CN106729686A

CN106729686A - A kind of haemophilus parasuis tetravalent inactivated vaccine

Info

Publication number: CN106729686A
Application number: CN201611163456.5A
Authority: CN
Inventors: 郭莉莉; 胡潇; 宫晓; 刘新文; 申洪银
Original assignee: Qingdao Yebio Bioengineering Co Ltd
Current assignee: Qingdao Yebio Bioengineering Co Ltd
Priority date: 2016-12-16
Filing date: 2016-12-16
Publication date: 2017-05-31

Abstract

The present invention provides a kind of haemophilus parasuis tetravalent inactivated vaccine, and wherein antigen is haemophilus parasuis YBH04, YBH05, YBH12 plant and YBH13 plants of inactivation.4 type YBH04 plants deposit number for CGMCC No.5479,5 type YBH05 plant deposit number for CGMCC No.5480,12 type YBH12 plant deposit number be CCTCC M 2016636,13 type YBH13 plants deposit number be CGMCC No.5501.The present invention filtered out from advantage epidemic link type YBH04 plants good of haemophilus parasuis 4 of immunogenicity, 5 type YBH05 plants, 12 type YBH12 plants and 13 type YBH13 plants prepare bacterial strain as vaccine, it is inoculated in respectively on TSB nutrient broth mediums and cultivates, harvest culture, inactivated through formalin, add 201 adjuvant mixing and emulsifyings to be made；For the haemophilus parasuis infection disease for preventing to be caused by 4,5,12,13 type haemophilus parasuises；This vaccine has the advantages that security is good, immune efficacy is high and duration of immunity is long.

Description

A kind of haemophilus parasuis tetravalent inactivated vaccine

Technical field

The invention belongs to veterinary biologicses technical field, and in particular to a kind of haemophilus parasuis tetravalent inactivated vaccine.

Background technology

Haemophilus suis (Haemophilus parasuis, HPS) is the pathogen of pig Glasser's diseases, causes pig Polyserositis, arthritis and meningitis etc..Foreign countries show that HPS epidemiology surveys the separation rate of clinical sick pig is up to 20% or so.As the development of China's Compact Develop, the generation of Haemophilus parasuis are in rising trend, caused to pig industry Huge economic loss, is one of bacterial infectious disease of serious harm pig industry development in recent years.Haemophilus parasuis is extremely Rare 15 serotype, the popular predominant serotype of country variant or area is different, the pathogenic and immunology of different serotypes Property difference is notable.At present, it has already been proven that haemophilus parasuis exists and popular on China overwhelming majority pig farm.

The more flourishing country of the pig industrys such as the U.S., Japan, Canada, Germany, Australia, Denmark, Spain is right The haemophilus parasuis epidemiology of this country is investigated, and shows that the separation rate of haemophilus parasuis is up to 20% or so, and And the serotype of prevalence generally all virulence are stronger, such as the type of serotype 4,5 types and 13 types.Japan, the U.S. and Hispanic serotype It is the most popular with 4 types and 5 types；And the isolated strains of Denmark and Australia are mainly based on Serotype 5 and 13 types；It is German main It is the type of serum 2 and 4 types.The prevalence study result of various countries shows that various countries' prevalence serotype respectively has difference, with otherness.

In recent years, Chinese scholar has carried out epidemiology survey to the haemophilus parasuis of different regions, roundup Result of study display serum 4 type, 5 types, 12 types and 13 types are current China serotype the most popular.We to the whole nation differently The haemophilus parasuis in area carries out epidemiological investigation, as a result shows currently to bring the pair of harm to China's pig industry development The predominant serotype of haemophilus suis is 4 types, 5 types, 12 types and 13 types.

The content of the invention

It is an object of the invention to provide a kind of haemophilus parasuis tetravalent inactivated vaccine, thus for preventing by 4,5,12, The haemophilus parasuis infection disease that 13 type haemophilus parasuises cause.

Haemophilus parasuis tetravalent inactivated vaccine of the present invention, wherein antigen be inactivation haemophilus parasuis YBH04, YBH05, YBH12 plant and YBH13 plants；

Wherein 4 type YBH04 plants deposit number for CGMCC No.5479,5 type YBH05 plant deposit number be CGMCC No.5480；13 type YBH13 plants deposit number be CGMCC No.5501；

Haemophilus parasuis YBH12 (Haemophilus parasuis YBH12) strain, in the preservation of November 10 in 2016 In Wuhan, China, the China typical culture collection center of Wuhan University, deposit number is CCTCC NO:M 2016636.

