KR100834026B1 - Riemerella vaccine for preventing riemerella infection, vaccine using the same and method for producing the same - Google Patents

Abstract

A riemerella vaccine for preventing riemerella infection is provided to defend the infection of field pathogenic Riemerella anatipestifer which occurs frequently in a country and prevent ducks from being perished by the riemerella infection, thereby minimizing economical loss of a duck industry. A riemerella vaccine for preventing riemerella infection comprises an inactivated Riemerella anatipestifer and an immuno-enhancing agent, wherein the inactivated Riemerella anatipestifer is a strain selected from the group consisting of serovar type I, serovar type IV, serovar type VII, serovar type XVI and a mixture strain thereof. A method for preparing the riemerella vaccine comprises the steps of: (a) culturing Riemerella anatipestifer isolated from a suck; (b) adding an inactivating agent such as 0.3% formalized physiological saline solution(FPSS) to a culture solution obtained from the step(a) to inactivate the Riemerella anatipestifer; and (c) adding an immuno-enhancing agent to the inactivated Riemerella anatipestifer culture solution to prepare a mixture vaccine. Further, the immuno-enhancing agent is ISA70.

Description

Rimerella vaccine for preventing Riemerella infection, Vaccine using the same and method for producing the same}

1 is a manufacturing process of the inactivated rimerella mixed vaccine according to a preferred embodiment of the present invention.

The present invention relates to a serimella type 1 and 7 isolates in Korea and a rimerella vaccine for the prevention of rimerella infection comprising a cotton enhancer, and a method for producing the same.

Rimerella infection is a rapid and chronic bacterial disease that occurs mainly in ducks 3 to 5 weeks of age, and presents symptoms such as fibrosis pericarditis, mesentitis, cystitis, meningitis, and meningitis, and causes severe weight loss with high mortality. It is known.

Rimerella anatifestifer, a bacterial cause causing the infectionRiemerella anatipestifer) Is a strain belonging to Gram-negative bacillus and is currently in vogue worldwide.

The disease was first reported in the United States in 1932 at the time of the discovery of the pathogen Pasteurella Pepe Ana Tee Festiva flops (Pfeiferrella anatipestifer) as a multiple of bulrida made in 1993, the name currently used rime Relais Ana Tee Festiva flops (Riemerella anatipestifer ).

Currently, the serotypes of a total of 21 species of Rimerella are known throughout the world, and if the serotypes of the causative agent causing the disease and the serotype of the vaccine strain are different from each other, the defense against Rimerella infection is not known. .

Identification of serotypes of isolated strains is carried out using plate agglomeration or agar sedimentation reactions. The four major strains of Rimerella that cause the epidemic of Rimerella infection in Korea are 4, namely serotypes 1, 4 and 7 And 16 were found to be present.

Globally, inactivated Rimerella vaccines are prepared and used using serotypes that are particularly prevalent in each country. In general, a mixed vaccine of Rimerella prepared by mixing two or more serotypes is used. have.

In the United States and Canada, mixed vaccines containing serotypes 1, 2 and 5 have been prepared and vaccinated for 2 to 3 week old ducks. There is no investigation, and there are many difficulties in developing a vaccine using the same.

Rimerella infection is an infectious disease and has been reported to have been isolated from wild birds as well as various species of birds, including ducks. Infection with Rimerella causes severe sepsis in ducks, which is why it is called duck sepsis and may be called slightly differently depending on the findings or symptoms. When the duck is infected by Rimerella bacteria, it progresses acutely or chronically. This results in pathological findings such as fibrosis pericarditis and mesentitis, and specifically causes meningitis and neurological symptoms. In addition, Rimerella infection is recognized as a serious disease in the duck industry all over the world because it causes the above-described lesions to cause a high mortality, weight loss, deterioration, and the like.

