CN102908615A - Novel haemophilus parasuis disease trivalent inactivated vaccine and preparation method thereof - Google Patents
Novel haemophilus parasuis disease trivalent inactivated vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN102908615A CN102908615A CN2011102188312A CN201110218831A CN102908615A CN 102908615 A CN102908615 A CN 102908615A CN 2011102188312 A CN2011102188312 A CN 2011102188312A CN 201110218831 A CN201110218831 A CN 201110218831A CN 102908615 A CN102908615 A CN 102908615A
- Authority
- CN
- China
- Prior art keywords
- haemophilus parasuis
- type
- strain
- adjuvant
- inactivated vaccine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention provides a novel haemophilus parasuis disease trivalent inactivated vaccine and a preparation method thereof. The vaccine comprises equal proportions of antigens of: inactivated haemophilus parasuis serotype 4 JS strain, inactivated haemophilus parasuis serotype 5 ZJ strain, and inactivated haemophilus parasuis serotype 12 HeB strain. According to the invention, the haemophilus parasuis serotype 4, serotype 5, and serotype 12 strains are obtained by separation. Concentration contents and relative proportions of the antigens prepared from the three strains are subjected to large amounts of woks and practices, such that the trivalent vaccine with an appropriate antigen ratio and an appropriate concentration is obtained. The trivalent vaccine has good preventing and treating effects against haemophilus parasuis diseases caused by various epidemic haemophilus parasuis serotypes in our nation. Especially, the trivalent vaccine can solve a problem of poor treatment effect of existing vaccines caused by novel haemophilus parasuis pathogens.
Description
Technical field
The present invention relates to a kind of haemophilus parasuis polyvalent vaccine, refer to especially tervalence inactivated vaccine and the preparation method of a kind of haemophilus parasuis 4 type JS strains, 5 type ZJ strains, 12 type HeB strains.
Background technology
Haemophilus parasuis (Haemophilus parasuis, Hps) cause polyserositis and the arthritis of pig, this disease is called again pig leather and draws Ze Shi sick (Glsser ' s Disease), once once be considered to by stress caused sporadic disease, be proved to be by haemophilus parasuis afterwards and caused.Haemophilus parasuis can affect from the young pig at 2 age to 4 monthly ages in week, mainly falls ill with the child care stage after wean, usually sees the pig in 5～8 ages in week, and sickness rate is generally 10%～15%, and mortality rate can reach 50% when serious.Main clinic symptoms shows as cough, dyspnea, becomes thin, walks lamely and the thick unrest of quilt hair; Mainly cut open the inspection pathological changes and show as fibrinous pleurisy, pericarditis, peritonitis, arthritis and meningitis etc.In addition, haemophilus parasuis also can cause septicemia, and may stay sequela after actute infection, i.e. sow miscarriage, the chronic limping of boar.
As far back as 1910, Germany scientist Glsser just found contacting between the polyserositis of haemophilus parasuis and pig and the arthritis.Along with the development of world's pig industry, this disease has become the typical bacteria disease that affects pig industry in the global range.Western developed country is on the basis of a large amount of separation haemophilus parasuises, the serotype complexity of finding this bacterium is various, press Kieletein-Rapp-Gabriedson (KRG) agar diffusion serotype method, at least haemophilus parasuis can be divided into 15 serotypes, other has the separated strain serotype more than 20% to decide; According to the seroepidemiological survey of the country such as Germany, the U.S., Canada, Japan and Spain, the most popular with serotype 4,5,13 types; According to the evaluation of China's seroepidemiological survey and isolated strains, the most popular with 4,5 types, secondly be 12 types, 13 types.In recent years, Immunogenicity power is not strong between each haemophilus parasuis sero-group, and the cause of disease sero-group of the Haemophilus parasuis that the vaccine strains sero-group of inoculation and there and then are popular does not conform to, and has brought certain difficulty for prevention and the treatment of this disease.Domestic vaccine for the Haemophilus parasuis immunoprophylaxis has the Haemophilus parasuis inactivated vaccine (Z-1517 strain) that Boehringer Ingelheim animal health (U.S.) company limited is produced at present; Haemophilus parasuis 4 types and 5 type inactivated vaccines that pig Haemophilus parasuis 1 type that the biological large pharmaceutical factory of Spain Hai Bolai produces and 6 type inactivated vaccines and Hua Zhong Agriculture University, Wuhan Keqian Animal Biological Products Co., Ltd., Zhongmu Industry Co.,Ltd produce; Certain preventive effect has been played in the application of these vaccines, and cause of disease epidemic strain that should disease and various serotype infect and have made existing vaccine be difficult to reach the immune effect of expection, especially also there are not Haemophilus parasuis 12 types, owing to there is not suitable strain preparation, add between each antigen to exist and disturb that it is aquaculture problem in the urgent need to address that a pin can be prevented and treated the Haemophilus parasuis that the new cause of disease of haemophilus parasuis causes.
Summary of the invention
In view of this, main purpose of the present invention is to provide a kind of Haemophilus parasuis that solves present technological deficiency, formed by the acceptable adjuvant of three kinds of antigens and veterinary, wherein, described three kinds of antigens haemophilus parasuis serum 4 type JS strains that are deactivation, the haemophilus parasuis Serotype 5 ZJ strain of deactivation, the haemophilus parasuis serum 12 type HeB strain antigens of deactivation.
The antigen that the present invention uses is from the 4 type JS strains of haemophilus parasuis serum, and preserving number is CCTCC NO:M 2011172; Haemophilus parasuis Serotype 5 ZJ strain, preserving number is CCTCC NO:M 2011173; The 12 type HeB strains of haemophilus parasuis serum, preserving number is CCTCC NO:M 2011174.
Preferably, being in equal proportions of three kinds of antigens of the present invention, haemophilus parasuis serum 4 types, 5 types, 12 type antigenic contents respectively are 2.0 * 10
9CFU/mL.
Preferably, the acceptable adjuvant of veterinary of the present invention is one or more compositions of white-oil adjuvant, aluminium hydroxide gel adjuvant and Montanide IMS1313 VG, Montanide IMS 251C VG, Montanide ISA15A VG and Montanide GEL PR polymer adjuvant.
Preferably, the acceptable adjuvant of veterinary of the present invention is aqueous adjuvant Montanide GEL PR.
