CN106520623B - A kind of serum 7-type haemophilus parasuis low virulent strain and its application - Google Patents

A kind of serum 7-type haemophilus parasuis low virulent strain and its application Download PDF

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CN106520623B
CN106520623B CN201611091197.XA CN201611091197A CN106520623B CN 106520623 B CN106520623 B CN 106520623B CN 201611091197 A CN201611091197 A CN 201611091197A CN 106520623 B CN106520623 B CN 106520623B
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vaccine
haemophilus parasuis
serum
virulent strain
low virulent
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贾爱卿
廖明
王贵平
樊惠英
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Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd
South China Agricultural University
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Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd
South China Agricultural University
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants

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Abstract

The invention discloses a kind of serum 7-type haemophilus parasuis low virulent strain and its application, serum 7-type haemophilus parasuis low virulent strain is preserved in China typical culture collection center on October 13rd, 2016, and preserving number is CCTCC No:M2016560;The pathogenicity of the low virulent strain weakens significantly, is suitable for preparing the medicine for the treatment of Haemophilus parasuis;The application of the serum 7-type haemophilus parasuis low virulent strain, to prepare the medicine for the treatment of Haemophilus parasuis using serum 7-type haemophilus parasuis low virulent strain;The medicine is vaccine, and the preparation process of vaccine is prepared including 1) primary seed solution;2) prepared by secondary seed solution;3) prepared by original bacteria liquid;4) vaccine is prepared.

Description

A kind of serum 7-type haemophilus parasuis low virulent strain and its application
Technical field
The present invention relates to a kind of serum 7-type haemophilus parasuis low virulent strain and its application, belong to haemophilus parasuis application Technical field.
Background technology
Haemophilus parasuis is the new hair bacterial infectious disease for seriously endangering pig breeding industry in recent years, causes the multiple of pig Scrositis, arthritis and meningoencephalitis etc., incidence are generally 10-15%, and the death rate is brought up to more than 50% to pig breeding industry Huge economic loss, causes the pay attention to day by day of people.The serotype of the sick pathogenic bacteria haemophilus parasuis (Hps) is answered It is miscellaneous various, can be at least 15 by Hps points by Kieleetin.Rapp-Gbarideosn (KRG) AGP test serotype method A serotype, the separation strains serotype for separately having more than 20% can not determine.Existing data shows that Hps has obvious region Feature.
Because haemophilus parasuis easily produces drug resistance, so the effective means for controlling this sick is vaccine inoculation.Document report The use of road commercial vaccine or oneself seedling can control the infection of haemophilus parasuis.But also have and prevent to lose using inactivated vaccine The example lost, this is probably since pathogenic strain is different from vaccine strains serotype thus lacks cross protection.Protected although intersecting Shield is the basic demand to commodity inactivated vaccine, but strain specificity inactivated vaccine may be because that there are more than one in swinery Bacterial strain or serotype and lack effect, it is also possible to because later swinery in introduce new bacterial strain and lose effect.Due to secondary pig The diversity of haemophilus serotype and account for significant proportion be unable to parting bacterial strain, the difference of Strain Virulence, and it is current right Protective antigens and virulence factor lack heightened awareness, it has not been possible to have a kind of inactivated vaccine to be produced at the same time to all pathogenic strains Raw cross-protection.It is prevention and the inevitable requirement and the general trend of events that control Haemophilus parasuis to develop the vaccine with cross protection Become.
The exploitation of vaccine product is in addition to efficient technical requirements, and cost is immunized must be cheap, it is impossible to beyond holding for aquaculture By ability.Subtract (weak) virus live vaccine because with immunizing potency high (antigen dose can be reduced), it is of low cost, broad-spectrum vaccine can be developed The new technology advantage of (live bacterial vaccines have intersecting protective), is the research hotspot of Most current infectious disease.
The content of the invention
For overcome the deficiencies in the prior art, of the invention first purpose is to provide a kind of serum 7-type pair pig bloodthirsty Bacillus HPKP-7 (Latin names:Haemophilus parasuis HPKP-7) natural low virulent strain, the pathogenicity of the low virulent strain Weaken significantly, be suitable for preparing the medicine for the treatment of Haemophilus parasuis.