In order to prevent Rimerella infection, there is an active development of a vaccine that can prevent its infection. Currently, the United States uses a multivalent bacterium vaccine obtained by mixing serotypes 1, 2, and 5, and identified and inactivated serotypes that are prevalent in countries in countries suffering from Rimerella infection. Later, using this to develop a multivalent vaccine. In Korea, damage to Rimerella infectious diseases continues to appear. Recently, with the rapid development of the duck industry, damage to diseases caused by infection with Rimerella is increasing, but there is no effective way to prevent it. Accordingly, in order to reduce the damage to Rimerella infection in Korea, the development of serotypes and the development of vaccines using them are urgently needed in Korea.

Therefore, the present inventors investigated and identified the serotypes for Rimerella bacteria mainly invented in Korea, and investigated the pathogenicity of the main serotypes. In addition, after inactivating the isolated strain is mixed with an adjuvant to prepare a mixed solution of inactivated Rimerella, using this to prepare a vaccine that can prevent the infection of highly pathogenic Rimerella, the prepared vaccine is highly pathogenic The present invention was completed by confirming that it exhibited efficacy against Rimerella strains.

The present invention has been proposed to solve the problems of the prior art as described above, an object of the present invention is to use the serotype of the Rimerella bacteria which is a bacterial disease that mainly occurs in the country and causes a lot of damage to the duck industry. To provide an activated rimerella combination vaccine.

In order to achieve the above object, the present invention is inactivated Rimerella anatifestifer ( Reemerella) isolated and domestically highly pathogenic Provided is a rimerella vaccine for the prevention of rimerella infection comprising anatipestifer ) and a cotton enhancer.

In the present invention, after investigating the predominantly Rimeella bacteria serotypes in Korea, the isolates are isolated from ducks, and the serotypes of these strains are examined and classified by examining the characteristics of the isolates.

At this time, the Rimerella bacteria used herein include serotypes 1, 4, 7 and 16 of Rimerella and mixed strains thereof, preferably serotypes 1 and 7 of Rimerella and mixed thereof Strains are preferred.

In addition, to achieve the above object, the present invention comprises the steps of (1) culturing the Rimerella bacteria isolated from ducks; (2) inactivating the Rimerella bacteria by adding an inactivating agent to the culture solution obtained in step 1; And (3) adding an immunopotentiator to the inactivated rimerella culture medium to produce an inactivated rimerella mixed vaccine.

 Immunity enhancers used in the rimerella vaccine for preventing rimerella infection according to the present invention include (ISA70), and the inactivating agent includes (0.3% FPSS, formalized physiological saline solution).

Hereinafter, the structure of the present invention will be described in more detail with reference to Examples and Test Examples, but the scope of the present invention is not limited thereto.

Example  One: Rimerella  Isolation and Serotype Investigation

(1) Isolation of Rimerella anatifestifer

In order to isolate and identify Rimerella strains, ducks showing clinical symptoms such as neurological symptoms and weakness were selected from ducks of two or more weeks in an outdoor duck breeding farm, and the selected ducks were autopsied to examine brains, organs, liver, and kidneys. The back organs were removed. After screening clear visual findings from the isolated organs, Rimerella strains were cultured using a sterile cotton swab in a blood medium (5% fibrin-free cotton sheep blood, basic blood agar medium, Difco) and MacConkey agar (MacConkey agar, Each was plated in Difco) and then cultured. At this time, strains that grew in blood medium but not grown in McConkey medium were identified to carry out biochemical and genetic tests to identify the Rimerella anatifestifer strain.

(2) antiserum production

Antisera against 21 known Rimerella anaetifestifer serotype reference strains was prepared using chicken and the method was followed by conventional serum preparation methods. The 21 serotype reference strains were cultured in blood medium for 48 hours at 37 ℃ to obtain a single colony.

The single colony was first cultured in TSBY (Tryptic soy broth, Difco; 3% yeast extract, Difco) for 24 hours at 37 ° C., and then the cultured portion was second cultured for 24 hours at 37 ° C. in fresh TSBY medium. The cultures of the 21 serotype reference strains were centrifuged to obtain individual bacteria, which were suspended in 0.3% FPSS (0.3%, formalized physiological saline solution) and then centrifuged overnight at room temperature for 21 serotypes. Base stocks were each inactivated.