More preferably, the thimerosal that also contains vaccine total amount 0.01%V in the Haemophilus parasuis tervalence inactivated vaccine of the present invention.
The present invention also provides a kind of preparation method of aforesaid Haemophilus parasuis tervalence inactivated vaccine, may further comprise the steps:
A. respectively the 4 type JS strains of haemophilus parasuis serum, haemophilus parasuis Serotype 5 ZJ strain, haemophilus parasuis serum 12 type HeB strain culture propagations are cultivated, obtained 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid, 12 type HeB strain bacterium liquid;
B. respectively step a is cultivated the 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid, the 12 type HeB strain bacterium liquid that obtain and carry out deactivation, and concentrated bacterium liquid;
C. above-mentioned deactivation, concentrated 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid, 12 type HeB strain bacterium liquid equal-volume ratios are mixed, and the thimerosal of adding vaccine total amount 0.01%V, then carry out emulsifying with the pharmaceutically acceptable adjuvant of beast, wherein, Haemophilus parasuis tervalence inactivated vaccine antigenic content is that haemophilus parasuis serum 4 types before the deactivation, 5 types, 12 type viable counts respectively are 2.0 * 10
9CFU/mL.
D. the Haemophilus parasuis triple inactivated vaccine that step c is obtained carries out packing.
Preferably, in the preparation method of Haemophilus parasuis tervalence inactivated vaccine of the present invention, the acceptable adjuvant of described veterinary is one or more compositions of white-oil adjuvant, aluminium hydroxide gel adjuvant and Montanide IMS1313 VG, Montanide IMS 251C VG, Montanide ISA15A VG and Montanide GEL PR polymer adjuvant.
Preferably, Haemophilus parasuis antibacterial of the present invention is the 4 type JS strains of haemophilus parasuis serum, and preserving number is CCTCC NO:M 2011172; Haemophilus parasuis Serotype 5 ZJ strain, preserving number is CCTCC NO:M 2011173; The 12 type HeB strains of haemophilus parasuis serum, preserving number is CCTCC NO:M 2011174.
Another object of the present invention is to provide the application of Haemophilus parasuis triple inactivated vaccine described above in prevention and treatment haemophilus parasuis related diseases.
Technique effect
The inventor unexpectedly separates the bacterial strain that obtains three strain haemophilus parasuis serum, 4 types, 5 types and 12 types, find this three strains haemophilus parasuis by test: 4 type JS strain bacterium, 5 type ZJ strain bacterium, 12 type HeB strain bacterium prepare triple inactivated vaccine can to China at present the Haemophilus parasuis that causes of popular multiple haemophilus parasuis serotype preferably prevention and therapeutic effect be arranged, particularly can solve at present the problem of the existing vaccine therapy poor effect that causes owing to the new cause of disease of haemophilus parasuis.
Secondly, polyvalent vaccine is usually owing to influencing each other of antigen causes immuning failure, the disease that particularly causes for the haemophilus parasuis of many serotypes, because serotype is numerous, select kind and the ratio of antigen more difficult, and for the immunological experiment of large animal (pig) very long, somewhat expensive of time often, this also is the reason that does not all the time occur effective haemophilus parasuis polyvalent vaccine on the market.And the present invention is obtaining on the new haemophilus parasuis basis of three strains, by to the concentration content of the antigen of this three kinds of strains preparation and mutually ratio carried out extensive work and practice, proportioning and the suitable trivalent vaccine of concentration between a kind of antigen have been obtained, the immune effect of vaccine is good, can effectively treat and prevent the haemophilus parasuis relevant disease, the polyvalent vaccine that has solved many serotype haemophilus parasuis relevant disease for a long time prepares hard problem.
At last, tervalence inactivated vaccine of the present invention has used oxidation aluminium glue or water type adjuvant, found that especially the aqueous adjuvant prepares tervalence inactivated vaccine) safety and immune effect the best, and it is simple to join Seedling technique, vaccine is easily injected, safe and reliable, overcome the in the past shortcoming of oil adjuvant commonly used.
Bacterial strain preservation explanation
The used bacterial strain of the present invention is:
The 4 type JS strains of Haemophilus parasuis serum:
Classification And Nomenclature: Haemophilus parasuis JS (Haemophilus parasuis JS),
Preserving number is: CCTCC NO:M 2011172;
Haemophilus parasuis Serotype 5 ZJ strain:
Classification And Nomenclature: Haemophilus parasuis ZJ (Haemophilus parasuis ZJ),
Preserving number is: CCTCC NO:M 2011173;
The 12 type HeB strains of Haemophilus parasuis serum:
Classification And Nomenclature: Haemophilus parasuis HeB (Haemophilus parasuis HeB),
Preserving number is: CCTCC NO:M 2011174;
More than the preservation date of three bacterial strains be on May 18th, 2011, the preservation address is Wuhan, China, Wuhan University, Chinese Typical Representative culture collection center, depositary institution is: Chinese Typical Representative culture collection center (being called for short CCTCC).
Description of drawings
Fig. 1 is the trivalent inactivated vaccine against Haemophilus parasuis infection process chart.
The specific embodiment
Novel Haemophilus parasuis tervalence inactivated vaccine provided by the invention, described Haemophilus parasuis inactivated vaccine comprises antigen and adjuvant; Wherein antigen comprises the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains, the 12 type HeB strain antigens of deactivation; Described adjuvant is the acceptable adjuvant of veterinary;
As the preferred embodiment of Haemophilus parasuis tervalence inactivated vaccine of the present invention, be in equal proportions (namely 1: 1: 1) of described three kinds of antigens, haemophilus parasuis serum 4 types, 5 types, 12 type antigenic contents are 2.0 * 10
9CFU/mL.Wherein, antigenic content of the present invention refers to the content of the viable count before deactivation.
As the preferred embodiment of Haemophilus parasuis tervalence inactivated vaccine of the present invention, described adjuvant is white-oil adjuvant, aluminium hydroxide gel adjuvant and take Montanide IMS1313 VG (comprise homologous series adjuvant), Montanide IMS 251C VG (comprise homologous series adjuvant), Montanide ISA15A VG (comprise homologous series adjuvant) and Montanide GEL PR (comprise homologous series adjuvant) the polymer adjuvant of carbomer (Carbomer) as the basis.
As the preferred embodiment of Haemophilus parasuis tervalence inactivated vaccine of the present invention, also contain the thimerosal of vaccine total amount 0.01%v in the described vaccine.
