CN105106946A - Oil emulsion vaccine for cattle and sheep foot rot and preparation method thereof - Google Patents

Abstract

The invention provides an oil emulsion vaccine for cattle and sheep foot rot and a preparation method thereof. The oil emulsion vaccine is prepared from an oil phase and a water phase in parts by weight of (1.5-3):1 by adding a 0.01% thimerosal solution, wherein the oil phase is prepared from autoclaved medicinal white oil, 10% Span-80 white oil mixed liquor and 10% mannitol monooleate white oil mixed liquor according to the ratio of (1.8-2.2):1:1; the water phase is prepared by dissolving 4-7% sucrose ester into a water solution containing 10% tween-80 and 10% polyoxyethylene castor oil derivatives and then mixing with an antigen solution according to the ratio of 1:(2.5-3); and the antigen solution is a bacterial solution obtained by carrying out subculture on fusobacterium necrophorum through a fusobacterium necrophorum subculture medium, carrying out amplification culture through a fusobacterium necrophorum amplification culture medium and then carrying out concentration and inactivation, wherein the bacterial count of the bacterial solution per milliliter is not lower than 4*10<10> CFU. The invention also provides the preparation method of the oil emulsion vaccine. The oil emulsion vaccine provided by the invention has the characteristics of low viscosity, good stability, good immune effect, small clinical adverse reaction and low cost.

Description

A kind of cattle and sheep foot rot oil emulsion vaccine and preparation method thereof

Technical field

The present invention relates to a kind of cattle and sheep foot rot vaccine and preparation method thereof, be specifically related to a kind of cattle and sheep foot rot oil emulsion vaccine and preparation method thereof.

Background technology

Foot rot (Footrot) is a kind of commonly encountered diseases of ruminant, in China except relatively low with northwest and Tibet, Inner Mongol milk claw ill sickness rate northeastward, all the other areas are especially in south, milch cow, goat, sheep foot rot are widely current, its sickness rate up to 8% ~ 20%, even 30% ~ 50%.Due to the factors such as unscientific feeding and management, bad environmental condition, puerperal disease, heredity cause milch cow and sheep sand crack, hoof leaks, at the bottom of hoof or heel ulcer equivalent damage, reduce hoof resistance, finally cause pathogenic bacterial infection, increase the weight of hoof damage.And cattle and sheep foot rot cure rate is low, mortality is high, treatment time is long, expenses for medicine is high, and treatment phase medication causes discarded milk and causes economic loss serious.Visible, cattle and sheep foot rot is the serious problems of puzzlement milch cow, sheep cultivation.

The cause of disease of milch cow and foot rot of sheep has multiple, existing aerobe has again anaerobe and amphimicrobe, most scholar thinks that the main pathogen of cattle and foot rot of sheep is actinomyces pseudonecrophorus (Fusobacteriumnecrophorum) and comb tumor bacteroid (Bacteroidesnodosus), and both generations mainly co-infection of foot rot causes.Wherein, actinomyces pseudonecrophorus is the most important cause of disease of cattle and sheep foot rot, and multiple organ suppuration pathological changes (disease such as Lemierre ' s syndrome, encephalitis as Hepar Bovis seu Bubali abscess, people) and other hoofs artiodactylous can be caused to infect.It is a kind of pleomorphism antibacterial that actinomyces pseudonecrophorus belongs to Fusobacterium (FusobacteriumKnorr1922), cultural character strictly anaerobic, the reports such as Feldman, in 14 strains from the isolated actinomyces pseudonecrophorus of the liver abscess of cattle, have the antigen group that 4 kinds different.

