant strain of Salmonella Typhimurium) was attempted in the egg contents. The eggs were soaked in 95% ethanol (95%) for 1 minute and dried. The eggs were mixed well, and some were applied to brilliant green agar (BGA) for counting. The rest was 40 ml of buffered peptone water (BD science, Becton, Dickinson). and company, Sparks, France) and incubated for 16 hours at 37 ° C. and then inoculated in Rappaport-Vassiliadis R10 broth (BD science, Becton, Dickinson and Company Sparks, France) and incubated at 42 ° C for 48 hours. The enrichment broth was inoculated in BGA and incubated at 37 ° C for 24 hours, followed by final confirmation by PCR method confirming Salmonella type colonies. As a result, no vaccine strains were detected, including all enrichment cultures. Therefore, when the vaccine of the present invention is inoculated at 15 and 18 weeks of age it could be seen that egg contamination due to the vaccine strain does not occur.

Example 6 Vaccine Program for Laying Hens

6.1 Defense Comparison by First Inoculation Period

Sixty-day-old Brown Nick chicks were divided into three groups: unvaccinated (group A), vaccinated group (group B) at day 1, and vaccinated (group C) at 7 days (n). = 20). Groups B and C were orally inoculated with 100 μl of PBS suspension containing 1 x 10 ⁷ cfu of all strains of JOL924, JOL928, JOL930, JOL954, JOL953, JOL960, JOL952, JOL955, JOL956, JOL906 (Table 8). . All animals were injected with PBS suspension containing 1 × 10 ⁸ cfu of field strain JOL718 at 4 weeks of age in the same manner as in Example 5 (5-3) to compare the protection against the vaccine. Mortality, morbidity, and gross lesion evaluation were performed in the same manner as in Example 3.

 As a result, the vaccinated group (Group A) had a mortality rate of 20% and morbidity rate of 60%, 10% and 30% for group B (vaccinated at 1 day of age), and 0% and 15% for group C (vaccinated at 7 days of age), respectively. In group C, the protection by vaccine was statistically shown. Therefore, in the present invention, it was confirmed that the first inoculation at 7 days of age was most effective in the laying hens having a long life.

Table 8 Defense of first inoculation time in laying hens

group	n	Vaccination	Observation after challenge with outdoor strain (4 weeks old)
		Inoculation time	Waste water (%)	Yi Hwansoo (%)	Gross lesions (mean ± SE)
A	20	-	4 (20)	12 (60)	3.4 ± 3.6
B	20	1 day old	2 (10)	6 (30)	1.8 ± 3.2
C	20	7 days old	0 (0) *	3 (15) **	0.5 ± 1.3

6-2 Prevention of Escherichia Coli in Laying Hens

Forty days old Brown Nick chicks were divided into two groups: unvaccinated group (Group A) and vaccinated group (Group B) (n = 20, Table 9). The vaccinated groups were all strains of JOL924, JOL928, JOL930, JOL954, JOL953, JOL960, JOL952, JOL955, JOL956, JOL906 at 1 week of age, all strains except JOL906 in the above strains at 5 weeks of age, 1 x 10 ⁷ cfu, respectively. 100 μl of the PBS suspension containing oral was inoculated orally.

 In order to observe the immune-inducing effect of vaccination, the plasma IgG response and secretory IgA response in the small intestine were observed weekly with E. coli pathogenic antigen-specific responses. After each week, measurements were taken. The above experiment was carried out in the same manner as in Example 3. In addition, the concentrations of interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-6 (IL-6) were measured to further examine the immune response after 3 weeks of the first and second doses. Chicken IFN-γ, IL-2 and IL-6 ELISA sets (CUSABIO BIOTECH CO., Ltd, Newark, USA) were used to measure plasma and antigen-stimulated lymphocyte cultures (supernatant) following the directions in the product instructions. .