The preparation method of novel Haemophilus parasuis inactivated vaccine comprises following steps:
A. respectively the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains, 12 type HeB strain culture propagations are cultivated, obtained 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid, 12 type HeB strain bacterium liquid;
B. respectively step a is cultivated in the 4 type JS strain bacterium liquid obtain, 5 type ZJ strain bacterium liquid, the 12 type HeB strain bacterium liquid and to add 0.3% formalin by total amount, places 37 ℃ of deactivations, during stir 3～5 times, concentrate bacterium liquid according to count results;
C. above-mentioned deactivation, concentrated 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid, 12 type HeB strain bacterium liquid equal proportions are mixed, and the thimerosal of adding vaccine total amount 0.01%, then carry out emulsifying with different adjuvants, finished product Haemophilus parasuis killed vaccine antigen content is that haemophilus parasuis serum 4 types before the deactivation, 5 types, 12 type viable counts respectively are 2.0 * 10
9CFU/mL;
D. with an amount of volume packing Haemophilus parasuis inactivated vaccine, cover bottle cap, and the jewelling lid.
In the preparation process of above-mentioned novel Haemophilus parasuis tervalence inactivated vaccine, from the cultured 4 type JS strain bacterium liquid of step a, 5 type ZJ strain bacterium liquid, the sampling of 12 type HeB strain bacterium liquid, carry out the check of pure property and count plate; Carry out deactivation check and safety check from 4 good type JS strain bacterium liquid of step b deactivation, 5 type ZJ strain bacterium liquid, the sampling of 12 type HeB strain bacterium liquid; The Haemophilus parasuis inactivated vaccine that step c is obtained carries out steriling test, safety verification and efficacy test, with safety, the reliability of guaranteeing vaccine.
Novel Haemophilus parasuis tervalence inactivated vaccine of the present invention has 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 present invention has adopted different vaccine adjuvants and the suitable Seedling technology of joining to prepare the Haemophilus parasuis tervalence inactivated vaccine, can prevent simultaneously the Haemophilus parasuis that is caused by the haemophilus parasuis various serotype.Can satisfy the demand of different breeding family and large-scale plant, and reach the effect that a pin is prevented more.
For making the present invention easier to understand, the below will further set forth specific embodiments of the invention.Such as special declaration not, general experimental technique of the present invention can use this area test method commonly used to carry out, or finishes with reference to " People's Republic of China's veterinary drug allusion quotation (in 2005 version) " and the test method of appendix.
Preparation and the potency test of embodiment 1 Haemophilus parasuis tervalence inactivated vaccine
The Haemophilus parasuis tervalence inactivated vaccine, by deactivation, concentrate and qualified Haemophilus parasuis serum 4 types of safety verification, 5 types, 12 type bacterial strains prepare, namely use haemophilus parasuis serum 4 types, 5 types, 12 type bacterial strains prepare antigen, respectively with Montanide IMS1313 VG (comprising the homologous series adjuvant), Montanide IMS 251C VG (comprising the homologous series adjuvant), Montanide ISA15A VG (comprising the homologous series adjuvant), Montanide GEL PR (comprising the homologous series adjuvant), white-oil adjuvant, aluminium hydroxide gel adjuvant mixed preparing vaccine, wherein, described three kinds of antigen concentrations are in equal proportions.
1. strain
A. haemophilus parasuis serum 4 type JS strains are separated, are identified that carry out preservation, preservation date at Chinese Typical Representative culture center: on May 18th, 2011, preserving number is: CCTCC M 2011172 by Pulaike Biological Engineering Co., Ltd..
B. haemophilus parasuis Serotype 5 ZJ strain is separated, is identified that carry out preservation, preservation date at Chinese Typical Representative culture center: on May 18th, 2011, preserving number is: CCTCC M 2011173 by Pulaike Biological Engineering Co., Ltd..
C. haemophilus parasuis serum 12 type HeB strains are separated, are identified that carry out preservation, preservation date at Chinese Typical Representative culture center: on May 18th, 2011, preserving number is: CCTCC M 2011174 by Pulaike Biological Engineering Co., Ltd..
2. the breeding of strain
A. the breeding of first order seed
Above-mentioned freeze-drying lactobacillus, streak inoculation are put 37 ℃ and were cultivated 18～24 hours on the TSA/NAD flat board, choose satisfactory bacterium colony, are inoculated in the TSB/NAD fluid medium, cultivate 12 hours for 37 ℃, as first order seed.
B. the breeding of secondary seed
Get the culture of first order seed, the amount of pressing 1%V adds in the TSB/NAD fluid medium, cultivated 12 hours for 37 ℃, through check pure after as secondary seed.
3. the cultivation of bacterium liquid
In haemophilus parasuis culture medium (merchant sells TSB), the glucose of the serum, 0.1%～5% (V/V) of the NAD, 5%～10% (V/V) of adding 0.01%～0.05% (V/V), haemophilus parasuis serum 4 types, 5 types, 12 type strains are added respectively cultivation in the culture medium by the amount of 1% (V/V) respectively, the rearmounted 37 ℃ of cultivations of mixing 18 hours obtain three kinds of bacterium liquid.
4. pure check
Take a sample from above-mentioned cultured three kinds of bacterium liquid, purely check according to " People's Republic of China's veterinary drug allusion quotation " appendix with the TSA/NAD culture medium, the result is pure.
5. count plate
From above-mentioned cultured three kinds of bacterium liquid sampling, carry out count plate with dull and stereotyped cultivation of TSA/NAD, with the bacterium number of determining to cultivate.
6. deactivation
In above-mentioned three kinds of cultured bacterium liquid, add formalin by 0.3% (V/V) of total amount, be positioned over 37 ℃ of deactivation 24h, during stir 3～5 times.
7. deactivation check
After the deactivation, the bacterium liquid 5mL that gets above-mentioned three kinds of deactivations is inoculated in the haemophilus parasuis TSB culture medium of glucose of serum, 0.1%～5% (V/V) that 100mL contains 0.01%～0.05% (V/V) NAD, 5%～10% (V/V), cultivated 18 hours for 37 ℃, transplant again in above-mentioned 100mL culture medium 37 ℃ and cultivated 18 hours.Get the bacterium liquid 0.1mL streak inoculation of deactivation in the TSA/NAD plating medium, cultivated 24 hours for 37 ℃.More than behind two kinds of culture medium culturings, equal asepsis growths.