At present, prevent cattle both at home and abroad, the generation of foot rot of sheep mainly regularly adopts the dippings such as copper sulfate, zinc sulfate, paraformaldehyde or formalin and regular foot paring, reduces hoof damage etc.Treatment adopts copper sulfate or iodine wrapping, systemic injection antibiotic, but is permanently connected to ground due to its hoof, and be in Infection Status, therapeutic effect is poor.Have commodity inactivated vaccine abroad, but because foot rot cause of disease is complicated large in different geographical result of use difference.Oil emulsion vaccine is the inactivated vaccine that on domestic and international vaccine marketplaces, output is maximum; the immunizing dose that its immune performance is stablized, biological safety is high, less and less immune time just can reach and extend the immunostimulation time, make body obtain enough protection antibodies.And span-80 adds in white oil as oil phase by traditional oils emulsion vaccine in process of production, tween 80, aluminium stearate add in antigen aqueous solution as aqueous phase, the oil emulsion vaccine stability that the two mixing and emulsifying obtains is high, can obtain the immune antibody of lasting higher level after immunity; But this oil emulsion vaccine viscosity is large, the stress that inoculation causes is comparatively large, can form aseptic suppuration stove, occur swelling, scleroma at inoculation position, or forms granuloma, and immune effect is poor; Bad impact is also created on animal meat product quality simultaneously.As can be seen here, aluminium stearate content, oil phase, aqueous phase, oil water phase ratio, shear time, be key factor affect oil emulsion vaccine viscosity from the quality of oil, oil emulsion vaccine also exist that bacterial strain mass propgation is difficult, the shortcoming such as immune effect and the bad side reaction that causes.Therefore, explore and a kind of there is the little cattle and sheep foot rot oil emulsion vaccine of low viscosity, good stability, good immune effect, side effect, will have great importance to the sound development of cattle, sheep aquaculture and China's animal husbandry.

Summary of the invention

Technical problem to be solved by this invention is, provides a kind of and has low viscosity, good stability, good immune effect, clinical adverse is little, cost is low cattle and sheep foot rot oil emulsion vaccine and preparation method thereof.

The technical scheme that the present invention solves the employing of its technical problem is, a kind of cattle and sheep foot rot oil emulsion vaccine, be 1.5-3: 1 (preferably 2: 1) by weight by oil phase and aqueous phase, final concentration is that 0.005% ~ 0.01% (preferably 1%) thimerosal solution is formed, wherein, described oil phase by autoclaved medicinal white oil, the Span-80 white oil mixed liquor of 10%, the Mannitol monooleate. white oil mixed liquor of 10% according to the composition of proportions of 1.8 ~ 2.2: 1: 1 (preferably 2: 1: 1); The sucrose ester of 4% ~ 7% (preferably 5.7%) is dissolved in containing 10% Tween 80 and 10% castor oil derivatives aqueous solution by described aqueous phase, then with the composition of proportions of antigenic solution according to 1:2.5 ~ 3 (preferred 1:2.8); Described antigenic solution is gone down to posterity through actinomyces pseudonecrophorus Secondary Culture culture medium by actinomyces pseudonecrophorus, after actinomyces pseudonecrophorus amplification culture culture medium amplification culture, concentrates, and deactivation obtains every milliliter of bacterium liquid bacterium number and is not less than 4 × 10 ¹⁰the bacterium liquid of CFU.

Further, described actinomyces pseudonecrophorus Secondary Culture culture medium is cattle brain-heart infusion medium; Described actinomyces pseudonecrophorus amplification culture culture medium is improvement brain-heart-infusion medium.

The technical scheme that the present invention solves the employing of its technical problem is further that a kind of preparation method of cattle and sheep foot rot oil emulsion vaccine, comprises the following steps:

(1) preparation of production seed: adopt actinomyces pseudonecrophorus Secondary Culture culture medium first actinomyces pseudonecrophorus to be carried out first order seed breeding, after qualified, then carry out secondary seed breeding and obtains secondary seed;

(2) preparation of antigenic solution: 5 ~ 10% inoculation secondary seeds by culture medium total amount in the actinomyces pseudonecrophorus amplification culture culture medium in fermentation tank are cultivated, results bacterium liquid, sampling is checked purely, should be pure, make count plate, viable count should be not less than 4 × 10 simultaneously ⁹cFU; Then bacterium liquid concentrates, and makes every milliliter of bacterium liquid bacterium number be not less than 4 × 10 ¹⁰cFU; Deactivation again;