Achieving the object of the present invention can be reached by adopting the following technical scheme that:A kind of serum 7-type haemophilus parasuis Low virulent strain, it is characterised in that:The serum 7-type haemophilus parasuis low virulent strain was preserved in Chinese Typical Representative on October 13rd, 2016 Culture collection, depositary institution address:In Wuhan, China Wuhan University;Preserving number is CCTCC No:M2016560; The Classification And Nomenclature is haemophilus parasuis HPKP-7, Latin name:Haemophilus parasuis HPKP-7.
Second object of the present invention is to provide a kind of application of above-mentioned serum 7-type haemophilus parasuis low virulent strain, sharp The medicine for the treatment of Haemophilus parasuis is prepared with serum 7-type haemophilus parasuis low virulent strain.
Third object of the present invention is to provide a kind of medicine for treating Haemophilus parasuis, to utilize serum 7-type Haemophilus parasuis low virulent strain prepares the vaccine for the treatment of Haemophilus parasuis.
Achieving the object of the present invention can be reached by adopting the following technical scheme that:A kind of secondary pig for the treatment of as described above is thermophilic The medicine of blood bacillosis, the medicine are vaccine.
Preferably, the vaccine is obtained by following preparation process:
1) prepared by primary seed solution:Take the serum 7-type haemophilus parasuis low virulent strain of lyophilized preservation to be inoculated in TSA solids to put down Plate, 18~24h is cultivated under conditions of being 37 DEG C in temperature;Then picking single bacterium is fallen within TSB culture mediums, is 37 DEG C in temperature Under the conditions of 16~18h of shaken cultivation, carry out purely after the assay was approved, as primary seed solution, put 2~8 DEG C and save backup;
2) prepared by secondary seed solution:Take the primary seed solution that step 1) obtains to be inoculated in TSB culture mediums, be 37 in temperature 16~18h of shaken cultivation under conditions of DEG C, purely after the assay was approved, as secondary seed solution, put 2~8 DEG C and save backup;
3) prepared by original bacteria liquid:The secondary seed nutrient solution that step 2) obtains is taken, is inoculated in TSB culture mediums, is in temperature Under conditions of 37 DEG C after 24~28h of shaken cultivation, original bacteria liquid is obtained;
4) vaccine is prepared:Sampled in the original bacteria liquid that step 3) obtains, according to《Chinese veterinary pharmacopoeia》Annex is purely examined Test and count plate;Then thalline original bacteria liquid centrifuging and taking precipitated, is washed after thalline is resuspended with sterile saline, washed Thalline normal saline dilution into concentration be 3 × 107~3 × 1010The seedling bacterium solution of CFU/mL, seedling bacterium solution is mixed with adjuvant Close, emulsification, the vaccine for the Haemophilus parasuis that obtains medical treatment, 2~8 DEG C save backup.
Wherein, more preferably, step 1)~3) in, the rotating speed of the shaken cultivation is 120~180rpm.
More preferably, the holding time of the primary seed solution and secondary seed solution is no more than 3 days.
More preferably, in step 2), by seed liquor and the volume ratio of culture medium 1:20, primary seed solution is inoculated in TSB In culture medium.
More preferably, in step 3), by seed liquor and the volume ratio of culture medium 1:100, secondary seed solution is inoculated in TSB In culture medium.
More preferably, in step 4), original bacteria liquid is centrifuged into 10min under conditions of rotating speed is 8000~10000rpm, is taken The thalline of precipitation.
The vaccine of the present invention includes but not limited to oil in water vaccine, water-in-oil type vaccine, water-in-oil-in water vaccine, can Selection adjuvant as needed;As the preferred embodiment of the present invention, in step 4), the adjuvant is aluminium glue adjuvant, and by seedling bacterium Liquid and aluminium glue adjuvant are with volume ratio 4:1 mixes, and 10~20min is emulsified under conditions of 5000~8000rpm of rotating speed.
Compared with prior art, the beneficial effects of the present invention are:
1st, serum 7-type haemophilus parasuis low virulent strain in the present invention, the pathogenicity of the low virulent strain weaken, are suitable for significantly Prepare the medicine for the treatment of Haemophilus parasuis;
2nd, need not when preparing medicine in terms of serum 7-type haemophilus parasuis low virulent strain is applied to medicine preparation by the present invention Inactivation, without carrying out inactivation inspection, condition is simple, the production efficiency higher of medicine.