The inactivated base strain was uniformly stirred with ISA70 (Seppic) oil to obtain a weight ratio of 70:30 to obtain a mixed solution, which was inoculated into the thigh muscles of 12-week-old chickens. Three weeks after the inoculation, blood was collected from chickens to separate serum and they were refrigerated until used in the experiment.

(3) Serum type investigation of Rimerella bacteria isolated in Korea

In order to investigate the serotypes of Rimerella bacteria isolated in Korea, plate aggregation reaction was performed at room temperature for 3 minutes to confirm that they were primary Rimerella bacteria, and then serotypes were examined using AGP method.

In the AGP method, it was determined that there was a distinct sedimentation (sedimentation line) between the antigen of domestic isolate and the serotype of Rimerella base strain.

Strains mainly used for identification of anti-Serimella bacteria, antiserum production and serotype determination of isolated strains in Korea are shown in Table 1 below.

ATCC RA strain ATCC 21845 and 3 weeks Reference stock Serotype 1, D-24105 (DRL) to Serotype 21, 1062/91 (NIAHT) Domestic isolated strain RA 32 outdoor isolates

Test Example 1:

The AGP (Agar gel preparation) method used in this test example is as follows. Purely purified 6 g of Agar powder and 48 g of sodium were dissolved in 600 g (600 mL) of distilled water, which was added to the plate and then solidified at room temperature to form an agar plate.

One hole of the agar plate was cut out and six holes were used as a center, and one group was used as a test group, and a plurality of test groups were made in one plate. In the center hole, put the antigen of Rimerella strain isolated from Korea, put the normal serum in one of the six holes around it, and put the serum of different serotypes in the other five places. The reaction was carried out for a time.

After the reaction was completed, a strain having a distinct sedimentation line between the antigen of each of the domestically isolated rimerella strains and each serotype was determined as a serotype that can be used in the present invention. Serum-type investigation of 32 rimerella strains isolated from Korea was carried out as described above, and the results are shown in Table 2.

Serotype 0 Separation bacteria % Separation One 7 22 4 One 3 7 22 69 16 2 6 sum 32 100

As can be seen from the results of Table 2, there are four serotypes of Rimerella bacteria, which are most prevalent in Korea, among which serotype 7 is the most distributed and serotype 1 is the next most distributed. I knew it was.

Example  2: Domestic separation Rimerella  Investigation of pathogenicity of strains Vaccine Caution  Produce

(1) Pathogenicity investigation of serotype 7

In order to investigate the pathogenicity of the ducks of the Rimerella strain belonging to the serotype 7, domestically isolated Rimerella strains were inoculated into ducks of 2 weeks of age, and the inoculation site of the duck was the right femoral muscle.

The primary pathogenicity test was performed to screen for pathogenicity among several strains, and the primary pathogenicity test was performed for the second time to confirm mortality and bacterial isolation rate.

The pathogenicity test was performed by inoculating 0.5 ml of 2.0 x 109 CFU / ml of bacterial count with two-week-old duck thigh muscle as the inoculation site. The pathogenic test method was performed in the same manner as the first and second tests.

(2) Pathogenicity investigation about serotype 1

In order to investigate the pathogenicity of ducks of the Rimerella strain belonging to serotype 1, domestically isolated Rimerella strains were inoculated into ducks of 2 weeks old, and the inoculation site of the duck was the right femoral muscle. The test method was performed in the same manner as serotype 7 to confirm the pathogenicity.

(3) Pathogenicity investigation about mixed inoculation of serotype 7 and type 1

In order to investigate the pathogenicity of ducks of the mixed strain of Rimerella of serotype 7 and serotype 1, the strains of serotype 7 and type 1 which were found to be highly pathogenic, respectively, were inoculated into 2 week old ducks, respectively. The number of bacteria was 1.0 x 109 CFU / ml, mixed at the same dose, and inoculated 0.5 m at each right thigh muscle area. The following pathogenicity test method was performed in the same manner as described above to confirm the pathogenicity of the mixed strain of the two serotypes.

