CN103191421B - Application of serotype 5 haemophilus parasuis (HPs) vaccine strain - Google Patents
Application of serotype 5 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 application of a serotype 5 HPs vaccine strain. The class name of the vaccine strain is HPs. The strain number is XX0306. The vaccine strain has been collected in the China Center for Type Culture Collection, with collection number being CCTCC No:M2013095 and collection date being March 21, 2013. The serotype 5 HPs strain XX0306 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 the application of a strain Serotype 5 haemophilus parasuis strain.
Background technology
Haemophilus parasuis (Haemophilus parasuis, HPs) be a kind of gram-negative coccobacillus in pasteurellosis bacillus section haemophilus, it is a kind of conditionality pathogen of pig, be worldwide distribution, infect and to cause after piglet and take the pig Ge Lazeshi that polyserositis, arthritis and meningitis be feature (Glasser ' s) disease.The piglet in 2～8 week age of main harm, sickness rate is generally 10%～15%, and mortality rate can reach more than 50%.
Haemophilus parasuis is harsh to nutritional requirement, and growth strictly needs the V factor (NAD), thinks that at present tryptose soya agar (TSA) culture medium and tryptone Semen sojae atricolor (TSB) culture medium are to cultivate the optimum medium of haemophilus parasuis.On the TSA plating 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, on chocolate flat board, 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% carbon dioxide (CO during first separation and Culture
2) can promote growth.During liquid culture, need in meat soup or TSB culture medium, add serum and the V factor (NAD), but interpolation V factor concentration constantly increases bacterial concentration, can not increase.Owing to self metabolite V factor can being discharged in culture medium in aureus growth process, so HPs and staphylococcus aureus co-inoculation are in blood agar plate culture, HPs is at staphylococcus aureus both sides well-grown, along with staphylococcus aureus apart from increase, bacterium colony diminishes gradually, and this phenomenon is called as satellitosis.HPs to external world environment resistance extremely a little less than, In 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 by propositions in 1992 such as Kielstein is worldwide accepted, the method is divided into 15 serotypes by HPs, yet has 15.2%～41% separated strain 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 China is generally popular, at present, and in Hebei, Henan, Hubei, Hunan, Anhui, Shanghai, Guangdong, Jiangxi Deng12Ge provinces and cities all has 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 antibacterial under medicine pressure, to select drug 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 antibiotic 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 pathogenicity 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 the application of a strain Serotype 5 haemophilus parasuis vaccine strain (XX0306 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 Serotype 5 haemophilus parasuis vaccine strain, its Classification And Nomenclature is haemophilus parasuis (Haemophilusparasuis), bacterial strain number is XX0306, bacterial strain number is XX0306 strain, be preserved in Chinese Typical Representative culture collection center, address: China. Wuhan. Wuhan University, postcode 430072, deposit number: CCTCC NO:M2013095, preservation date: on March 21st, 2013.This bacterial strain is that inventor gathers voluntarily screening and obtains a strain and have stronger virulence and very high immunogenic bacterial strain in April, 2003 in Hebei province's Hengshui City.
The application of above-mentioned Serotype 5 haemophilus parasuis vaccine strain in preparation treatment pig Haemophilus parasuis medicine.
Wherein, described medicine is vaccine, preferred oil water-in type vaccine or oil in water vaccine or W/O/W type vaccine.
Wherein, Serotype 5 haemophilus parasuis vaccine strain and pharmaceutically acceptable carrier or auxiliary material combination form medicine, as unit price Seedling, multivalence Seedling or connection Seedling etc.
Wherein, described Serotype 5 haemophilus parasuis vaccine strain bacteria containing amount in medicine (preferred vaccine) is 3.5 * 10
9more than CFU/mL.
Beneficial effect: Serotype 5 haemophilus parasuis XX0306 of the present invention strain has 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; duration is long; and the pig of homology strain counteracting toxic substances is had to 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.
The specific embodiment
According to following embodiment, the present invention may be better understood.Yet, 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 evaluation of Serotype 5 haemophilus parasuis XX0306 strain.