Above-mentioned haemophilus parasuis YBH04, YBH05, YBH12 plant and YBH13 plants is gone out using formalin It is living；

In above-mentioned inactivated vaccine the quantity of YBH04, YBH05, YBH12 plant and YBH13 bacterial strains be preferably 2.5-3.0 × 10⁹CFU/ml。

The present invention filters out immunogenicity good type YBH04 plants of haemophilus parasuis 4,5 types from advantage epidemic link YBH05 plants, 12 type YBH12 plants and 13 type YBH13 plants prepare bacterial strain as vaccine, TSB nutrient broth mediums are inoculated in respectively Upper culture, harvests culture, is inactivated through formalin, adds 201 adjuvant mixing and emulsifyings to be made；For preventing by 4,5,12,13 The haemophilus parasuis infection disease that type haemophilus parasuis causes；This vaccine has security is good, immune efficacy is high and duration of immunity is long etc. Advantage.

Specific embodiment

The information of antigen bacterial strain used in the present invention is as follows：

First, the screening of type YBH12 plants of haemophilus parasuis serum 12

1. since two thousand eight, our epidemiology to haemophilus parasuis are investigated, and right for epidemiology survey Selected swine farms are tracked investigation.Investigation result finds that the type of serum 4,5,12,13 is currently a popular serotype.And 4,5,13 types Suitable Vaccine strains have been filtered out, and tervalence inactivated vaccine has been developed；But the bacterium of the type of haemophilus parasuis serum 12 Strain is also screened.

2. bacteria distribution gathers lungs, pleural effusions and ascites, hydropericardium, painstaking effort, joint fluid and the brain tissue of doubtful sick pig Deng pathological material of disease, TSA culture mediums are inoculated with, in 5%CO₂Under the conditions of, 37 DEG C of 24~48h of culture, picking single bacterium colony is cultivated and is reflected It is fixed.

3. Bacteria Identification

3.1 morphologic observations take doubtful bacterium colony, gram stain microscopy after smear, observe its ne ar.

3.2 biochemical characteristics identification take doubtful bacterium colony routinely carry out hemolytic experiment, urease test, oxidase test, V-P experiments, catalase test, nitrate reduction test, indole test, satellite growth test；Sugar fermentating test, including grape Sugar, sucrose, fructose, galactolipin, ribose and maltose.Bacterial strain is compared with the type International Reference bacterial strains of HPS 12 simultaneously, in 37 DEG C 24~48h of culture, observes result.

According to haemophilus parasuis 16S rRNA primers, primer is (F for 3.3 PCR identifications：5’-GGC TTC GTC ACC CTC TGT -3 ', R：5’-GTG ATG AGG AAG GGT GGT GT–3’).Doubtful HPS to being separated to is separated Bacterial strain enters performing PCR identification, and the product of PCR amplifications should be the fragment of 822bp sizes.

The identification of 3.4 serological characteristics carries out serotype mirror using agar gel diffusion test to haemophilus parasuis isolated strains It is fixed.

4. the type haemophilus parasuis of 10 plants of serum 12 difference intraperitoneal injection 8~10 week old health that virulence test will be separate Each 5 of susceptible piglet, 3.0ml/ (viable bacteria amount is each about 5.0 × 10⁹CFU), separately take 5 and do not attack poison as control.It is continuous to see Examine 14, record morbidity and the death condition of test pig.

5. immunogenicity is according to virulence test result, and YBH12 plant stronger isolated strains bacterium solution of the virulence that will be screened is distinguished Adjust to 2.0 × 10⁹CFU/ml, with 201 adjuvants in mass ratio 1 after formalin-inactivated:1 mixing and emulsifying, prepares univalent inactivated vaccine, The week old piglet of intramuscular injection 3~5,2.0ml/ heads, every group 5.Two are carried out by same dose same procedure within 21 days after primary immune response Secondary immune, all piglets on the 14th are taken a blood sample and separate serum after secondary immunity, and the antibody for determining serum using micro-agglutination method is imitated Valency；And carry out with this bacterial strain intraperitoneal injection respectively and attack poison (viable bacteria amount is each about 6.0 × 10⁹CFU).The morbidity of viewing test pig and Death condition, compares immunogenicity of the different strains to pig.Result of the test shows that micro-agglutination antibody is 5/5 more than or equal to 1: 32；Immune group is 5/5 protection, and control group is 5/5 morbidity.