Brief description of the drawings
Fig. 1 is the growthform figure on tablet of 1 bacterium colony of embodiment;
Fig. 2 is the colonial morphology figure under 1 microscope of embodiment;
Fig. 3 is the gel electrophoresis figure of embodiment 1PCR amplifications;
It is described that a kind of serum 7-type haemophilus parasuis natural low virulent strain is provided, it is preserved in China on October 13rd, 2016 Type Tissue Collection, depositary institution address:In Wuhan, China Wuhan University;Preserving number is CCTCC No: M2016560;The Classification And Nomenclature is haemophilus parasuis HPKP-7, Latin name:Haemophilus parasuis HPKP- 7。
Embodiment
In the following, with reference to attached drawing and embodiment, the present invention is described further:
A kind of serum 7-type haemophilus parasuis low virulent strain, the serum 7-type haemophilus parasuis low virulent strain was in 2016 10 The moon is preserved in China typical culture collection center on the 13rd, and preserving number is CCTCC No:M2016560.
Above serum 7-type haemophilus parasuis low virulent strain is prepared to the vaccine for the treatment of Haemophilus parasuis.
The preparation method of vaccine comprises the following steps:
1) prepared by primary seed solution:Take the serum 7-type haemophilus parasuis low virulent strain of lyophilized preservation to be inoculated in TSA solids to put down Plate, 18~24h is cultivated under conditions of being 37 DEG C in temperature;Then picking single bacterium is fallen within TSB culture mediums, is 37 DEG C in temperature Under the conditions of, with rotating speed 120~180rpm, 16~18h of shaken cultivation, carry out purely after the assay was approved, as primary seed solution, putting 2 ~8 DEG C save backup, and the holding time is no more than 3 days;
2) prepared by secondary seed solution:The primary seed solution that step 1) obtains is taken, by seed liquor and the volume ratio of culture medium 1: 20 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, with rotating speed 120~180rpm, 16~18h of shaken cultivation, into Row purely after the assay was approved, as secondary seed solution, is put 2~8 DEG C and is saved backup, and the holding time is no more than 3 days;
3) prepared by original bacteria liquid:The secondary seed nutrient solution that step 2) obtains is taken, by seed liquor and the volume ratio of culture medium 1: 100 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, after 24~28h of rotating speed 120~180rpm shaken cultivations, Obtain original bacteria liquid;
4) vaccine is prepared:Sampled in the original bacteria liquid that step 3) obtains, according to《Chinese veterinary pharmacopoeia》Annex is purely examined Test and count plate;By original bacteria liquid, centrifugation 10min takes the thalline of precipitation under conditions of rotating speed is 8000~10000rpm, with nothing Bacterium physiological saline be resuspended thalline after wash, washed thalline normal saline dilution into concentration be 3 × 107~3 × 1010CFU/ The seedling bacterium solution of mL, seedling bacterium solution is mixed with aluminium glue adjuvant, and 10~20min is emulsified under conditions of 5000~8000rpm of rotating speed, Obtain medical treatment the vaccine of Haemophilus parasuis, and 2~8 DEG C save backup.
Embodiment 1
The separation identification of haemophilus parasuis low virulent strain:
Level land area slaughterhouse collection pig tonsil is opened from Guangdong within 2016, under sterile working, wiped with cotton ball soaked in alcohol whole Tonsil, cuts a fritter, and the TSA containing 10% calf serum of percent by volume and 0.1%NAD is first inoculated in section and is put down Plate, 37 DEG C of overnight incubations.
The growthform on tablet of bacterium colony as shown in Figure 1, picking well-grown on tablet, colonial morphology is smaller, Canescence, translucent, neat in edge, slightly slime mould fall, through Gram's staining, micro- Microscopic observation, and the colonial morphology under microscope As shown in Fig. 2, in pale red, in long-chain or the bacterium colony of short chain, to expand the mirror that 16S rRNA are carried out after cultivating to suspicious bacterium colony It is fixed.The primer sequence used is:
Above-mentioned bacterial strains 16S rRNA PCR products serve Hai Shenggong bioengineering Co., Ltd sequencing, sequencing result with Known array carries out Blast comparisons in GenBank, and comparison result shows that the genetic fragment that PCR is obtained is 822bp, genetic fragment Sequence as shown in sequence table SEQ ID No.1, with the homology of known haemophilus parasuis 98.4~100%, determine point From to bacterial strain be haemophilus parasuis.
The haemophilus parasuis of above-mentioned identification is subjected to serotype PCR identifications, primer sequence:
As shown in figure 3, obtaining the purpose fragment that size is about 600bp through PCR amplification, amplified production is through sequencing, sequencing knot For fruit as shown in sequence table SEQ ID No.2, it is 100% to compare with the homology of the funQ genes of haemophilus parasuis serum 7-type, It is thus determined that separated bacterial strain is serum 7-type, most at last strain was named HPKP-7.
