CN103463632A - Preparation method for D type clostridium perfringen toxoid vaccine of cattle and sheep enterotoxaemia - Google Patents
Preparation method for D type clostridium perfringen toxoid vaccine of cattle and sheep enterotoxaemia Download PDFInfo
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Abstract
The invention relates to a preparation method for a D type clostridium perfringen toxoid vaccine of cattle and sheep enterotoxaemia. The preparation method comprises the following steps: carrying out strain recovery, strain enrichment, virus culture and inactivation on D type clostridium perfringen strains to prepare a toxoid solution; and emulsifying the toxoid solution and a white oil adjuvant to prepare the D type clostridium perfringen toxoid vaccine. When in use, 4ml of the D type clostridium perfringen toxoid vaccine is injected to muscles of cattle and sheep for immunization once per half a year, so that the cattle and sheep enterotoxaemia can be effectively prevented. According to the preparation method, the D type clostridium perfringen toxoid vaccine which is full in titer, high in immunizing power, good in pertinence and free from toxic and side effects is developed and prepared according to the pathogenesis of the cattle and sheep enterotoxaemia and is used for preventing D type clostridium perfringen diseases of animals and controlling the prevalence of the D type clostridium perfringen diseases.
Description
(1) technical field
The present invention relates to a kind of preparation method of D type bacillus perfringens toxoid vaccine of cattle and sheep enterotoxemia.
(2) background of invention
Bacillus perfringens is the gastral normal floras of people, animal, be distributed widely in occurring in nature, this bacterium can produce multiple extracellular toxin, the extracellular toxin of having found at present reaches 12 kinds, wherein α, β, ε and ι are that main lethal toxin is also the typing toxin, according to these four kinds of toxin, bacillus perfringens is divided into A, B, C, D and five toxin types of E, and each toxin type all can infect people or poultry and cause specific disease.D type bacterium mainly produces α, ε extracellular toxin, can cause the enterotoxemia of the animals such as lamb, sheep, goat, cattle and squirrel.The ground such as Heilungkiang, Qinghai, Yunnan, Ningxia all have D type bacterium to cause the toxemic report of Intestinum Bovis seu Bubali in recent years, and Adult Bovine and calf all have generation.Except infecting poultry, D type bacterium causes for example death of the blue or green deer of national second class protection animal in zoo, Shanghai of national rare animal enterotoxemia.This disease is fallen ill generally relatively suddenly, mortality rate is high, very harmful, fundamentally control the propagation of primary disease, and the tremendous economic loss that reduces or avoid bringing to animal husbandry production, must take the efficiently and effectively preventive measure.Mueller (1988) points out: " it is to form toxin and the metabolite of secretion due to them that clostridieum welchii causes a disease, and without toxin, produces and just not there will be clinical symptoms "; Chai Tongjie (1999,2001) also proposing same viewpoint is that bacillus perfringens infects the Sudden Death Syndrome caused, due under some stressed condition, these pathogen are " explosivity " breeding in animal body, a large amount of extracellular toxins that secretion produces enter blood circulation, cause Intestinal source toxemia, to histoorgan generation toxic action, cause quick death.According to above-mentioned pathogenesis, the key preventive of vaccine also should be placed on the toxemic immunity caused for toxin.The main ectotoxic chemical composition that should on the basis to the research of clostridieum welchii extracellular toxin, develop the formation of toxoid vaccine clostridieum welchii is protein, has immunogenicity preferably.Therefore, according to the pathogeny of clostridieum welchii, preparation D type clostridieum welchii toxin, will make toxoid vaccine after its deactivation, then prepares on this basis antitoxic serum, for poultry prevention and treatment, is immunity and the therapeutic effect that can reach infallible.
(3) summary of the invention
In order to address the above problem, the invention provides to a kind of preparation method of D type bacillus perfringens toxoid vaccine of cattle and sheep enterotoxemia.
A kind of preparation method of D type bacillus perfringens toxoid vaccine of cattle and sheep enterotoxemia, concrete steps are as follows:
(1) D type bacillus perfringens strain is inoculated on 5% Sheep Blood-1% glucose-agar plate to 37 ℃ of anaerobism (anaerobic environment gas composition composition 88%N
2, 7%H
2, 5%CO
2) cultivate recovery in 36 hours;
(2) the single colony inoculation after the picking recovery increases bacterium 10h in FT45 ℃ of anaerobism of THIOGLYCOLLIC ACID salt fluid culture medium; By the FT enrichment culture medium according to inoculum concentration 5%(volume ratio) inoculation anaerobism liver bouillon, 43 ℃ of anaerobism are cultivated 6h; Get 4 ℃ of centrifugal 15min of 10000r/min of anaerobism liver bouillon bacterium liquid, seitz filter is crossed the leaching supernatant and is obtained toxin soiutions;
(3) toxin soiutions is adjusted to pH to 7.2 with 1M NaOH, then add formaldehyde to make concentration of formaldehyde in toxin soiutions reach 0.3%, fully vibration mixes, and puts 37 ℃ of 96h deactivations, and interval 5-6h jolting once, obtains toxoid solution;
(4) white oil and Span-80 are mixed according to the volume ratio of 94:6, then add the aluminium stearate of white oil and Span-80 mixed liquor mass ratio 2% to dissolve to mix, 121 ℃ of autoclavings are oil phase in 30 minutes; Get toxoid solution, add Tween-80s to make Tween-80s concentration in toxoid solution reach 2%, mix, be water; By oil phase and the water ratio emulsifying of 1:1 by volume, in emulsion, adding thimerosal to make the thimerosal final concentration is 0.01%, obtains the D type bacillus perfringens toxoid vaccine toxoid vaccine of cattle and sheep enterotoxemia.