8. safety detection
Get above-mentioned three kinds of 5 of Balb/C mices of the bacterium vena axillaris injection 18～22g of deactivation, every 0.2mL, the result is 5/5 strong living in three days, for deactivation thorough.
9. the preparation of vaccine
A. the preparation of oily adjuvant Seedling
Concrete composition proportion such as following table:
| The main material component | Content (%) |
| White oil for animals | 93 |
| Si Ben-80 | 6 |
| Aluminium stearate | 1 |
| Tween 80 | 4 |
| Water: oil phase (volume ratio) | 1∶1.5 |
The Haemophilus parasuis tervalence inactivated vaccine of making take white oil as adjuvant.Concrete grammar is as follows:
The preparation of oil phase: 93 parts of white oils, add 1 part of aluminium stearate, the limit edged stirs, and adds 6 parts of Si Ben-80 again, fully be mixed, 121 ℃, 30 minutes.Water preparation: with reference to the colony counting result, with each bacterium liquid with ultrafilter simmer down to 5.0 * 10
10CFU/ml.Three kinds of serotype deactivations and the bacterium liquid equal proportion that is up to the standards are mixed, and adding final concentration is 4% sterilization tween 80, and the limit edged stirs, to fully dissolving.Emulsifying: the ratio of water and oil phase is 1: 1.5, and namely 1 part of water is joined 1.5 parts of oil phases in high speed agitator emulsifying 2-5 minute, and the thimerosal of the 0.01%V of adding vaccine total amount before stopping stirring is so that the content of three kinds of antigens is 2.0 * 10 in the vaccine
9CFU/ml.
B. the preparation of aluminium glue adjuvant Seedling
Concrete composition proportion is as follows:
| The main material component | Content (%) |
| Antigen (1: 1: 1) | 75～85 |
| Aluminium hydroxide gel | 15～25 |
Be the Haemophilus parasuis tervalence inactivated vaccine that adjuvant is made with aluminium hydroxide gel.Concrete grammar is as follows:
Bacterium liquid is concentrated with hollow fiber membrane ultrafiltration device, and supernatant discarded, and with the resuspended precipitate of normal saline with reference to the colony counting result, is adjusted bacterial concentration and is haemophilus parasuis serum 4 types, 5 types, 12 types respectively 1.0 * 10
10CFU/ml mixes three kinds of antigen equal proportions.The aluminium hydroxide gel that adds 15～25% (V/V) sterilization in haemophilus parasuis serum 4 types, 5 types, three kinds of bacterium liquid that mix of 12 types shakes up; And the thimerosal of 0.01% (V/V) of adding vaccine total amount, so that the content of three kinds of antigens is 2.0 * 10 in the vaccine
9CFU/ml fully stirs, and obtains the Haemophilus parasuis tervalence inactivated vaccine of aluminium hydroxide gel adjuvant.
C. the preparation of water type Adjuvanted vaccines
Concrete composition proportion is as follows:
The Haemophilus parasuis tervalence inactivated vaccine of making for adjuvant with Montanide IMS1313 VG (comprising the homologous series adjuvant) or Montanide IMS251C VG (comprising the homologous series adjuvant) or Montanide GEL PR (comprising the homologous series adjuvant).Detailed process is as follows: bacterium liquid is concentrated with hollow fiber membrane ultrafiltration device, and supernatant discarded, and with the resuspended precipitate of normal saline with reference to the colony counting result, is adjusted bacterial concentration and is haemophilus parasuis serum 4 types, 5 types, 12 types respectively 2.0 * 10
10CFU/ml mixes three kinds of antigen equal proportions.According to the Montanide IMS1313 VG (comprising the homologous series adjuvant) or Montanide IMS 251C VG (comprising the homologous series adjuvant) or the Montanide GEL PR (comprising the homologous series adjuvant) that join Seedling standard adding 10%～50% (V/V), stirring at low speed emulsifying, the thimerosal that adds 0.01% (V/V) of vaccine total amount before stopping stirring is so that the content of three kinds of antigens is 2.0 * 10 in the vaccine
9CFU/ml.
D. the preparation of oil in water vaccine:
Concrete composition proportion is as follows:
| The main material component | Content (V/V%) |
| Antigen (1: 1: 1) | 80～90 |
| Montanide ISA15A VG | 10～20 |
The Haemophilus parasuis tervalence inactivated vaccine of making take Montanide ISA15A VG as adjuvant.Specific as follows: bacterium liquid is concentrated with hollow fiber membrane ultrafiltration device, supernatant discarded, and with the resuspended precipitate of normal saline, with reference to the colony counting result, adjusting bacterial concentration is that haemophilus parasuis serum 4 types, 5 types, 12 types respectively are 2.0 * 10
10CFU/ml mixes three kinds of antigen equal proportions.According to the Montanide ISA15A VG (comprising the homologous series adjuvant) that joins Seedling standard adding 10～20% (V/V), stirring at low speed emulsifying, the thimerosal that adds 0.01% (V/V) of vaccine total amount before stopping stirring is so that the content of three kinds of antigens is 2.0 * 10 in the vaccine
9CFU/ml.
10. the steriling test of vaccine
Get above-mentioned vaccine, carry out steriling test, as a result asepsis growth according to " People's Republic of China's veterinary drug allusion quotation " appendix.
11. packing
With 20mL/ bottle, 50mL/ bottle, the packing of 100mL/ bottle, cover bottle cap, and the jewelling lid.
12. usage and consumption
The musculi colli injection makes vaccine balance to room temperature also fully shake up before the use, no matter pig is big or small, and per injection 2ml.The recommendation immune programme for children is: replacement gilt is exempted from antenatal 8～9 all head, and two exempt from after 3 weeks, and later every tire antenatal 4～5 all immunity once; Piglet 3～4 age in week head exempt from, two exempt from after 3 weeks.