Pathogenicity of major isolates by serotype Serotype Isolated strain Number of deaths / test (mortality rate,%) Bacterial Separation / Test Water (Separation Rate,%) 7 05Q047 11/10 (100) 11/10 (100) One FS1124-44 11/10 (100) 11/10 (100) 1 + 7 05Q047 / FS1124-44 11/10 (100) 11/10 (100) Conditions: Inoculation age: 2 weeks, number of inoculation strains: 2.0 × 10 ⁹ CFU / ml Inoculation site: muscle of the thigh, inoculation amount: 0.5 ml

As can be seen in Table 3, all of the ducks inoculated with serotype 7 strains, serotype 1 strains and serotype 7 and type 1 mixed strains were found to exhibit 100% lethality (pathogenicity). The strain was isolated from all dead ducks. These pathogenic strains were selected as vaccine candidate strains.

(4) Preparation of vaccine strain

Rimerella strains selected as candidate vaccine strains were cultured in blood medium (5% fibrin depleted sheep blood, basal blood agar medium, Difco) for 48 hours at 37 ℃ to obtain a Rimerella strain having a single colony, which is a yeast extract After shaking overnight in TSB (tryptic soy broth) containing the number of strains was measured.

Cells were harvested by centrifugation of appropriately cultured Rimerella strains, suspended in sterile saline solution containing 0.3% formalin, centrifuged and resuspended, and then inactivated with shaking at room temperature overnight.

A portion of the Rimerella strain culture was incubated overnight in a blood medium to confirm that the Rimerella strain was inactivated, and no colonies were formed in the Rimerella strain culture. Inactivated Rimerella strains were homogeneously mixed with ISA70 oil and then used as vaccine strains.

Test Example  1: oil and Vaccine Caution  safety

To ascertain the safety of ducks of the Rimerella vaccine and oil prepared in Example 2, the inoculated subcutaneous and femoral muscles of the duck neck at 2 weeks of age were inoculated into the duck neck and 6 weeks of age. Until then, the inflammatory response at the inoculation site of the duck and the residual oil were confirmed.

The material used was 0.3% FPSS, oil and oil-limerella vaccine strains were inoculated subcutaneously and muscle respectively (see Table 4).

Safety test on the site of inoculation of oil and manufactured beet Experimental group Observation time Inoculation method Population with clinical symptoms / inoculation population 0.3% FPSS 4 SC 0/10 (0) IM 0/10 (0) 0.3% FPSS 4 SC 0/15 (0) IM 0/15 (0) Vaccine TQ (0.3% FPSS + Oil + 2.0 × 2.0 CFU / mL) 4 SC 0/45 (0) IM 0/45 (0)

As can be seen in Table 4, no clinical symptoms were observed at the inoculation site of the oil-based vaccine strain, and no lesions were observed at the inoculation site at the time of necropsy. Therefore, it was judged that the vaccine strain which added this oil is stable.

Example  3: In bacteria  According Vaccine Caution Defense  Research

(1) Protective ability test according to the number of bacteria of the manufactured vaccine

In order to investigate the defense ability according to strain numbers contained in the vaccine strain, serotype 1 and serotype 7 were mixed to prepare vaccines having various bacterial counts. Two-week-old ducks were inoculated with the vaccine prepared above, and were observed for one week after challenge inoculation with an outdoor strain of 2.0 × 10 9 CFU / ml at 4 weeks of age. At this time, the inoculation site was the right femoral muscle and the inoculation amount was 0.5 ml per horse (see Table 5).