Described 5% Sheep Blood-1% glucose-agar plate composition is: 1 gram glucose, 3.8 gram Semen Glycines powder agar, 5 milliliters of Sheep Bloods and 100 ml deionized water;
During use, to the cattle and sheep intramuscular injection, once, each 4ml, can effectively prevent the generation of cattle and sheep enterotoxemia in immunity every half a year.
Beneficial effect
On market, prevention cattle and sheep enterotoxemia vaccine is all the full vaccine of deactivation now, the popular of this disease is had to certain control action, but also come with some shortcomings, and during due to deactivation, concentration of formaldehyde is large, and the domestic animal reaction of inoculation is heavier.And the exploitation of the present invention's pathogenesis sick according to this prepared tire complete, immunity is strong, specific aim good, the D type bacillus perfringens toxoid vaccine that has no side effect, be used for the prevention of the D type bacillus perfringens disease of animal, control the popular of D type bacillus perfringens disease
(4) specific embodiment
The D type bacillus perfringens toxoid vaccine preparation of embodiment 1 cattle and sheep enterotoxemia
The recovery of strain is by (the National Collection of Type Cultures Britain microorganism fungus kind preservation center preservation of D type bacillus perfringens kind, preserving number: NCTC8346) be inoculated in 5% Sheep Blood-1% glucose-agar plate (1 gram glucose, 3.8 gram Semen Glycines powder agar, 5 milliliters of Sheep Bloods and 100 ml deionized water) upper, 37 ℃ of anaerobism (anaerobic environment gas composition composition 88%N
2, 7%H
2, 5%CO
2) cultivate recovery in 36 hours;
Antibacterial increases the single colony inoculation of bacterium picking in THIOGLYCOLLIC ACID salt fluid culture medium (FT, purchased from Qingdao day aquatic organism technology company limited) after 45 ℃ of anaerobism increase bacterium 10h, get the 5mlFT enrichment culture medium and join 43 ℃ of anaerobism cultivation 6h of 100ml anaerobism liver bouillon (purchased from Qingdao day aquatic organism technology company limited), obtain anaerobism liver bouillon bacterium liquid;
The preparation of toxin is 4 ℃ of centrifugal 15min of 10000r/min of anaerobism liver bouillon bacterium liquid, then crosses the leaching supernatant with aperture 0.22 μ m seitz filter and obtain toxin soiutions;
The existence of checking toxin α, ε is used indirect enzyme-linked immunosorbent adsorption experiment (ELISA) and polyacrylamide gel electrophoresis (SDS-PAGE) to detect in extracellular toxin and is had α, ε;
The deactivation of toxin is adjusted pH to 7.2 by above-mentioned toxin soiutions with 1M NaOH, then adds formaldehyde to make concentration of formaldehyde in toxin soiutions reach 0.3%, and fully vibration mixes, and puts 37 ℃ of 96h deactivations, and interval 5-6h jolting once, obtains toxoid solution;
The preparation of toxoid vaccine mixes white oil and Span-80 according to the volume ratio of 94:6, add again the aluminium stearate of mass ratio 2% in white oil and Span-80 mixed liquor, making the aluminium stearate final concentration is 2%, dissolves and mixes, and 121 ℃ of autoclavings are oil phase in 30 minutes; At toxoid solution, add Tween-80s to make the Tween-80s final concentration reach 2%, mix, be water; The volume ratio emulsifying that oil phase and water are pressed to 1:1, in emulsion, adding thimerosal to make the thimerosal final concentration is 0.01%, obtains toxoid vaccine.
Implement 2 cattle and sheep enterotoxemia D type bacillus perfringens toxoid vaccine validity checks
Steriling test sulphur glycollate culture medium (T.G) is for anaerobic and aerobism bacteriologic test; Peptone from casein agar (G.A) solid medium is for the aerobism bacteriologic test; Glucose peptone soup (G.P) is for mycete and saprophytic bacteria check.After the toxoid detoxification is filtered, each two, inoculation TG tubule, GA inclined-plane, every 0.2ml, put 25 ℃ of cultivations for one, and one is placed in 37 ℃ of cultivations, separately gets 1 GP tubule of 0.2ml inoculation and puts 25 ℃ of cultivations.All cultivate asepsis growth (main with reference to " People's Republic of China's veterinary biologics quality standard ") 7
Safety verification is got 4 of 1.5-2kg Healthy Rabbits, difference intramuscular inoculation 5ml D type bacillus perfringens toxoid vaccine, Continuous Observation 10 days.Observation is without part or whole body abnormal response (main with reference to " People's Republic of China's veterinary biologics quality standard ")
The minimum lethal dose of sheep (MLD) is measured 8 of sheep getting 6-12 monthly age, 30-40kg, 2 one group, the D type clostridium perfringens toxoid solution prepared with 4ml, 8ml, 12ml, 16ml the present invention carries out intravenous injection to sheep, and the MLD that observes the 24h. survey is 12ml.