(3) preparation of oil phase: autoclaved medicinal white oil, the Span-80 white oil mixed liquor of 10%, the Mannitol monooleate. white oil mixed liquor of 10% are mixed according to the ratio of 1.8 ~ 2.2: 1: 1,35 ~ 38 DEG C of water-baths for a moment, fully shake up, obtain oil phase;

(4) preparation of aqueous phase: be dissolved in by the sucrose ester of 4% ~ 7% containing 10% Tween 80 and 10% castor oil derivatives aqueous solution, then mixes with the ratio of antigenic solution according to 1:2.5 ~ 3, obtains aqueous phase; Wherein, castor oil derivatives is a series of materials be obtained by reacting by not commensurability oxirane and Oleum Ricini or castor oil hydrogenated, two kinds of Main Derivatives are CREMOPHORE EL (EL-35) and polyoxyl 40 hydrogenated castor oil (RH-40), its HLB value is 13 ~ 15, be mainly used in solubilizing agent and the emulsifying agent of water-insoluble drug or other fat-soluble medicines, haemolysis can be prevented, and urine drains can be passed through very soon, Chinese Pharmacopoeia does not record, and includes in European Pharmacopoeia and American Pharmacopeia.TW-80 has taken in multinational pharmacopeia as solubilizing agent, emulsifying agent and stabilizing agent, along with going deep into of pharmaceutical research in recent years, people recognize that Tween 80 is the material with certain biology and pharmacologically active, and relevant to the untoward reaction that clinical drug occurs, and there is slight haemolysis when concentration is 4%.

(5) emulsifying: by oil phase and aqueous phase be by weight 1.5 ~ 3: 1 carry out shearing 10 ~ 15min after, before termination, add 2% thimerosal solution, make its ultimate density be 0.005% ~ 0.01%.

Further, the preparation method of described cattle and sheep foot rot oil emulsion vaccine, comprises the following steps:

(1) preparation of seed: actinomyces pseudonecrophorus is inoculated in cattle brain-heart infusion medium, 37 ~ 39 DEG C of Anaerobic culturel 48 hours, sampling inoculation brain-heart-infusion agar plate, 37 ~ 39 DEG C of Anaerobic culturel 48 ~ 96 hours, purely after the assay was approved, as first order seed; Get first order seed and be inoculated in pH7.6 brain-heart-infusion medium, 37 ~ 39 DEG C of Anaerobic culturel 36 ~ 48 hours, pure inspection is done in sampling, qualified rear as secondary seed;

(2) preparation of vaccine antigen solution: the improvement brain-heart-infusion medium in fermentation tank is preheated to 37 ~ 39 DEG C, by 5 ~ 10% inoculation secondary seeds of culture medium total amount, cultivates 10 hours for 37 ~ 39 DEG C; Results bacterium liquid, sampling is checked purely, should be pure, and make count plate, viable count should be not less than 4 × 10 simultaneously ⁹cFU; With the hollow fiber membrane ultrafiltration device of 0.22 μm, bacterium liquid qualified for pure inspection is concentrated, make every milliliter of bacterium liquid bacterium number be not less than 4 × 10 ¹⁰cFU; Regulate culture fluid pH to 6.8 ~ 7.2, add formalin, put 37 DEG C of deactivations 24 hours by 0.3% of bacterium liquid total amount, period stirred 1 time every 6 ~ 8 hours;

(3) preparation of oil phase: autoclaved medicinal white oil, the Span-80 white oil mixed liquor of 10%, the Mannitol monooleate. white oil mixed liquor of 10% are mixed according to the ratio of 2: 1: 1,37 DEG C of water-baths for a moment, fully shake up, obtain oil phase;

(4) preparation of aqueous phase: be dissolved in by the sucrose ester of 5.7% containing 10% Tween 80 and 10% polyoxyl 40 hydrogenated castor oil aqueous solution, then mixes with the ratio of vaccine antigen solution according to 1:2.8, obtains aqueous phase;

(5) emulsifying: get oil phase 2 parts and be put in colloid mill, slow rotation stirs, and slowly adds aqueous phase 1 part simultaneously, stirs 10 ~ 15 minutes after adding with 8000r/min again, adds 2% thimerosal solution, make its ultimate density be 0.01% before termination is stirred.