6. intersection is attacked poison and is protected according to virulence test result, YBH12 plants of stronger isolated strains bacterium solution of the virulence that will be screened Adjust to 2.0 × 10⁹CFU/ml, with 201 adjuvants in mass ratio 1 after formalin-inactivated:1 mixing and emulsifying, prepares univalent inactivated vaccine, Leg muscle injects piglet, 2.0ml/ heads.Secondary immunity is carried out by same dose same procedure within 21 days after primary immune response, it is secondary to exempt from All piglets on the 14th are carried out attacking poison (6.0 × 10 respectively with 10 plants of separate homotype bacterial strains after epidemic disease⁹CFU).Observation 14 days, record examination Test morbidity and the death condition of piglet.Result of the test shows that univalent inactivated vaccine prepared by YBH12 strain vaccines can resist YBH12 Strain and other 9 plants of attacks of separation strains, attack malicious protective rate and are 5/5, and malicious protection is attacked with preferably intersection.

Three haemophilus parasuises of other used in haemophilus parasuis tetravalent inactivated vaccine of the invention (Haemophilus parasuis) bacterial strain is YBH04, YBH05 and YBH13 plant；Wherein 4 type YBH04 plants (2009 from Shandong Save separation in the joint fluid of certain pig farm sick dead pig) and the 5 type YBH05 plants of (passes from Shandong Province Pingdu pig farm sick dead pig in 2008 Separated in section liquid), 13 type YBH13 plants (being separated from the heart of Jinan City, Shandong Province pig farm sick dead pig for 2009).4 type YBH04 Strain, 5 type YBH05 plants and 13 type YBH13 plants deliver Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on November 23rd, 2011 The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center of Institute of Microorganism, Academia Sinica, deposit number Respectively：CGMCC No.5479, CGMCC No.5480 and CGMCC No.5501；12 type YBH12 plants in November 10 in 2016 It is deposited in the China typical culture collection center of Wuhan, China, Wuhan University day, deposit number is CCTCC M 2016636.

2 strain features

YBH04, YBH05, YBH12 plant and YBH13 plants bacterium is for 2.1 forms and biochemical characteristic haemophilus parasuis bacterial strain The tiny bacillus of Gram-negative, with various different forms (from single coccobacillus to it is long, elongated, so that thread Thalline).Catalase test, nitrate reduction test are the positive, hemolytic experiment, urease test, oxidase test, V-P Experiment, indole test are feminine gender, and " satellite growth " phenomenon can be presented, can glucose fermentation, sucrose, fructose, galactolipin, ribose And maltose.

2.2 YBH04, YBH05, YBH12 plant of cultural character haemophilus parasuises and YBH13 plants are containing 0.01% coenzyme (NAD) and in the pancreas peptone soybean broth fluid nutrient medium of 5% NBCS grown in the muddy of uniformity；Containing On the tryptose soya agar solid medium of 0.01%NAD and 5% NBCS, 37 DEG C are cultivated 24~48 hours, shape Into 0.3~2.0mm of diameter, circle, it is smooth, moistening, water white transparency dewdrop sample bacterium colony.

2.3 serotype application agar gel diffusion tests carry out Serotype Identification, haemophilus parasuis YBH04, YBH05, YBH12 plants is respectively the types of HPS 4,5,12,13 with YBH13 plants of bacterium.

The week old of 2.4 Strain Virulence intraperitoneal injection 8~10 susceptible piglet of health, can make at least 4 and the morbidity of above piglet The poison amount of attacking of each bacterial strain is respectively YBH04 plants about 5.0 × 10⁹CFU/ heads, YBH05 plants about 5.0 × 10⁹CFU/, YBH12 Strain about 6.0 × 10⁹CFU/, YBH13 plants about 6.0 × 10⁹CFU/ heads.

2.5 immunogenicities take 3~5 week old susceptible piglet 30 of health, are randomly divided into eight groups of A, B, C, D, E, F, G, H, often Group 5, A, B, C, D group is immune group, and E, F, G, H group are nonimmune control group.The musculi colli injection respectively of A, B, C, D group YBH04 plants, YBH05 plants, YBH12 plants, YBH13 plants of univalent 201 adjuvant inactivated vaccine, 2.0ml/ (antigenic content be 0.5 × 10⁹CFU/ml).Two are carried out after immune by same procedure and dosage within 21st to exempt from, two exempt to attack poison within 14th afterwards.A, E group test pig abdominal cavity (viable bacteria amount is about 5.0 × 10 to YBH04 plants of bacterium solution 3.0ml of injection⁹CFU/)；B, F group YBH05 plants of bacterium solution of test pig intraperitoneal injection (viable bacteria amount is about 5.0 × 10 to 3.0ml⁹CFU/)；(viable bacteria amount is about for YBH12 plants of bacterium solution 3.0ml of C, G group test pig intraperitoneal injection It is 6.0 × 10⁹CFU/)；(viable bacteria amount is about 6.0 × 10 to YBH13 plants of bacterium solution 3.0ml of D, H group test pig intraperitoneal injection⁹CFU/ Head).Observation 14 days, immune group pig all should at least 4 head protections, control group pig all should at least 4 hairs disease.

