CN101342367A - A live attenuated vaccine against Edwardsiella tarda - Google Patents

Abstract

The invention is a technology for preventing and controlling aquaculture animal diseases, and relates to attenuated vaccines for aquaculture bacterial diseases, in particular to a live attenuated vaccine for Edwardsiella tarda. The esrB gene deletion attenuated mutant strain of Edwardsiella tarda strain LSE40, the mutant strain of Edwardsiella tarda is MZLSE40esrB, which is preserved in the General Microorganism Center CGMCC of the China Committee for the Collection of Microbial Cultures, and the preservation number is: CGMCC No.2087, The mutant strain MZLSE40esrB does not contain exogenous antibiotic selection markers and exogenous gene fragments. Compared with the wild-type Edwardsiella lentus, the attenuated vaccine of the present invention has obvious low toxicity and immune protection rate, and does not contain any exogenous antibiotic resistance markers and exogenous gene fragments. Tests have proved that the live attenuated vaccine of the invention can effectively protect susceptible fish from infection by pathogenic Edwardsiella tarda.

Description

A live attenuated vaccine against Edwardsiella tarda

technical field

The invention relates to aquaculture animal disease prevention technology, and relates to attenuated vaccines for aquaculture bacterial diseases, in particular to a live attenuated vaccine for Edwardsiella tarda.

Background technique

Edwardsiella tarda is a Gram-negative pathogen that infects a wide range of hosts and can infect a variety of lower and higher animals. In aquaculture animals, the bacterium is an important pathogenic bacterium of fish, amphibians and reptiles. The Edwardsiella disease caused by it is a common infectious disease in aquaculture fish, which is very harmful to the health of farmed animals and the aquaculture industry. In my country, pathogenic Edwardsiella has been isolated from various animals such as bullfrog, soft-shelled turtle, eel, flounder, turbot, and red sea bream.

Due to the use of various antibiotics for disease prevention and control in the production process, more and more drug-resistant strains of Edwardsiella have been isolated from all over the world, most of which are multi-drug resistant, which not only reduces the therapeutic effect, but also multi-resistant The existence of drug-resistant strains and drug-resistant plasmids increases the pressure on the environment and poses a serious threat to the environment. As early as 1981, the world "Alliance for Antibiotic Use with Caution" was established internationally, and its member countries have reached more than 90 countries. At present, many countries have banned the use of antibiotic additives in animal feed. After my country's accession to the WTO, it is urgent to solve the problems of antibiotic residues. In order to reduce the impact of antibiotics on the environment, it is undoubtedly the best way to prevent and treat Edward's disease through immunization. Much work has been done on various vaccine compositions (including inactivated somatic cell vaccines, lipopolysaccharide LPS, lipoid A-depleted polysaccharide, hemolysin) and routes of immunization (including injection, immersion, oral administration) of Edwardsiella tarda , but the protective effects of these inactivated vaccines or subunit vaccines are not satisfactory, and have not met the requirements for disease prevention and control.

The virulence factors of Edwardsiella tarda include hemolysin, chondroitinase, subcutaneous necrosis toxin, catalase, and type III secretion system, which are involved in bacterial adhesion, colonization, invasion, and survival in animal hosts. process of reproduction and dispersal. Edwardsiella tarda can invade the epithelial cells and phagocytes of the host, and grow and multiply in the cells. Therefore, as an effective vaccine, only by systematically stimulating the animal's humoral and cellular immune responses can the prevention and treatment of Edwardsiella disease be achieved. Inactivated vaccines or subunit vaccines have incomplete protective antigens and incomplete immune responses, especially they mainly cause humoral immune responses in animals and cannot effectively stimulate cellular immune responses, which is the root cause of unsatisfactory disease prevention and control effects; Secondly, inactivated vaccines and subunit vaccines are mainly used to immunize fish by injection, which has side effects such as high labor costs, inconvenient administration, and easy to cause animal stress. So far, no vaccine for Edwardsiella tarda is available on the market.

