JP2002080396A - Vaccine preparation for chicken - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vaccine preparation for chicken, usable without inducing excessive stress to the inoculated chicken, causing extremely slight residence of the injected substance and formation of granuloma at the injected part, keeping high safety and enabling easy inoculation work. SOLUTION: The vaccine preparation for chicken is a W/O/W-type emulsion containing an antigen derived from Salmonella and a hydrophilic oily adjuvant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a chicken vaccine preparation containing an antigen derived from Salmonella, and more particularly, to a chicken vaccine which exhibits high safety and excellent efficacy, and can save labor for injection work. Formulation.

[0002]

2. Description of the Related Art Salmonella food poisoning in humans due to chicken meat and eggs has increased in recent years and has become a problem. One of the means to prevent this Salmonella food poisoning is to immunize chickens with a vaccine containing Salmonella antigens to suppress the spread of Salmonella among the flocks and the colonization of the bacteria in the chicken body, and as a result, Salmonella to chicken eggs and chicken meat Attempts have been made to reduce pollution.

[0003] Among the chicken salmonella vaccines, inactivated vaccines are classified into two types, oily and aluminum hydroxide gel, depending on the adjuvant used. Among them, oil-based adjuvant vaccine induces excellent immune response, but on the other hand, it induces strong stress in vaccinated chickens, and causes high inoculation reaction at the site of inoculation, leaving inoculum and inoculation marks. There is. For this reason, in Japan, the site of vaccination with Salmonella oil vaccine is currently limited to the subcutaneous region of the shoulder. In addition, oil-based adjuvants generally have a problem that the viscosity is high and the stroke of the continuous syringe is heavy.
For these reasons, the labor and labor involved in the operation of inoculating an oil-based adjuvant vaccine tend to be large.

[0004] On the other hand, although aluminum hydroxide gel does not have the above-mentioned stress and residual problems, its adjuvant effect is generally weaker than oil-based adjuvant.

[0005]

As described above, in the Salmonella inactivated chicken vaccine, a formulation containing an adjuvant which exhibits high safety and excellent efficacy and which can save labor for injection work is desired. ing. In view of such circumstances, the present invention does not induce excessive stress in inoculated chickens,
It is an object of the present invention to provide a vaccine preparation in which injection substance remains at an injection site and granuloma formation is extremely mild, and maintains high safety.

[0006] Another object of the present invention is to provide a vaccine preparation which can be inoculated into muscle, which is relatively easy to inoculate.

It is a further object of the present invention that two injections of the vaccine can provide high antibody responsiveness and long-term immunity.

[0008]

Means for Solving the Problems In order to achieve the above object, the present inventors have tried to apply a hydrophilic oil-based adjuvant to a Salmonella vaccine, and conducted intensive research. It has been found that the above-mentioned problems can be solved by using the emulsion of the present invention, and the present invention has been completed. That is, the present invention is as follows. (1) A vaccine preparation for chicken, which is a water-in-oil-in-water emulsion containing a Salmonella-derived antigen and a hydrophilic oil-based adjuvant. (2) The vaccine preparation for chickens according to the above (1), wherein the Salmonella-derived antigen is an inactivated cell of Salmonella enteritidis and / or a component derived from a cell of Salmonella enteritidis. (3) The chicken according to the above (1) or (2), which is a mixed vaccine containing one or more antigens derived from chicken or viral or bacterial pathogens in addition to the Salmonella derived antigen. Vaccine preparation. (4) The chicken vaccine preparation according to any one of (1) to (3), which is a vaccine for intramuscular injection.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In order to solve the above problems, the vaccine preparation for animals provided by the present invention is characterized by a water in oil in water containing an antigen derived from Salmonella and a hydrophilic oily adjuvant. , Below "w / o / w"
Emulsion), particularly where the vaccine formulation is for chickens.

The hydrophilic oily adjuvant used in the present invention is a composition acting as an adjuvant, and is a mixture containing an emulsifier and an oil as components.

As the emulsifier, for example, esters or ethers of fatty acids and sugars (sorbitol, mannitol, sucrose, glucose, etc.), esters of fatty acids with glycerol or polyol, and the like are used.

As the oil, for example, Marcol 52
Mineral oils such as Drakeol 6VR (manufactured by PENREKO, USA), vegetable oils such as peanut oil, olive oil, sesame oil, soybean oil, wheat malt oil, and animal oils such as squalene, squalane, whale wax oil, etc. it can.