The production method of haemophilus parasuis tetravalent inactivated vaccine, mainly：

1 is cultivated four plants of strains respectively using fermentation tank：Cultivated by 60%~80% addition TSB of fermenter volume Base, while adding defoamer, is passed through high steam and is sterilized, and when its temperature is down to 37~38 DEG C, is separately added into 5% newborn Cow's serum, 0.01%NAD and 3%~5% secondary seed solution carry out fermented and cultured.36~37 DEG C of cultivation temperature, speed of agitator 100r/min, pH value 7.2~7.4, it is 60%~80%, bacterium solution that whole incubation controls dissolved oxygen amount by adjusting air inflow After culture 12~14 hours, sampling carries out count plate and purely inspection.Respectively by the bacterium solution of completion of fermenting, pillar centrifugation is imported Machine is centrifuged, and harvests bacterium mud, 2~8 DEG C of preservations is placed in, no more than 48 hours.According to count plate result, bacterium mud is resuspended in respectively In the physiological saline of appropriate sterilizing, 1.6 × 10 are diluted to¹⁰CFU/ml, metered 10% formalin, makes it dense eventually respectively It is 0.2% to spend, and 37 DEG C of stirrings are inactivated 16 hours, carry out inactivation inspection, and remaining bacterium solution put 2~8 DEG C of preservations, no more than 14 days.Will The type antigen liquid mixed in equal amounts of haemophilus parasuis 4,5,12,13 of inactivation, as water phase.

2 vaccines prepare water phase and oil phase is preheated to 31 DEG C, in mass ratio 1:1 mixing, 4000r/min stirs 30~40 points Clock, prepares inactivated vaccine.

The present invention is described in detail with reference to embodiment.

Embodiment 1：

First, prepared by vaccine

1 production is prepared with seed

The recovery of 1.1 production strains and first order seed are bred respectively by YBH04 plants, YBH05 plants, YBH12 plants, YBH13 plants Freeze-drying lactobacillus dissolve, and streak inoculation puts 5%CO in TSA culture mediums₂, 37 DEG C of cultures 24~48 hours, single typical case is chosen respectively In TSA culture mediums, 37 DEG C are cultivated 16~24 hours the intensive streak inoculation of bacterium colony；Picking lawn is inoculated in TSB culture mediums, 37 respectively DEG C 150r/min shaken cultivations 12~16 hours, through purely after the assay was approved, as first order seed.Less than -15 DEG C preserve, should not More than 30 days.

In 3%~5% ratio be inoculated with TSB culture mediums first order seed by the breeding of 1.2 secondary seeds, and 37 DEG C of 150r/min shake Culture is swung 12~16 hours, after microscopy is pure, as secondary seed.2~8 DEG C of preservations, should be no more than 24 hours.

2 seedling culture mediums are TSB culture mediums.

The preparation of 3 seedling bacterium solutions

3.1 bacterium solution cultures are cultivated four plants of strains respectively using fermentation tank：By the 60%~80% of fermenter volume TSB culture mediums are added, while adding defoamer, high steam is passed through and is sterilized, when its temperature is down to 37~38 DEG C, respectively Adding 5% NBCS, 0.01%NAD and 3%~5% secondary seed solution carries out fermented and cultured.36~37 DEG C of cultivation temperature, Speed of agitator 100r/min, pH value 7.2~7.4, whole incubation controlled by adjusting air inflow dissolved oxygen amount for 60%~ 80%, after bacterium solution culture 12~14 hours, sampling carries out count plate and purely inspection.Respectively by the bacterium solution of completion of fermenting, lead Enter pillar centrifuge, harvest bacterium mud, 2~8 DEG C of preservations are placed in, no more than 48 hours.

Respectively be resuspended in bacterium mud in the physiological saline of appropriate sterilizing by 3.2 inactivations according to count plate result, dilutes To 1.6 × 10¹⁰CFU/ml, metered 10% formalin respectively, make its final concentration of 0.2%, 37 DEG C of stirring inactivations 16 are small When, inactivation inspection is carried out, remaining bacterium solution put 2~8 DEG C of preservations, no more than 14 days.