The present invention, by medium optimization, filters out the condition of actinomyces pseudonecrophorus amplification culture, reduces the toxigenic capacity of actinomyces pseudonecrophorus; On vaccine oil emulsion phase formula, select high-quality and efficient emulsifying agent sucrose ester and monoglyceride composite, substitute aluminium stearate, reduce the side reaction of injecting and causing, in addition, the sucrose ester of high hlb also has the effect suppressing thermostability sporeformer, and this is also very favourable to the storage of product; On aqueous phase formula, reduce the consumption of TW-80, increase polyoxyl 40 hydrogenated castor oil, reduce the clinical adverse that may occur.

The present invention has better specific aim to endemy cattle and sheep foot rot control, can reach effective protective rate.

Accompanying drawing explanation

Fig. 1 is particle size distribution figure.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is illustrated further.

Embodiment 1: the preparation of cattle and sheep foot rot oil emulsion vaccine

1, strain: actinomyces pseudonecrophorus is open in the following documents: Xie Jing, Liao Dangjin, Wang Ming, He Wanping, Cao Ye, Zhao Sujun, Zeng Wei, Li Jiangling, Pan Baoliang, Ye Yonggang, Luo Dandan. " Chinese Veterinary Journal ", the investigation of Sichuan milk cattle footrot the main pathogenic fungi .2013,4:35-37; Xie Jing, Liao Dangjin, Wang Ming, Ye Yonggang, Cao Ye, Zhao Sujun, Li Jiangling, Luo Dandan, Wang Qiushi, Pan Baoliang. milch cow source actinomyces pseudonecrophorus strain from Sichuan isolation identification and biological property analysis, " herding and veterinary ", 2013,45 (3): 64-66.

Actinomyces pseudonecrophorus is separated from Sichuan Province's large-scale milch cow farms, is the polymorphic bacillus of Gram-negative, and tissue smear or children bacterium in age are long filament shape, and artificial culture is shorter individuality, shaft-like or club shape more than 24 hours thalli morphologies.This bacterium strictly anaerobic, grows in lonely liver bouillon, be just even muddiness, and rear change clearly and produce flocculent deposit.On Cor Bovis seu Bubali brain infusion agar plate, 37 ~ 39 DEG C of Anaerobic culturel 48 ~ 96 hours, form canescence or translucent, neat in edge, smooth colony, on egg yolk agar plate, under anaerobic cultivate, and form " fried egg " sample, the positive bacterium colony of lipase.

2, vaccine manufacture and the inspection of semifinished product (1) actinomyces pseudonecrophorus Secondary Culture culture medium are cattle brain-heart infusion medium, and concrete formula is as table 1.

Table 1-actinomyces pseudonecrophorus Secondary Culture culture medium prescription (1L)

Method for making is as follows:

1. the preparation of Medulla Bovis seu Bubali, beef heart infusion: fascia will be removed and the Medulla Bovis seu Bubali rubbed and each 500 grams of Cor Bovis seu Bubali, put respectively in the triangular flask of 2000ml, each adding distil water 1000ml, put 4 DEG C to spend the night, next day skims oil slick, puts respectively in 45 DEG C of water-baths and heats 1h, then boil 30 minutes, with filtered through gauze (Medulla Bovis seu Bubali immersion not easily filtering, 4 DEG C of refrigerators can be put leave standstill, after making contamination precipitation, draw supernatant), mend to 1000ml with distilled water, for subsequent use after autoclaving.

2., after being mixed by mentioned component, heating for dissolving, adjusts pH for 7.6 ~ 7.8 with sodium hydroxide solution after cooling, for subsequent use with 116 DEG C of sterilizings, 30 ~ 40 minutes autoclavings after subpackage.

(3) add 1.5%-2% agar in above-mentioned brain-heart-infusion and be brain-heart-infusion agar.

(2) actinomyces pseudonecrophorus amplification culture culture medium is the cattle brain-heart infusion medium of improvement, and concrete formula is as table 2.

