JPH0662433B2 - Method for producing powdered vaccine of Haemophilus paragarinarum - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to Haemophilus haemophilus.
paragallinarum) powdered vaccine production method.

Here, the powdered vaccine means an antigen that inactivates the infectivity of Haemophilus paragallinarum and is dehydrated to be a dry powder.

Haemophilus paragallinarum is known as one of the gram-negative bacteria that is a pathogenic bacterium of chicken infectious coryza, and chickens infected and infected with the bacterium exhibit facial swelling, nasal discharge, and tearing as clinical symptoms, and also the egg laying rate. It is known to cause a decline.

In addition, as a vaccine against this disease, there are two types that are commercially available in a liquid form and a freeze-dried form, and its infection control effect is recognized. The vaccine produced according to the general method here kills pathogenic microorganisms by chemical means such as formalin, or kills them by physical means such as ultraviolet irradiation. There are vaccines and live attenuated vaccines that attenuate pathogenic microorganisms using biological means, and the commercial vaccines for the Haemophilus paragarinarum strains also belong to these.

However, these vaccines
It is difficult to maintain antigenicity for a long period of time because it contains sugar, lipid, etc. Therefore, for long-term storage, conditions such as freeze-drying, freezing, and refrigerating storage are adopted, but the freeze-drying method, which has the best storage effect, requires a large amount of equipment to carry out this. There is a problem that investment is required and power consumption is large.

Under such circumstances, it is desired to provide a simple method for producing a vaccine that has a long antigenic life span.

Another problem that is desired in the production of a vaccine against the above-mentioned pathogenic bacteria is to realize easy transportation and handling of the produced vaccine. Conventional liquid or freeze-dried vaccines are difficult to carry in large quantities or require special transportation methods. In addition, three serotypes of Haemophilus paragallinarum (1962, Page of the United States was formed by plate agglutination reaction). This is because the selection of the use mode of the inoculated vaccine, which should be performed according to the epidemic situation of another), is conventionally restricted in the vaccine.

The present invention is different from the above-mentioned conventional vaccines, a culture solution of Haemophilus paragarinarum bacterium, or a bacterium solution obtained by suspending the above-described bacterium precipitated and separated in a NaCl addition buffer or a physiological saline solution, A step of adding 2 to 6 times as much alcohol or acetone as that of the bacterial solution to collect the formed precipitate, and suspending the collected precipitate again in alcohol or acetone, and then collecting the precipitate and drying it under reduced pressure. A powdered vaccine of Haemophilus paragallinarum is produced by a method comprising the steps of:

The powdered vaccine produced according to the present invention has a characteristic that it can be stored even at room temperature, and not only this but also its usage, it is dissolved in physiological saline or the like and inoculated into chickens, and it is powdered. Therefore, it can be used as a tablet or filled in a capsule.

The present invention will be described in detail below.

In the present invention, the infectious inactivated antigen is suitably produced as a dehydrated powdery substance by sequentially performing the following operations using a liquid containing Haemophilus paragarinarum bacterium as a production material.

(a) Recovery of Bacteria Using Polar Solvent (b) Dehydration and Drying of Bacteria Using Alcohol or Acetone Manufacturing Material As a manufacturing material used in the present invention, a culture solution of hemophilus paragarinarum is used.

When positively separating and removing the media components that are mixed in the vaccine, the media components in the culture fluid are removed by centrifugation, and the obtained bacterial fraction is added to a buffer solution containing NaCl (phosphate. Buffer solution, Tris-hydrochloric acid buffer solution, etc.) or a bacterial solution suspended in physiological saline may be used.

Cultivation of the bacterium is performed according to a general method, and an example of the medium used is as follows.

Medium: 1000 ml Chicken broth 200 ml Casamino acid (Difco) 5 g Sodium chloride 5 g Yeast extract ^* 30 ml Chicken serum ^* 15 ml Purified water Remaining pH: 7.0-7.4 (*: Over-sterilized) Medium Chicken broth is usually prepared using the pectoral muscles of broilers (laying hens are acceptable) as follows.

Extracted overnight at 4 ° C.

2) Boil for 30 minutes 3) Metushirou filter 4) No.2 filter paper 5) Tris (hydroxyl) aminomethane pH 7.2
Corrected to 6) Store at -20 ℃ until use.

Chicken sera, regardless of type, are used that do not have antibodies against Coryza.

The yeast extract is prepared as follows using dry yeast used for bread making, for example.