4 inspections of semifinished product

4.1 purely inspections are by existing《Chinese veterinary pharmacopoeia》Annex is tested.

4.2 count plates are by existing《Chinese veterinary pharmacopoeia》Annex is counted.

4.3 inactivation inspections are by existing《Chinese veterinary pharmacopoeia》Annex is tested.

It is prepared by 5 vaccines

5.1 water are mutually prepared will check YBH04 plants qualified, YBH05 plants, YBH12 plants, YBH13 plants of bacterium solution mixed in equal amounts.

5.2 oil phase MONTANIDETM ISA 201VG adjuvants (hereinafter referred to as 201 adjuvants).

5.3 emulsified water phases and oil phase are preheated to 31 DEG C, in mass ratio 1:1 mixing, 4000r/min is stirred 30~40 minutes.

6 packing quantitative separatings, seal, and adhesive label, put 2~8 DEG C of preservations.

2nd, the safety test of vaccine

1 pair of minimum single dose and single dose using age in days repeats the experiment 21 ages in days susceptible piglet 15 of health, is divided into 3 groups, every group 5.A, B group are immunized again on the 14th per incidence 201501 batches of vaccine 2.0ml, B groups of intramuscular injection after primary immune response Once.C groups are not immunized controls group.Observation 14 days, the injection site for observing piglet whether there is the adverse reactions such as red and swollen, scleroma.Examination Test result to show, piglet single dose immunization and single dose repeat immune spirit, diet normally, and injection site is without red and swollen, scleroma Deng adverse reaction.

3 batches of epidemic diseases that the safety testing of 2 pairs of overdose injections of piglet, farrowing sow and herd boar is manufactured experimently with laboratory Seedling (201501,201502,201503 batches) takes 21 age in days piglets, antenatal 6-7 week old farrowing sow, 1-3 herd boars each 5 respectively Head, per incidence intramuscular injection 4.0ml/ heads, observes 14, and observing the injection site of piglet, to whether there is red and swollen, scleroma etc. bad anti- Should.Result of the test shows that overdose is immunized the spirit of piglet, farrowing sow and herd boar, diet normally, and injection site is without red The adverse reactions such as swollen, scleroma.

3rd, vaccine micro-agglutination antibody titer and attack poison protection correlation test

The pig Haemophilus parasuis tetravalent inactivated vaccine (hereinafter referred to as " inactivated vaccine ") manufactured experimently with this seminar laboratory, 0.5ml/, 1ml/, the 2ml/ various dose susceptible piglet of immune 3 week old health are pressed respectively, carry out minimum immune dosage examination Test and antibody protects correlation test with poison is attacked.Minimum immune dosage result shows, the immune son of the dosage group of more than 1ml/ The type antibody titers of HPS 4,5,12,13 are 1 in Swine serum:32 and more than, attack poison can reach 4/5 and above protect, therefore should The immune amount of the minimum of vaccine is 1ml/ heads, it is contemplated that influence factor is more during clinical practice, to ensure the immune effect of vaccine, will The immunizing dose of inactivated vaccine is defined as 2.0ml/ heads.

Antibody shows with poison protection correlation test result is attacked, when antibody titer≤1 of the types of HPS 4,5,12,13:When 8, It is≤2/5 to attack malicious protective rate；Antibody titer is 1:When 16, it is≤3/5 to attack malicious protective rate；Antibody titer >=1:When 32, malicious guarantor is attacked Shield rate is 4/5~5/5.Therefore antibody and attack poison protection between be proportionate.Result of the test refers to table 1-2.

The antibody titer of the immune swine of table 1

Table 2 is immunized the protest test result of piglet

4th, antibody dynamic regularity, immune duration and piglet maternal antibody duration experiment