Strain Protection of Inoculated Vaccines Vaccine Attack strain Vaccine strain (CFU / ml) Mortality Rate (Number of Deaths / Tests,%)  1 + 7 type   Type 1 2.0 × 10 ⁸ 0/10 (0) 2.0 × 10 ⁷ 10/10 (100) 2.0 × 10 ⁶ 10/10 (100) 2.0 × 10 ⁵ 10/10 (100) Control Vaccine Unvaccinated 5/5 (100)  1 + 7 type   7 inch 2.0 × 10 ⁸ 0/10 (0) 2.0 × 10 ⁷ 5/10 (50) 2.0 × 10 ⁶ 90/10 (90) 2.0 × 10 ⁵ 10/10 (100) Control Vaccine Unvaccinated 5/5 (100)

As can be seen in Table 5, when the number of bacteria contained in the vaccine is more than 1.0 x 108 CFU / ml per horse was confirmed to exhibit a 100% protective capacity.

(2) Protective ability test according to age of vaccine

After 3 weeks of inoculation into 2 weeks of ducks inoculated with 2 ml of duck, 6 weeks at intervals of 1 week of challenge were inoculated with the pathogenic rimerella strains to examine the protective ability by age after inoculation.

At this time, the inoculation site was the right femoral muscle and the inoculation amount was 0.5 ml per horse (see Table 6).

Investigation of the protective ability against Rimerella strains according to the age of the prepared vaccine Attack age Attack strain (CFU / mL) % Mortality Non-vaccinated group (dead number / test number) Vaccination group (the number of the dead / tests) 3 weeks old Type 1 (2.0 × 10 ⁹ ) 10/10 (100) 0/10 (0) 7 type (2.0 × 10 ⁹ ) 10/10 (100) 0/10 (0) 4 weeks old Type 1 (2.0 × 10 ⁹ ) 10/10 (100) 0/10 (0) 7 type (2.0 × 10 ⁹ ) 9/10 (90) 0/10 (0) 5 weeks old Type 1 (2.0 × 10 ⁹ ) 8/10 (80) 0/10 (0) 7 type (2.0 × 10 ⁹ ) 9/10 (90) 0/10 (0) 6 weeks old Type 1 (2.0 × 10 ⁹ ) 7/10 (70) 0/10 (0) 7 type (2.0 × 10 ⁹ ) 9/10 (90) 0/10 (0)

As can be seen in Table 6, the vaccinated control group had protection until the 6-week-old time of inoculation when the vaccine was inoculated to two-week-old ducks in both serotype 7 and serotype 1 It was confirmed.

As described above, it has been confirmed that the rimerella vaccine for preventing the rimerella infection according to the present invention can effectively protect against the infection of outdoor pathogenic rimerella, which occurs mainly in Korea. Therefore, the rimerella vaccine for preventing the remerella infection according to the present invention can prevent the death of duck due to the remerella infection, thereby minimizing the economic damage in the current duck industry.

Claims (8)

Including inactivated rimerella and immunopotentiators, The inactivated Rimerella bacteria are selected from the group consisting of serotype 1, serotype 4, serotype 7, serotype 16, and mixed strains thereof. vaccine. delete According to claim 1, wherein the Rimerella bacterium is one of serotype 1, serotype 7 or mixed strains of serotype 1 and serotype 7 type Rimerella vaccine for preventing infection of Rimerella. According to claim 1, wherein the immunostimulating agent Rimerella vaccine for preventing the remerella infection, characterized in that consisting of ISA70. (1) culturing the Rimerella bacteria isolated from the duck; (2) inactivating the Rimerella bacteria by adding an inactivating agent to the culture solution obtained in step 1; And (3) adding an immunopotentiator to the inactivated Rimerella culture medium to prepare an inactivated Rimerella bacteria mixed vaccine; Said Rimerella bacteria serotype 1, serotype 7 or serotype 1 and serotype 7 a method of producing a mixed vaccine of any one of the characterized in that the mixed strain of serotype 7. delete 6. The method of claim 5, wherein the immunopotentiator is composed of ISA70. 6. The method of claim 5, wherein the inactivating agent is made of 0.3% formalized physiological saline solution (FPSS).

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