Immunizing dose is determined and to be got 8 of 6-12 monthly age, 30-40kg sheep, every 2 is one group (distinguishing intramuscular injection 2ml, 4ml, 6ml, 8ml D type bacillus perfringens toxoid vaccine for every group), all with the clostridium perfringens toxoid solution of a MLD, carry out the intravenous inoculation counteracting toxic substances again after 21 days, observe 72h, record the Mortality situation.Morbidity of immune group sheep, the death of injection 2ml, the immune group sheep of 4ml, 6ml, 8ml is without morbidity.Therefore immunizing dose is 4ml.
Efficacy test, with 18 of the sheep in 1-3 year, is divided into 3 groups, and 6 every group, wherein only, another 2 vaccinate is not in contrast for every group 4 intramuscular injection D type bacillus perfringens toxoid vaccine 4ml/.Inoculate after 21 days, every group every sheep is injected 1 MLD(12ml again) D type clostridium perfringens toxoid solution.The sheep of matched group is all dead, dead one of the sheep of vaccinate, and all the other are all survived.
The antibody Fluctuation of toxoid vaccine is got 4 of the sheep (not carrying out the clostridieum welchii vaccine immunization) of 6-12 monthly age, 30-40kg left and right, three D type bacillus perfringens toxoid vaccines prepared by each intramuscular injection 4ml, the normal saline of an intramuscular injection same dose in contrast.Respectively at after injection, four sheep being carried out to the jugular vein blood sampling in 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 17 weeks, 20 weeks, 22 weeks, 24 weeks, α, ε toxin antibody that the test of application indirect ELISA detects in its serum are tired.According to the known effective protection period of antibody titer of surveying, it is 6 months.
Claims (1)
1. the preparation method of the D type bacillus perfringens toxoid vaccine of a cattle and sheep enterotoxemia is characterized in that concrete steps are as follows:
1) D type bacillus perfringens strain is inoculated on 5% Sheep Blood-1% glucose-agar plate, 37 ℃ of anaerobism are cultivated recovery in 36 hours;
2) the single colony inoculation after the picking recovery increases bacterium 10h in FT45 ℃ of anaerobism of THIOGLYCOLLIC ACID salt fluid culture medium; By the FT enrichment culture medium, according to inoculum concentration 5% volume ratio inoculation anaerobism liver bouillon, 43 ℃ of anaerobism are cultivated 6h; Get 4 ℃ of centrifugal 15min of 10000r/min of anaerobism liver bouillon bacterium liquid, seitz filter is crossed the leaching supernatant and is obtained toxin soiutions;
3) toxin soiutions is adjusted to pH to 7.2 with 1M NaOH, then add formaldehyde to make concentration of formaldehyde in toxin soiutions reach 0.3%, fully vibration mixes, and puts 37 ℃ of 96h deactivations, and interval 5-6h jolting once, obtains toxoid solution;
4) white oil and Span-80 are mixed according to the volume ratio of 94:6, then add the aluminium stearate of white oil and Span-80 mixed liquor mass ratio 2% to dissolve to mix, 121 ℃ of autoclavings are oil phase in 30 minutes; Get toxoid solution, add Tween-80s to make Tween-80s concentration in toxoid solution reach 2%, mix, be water; By oil phase and the water ratio emulsifying of 1:1 by volume, in emulsion, adding thimerosal to make the thimerosal final concentration is 0.01%, obtains the D type bacillus perfringens toxoid vaccine toxoid vaccine of cattle and sheep enterotoxemia;
Described 5% Sheep Blood-1% glucose-agar plate composition is: 1 gram glucose, 3.8 gram Semen Glycines powder agar, 5 milliliters of Sheep Bloods and 100 ml deionized water.
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Cited By (2)
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| CN104829712A (en) * | 2015-04-23 | 2015-08-12 | 山东农业大学 | C and D type C.perfringens antitoxin serum and preparation method thereof |
| CN107789622A (en) * | 2017-10-18 | 2018-03-13 | 山东农业大学 | A kind of calf enterotoxemia C.perfringens β2The preparation method of toxin toxoid vaccine |
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| CN107789622A (en) * | 2017-10-18 | 2018-03-13 | 山东农业大学 | A kind of calf enterotoxemia C.perfringens β2The preparation method of toxin toxoid vaccine |
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