2) Mix with a mixer 3) Extract at 4 ° C for 2 nights 4) Centrifuge with a cooling centrifuge and collect the supernatant.

5) Correct the pH to 7.6-7.8 with 10% NaOH.

6) Zyturo and Millipore oversterilization 7) Store at -20 ° C until use.

For the recovery of bacteria and inactivation of infectious agents, in the process of recovering bacteria, a polar solvent was added to the bacterial solution as the above-mentioned manufacturing material, and the mixture was stirred and allowed to stand for a predetermined time, causing precipitation and clearly separating the precipitate from the supernatant. Remove the supernatant when possible,
Collect the precipitate.

Examples of the polar solvent include methanol, ethanol, and acetone. Of these, methanol is most typically used. When the bacteria come into contact with the polar solvent, hemophilus
Infectious inactivation of Paragalinarum is achieved at the same time.

It is desirable to cool the polar solvent to be added to the bacterial solution before use. The amount to be added to the bacterial solution is usually 2
The amount is 6 to 6 times, preferably 2 to 3 times.

When a polar solvent is added, the suspension in the bacterial solution causes coagulation and precipitation with a decrease in the dielectric constant.However, if the amount of the polar solvent added is small, the precipitation is not sufficient, but on the contrary, if it is too large. In addition, the obtained precipitate becomes a lump, and sufficient dehydration cannot be achieved in the subsequent dehydration / drying operation.

The precipitate fraction produced by adding the polar solvent is generally recovered by centrifugation, but it may also be recovered by filtration using a paper filter, hollow fiber, membrane filter or the like.

In order to obtain a clear distinction between the precipitate and the supernatant, the solution containing the polar solvent is added for at least 1 hour or more, preferably 2-3.
It is desirable to leave it for about a time.

Dehydration and Drying of Bacteria In dehydration and drying of bacteria, the precipitate fraction of the bacteria recovered as described above is suspended in a specific organic solvent, and the precipitate is recovered and dried under reduced pressure.

As the solvent, a polar solvent (methanol, ethanol, acetone) used in a bacterium recovery operation is used, but it does not have to be the same as that used in the bacterium recovery.

It is desirable that the medium is cooled before use. The amount of the solvent for suspending the bacterial precipitate is not particularly limited, but it is often desirable to set the amount to about half the amount of the bacterial solution used in the production.

Further, the precipitation is generally recovered by centrifugation or filtration. The suspension may be allowed to stand for about 1 hour or longer, preferably 2 to 3 hours, prior to collecting the precipitate.

Drying under reduced pressure evaporates the solvent from the recovered precipitate, and is carried out by storing the recovered precipitate in a vacuum (vacuum) container.

The above-mentioned dehydration / drying operation of the bacteria realizes suitable drying of the vaccine to be produced in combination with the gentle dehydration action by the operation of collecting the bacteria in the preceding stage, but if necessary, the dehydration / drying operation is performed twice or more. It may be repeated.

It was confirmed that the powdered vaccine produced by recovering the above-mentioned bacteria and dehydrated and dried exhibited a suitable infection control effect in a clinical test conducted by inoculating the chicken with the powdered vaccine.

Further, the powdered vaccine can be stably stored antigenically under the condition of not exceeding 22 ° C, and can be stored for a long period of 1 year or more under the optimum condition of 4 ° C or less.

The flow sheet of one embodiment of the present invention is as follows.

* 1 The entire process is performed at 0-4 ° C.

* 2 Medium components other than bacteria are almost removed.

* 3 Phosphate buffer solution (PBS) NaCl 8.0 g KCl 0.2 g Na ₂ HPO ₄ 1.15 g KH ₂ PO ₄ 0.2 g Dissolve the above reagents in distilled water to make 1000 ml, 50 of which
The bacteria are suspended in 0 ml.

* 4 A water-containing precipitate in which approximately 100% of the bacteria are recovered.

* 5 A water-containing precipitate that collects almost 100% of bacteria and contains some medium components other than bacteria.

* 6 After this operation, the same applies to precipitation ^{* 4} and precipitation ^{* 5} .

* 7 Stored in a vacuum desiccator overnight.

* 8 As a dry powder vaccine.

Next, an example of the present invention and a test example of the anti-infective effect thereof will be shown.

(Culture Bacterial Fluid) A type A and C type serotypes of Haemophilus paragallinarum were cultured for 17 hours under the conditions of 37 ° C., aeration and static standing in the liquid medium shown as the above example to prepare a culture medium. did.