Particularly preferred is octadecenoic anhydride mannitol ether 10 to 20% (V / V).
And a mixture of liquid paraffin and 80 to 90% (V / V) can be mentioned as a typical example of the hydrophilic oil-based adjuvant.

The amount of the oil adjuvant added to the antigen solution is preferably in the range of 25 to 70% (V / V), and particularly preferably 50 to 60% (V / V).

As the Salmonella-derived antigen to be used in the present invention, those derived from cells belonging to the genus Salmonella and acting as antigens can be used. Specifically, inactivated cells of the serotype of Salmonella Or a component derived from the cell body is included. Salmonella serotypes include, for example, Salmonella enteritidis
is, hereinafter abbreviated as “SE”), Salmonella Typhimurium, Salmonella Infantis, and the like, and SE is particularly preferred. The cell-derived components may be those extracted and purified from Salmonella spp. (Subunits) or recombinant proteins expressed in other organisms or cells such as E. coli based on Salmonella spp. Genes. .

The vaccine preparation of the present invention may contain a substance having antigenicity in addition to the Salmonella-derived antigen. Specifically, as other substances to be mixed with Salmonella-derived antigens, for example, antigens derived from viral or bacterial pathogens are preferable, and more specifically, Haemophilus paragallinarum (Haemophilus paragallinarum), ornithobacteria Ornithobacter
ium rhinotracheale), Mycoplasma gallisepticum, Mycoplasma synoviae, Newcastle disease virus, chicken infectious bronchitis virus, chicken infectious bursal disease virus, egg-laying syndrome virus, chicken adenovirus, chicken leo. Viruses and the like. The pathogen-derived antigen may be the bacterium or virus itself, an antigen extracted and purified from the bacterium or virus, or a recombinant protein expressed in another organism such as Escherichia coli based on its gene.

Further, the vaccine preparation of the present invention may contain other components which are generally mixed with the vaccine preparation.

The vaccine preparation of the present invention can be obtained by blending each component and emulsifying to prepare a w / o / w emulsion. The order of blending each component is not limited as long as it can have a w / o / w structure.For example, a hydrophilic oily adjuvant is prepared by mixing an emulsifier and an oil, and a hydrophilic oily adjuvant and an antigen derived from Salmonella. Are mixed and emulsified to form a water-in-oil-in-water emulsion. w / o / w emulsions are generally w / o
It can be prepared according to emulsification conditions for preparing a / w emulsion.

The vaccine preparation of the present invention does not cause an excessive inoculation reaction in the vaccinated chickens even when intramuscularly inoculated, and the survivability of the inoculum is very slight, and is useful as a vaccine for intramuscular inoculation. is there.

[0020]

The present invention will be described below with reference to examples.
The present invention is not limited to the following examples.

Example 1 Preparation of Chicken Salmonella Hydrophilic Oil-Based Adjuvant Vaccine (1) Preparation of Antigen Solution SE KEHL strain (collected in the field and held by the Institute of Biological Sciences, Japan) at 37 ° C. in a liquid medium. The culture was performed for 4 to 10 hours. 1.0% (V / V) of formalin was added to the culture at a ratio of 1.0% (V / V), and sensitized at 37 ° C. for 9 hours or more to inactivate. After inactivation, the cells were collected by centrifugation, and the obtained cells were suspended in a phosphate buffered saline solution (hereinafter abbreviated as PBS).
What was uniformly suspended in 1/50 volume of PBS was used as an antigen solution. At the time of vaccine preparation, a predetermined total bacterial count (8.0 ×
(10 ⁸ cells / mL or more). (2) Preparation of hydrophilic oil-based adjuvant Liquid paraffin and octadecenoic anhydride mannitol ether were mixed at a ratio of 4: 1 (volume) to prepare a hydrophilic oil-based adjuvant. (3) Preparation of vaccine preparation The prepared antigen solution and hydrophilic oil adjuvant were heated to 30 ° C and mixed in equal amounts, respectively, and then homogenized (Silverson L4R, L4R) at 2,000 rpm for 10 minutes. Emulsified.

Example 2 Evaluation of Characteristics of Chicken Salmonella Hydrophilic Oil Adjuvant Vaccine The vaccine preparation prepared in Example 1 was a milky white to white opaque homogeneous liquid. The viscosities are shown in Table 1. The measurement was carried out using a Brookfield LVF model at a temperature of 20 ° C. ± 0.1 ° C. and a rotation speed of 30 rpm.

