CN102614511B - Propolis-adjuvant inactivated vaccine for mink pasteurellosis - Google Patents

Abstract

The invention provides a propolis-adjuvant inactivated vaccine for mink pasteurellosis and a method for preparing the propolis-adjuvant inactivated vaccine. The method includes: vaccinating mink original pasteurella onto a blood agar slant, culturing for 24 hours at 37 DEG C, checking purity, using as seed pasteurella liquid vaccinated blood agar matrix, inactivating to obtain an aqueous phase to be fully mixed with propolis adjuvant to obtain the propolis-adjuvant inactivated vaccine for mink pasteurellosis. The propolis-adjuvant inactivated vaccine can effectively prevent mink pasteurellosis, and adverse reactions after vaccination and influences on fur quality can be evidently reduced to create conditions for control of fur-bearing animal diseases.

Description

Propolis inactivated vaccine for mink pasteurellosis

technical field

The invention relates to an inactivated mink pasteurellosis vaccine with propolis as an adjuvant and a preparation method thereof, belonging to the technical field of veterinary biological products.

Background technique

Pasteurellosis is mainly a bacterial hemorrhagic and septic infectious disease of livestock, poultry, wild animals and fur animals caused by Pasteurella multocida and Pasteurella hemolyticus. The disease is widely distributed and occurs all over the world. Pasteurella in mink is also prevalent in recent years. It has a high morbidity rate and a high mortality rate. It is very harmful to the fur animal breeding industry and often causes serious economic losses.

At present, the main treatment for mink pasteurellosis in my country is antibiotics, but there are certain defects in the drug treatment. Once the drug is stopped, it is easy to relapse, and it is easy to produce drug resistance. An effective measure to prevent the occurrence of the disease is to develop a corresponding vaccine.

In order to enhance the immunogenicity of vaccines, immune adjuvants are often added in the development of inactivated bacterial vaccines. Immune adjuvant is a kind of immunomodulator, which can be injected into animals before antigen or mixed with antigen or at the same time, which can specifically enhance the immunogenicity of antigen and improve the immune effect. Immune adjuvants can enhance the antibody response stimulated by the antigen, improve the immunogenicity, and enhance the non-specific killing function of phagocytes and the stimulation of specific cellular immunity. Therefore, adjuvants are essential components of vaccines, especially inactivated vaccines. The advantages of propolis as a biologically active immune adjuvant have been gradually recognized. Propolis has broad-spectrum biological activity and is a good immune enhancer and stimulator. It can not only cause specific immune response, but also start non-specific defense mechanism, and has obvious effects of enhancing immune antibody production and improving white blood cell phagocytosis , After using this adjuvant, it can effectively protect the quality of fur.

Contents of the invention

The object of the present invention is to provide a kind of preparation method of the mink source Pasteurellosis inactivated vaccine that uses propolis as adjuvant, so that the side effects after immunizing the mink are small, the influence on fur quality is small, the immune generation time is early, and the immunity is strong. The immunity period is long.

Specific implementation steps:

1. Preparation of inactivated stock solution of Pasteurella derived from mink.

1) Pasteurella multocida from mink was inoculated on the blood agar slant, cultured at 37°C for 24 hours, and it was tested to be pure and used as a strain;

2) Inoculate the blood agar slant with an appropriate amount of bacterial culture, culture it at 37°C for 18-24 hours, and collect the culture for inspection;

3) Take the cultured bacteria solution and add formaldehyde solution to make the final concentration of formaldehyde to 0.4%, inactivate on a shaker at 37°C for 24 hours.

2. Preparation of propolis solution: Select propolis of known purity, crush it, sieve it, dissolve it in 95% ethanol solution, and then prepare a solution of appropriate concentration according to requirements and store it at 4°C for later use.

3. Preparation of finished vaccine: Add propolis solution and stir for 15-20 minutes to make it fully mixed and emulsified according to the standard that the dry matter content of propolis per ml of vaccine is 8-15mg.

4. As a preferred embodiment of the preparation method of mink pasteurellosis propolis inactivated seedlings of the present invention, in step 1 and 3), after the inactivation, the Pasteurella culture solution can also be subjected to inactivation test and toxin test. The role of inactivation test and toxin test is to control the quality of vaccines in the manufacturing process.

The Pasteurella of the present invention is a strain of Pasteurella multocida C52-17 purchased from the strain room of the China Veterinary Drug Control Institute as a strain for producing vaccines, serotype A: 1, lyophilized on April 2008, and valid for 10 years.

Detailed ways

Example 1:

Vaccine preparation: this product is to select a strain of Pasteurella multocida C52-17 purchased by the inventor from the bacterial strain room of China Veterinary Drug Supervision Institute as the bacterial strain for producing the vaccine. After resuscitation, the blood agar slant medium was inoculated for 18 hours. After checking for no miscellaneous bacteria, use it as a seed liquid, inoculate the blood agar slant with an appropriate amount of bacterial culture, and incubate at 37°C for 18-24 hours, take the cultured bacteria liquid and add formaldehyde solution to make the final concentration of formaldehyde 0.4%, at 37°C Shaker inactivation for 24 hours.