The attenuated live vaccine has low toxicity to animals, and can effectively stimulate the humoral and cellular immune responses of animals, and provide comprehensive immune protection for animals. It is one of the main development directions in the field of vaccine development at present; The development of multivalent vaccine based on the carrier of source antigen is the frontier and hot spot in the world. At present, the U.S. patent has a method of using rifampicin (rifampicin) medium to carry out multiple subcultures of Edwardsiella tarda to screen attenuated strains as attenuated live vaccines (United States Patent 7067122). The genetic background of the attenuated strains obtained by this method is unclear. The mechanism of bacterial virulence weakening is not clear, and it is easy to cause virulence recovery during use, and it is difficult to monitor the environment of vaccine strains. Attenuated vaccines constructed using transposon or gene insertion inactivation technology carry antibiotics and other foreign gene fragments, which are easy to release antibiotics and foreign gene fragments to the environment when administered, and there is gene transfer or integration into other pathogens or other organisms potential danger. Therefore, it is difficult for these attenuated live vaccines to pass the approval regulations of the biological product safety inspection and management agencies of various countries, so it is difficult to realize commercialization.

Marker-free gene mutation technology can perform deletion mutation on the target gene. Through the deletion of large fragments of multiple target genes, the risk of environmental transmission caused by virulence recovery during administration is greatly reduced. At the same time, the attenuated strain does not carry any exogenous antibiotic screening markers and exogenous gene fragments. The genetic background is clear and the mechanism of virulence attenuation is clear. It is easy to distinguish vaccine strains from wild-type strains, which is convenient for environmental monitoring of vaccine strains and improves the safety of vaccines. and controllability. At the same time, using markerless gene mutation attenuated mutants as vaccine carriers to express heterologous antigens to prepare multivalent vaccines has the potential to prevent and treat a variety of infectious diseases, so it is currently an international research frontier and hot spot. At present, there are no literature reports on the development of attenuated live vaccines against Edwardsiella tarda using marker-free gene mutation technology at home and abroad.

Contents of the invention

The purpose of the present invention is to provide a live attenuated Edwardia tarda vaccine without exogenous antibiotic resistance markers and exogenous gene fragments.

In order to realize the purpose of the above invention, the technical scheme adopted in the present invention is: the esrB gene deletion attenuated mutant strain of the Edwardsiella tarda strain wild-type strain LSE40, and the Edwardsiella tarda mutant strain is MZLSE40esrB, which was released in June 2007 On the 13th, it was preserved in CGMCC, General Microbiology Center of China Committee for Culture Collection of Microorganisms. The preservation number is: CGMCC No.2087, and the classification name is Edwardsiella tarda. Its mutant strain MZLSE40esrB does not contain exogenous antibiotic selection markers and exogenous antibiotics. Gene fragment.

Attenuated live vaccine preparation: bacterial cells containing attenuated Edwardsiella tarda MZLSE40esrB at a concentration of 10 ⁶ -10 ⁹ cfu/mL and PBS phosphate buffer.

The Edwardsiella tarda strain LSE40 strain of the present invention is a wild-type strain, which is isolated from a turbot fish in which Edward's disease broke out in seawater fish farms in the Yellow Sea of my country, and is a pathogenic strain. Its biochemical identification characteristics conform to Bergey's Bacterial System Identification Manual (Ninth Edition). In the method of the present invention, tryptone medium can be selected as the medium for cultivating the attenuated vaccine strain.

The beneficial effect that the present invention has

Compared with the wild-type strain LSE40, the attenuated Edwardsiella lentus vaccine strain MZLSE40esrB provided by the present invention has significantly lower toxicity. The vaccine prepared with the attenuated strain can not only stimulate fish to produce humoral immunity, but also can potentially stimulate fish to produce cellular immunity, and effectively protect test fish from infection by strong pathogenic Edwardsiella wild-type strain. The attenuated vaccine has a wide range of applications and can be used for the prevention and treatment of Edwardsiella in various vertebrates, including various economical marine and freshwater cultured fish, ornamental fish, soft-shelled turtles, frogs and the like.

The Edwardsiella tarda attenuated vaccine provided by the invention does not contain any exogenous gene fragments and exogenous antibiotic selection markers, and there is no risk of exogenous gene transfer and antibiotic gene transmission to the environment. Due to the deletion of large fragments of virulence genes by means of gene deletion mutation, it is theoretically believed that the virulence of the attenuated vaccine cannot be restored, which greatly eliminates the risk of spreading a large number of pathogens to the environment; the genetic background of the mutation is clear, and the attenuation mechanism is clear. It is easy to distinguish vaccine strains from wild-type strains, facilitate environmental monitoring of vaccine strains, improve the safety and controllability of vaccines, and obtain effective attenuated strains of Edwardsiella tarda without marker gene mutations, which can be used to control Edwardsiella in aquaculture animals sick. The above characteristics provide support and guarantee for the commercial development of the unmarked attenuated live vaccine of Edwardsiella tarda.