With (4 type YBH04 plants+5 type YBH05 plants of 3 batches of standby pig Haemophilus parasuis tetravalent inactivated vaccines of this project team system + 12 type YBH12 plants+13 type YBH13 plants) (lot number is respectively 201501,201502,201503), musculi colli injects 3~5 weeks The susceptible piglet of age health, 1~3 year herd boar and antenatal 6~7 weeks farrowing sows.Piglet head carries out two for 3 weeks and exempts from after exempting from, 2.0ml/ Head.Piglet exempt from one after 21 days, two exempt from after randomly select within 7 days, 14 days, 1,2,4,6,7 months 5 blood samplings, HPS in measure serum 4th, the antibody titer of 5,12,13 types；1,3,6,7 months after immune, blood sampling respectively determines its antibody titer to herd boar, and observation is anti- The Fluctuation of body level；Sow was immunized at antenatal 6~7 weeks, and 2,4,6,7 months blood sampling detection serum resists after immune Body, after farrowing, blood sampling on the 7th, 14,21,28th determines the antibody level of serum after piglet is born respectively, and in 21 days after birth Protest test was carried out with 28 days.Result of the test shows that piglet is being immunized rear 7 months antibody still >=1:32；Herd boar exempts from rear 7 Individual month antibody is still >=1:32；7 months antibody >=1 after sow is immune:32, the pig that farrows birth after antibody on the 21st still >=1:32,21 The malicious protective rate of attacking of day is 4/5~5/5, and the antibody of 28 days is respectively less than 1 after birth:The malicious protective rate of attacking of 32,28 days is 2/5~3/ 5.According to data above, final to determine that vaccine is 6 months to the duration of immunity of piglet and boar, piglet maternal antibody is protected to birth 21 days afterwards.Refer to table 3- tables 6.

The measure of micro-agglutination antibody dynamic regularity after the piglet immunological of table 3

The measure of micro-agglutination antibody dynamic regularity after the antenatal 6-7 weeks farrowing sow of table 4 is immune

The piglet maternal antibody Fluctuation measurement result of table 5

Test pig age in days	The types of HPS 4	The types of HPS 5	The types of HPS 12	The types of HPS 13
					1 week old piglet	≥1:32	≥1:32	≥1:32	≥1:32
2 week old piglets	≥1:32	≥1:32	≥1:32	≥1:32
					3 week old piglets	≥1:32	≥1:32	≥1:32	≥1:32
4 week old piglets	≤1:8	≤1:16	≤1:8	≤1:16
					Nonimmune control group	≤1:4	≤1:4	≤1:4	≤1:4

The passive immunity piglet protest test result of table 6

Also, it is immunized with the haemophilus parasuis vaccine sold in the market, then the YBH04 used with the present invention Strain, YBH05 plants YBH12 plants YBH13 plants carry out challenge viral dosage；Result shows that the immune effect of the vaccine sold in the market is remote Less than tetravalent vaccine effect prepared by the present invention.

5th, vaccine product inspection

1 proterties

Appearance milky white emulsion.

Formulation water-in-oil-in water.A cleaning suction pipe is taken, a small amount of vaccine is drawn and is dripped in cold water surface, should expanded in cloud Dissipate.

Stability is drawn in vaccine 10ml addition centrifuge tubes, is centrifuged 15 minutes with 3000r/min, the water phase that ttom of pipe is separated out 0.5ml should be no more than.

Viscosity is by existing《Chinese veterinary pharmacopoeia》Annex is carried out, and should meet regulation.

The inspection of 2 loading amounts is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and should meet regulation.

3 steriling tests are by existing《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.

4 safety verifications, per incidence intramuscular injection vaccine 4.0ml, observe 14 with 3~5 week old susceptible piglet 5 of health Day, should occur without because of the locally or systemically adverse reaction that vaccine causes.

5 efficacy tests are first checked with serological method, during serological method disqualified upon inspection, then are entered with Immunization method Performing check.

5.1 serological methods take 3~5 week old susceptible piglet 10 of health, wherein 5 incidence intramuscular injection vaccines 2.0ml/ heads, remaining 5 are made nonimmune control.21 days after primary immune response, secondary immunity is carried out by same dose and same procedure, 14 days after secondary immunity serum of being taken a blood sample to all piglets and separated, detects the type antibody titer of HPS4,5,13.The blood of immune group piglet In clear 4,5,12,13 type antibody titers all should >=1:32, the type antibody titer of serum 4,5,12,13 of control group piglet all should≤1: 4。

5.2 Immunization methods take 3~5 week old susceptible piglet 40 of health, are randomly divided into eight groups of A, B, C, D, E, F, G, H, Every group 5, A, B, C, D group are immune group, and E, F, G, H group are nonimmune control group.The musculi colli injection respectively of A, B, C, D group YBH04 plants, YBH05 plants, YBH12 plants, YBH13 plants of univalent 201 adjuvant inactivated vaccine, 2.0ml/ (the preceding bacterium about content of inactivation It is 2.0 × 10⁹CFU/ml).Two are carried out after immune by same procedure and dosage within 21st to exempt from, two exempt to attack poison within 14th afterwards.A, E group are tested (viable bacteria amount is about 5.0 × 10 to YBH04 plants of bacterium solution 3.0ml of pig intraperitoneal injection⁹CFU/)；B, F group test pig intraperitoneal injection YBH05 (viable bacteria amount is about 5.0 × 10 to strain bacterium solution 3.0ml⁹CFU/)；YBH12 plants of bacterium solution 3.0ml of C, G group test pig intraperitoneal injection is (living Bacterium amount is about 6.0 × 10⁹CFU/)；YBH13 plants of bacterium solution 3.0ml of D, H group test pig intraperitoneal injection (viable bacteria amount is about 6.0 × 10⁹CFU/).Observation 14 days, immune group pig all should at least 4 head protections, control group pig all should at least 4 hairs disease.