Table 2-actinomyces pseudonecrophorus amplification culture culture medium prescription (1L)

(3) production Spawn preparation

1. first order seed breeding and qualification: actinomyces pseudonecrophorus is inoculated in cattle brain-heart infusion medium, 37 ~ 39 DEG C of Anaerobic culturel 48 hours, sampling inoculation brain-heart-infusion agar plate, 37 ~ 39 DEG C of Anaerobic culturel 48 ~ 96 hours, purely after the assay was approved, as first order seed.25 DEG C of anaerobism are preserved, and are no more than 7.

2. secondary seed breeding: get first order seed and be inoculated in pH7.6 brain-heart-infusion medium, 37 ~ 39 DEG C of Anaerobic culturel 36 ~ 48 hours, pure inspection is done in sampling, qualified rear as secondary seed.25 DEG C of preservations, the operating period is no more than 2.

(4) the seedling preparation of bacterium liquid

1. seedling culture medium: improvement brain-heart-infusion medium.

2. seedling bacterium liquid is cultivated: the improvement brain-heart-infusion medium in fermentation tank is preheated to 37 ~ 39 DEG C, by 5 ~ 10% inoculation secondary seeds of culture medium total amount, cultivates 10 hours for 37 ~ 39 DEG C.Results bacterium liquid, puts 2 ~ 8 DEG C and saves backup.

3. pure inspection and count plate: cultivate after terminating, sampling is checked purely, should be pure.Make count plate, viable count should be not less than 4 × 10 simultaneously ⁹cFU.

4. concentrate: with the hollow fiber membrane ultrafiltration device of 0.22 μm, bacterium liquid qualified for pure inspection is concentrated, make every milliliter of bacterium liquid bacterium number be not less than 4 × 10 ¹⁰cFU.

5. deactivation: regulate culture fluid pH to 6.8 ~ 7.2, add formalin, put 37 DEG C of deactivations 24 hours by 0.3% of bacterium liquid total amount, period stirred 1 time every 6 ~ 8 hours.

6. deactivation inspection: sampling is inoculated in brain-heart-infusion agar plate, 37 ~ 39 DEG C of Anaerobic culturel 48 ~ 96 hours, answer asepsis growth.

(5) preparation of vaccine

1. oil phase preparation: autoclaved medicinal white oil, the Span-80 white oil mixed liquor of 10%, the Mannitol monooleate. white oil mixed liquor of 10% are mixed according to the ratio of 2: 1: 1,37 DEG C of water-baths for a moment, fully shake up.

2. aqueous phase preparation: be dissolved in by sucrose ester (final concentration 5.7%) containing 10% Tween 80 and 10% polyoxyl 40 hydrogenated castor oil aqueous solution, then this solution and antigenic solution mix for subsequent use according to the ratio of 1:2.8.

3. emulsifying: get oil phase 2 parts and be put in colloid mill, starts motor slow rotation and stirs, slowly add aqueous phase 1 part simultaneously, stir 10-15 minute again after adding with 8000r/min, before termination is stirred, add 2% thimerosal solution, and make its ultimate density be ten thousand/.

4. the inspection of semifinished product: undertaken by existing " Chinese veterinary pharmacopoeia " annex, answer asepsis growth.

(5) subpackage: after qualified without inspection, quantitative separating, and adhesive label, put 2 ~ 8 DEG C of preservations.

Embodiment 2: cattle and sheep foot rot oil emulsion vaccine finished product check analysis

1, the cattle and sheep foot rot oil emulsion vaccine finished product prepared of the present embodiment

(1) physical behavior

Outward appearance: milky Emulsion.

Dosage form: water-in-oil type.Get a clean suction pipe, draw in a small amount of vaccine instillation cold water, except the 1st, all the other equal indiffusion.

Stability: draw vaccine 10ml and add in conical centrifuge tube, centrifugal 15 minutes with 4000r/min minute, the aqueous phase of separating out at the bottom of pipe answers≤0.5ml, and result of the test is 0.05ml.Oil emulsion is placed on 4 DEG C, room temperature, 37 DEG C of its breakdown of emulsion situations of environment perception see the following form 3.