[0023]

[Table 1]

As shown in Table 1, the viscosity of the three lots was 1
It was extremely low at 8.7 to 19.8 cps. With such a viscosity, the stroke of the syringe is very light at the time of injection of the present vaccine, and the workability at the time of continuous injection can be improved.

Example 3 Safety of Chicken Salmonella Hydrophilic Oil-Adjuvant Vaccine Against Chickens 0.25 mL of the vaccine prepared in Example 1 was injected into the lower leg muscles of 10 35-day-old chickens at intervals of 3 weeks. Was injected twice. Clinical abnormalities were observed from the first to 2 weeks after the second injection. At the time of each injection and at necropsy, the body weight of the test group was measured,
Comparison was made with a non-injected control group measured at the same time. Necropsy of injection sites was performed two weeks after the second injection, and for each injection site, residual injection material and granulation tissue formation were observed. Table 2 shows the results
And 3.

[0026]

[Table 2]

In Table 2, the “number of clinically abnormally occurring birds” is represented by the number of clinically abnormally occurring birds / number of test birds.

As can be seen from Table 2, none of the chickens injected with the prototype vaccine developed clinical abnormalities, and the average body weight at each injection and at the time of necropsy increased steadily as in the control group.

[0029]

[Table 3]

In Table 3, the score of "swelling" is an average value obtained by scoring the swelling at each injection site from 0 to 3 (0: none, 1: mild, 2: moderate, 3: severe). Indicated by Also,
The “granulation area” was represented by the average value of the area (area; cm ² ) of the remaining granules and the formed granulation tissue of the injection at each injection site. “Size” indicates the size of granulation tissue formed at each injection site from 0 to 3 (0: none, 1: millet size, 2: rice size,
3: azuki bean size).

As shown in Table 3, there was no swelling and only slight formation of granulation tissue was observed at the first injection site (5 weeks after inoculation at necropsy) in the examples. Even at the second injection topic (two weeks after vaccination at necropsy), slight swelling and formation of mild to moderate granulation tissue were observed, and the reaction at the site of inoculation was smaller than that of the conventional oil adjuvant vaccine. It was very weak.

Example 4 Efficacy of Chicken Salmonella Hydrophilic Oil Adjuvant Vaccine 0.25 mL of the vaccine prepared in Example 1 was intramuscularly injected into the lower leg muscles of 10 35-day-old chickens at intervals of 3 weeks. Injections. Non-injected control group challenged orally with SE virulent strain 2 weeks after the second injection, and then detected and tested for viable bacteria in cecum, liver and spleen 1 week after challenge. The efficacy was determined by comparing with. The results are shown in Table 4 below.
It was shown to.

[0033]

[Table 4]

In Table 4, the "antibody titer at the time of challenge" is shown as a geometric mean value of the antibody titer by a microplate method using a 100-fold diluted solution of a bacterium for rapid diagnosis of chick diarrhea as an antigen. “Detection of bacteria” indicates the number of positive bacteria detection / number of test birds. When the number of viable bacteria is below the detection limit (3.2 of the cecum and 2.2 of the parenchymal organ), the average value (logarithmic value) obtained by calculating 2.2 for the cecum and 1.2 for the parenchymal organ , CFU /
g).

As can be seen from Table 4, two weeks after the second injection, the average agglutinated antibody titer was 121 to 226 only in the vaccine injection group.
A two-fold antibody response was observed. The bacterial detection rate and viable cell count in the cecum after the live bacterial challenge, and the bacterial detection rate from the spleen and parenchyma were significantly lower in the vaccine-injected group than in the control group. As described above, it has been revealed that the vaccine injection can exert an effect of inducing an antibody response to Salmonella and suppressing colonization of Salmonella in the cecum and transfer to a parenchymal organ.

Example 5 Search for Persistence of Immunity and Remaining Injection of Chicken Salmonella Hydrophilic Oil-based Adjuvant Vaccine Vaccine prepared in Example 1 in the same manner as in Example 4
mL was injected twice into the lower leg muscles of 10 35-day-old chickens at 3 week intervals. Blood was collected at 5, 9, 21, and 29 weeks after the second injection, and the persistence of immunity was examined by measuring the aggregated antibody titer. Blood was simultaneously collected from 10 control group birds of the same age, and the agglutinating antibody titer was measured. In addition, 29 weeks after the second injection,
Challenge tests with SE virulent strains were performed on the vaccine injection group and the control group. The results are shown in Tables 5 and 6.

