CN104248755A - Haemophilus parasuis disease vaccine composition, preparation method and application thereof - Google Patents
Haemophilus parasuis disease vaccine composition, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a Haemophilus parasuis disease vaccine composition, which contains an immune dose of capsule removed Haemophilus parasuis whole bacterium antigen and a veterinarily acceptable adjuvant. The capsule removed Haemophilus parasuis disease vaccine composition provided by the invention not only provides good immunoprotection to haemophilus parasuis of one same serotype, but also can have good preventive and therapeutic effects on Haemophilus parasuis diseases caused by currently epidemic Haemophilus parasuis of a variety of serotypes in China.
Description
Technical field
The present invention relates to a kind of Haemophilus parasuis vaccine, and its preparation method and application.
Background technology
Haemophilus parasuis, also known as multiple cellulosic oromeningitis and arthritis, is a kind of clinically with the infectious disease that body temperature rising, arthroncus, breathing suffering, polyserositis, arthritis and high mortality are feature.This disease is also called pig leather and draws Ze Shi disease (Glsser ' s Disease), be once once considered to by stress be caused sporadic disease, be proved to be afterwards caused by haemophilus parasuis (Haemophilus parasuis, Hps).
Haemophilus parasuis can affect the young pig from 2 week age to 4 monthly ages, and mainly fall ill with the child care stage after wean, usually see the pig in 5 ~ 8 week age, sickness rate is generally 10% ~ 15%, and time serious, mortality rate can reach 50%.Main clinic symptoms shows as cough, dyspnea, become thin, walk lamely and by hair slightly disorderly; Mainly cut open inspection pathological changes and show as fibrinous pleurisy, pericarditis, peritonitis, arthritis and meningitis etc.In addition, haemophilus parasuis also can cause septicemia, and may leave sequela after actute infection, i.e. Sow abortion, the chronic limping of boar.Along with the development of world's pig industry, this disease has become the typical bacteria disease affecting pig industry in global range.
Haemophilus parasuis belongs to Gram-negative bacillus pumilis, and form is changeable.As far back as 1910, Germany scientist Glsser just found contacting between the polyserositis of haemophilus parasuis and pig and arthritis.Western developed country is on a large amount of basis being separated haemophilus parasuis, find that the serotype complexity of this bacterium is various, by Kieletein-Rapp-Gabriedson (KRG) agar diffusion serotype method, at least haemophilus parasuis can be divided into 15 serotypes, separately have the separated strain serotype of more than 20% to determine; According to the seroepidemiological survey of the countries such as Germany, the U.S., Canada, Japan and Spain, the most popular with serotype 4,5,13 type; According to the qualification of China's seroepidemiological survey and isolated strains, the most popular with 4,5 types, be secondly 12 types, 13 types.
In recent years, between each haemophilus parasuis sero-group, Immunogenicity power is strong, and the cause of disease sero-group of the Haemophilus parasuis that the vaccine strains sero-group of inoculation is popular with there and then does not conform to, and brings certain difficulty to the prevention and therapy of this disease.Haemophilus parasuis 4 type that pig Haemophilus parasuis 1 type that the domestic vaccine for Haemophilus parasuis immunoprophylaxis has Boehringer Ingelheim animal health (U.S.) company limited to produce at present Haemophilus parasuis inactivated vaccine (Z-1517 strain), the biological large pharmaceutical factory of Spain Hai Bolai produce and 6 type inactivated vaccines and Hua Zhong Agriculture University, Wuhan Keqian Animal Biological Products Co., Ltd., Zhongmu Industry Co., Ltd produce and 5 type inactivated vaccines.The application of these vaccines serves certain preventive effect to Haemophilus parasuis.But the cause of disease epidemic strain of Haemophilus parasuis and various serotype infect and have made existing vaccine be difficult to reach the immune effect of expection.Owing to there is no the preparation of suitable strain, add to there is interference between each antigen, provide a kind of vaccine effectively can preventing and treating the Haemophilus parasuis caused by the new cause of disease of haemophilus parasuis to be aquaculture problem in the urgent need to address.