1. production culture medium
TSA solid medium preparation: accurately take TSA powder 40g, be dissolved in 940mL distilled water, fully shake up rear 121 ℃ of high pressure steam sterilization 15min, while being cooled to 50～60 ℃, add the Ox blood serum 50mL of filtration sterilization, the 0.01%NAD of 10mL filtration sterilization, after mix homogeneously, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices is standby.
TSB fluid medium preparation: accurately take TSB powder 30g, be dissolved in 940mL distilled water, fully shake up rear 121 ℃ of high pressure steam sterilization 15min, add before use the Ox blood serum 50mL of filtration sterilization, the 0.01%NAD of 10mL filtration sterilization.
2. haemophilus parasuis separation and purification and evaluation
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 in Jiangsu Province, China area; take persistent fever, pleuritis, peritonitis, arthritis etc. is feature; choose lung, hydrothorax, pericardial fluid, the joint fluid inoculation TSA/NAD solid medium of disease pig; put under 37 ℃, 5%CO2 condition and cultivate 24～48 hours; the petite smear of the translucent protuberance of picking; gram stain microscopy; choose the little shaft-like or pleomorphism dialister bacterium of Gram-negative, then carry out twice TSA/NAD solid medium sub-clone 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 ℃, 5%CO
2under condition, cultivate 24～48 hours.Observe colonial morphology, visible needle point size, circle, neat in edge, flat, the petite of the translucent protuberance of canescence.Getting single bacterium colony smear Gram’s staining, is Gram-negative, is pleomorphism, is the single little coccobacillus or elongated thread bacillus.
Pure culture bacterium liquid is done to intersect line with staphylococcus aureus on de-fine sheep blood Solid agar culture, put 37 ℃, 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 the 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 ℃, 5%CO
2under condition, cultivate 24～48 hours, result only has colony growth around at 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 tube, supplement respectively the NAD of final concentration 0.01%, put 37 ℃ of standing cultivations, after 48h, judge biochemical characteristic result.Result shows that isolated strains is to glucose, sucrose and fructose fermentation, to maltose, galactose, xylose and fructose azymic, indole test, oxidase test and urease test are negative, and nitrate reduction test and catalase test are positive, meet the biochemical characteristic of haemophilus parasuis.According to the source place of this bacterial strain by its called after XX0306 strain.
2.3OmpA gene sequencing and analysis
2.3.1 design of primers is according to two primers of haemophilus parasuis OmpA gene order design in GenBank, primer sequence is: P1:5 '-ctccacaagctaacactttc-3 ', P2:5 '-catagaaacttcttttgaacc-3 ', synthetic by Shanghai Sheng Gong biological engineering company limited, this primer amplification clip size is 1038bp.
2.3.2 XX0306 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 ℃ of water-bath 10min, the centrifugal 5min of 12000r/min collects supernatant, be pcr template ,-20 ℃ 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 condition is: 95 ℃ of denaturation 5min, and 94 ℃ of degeneration 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min eventually.Get 10 μ L pcr amplification products, agarose gel with 1% electrophoresis under 120V voltage is identified, using type strain as positive control, and having there is the specific band of the 1038bp size identical with type strain in result, illustrates that the XX0306 strain being separated to is haemophilus parasuis simultaneously.
Through recovery, be accredited as positive pcr amplification product and transfer to the order-checking of Shanghai Sheng Gong biological engineering company limited.Haemophilus parasuis XX0306 strain OmpA gene sequencing the results are shown in 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 XX0306 strain serological Identification
By Kleletein-Rapp-Gabriedson (KRG) agar diffusion serotype method, the serotype of haemophilus parasuis XX0306 strain is identified.Haemophilus parasuis XX0306 strain is inoculated in to TSB culture medium, 37 ℃, 5%CO in 5% ratio
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 supernatant for the typing antigen of agar gel 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 heat treatment typing antigen 6 μ l of bacterial strain to be checked, 37 ℃, wet box is hatched, at 24h, 48h, 72h, observe and record result respectively, establish the contrast of reference culture simultaneously.Agar gel diffusion test shows, the high pressure extract antigen of haemophilus parasuis XX0306 strain only has precipitation line with the positive serum of 5 type haemophilus parasuis type strains, without precipitation line, illustrate that haemophilus parasuis XX0306 strain is Serotype 5 with the serotype of other type type strain.
