CN103432578A - Preparation method of C-type clostridium perfringens toxin vaccine of piglet red dysentery - Google Patents

Abstract

The invention relates to a preparation method of a C-type clostridium perfringens toxin vaccine of piglet red dysentery. The preparation method comprises the following steps of inoculating strains NCTC (National Collection Of Type Culture) 3180 of the C-type clostridium perfringens into a blood plate culture medium for carrying out anaerobic cultivation for 36 hours at 37 DEG C; carrying out shake cultivation on the culture by adopting a homemade toxin-producing medium (100 ml of PBS (Phosphate Buffer Solution), 2g of peptones, 1g of dextrin, 2g of yeast extracts, and 1.2g of L- arginine) under an anaerobic environment; inactivating the culture, filtering and adding an adjuvant to prepare the C-type clostridium perfringens toxin vaccine of the piglet red dysentery. During a using process, intramuscular injection is respectively adopted once a month and half a month before the delivery of a sow, wherein the dosage is 5-10 ml at a time. The C-type clostridium perfringens toxin vaccine of the piglet red dysentery is used for the sow at the later immunological gestation period, so that piglets obtain passive immunity through colostrums, and therefore, piglet red dysentery can be prevented. Moreover, the C-type clostridium perfringens toxin vaccine of the piglet red dysentery can be used for preventing sheep struck and calf enterotoksemia, so that the popularization of the C-type clostridium perfringens disease can be effectively controlled.

Description

A kind of preparation method of piglet red dysentery C type bacillus perfringens toxoid vaccine

(1) invention field

The present invention relates to a kind of preparation method of piglet red dysentery C type bacillus perfringens toxoid vaccine.

(2) background of invention

Clostridieum welchii claims again bacillus perfringens, according to toxogenic difference, can be divided into ABCDE five types.C type bacterium is mainly the cause of disease of struck, and the newborn piglet susceptible shows as dysentery and high mortality, also can cause enterotoxemia and the necrotic enteritis of lamb, calf, sheep, and this bacterium mainly produces α, two kinds of extracellular toxins of β.From the eighties mid-term, at first the whole nation approximately 20 provinces and cities Sudden Death Syndrome occurs in pig, cattle and sheep rabbit group, to animal husbandry production, has brought very large loss.In recent years, the sickness rate of ruminant necrotic enteritis in all parts of the country and enterotoxemia also has obvious ascendant trend, becomes a great problem of puzzlement China animal husbandry development, has seriously affected the development of animal husbandry.

In order to control the popular of bacillus perfringens disease, many scholars once developed the inactivated vaccine of bacillus perfringens for prevention.Wang Keling (1998) etc. is studied rabbit hemorrhagic disease, clostridieum welchii disease, Bacillus pasteurii disease trigeminy vaccine; Tan Shiwen etc. (2000) had once carried out the development of lamb dysentery, colibacillosis, sheep struck, Intestinum caprae seu ovis toxemia tetrad oil adjuvant inactivated vaccine.(1985) such as the uncommon Zhe of literary composition have also developed the multi-joint dry powder inactivated vaccine of clostridieum welchii.Also have in addition sheep braxy, struck, enterotoxemia triple inactivated vaccine, the sick A type of rabbit bacillus perfringens inactivated vaccine, these vaccines have certain control action to this disease popular, but also come with some shortcomings, as heavier as the domestic animal reaction of inoculation, the injected dose of multi-joint Seedling is larger, and the immune effect of some vaccine is uncertain, and quality needs further to improve.

The pathogenesis of C type bacterium is that extraneous factor causes bacillus perfringens in intestinal to be bred in a large number, produces a large amount of extracellular toxins and stick on epithelium of intestinal mucosa in intestinal, makes the intestinal wall permeability increase, and toxin enters blood circulation, causes toxemia.Bohenel 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 ".According to above-mentioned pathogenesis, the key preventive of C type bacillus perfringens vaccine should be on the basis of external toxin study, the development toxoid vaccine.

(3) summary of the invention

In order to address the above problem, the invention provides a kind of preparation method of piglet red dysentery C type bacillus perfringens toxoid vaccine.