6 residual formaldehydes are determined by existing《Chinese veterinary pharmacopoeia》Annex is measured, and should meet regulation.

6th, the effect of vaccine and purposes

For the Haemophilus parasuis for preventing to be caused by 4,5,12,13 type haemophilus parasuises.Duration of immunity is 6 months.

7th, usage and consumption musculi colli are injected.Piglet：3~5 week old head exempt from, booster immunization 1 time after 3 weeks, every time 2.0ml/ heads；Sow：Antenatal 6~7 weeks immune 1 time, each 2.0ml/ heads；Herd boar：Every 6 months immune 1 time, each 2.0ml/ Head.

Embodiment 2

1. production is prepared with seed

(1) first order seed breeding with identification by YBH04, YBH05, YBH12 plant, YBH13 plants of freeze-drying lactobacillus streak inoculation in On TSA agar plates, in 5%~10%CO₂Under the conditions of, 37 DEG C are cultivated 24~48h, each picking colonies typical or lawn, inoculation In 100mlTSB meat soups, 37 DEG C of 24~48h of shaken cultivation are put.2ml bacterium solutions are respectively taken through purely after the assay was approved, as one-level kind Son.Preserved at -20 DEG C, no more than 1 month；Preserved at -70 DEG C, no more than 3 months (referring to table 7).

The preparation of the first order seed of table 7

(2) secondary seed breeding takes YBH04, YBH05, YBH12 plant, YBH13 bacterial strain first order seeds, streak inoculation TSA fine jades Fat flat board, containing 5%~10%CO₂Under the conditions of, 37 DEG C of 24~48h of culture；Colonies typical or lawn are respectively selected, is inoculated in In 800mlTSB meat soups, 37 DEG C of 24~48h of shaken cultivation are put, respectively take 2ml bacterium solutions warp purely after the assay was approved, as two grades kinds Son.In 2~8 DEG C of preservations, 24h (referring to table 8) should be no more than.

The preparation of the secondary seed of table 8

2. bacterium solution culture cultivates 4,5,12,13 type bacterium solutions respectively using fermentation tank.By the 60%~70% of fermenter volume Add the semisynthetic medium 20000ml of production, while add defoamer (bubble enemy) to carry out disinfection, treat its temperature be down to 37~ At 38 DEG C, the secondary seed solution (monotype) of 2~8 DEG C of preservations is added in fermentation tank by 2%~2.5% each 400ml.Culture temperature Degree control 37~38 DEG C, 100~200r/min of speed of agitator, pH 7.2~7.4,0.03~0.05Pa of tank pressure, dissolved oxygen amount 40%, 12~14h is cultivated, bacterium solution 24300 is harvested, formalin 672ml is added by the 0.3% of bacterium solution volume, 37 DEG C of stirrings are gone out 24h living, sampling carries out inactivation inspection, and the bacterium solution after inactivation puts 2~8 DEG C of preservations.(referring to table 9).

Haemophilus parasuis YBH04, YBH05, YBH12 plant of table 9 and YBH13 plants of Counting alive microbial result

4. vaccine formulation

4.1 water are mutually prepared will check YBH04 plants, YBH05 plants, YBH12 plants and YBH13 plants qualified bacterium solution mixed in equal amounts.

4.2 oil phase MONTANIDETM ISA 201VG adjuvants (hereinafter referred to as 201 adjuvants).

4.3 emulsified water phases and oil phase are preheated to 31 DEG C, in mass ratio 1:1 mixing, 4000r/min is stirred 30 minutes.

4.4 packing quantitative separatings, seal, and adhesive label, put 2~8 DEG C of preservations.Refer to table 10.

The vaccine of table 10 is emulsified and dispensed

Vaccine product inspection

(1) proterties

Appearance milky white emulsion.

Formulation water-in-oil type.A cleaning suction pipe is taken, it is in oil droplet shape indiffusion to draw 2ml vaccines and drip in cleaning cold water surface.