Summary of the invention
In view of this, main purpose of the present invention is to provide a kind of modified model Haemophilus parasuis vaccine combination solving current technological deficiency, and described Haemophilus parasuis vaccine combination comprises the haemophilus parasuis full bacterium antigen and veterinarily acceptable carrier of removing pod membrane.
The haemophilus parasuis full bacterium antigen of described removal pod membrane comprises the street strain of clinical separation well known to those skilled in the art, comprises the full bacterium antigen that 1 ~ 15 serotype identified at present removes pod membrane.
Preferably, the full bacterium antigen of haemophilus parasuis removing pod membrane is deactivated form.The haemophilus parasuis full bacterium antigen of the removal pod membrane after deactivation and veterinarily acceptable carrier are prepared and can be obtained a kind of haemophilus parasuis inactivated vaccine.Preferably, the full bacterium antigen of haemophilus parasuis of the present invention is from haemophilus parasuis serum 4 type antigen, 5 type antigens or 12 type antigens.
More preferably, the full bacterium antigen of haemophilus parasuis of the present invention is selected from haemophilus parasuis serum 4 type bacterial strain JS strain, the ZJ strain of haemophilus parasuis Serotype 5 bacterial strain or haemophilus parasuis serum 12 type bacterial strain HeB strain.
Haemophilus parasuis serum 4 type (Haemophilus Parasuis, HPs) bacterial strain JS strain, carries out preservation, preservation date in China typical culture collection center (CCTCC): on May 18th, 2011, preserving number is CCTCC No:M2011172.
Haemophilus parasuis Serotype 5 (Haemophilus Parasuis, HPs) bacterial strain ZJ strain, carries out preservation, preservation date in China typical culture collection center (CCTCC): on May 18th, 2011, preserving number is CCTCC No:M2011173.
Haemophilus parasuis serum 12 type (Haemophilus Parasuis, HPs) bacterial strain HeB strain, carries out preservation, preservation date in China typical culture collection center (CCTCC): on May 18th, 2011, preserving number is CCTCC No:M2011174.
Preferably, the full bacterium antigenic content of haemophilus parasuis described in vaccine combination of the present invention is 10
8cFU/ml ~ 10
10cFU/ml; More preferably, the full bacterium antigenic content of described haemophilus parasuis is 10
9cFU/ml.
Term used herein " vaccine " or " vaccine combination " are used interchangeably, and refer to the pharmaceutical composition comprising at least one immunogenic substance of induce immune response in animal.Vaccine or vaccine combination can comprise other compositions typical of vaccine or vaccine combination in addition, such as adjuvant etc.
Term used herein " antigenic content " refers to the content of the viable count before the full bacterium antigens inactive of described haemophilus parasuis.
Term used herein " inactivated vaccine ", also referred to as inactivated vaccines, refers to as antigen to produce the suspension of the inactivation of bacterial of immunity.The example of inactivated vaccine comprises whole-bacterial-vaccine and cracking type vaccine.Use known method can produce inactivated vaccine easily.Such as, by full bacterium inactivated vaccine can be obtained with formalin process antibacterial.
Term used herein " carrier " refers to the diluent of administration together with bacterium antigen complete in described haemophilus parasuis, adjuvant, excipient or solvent etc.
Preferably, described carrier is adjuvant, described adjuvant is white-oil adjuvant, aluminium hydroxide gel, Montanide IMS1313VG based on carbomer (Carbomer), Montanide IMS251C VG, Montanide ISA15A VG or Montanide GEL PR polymer, mineral oil, propolis, ISA206, ISA760VG, aluminium hydroxide, aluminum phosphate, saponin, vegetable oil, ethyl oleate, two (caprylic/capric) propylene glycol ester, three (caprylic/capric) glyceride, two oleic acid propylene glycol esters, isostearate, sorbitan, mannide, glycerol, polyglycereol, propylene glycol, oleic acid, isostearic acid, castor oil acid, hydroxy stearic acid ester, Pluronic L121, acrylic or methacrylic acid polymer, maleic anhydride and alkenyl derivative copolymer, the compositions of one or more of the polymer of the acrylic or methacrylic acid that polyalkenyl ether that is sugared or polyhydric alcohol is cross-linked.