Embodiment 2: the screening of the best adjuvant of Haemophilus parasuis vaccine
(1) safety: get 5 of healthy susceptible pigs of each intramuscular injection of vaccine of embodiment 1 preparation, every 4mL, in 14 days without local response and whole strong living.Assay such as following table 1:
Table 1 vaccine test result
(2) potency test:
Test in Pulaike Biological Engineering Co., Ltd. experimental animal room.Get the vaccine of embodiment 1 preparation, every group of each 15 of healthy susceptible pig (Pulaike Biological Engineering Co., Ltd. experimental animal room is raised and provided) with 28 ages in days of vaccine, each intramuscular injection 2mL, contain 1 using dosage, after 28 days, the immunity test pig of having injected every kind of vaccine is divided into three groups at random, uses respectively described haemophilus parasuis serum 4 types, 5 types, 12 type bacterial strain counteracting toxic substances.15 of contrast pigs are divided three groups, use respectively described haemophilus parasuis serum 4 types, 5 types, 12 type bacterial strain counteracting toxic substances.
Haemophilus parasuis serum 4 type JS strains: each 5 of haemophilus parasuis Seedling immune swines choosing respectively embodiment 1 preparation; together with 5 of the identical contrast pigs of condition, the serum 4 type JS strain toadstool liquid of 1 morbidity amount of lumbar injection were observed 10 days; immune swine is protected more than 4/5, and the contrast pig falls ill more than 4/5.
Haemophilus parasuis Serotype 5 ZJ strain: each 5 of haemophilus parasuis Seedling immune swines choosing respectively embodiment 1 preparation; together with 5 of the identical contrast pigs of condition; the strong toadstool liquid of Serotype 5 ZJ strain of 1 lethal dose of lumbar injection; observed 10 days; immune swine is protected more than 4/5, and the contrast pig is dead more than 4/5.
Haemophilus parasuis serum 12 type HeB strains: each 5 of haemophilus parasuis Seedling immune swines choosing respectively embodiment 1 preparation; together with 5 of the identical contrast pigs of condition; the strong toadstool liquid of serum 12 type HeB strains of 1 lethal dose of lumbar injection; observed 10 days; immune swine is protected more than 4/5, and the contrast pig is dead more than 4/5.Assay is as shown in table 2.
Table 2 vaccine potency assay
Annotate: Haemophilus parasuis morbidity standard: heating (body temperature continues 1～5 more than 40.5 ℃), lethargy appear in the morbidity pig, cough, dyspnea, become thin, walk lamely and by the thick clinical symptoms such as disorderly of hair.Dying pig is cutd open inspection, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, serosity or fibrinous exudate appear in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
According to the comparison by above safety and potency test of existing animal vaccines adjuvant and new vaccine adjuvant, (the 4 type JS strains+5 type ZJ strains+12 type HeB strains) safety of Haemophilus parasuis tervalence inactivated vaccine and the immune effect of as a result aqueous adjuvant Montanide GEL PR preparation are best, and it is simple to join Seedling technique, vaccine is easily injected, safety, reliable.
Preparation and the contrast test of embodiment 3 aqueous adjuvant Haemophilus parasuis tervalence inactivated vaccines (4 type JS strains+5 type ZJ strains+12 type HeB strains)
According to the selection result, select safety, the best Montanide GEL PR adjuvant of immune effect to prepare three batches of laboratory products, after qualified through safety, efficacy test and the Haemophilus parasuis inactivated vaccine of selling on the market compare test, further inspection security and immune effect.
I adopts the Montanide GEL PR water adjuvant of import to prepare by the following method vaccine (flow chart is seen appendix) at first according to the requirement of " Ministry of Agriculture's new biological product management method for animals ":
1, strain
A. haemophilus parasuis serum 4 type JS strains are separated, are identified that carry out preservation, preservation date at Chinese Typical Representative culture center: on May 18th, 2011, preserving number is: CCTCC M 2011172 by Pulaike Biological Engineering Co., Ltd..
B. haemophilus parasuis Serotype 5 ZJ strain is separated, is identified that carry out preservation, preservation date at Chinese Typical Representative culture center: on May 18th, 2011, preserving number is: CCTCC M 2011173 by Pulaike Biological Engineering Co., Ltd..
C. haemophilus parasuis serum 12 type HeB strains are separated, are identified that carry out preservation, preservation date at Chinese Typical Representative culture center: on May 18th, 2011, preserving number is: CCTCC M 2011174 by Pulaike Biological Engineering Co., Ltd..
2. the breeding of strain
A. the breeding of first order seed
Above-mentioned freeze-drying lactobacillus, streak inoculation are put 37 ℃ and were cultivated 18～24 hours on the TSA/NAD flat board, choose satisfactory bacterium colony, are inoculated in the TSB/NAD fluid medium, cultivate 12 hours for 37 ℃, as first order seed.
B. the breeding of secondary seed
Get the culture of first order seed, add in the TSB/NAD fluid medium by 1% amount, cultivated 12 hours for 37 ℃, through check pure after as secondary seed.
3. the cultivation of bacterium liquid
In haemophilus parasuis culture medium (merchant sells TSB), add 0.01%～0.05%NAD, 5%～10% serum, 0.1%～5% glucose, haemophilus parasuis serum 4 types, 5 types, 12 type strains are added respectively cultivation in the culture medium by 1% amount respectively, the rearmounted 37 ℃ of cultivations of mixing 18 hours obtain three kinds of bacterium liquid.
4. pure check
Take a sample from above-mentioned cultured three kinds of bacterium liquid, purely check according to " People's Republic of China's veterinary drug allusion quotation " appendix with the TSA/NAD culture medium, the result is pure.
5. count plate
From above-mentioned cultured three kinds of bacterium liquid sampling, carry out count plate with dull and stereotyped cultivation of TSA/NAD, take definite bacterium number of cultivating as 2.5 * 10
9CFU/mL.
6. deactivation
In above-mentioned three kinds of cultured bacterium liquid, add formalin by 0.3% (V/V) of total amount, be positioned over 37 ℃ of deactivation 24h, during stir 3～5 times.
7. deactivation check
After the deactivation, the bacterium liquid 5mL that gets above-mentioned three kinds of deactivations is inoculated in the haemophilus parasuis TSB culture medium of glucose of serum, 0.1%～5% (V/V) that 100mL contains 0.01%～0.05% (V/V) NAD, 5%～10% (V/V), cultivated 18 hours for 37 ℃, transplant again in above-mentioned 100mL culture medium 37 ℃ and cultivated 18 hours.Get the bacterium liquid 0.1mL streak inoculation of deactivation in the TSA/NAD plating medium, cultivated 24 hours for 37 ℃.After two kinds of methods are cultivated, all should asepsis growth.
