CN103432579A - Preparation method of lamb-dysentery clostridium perfringens type B toxoid vaccine - Google Patents

Abstract

The invention relates to a preparation method of a preparation method of a lamb-dysentery clostridium perfringens type B toxoid vaccine. The preparation method comprises the following steps of inoculating the clostridium perfringens type B in a thioglycollate fluid culture medium for enriched culture under the condition of an anaerobic environment at 38 DEG C for 14 h; adding the culture to a culture medium of tryptone, dextrin, yeast extract and L-arginine which are dissolved by a buffer solution, carrying out shaking culture in the anaerobic environment for 5 h, and then performing 3800 g centrifuging at 4 DEG C for 15 min, and filtering to remove bacteria in a Seitz filter; adding a toxoid inactivated previously to a sterile white oil adjuvant in the ratio of 1: 1, and adding 0.01% of merthiolate, thereby obtaining the toxoid vaccine. The vaccine is free of toxic and side effects, high in valence, strong in immunizing power, and capable of effectively preventing and controlling clostridium perfringens type B induced lamb dysentery and enterotoksemia or necroticenteritis of sheep, goat, foal and calf.

Description

A kind of preparation method of lamb dysentery Type B bacillus perfringens toxoid vaccine

(1) technical field

The present invention relates to a kind of preparation method of lamb dysentery Type B bacillus perfringens toxoid vaccine.

(2) background of invention

The Type B bacillus perfringens can cause enterotoxemia or the necrotic enteritis of lamb dysentery, sheep, goat, coltfoal and calf, and main nosotoxin is α, β, ε toxin.The necrotic enteritis caused by β-toxin, be commonly considered as because the trypsin in food has stoped the decomposition of this toxin in the intestinal, thereby cause the generation of this disease.In addition, this toxin possibility or a kind of neurotoxin.And the secretory volume of Type B ε-toxin is few, it can impel the aorta of animal to cause hypertension with other tremulous pulse contraction; In addition, also have with blood brain barrier in the ability of blood vessel epithelium receptors bind, thereby cause that vascular permeability increases, finally cause the lethal edema, but ε-toxin thermo-labile (Mcdonel, 1986).

For many years, what during China produces, use is inactivated vaccine, and as far back as 1989, old stannum congruence was carried out the research of " rabbit hemorrhagic disease and A type clostridieum welchii connection Seedling " and achieved success; Tong Cheng has just waited the clostridieum welchii list Seedling of (1991) development rabbit, can produce 82.01% protective rate to laboratory animal; Lu Cheng (1997) etc. is studied rabbit Pestivirus and A type clostridieum welchii bigeminy Seedling; Yang Ying (1997) is studied A type clostridieum welchii aluminium hydroxide inactivated vaccine, and vaccine 0.6ml, 2ml, 4ml can protect the attack of mice, rabbit and pig opposing standard A type 1MLD toxin; Yao Xiangyan etc. (1998) adopt the concentrated AL (OH) of cattle source clostridieum welchii separated strain development cattle clostridieum welchii of three separate sources ₃inactivated vaccine; Wang Keling (1998) etc. is studied rabbit hemorrhagic disease, clostridieum welchii disease, Bacillus pasteurii disease trigeminy vaccine; Tan Shiwen etc. (2000) have carried out the development of lamb dysentery, Intestinum caprae seu ovis toxemia, colibacillosis, sheep struck tetrad oil adjuvant inactivated vaccine.These vaccines have certain effect to the prevention of this disease, also come with some shortcomings: most critical ground is that the effective ingredient in vaccine is mainly somatic antigen simultaneously, induce antibody that body produces be merely able in and somatic antigen, but be difficult to the antigen that neutralizes a toxin, entirely do not conform to domestic animal " Sudden Death Syndrome " pathogenesis.Therefore, also there are some queries in the effectiveness of inactivated vaccine.

Chai Tongjie (2001) finds the research of domestic animal " Sudden Death Syndrome " pathogenesis: under certain stress state, in animal intestinal, microecological balance destroys, make clostridieum welchii be the breeding of ' explosivity ', moment produces a large amount of extracellular toxins, the rising of intestinal mucosal permeability makes toxin or antibacterial+toxin enter blood circulation to cause toxemia (sometimes with bacteremia), thereby cause the exhaustion of vitals, cause animal dead; Thus, the theory of " the quick main causes of death of clostridieum welchii Infective and animal are Intestinal source toxemias, and the key of prevention is the toxemia caused for toxin, and the antigenic component of vaccine should be corresponding toxoid " is further proposed.