Stability is drawn in vaccine 10ml addition centrifuge tubes, and 3000r/min is centrifuged 15 minutes, and anhydrous phase is separated out.

Viscosity measurement result is respectively 25.8cP, 26.2cP, 26.4cP.

(2) steriling test is pressed《Chinese veterinary pharmacopoeia》Method is carried out, asepsis growth.

(3) safety verification takes 3~5 week old susceptible piglet 5 of health, and musculi colli is vaccinated, 4.0ml/ heads, observation 14, all test pig spirit, feeding and drinking-water were showed no obvious abnormalities, and injection site and whole body have no adverse reaction.Refer to Table 11.

The pig Haemophilus parasuis tetravalent inactivated vaccine safety examination result of table 11

(4) residual formaldehyde is determined and pressed《Chinese veterinary pharmacopoeia》Method is carried out, and content of formaldehyde meets for 0.06%~0.07% Regulation.

(5) efficacy test

3~5 week old susceptible piglet 40 of health, is randomly divided into eight groups of A, B, C, D, E, F, G, H, every group 5, wherein A, B, C, D group are immune group, and equal musculi colli is vaccinated, 2.0ml/ heads；E, F, G, H group are nonimmune control group, equal musculi colli Injecting normal saline, 2.0ml/ heads.Carry out secondary immunity by same dose and same procedure within 21 days after immune, two exempt from after 14 days it is right All piglets are taken a blood sample and separate serum, detect the type antibody titers of HPS 4,5,12,13；A, E group test pig intraperitoneal injection after blood sampling (viable bacteria amount is about 5.0 × 10 to YBH04 plants of bacterium solution 3.0ml⁹CFU/)；B, F group YBH05 plants of bacterium solution of test pig intraperitoneal injection (viable bacteria amount is about 5.0 × 10 to 3.0ml⁹CFU/)；(viable bacteria amount is about for YBH12 plants of bacterium solution 3.0ml of C, G group test pig intraperitoneal injection It is 5.0 × 10⁹CFU/)；(viable bacteria amount is about 6.0 × 10 to YBH13 plants of bacterium solution 3.0ml of D, H group test pig intraperitoneal injection⁹CFU/ Head).Observe morbidity and the death condition of 14 day entry test pigs.Result shows, the Serologic detection side that 3 batches of vaccines are carried out with pig Method result shows, 14 days after 3 batches of vaccine secondary immunity pigs, the micro-agglutination antibody titer of 4,5,12,13 types of immune group pig is equal ≥1:32, the antibody of control group pig is ≤1:4.Attack poison protection result and show that the malicious protective rate of attacking of immune group pig is 4/5~5/ 5, the incidence of disease for compareing pig is 4/5~5/5.Refer to table 12.

The pig Haemophilus parasuis tetravalent inactivated vaccine protest test result of table 12

Note:Molecule is noted:Molecule refers to protection piglet number, and denominator refers to attacks malicious piglet sum.

The above results show that the vaccine of present invention preparation has good immune effect to pig haemophilus parasuis.

Claims

1. a kind of haemophilus parasuis tetravalent inactivated vaccine, it is characterised in that described antigen is the haemophilus parasuis of inactivation YBH04, YBH05, YBH12 plant and YBH13 plants.

2. haemophilus parasuis tetravalent inactivated vaccine as claimed in claim 1, it is characterised in that the guarantor of described YBH04 plants It is CGMCC No.5479 to hide numbering.

3. haemophilus parasuis tetravalent inactivated vaccine as claimed in claim 1, it is characterised in that the guarantor of described YBH05 plants It is CGMCC No.5480 to hide numbering.

4. haemophilus parasuis tetravalent inactivated vaccine as claimed in claim 1, it is characterised in that the guarantor of described YBH13 plants It is CGMCC No.5501 to hide numbering.

5. haemophilus parasuis tetravalent inactivated vaccine as claimed in claim 1, it is characterised in that the guarantor of described YBH12 plants It is CCTCC M 2016636 to hide numbering.

6. haemophilus parasuis tetravalent inactivated vaccine as claimed in claim 1, it is characterised in that described haemophilus parasuis YBH04, YBH05, YBH12 plant and YBH13 plants is inactivated using formaldehyde.

7. haemophilus parasuis tetravalent inactivated vaccine as claimed in claim 1, it is characterised in that described YBH04, YBH05, YBH12 plants is 2.5-3.0 × 10 with the quantity of YBH13 bacterial strains⁹CFU/ml。