8. safety check
Get above-mentioned three kinds of 5 of Balb/C mices of the bacterium vena axillaris injection 18～22g of deactivation, every 0.2mL, 5/5 strong living in three days, the side is thorough for deactivation.
9. the preparation of vaccine
Concrete composition proportion is as follows:
| The main material component | Content (V/V%) |
| Antigen (1: 1: 1) | 80 |
| Montanide GEL PR | 20 |
Be the Haemophilus parasuis tervalence inactivated vaccine that adjuvant is made with Montanide GEL PR.Detailed process is as follows: bacterium liquid is concentrated with hollow fiber membrane ultrafiltration device, and supernatant discarded, and with the resuspended precipitate of normal saline with reference to the colony counting result, is adjusted bacterial concentration and is haemophilus parasuis serum 4 types, 5 types, 12 types respectively 2.5 * 10
9CFU/ml mixes three kinds of antigen equal proportions.According to the Montanide GEL PR that joins Seedling standard adding 20%V/V, stirring at low speed (800 rev/mins) emulsifying, the thimerosal of the 0.01%V of adding vaccine total amount before stopping stirring.The antigen concentration of final vaccine finished product is that haemophilus parasuis serum 4 types, 5 types, 12 types are 2.0 * 10
9CFU/ml
10. physical behavior detection, steriling test: the Haemophilus parasuis inactivated vaccine of trial-production (4 type JS strains+5 type ZJ strains+12 type HeB strains) 3 batches (lot number 0801,0802,0803).Through physical behavior detect, steriling test is qualified, detailed results sees Table 3;
The check of table 3 tervalence inactivated vaccine physical behavior and steriling test
Annotate: T.G represents sulphur glycollate culture medium, and G.A represents the peptone from casein agar culture medium, the G.P dextrose peptone medium; "-" expression asepsis growth.
11. safety verification: get 5 of healthy susceptible pigs of each intramuscular injection of 3 batches of (lot number 0801,0802,0803) vaccines of said method preparation, every 4mL without local response and whole strong living, was up to the standards in 14 days.
12. efficacy test: test in our company experimental animal room.Get 3 batches of (lot number 0801,0802,0803) vaccines of preparation, the every batch of vaccine each with 15 of the healthy susceptible pigs of 28 ages in days, each intramuscular injection 2mL, contain 1 using dosage, after 28 days, the immunity test pig of having injected every kind of vaccine is divided into three groups at random, uses respectively described haemophilus parasuis serum 4 types, 5 types, 12 type bacterial strain counteracting toxic substances.15 of contrast pigs are divided three groups, use respectively described haemophilus parasuis serum 4 types, 5 types, 12 type bacterial strain counteracting toxic substances.
Haemophilus parasuis serum 4 type JS strains: each 5 of haemophilus parasuis Seedling immune swines choosing respectively embodiment 1 preparation; together with 5 of the identical contrast pigs of condition, the serum 4 type JS strain toadstool liquid of 1 morbidity amount of lumbar injection were observed 10 days; immune swine is protected more than 4/5, and the contrast pig falls ill more than 4/5.
Haemophilus parasuis Serotype 5 ZJ strain: each 5 of haemophilus parasuis Seedling immune swines choosing respectively embodiment 1 preparation; together with 5 of the identical contrast pigs of condition; the strong toadstool liquid of Serotype 5 ZJ strain of 1 lethal dose of lumbar injection; observed 10 days; immune swine is protected more than 4/5, and the contrast pig is dead more than 4/5.
Haemophilus parasuis serum 12 type HeB strains: each 5 of haemophilus parasuis Seedling immune swines choosing respectively embodiment 1 preparation; together with 5 of the identical contrast pigs of condition; the strong toadstool liquid of serum 12 type HeB strains of 1 lethal dose of lumbar injection; observed 10 days; immune swine is protected more than 4/5, and the contrast pig is dead more than 4/5.Assay is qualified as shown in table 4.
Table 4 tervalence inactivated vaccine efficacy test result
Annotate: Haemophilus parasuis morbidity standard: heating (body temperature continues 1～5 more than 40.5 ℃), lethargy appear in the morbidity pig, cough, dyspnea, become thin, walk lamely and by the thick clinical symptoms such as disorderly of hair.Dying pig is cutd open inspection, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, serosity or fibrinous exudate appear in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
II vaccine contrast test
Randomly drawing the above-mentioned Haemophilus parasuis inactivated vaccine that is up to the standards (4 type JS strains+5 type ZJ strains+12 type HeB strains) lot number and be Haemophilus parasuis inactivated vaccine (4 types+5 types) lot number by biotech firm's production before the section of Wuhan of selling on 0801 some bottles of vaccine and the market is that 100409 immune swines compare test, and result of the test sees Table 5,6:
Table 5 safety testing result
Table 6 Immunization result of the test
Annotate: Haemophilus parasuis morbidity standard: heating (body temperature continues 1～5 more than 40.5 ℃), lethargy appear in the morbidity pig, cough, dyspnea, become thin, walk lamely and by the thick clinical symptoms such as disorderly of hair.Dying pig is cutd open inspection, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, serosity or fibrinous exudate appear in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
Can find out according to result of the test; the Haemophilus parasuis tervalence inactivated vaccine (4 type JS strains+5 type ZJ strains+12 type HeB strains) of making for adjuvant with Montanide GEL PR (comprising the homologous series adjuvant) is more safer and have a good protection effect than the like product of selling on the market; can overcome the phase mutual interference between the various strains; protective rate can reach more than 80%, the Haemophilus parasuis that prevents simultaneously haemophilus parasuis 4 types, 5 types, 12 types to cause.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (11)
1. a Haemophilus parasuis tervalence inactivated vaccine is characterized in that, is comprised of the acceptable adjuvant of three kinds of haemophilus parasuis antigens and veterinary.
2. the wherein said antigen of Haemophilus parasuis tervalence inactivated vaccine according to claim 1 is the haemophilus parasuis serum 4 type JS strains of deactivation, the haemophilus parasuis Serotype 5 ZJ strain of deactivation, the haemophilus parasuis serum 12 type HeB strain antigens of deactivation.
3. Haemophilus parasuis tervalence inactivated vaccine according to claim 1 is characterized in that, described haemophilus parasuis serum 4 type JS strains, and preserving number is CCTCC NO:M 2011172; Haemophilus parasuis Serotype 5 ZJ strain, preserving number is CCTCC NO:M2011173; The 12 type HeB strains of haemophilus parasuis serum, preserving number is CCTCC NO:M 2011174.