Table 3-oil emulsion stability observing

Viscosity: with 1ml suction pipe (lower internal diameter is 1.2mm, and upper internal diameter is 2.7mm), draws about 25 DEG C vaccine 1ml, make its vertical natural flow out, 3 groups of times required for 0.4ml of flowing out are respectively 4.7 seconds, 4.8 seconds, 4.7 seconds.By NDJ-5S rotary viscosity measurement oil emulsion viscosity, meansigma methods is 39, and viscosity is low.

Particle diameter is tested: aqueous emulsion is prepared rear LS-POP (6) laser fineness gage and measure size, result is as table 4, Fig. 1, and particle diameter is less than 500um, meets oil emulsion Particle size requirements.

Table 4-particle size distribution table

(2) steriling test: undertaken by existing " Chinese veterinary pharmacopoeia " annex, asepsis growth.

(3) safety verification: with body weight 1.5 ~ 2.0kg rabbit 4, each intramuscular injection vaccine 5.0ml, observe 10, all should be good for work, injection site should not necrose.

(4) efficacy test: with body weight 1.5 ~ 2.0kg rabbit 4, each intramuscular injection vaccine 1.0ml, immunity is after 21 days, and together with the contrast rabbit 4 that condition is identical, injection lethal dose bacterium liquid, contrast rabbit is all dead, and immunize rabbit at least protects 3 for qualified.

(5) formaldehyde, the thimerosal determination of residual amount: undertaken by existing " Chinese veterinary pharmacopoeia " annex, the pertinent regulations of veterinary biologics general rule should be met.

2, the finished product of cattle and sheep foot rot oil emulsion vaccine prepared of conventional method

Prepared by oil phase: white oil, span-80 and aluminium stearate are according to the proportions of 94ml:6ml:1.5g.The white oil that first takes a morsel heats, and is added by aluminium stearate wherein, fully mixes dissolving, then mixs homogeneously with residue white oil and whole span-80, and 121.5Pl16 DEG C of autoclaving 30min, namely obtains oil phase.

Prepared by aqueous phase: a certain amount of Tween 80 is dissolved in antigenic solution, makes it account for 2.5% of vaccine cumulative volume.

Emulsifying: get oil phase 2 parts and be put in colloid mill, starts motor slow rotation and stirs, slowly add aqueous phase 1 part simultaneously, stir 10-15 minute again after adding with 8000r/min, before termination is stirred, add 2% thimerosal solution, and make its ultimate density be ten thousand/.

The inspection of semifinished product: undertaken by existing " Chinese veterinary pharmacopoeia " annex, answer asepsis growth.

Subpackage: after qualified without inspection, quantitative separating, and adhesive label, put 2 ~ 8 DEG C of preservations.

Finished product through cattle and sheep foot rot oil emulsion vaccine prepared by inspection conventional method:

(1) physical behavior

Outward appearance: milky Emulsion.

Dosage form: water-in-oil type.Get a clean suction pipe, draw in a small amount of vaccine instillation cold water, except the 1st, all the other equal indiffusion.

Stability: draw vaccine 10ml and add in conical centrifuge tube, centrifugal 15 minutes with 4000r/min minute, the aqueous phase of separating out at the bottom of pipe answers≤0.5ml, and result of the test is 0.1ml.Oil emulsion is placed on 4 DEG C, room temperature, 37 DEG C of its breakdown of emulsion situations of environment perception see the following form 3.

Table 5-oil emulsion stability observing

Viscosity: with 1ml suction pipe (lower internal diameter is 1.2mm, and upper internal diameter is 2.7mm), draws about 25 DEG C vaccine 1ml, make its vertical natural flow out, 2 groups of times required for 0.4ml of flowing out are respectively 5.7 seconds, 6.1 seconds.By NDJ-5S rotary viscosity measurement oil emulsion viscosity, meansigma methods is 52, and viscosity is low.

(2) steriling test: undertaken by existing " Chinese veterinary pharmacopoeia " annex, asepsis growth.

