CN103463632B - The preparation method of the D type bacillus perfringens toxoid vaccine of cattle and sheep enterotoxemia - Google Patents

Abstract

The invention relates to a preparation method of a type D Clostridium perfringens toxoid vaccine for cattle and sheep enterotoxemia. Prepare toxoid solution by inactivation; emulsify toxoid solution with white oil adjuvant; when used, intramuscular injection to cattle and sheep, immunization once every six months, 4ml each time, can effectively prevent the occurrence of enterotoxemia in cattle and sheep . According to the pathogenic mechanism of the disease, the present invention develops and prepares a D-type Clostridium perfringens toxoid vaccine with full potency, strong immunity, good pertinence, and no side effects, which is used for D-type perfringens of animals The prevention of clostridial disease, control the prevalence of D-type Clostridium perfringens.

Description

Preparation method of Clostridium perfringens type D toxoid vaccine for cattle and sheep enterotoxemia

(1) Technical field

The invention relates to a preparation method of a type D Clostridium perfringens toxoid vaccine for cattle and sheep enterotoxemia.

(2) Background of the invention

Clostridium perfringens is a normal flora of the digestive tract of humans and animals, widely distributed in nature, the bacteria can produce a variety of exotoxins, up to 12 kinds of exotoxins have been found, of which α, β, ε and ι It is the main lethal toxin and also a typing toxin. According to these four toxins, Clostridium perfringens is divided into five toxin types: A, B, C, D and E. Each toxin type can infect humans or livestock cause specific diseases. Type D bacteria mainly produce α and ε exotoxins, which can cause enterotoxemia in lambs, sheep, goats, cattle and chinchillas. In recent years, there have been reports in Heilongjiang, Qinghai, Yunnan, Ningxia and other places that type D bacteria cause bovine enterotoxemia, both in adult cattle and calves. In addition to infecting livestock and poultry, type D bacteria caused enterotoxemia in rare animals in the country, such as the death of Qinglu, a second-class national protected animal in Shanghai Zoo. The onset of the disease is generally rapid, the mortality rate is high, and the harm is great. In order to fundamentally control the spread of the disease and reduce or avoid the huge economic losses brought to animal husbandry production, effective preventive measures must be taken. Mueller (1988) pointed out: "Clostridium welchii causes disease because they form secreted toxins and metabolites, and no clinical symptoms will appear without toxin production"; Sudden death caused by Clostridium mycobacterium infection is due to the "explosive" reproduction of these pathogenic bacteria in animals under certain stress conditions, and a large amount of exotoxin secreted into the blood circulation, leading to intestinal toxemia and damage to tissues and organs Toxic effect occurs, causing rapid death. According to the above pathogenic mechanism, the focus of vaccine prevention should also be on immunity against toxemia caused by toxins. The toxoid vaccine should be developed on the basis of the research on the exotoxin of Clostridium welchii. The main chemical composition of the exotoxin formed by Clostridium welchii is protein, which has good immunogenicity. Therefore, according to the pathogenesis of Clostridium welchii, it is possible to prepare D-type Clostridium welchii toxin, inactivate it and make a toxoid vaccine, and then prepare antitoxin serum on this basis for the prevention and treatment of livestock and poultry. To achieve a reliable immune and therapeutic effect.

(3) Contents of the invention

In order to solve the above problems, the present invention provides a preparation method of Clostridium perfringens type D toxoid vaccine for cattle and sheep enterotoxemia.

A kind of preparation method of the D-type Clostridium perfringens toxoid vaccine of cattle and sheep enterotoxemia, concrete steps are as follows:

(1) Inoculate Clostridium perfringens type D on 5% sheep blood-1% glucose-agar plate, anaerobic at 37°C (anaerobic environment gas composition 88%N ₂ , 7%H ₂ , 5% CO ₂ ) for 36 hours to recover;

(2) Pick a single recovered colony and inoculate it in thioglycolate fluid medium FT45°C for 10 hours of anaerobic enrichment; inoculate the FT enrichment culture medium with an inoculum volume of 5% (volume ratio) into anaerobic broth, 43 Centrifuge anaerobically at 10000r/min for 15min at 10000r/min at 4℃, and filter the supernatant to obtain the toxin solution;

(3) Adjust the pH of the toxin solution to 7.2 with 1M NaOH, then add formaldehyde to make the formaldehyde concentration in the toxin solution reach 0.3%, shake and mix well, put it at 37°C for 96 hours to inactivate, and shake once every 5-6 hours to obtain the toxoid solution;