2.5 animal challenge tests
Serotype 5 haemophilus parasuis XX0306 strain is cultivated through TSB/NAD fluid medium, carries out count plate, after concentrating, makes infection titer reach 1 * 10
9cFU/ml, through 5 of the healthy susceptible pigs of lumbar injection 50～60 ages in days, 5ml/ head.Separately establish 1 group of matched group, 5, lumbar injection TSB/NAD culture medium, 5ml/ head.After counteracting toxic substances, raise routinely antibiotic-free in feedstuff.Observe 14, survey body temperature every day, observe clinical symptoms.Counteracting toxic substances cutd open inspection after 14 days, according to clinical symptoms with cut open inspection pathological changes and calculate morbidity number.Meanwhile, the pathological material of disease of organizing of getting morbidity pig is done antibacterial separation and identifies.
The demonstration of counteracting toxic substances result, Continuous Observation is after 14 days, and 5 experiment pig of Serotype 5 haemophilus parasuis XX0306 strain counteracting toxic substances group are first sequela all; Contrast pig is acted normally.Clinical symptoms shows as, and experiment pig infection haemophilus parasuis separated strain fervescence after 1 day, engenders appetite depression subsequently, and disorderly thick by hair, ear and whole skin are rubescent, and nose liquid increases, and 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, articular cavity mucus increase, Pulmonary hemorrhage, the pathological changes such as swollen lymph node.The results are shown in Table 1.
Utilize the pathological material of disease of organizing of isolated strains counteracting toxic substances to carry out antibacterial separation.In the sick pig of 4 hairs of result from XX0306 strain counteracting toxic substances group, be separated to the bacterium colony antibacterial similar to haemophilus parasuis type strain with thalli morphology.All antibacterials that are separated to are carried out to PCR evaluation, 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 XX0306 strain
"-" represents not immunity or inapplicable.
2.6 immunogenic mensuration
With TSB/NAD fluid medium, cultivate Serotype 5 haemophilus parasuis XX0306 strain, after count plate, viable bacteria culture is used respectively final concentration 0.3% formalin deactivation 12h, and bacterium number is condensed into 4 * 10
9cFU/ml, through deactivation after the assay was approved, prepares vaccine by 1 ︰ 3 emulsifyings 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.Distinguish immune XX0306 strain vaccine 0.5ml, 1ml, 2ml for 1st～3 groups every group, the 4th group is counteracting toxic substances matched group, and the 5th group is normal healthy controls group.Each is organized vaccine immunity group first immunisation and after 3 weeks, uses Isodose vaccine booster immunization once.Head exempts from latter 42 days, and except 5 of normal healthy controls groups, the pig of all immune group and counteracting toxic substances matched group forwards counteracting toxic substances Animal House to.1st～3 groups and the 4th group every group respectively intraperitoneal injection through concentrated XX0306 strain viable bacteria culture, (bacterium number is (1 * 10
9cFU/ml), 5ml/ 4; The 5th group of intraperitoneal injection TSB/NAD culture medium, 5ml/ head.After counteracting toxic substances, observe 14, cut open inspection.According to clinical symptoms with cut open inspection pathological changes and calculate morbidity number and counteracting toxic substances protective rate.The demonstration of Immunization protection test result, 5 pigs of counteracting toxic substances matched group all fall ill (wherein dead 2), occur typical Haemophilus parasuis clinical symptoms and significantly pathological change.The experiment pig of normal healthy controls group is not morbidity all.In 5 pigs of XX0306 strain 0.5mL immune group, have 2 to occur clinical symptoms and pathological change, in 5 pigs of 1mL immune group, only have 1 to occur cuing open inspection pathological changes, counteracting toxic substances protective rate reaches respectively 3/5 and 4/5; And the experiment pig of 2mL immune group all less than morbidity, counteracting toxic substances protective rate reaches 5/5.Immunization protection situation refers to table 2.
The immunogenicity determining result of the test of table 2 haemophilus parasuis XX0306 strain
"-" represents inapplicable.
From table 2 result, it is 1 * 10 containing viable count that the minimum immunoprotection dosage of haemophilus parasuis XX0306 strain is 1mL
9cFU/mL.