A kind of preparation method of piglet red dysentery C type bacillus perfringens toxoid vaccine, comprise the steps:

The strain of C type bacillus perfringens is inoculated on the blood plate culture medium, and 37 ℃ of anaerobism are cultivated the 36h recovery; Get a single colony inoculation after recovery in THIOGLYCOLLIC ACID salt fluid culture medium FT, 37 ℃ of anaerobism are cultivated 12h and are increased bacterium; To increase the bacterium culture according to inoculum concentration 5%(volume ratio) be inoculated in toxin producing medium, 40 ℃ of anaerobism shaken cultivation 5h; Medium centrifugal, get supernatant, and the seitz filter filtration sterilization obtains extracellular toxin solution; In toxin soiutions, add outside the extracellular toxin liquor capacity than 0.3% formaldehyde, fully vibration mixes, 37 ℃, deactivation 8 days, during once obtain every the 5-6h jolting toxoid solution that deactivation is good; After white oil and span-80 are mixed with volume ratio 32:3, then add the aluminium stearate of white oil and span-80 mixed liquor mass ratio 2%, autoclaving obtains oil phase; In deactivation, in good toxoid solution, add the Tween-80 of volume ratio 2% to be mixed to get water; Oil phase and water, with volume ratio 1:1 emulsifying, are added to 0.01 gram thimerosal in every 100ml emulsion, after mixing, make C type bacillus perfringens toxoid vaccine;

Described blood plate medium component is: 100 ml distilled waters, 1 gram glucose, 3.8 gram bean agar powders and 5 milliliters of Sheep Bloods; Described toxin producing medium composition is: 100 milliliters of PBS buffer, 2 gram peptones, 1 gram dextrin, 2 gram yeast extracts and 1.2 gram L-arginines; The composition of described PBS buffer is: 100 ml deionized water, 0.8 gram sodium chloride, 0.02 gram potassium chloride, 0.358 gram disodium hydrogen phosphate dodecahydrate and 0.027 gram potassium dihydrogen phosphate.

During use, intramuscular injection, sow before childbirth each injection of January and first quarter moon once, dosage is each 5-10 milliliter.

Beneficial effect

These research and development have prepared the height of tiring, immunity is strong, therapeutic effect good, the C type bacillus perfringens toxoid vaccine of the struck had no side effect, piglet red dysentery, calf enterotoxemia, sow for immune latter half of gestation, newborn piglet obtains passive immunity by colostrum, can prevent piglet red dysentery.Can also prevent struck, the calf enterotoxemia, thus can effectively control the popular of C type bacillus perfringens disease.

(4) specific embodiment

The preparation of embodiment 1 piglet red dysentery C type bacillus perfringens toxoid vaccine

Antibacterial increases inoculating loop picking three ring C type bacillus perfringens strain (National Collection of Type Cultures Britain microorganism fungus kind preservation center preservations for bacterium, preserving number: NCTC3180) be inoculated in a blood plate culture medium (after 100 ml distilled waters, 1 gram glucose and 3.8 gram bean agar powder sterilizings, adding 5 milliliters of Sheep Bloods to divide installs on six blood plates) upper, 37 ℃ of anaerobism are cultivated 36h.Get single colony inoculation and contain 7-8 milliliter THIOGLYCOLLIC ACID salt fluid culture medium (FT) (medium component is general) in vitro, 37 ℃ of anaerobism cultivation ((88%N in one ₂, 7%H ₂and 5%CO ₂)) 12h increases bacterium.Getting 5 milliliters of liquid cultures is inoculated in 100 milliliters of toxin producing mediums (100 milliliters of PBS buffer, 2 gram peptones, 1 gram dextrin, 2 gram yeast extracts, 1.2 gram L-arginines), the composition of described PBS buffer is: 100 ml deionized water, 0.8 gram sodium chloride, 0.02 gram potassium chloride, 0.358 gram disodium hydrogen phosphate dodecahydrate, 0.027 gram potassium dihydrogen phosphate, 40 ℃ of anaerobism shaken cultivation 5h.

Extracellular toxin is produced 4 ℃ of centrifugal 30min of 10000r/min of culture fluid, gets supernatant, and the seitz filter filtration sterilization with aperture 0.22 μ m, obtain extracellular toxin solution.

The toxin deactivation adds the extracellular toxin liquor capacity than 0.3% formaldehyde in the extracellular toxin solution of producing, and fully vibration mixes, and then puts 37 ℃, 8 days complete inactivations, during every the 5-6h jolting once, obtain the toxoid that deactivation is good.