(4) Mix the white oil and Span-80 according to the volume ratio of 94:6, then add the white oil and Span-80 mixture with a mass ratio of 2% aluminum stearate to dissolve and mix, and then autoclave at 121°C for 30 minutes. It is the oil phase; take the toxoid solution, add Tween-80s to make the concentration of Tween-80s in the toxoid solution reach 2%, mix well, it is the water phase; emulsify the oil phase and the water phase at a volume ratio of 1:1 Adding thimerosal to the emulsion to make the final concentration of thimerosal 0.01%, to obtain the toxoid vaccine of Clostridium perfringens type D toxoid vaccine for cattle and sheep enterotoxemia.

The composition of the 5% sheep blood-1% glucose-agar plate is: 1 gram of glucose, 3.8 grams of soy flour agar, 5 milliliters of sheep blood and 100 milliliters of deionized water;

When in use, inject intramuscularly to cattle and sheep, and immunize once every six months, 4ml each time, which can effectively prevent the occurrence of enterotoxemia in cattle and sheep.

Beneficial effect

The vaccines for preventing enterotoxemia in cattle and sheep on the market are all inactivated whole vaccines, which have a certain control effect on the epidemic of the disease, but there are also some shortcomings. Due to the high concentration of formaldehyde during inactivation, the vaccination reaction of livestock is relatively serious. . And the present invention develops and prepares the D-type Clostridium perfringens toxoid vaccine with full potency, strong immunity, good pertinence, and no side effects according to the pathogenic mechanism of the disease, which is used for the D-type perfringens of animals. Prevention of Clostridium perfringens and control of the prevalence of Clostridium perfringens type D

(4) Specific implementation methods

Example 1 Clostridium perfringens type D toxoid vaccine preparation for cattle and sheep enterotoxemia

The recovery of bacterial strains Clostridium perfringens type D (preserved by National Collection of Type Cultures British Microorganism Culture Collection Center, preservation number: NCTC8346) was inoculated on 5% sheep blood-1% glucose-agar plate (1 gram of glucose, 3.8 grams of soybean Powder agar, 5ml sheep blood and 100ml deionized water), 37℃ anaerobic (anaerobic environment gas composition 88%N ₂ , 7%H ₂ , 5%CO ₂ ) culture for 36 hours recovery;

Bacterial enrichment Pick a single colony and inoculate it in thioglycolate fluid medium (FT, purchased from Qingdao Rishui Biotechnology Co., Ltd.) After anaerobic enrichment at 45°C for 10 hours, take 5ml FT enrichment culture solution and add it to 100ml anaerobic meat Liver soup (purchased from Qingdao Rishui Biotechnology Co., Ltd.) was cultured anaerobically at 43°C for 6 hours to obtain anaerobic broth broth;

Preparation of toxin: Centrifuge the anaerobic broth broth at 10,000 r/min for 15 minutes at 4°C, then filter the supernatant with a 0.22 μm Chua filter to obtain the toxin solution;

Verify the presence of toxin α, ε using indirect enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (SDS-PAGE) to detect the presence of α, ε in exotoxin;

Toxin inactivation Adjust the pH of the above toxin solution to 7.2 with 1M NaOH, then add formaldehyde to make the formaldehyde concentration in the toxin solution reach 0.3%, shake and mix well, place at 37°C for 96h to inactivate, shake once every 5-6h to get Toxoid solution;

Preparation of toxoid vaccine Mix white oil and Span-80 according to the volume ratio of 94:6, and then add 2% aluminum stearate by mass ratio to the mixture of white oil and Span-80 to make the final concentration of aluminum stearate It is 2%, melted and mixed, and sterilized under high pressure at 121℃ for 30 minutes to form the oil phase; add Tween-80s to the toxoid solution to make the final concentration of Tween-80s reach 2%, and mix well to form the water phase; mix the oil phase with The water phase is emulsified at a volume ratio of 1:1, and thimerosal is added to the emulsion so that the final concentration of thimerosal is 0.01%, and the toxoid vaccine is obtained.