Embodiment 2: the application of Serotype 5 haemophilus parasuis XX0306 strain in Haemophilus parasuis vaccine.
1 strain
Seedling strain is Serotype 5 haemophilus parasuis XX0306 strain, its deposit number is that CCTCC NO:M2013095 thalline, colonial morphology meet haemophilus parasuis type strain form, biochemical characteristic and cultural character are stable, piglet is had to stronger virulence, and have good immunogenicity.
2 productions are prepared with strain
2.1 one-level seedings are cultivated the secondary pig of 5 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 ℃, cultivate 24 hours, 5 above colonies typicals are selected in every strain, be inoculated in respectively in TSB/NAD fluid medium, 37 ℃ 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 ℃ 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 fluid medium, and 37 ℃ of vibration 180r/min cultivate 18～24 hours.Culture medium, through purely after the assay was approved, as secondary seeding, is put at 2～8 ℃ and is preserved, and preserves and is no more than 2.
Secondary seeding culture fluid 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 fluid medium, 37 ℃ of shaken cultivation 24 hours, gather in the crops and sample, according to the existing < < veterinary drug allusion quotation > > of People's Republic of China (PRC) appendix, purely check and count plate.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% formalin, 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 ℃ of deactivations, and every 2h jolting once.With liquid in container temperature, reach 37 ℃ and start timing, 6h, 9h, 12h, 15h, 18h, 21h, 24h draw samples after starting respectively at deactivation, carry out pure and deactivation check.During each point in time sampling of four concentration of formaldehyde, equal asepsis growths.Final concentration is that 0.3% and 0.4% formalin deactivation haemophilus parasuis XX0306 strain bacterium liquid does not all detect viable bacteria respectively when 18h and 12h, as shown in table 3.Therefore, it is 0.3% formalin deactivation 18h that the ablation method of bacterium liquid is selected final concentration, and every 2h jolting once.Inactivated bacterial liquid is qualified through check asepsis growth, usings this inactivated bacterial liquid as antigen for vaccine.
The result of table 3 variable concentrations formalin-inactivated haemophilus parasuis XX0306 strain culture fluid
"+" 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 ℃ of sterilizings 30 minutes, are cooled to room temperature standby.
4.1.2 water prepares to get 96 parts of the haemophilus parasuis XX0306 strain bacterium liquid that deactivation is up to the standards, 4 parts of tween 80s, and stirring and evenly mixing is standby.
4.1.3 seedling and subpackage are got 2.5 parts of oil phases and are joined in aseptic beaker, start homogenizer, 10000r/min, after homogenizing 30～60s, slowly add 1 part of water, continue emulsifying, 12000～13000r/min, 5～8 minutes, before emulsifying finishes, adding final concentration was 0.005% thimerosal 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 emulsifying is qualified.The vaccine qualified to emulsifying carries out quantitative separating, seals, and labels, and puts at 2～8 ℃ 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 ℃, standby.
4.2.2 antigen is prepared to get the haemophilus parasuis XX0306 strain bacterium liquid that deactivation is up to the standards, and is heated to 31 ℃, standby.
4.2.3 seedling and subpackage are pressed ISA201VG adjuvant and antigenic quality than being 1:1 proportioning (as haemophilus parasuis XX0306 strain bacterium liquid 10g, ISA201VG adjuvant 10g).Each all needs to be heated to 31 ℃ before mixing.This water antigen medium is added in ISA201VG, maintain the temperature at 30 ° of C, with low-shearing force, mix, obtain stabilization formulations.To quantitative separating after the qualified vaccine mix homogeneously of emulsifying, seal, label, put at 2～8 ℃ and preserve.
5 vaccine product inspections
5.1 outward appearance haemophilus parasuis oil adjuvant killed vaccine outward appearances are milky homogeneous latex emulsion.Haemophilus parasuis water adjuvant inactivated vaccine outward appearance is milky emulsion, and bottom goes out without obvious water liquation.
5.2 dosage forms 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, each equal indiffusion later, illustrate that dosage form stablizes.Haemophilus parasuis water adjuvant inactivated vaccine is W/O/W 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 milky foreign country, be illustrated as W/O/W type.