More preferably, described adjuvant is the one or more combination thing in white-oil adjuvant, aluminium hydroxide gel or Montanide IMS1313VG, Montanide IMS251C VG based on carbomer (Carbomer), Montanide ISA15A VG and Montanide GEL PR polymer.
Preferably, described haemophilus parasuis full bacterium antigen is described vaccine combination 50% ~ 90% percent by volume.
Preferably, vaccine combination of the present invention can be prepared into peroral dosage form or non-oral dosage forms.More preferably, described vaccine combination is prepared into the non-oral dosage forms given by Intradermal, muscle, intraperitoneal, intravenous, subcutaneous, intranasal or epidural pathways.
Another object of the present invention is the preparation method providing Haemophilus parasuis inactivated vaccine as above, comprises and cultivates haemophilus parasuis bacterial strain; Remove pod membrane, and deactivation, concentrated; Antigenic component is aided with in proportion carrier and is prepared into vaccine.Specifically comprise the steps:
(1) breeding of haemophilus parasuis spawn culture is obtained bacterium liquid;
(2) the bacterium liquid described cultivation obtained removes pod membrane;
(3) the bacterium liquid of described removal pod membrane is carried out deactivation, and concentrated bacterium liquid;
(4) bacterium liquid also concentrated for described deactivation and veterinarily acceptable carrier are carried out emulsifying.
Preferably, remove pod membrane described in above-mentioned steps (2) within 30 minutes, to realize by bacterium liquid being placed in 60 DEG C of water-baths.
Preferably, concentrated employing ultrafiltration described in above-mentioned steps (3), cycles of concentration is 3 times.
Preferably, deactivation described in above-mentioned steps (3) adopts formalin-inactivated method; More preferably, adopt the final concentration of formalin to be 0.1% ~ 0.3%(v/v), inactivation time is 24 ~ 48h, and inactivation temperature is 37 DEG C.
In the preparation process of above-mentioned modified model Haemophilus parasuis inactivated vaccine, to the cultured bacterium liquid sampling of step (1), carry out the inspection of pure property and count plate; Deactivation inspection and safety verification are carried out in the bacterium liquid sampling good to step (3) deactivation; Steriling test, safety verification and efficacy test are carried out to the Haemophilus parasuis inactivated vaccine that step (4) obtains, to guarantee safety, the reliability of vaccine.
Another object of the present invention is to provide the application of Haemophilus parasuis vaccine combination as above in the medicine preparing prevention and therapy haemophilus parasuis infection relevant disease.
Term used herein " haemophilus parasuis infection relevant disease " refers to infect by haemophilus parasuis the disease caused.Its example comprises main clinic symptoms and shows as cough, dyspnea, becomes thin, to walk lamely and by hair slightly disorderly; Mainly cut open inspection pathological changes and show as fibrinous pleurisy, pericarditis, peritonitis, arthritis and meningitis etc.In addition, haemophilus parasuis also can cause septicemia, and may leave sequela after actute infection, i.e. Sow abortion, the chronic limping of boar.But be not limited thereto.
Term used herein " prevention " refers to by giving suppress haemophilus parasuis to infect according to vaccine combination of the present invention or postpone all behaviors of seizure of disease.Term " treatment " refers to all behaviors making haemophilus parasuis infect the symptom caused to alleviate or take a turn for the better by giving vaccine combination according to the present invention.