The disease incidences such as lamb dysentery that the Type B bacillus perfringens causes are anxious, and dead fast, symptom not too obviously and at all has little time treatment sometimes.To fundamentally control the propagation of primary disease, reduce or avoid economic loss, must take effective preventive measure.The main ectotoxic chemical composition that bacillus perfringens forms is protein, has immunogenicity preferably.Therefore, according to the pathogeny of bacillus perfringens, prepare the bacillus perfringens extracellular toxin, will after its deactivation, make toxoid vaccine, prevent for poultry.

(3) summary of the invention

In order to address the above problem, the preparation method of the lamb dysentery Type B bacillus perfringens toxoid vaccine that the present invention's invention provides a kind of and tired entirely, immunity is strong, specific aim good, have no side effect, for the particularly prevention of lamb dysentery of Type B bacillus perfringens disease of animal, control the popular of the Type B bacillus perfringens disease such as lamb dysentery.

The preparation method of the Type B bacillus perfringens toxoid vaccines such as lamb dysentery comprises the following steps:

1B type bacillus perfringens strain is inoculated on the blood plate culture medium in the lower 37 ℃ of 46h recovery of anaerobic environment (gas consist of 85% nitrogen 9% hydrogen 6% carbon dioxide).

2B type bacillus perfringens is seeded in THIOGLYCOLLIC ACID salt fluid culture medium, at the lower 38 ℃ of 14h of anaerobic environment (gas consist of 85% nitrogen 9% hydrogen 6% carbon dioxide), cultivates and increases bacterium.

3 join toxin producing medium by the above-mentioned culture of 8ml, and (every 100mlPBS buffer dissolves 2g tryptone, 1g dextrin, 2g yeast extract and 1.2gL- arginine) in, shaken cultivation under anaerobic environment, 221 pendulum/min, cultivate 5h for 38 ℃, then at 4 ℃ of centrifugal 15min of 3800g, then uses filtration sterilization in the seitz filter of aperture 0.22 μ m, obtains extracellular toxin solution.

4 add the extracellular toxin liquor capacity than the formaldehyde that is 0.3% in toxin soiutions outside, and fully vibration mixes, and puts 37 ℃ of 48h deactivations, and interval 5-6h jolting once, obtains the toxoid solution that deactivation is good.

5 add the white-oil adjuvant emulsifying of sterilizing in toxoid solution by the 1:1 volume ratio, in every 100 milliliters of emulsions, add 0.01 gram thimerosal to make Type B bacillus perfringens toxoid vaccine;

Described white-oil adjuvant forms: 9 parts of white oils and a Span-80 add white oil and Span-80 mixeding liquid volume than 2% Tween-20 and the aluminium stearate of mass ratio 1% after mixing;

Described blood plate medium component is: 100 ml distilled waters, 3.7g Semen Glycines powder agar, 1g glucose and 5% volume ratio Sheep Blood;

Described toxin producing medium composition is: every 100mlPBS buffer dissolves 2g tryptone, 1g dextrin, 2g yeast extract and 1.2gL- arginine;

Described PBS buffer composition is: 1L deionized water, 8g NaCl, 0.2g KCl, 3.58g Na ₂hPO ₄12H ₂o, 0.27g KH ₂pO ₄.

During use, intramuscular injection, one time 5 milliliters.

Beneficial effect

This vaccine has no side effect, the height of tiring, immunity is strong, can effectively prevent, control enterotoxemia or the necrotic enteritis of the lamb dysentery, sheep, goat, coltfoal and the calf that are caused by the Type B bacillus perfringens, can reduce the loss for aquaculture, bring visual interests.

(4) specific embodiment

Detailed process prepared by embodiment 1 vaccine

At first, preparation bacterium liquid.(National Collection of Type Cultures-Britain microorganism fungus kind preservation center preservation preserving number: NCTC6121) picking one ring (approximately 3 μ l) is inoculated in blood plate culture medium (100ml distilled water, 3.7g Semen Glycines powder agar, 1g glucose, 5 milliliters of Sheep Bloods) (each gas concentration is 85%N to anaerobic environment Type B bacillus perfringens strain NCTC6121 ₂, 9%H ₂, 6%CO ₂) 37 ℃ of 46h recoveries.Type B bacillus perfringens colony inoculation of picking is produced at THIOGLYCOLLIC ACID salt fluid culture medium FT(Qingdao day aquatic organism technology company limited) in, in gas concentration, be 85%N ₂, 9%H ₂, 6%CO ₂the lower 38 ℃ of 14h of anaerobic environment condition cultivate and increase bacterium.