4. Haemophilus parasuis tervalence inactivated vaccine according to claim 1 is characterized in that, being in equal proportions of described three kinds of antigens, and the content of haemophilus parasuis serum 4 types, 5 types, 12 type antigens respectively is 2.0 * 10
9CFU/mL.
5. Haemophilus parasuis tervalence inactivated vaccine according to claim 1, it is characterized in that the acceptable adjuvant of described veterinary is one or more compositions of white-oil adjuvant, aluminium hydroxide gel adjuvant and Montanide IMS1313 VG, Montanide IMS 251C VG, Montanide ISA15A VG, Montanide GEL PR adjuvant.
6. Haemophilus parasuis tervalence inactivated vaccine according to claim 1 is characterized in that, the acceptable adjuvant of described veterinary is aqueous adjuvant Montanide GEL PR.
7. Haemophilus parasuis tervalence inactivated vaccine according to claim 1 is characterized in that, also contains the thimerosal of vaccine total amount 0.01%V/V in the described Haemophilus parasuis tervalence inactivated vaccine.
8. the preparation method of a Haemophilus parasuis tervalence inactivated vaccine claimed in claim 1 is characterized in that, may further comprise the steps:
A. respectively the 4 type JS strains of haemophilus parasuis serum, haemophilus parasuis Serotype 5 ZJ strain, haemophilus parasuis serum 12 type HeB strain culture propagations are cultivated, obtained 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid, 12 type HeB strain bacterium liquid;
B. respectively step a is cultivated the 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid, the 12 type HeB strain bacterium liquid that obtain and carry out deactivation, and concentrated bacterium liquid;
C. above-mentioned deactivation, concentrated 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid, 12 type HeB strain bacterium liquid equal-volume ratios are mixed, and the thimerosal of adding vaccine total amount 0.01% (V/V), then carry out emulsifying with the pharmaceutically acceptable different adjuvants of beast, wherein, Haemophilus parasuis tervalence inactivated vaccine antigenic content is that haemophilus parasuis serum 4 types before the deactivation, 5 types, 12 type viable counts respectively are 2.0 * 10
9CFU/mL.
9. the preparation method of Haemophilus parasuis tervalence inactivated vaccine according to claim 6, it is characterized in that the acceptable adjuvant of described veterinary is one or more compositions of white-oil adjuvant, aluminium hydroxide gel adjuvant and Montanide IMS1313 VG, Montanide IMS 251C VG, Montanide ISA15A VG and Montanide GEL PR polymer adjuvant.
10. a haemophilus parasuis is characterized in that, described haemophilus parasuis is the 4 type JS strains of haemophilus parasuis serum, and preserving number is CCTCC NO:M 2011172; Haemophilus parasuis Serotype 5 ZJ strain, preserving number is CCTCC NO:M 2011173; The 12 type HeB strains of haemophilus parasuis serum, preserving number is CCTCC NO:M 2011174.
11. according to claim 1-7 application of the described triple inactivated vaccine of any one in the medicine of preparation prevention and treatment haemophilus parasuis related diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110218831.2A CN102908615B (en) | 2011-08-01 | 2011-08-01 | Novel haemophilus parasuis disease trivalent inactivated vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110218831.2A CN102908615B (en) | 2011-08-01 | 2011-08-01 | Novel haemophilus parasuis disease trivalent inactivated vaccine and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102908615A true CN102908615A (en) | 2013-02-06 |
| CN102908615B CN102908615B (en) | 2014-11-19 |
Family
ID=47607347
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110218831.2A Active CN102908615B (en) | 2011-08-01 | 2011-08-01 | Novel haemophilus parasuis disease trivalent inactivated vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102908615B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194413A (en) * | 2013-04-11 | 2013-07-10 | 江苏省农业科学院 | Serotype 4 haemophilus parasuis (HPs) vaccine strain |
| CN103948918A (en) * | 2014-03-21 | 2014-07-30 | 母连志 | Method for preparing trivalent inactivated vaccine for preventing mink hemorrhagic pneumonia |
| CN104450556A (en) * | 2014-10-09 | 2015-03-25 | 扬州优邦生物制药有限公司 | Serum-12 type haemophilus lus paradis vaccine strain and application thereof |
| WO2015051371A3 (en) * | 2013-10-04 | 2015-07-09 | Merial Limited | Haemophilus parasuis vaccine serovar type four |
| CN105420141A (en) * | 2015-09-17 | 2016-03-23 | 龙岩学院 | Haemophilus parasuis strain |
| CN105617373A (en) * | 2014-11-06 | 2016-06-01 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and applications thereof |
| WO2016119078A1 (en) * | 2015-01-29 | 2016-08-04 | 山东省农业科学院畜牧兽医研究所 | Combined use of haemophilus parasuis lc strain and lz-20100109 strain |
| CN106350571A (en) * | 2016-08-30 | 2017-01-25 | 北京市农林科学院 | Method for rapidly screening sensitive antibiotic for treating cow mastitis |
| 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 |
| CN107589256A (en) * | 2017-09-30 | 2018-01-16 | 天津瑞普生物技术股份有限公司 | The method of inspection of the type of Haemophilus parasuis 4,5 type bivalent inactivated vaccine effect |
| CN109806389A (en) * | 2019-02-22 | 2019-05-28 | 河南省农业科学院畜牧兽医研究所 | A kind of trivalent inactivated vaccine against Haemophilus parasuis infection and its application |
| CN112870342A (en) * | 2021-01-25 | 2021-06-01 | 兆丰华生物科技(南京)有限公司 | Preparation method of trivalent inactivated vaccine of haemophilus parasuis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101721696A (en) * | 2009-12-18 | 2010-06-09 | 西南民族大学 | Trivalent oil emulsion inactivated vaccine for 4 type, 5 type and 13 type serum of haemophilus parasuis |
-
2011
- 2011-08-01 CN CN201110218831.2A patent/CN102908615B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101721696A (en) * | 2009-12-18 | 2010-06-09 | 西南民族大学 | Trivalent oil emulsion inactivated vaccine for 4 type, 5 type and 13 type serum of haemophilus parasuis |