As a result, plasma IgG concentration was elevated after 2 weeks of the first inoculation, and further increased after 3 weeks. It increased again 4 weeks after the second inoculation (FIG. 15). The secreted IgA concentration in the small intestine was increased two weeks after the first inoculation, decreased at the second inoculation, and then again after the second inoculation (FIG. 16). In addition, it was confirmed that lymphocyte proliferation response to each antigen was induced after each 3 weeks of the first and second inoculations (FIG. 17). Plasma IFN- [gamma] concentrations were found to be elevated at 7 and 14 days after the first inoculation and 3 days after the second inoculation, and IL-2 was also at 7 and 14 days after the first inoculation and 3 and 7 after the second inoculation. It was elevated after days (FIG. 18). IL-6 was elevated 10 days after the first inoculation and 7 days after the second inoculation. IFN-γ concentrations in peripheral lymphocyte cultures stimulated with E. coli pathogenic antigens were elevated 3 days after the first and 3 days after the second inoculation for the antigens tested, respectively, and IL-2 was increased after 3 days after the first inoculation and It was elevated 7 days after the second inoculation (FIG. 19). IL-6 was elevated 7 days after the first inoculation and 7 days after the second inoculation. Where FN-γ is secreted from CD4 + T cells and NK cells and activates macrophage. IL-2 is secreted from Th-1 cells and is required for the differentiation of T cells. IL-6 is also secreted from macrophage or Th-2 cells, which are required for plasma cell maturation and antibody production. Therefore, it was confirmed that cellular immunity is well induced from the increased secretion of these cytokines.

All animals were injected with PBS suspension containing 1 × 10 ⁸ cfu of outdoor strain JOL718 at 8 weeks of age in the same manner as in Example 5 (5-3) to compare the protection against the vaccine. Mortality, morbidity, and gross lesion evaluation were performed in the same manner as in Example 3. As a result, unvaccinated group (Group A) had mortality rate of 15% and morbidity rate of 55%. Group B (inoculation at 1 day of age) was 0% and 15%, respectively.

Table 9 E. Coli Defense Program in Laying Hens

group	n	Vaccination		Observations after challenge with outdoor strain (8 weeks old)
		1st inoculation	2nd inoculation	Waste water (%)	Yi Hwansoo (%)	Gross lesions (mean ± SE)
A	20	PBS	PBS	3 (15)	11 (55)	2.7 ± 3.5
B	20	1 week old	5 weeks old	0 (0)	3 (15) **	0.5 ± 1.3

The present invention relates to a poultry vaccine and vaccine composition comprising an attenuated Salmonella strain expressing the pathogenic factor of poultry Escherichia coli, poultry vaccine and vaccine composition that can effectively protect against coliform and Salmonella bacteria of poultry It is available to industries that increase the immune response of poultry using them.

Claims (13)

1. Pathogenic factors papA, papG, f17aA, f17aG, iron-regulated aerobactin receptor (iutA), CS31A surface antigen (clpG), afa8D (afimbrial adhesion), afa8E (afimbrial adhesion) and intimin (eaeA) An attenuated Salmonella mutant strain into which the gene of.
2. Poultry vaccine comprising at least one attenuated Salmonella mutant strain according to claim 1.
3. Poultry vaccine comprising all nine attenuated Salmonella mutants according to claim 1.
4. The poultry vaccine of claim 2, wherein the poultry vaccine further comprises an attenuated Salmonella mutant strain into which the eltB gene is introduced.
5. 4. The poultry vaccine of claim 3, wherein the poultry vaccine further comprises an attenuated Salmonella mutant strain into which the eltB gene has been introduced.
6. The poultry vaccine according to any one of claims 1 to 5, wherein the Salmonella mutant strain is live.
7. The poultry vaccine of claim 2, wherein the poultry vaccine is characterized in that each Salmonella mutant is mixed in the same ratio.
8. A composition for preventing and treating pathogenic Escherichia coli and Salmonella bacterium in poultry comprising the vaccine of any one of claims 2 to 5.
9. The composition of claim 8, wherein the poultry is broiler, laying hen, duck, quail.
10. A method for increasing the immune response of poultry, characterized by inoculating a vaccine according to any one of claims 2 to 5.
11. The method of claim 10, wherein the inoculation is an oral inoculation.
12. A method of increasing the immune response of a chick, characterized by inoculating the laying hens with the vaccine of claim 2.
13.  A method for increasing the immune response of an egg, characterized by inoculating a breeder with a vaccine of any one of claims 2 to 5.

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