Detailed ways

Embodiment 1 attenuated live vaccine preparation:

1) TSB medium: tryptone 17.0g/L, soybean protein 3.0g/L, NaCl 5.0g/L, K ₂ HPO 42.5g/L, glucose 2.5g/L, pH 7.3.

Dissolve the above ingredients in 1000ml of distilled water, adjust the pH with 10% NaOH, sterilize at 121 lbs, 125°C for 15 minutes, cool down at room temperature for later use.

2) TSA medium: tryptone 17.0g/L, soybean protein 3.0g/L, NaCl 5.0g/L, K ₂ HPO 42.5g/L, glucose 2.5g/L, agar powder 1.5g/L, pH 7.3.

Dissolve the above components in 1000ml of distilled water, adjust the pH with 10% NaOH, sterilize at 121 pounds, 125°C for 15 minutes, cool to 60°C to make a plate for use.

3) PBS buffer solution: Na ₂ HPO ₄ ·2H ₂ O 2.74g/L, NaH ₂ PO ₄ ·H ₂ O 0.63g/L, the components were dissolved in 1000ml of distilled water, sterilized at 121 lbs, 125°C for 15 minutes, cool at room temperature for later use.

Vaccine preparation: The seeds of MZLSE40esrB attenuated vaccine strain stored at -80°C with the preservation number: CGMCC No. 2087 were streaked and inoculated on a TSA plate, and incubated at 25-30°C for 48 hours. Pick 3-5 colonies, inoculate them in 100mL of TSB medium in step 1), and culture them on a shaker (200 rpm) at 25-30°C for 12-18 hours, and take 5-10% (V/V) Inoculate 500mL of TSB medium in an appropriate amount, and cultivate on a shaker (200 rpm) at 25-30°C for 12-18 hours. Collect live bacterial liquid, wash with PBS three times, collect bacterial cells by centrifugation, the centrifugation condition is 4000×g, 10 minutes, 4°C, and then dilute with PBS to a bacterial suspension with a concentration of 10 ⁵ -10 ⁹ cfu/mL, Stored at 4°C, the Edwardsiella tarda attenuated live vaccine is ready for use.

Embodiment 2 takes turbot (Scophthalmus maximus) as the mensuration of the median lethal dose LD ₅₀ of experimental animals:

Healthy test fish with a body length of 8-10cm are temporarily raised in a 1000L aquarium, equipped with a flowing circulating water treatment and purification system, and the breeding water temperature is maintained at 16-18°C. After 4 weeks of temporary breeding, they were randomly divided into 20L experimental aquariums, and each group was set up with two parallel experimental aquariums, with 10 fish in each aquarium, and continued to be temporarily raised for 2 weeks. In the test aquarium, the water was changed once a day in the morning and afternoon, the amount of water changed was 1/2, and the water temperature was maintained at 16-18°C.

During the artificial infection test, each group of experimental fish was injected with the attenuated vaccine strain and the wild-type strain obtained in Example 1 with different dilution concentrations of 10 ² -10 ⁹ cfu/mL, and each fish was intramuscularly injected with 0.1 mL, and observed for 14 days. Record the number of dead fish per day in each group, calculate the cumulative number of deaths and mortality, and calculate the LD ₅₀ value (see Table 1).

Table 1 Pathogenicity test of wild-type and attenuated strains of Edwardsiella tarda to turbot

The above results indicated that the attenuated strain of Edwardsiella tarda had obvious attenuation effect compared with the wild-type strain, and the LD ₅₀ of turbot was increased by 3 orders of magnitude.