Vaccine inactivation test: Take 100 microliters of formaldehyde-inactivated bacterial solution and put it in 3ml liquid medium, take three samples for one culture, and inoculate each sample in three test tubes for five days for inactivation safety inspection. If the inoculated liquid culture medium is still clear and free of turbidity, the safety inspection is qualified.

Preparation of finished vaccine: add propolis solution and stir for 15-20 minutes according to the standard that the dry matter content of propolis per ml of vaccine is 8-15 mg, to make it fully mixed and emulsified.

Example 2

Three batches of vaccines prepared in Example 1 were injected into healthy minks in single dose, single dose repetition and overdose, and inoculated into the muscles of the hind limbs. After 14 days of observation, the body temperature and appetite of the inoculated minks were normal, there was no swelling and inflammation at the inoculation site, and no local and systemic reactions occurred: 3 animals were selected for each batch of vaccine, and the skin at the injection site was cut to observe the pathological changes of the injection site after necrosis. There was no abnormal change, and the muscle tissue at the inoculation site of the inner muscle of the hind limb was normal, only the injection site was dark brown, the size of a corn kernel, which was different from the surrounding reddish-brown muscle, and there was no inflammatory reaction at the injection site. The 60-70-day-old mink continued to be observed until 30 days after inoculation, and the mink developed well. It shows that the vaccine has good safety for mink of different ages.

Example 3

Three batches of mink pasteurellosis propolis inactivated vaccines were used to immunize 20 mice of 16-18g respectively, 10 mice in the experimental group and 10 mice in the control group, 0.2mL/mouse, and 21 days after the immunization, 20 mice in the control group were treated with Pasteurella After challenge and observation for 7 days, 80% of the mice in the three batches of vaccine-immunized groups were protected, and 100% of the mice in the control group were infected.

Three batches of mink pasteurellosis propolis inactivated vaccine were used to immunize healthy minks aged 2-3 months respectively. Each batch of vaccine was inoculated with 10 minks, 1mL/mink. After 21 days after immunization, 10 minks in the control group were passed with Pasteurella. Intratracheal instillation of challenge virus (5 rats in each group), observation for 7 days, 80% of the minks in the three batches of vaccine immunized groups were protected, and 100% of the minks in the control group were infected.

Example 4

Three batches of propolis inactivated vaccines were used to determine the immune protection period. Divided into experimental group and matched group, experimental group six groups, every group of 30 minks, with three batches of propolis inactivated vaccine immunization of the present invention, each batch of vaccine immunization 10 mink, every hind limb inner muscle injection 1mL; control group Divided into six groups, 10 minks in each group, intramuscular injection of 1mL of normal saline in the hind limbs, 1-6 months after immunization, challenged with the isolated strain of Pasteurella every month, and determined the protection of the vaccine against the strain . Experimental results show that the propolis inactivated vaccine of the present invention has a protection period of 6 months against Pasteurella mink, and the immune protection rate is above 80%.

Claims (1)

1. a preparation method for mink Bacillus pasteurii disease inactivated propolis vaccines, comprises the steps:

Prepared by vaccine: be selected to a strain pasteurella multocida C52-17 of China Veterinery Drug Inspection Office's strain room purchase as the bacterial strain producing vaccine, inoculation blood agar slant medium 18 hours after recovery, microscopy without after miscellaneous bacteria as seed liquor, 18-24 hour is cultivated with appropriate microbial strain culture inoculation 37 DEG C, blood agar inclined-plane, get cultivation bacterium liquid and add formalin, its ultimate density containing formaldehyde is made to be 0.4%, 37 DEG C of shaking table deactivations 24 hours;

Vaccine inactivation is checked: the bacterium liquid after formalin-inactivated is got 100 microlitres and is connected in 3ml fluid medium, once cultivate and get three samples, each sample is inoculated in three test tubes to observe and carries out deactivation safety verification in five days, and the fluid medium inoculated afterwards for five days still clarifies without muddiness that then safety verification is qualified;

Finished product vaccine prepare: by every ml vaccine propolis-containing dry matter content be 8-15mg standard add propolis solution stir 15-20 minute, make its abundant mixing and emulsifying;

Adopt three batches of inactivated propolis vaccines to carry out immune period mensuration: to be divided into experimental group and matched group, experiment component six groups, often organize 30 minks, with described three batches of inactivated propolis vaccines immunity, often criticize vaccine immunity 10 minks, intramuscular injection 1mL inside every hind leg; Matched group is divided into six groups; often organize 10 minks; hind leg internal memory intramuscular injection normal saline is to 1mL; 1-6 after immunity month; monthly use pasteurellosis bacillus isolated strain counteracting toxic substances; measure vaccine to the protection of strain, described inactivated propolis vaccines is 6 months to the protection period of mink pasteurellosis bacillus, and immune protective rate is more than 80%.