Technique effect
Inventor unexpectedly passes through to remove pod membrane by being separated the haemophilus parasuis obtained, prepare Haemophilus parasuis inactivated vaccine, by test find this inactivated vaccine not only immunogenicity do not reduce, the haemophilus parasuis of same serotype is infected good immanoprotection action is provided, good prevention and therapy effect can also be had to the Haemophilus parasuis that the haemophilus parasuis of the popular at present various serotype of China causes, particularly can solve at present former due to haemophilus parasuis neopathy and the problem of existing vaccine therapy poor effect that is that cause, overcome the deficiency that haemophilus parasuis that existing commercial vaccine can only prevent a kind or 2 kinds of haemophilus parasuis epidemic isolates to cause infects, the demand of different breeding family and large-scale plant can be met, and reach the how anti-effect of a pin.
Secondly, polyvalent vaccine usually causes immuning failure due to influencing each other of antigen, particularly for the disease that the haemophilus parasuis of many serotype causes, because serotype is numerous, kind and the ratio of selection antigen are more difficult, and for immunological experiment often time very long, the somewhat expensive of large animal (pig), this is also reason market not occurring the vaccine of the Haemophilus parasuis effectively preventing various serotype haemophilus parasuis to infect all the time.And the present invention is on the strain haemophilus parasuis basis obtained, good by the immune effect removing inactivated vaccine prepared by pod membrane, effectively can treat and prevent haemophilus parasuis relevant disease, solving the problem that the polyvalent vaccine effect of many serotype haemophilus parasuis relevant disease is for a long time undesirable.
Detailed description of the invention
For making the present invention easier to understand, specific embodiments of the invention will be set forth further below.As non-special declaration, general experimental technique of the present invention can use the conventional test method in this area to carry out, or completes with reference to " People's Republic of China's veterinary drug allusion quotation (two 〇 mono-〇 versions) " and the test method of annex.
The preparation of embodiment 1 Haemophilus parasuis inactivated vaccine and potency test
1. strain
A. haemophilus parasuis serum 4 type JS strain, Classification And Nomenclature: Haemophilus parasuis JS(Haemophilus parasuis JS), be separated by Pulaike Biological Engineering Co., Ltd., identify, carry out preservation at Chinese Typical Representative culture center, preservation date: on May 18th, 2011, preserving number is: CCTCC M2011172.
B. haemophilus parasuis Serotype 5 ZJ strain, Classification And Nomenclature: Haemophilus parasuis ZJ(Haemophilus parasuis ZJ), be separated by Pulaike Biological Engineering Co., Ltd., identify, carry out preservation at Chinese Typical Representative culture center, preservation date: on May 18th, 2011, preserving number is: CCTCC M2011173.
C. haemophilus parasuis serum 12 type HeB strain, Classification And Nomenclature: Haemophilus parasuis HeB(Haemophilus parasuis HeB), be separated by Pulaike Biological Engineering Co., Ltd., identify, carry out preservation at Chinese Typical Representative culture center, preservation date: on May 18th, 2011, preserving number is: CCTCC M2011174.
2. the breeding of strain
A. the breeding of first order seed
Above-mentioned freeze-drying lactobacillus, streak inoculation, on TSA/NAD flat board, is put 37 DEG C and is cultivated 18 ~ 24 hours, choose satisfactory bacterium colony, be inoculated in TSB/NAD fluid medium, cultivates 12 hours, as first order seed for 37 DEG C.
B. the breeding of secondary seed
Get the culture of first order seed, add in TSB/NAD fluid medium by the amount of 1%V, cultivate 12 hours for 37 DEG C, through checking purely as secondary seed.
3. the cultivation of bacterium liquid
In haemophilus parasuis culture medium (commercially TSB), add 0.01% ~ 0.05%(V/V) NAD, 5% ~ 10%(V/V) serum, 0.1% ~ 5%(V/V) glucose, by haemophilus parasuis serum 4 type, 5 types, 12 type strains respectively by 1%(V/V) amount add in culture medium and cultivate respectively, mix rearmounted 37 DEG C to cultivate 18 hours, obtain three kinds of bacterium liquid.
4. purely check
Sample from above-mentioned cultured three kinds of bacterium liquid, purely check according to " People's Republic of China's veterinary drug allusion quotation " annex by TSA/NAD culture medium, result is pure.