Embodiment 3 takes flounder (Paralichthys olivaceus) as the mensuration of the median lethal dose LD ₅₀ of experimental animals:

Healthy test fish with a body length of 8-10cm are temporarily kept in a 1000L aquarium, equipped with a flowing circulating water treatment and purification system, and the temperature of the breeding water is maintained at 18-20°C. After 4 weeks of temporary breeding, they were randomly divided into 20L experimental aquariums, and each group was set up with two parallel experimental aquariums, with 10 fish in each aquarium, and continued to be temporarily raised for 2 weeks. In the test aquarium, the water was changed once a day in the morning and afternoon, the amount of water change was 1/2, and the water temperature was maintained at 18-20°C.

During the artificial infection test, each group of experimental fish was injected with the attenuated vaccine strain and the wild-type strain obtained in Example 1 with different dilution concentrations of 10 ² -10 ⁹ cfu/mL, and each fish was intramuscularly injected with 0.1 mL, and observed for 14 days. Record the number of dead fish per day in each group, calculate the cumulative number of deaths and mortality, and calculate the LD ₅₀ value (see Table 2).

Table 2 Pathogenicity test of wild-type and attenuated strains of Edwardsiella tarda to flounder fish

The above results indicated that the attenuated strain of Edwardsiella tarda had a significant attenuation effect compared with the wild-type strain, and the LD ₅₀ of flounder was reduced by about 3 orders of magnitude.

Embodiment 4 takes turbot as the injection immunization dose test of experimental animal:

The healthy test fish were randomly divided into 4 groups, 30 fish in each group, and two parallel groups were set up in each test group. The prepared attenuated vaccine was immunized by abdominal intramuscular injection, and the attenuated vaccine strain obtained in Example 1 with concentrations of 10 ³ cfu/mL, 10 ⁵ cfu/mL, and 10 ⁷ cfu/mL were injected respectively, 0.1 mL per tail. The fish in the control group were injected with 0.1 mL of PBS. After 4 weeks of immunization, the turbot in various experimental groups were challenged with live wild-type Edwardsiella tarda LSE40 at a dose of 3×10 ⁶ cfu/tail. The number of deaths in the immunization group and the control group was counted within 7 days, and the average value of the two parallel groups was taken to calculate the immune protection rate (see Table 3).

Calculate the immune protection rate according to the following formula: immune protection rate (RPS)%=(control group death rate-immune group death rate)/control group death rate×100%

Table 3 The immune protection rate of turbot 4 weeks after immunization with different immunization doses of Edwardsiella tarda attenuated vaccine

It can be seen from the above that as the inoculated dose increases, the immune protection rate of the attenuated vaccine also increases. The inoculation dose range from 10 ⁵ cfu/tail to 10 ⁷ cfu/tail showed a good effect on preventing and controlling the infection of wild-type Edwardsiella tarda.

Embodiment 5 takes turbot as the immersion immune efficacy test of experimental animal:

Healthy test fish were randomly divided into groups, 30 fish in each test group, and two parallel sets were set up in each group, soaked in concentrations of 10 ⁶ cfu/mL, 10 ⁷ cfu/mL, and 10 ⁸ cfu/mL for 30 minutes, and the control group was treated with no bacteria soaked in TSB for 30 minutes as a control. After 30 days of immunization, the fish in each test group were challenged with 0.1 mL intraperitoneally, 10 ⁷ cfu/tail. The number of deaths in the immunization group and the control group was counted within 7 days, and the average value of the two parallel groups was taken to calculate the immune protection rate (see Table 4).

Table 4 Immune protection effect of turbot 30 days after immunization with Edwardsiella tarda attenuated vaccine

It can be seen from the above that as the concentration of soaking inoculum increases, the immune protection rate of the attenuated vaccine also increases, and the immune protection effect obtained with the soaking inoculation concentration of 10 ⁸ cfu/mL is the best.

Claims (2)

1. Edwardsiella tarda attenuated vaccine, it is characterized in that: the weak malicious mutant of the esrB gene delection of Edwardsiella tarda strain LSE40, described Edwardsiella tarda mutant is MZLSE40esrB, it is stored in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, deposit number is: CGMCC No.2087 does not contain external source antibiotic-screening labelling and exogenous genetic fragment among its mutant MZLSE40esrB.

2. an Edwardsiella tarda attenuated live vaccine preparation is characterized in that: contain 10 ⁶-10 ⁹Bacterial cell and the phosphate buffer of the Edwardsiella tarda attenuated strain MZLSE40esrB of cfu/mL.