Embodiment 2
The serum 7-type haemophilus parasuis low virulent strain HPKP-7 that embodiment 1 is obtained carries out pathogenicity detection:
1) pathogenicity of cavy is detected
Virulence determination is carried out to the HPKP-7 that separation obtains using cavy as model animal.
Cavy is observed three days after buying, and after situation without exception, is randomly divided into 12 groups, every group 5,12 groups of isolation are raised Support, keep same experimental conditions.
5 groups of cavys of artificial challenge are carried out by being injected intraperitoneally with the HPKP-7 that embodiment 1 obtains during experiment, in addition 6 Group cavy is infected using haemophilus parasuis with reference to velogen strain Serotype 5 Nagasaki as control strain, and 1 group of cavy is as cloudy Property control, injecting normal saline, continuous observation 10 days observes and records cavy morbidity and death condition, as shown in table 1.
1 cavy of table attacks malicious result
Result of the test shows that Nagasaki plants of inoculum concentrations of strong poison control group are 1.5 × 108、1.5×109、1.5×1010、 1.5×1011、1.5×1012During CFU, the death of test group whole cavy is responsible for, and separated strain HPKP-7 is being inoculated with Dosage is 1.5 × 108、1.5×109、1.5×1010、1.5×1011During CFU, cavy does not show any clinical symptoms, growth In good condition, dosage of inoculation is 1.5 × 1012During CFU, when cavy to the off-test for having 2/5, which dissects, visible intraperitoneal has fiber Disposition is oozed out, but does not cause death.
2) pathogenicity of piglet is detected
25 age in days weanling pigs 30, are randomly divided into 6 groups, every group 5, wherein 5 are used as negative control, inject physiology salt Water, 1 group of Nagasaki plants of intraperitoneal injection haemophilus parasuis, 4 groups of HPKP-7 plants of injections, continuous observation 10 days, observes and records examination Piglet morbidity and death condition are tested, the results are shown in Table 2.
2 piglet of table attacks malicious result
Result of the test is shown, is injected intraperitoneally 3 × 109After Nagasaki plants of CFU, 5 experiment piglets fall ill, experiment knot Dead 3 during beam, and inject 3 × 109、3×1010、3×1011During HPKP-7 plants of CFU, piglet does not cause morbidity, and injection 3 × 1012There are slight clinical symptoms in 1 piglet during CFU, does not occur the phenomenon of piglet death.Cavy and piglet challenge test result Confirm, haemophilus parasuis HPKP-7 pathogenicities weaken significantly.
Embodiment 3
The immune efficacy detection of haemophilus parasuis low virulent strain HPKP-7:
Vaccine is prepared by following steps:
1) prepared by primary seed solution:Take the serum 7-type haemophilus parasuis low virulent strain of lyophilized preservation to be inoculated in TSA solids to put down Plate, 18h is cultivated under conditions of being 37 DEG C in temperature;Then picking single bacterium is fallen within TSB culture mediums, in the condition that temperature is 37 DEG C Under, with rotating speed 120rpm shaken cultivation 18h, carry out purely after the assay was approved, as primary seed solution, it is standby putting 2~8 DEG C of preservations With the holding time is no more than 3 days;
2) prepared by secondary seed solution:The primary seed solution that step 1) obtains is taken, by seed liquor and the volume ratio of culture medium 1: 20 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, with rotating speed 120rpm shaken cultivation 16h, are purely examined After qualification, as secondary seed solution, put 2~8 DEG C and save backup, the holding time is no more than 3 days;
3) prepared by original bacteria liquid:The secondary seed nutrient solution that step 2) obtains is taken, by seed liquor and the volume ratio of culture medium 1: 100 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, after rotating speed 120rpm shaken cultivations 24h, obtain opportunistic pathogen Liquid;
4) vaccine is prepared:Sampled in the original bacteria liquid that step 3) obtains, according to《Chinese veterinary pharmacopoeia》Annex is purely examined Test and count plate;By original bacteria liquid, centrifugation 10min takes the thalline of precipitation under conditions of rotating speed is 8000rpm, uses sterile physiological Brine be resuspended thalline after wash, washed thalline normal saline dilution into concentration be 3 × 1010The seedling bacterium solution of CFU/mL, By seedling bacterium solution and aluminium glue adjuvant with volume ratio 4:1 mixes, and emulsifies 20min under conditions of rotating speed 8000rpm, obtain medical treatment secondary pig The vaccine of haemophilus disease, 2~8 DEG C save backup.
The vaccine of obtained treatment Haemophilus parasuis is subjected to Immunoprotection test:
A. to the Immunoprotection test of cavy
Cavy 15 is taken, is randomly divided into 3 groups, every group 5, the haemophilus parasuis vaccine muscle of the above-mentioned preparation of test group Cavy is injected, only, two exempt from 2mL/ after 14 days, and 2mL/ only, after two exempt from 14 days, attacks poison control and physiological saline is immunized in blank control, To immune group and attack malicious control group cavy and carry out attacking poison for Nagasaki plant with Serotype 5 velogen strain, intraperitoneal injection, 1.5 × 109Only, continuous observation 10 days, observes and records cavy morbidity and death condition, calculates every group of protective rate, as shown in table 3 CFU/.
3 vaccine immunity protection test result of table
Result of the test shows, vaccine immunity group, which is attacked after poison, not to be occurred falling ill and dead, and 5 cavys of control group fall ill, 4 It is only dead, it was demonstrated that vaccine has certain protective efficacy to velogen strain.
B. to the cross-protection test of different serotypes
Because of 4,5,12,13 type haemophilus parasuis of Major Epidemic serum in China's clinic, thus we to vaccine to this 4 The cross-protection test of serotype is verified.40 cavys are taken, are randomly divided into 8 groups, wherein 4 groups of intramuscular injection pair pigs are bloodthirsty Bacillus attenuated vaccine, only, two exempt from 2mL/ after 14 days, and 2mL/ only, attacks malicious control group injecting normal saline, two exempt from 14 days after use respectively 4,5,12,13 type haemophilus parasuis of serum attack poison, and 2 × 109Only, continuous observation 10 days, observes and records cavy hair to CFU/ Disease and death condition, calculate every group of protective rate, as shown in table 4.
Table 4 attacks different serotypes bacterial strain the immunoprotection result of poison
Result of the test shows that the haemophilus parasuis attenuated vaccine of preparation is capable of providing different serotypes preferable friendship Fork protection.
For those skilled in the art, technical solution that can be as described above and design, make other each Kind is corresponding to be changed and deforms, and all these change and deform the protection model that should all belong to the claims in the present invention Within enclosing.
SEQUENCE LISTING
<110>Guangdong Haida Husbandry and Veterinary Institute Co., Ltd. of Agricultural University Of South China
<120>A kind of serum 7-type haemophilus parasuis low virulent strain and its application
<130> 2016
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 822
<212> DNA
<213>Haemophilus parasuis HPKP-7
<400> 1
tgaccagcca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga 60
atattgcrca atggggggaa ccctgatgca gccatgccgc gtgaatgaag aaggccttcg 120
ggttgtaaag ttctttcggt gangaggaag ggtggtgttt taatagaaca ctacattgac 180
gttagtcaca gaagaagcac cggctaactc cgtgccagca gccgcggtaa tacggagggt 240
gcgagcgtta atcggaatga ctgggcgtaa agggcacgca ggcggtgact taagtgagat 300
gtgaaagccc cgagcttaac ttgggaattg catttcatac tgggttgcta gagtatttna 360
gggagggnta gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaataccgaa 420
ggcgaaggca gccccttggg aaaatactga cgctcatgtg cgaaagcgtg gggagcaaac 480
aggattagat accctggtag tccacgctgt aaacgctgtc gattnnggga ttgggctnag 540
agcttggtgc ccgtagctaa cgtgataaat cgaccgcctg gggagtacgg ccgcaaggtt 600
aaaactcaaa tnaattgacg ggggccgcac aagcggtgga gcatgtggtt taattcgatg 660
naacgcgaag aaccttacct actcttgaca tcctaagaag aactcagaga tgagtttgtg 720
ccttcgggaa cttagagaca ggtgctgcat ggctgtcgtc agctcgtgtt gtgaaatgtt 780
gggttnagtc ccgcaacgag cgcaaccctt atcctttgtt gc 822
<210> 2
<211> 600
<212> DNA
<213>Haemophilus parasuis HPKP-7
<400> 2
tagttggtat attattttct aaacgttatc ctataaaatg gaataatgtt aatttagaat 60
ttaaaattcc gtactgtata agtataataa taattttatt ttcaatattt attagttttc 120
ttgcatttga ttttaatgag ttactttatc gtcagaagta tgttactaat gaaactcata 180
gggttttagt tattttattt aaagtgttat ctccgatttc atcttttcta tgtggcttta 240
ttcgaaataa atatttgaag tataactttc tatttcttat tctaagtgtt ccttttagcc 300
tcaattctag aataatatcc ttagatttaa tgatgtttta tattgggagt gttttgtgtt 360
ttaaaagagg ttttttgtat aaatccataa ttatttttag tgtgattttt tctatatcct 420
ctcttatttt aagggggatg tcagagcaag gttttatata taacatatta aatttatctt 480
acatttttga tattagctta gatgtttttt ttagaggggt gaattatgtg accagttttt 540
cagtatatga aatggaatat gctttagaac agaaacttag tggtacagat gcattctaat 600

Claims (9)

  1. A kind of 1. serum 7-type haemophilus parasuis low virulent strain, it is characterised in that:The serum 7-type haemophilus parasuis (Haemophilus parasuis)Low virulent strain was preserved in China typical culture collection center, preservation on October 13rd, 2016 Number it is CCTCC No: M2016560.
  2. A kind of 2. medicine for treating Haemophilus parasuis, it is characterised in that:The medicine is to utilize blood as claimed in claim 1 Vaccine prepared by clear 7 type haemophilus parasuis low virulent strain.
  3. 3. a kind of preparation method of vaccine as claimed in claim 2, it is characterised in that the preparation process of the vaccine is:
    1)It is prepared by primary seed solution:The serum 7-type haemophilus parasuis low virulent strain of lyophilized preservation is taken to be inoculated in TSA solid plates, 18~24h is cultivated under conditions of being 37 DEG C in temperature;Then picking single bacterium is fallen within TSB culture mediums, in the bar that temperature is 37 DEG C 16~18h of shaken cultivation under part, purely after the assay was approved, as primary seed solution, put 2~8 DEG C and save backup;
    2)It is prepared by secondary seed solution:Take step 1)Obtained primary seed solution is inoculated in TSB culture mediums, is 37 DEG C in temperature Under the conditions of 16~18h of shaken cultivation, carry out purely after the assay was approved, as secondary seed solution, put 2~8 DEG C and save backup;
    3)It is prepared by original bacteria liquid:Take step 2)Obtained secondary seed nutrient solution, is inoculated in TSB culture mediums, is 37 DEG C in temperature Under conditions of after 24~28h of shaken cultivation, obtain original bacteria liquid;
    4)Prepare vaccine:In step 3)Sampled in obtained original bacteria liquid, according to《Chinese veterinary pharmacopoeia》Annex carry out purely examine and Count plate;Then thalline original bacteria liquid centrifuging and taking precipitated, is washed, washed bacterium after thalline is resuspended with sterile saline Body normal saline dilution into concentration be 3 × 107~3 × 1010The seedling bacterium solution of CFU/mL, seedling bacterium solution is mixed with adjuvant, Emulsification, the vaccine for the Haemophilus parasuis that obtains medical treatment, 2~8 DEG C save backup.
  4. 4. the preparation method of vaccine as claimed in claim 3, it is characterised in that:Step 1)~3)In, the shaken cultivation turns Speed is 120~180rpm.
  5. 5. the preparation method of vaccine as claimed in claim 3, it is characterised in that:The primary seed solution and secondary seed solution Holding time is no more than 3 days.
  6. 6. the preparation method of vaccine as claimed in claim 3, it is characterised in that:Step 2)In, by seed liquor and culture medium Volume ratio 1:20, primary seed solution is inoculated in TSB culture mediums.
  7. 7. the preparation method of vaccine as claimed in claim 3, it is characterised in that:Step 3)In, by seed liquor and culture medium Volume ratio 1:100, secondary seed solution is inoculated in TSB culture mediums.
  8. 8. the preparation method of vaccine as claimed in claim 3, it is characterised in that:Step 4)In, it is in rotating speed by original bacteria liquid 10min is centrifuged under conditions of 8000~10000rpm, takes the thalline of precipitation.
  9. 9. the preparation method of vaccine as claimed in claim 3, it is characterised in that:Step 4)In, the adjuvant is aluminium glue adjuvant, And by seedling bacterium solution and aluminium glue adjuvant with volume ratio 4:1 mixes, and 10~20min is emulsified under conditions of 5000~8000rpm of rotating speed.
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