5. count plate
Sample from above-mentioned cultured three kinds of bacterium liquid, carry out count plate with TSA/NAD slat chain conveyor, to determine the bacterium number cultivated.
6. remove pod membrane
Above-mentioned cultured three kinds of bacterium liquid are placed in 60 DEG C of water-baths 30 minutes respectively, remove pod membrane.
7. deactivation
Remove in the bacterium liquid of pod membrane to above-mentioned three kinds, the 0.3%(V/V by total amount) add formalin, be positioned over 37 DEG C of deactivation 24h, period stirs 3 ~ 5 times.
8. deactivation inspection
After deactivation, the bacterium liquid 5mL getting above-mentioned three kinds of deactivations is inoculated in 100mL containing 0.01% ~ 0.05%(V/V) NAD, 5% ~ 10%(V/V) serum, 0.1% ~ 5%(V/V) glucose haemophilus parasuis TSB culture medium in, cultivate 18 hours for 37 DEG C, then to transplant in above-mentioned 100mL culture medium 37 DEG C and cultivate 18 hours.Get the bacterium liquid 0.1mL streak inoculation of deactivation in TSA/NAD plating medium, cultivate 24 hours for 37 DEG C.After above two kinds of culture medium culturings, equal asepsis growth.
9. safety detection
Get the Balb/C mice 5 of above-mentioned three kinds of bacterium vena axillaris injection 18 ~ 22g of deactivation, every 0.2mL, result is 5/5 strong to live, for deactivation is thorough in three days.
10. the preparation of vaccine
Table 1 is group vaccine strains and antigenic content respectively
Be that Haemophilus parasuis inactivated vaccine made by adjuvant with white oil.Concrete grammar is as follows:
Prepared by oil phase: white oil 94 parts, then adds 6 parts of Si Ben-80, is fully mixed, 121 DEG C, 30 minutes;
Prepared by aqueous phase: with reference to colony counting result, and by removal pod membrane, deactivation the bacterium liquid be up to the standards, adding final concentration is 4% sterilizing tween 80, and limit edged stirs, to dissolving completely;
Emulsifying: the ratio of aqueous phase and oil phase is 1:1.5, namely 1 part of aqueous phase joins 1.5 parts of oil phases emulsifying 2-5 minute in high speed agitator.
Make 9 groups of vaccines altogether according to adopted bacterial strain is different with antigenic content, bacterial strain and the antigenic content of each group vaccine are as shown in table 1.
11. potency tests
Test in Pulaike Biological Engineering Co., Ltd.'s experimental animal room.Get the vaccine of above-mentioned preparation, often organize the healthy susceptible pig 5 (Pulaike Biological Engineering Co., Ltd.'s experimental animal room is raised and provides) that 28 ages in days used by vaccine, each intramuscular injection 2mL, containing 1 using dosage, after 28 days, by injected often organize vaccine immunity test pig respectively with described haemophilus parasuis serum 4 type, 5 types, 12 type bacterial strain counteracting toxic substances.Contrast pig 15, divides three groups, respectively with described haemophilus parasuis serum 4 type, 5 types, 12 type bacterial strain counteracting toxic substances.
Haemophilus parasuis serum 4 type JS strain: each 5 of the immunity test pig choosing the haemophilus parasuis Seedling having injected above-mentioned preparation respectively; together with the contrast pig 5 that condition is identical; the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount; observe 10 days; immune swine more than 5/5 is protected, and contrast pig 5/5 falls ill.
Haemophilus parasuis Serotype 5 ZJ strain: each 5 of the immunity test pig choosing the haemophilus parasuis Seedling having injected above-mentioned preparation respectively; together with the contrast pig 5 that condition is identical; the strong toadstool liquid of Serotype 5 ZJ strain of lumbar injection 1 lethal dose; observe 10 days; immune swine more than 5/5 is protected, and contrast pig 4/5 is dead.