(3) safety verification: with body weight 1.5 ~ 2.0kg rabbit 4, each intramuscular injection vaccine 5.0ml, observe 10, all should be good for work, injection site should not necrose.

(4) efficacy test: with body weight 1.5 ~ 2.0kg rabbit 4, each intramuscular injection vaccine 1.0ml, immunity is after 21 days, and together with the contrast rabbit 4 that condition is identical, injection lethal dose bacterium liquid, contrast rabbit is all dead, and immunize rabbit at least protects 3 for qualified.

(5) formaldehyde, the thimerosal determination of residual amount: undertaken by existing " Chinese veterinary pharmacopoeia " annex, the pertinent regulations of veterinary biologics general rule should be met.

From finished product results of comparison, cattle and sheep foot rot oil emulsion vaccine end properties prepared by the present embodiment is better than oil emulsion vaccine finished product prepared by conventional method.

Embodiment 3: Immunoprotection test

Matched group 15, not immune, experimental group 15 sheep every subcutaneous vaccination inactivated vaccine 2ml, exempt from for the 22nd day two.Within 35th day, use 7 × 10 respectively ⁹/ only, 3.5 × 10 ⁸/ only with 3.5 × 10 ⁷/ various dose bacterium liquid carries out counteracting toxic substances to corresponding experimental group and matched group sheep, observes 7 days, record sheep hoof infection conditions.According to sheep hoof lesion extent after counteracting toxic substances, when counteracting toxic substances dosage is 7 × 10 ⁹/ time, immune group hoof damage total mark reduce by 4 points, protective efficacy is 26.67%; When counteracting toxic substances dosage is 3.5 × 10 ⁸/ time, immune group hoof damage total mark reduce by 6 points, protective efficacy is 40%, when counteracting toxic substances dosage is 3.5 × 10 ⁷/ time, matched group sickness rate is 40%, and immune group sickness rate is 0.

Claims (5)

1. a cattle and sheep foot rot oil emulsion vaccine, it is characterized in that, be 1.5 ~ 3: 1 by weight by oil phase and aqueous phase, final concentration is that 0.005% ~ 0.01% thimerosal solution is formed, wherein, described oil phase by autoclaved medicinal white oil, the Span-80 white oil mixed liquor of 10%, the Mannitol monooleate. white oil mixed liquor of 10% according to 1.8 ~ 2.2: 1: 1 composition of proportions; The sucrose ester of 4% ~ 7% is dissolved in containing 10% Tween 80 and 10% castor oil derivatives aqueous solution by described aqueous phase, then with the composition of proportions of antigenic solution according to 1:2.5 ~ 3; Described antigenic solution is gone down to posterity through actinomyces pseudonecrophorus Secondary Culture culture medium by actinomyces pseudonecrophorus, after actinomyces pseudonecrophorus amplification culture culture medium amplification culture, concentrates, and deactivation obtains every milliliter of bacterium liquid bacterium number and is not less than 4 × 10 ¹⁰the bacterium liquid of CFU.

2. cattle and sheep foot rot oil emulsion vaccine according to claim 1, it is characterized in that, be 2: 1 by weight by oil phase and aqueous phase, final concentration is that 0.01% thimerosal solution is formed, wherein, described oil phase by autoclaved medicinal white oil, the Span-80 white oil mixed liquor of 10%, the Mannitol monooleate. white oil mixed liquor of 10% according to 2: 1: 1 composition of proportions; The sucrose ester of 5.7% is dissolved in containing 10% Tween 80 and 10% castor oil derivatives aqueous solution by described aqueous phase, then with the composition of proportions of antigenic solution according to 1:2.8.

3. cattle and sheep foot rot oil emulsion vaccine according to claim 1 and 2, is characterized in that, described actinomyces pseudonecrophorus Secondary Culture culture medium is cattle brain-heart infusion medium; Described actinomyces pseudonecrophorus amplification culture culture medium is improvement brain-heart-infusion medium.