Toxoid vaccine preparation mixes white oil and span-80 with the volume ratio of 32:3 after, then add the aluminium stearate of white oil and span-80 mixed liquor mass ratio 2%, autoclaving, be oil phase.In deactivation, good toxoid solution adds the Tween-80 of volume ratio 2% to mix, and is water.Oil phase and water, with volume ratio 1:1 emulsifying, add 0.01 gram thimerosal in every 100ml emulsion, after mixing, make C type bacillus perfringens toxoid vaccine.

The steriling test of vaccine carries out with reference to 301 pages of " People's Republic of China's veterinary biologics quality standard " (calendar year 2001 version) appendix, asepsis growth.

4 of the rabbit of the safety verification use body weight 1.5-2 kilogram of vaccine, 5 milliliters of each intramuscular injection C type bacillus perfringens toxoid vaccines, observe 10, and all strong alive, injection site does not necrose.

6 of the vaccine potency check healthy susceptible pigs above with body weight 40kg, wherein every intramuscular injection C type bacillus perfringens toxoid vaccine of 4 pigs is 5 milliliters, and 2 vaccinate is not as a control group; After 14 days, 6 pigs are all injected C type bacillus perfringens extracellular toxin solution prepared by 10 milliliters of the present invention, observe 3-5 day, and 2 pigs of matched group are all dead, and 4 of immune group pigs are all not dead; Continue to observe 7 days, the diseases such as struck, piglet red dysentery, calf enterotoxemia do not occur, illustrate that this vaccine has reached 100% to the protection of pig.

Immunizing dose definite selects 8 of the above healthy susceptible pigs of body weight 40kg, be equally divided into four groups, be numbered respectively A group, B group, C group, D group, every group is injected respectively C type bacillus perfringens toxoid vaccine dosage is 1ml, 3ml, 5ml, 7ml, and after two weeks, 8 pigs are all injected the C type bacillus perfringens extracellular toxin solution of 10 milliliters, observe 3-5 day, A group and B group pig are all dead, all healthy survivals of C group and D group, so immunizing dose is decided to be 5 milliliters.

Claims (1)

1. the preparation method of a piglet red dysentery C type bacillus perfringens toxoid vaccine, is characterized in that comprising the steps:

The strain of C type bacillus perfringens is inoculated on the blood plate culture medium, and 37 ℃ of anaerobism are cultivated the 36h recovery; Get a single colony inoculation after recovery in THIOGLYCOLLIC ACID salt fluid culture medium FT, 37 ℃ of anaerobism are cultivated 12h and are increased bacterium; To increase the bacterium culture and be inoculated in toxin producing medium according to inoculum concentration 5% volume ratio, 40 ℃ of anaerobism shaken cultivation 5h; Medium centrifugal, get supernatant, and the seitz filter filtration sterilization obtains extracellular toxin solution; In toxin soiutions, add outside the extracellular toxin liquor capacity than 0.3% formaldehyde, fully vibration mixes, 37 ℃, deactivation 8 days, during once obtain every the 5-6h jolting toxoid solution that deactivation is good; After white oil and span-80 are mixed with volume ratio 32:3, then add the aluminium stearate of white oil and span-80 mixed liquor mass ratio 2%, autoclaving obtains oil phase; In deactivation, in good toxoid solution, add the Tween-80 of volume ratio 2% to be mixed to get water; Oil phase and water, with volume ratio 1:1 emulsifying, are added to 0.01 gram thimerosal in every 100ml emulsion, after mixing, make C type bacillus perfringens toxoid vaccine;

Described blood plate medium component is: 100 ml distilled waters, 1 gram glucose, 3.8 gram bean agar powders and 5 milliliters of Sheep Bloods; Described toxin producing medium composition is: 100 milliliters of PBS buffer, 2 gram peptones, 1 gram dextrin, 2 gram yeast extracts and 1.2 gram L-arginines; The composition of described PBS buffer is: 100 ml deionized water, 0.8 gram sodium chloride, 0.02 gram potassium chloride, 0.358 gram disodium hydrogen phosphate dodecahydrate and 0.027 gram potassium dihydrogen phosphate.