Haemophilus parasuis serum 12 type HeB strain: each 5 of the immunity test pig choosing the haemophilus parasuis Seedling having injected above-mentioned preparation respectively; together with the contrast pig 5 that condition is identical; the strong toadstool liquid of serum 12 type HeB strain of lumbar injection 1 lethal dose; observe 10 days; immune swine more than 5/5 is protected, and contrast pig 4/5 is dead.
Efficacy test results is as shown in table 2.
Table 2 efficacy test results
Note: Haemophilus parasuis morbidity standard: heating (body temperature more than 40.5 DEG C, continue 1 ~ 5 day), lethargy, cough, dyspnea appear in morbidity pig, become thin, walk lamely and by the thick clinical symptoms such as disorderly of hair.Cut open inspection to dying pig, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, there is serosity or fibrinous exudate in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
Embodiment 2 cross-protection test
Test in Pulaike Biological Engineering Co., Ltd.'s experimental animal room.Get the vaccine 2 of above-mentioned preparation, vaccine 5 and vaccine 8, often organize the healthy susceptible pig 15 (Pulaike Biological Engineering Co., Ltd.'s experimental animal room is raised and provides) that 28 ages in days used by vaccine, each intramuscular injection 2mL, containing 1 using dosage, after 28 days, three groups are divided at random, respectively with described haemophilus parasuis serum 4 type, 5 types, 12 type bacterial strain counteracting toxic substances by having injected the immunity test pig often organizing vaccine.Select contrast pig 15 simultaneously, divide three groups, respectively with described haemophilus parasuis serum 4 type, 5 types, 12 type bacterial strain counteracting toxic substances.Specific as follows:
Choose haemophilus parasuis vaccine 2 immunity test pig 15 prepared by embodiment 1; be divided into three groups at random; distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose; observe 10 days, immune swine more than 4/5 is protected.
Choose haemophilus parasuis vaccine 5 immunity test pig 15 prepared by embodiment 1; be divided into three groups at random; distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose; observe 10 days, immune swine more than 4/5 is protected.
Choose haemophilus parasuis vaccine 8 immunity test pig 15 prepared by embodiment 1; be divided into three groups at random; distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose; observe 10 days, immune swine more than 4/5 is protected.
Set up matched group simultaneously, select contrast pig 15, three groups are divided at random under equal conditions, distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose, observe 10 days, matched group more than 4/5 is fallen ill or death.
Result of the test is as shown in table 3.
Table 3 cross-protection test result
Note: Haemophilus parasuis morbidity standard: heating (body temperature more than 40.5 DEG C, continue 1 ~ 5 day), lethargy, cough, dyspnea appear in morbidity pig, become thin, walk lamely and by the thick clinical symptoms such as disorderly of hair.Cut open inspection to dying pig, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, there is serosity or fibrinous exudate in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
The novel Haemophilus parasuis inactivated vaccine of the different adjuvant of embodiment 3 and potency test
Utilize different adjuvant Haemophilus parasuis inactivated vaccine: by embodiment 1 prepare remove pod membrane, deactivation, concentrated and safety verification is qualified Haemophilus parasuis serum 4 type bacterial strain prepare, namely antigen is prepared with haemophilus parasuis serum 4 type bacterial strain, respectively with Montanide IMS1313VG, Montanide IMS251C VG, Montanide ISA15A VG, Montanide GEL PR or aluminium hydroxide gel adjuvant mixed preparing vaccine.Concrete formula is in table 4.
The preparation of aqueous adjuvants vaccine: be the Haemophilus parasuis inactivated vaccine that adjuvant is made with Montanide IMS1313VG or Montanide IMS251C VG or Montanide GEL PR.Concrete grammar is as follows: concentrated by bacterium liquid hollow fiber membrane ultrafiltration device, supernatant discarded, and with the resuspended precipitate of normal saline, with reference to colony counting result, according to joining Seedling Standard entertion 10% ~ 50%(V/V) Montanide IMS1313VG or Montanide IMS251C VG or Montanide GEL PR, stirring at low speed emulsifying, makes the content of antigen in vaccine be 10
9cFU/ml.