5.3 stability are drawn vaccine 10ml and are added centrifuge tube, and with 3000r/min centrifugal 15 minutes, the water that separate out at the pipe end did not surpass the regulation of 0.5ml.
The existing < < of 5.4 viscosity Chinese veterinary pharmacopoeia > > appendix is tested, should be up to specification.
5.5 steriling tests are tested by existing < < Chinese veterinary pharmacopoeia > > appendix, the equal asepsis growth of vaccine of preparation.
5.6 thimerosal residual quantities are tested by existing < < Chinese veterinary pharmacopoeia > > appendix, in vaccine, thimerosal residual quantity is 0.006～0.007%, meets the regulation of veterinary biologics general rule.
5.7 content of formaldehyde are measured and are tested by existing < < Chinese veterinary pharmacopoeia > > appendix, in vaccine, content of formaldehyde is 0.03～0.11%, meets the regulation of veterinary biologics general rule.
The Haemophilus parasuis inactivated vaccine single dose (2mL/ head) that 5.8 safety examinations are prepared with laboratory, single dose 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 general reaction of piglet and sow, and the farrowing situation of sow; Respectively before inoculation, inoculate in latter 14 days, survey anus temperature every day, heavy dose of inoculation safety 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 fetus and miscarriage; After vaccine immunity, 24-48 hour piglet and sow fervescence are all no more than 1 ℃, but be obvious one cross property, time of occurrence is of short duration, and pig is not produced to harmful effect.Head exempts from latter 30 days Haemophilus parasuis bivalent inactivated vaccine immune group and observes obvious inflammatory reaction, and after head exempts from, tissue slice on the 60th, 90 is not all observed obvious inflammatory reaction.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 cervical region intramuscular injection vaccine (2mL), after 21 days with Isodose booster immunization once.Set up pig that condition is identical to make each 5 of counteracting toxic substances matched group, normal healthy controls groups, head exempts from latter 42 days counteracting toxic substances, observes 14 after counteracting toxic substances, cuts open inspection simultaneously, according to clinical symptoms with cut open inspection pathological changes and calculate morbidity number and counteracting toxic substances protective rate.Result demonstration, two counteracting toxic substances matched group test pig are all fallen ill, immune group counteracting toxic substances protective rate 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 (3)
1. the Serotype 5 haemophilus parasuis vaccine strain application in preparation pig Haemophilus parasuis medicine;
Wherein, described Serotype 5 haemophilus parasuis vaccine strain, its Classification And Nomenclature is haemophilus parasuis (Haemophilus parasuis), bacterial strain number is XX0306, be preserved in Chinese Typical Representative culture collection center, deposit number: CCTCC NO:M2013095, preservation date: on March 21st, 2013;
Wherein, described medicine is inactivated vaccine.
2. application according to claim 1, is characterized in that, described vaccine is water-in-oil type vaccine or oil in water vaccine or W/O/W type vaccine.
3. according to the application described in any one in claim 1 to 2, it is characterized in that, described Serotype 5 haemophilus parasuis vaccine strain bacteria containing amount in medicine is 3.5 * 10
9more than CFU/mL.
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| CN104450557A (en) * | 2014-10-09 | 2015-03-25 | 扬州优邦生物制药有限公司 | Serum-5 type haemophilus lus paradis vaccine strain and application thereof |
| CN104450555A (en) * | 2014-10-09 | 2015-03-25 | 扬州优邦生物制药有限公司 | Serum-13 type haemophilus lus paradis vaccine strain and application thereof |
| CN104450556A (en) * | 2014-10-09 | 2015-03-25 | 扬州优邦生物制药有限公司 | Serum-12 type haemophilus lus paradis vaccine strain and application thereof |
| CN104388340A (en) * | 2014-10-28 | 2015-03-04 | 中国动物卫生与流行病学中心 | Blood serum 5 type haemophilus parasuis and application thereof |
| CN115414350B (en) * | 2022-09-22 | 2023-08-04 | 湖北省农业科学院畜牧兽医研究所 | Application of mangiferin in preparing medicine for inhibiting haemophilus parasuis |
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