The A type used the No. 221 strain distributed by the Veterinary Drug Laboratory, and the C type used the G-1 strain isolated from the diseased chicken at the poultry farm in Gunma prefecture by the Japan Institute of Biological Sciences. .

(Production of Dry Powdered Vaccine) A powdered vaccine was produced according to the procedure of the above-mentioned flow sheet using the above-mentioned cultured bacterial solution as a production material.

The results are shown in Tables 1 and 2. Table 1 shows the case where acetone was used for both the recovery of bacteria and dehydration drying, and Table 2 shows the case where methanol was used for both.

However, in the production, the culture bacterium solution was directly used except for 3 ^* and 13 ^* . For 3 ^* and 13 ^*, a bacterial solution in which a precipitate recovered from the cultured bacterial solution was suspended in a phosphate buffer was used.

Infection control efficacy test of powdered vaccine (Test method) 0.2 mg and 0.8 mg of the manufactured powdered vaccine,
It is dissolved in 0.5 ml of a phosphate buffer solution ^{* 2} (pH 7.2-7.4) containing 2.5 mg of aluminum hydroxide gel ^{* 1} , and this is intramuscularly injected into the thigh of a chicken ^{* 3} . 1 in the 3rd week after injection
~ 2 x 10 ⁸ CFU of Haemophilus paragallinarum
^{* 4} was infected from the nasal cavity of chicken, and the clinical symptoms were observed for 1 week thereafter.

* 1 Aluminum hydroxide gel Aluminum sulfate 149.4 g Ammonia water 85.3 ml Dissolve the above reagents in distilled water to make 1000 ml.

* 2 Phosphate buffer solution (PBS) NaCl 8.0 g KCl 0.2 g Na ₂ HPO ₄ 1.15 g KH ₂ PO ₄ 0.2 g Dissolve the above reagents in distilled water to make 1000 ml.

* 3 SPF (Specific pathogen fiee) Line M strain chicken.

* 4 The strain used for infection is the same as that used for the production of powdered vaccine.

(Determination of effect) The results of clinical symptoms were determined by comparing the clinical symptoms with those of the non-vaccinated group that had the same bacterial infection as controls.

Further, in order to determine the long-term stability of the antigenicity of the powdered vaccine, the above-mentioned test was similarly performed on the powdered vaccine after storage for a certain period.

Since the serotype A strain No. 221 has hemagglutinin (abbreviated as HAEmagglutinin: HA) that aggregates erythrocytes of chickens, the powdery vaccine produced from this strain has the above-mentioned clinical symptoms and HA-blocking antibody. Production of (abbreviated as HI)
It was measured by HI reaction.

These results are shown in Tables 3-1, 3-2 and 4-1, 4 below.
-2, and the powdered vaccine derived from the serotype A No. 221 strain was confirmed to be antigenic in both clinical symptoms and HI value in comparison with the control, and after long-term storage. However, the antigenicity was maintained.

Also, regarding the powdered vaccine derived from the serotype C type G-1 strain, the antigenicity was confirmed in clinical symptoms in comparison with the control, and the antigenicity was maintained even after long-term storage.

According to the present invention, a vaccine of infectious coryza, which causes pathological swelling, nasal discharge, and lacrimation as clinical symptoms and causes a drop in egg production and which causes Haemophilus paragallinarum, is maintained for a long period of time. In addition, it has an effect that it can be provided as a product excellent in handleability and transportability.

Further, according to the method of the present invention, compared to the freeze-drying method, the burden on the equipment is extremely small and the power consumption can be reduced.

 ─────────────────────────────────────────────────── ─── Continuation of front page (56) References JP-A-49-6118 (JP, A) JP-B-40-8152 (JP, B1) Vaccine of animals Kitasato Institute, Ltd. Yokendo P Co., Ltd. ． 220-230 (issued in 1978) P. 11, 12

Claims (1)

[Claims] 1. A culture solution of Haemophilus paragallinarum or a bacterium obtained by precipitating and separating it from NaCl.
A step of adding 2 to 6 times the amount of alcohol or acetone of the bacterial solution to a bacterial solution suspended in an addition buffer or physiological saline to recover a precipitate formed, and again collecting the recovered precipitate with alcohol or acetone. And the step of collecting the precipitate and drying it under reduced pressure, the method for producing a powdered vaccine of Haemophilus paragallinarum.