The preparation of oil-in-water vaccine: the Haemophilus parasuis inactivated vaccine made for adjuvant with Montanide ISA15A VG.Concrete grammar is as follows: concentrated by bacterium liquid hollow fiber membrane ultrafiltration device, supernatant discarded, and with the resuspended precipitate of normal saline, with reference to colony counting result, according to joining Seedling Standard entertion 10% ~ 20%(V/V) Montanide ISA15A VG, stirring at low speed emulsifying, makes the content of antigen in vaccine be 10
9cFU/ml.
The preparation of aluminium glue adjuvant: be the Haemophilus parasuis inactivated vaccine that adjuvant is made with aluminium hydroxide gel.Concrete grammar is as follows: concentrated by bacterium liquid hollow fiber membrane ultrafiltration device, supernatant discarded, and with the resuspended precipitate of normal saline, with reference to colony counting result, according to joining Seedling Standard entertion 15% ~ 25%(V/V) aluminium hydroxide gel of sterilizing shakes up; The content of antigen in vaccine is made to be 10
9cFU/ml, fully stirs, and obtains the Haemophilus parasuis inactivated vaccine of aluminium hydroxide gel adjuvant.
The formula of the different adjuvant vaccine of table 4
Potency test
Test in Pulaike Biological Engineering Co., Ltd.'s experimental animal room.Get the vaccine of above-mentioned preparation, often organize the healthy susceptible pig 15 (Pulaike Biological Engineering Co., Ltd.'s experimental animal room is raised and provides) of each immune 28 ages in days of vaccine, each intramuscular injection 2mL, containing 1 using dosage, after 28 days, by injected often organize vaccine immunity test pig respectively with described haemophilus parasuis serum 4 type, 5 types, 12 type bacterial strain counteracting toxic substances.Contrast pig 15, divides three groups, respectively with described haemophilus parasuis serum 4 type, 5 types, 12 type bacterial strain counteracting toxic substances.Specific as follows:
Choose the haemophilus parasuis vaccine A immune swine 15 of above-mentioned preparation; be divided into three groups at random; distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose; observe 10 days, immune swine more than 4/5 is protected.
Choose the haemophilus parasuis vaccine B immune swine 15 of above-mentioned preparation; be divided into three groups at random; distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose; observe 10 days, immune swine more than 4/5 is protected.
Choose the haemophilus parasuis vaccine C immune swine 15 of above-mentioned preparation; be divided into three groups at random; distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose; observe 10 days, immune swine more than 4/5 is protected.
Choose the haemophilus parasuis vaccine D immune swine 15 of above-mentioned preparation; be divided into three groups at random; distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose; observe 10 days, immune swine more than 4/5 is protected.
Choose the haemophilus parasuis vaccine E immune swine 15 of above-mentioned preparation; be divided into three groups at random; distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose; observe 10 days, immune swine more than 4/5 is protected.
Set up matched group simultaneously, select contrast pig 15, three groups are divided at random under equal conditions, distinguish the serum 4 type JS strain toadstool liquid of lumbar injection 1 morbidity amount, the Serotype 5 ZJ strain strong toadstool liquid of 1 lethal dose and the strong toadstool liquid of serum 12 type HeB strain of 1 lethal dose, observe 10 days, matched group more than 4/5 is fallen ill or death.
Result of the test is as shown in table 5.
The efficacy test results of the different adjuvant vaccine of table 5
Note: Haemophilus parasuis morbidity standard: heating (body temperature more than 40.5 DEG C, continue 1 ~ 5 day), lethargy, cough, dyspnea appear in morbidity pig, become thin, walk lamely and by the thick clinical symptoms such as disorderly of hair.Cut open inspection to dying pig, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, there is serosity or fibrinous exudate in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a Haemophilus parasuis vaccine combination, described Haemophilus parasuis vaccine combination comprises the haemophilus parasuis full bacterium antigen of the removal pod membrane of immunity amount and veterinarily acceptable carrier.