Above-mentioned culture is joined to homemade toxin producing medium, and (every 100mlPBS buffer dissolves 2g tryptone, 1g dextrin, 2g yeast extract and 1.2gL- arginine) in, shaken cultivation under anaerobic environment, 221 pendulum/min, 38 ℃ of 5h, 4 ℃ of 15min3800g are centrifugal, use again filtration sterilization in the seitz filter of aperture 0.22 μ m, obtain extracellular toxin, add formaldehyde to make the formaldehyde final concentration reach 0.3% prepared extracellular toxin, fully vibration mixes, put 37 ℃ of 48h deactivations, interval 5-6h jolting once.

Add the white-oil adjuvant of sterilizing in deactivation in good toxoid solution by the 1:1 volume ratio, add thimerosal (in every 100 milliliters of toxoid solution He in white-oil adjuvant vaccine mixed liquor, adding 0.01 gram thimerosal), obtain toxoid vaccine.During use, intramuscular injection, one time 5 milliliters.

The checking of embodiment 2 vaccine safeties and effect

12 of definite healthy sheep with body weight 30kg of minimum lethal dose, be divided at random four groups, inject respectively Type B bacillus perfringens extracellular toxin prepared by 5ml, 7ml, 9ml, 11ml, after one day, injection 5ml and the ectotoxic sheep diarrhea of 7ml but not dead, dead after the sheep severe diarrhea of injection 9ml and 11ml toxin, thereby definite 9ml is the sheep minimum lethal dose.

Safety examination is got 4 of 1.5kg Healthy Rabbits, the prepared Type B bacillus perfringens toxoid vaccine of difference intramuscular inoculation 5ml, Continuous Observation 10 days.Local without untoward reaction such as granulomas, whole body is also without abnormal response.(with reference to " People's Republic of China's veterinary biologics quality standard ")

12 of the healthy sheep that using dosage definite is 30kg by body weight, be divided at random four groups, inject respectively 4,5,6, the prepared vaccine of 7ml, four groups of Type B bacillus perfringens extracellular toxins of all injecting 9ml after 18d, observe 3d, inject 5,6, the sheep of 7ml vaccine is not all dead, and without the symptom of lamb dysentery, thereby immunizing dose is decided to be 5ml.

6 of the healthy sheep of immune efficacy check use body weight 30kg, wherein 4 toxoid vaccines prepared by intramuscular injection 5ml, do not inject toxoid vaccine as a control group for other two.After 18d, 6 sheep are all injected the Type B bacillus perfringens extracellular toxin of 9ml.After 24h, 2 sheep severe diarrheas of matched group are also dead, and the sheep that immunity is crossed is all not dead, and continue to observe 3d, without disease symptoms such as lamb dysenteries.Illustrate that this vaccine has reached 100% to the protection of sheep.

Claims (1)

1. the preparation method of a lamb dysentery Type B bacillus perfringens toxoid vaccine is characterized in that comprising the following steps:

1) Type B bacillus perfringens strain is inoculated in 37 ℃ of 46h recoveries under anaerobic environment on the blood plate culture medium;

2) the Type B bacillus perfringens after the recovery is seeded in THIOGLYCOLLIC ACID salt fluid culture medium, and under anaerobic environment, 38 ℃ of 14h cultivate and increase bacterium;

3) by step 2) increase the bacterium culture according to inoculum concentration 8% volume ratio inoculation toxin producing medium, shaken cultivation under anaerobic environment, cultivate 5h for 38 ℃, and then, at 4 ℃ of centrifugal 15min of 3800g, the seitz filter filtration sterilization, obtain extracellular toxin solution;

4) in toxin soiutions, add the extracellular toxin liquor capacity than the formaldehyde that is 0.3% outside, fully vibration mixes, and puts 37 ℃ of 48h deactivations, and interval 5-6h jolting once, obtains the toxoid solution that deactivation is good;

5) add the white-oil adjuvant emulsifying of sterilizing by the 1:1 volume ratio in toxoid solution, in every 100 milliliters of emulsions, add 0.01 gram thimerosal to make Type B bacillus perfringens toxoid vaccine;

Described white-oil adjuvant forms: 9 parts of white oils and a Span-80 add white oil and Span-80 mixeding liquid volume than 2% Tween-20 and the aluminium stearate of mass ratio 1% after mixing;

Described blood plate medium component is: 100 ml distilled waters, 3.7g Semen Glycines powder agar, 1g glucose and 5% volume ratio Sheep Blood;

Described toxin producing medium composition is: every 100mlPBS buffer dissolves 2g tryptone, 1g dextrin, 2g yeast extract and 1.2gL-arginine;

Described PBS buffer composition is: 1L deionized water, 8g NaCl, 0.2g KCl, 3.58g Na ₂hPO ₄12H ₂o, 0.27g KH ₂pO ₄.