Regarding the residual injection, 5 days after the second injection
Necropsy was performed at weeks and 30 weeks, and observations were made on each injection site for persistence of the injected substance and granulation tissue formation. Table 7 shows the results
It was shown to.

[0038]

[Table 5]

In Table 5, "Positive wing count" was determined to be positive for individuals having an antibody titer of 20 times or more.

As shown in Table 5, the antibody titers of the vaccine injection group peaked at 5 weeks after the second injection, and thereafter were 9 and 21 days.
Decreased in weeks. Even after 29 weeks, an effective antibody titer was maintained, and the antibody positive rate remained at 100% throughout the test period. On the other hand, in the control group, the antibody titer was 10 times or less and the antibody positive rate was 0% throughout the entire period.

[0041]

[Table 6]

In Table 6, "Bacteria detection" means the number of positive bacteria detection /
The number of test birds was shown. When the “viable cell count” is below the detection limit (3.2 of the cecum and 2.2 of the parenchymal organ), the average value (calculated as 2.2) is calculated for the cecum and 1.2 for the parenchymal organ. Numerical values, CFU / g).

As shown in Table 6, in the oral challenge test 29 weeks after the second injection, there was no difference in the rate of bacterial detection in the cecum, but the number of live bacteria in the vaccine injection group was higher than that in the control group. Significantly lower, and an effect of inhibiting bacterial colonization on the cecum was observed.
In the parenchyma organs, the vaccine injection group tended to have a lower detection rate of the liver and spleen bacteria than the control group, but there was no significant difference. However, if they sum,
The vaccine injection group was found to be significantly lower. In comparison of the viable cell count, the liver, spleen and their total were significantly lower in the vaccine injection group than in the control group. As a result, the effect of this vaccine injection on the transfer to the parenchyma organs was confirmed.
From the above results, it was revealed that in the challenge test as well as the antibody measurement test, the vaccine injection effect was maintained until 29 weeks after the second injection.

[0044]

[Table 7]

In Table 7, the score of "swelling" is an average value obtained by scoring the swelling at each injection site from 0 to 3 (0: none, 1: mild, 2: moderate, 3: severe). Indicated by Also,
The “granulation area” was represented by the average value of the area (area: cm ² ) of the remaining granules and the formed granulation tissue of the injection at each injection site. The “size” was obtained by scoring the size of granulation tissue formed at each injection site on a scale of 0 to 3 (0: none, 1: millet size, 2: rice size, 3: red bean size). The average value was shown.

As can be seen from Table 7, the injection site and granulation tissue disappeared at the first injection site 5 weeks after the second injection (8 weeks after the first injection). At the second injection topic, a mild granulation tissue reaction was observed 5 weeks after the injection, and disappeared in 6 out of 10 birds 30 weeks after the injection. The granulation tissue was only slightly observed in the other four birds, and the survivability of the local injection reaction was found to be extremely slight.

As shown in the above Examples, it was revealed that the vaccine preparation prepared according to the present invention was excellent in the following items. 1) The viscosity is lower than that of an oil adjuvant preparation, and labor is saved during a continuous injection operation. 2) In the case of intramuscular inoculation, the inoculated chicken does not cause an inoculation reaction and the inoculum remains very mild.
Intramuscular inoculation is possible, and the inoculation work becomes easy. 3) Booster immunization is possible because the stress on chickens due to vaccine injection is small. 4) The protective effect as determined by the antibody and challenge test persists for longer than 29 weeks after the second injection.

[0048]

According to the present invention, it is possible to provide a chicken vaccine preparation which exhibits high safety and excellent efficacy and which can save labor for injection work.

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Claims (4)

[Claims] 1. A chicken vaccine preparation which is a water-in-oil-in-water emulsion containing a Salmonella-derived antigen and a hydrophilic oil-based adjuvant. 2. The method according to claim 1, wherein the Salmonella-derived antigen is an inactivated cell of Salmonella enteritidis and / or a component derived from a cell of Salmonella enteritidis.
A vaccine preparation for chickens according to the above. 3. In addition to the Salmonella-derived antigen, one of the chicken-derived viral or bacterial pathogen-derived antigens
2. A combination vaccine comprising one or more species.
Or the chicken vaccine formulation according to 2. 4. The chicken vaccine preparation according to claim 1, which is a vaccine for intramuscular injection.