2. Haemophilus parasuis vaccine combination according to claim 1, is characterized in that, the full bacterium antigen of haemophilus parasuis of described removal pod membrane is deactivated form.
3. Haemophilus parasuis vaccine combination according to claim 1, is characterized in that, the full bacterium antigen of described haemophilus parasuis is haemophilus parasuis serum 4 type, 5 types or 12 type antigens.
4. Haemophilus parasuis vaccine combination according to claim 1, is characterized in that, the full bacterium antigen of described haemophilus parasuis is haemophilus parasuis serum 4 type bacterial strain JS strain, 5 type bacterial strain ZJ strains or 12 type bacterial strain HeB strain antigens.
5. Haemophilus parasuis vaccine combination according to claim 1, is characterized in that, the full bacterium antigenic content of described haemophilus parasuis is 10
8~ 10
10cFU/ml, preferably 10
9cFU/ml.
6. Haemophilus parasuis vaccine combination according to claim 1 and 2, it is characterized in that, described carrier is white-oil adjuvant, aluminium hydroxide gel adjuvant or the Montanide IMS1313VG based on carbomer, Montanide IMS251C VG, Montanide ISA15A VG or Montanide GEL PR polymer adjuvants.
7. Haemophilus parasuis vaccine combination according to claim 6, is characterized in that, the full bacterium antigen of described haemophilus parasuis is vaccine combination 50% ~ 90% percent by volume.
8. prepare a method for described Haemophilus parasuis vaccine combination, described method comprises the steps:
(1) haemophilus parasuis spawn culture propagation is obtained bacterium liquid;
(2) the bacterium liquid described cultivation obtained removes pod membrane;
(3) the bacterium liquid of described removal pod membrane is carried out deactivation, and concentrated bacterium liquid;
(4) bacterium liquid also concentrated for described deactivation and veterinarily acceptable carrier are carried out emulsifying.
9. the preparation method of Haemophilus parasuis vaccine combination according to claim 8, is characterized in that, removes pod membrane by being placed in 60 DEG C of water-baths realization in 30 minutes by cultivating the bacterium liquid obtained described in step (2).
10. the application of the Haemophilus parasuis vaccine combination as described in claim 1 ~ 7 in the medicine preparing prevention and therapy haemophilus parasuis infection relevant disease.
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| CN104488823A (en) * | 2015-01-09 | 2015-04-08 | 武汉轻工大学 | Method for building model of piglets infected by haemophilus parasuis (HPS) |
| CN105420141A (en) * | 2015-09-17 | 2016-03-23 | 龙岩学院 | Haemophilus parasuis strain |
| CN106421769A (en) * | 2016-12-16 | 2017-02-22 | 青岛易邦生物工程有限公司 | Haemophilus parasuis disease vaccine |
| CN106591189A (en) * | 2016-12-16 | 2017-04-26 | 青岛易邦生物工程有限公司 | Haemophilus parasuis strain |
| CN106729686A (en) * | 2016-12-16 | 2017-05-31 | 青岛易邦生物工程有限公司 | A kind of haemophilus parasuis tetravalent inactivated vaccine |
| CN107245459A (en) * | 2017-04-11 | 2017-10-13 | 河南省农业科学院畜牧兽医研究所 | Haemophilus parasuis and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104488823A (en) * | 2015-01-09 | 2015-04-08 | 武汉轻工大学 | Method for building model of piglets infected by haemophilus parasuis (HPS) |
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| CN106729686A (en) * | 2016-12-16 | 2017-05-31 | 青岛易邦生物工程有限公司 | A kind of haemophilus parasuis tetravalent inactivated vaccine |
| CN107245459A (en) * | 2017-04-11 | 2017-10-13 | 河南省农业科学院畜牧兽医研究所 | Haemophilus parasuis and application thereof |
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Application publication date: 20141231 |