Non-Patent Citations (1)
| Title |
|---|
| 蔡旭旺: "副猪嗜血杆菌的分离鉴定及诊断方法与灭活疫苗的研究", 《中国优秀博硕士学位论文全文数据库（博士）农业科技辑》 * |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194413B (en) * | 2013-04-11 | 2014-12-03 | 江苏省农业科学院 | Serotype 4 haemophilus parasuis (HPs) vaccine strain |
| CN103194413A (en) * | 2013-04-11 | 2013-07-10 | 江苏省农业科学院 | Serotype 4 haemophilus parasuis (HPs) vaccine strain |
| WO2015051371A3 (en) * | 2013-10-04 | 2015-07-09 | Merial Limited | Haemophilus parasuis vaccine serovar type four |
| CN105992592A (en) * | 2013-10-04 | 2016-10-05 | 梅里亚股份有限公司 | Haemophilus parasuis vaccine serovar type four |
| JP2016533353A (en) * | 2013-10-04 | 2016-10-27 | メリアル インコーポレイテッド | Hemophilus paraswiss vaccine serotype 4 |
| US9504740B2 (en) | 2013-10-04 | 2016-11-29 | Merial, Inc. | Haemophilus parasuis vaccine serovar type four |
| CN103948918A (en) * | 2014-03-21 | 2014-07-30 | 母连志 | Method for preparing trivalent inactivated vaccine for preventing mink hemorrhagic pneumonia |
| CN104450556A (en) * | 2014-10-09 | 2015-03-25 | 扬州优邦生物制药有限公司 | Serum-12 type haemophilus lus paradis vaccine strain and application thereof |
| CN105617373B (en) * | 2014-11-06 | 2019-07-09 | 普莱柯生物工程股份有限公司 | A kind of vaccine composition and its preparation method and application |
| CN105617373A (en) * | 2014-11-06 | 2016-06-01 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and applications thereof |
| WO2016119078A1 (en) * | 2015-01-29 | 2016-08-04 | 山东省农业科学院畜牧兽医研究所 | Combined use of haemophilus parasuis lc strain and lz-20100109 strain |
| CN105420141A (en) * | 2015-09-17 | 2016-03-23 | 龙岩学院 | Haemophilus parasuis strain |
| CN105420141B (en) * | 2015-09-17 | 2019-05-14 | 龙岩学院 | A kind of preparation method of five bivalent inactivated vaccine of haemophilus parasuis bacterial strain |
| CN106350571A (en) * | 2016-08-30 | 2017-01-25 | 北京市农林科学院 | Method for rapidly screening sensitive antibiotic for treating cow mastitis |
| CN106350571B (en) * | 2016-08-30 | 2019-09-24 | 北京市农林科学院 | A kind of method of the sensitive antibiotics of quick screening treatment mastitis for milk cows |
| 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 |
| CN107589256A (en) * | 2017-09-30 | 2018-01-16 | 天津瑞普生物技术股份有限公司 | The method of inspection of the type of Haemophilus parasuis 4,5 type bivalent inactivated vaccine effect |
| CN109806389A (en) * | 2019-02-22 | 2019-05-28 | 河南省农业科学院畜牧兽医研究所 | A kind of trivalent inactivated vaccine against Haemophilus parasuis infection and its application |
| CN109806389B (en) * | 2019-02-22 | 2022-03-22 | 河南省农业科学院畜牧兽医研究所 | Haemophilus parasuis trivalent inactivated vaccine and application thereof |
| CN112870342A (en) * | 2021-01-25 | 2021-06-01 | 兆丰华生物科技(南京)有限公司 | Preparation method of trivalent inactivated vaccine of haemophilus parasuis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102908615B (en) | 2014-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102908615B (en) | Novel haemophilus parasuis disease trivalent inactivated vaccine and preparation method thereof | |
| CN103157100B (en) | hemophilus parasuis disease, swine streptococcosis bivalent inactivated vaccine and preparation method thereof | |
| CN103263666B (en) | Porcine circovirus 2 type, porcine mycoplasmal pneumonia bivalent inactivated vaccine and preparation method thereof | |
| CN102949714B (en) | Swine Streptococcosis trivalent inactivated vaccine and preparation method thereof | |
| CN104530232A (en) | Preparation method of refined egg yolk antibody for duck viral hepatitis | |
| CN104248755A (en) | Haemophilus parasuis disease vaccine composition, preparation method and application thereof | |
| CN103908665B (en) | A kind of vaccine combination and its preparation method and application | |
| CN106177935A (en) | A kind of ruminant clostridial disease tetrad inactivated vaccine and preparation method thereof | |
| CN103191421A (en) | Application of serotype 5 haemophilus parasuis (HPs) vaccine strain | |
| CN104774796B (en) | Fowl enteropathogenic E. Coli inactivated vaccine and preparation method thereof | |
| CN104096222B (en) | A kind of vaccine combination and its preparation method and application | |
| CN103103163A (en) | Lentogenic CH60 strain of duck virual hepatitis virus and attenuated live vaccine thereof | |
| CN101766814A (en) | Swine pasteurellosis bivalent inactivated vaccine and preparation method thereof | |
| CN101507816A (en) | Riemerlla anatipestifer bivalent inactivated vaccine and preparation method thereof | |
| CN104069489B (en) | Newcastle disease and infectious bursa of Fabricius bivalent inactivated vaccine and preparation method thereof | |
| CN103157101B (en) | Combined inactivate vaccine for haemophilus parasuis disease and streptococcus suis disease and preparation method for same | |
| CN106492210A (en) | Goats contagious pleuropneumonia inactivated vaccine and production method thereof | |
| CN104511015A (en) | Vaccine composition, preparation method and application thereof | |
| CN105797149A (en) | Grass carp bacterial septicemia and grass carp bacterial red skin disease bigeminy propolis inactivated vaccine and preparing technology | |
| RU2549434C2 (en) | Method for making brucellosis diagnostic serum | |
| CN101703769A (en) | Novel tetravalent inactivated vaccine for streptococcus suis disease | |
| CN102139104A (en) | Production method for triple inactivated vaccine for newcastle disease, avian influenza (H9 subtype) and infectious bursal disease | |
| CN105582535A (en) | Preparation method of CSF (Classical Swine Fever) and PR (Pseudorabies) bivalent live vaccine and product of CSF and PR bivalent live vaccine | |
| CN101721695B (en) | Novel trivalent inactivated vaccine for swine streptococcosis | |
| CN103566364B (en) | Porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |