CN103194413B - Serotype 4 haemophilus parasuis (HPs) vaccine strain - Google Patents
Serotype 4 haemophilus parasuis (HPs) vaccine strain Download PDFInfo
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Abstract
The invention relates to the field of vaccines of Haemophilus parasuis (HPs) in veterinary biological products and discloses a serotype 4 HPs vaccine strain. The class name of the vaccine strain is HPs. The strain number is FS0307. The vaccine strain has been collected in the China Center for Type Culture Collection, with collection number being CCTCC NO:M2013094 and collection date being March 21, 2013. The serotype 4 HPs strain FS0307 has stronger pathogenicity toward swine and has good immunogenicity. The inactivated vaccines prepared from the vaccine strain are safe and reliable and have quite good protective effects on the swine challenging homological strains. The morbidity and mortality of the immunized swinery are obviously reduced. Either the single vaccine or the combined vaccine has the immune effects equivalent or superior to the immune effects of the existing commercialized vaccines and can effectively prevent prevalence of HPs.
Description
Technical field
The present invention relates to Haemophilus parasuis vaccine field in veterinary biological product, particularly relate to strain serum 4 type haemophilus parasuis strains.
Background technology
Haemophilus parasuis (Haemophilus parasuis, HPs) be a kind of gram-negative coccobacillus in pasteurellosis bacillus section hemophilus, it is a kind of conditionality pathogenic bacteria of pig, be worldwide distribution, infect the pig Ge Lazeshi that causes after piglet taking polyserositis, sacroiliitis and meningitis as feature (Glasser ' s) disease.The piglet in 2～8 week age of main harm, sickness rate is generally 10%～15%, and mortality ratio can reach more than 50%.
Haemophilus parasuis is to nutritional requirement harshness, and growth strictly needs the V factor (NAD), thinks that at present tryptose soya agar (TSA) substratum and Tryptones soybean (TSB) substratum are the optimum mediums of cultivating haemophilus parasuis.On the TSA plate culture medium that adds NAD, cultivate the visible needle point size of 24h canescence bacterium colony, be protuberance circular, smooth surface, moistening, neat in edge.HPs grows difficult on Chocolate Agar flat board, have report to show, chocolate dull and stereotyped upper HPs cultivations of can not going down to posterity, is not suitable for subculture in vitro separately cultivation, but Chocolate Agar flat board can, for separating of this bacterium, be supplied with 5%～10% carbonic acid gas (CO when first separation and Culture
2) can promote growth.When liquid culture, need in meat soup or TSB substratum, add serum and the V factor (NAD), can not increase but interpolation V factor concentration constantly increases bacterial concentration.Owing to self meta-bolites V factor can being discharged in substratum in aureus growth process, so HPs and streptococcus aureus co-inoculation are in blood agar plate culture, HPs is at streptococcus aureus both sides well-grown, along with streptococcus aureus apart from increase, bacterium colony diminishes gradually, and this phenomenon is called as satellitism.HPs to external world environment resistibility extremely a little less than, vitro culture is very easily dead.
Between HPs bacterial strain, antigenicity and Virulence Difference are very large, and serological typing is one of important method of difference different strains.At present, the serological typing method (KRG) based on heat stable antigen immunodiffusion being proposed for 1992 by Kielstein etc. is worldwide accepted, HPs is divided into 15 serotypes by the method, but have 15.2%～41% strain isolated serotype can not determine.According to the seroepidemiological survey of the states such as Japan, Germany, the U.S. and Spain, the most popular with serotype 4,5 and 13.HPs is generally popular in China, at present, and in Hebei, 12 provinces and cities such as Henan, Hubei, Hunan, Anhui, Shanghai, Guangdong, Jiangxi all have Haemophilus parasuis that the report of HPs occurs and isolates.
Owing to usually not even rationally not using in a large number antimicrobial drug on veterinary clinic, cause pig farm bacterium under medicine pressure, to select resistance flora.Therefore, increasing by the difficulty of medicine prevention and control Haemophilus parasuis, and along with the worry of people to food-safety problem, the application meeting of microbiotic aspect prevention and control Animal diseases still less, and is corresponding vaccine by what play a greater role.HPs serotype is more, and the virulence of different serotypes bacterial strain and virulence differ greatly, and between each serotype, immune cross-protection is very low, so existing domestic and international commercial Haemophilus parasuis vaccine all can not provide fully effective protection to this disease.Therefore, the universal HPs bacterial strain that separation screening immunogenicity is strong, and utilize it to prepare the most important thing that efficient vaccine is prevention and control Haemophilus parasuis.
Summary of the invention
Technical problem to be solved by this invention is to provide strain serum 4 type haemophilus parasuis vaccine strains (FS0307 strain), and this vaccine strain has stronger virulence and very high immunogenicity.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
One strain serum 4 type haemophilus parasuis vaccine strains, its Classification And Nomenclature is haemophilus parasuis (Haemophilus parasuis), bacterial strain number is FS0307, be preserved in Chinese Typical Representative culture collection center, address: China. Wuhan. Wuhan University, postcode 430072, deposit number: CCTCC NO:M2013094, preservation date: on March 21st, 2013.This bacterial strain be contriver gather voluntarily in April, 2003 in Hebei province's Hengshui City screening obtain a strain there is stronger virulence and very high immunogenic bacterial strain.
The application of above-mentioned serum 4 type haemophilus parasuis vaccine strains in preparation treatment pig Haemophilus parasuis medicine.
Wherein, described medicine is vaccine, preferred oil water-in type vaccine or oil in water vaccine or water-in-oil-in-water type vaccine.
Wherein, serum 4 type haemophilus parasuis vaccine strains and pharmaceutically acceptable carrier or auxiliary material combination form medicine, as unit price seedling, multivalence seedling or connection seedling etc.
Wherein, described serum 4 type haemophilus parasuis vaccine strains bacteria containing amount in vaccine is 2 × 10
9more than CFU/mL.
Beneficial effect: serum 4 type haemophilus parasuis FS0307 strains of the present invention have stable biological characteristics, has stronger pathogenicly to piglet, and have good immunogenicity.Apply its oil adjuvant killed vaccine of preparing safe and reliable; can produce the antibody of higher level; extended period is long; and homology bacterial classification is attacked to malicious pig and there is extraordinary protection effect; after immunity, the M & M of swinery all obviously reduces; its immune effect all reaches or is better than existing commercialized vaccine on market, can effectively prevent the popular of haemophilus parasuis.
Embodiment
According to following embodiment, the present invention may be better understood.But, those skilled in the art will readily understand, the described content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1: separation and purification and the qualification of serum 4 type haemophilus parasuis FS0307 strains.
1. production substratum
TSA solid medium preparation: accurately take TSA powder 40g, be dissolved in 940mL distilled water, fully shake up rear 121 DEG C of high pressure steam sterilization 15min, while being cooled to 50～60 DEG C, add the bovine serum 50mL of filtration sterilization, the 0.01%NAD of 10mL filtration sterilization, mix rear a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices for subsequent use.
TSB liquid nutrient medium preparation: accurately take TSB powder 30g, be dissolved in 940mL distilled water, fully shake up rear 121 DEG C of high pressure steam sterilization 15min, add before use the bovine serum 50mL of filtration sterilization, the 0.01%NAD of 10mL filtration sterilization.
2. haemophilus parasuis separation and purification and qualification
2.1 separation and purification
2003; the a large amount of sick pigs that occur that doubtful haemophilus parasuis infects of certain large-scale pig farm of China Hebei Province; taking persistent fever, pleuritis, peritonitis, sacroiliitis etc. as feature, choose lung, hydrothorax, pericardial fluid, the synovial fluid inoculation TSA/NAD solid medium of disease pig, put 37 DEG C, 5%CO
2under condition, cultivate 24～48 hours, the small colonies smear of the translucent protuberance of picking, gram stain microscopy, chooses the little shaft-like or polymorphism dialister bacterium of Gram-negative, then carries out twice TSA/NAD solid medium subclone and cultivate.Inoculate blood Solid agar culture and plain agar solid medium simultaneously, well-grown on result TSA/NAD solid medium, blood Solid agar culture and plain agar solid medium do not have bacterium colony to generate.
2.2 biochemical identification
Get pure culture bacterium liquid inoculation TSA/NAD solid medium, put 37 DEG C, 5%CO
2under condition, cultivate 24～48 hours.Observe colonial morphology, visible needle point size, circle, neat in edge, flat, the small colonies of the translucent protuberance of canescence.Getting single bacterium colony smear gramstaining, is Gram-negative, is polymorphism, is single little coccobacillus or elongated thread bacillus.
Pure culture bacterium liquid is done to intersect line with streptococcus aureus on de-fine sheep blood Solid agar culture, put 37 DEG C, 5%CO
2under condition, cultivate after 24～48 hours, be little and transparent bacterium colony, better the closer near growth staphylococcus, present typical satellite growth phenomenon, and there is not haemolysis in haemophilus parasuis periphery of bacterial colonies on blood agar plate.
Pure culture bacterium liquid is inoculated in to TSA/NAD solid medium, then sticks respectively the circular filter paper sheet that is impregnated with X, V, the X+V factor.Put 37 DEG C, 5%CO
2under condition, cultivate 24～48 hours, result only has colony growth around the scraps of paper containing V, the X+V factor, shows that this bacterial strain only relies on the V factor, and does not rely on the X factor.
By pure culture bacterium liquid inoculation micro biochemical reaction tubes, supplement respectively the NAD of final concentration 0.01%, put 37 DEG C and leave standstill cultivation, after 48h, judge biochemical characteristic result.Result shows that isolated strains is to glucose, sucrose and fructose fermentation, to maltose, semi-lactosi, wood sugar and fructose nonfermented, indole test, oxidase test and urease test are negative, and nitrate reduction test and the catalase test positive meet the biochemical characteristic of haemophilus parasuis.According to the source place of this bacterial strain by its called after FS0307 strain.
2.316S rRNA gene sequencing and analysis
2.3.1 design of primers is according to two primers of haemophilus parasuis P5 gene order design in GenBank, primer sequence is: P1:5 '-gctccacaagctaacactttc-3 ', P2:5 '-catagaaacttcttttgaacc-3 ', synthetic by Shanghai Sheng Gong biotechnology company limited, this primer amplification clip size is 1038bp.
2.3.2 FS0307 strain culture 1.5mL is got in the preparation of thallus DNA template, and the centrifugal 5min of 10000r/min, abandons supernatant, precipitate resuspendedly with 200 μ L distilled waters, after 100 DEG C of water-bath 10min, the centrifugal 5min of 12000r/min collects supernatant, be pcr template ,-20 DEG C save backup.
2.3.3PCR increase and check order with the thallus DNA extracting as pcr template.Pcr amplification system is: 10 × PCRBuffer5.0 μ L, MgCl
2(25mM) 2.5 μ L, dNTP(2.5mM) 4.0 μ L, primer P1(50 μ M) 0.5 μ L, primer P2(50 μ M) 0.5 μ L, template DNA 3.0 μ L, Taq enzyme (5U/ μ L) 0.5 μ L adds distilled water to 50 μ L.Reaction conditions is: 95 DEG C of denaturation 5min, and 94 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C are extended 1min, 30 circulations, 72 DEG C are extended 10min eventually.Get 10 μ L pcr amplification products, sepharose with 1% is electrophoresis qualification under 120V voltage, using type strain as positive control, having there is the specific band of the 1038bp size identical with type strain in result, illustrates that the FS0307 strain being separated to is haemophilus parasuis simultaneously.
Be accredited as positive pcr amplification product through recovery and transfer to the order-checking of Shanghai Sheng Gong biotechnology company limited.Haemophilus parasuis FS0307 strain P5 gene sequencing result is as shown in SEQ ID No.1, and the sequence of announcing in this sequence and GenBank is carried out BLAST and compared, and reaches more than 95% with the homology of domestic and international existing haemophilus parasuis corresponding sequence.2.4 haemophilus parasuis FS0307 strain serological identification
By Kleletein-Rapp-Gabriedson (KRG) agar diffusion serotype method, the serotype of haemophilus parasuis FS0307 strain is identified.Haemophilus parasuis FS0307 strain is inoculated in to TSB substratum in 5% ratio, 37 DEG C, 5%CO
2under condition, cultivate after 18～24 hours, it is 1 × 10 that bacterium liquid is concentrated into bacteria containing amount
10cFU/mL, the centrifugal 10min of bacterium liquid 5000r/min, gets the somatotype antigen of supernatant for agar diffusion test.Prepare 1% agar plate, punching (6, middle 1 hole periphery), aperture 5.0mm, pitch-row 3.0mm.。In 6 holes of periphery, add the anti-hyper-immune serum of 6 μ l haemophilus parasuis type strain rabbit (directly over hole be labeled as 1, add 1 type hyper-immune serum, the serum of all the other each serotypes is pressed serotype incremental order successively with clockwise direction application of sample), interstitial hole adds the thermal treatment somatotype antigen 6 μ l of bacterial strain to be checked, 37 DEG C, wet box is hatched, observe and record result at 24h, 48h, 72h respectively, establish the contrast of reference culture simultaneously.Agar diffusion test shows, the high pressure extract antigen of haemophilus parasuis FS0307 strain only has precipitation line with the positive serum of 4 type haemophilus parasuis type strains, without precipitation line, illustrate that haemophilus parasuis FS0307 strain is serum 4 types with the serotype of other type type strain.
2.5 animal challenge tests
Serum 4 type haemophilus parasuis FS0307 strains are cultivated through TSB/NAD liquid nutrient medium, carry out live bacterial count, after concentrating, make infection titer reach 1 × 10
9cFU/ml, through 5 of the healthy susceptible pigs of abdominal injection 50～60 ages in days, 5ml/ head.Separately establish 1 group of control group, 5, abdominal injection TSB/NAD substratum, 5ml/ head.After attacking poison, raise routinely antibiotic-free in feed.Observe 14, survey body temperature every day, observe clinical symptom.Attack poison and cut open inspection after 14 days, according to clinical symptom with cut open inspection pathology and calculate morbidity number.Meanwhile, the pathological material of disease of organizing of getting morbidity pig does bacterium separation and qualification.
Attack malicious result and show, Continuous Observation is after 14 days, and all first sequela of 5 experiment pig of poison group are attacked in serum 4 type haemophilus parasuis FS0307 strains; Contrast pig is acted normally.Clinical symptom shows as, and experiment pig infection haemophilus parasuis strain isolated fervescence after 1 day, engenders that appetite reduces subsequently, and thick random by hair, ear and whole skin are rubescent, and nose liquid increases, and expiratory dyspnea appears in the later stage, and arthroncus does not rise sleepingly.Cut open inspection and find, there is thoracic cavity, seroperitoneum in morbidity pig and the pig that dies of illness, has pleura, peritoneal adhesion in various degree, and arthroncus, joint cavity mucus increase, Pulmonary hemorrhage, the pathologies such as swollen lymph node.The results are shown in Table 1.
The pathological material of disease of organizing that utilizes isolated strains to attack poison carries out bacterium separation.Result is attacked in the sick pig of 4 hairs malicious group and is separated to the bacterium colony bacterium similar to haemophilus parasuis type strain with thalli morphology from FS0307 strain.All bacteriums that are separated to are carried out to PCR qualification, there is specific band at 1038bp place in result simultaneously, proves that bacterial isolate is haemophilus parasuis.
The virulence test result of table 1 haemophilus parasuis FS0307 strain
"-" represents not immunity or inapplicable.
2.6 immunogenic mensuration
Cultivate serum 4 type haemophilus parasuis FS0307 strains with TSB/NAD liquid nutrient medium, after live bacterial count, viable bacteria culture is used respectively final concentration 0.3% formaldehyde solution deactivation 12h, and bacterium number is condensed into 4 × 10
9cFU/ml, through deactivation after the assay was approved, prepares vaccine by 1 ︰ 3 emulsifications of the ratio of water oil phase.Choose about 2 week age 25 of healthy susceptible pigs, be divided at random 5 groups, 5 every group.1st～3 groups every group respectively immune FS0307 strain vaccine 0.5ml, 1ml, 2ml, the 4th group for attacking malicious control group, and the 5th group is normal healthy controls group.Each group vaccine immunity group first immunisation is used Isodose vaccine booster immunization once after 3 weeks.Head exempts from latter 42 days, and except 5 of normal healthy controls groups, all immune group forward to and attack malicious Animal House with the pig of attacking malicious control group.1st～3 groups and the 4th group every group respectively intraperitoneal injection through concentrated FS0307 strain viable bacteria culture, (bacterium number is (1 × 10
9cFU/ml), 5ml/ 4; The 5th group of intraperitoneal injection TSB/NAD substratum, 5ml/ head.After attacking poison, observe 14, cut open inspection.According to clinical symptom with cut open inspection pathology and calculate morbidity number and attack malicious protection ratio.Immunization protection test result shows, attacks 5 pigs of malicious control group and all falls ill (wherein dead 2), occurs typical Haemophilus parasuis clinical symptom and significantly pathological change.The experiment pig of normal healthy controls group is not morbidity all.In 5 pigs of FS0307 strain 0.5mL immune group, there are 2 to occur clinical symptom and pathological change, in 5 pigs of 1mL immune group, only have 1 to occur cuing open inspection pathology, attack malicious protection ratio and reach respectively 3/5 and 4/5; And the experiment pig of 2mL immune group is not all fallen ill, attack malicious protection ratio and reach 5/5.Immunization protection situation refers to table 2.
The immunogenicity determining test-results of table 2 haemophilus parasuis FS0307 strain
"-" represents inapplicable.
From table 2 result, it is 1 × 10 containing viable count that the minimum immunoprotection dosage of haemophilus parasuis FS0307 strain is 1mL
9cFU/mL.
Case study on implementation 2: serum 4 application of type haemophilus parasuis FS0307 strain in Haemophilus parasuis vaccine.
1 bacterial classification
Seedling bacterial classification is serum 4 type haemophilus parasuis FS0307 strains, its deposit number is that CCTCC No:M2013094 thalline, colonial morphology meet haemophilus parasuis type strain form, biochemical characteristic and cultural characters are stable, piglet is had to stronger virulence, and have good immunogenicity.
2 productions are prepared with bacterial classification
2.1 one-level seedings are cultivated the secondary pig of 4 types of getting and have a liking for after the dissolving of bacillus basic bacteria lyophilized products, be inoculated in TSA/NAD solid medium, at 37 DEG C, cultivate 24 hours, more than 5 colonies typical is selected in every strain, be inoculated in respectively in TSB/NAD liquid nutrient medium, 37 DEG C of vibration 180r/min cultivate 18～24 hours.Then gather in the crops culture, carry out purely after the assay was approved, as one-level seeding, put at 2～8 DEG C and preserve, preserve and be no more than 2.
One-level seeding culture is got in the breeding of 2.2 secondary seedings, 1: 100 by volume inoculation TSB/NAD liquid nutrient medium, and 37 DEG C of vibration 180r/min cultivate 18～24 hours.Substratum, through purely after the assay was approved, as secondary seeding, is put at 2～8 DEG C and is preserved, and preserves and is no more than 2.
Secondary seeding nutrient solution is got in the preparation of 2.3 seedling bacterium liquid, within 1: 100 by volume, be inoculated in the culture tank that fills TSB/NAD liquid nutrient medium, 37 DEG C of shaking culture 24 hours, gather in the crops and sample, purely check and live bacterial count according to existing " People's Republic of China's veterinary drug allusion quotation " annex.Every batch of seedling bacterium liquid is all without varied bacteria growing, and viable count reaches 4.5 × 10
9cFU/ml.
The deactivation of 3 bacterium liquid
Get haemophilus parasuis culture 300mL and join in 500mL bottle, divide 3 bottles.Every bottle drips 10% formaldehyde solution, and limit edged mixes, and makes the formaldehyde final concentration of 3 bottles of cultures be respectively 0.1%, 0.2%, 0.3% and 0.4%, then transfers in another bottle, puts 37 DEG C of deactivations, and every 2h jolting once.Reach 37 DEG C with liquid in container temperature and start timing, 6h, 9h, 12h, 15h, 18h, 21h, 24h draw samples after starting respectively at deactivation, carry out pure and deactivation inspection.When each point in time sampling of four concentration of formaldehyde, all asepsis growths.Final concentration is that 0.3% and 0.4% formaldehyde solution deactivation haemophilus parasuis FS0307 strain bacterium liquid does not all detect viable bacteria respectively in the time of 18h and 12h, as shown in table 3.Therefore, it is 0.3% formaldehyde solution deactivation 18h that the ablation method of bacterium liquid is selected final concentration, and every 2h jolting once.Inactivated bacterial liquid is qualified through inspection asepsis growth, using this inactivated bacterial liquid as antigen for vaccine.
The result of table 3 different concns formalin-inactivated haemophilus parasuis FS0307 strain nutrient solution
"+" positive, indicates haemophilus parasuis growth; "-" feminine gender, indicates to grow without haemophilus parasuis.
The preparation of 4 vaccines
The preparation of 4.1 haemophilus parasuis oil adjuvant killed vaccines
4.1.1 94 parts of injection white oils are got in oil phase preparation, and Si Ben-806 part, stir, and through 121 DEG C of sterilizings 30 minutes, are cooled to room temperature for subsequent use.
4.1.2 water prepares to get 96 parts of the haemophilus parasuis FS0307 strain bacterium liquid that deactivation is up to the standards, 4 parts of tween-80s, and stirring and evenly mixing is for subsequent use.
4.1.3 seedling and packing are got 2.5 parts of oil phases and are joined in aseptic beaker, start homogenizer, 10000r/min, after homogeneous 30～60s, slowly add 1 part of water, continue emulsification, 12000～13000r/min, 5～8 minutes, before emulsification finishes, adding final concentration was 0.005% Thiomersalate solution, makes water-in-oil emulsion.Get 10ml vaccine with the centrifugal 15min of 3000r/min, test tube lower floor separates out water and is less than 0.5ml, and emulsification is qualified.The vaccine qualified to emulsification carries out quantitative separating, seals, and labels, and puts at 2～8 DEG C and preserves.
The preparation of 4.2 haemophilus parasuis water adjuvant inactivated vaccines
4.2.1 Seppic ISA201VG adjuvant is got in adjuvant preparation, is heated to 31 DEG C, for subsequent use.
4.2.2 antigen is prepared to get the haemophilus parasuis FS0307 strain bacterium liquid that deactivation is up to the standards, and is heated to 31 DEG C, for subsequent use.
4.2.3 seedling and packing are pressed ISA201VG adjuvant and antigenic quality than being 1:1 proportioning (as haemophilus parasuis FS0307 strain bacterium liquid 10g, ISA201VG adjuvant 10g).Each all needs to be heated to 31 DEG C before mixing.This water antigen medium is added in ISA201VG, maintain the temperature at 30 ° of C, mix with low-shearing power, obtain stabilization formulations.The qualified vaccine of emulsification is mixed to rear quantitative separating, seal, label, put at 2～8 DEG C and preserve.
5 vaccine inspection after constructions
5.1 outward appearance haemophilus parasuis oil adjuvant killed vaccine outward appearances are oyster white homogeneous latex emulsion.Haemophilus parasuis water adjuvant inactivated vaccine outward appearance is milky emulsion, and bottom goes out without obvious water liquation.
5.2 formulations are water-in-oil-type.Get a clean suction pipe, draw a small amount of vaccine and drip in cold water, be cloud diffusion except first, later each equal indiffusion, illustrate that formulation stablizes.Haemophilus parasuis water adjuvant inactivated vaccine is water-in-oil-in-water type; Get a clean suction pipe, draw a small amount of vaccine and drip in cold water, drop part oneself dilution, and make water present oyster white foreign country, be illustrated as water-in-oil-in-water type.
5.3 stability are drawn vaccine 10ml and are added centrifuge tube, and with 3000r/min centrifugal 15 minutes, the pipe end water of separating out did not exceed the regulation of 0.5ml.
5.4 viscositys existing " Chinese veterinary pharmacopoeia " annex is tested, and should conform with the regulations.
5.5 steriling tests are tested by existing " Chinese veterinary pharmacopoeia " annex, the equal asepsis growth of vaccine of preparation.
5.6 Thiomersalate residual quantities are tested by existing " Chinese veterinary pharmacopoeia " annex, and in vaccine, Thiomersalate residual quantity is 0.006～0.007%, meets the regulation of veterinary biologics general rule.
5.7 formaldehyde content are measured and are tested by existing " Chinese veterinary pharmacopoeia " annex, and in vaccine, formaldehyde content is 0.03～0.11%, meets the regulation of veterinary biologics general rule.
Haemophilus parasuis inactivated vaccine single dose (2mL/ head), single dose that 5.8 safety examinations are prepared with laboratory repeat (2mL/ head, 2 times) and heavy dose of repetition (4mL/ head) musculi colli injection healthy susceptible piglet in 2 week age, and heavy dose of (4mL/ head) musculi colli injection gestation sow of 90 days, each group is 6.After immunity, observe the clinical manifestation of piglet and sow, record part, the systemic reaction of piglet and sow, and the farrowing situation of sow; Respectively before inoculation, inoculate in latter 14 days, survey rectal temperature every day, heavy dose of inoculation proof test group after immunity 30,60 and 90 days, extracts respectively 2 pigs, cuts open and kills, and injection site is carried out to local organization cut sections for microscopic examination.Result shows, experiment pig appetite is normal, mental status is good; Sows farrowing 100% is strong lives, and without stillborn foetus and miscarriage; After vaccine immunity, 24-48 hour piglet and sow fervescence are all no more than 1 DEG C, but be obvious one cross property, time of occurrence is of short duration, and pig is not produced to detrimentally affect.The vaccine safety that preparation is described is reliable.
5.9 potency tests are chosen 5 of healthy susceptible piglets in 2 week age, each 1 part of neck intramuscular injection vaccine (2mL), after 21 days with Isodose booster immunization once.Set up the pig that condition is identical to attack each 5 of malicious control group, normal healthy controls group, head exempts to attack poison in latter 42 days simultaneously, observes 14 after attacking poison, cuts open inspection, according to clinical symptom with cut open inspection pathology and calculate morbidity number and attack malicious protection ratio.Result demonstration, two attack malicious control group test pig all falls ill, immune group is attacked malicious protection ratio and is 100%(5/5).None morbidity of normal healthy controls group pig.Potency test is all qualified, illustrates that the vaccine of preparation has extraordinary immune protective efficiency to piglet.
Claims (1)
1. strain serum 4 type haemophilus parasuis vaccine strains, its Classification And Nomenclature be haemophilus parasuis (
haemophilus parasuis), bacterial strain number is FS0307, has been preserved in Chinese Typical Representative culture collection center, deposit number: CCTCC NO:M2013094, preservation date: on March 21st, 2013.
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|---|---|
| CN103194413A (en) | 2013-07-10 |
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