4. a preparation method for the cattle and sheep foot rot oil emulsion vaccine as described in one of claims 1 to 3, is characterized in that, comprise the following steps:

(1) preparation of production seed: adopt actinomyces pseudonecrophorus Secondary Culture culture medium first actinomyces pseudonecrophorus to be carried out first order seed breeding, after qualified, then carry out secondary seed breeding and obtains secondary seed;

(2) preparation of antigenic solution: 5 ~ 10% inoculation secondary seeds by culture medium total amount in the actinomyces pseudonecrophorus amplification culture culture medium in fermentation tank are cultivated, results bacterium liquid, sampling is checked purely, should be pure, make count plate, viable count should be not less than 4 × 10 simultaneously ⁹cFU; Then bacterium liquid concentrates, and makes every milliliter of bacterium liquid bacterium number be not less than 4 × 10 ¹⁰cFU; Deactivation again;

(3) preparation of oil phase: autoclaved medicinal white oil, the Span-80 white oil mixed liquor of 10%, the Mannitol monooleate. white oil mixed liquor of 10% are mixed according to the ratio of 1.8 ~ 2.2: 1: 1,35 ~ 38 DEG C of water-baths for a moment, fully shake up, obtain oil phase;

(4) preparation of aqueous phase: be dissolved in by the sucrose ester of 5% ~ 6% containing 10% Tween 80 and 10% castor oil derivatives aqueous solution, then mixes with the ratio of antigenic solution according to 1:2.5 ~ 3, obtains aqueous phase;

(5) emulsifying: by oil phase and aqueous phase be by weight 2 ~ 3: 1 carry out shearing 10 ~ 15min after, before termination, add 2% thimerosal solution, make its ultimate density be 0.005% ~ 0.01%.

5. the preparation method of cattle and sheep foot rot oil emulsion vaccine according to claim 4, is characterized in that, comprise the following steps:

(1) preparation of seed: actinomyces pseudonecrophorus is inoculated in cattle brain-heart infusion medium, 37 ~ 39 DEG C of Anaerobic culturel 48 hours, sampling inoculation brain-heart-infusion agar plate, 37 ~ 39 DEG C of Anaerobic culturel 48 ~ 96 hours, purely after the assay was approved, as first order seed; Get first order seed and be inoculated in pH7.6 brain-heart-infusion medium, 37 ~ 39 DEG C of Anaerobic culturel 36 ~ 48 hours, pure inspection is done in sampling, qualified rear as secondary seed;

(2) preparation of vaccine antigen solution: the improvement brain-heart-infusion medium in fermentation tank is preheated to 37 ~ 39 DEG C, by 5 ~ 10% inoculation secondary seeds of culture medium total amount, cultivates 10 hours for 37 ~ 39 DEG C; Results bacterium liquid, sampling is checked purely, should be pure, and make count plate, viable count should be not less than 4 × 10 simultaneously ⁹cFU; With the hollow fiber membrane ultrafiltration device of 0.22 μm, bacterium liquid qualified for pure inspection is concentrated, make every milliliter of bacterium liquid bacterium number be not less than 4 × 10 ¹⁰cFU; Regulate culture fluid pH to 6.8 ~ 7.2, add formalin, put 37 DEG C of deactivations 24 hours by 0.3% of bacterium liquid total amount, period stirred 1 time every 6 ~ 8 hours;

(3) preparation of oil phase: autoclaved medicinal white oil, the Span-80 white oil mixed liquor of 10%, the Mannitol monooleate. white oil mixed liquor of 10% are mixed according to the ratio of 2: 1: 1,37 DEG C of water-baths for a moment, fully shake up, obtain oil phase;

(4) preparation of aqueous phase: be dissolved in by the sucrose ester of 5.7% containing 10% Tween 80 and 10% polyoxyl 40 hydrogenated castor oil aqueous solution, then mixes with the ratio of vaccine antigen solution according to 1:2.8, obtains aqueous phase;

(5) emulsifying: get oil phase 2 parts and be put in colloid mill, slow rotation stirs, and slowly adds aqueous phase 1 part simultaneously, stirs 10 ~ 15 minutes after adding with 8000r/min again, adds 2% thimerosal solution, make its ultimate density be 0.01% before termination is stirred.