Carrying out the effect test of Clostridium perfringens type D toxoid vaccine for cattle and sheep enterotoxemia

Sterility test Thioglycollate medium (T.G) is used for anaerobic and aerobic bacteria test; Caseptone agar (G.A) solid medium is used for aerobic bacteria test; Glucose peptone soup (G.P) is used for mold And saprophytic bacteria test. After detoxification and filtration of toxoids, inoculate two TG small tubes and two GA slanted tubes, each with 0.2ml, culture at 25°C and one at 37°C, and inoculate a GP small tube with 0.2ml and culture at 25°C . All cultured for 7 days, sterile growth (mainly refer to "Quality Standards of Veterinary Biological Products of the People's Republic of China")

Safety inspection Take 4 healthy rabbits of 1.5-2kg, and inoculate 5ml type D Clostridium perfringens toxoid vaccine intramuscularly, and observe continuously for 10 days. No local or systemic abnormal reactions were observed (mainly refer to the "Quality Standards for Veterinary Biological Products of the People's Republic of China")

The minimum lethal dose (MLD) of sheep is determined to take 8 sheep of 6-12 months old and 30-40kg, 2 in a group, and use 4ml, 8ml, 12ml, 16ml of D-type Clostridium perfringens toxin prepared by the present invention The solution was injected intravenously to the sheep, and the MLD measured for 24 hours was 12ml.

Determine the immunization dose Take 8 sheep aged 6-12 months and 30-40kg, each 2 as a group (2ml, 4ml, 6ml, 8ml D-type Clostridium perfringens toxoid vaccine for each group respectively), after 21 days Then, an MLD Clostridium perfringens toxin solution was used for intravenous inoculation to challenge the virus, observed for 72 hours, and the incidence and death conditions were recorded. One sheep in the immunization group injected with 2ml developed disease and one died, and the sheep in the immunization group injected with 4ml, 6ml, and 8ml had no disease. Therefore the immunization dose is 4ml.

18 sheep aged 1-3 years were used for efficacy test, divided into 3 groups, 6 sheep in each group, 4 sheep in each group were intramuscularly injected with Clostridium perfringens type D toxoid vaccine 4ml/sheep, and the other 2 sheep were not injected with vaccine as comparison. 21 days after inoculation, each sheep in each group was injected with 1 MLD (12 ml) of Clostridium perfringens type D toxin solution. All the sheep in the control group died, one of the vaccinated sheep died, and the rest survived.

Toxoid Vaccine Antibody Growth and Decline Law Take 4 sheep (not immunized with Clostridium welchii vaccine) aged 6-12 months and about 30-40 kg, and inject 4ml of Clostridium perfringens D-type into each of the three sheep Fungal toxoid vaccine, one intramuscular injection of the same dose of saline as a control. Blood was collected from the jugular vein of four sheep at 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 17 weeks, 20 weeks, 22 weeks, and 24 weeks after injection, and the indirect ELISA test was used to detect Antibody titers of α and ε toxin in the serum. According to the measured antibody titer, the effective protection period is 6 months.

Claims (1)

1. a D-type Clostridium perfringens toxoid vaccine of cattle and sheep enterotoxemia, is characterized in that obtaining by following preparation method: 1) inoculate Clostridium perfringens type D on a 5% sheep blood-1% glucose-agar plate, and recover by anaerobic culture at 37°C for 36 hours; 2) Pick a recovered single colony and inoculate it in thioglycolate fluid medium FT45°C for anaerobic enrichment for 10 hours; inoculate the FT enrichment culture medium with an inoculum volume ratio of 5% to anaerobic broth, and inoculate it anaerobically at 43°C. Cultivate for 6 hours; take the anaerobic broth broth and centrifuge at 10,000 r/min at 4°C for 15 minutes, filter the supernatant with a Chua filter to obtain the toxin solution; 3) Adjust the pH of the toxin solution to 7.2 with 1M NaOH, then add formaldehyde to make the formaldehyde concentration in the toxin solution reach 0.3%, shake and mix well, place at 37°C for 96 hours to inactivate, shake once every 5-6 hours to obtain the toxoid solution ; 4) Mix white oil and Span-80 according to the volume ratio of 94:6, then add white oil and Span-80 mixed liquid with a mass ratio of 2% aluminum stearate to dissolve and mix, and autoclave at 121°C for 30 minutes. Oil phase; take the toxoid solution, add Tween-80s to make the concentration of Tween-80s in the toxoid solution reach 2%, mix well, it is the water phase; emulsify the oil phase and the water phase at a volume ratio of 1:1, Adding thimerosal to the emulsion so that the final concentration of thimerosal is 0.01%, the Clostridium perfringens type D toxoid vaccine for cattle and sheep enterotoxemia is obtained; The composition of the 5% sheep blood-1% glucose-agar plate is: 1 gram of glucose, 3.8 grams of soy flour agar, 5 milliliters of sheep blood and 100 milliliters of deionized water.