CN103194413A - 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 disease vaccine field in the veterinary biological product, particularly relate to a strain serum 4 type haemophilus parasuis strains.
Background technology
Haemophilus parasuis (Haemophilus parasuis, HPs) be a kind of gram-negative coccobacillus in the pasteurellosis bacillus section hemophilus, it is a kind of conditionality pathogenic bacteria of pig, be worldwide distribution, infect to cause with polyserositis, sacroiliitis and meningitis being pig Ge Lazeshi (Glasser ' s) disease of feature behind the piglet.The piglet in ages in main harm 2～8 week, sickness rate is generally 10%～15%, and mortality ratio can reach more than 50%.
Haemophilus parasuis is to the nutritional requirement harshness, and the growth strictness 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.Cultivate the visible needle point size of 24h canescence bacterium colony at the TSA plate culture medium that adds NAD, it is circular to be protuberance, smooth surface, moistening, neat in edge.HPs is in Chocolate Agar flat board growth difficulty, have report to show, chocolate dull and stereotyped HPs cultivations of can not going down to posterity of going up is not suitable for the subculture in vitro separately cultivation, but the Chocolate Agar flat board can be for separating of this bacterium, supplies with 5%～10% carbonic acid gas (CO during first separation and Culture
2) can promote growth.Need in meat soup or TSB substratum, add serum and the V factor (NAD) during liquid culture, can not increase but interpolation V factor concentration constantly increases bacterial concentration.Owing to self meta-bolites V factor can be discharged in the substratum in the aureus growth process, so HPs and streptococcus aureus co-inoculation are cultivated in the blood agar plate, 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 the environment resistibility extremely a little less than, vitro culture is very easily dead.
Antigenicity and virulence are widely different between the HPs bacterial strain, and serological typing is one of important method of difference different strains.At present, the serological typing method (KRG) based on the heat stable antigen immunodiffusion by propositions in 1992 such as Kielstein worldwide is accepted, this method is divided into 15 serotypes with HPs, yet has 15.2%～41% strain isolated serotype can not determine.According to the seroepidemiological survey of Japan, Germany, states such as the U.S. and Spain, with serotype 4,5 and 13 the most popular.HPs is generally popular in China, at present, and in Hebei, all there are the sick report that takes place and isolate HPs of haemophilus parasuis in 12 provinces and cities such as Henan, Hubei, Hunan, Anhui, Shanghai, Guangdong, Jiangxi.
Owing to usually not even rationally do not use antimicrobial drug in a large number on the veterinary clinic, cause the pig farm bacterium under medicine pressure, to select the resistance flora.Therefore, increasing with the difficulty of medicine prevention and control haemophilus parasuis disease, and along with the worry of people to food-safety problem, the application meeting of microbiotic aspect the prevention and control Animal diseases still less, and what will play a greater role is corresponding vaccine.HPs serotype is more, and the virulence of different serotypes bacterial strain and virulence differ greatly, and immune cross protection power is very low between each serotype, so existing domestic and international commercial haemophilus parasuis disease vaccine all can not provide protection in full force and effect to this disease.Therefore, the universal HPs bacterial strain that the separation screening immunogenicity is strong, and utilize its preparation efficient vaccine be the most important thing of prevention and control haemophilus parasuis disease.
Summary of the invention
Technical problem to be solved by this invention provides a 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 called after haemophilus parasuis (Haemophilus parasuis) of classifying, bacterial strain number is FS0307, be preserved in Chinese typical culture collection center, address: China. Wuhan. Wuhan University, postcode 430072, deposit number: CCTCC NO:M2013094, preservation date: on March 21st, 2013.This bacterial strain is that the contriver gathers screening voluntarily 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 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 constitute 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 the CFU/mL.
Beneficial effect: serum 4 type haemophilus parasuis FS0307 strains of the present invention have stable biological characteristics, piglet are had stronger pathogenic, and have good immunogenicity.The oil adjuvant killed vaccine of using its preparation is safe and reliable; can produce the antibody of higher level; extended period is long; and the pig of the homology bacterial classification being attacked poison has extraordinary protection effect; the M ﹠ M of immunity back swinery all obviously reduces; its immune effect all reaches or is better than existing commercialized vaccine on the market, can effectively prevent the popular of haemophilus parasuis.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described content of embodiment only is used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: separation and purification and the evaluation of serum 4 type haemophilus parasuis FS0307 strains.
1. produce and use substratum
TSA solid medium preparation: accurately take by weighing TSA powder 40g, be dissolved in 940mL distilled water, fully shake up back 121 ℃ of high pressure steam sterilization 15min, when being cooled to 50～60 ℃, the bovine serum 50mL, the 0.01%NAD of 10mL filtration sterilization that add filtration sterilization, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices is standby after mixing.
TSB liquid nutrient medium preparation: accurately take by weighing TSB powder 30g, be dissolved in 940mL distilled water, fully shake up back 121 ℃ of high pressure steam sterilization 15min, face the bovine serum 50mL with preceding adding filtration sterilization, the 0.01%NAD of 10mL filtration sterilization and get final product.
2. haemophilus parasuis separation and purification and evaluation
2.1 separation and purification
2003; the a large amount of sick pigs that doubtful haemophilus parasuis infection occurs of certain large-scale pig farm in area, China Hebei; be feature with persistent fever, pleuritis, peritonitis, sacroiliitis etc., choose lung, hydrothorax, pericardial fluid, the synovial fluid inoculation TSA/NAD solid medium of disease pig, put 37 ℃, 5%CO
2Cultivated under the condition 24～48 hours, the small colonies smear of the translucent protuberance of picking, gram stain microscopy is chosen the little shaft-like or polymorphism dialister bacterium of Gram-negative, carries out twice TSA/NAD solid medium subclone again and cultivates.Inoculate blood agar solid medium and plain agar solid medium simultaneously, well-grown on the TSA/NAD solid medium as a result, blood agar solid medium 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
2Cultivated under the condition 24～48 hours.Observe colonial morphology, visible needle point size, circle, neat in edge, flat, the small colonies of the translucent protuberance of canescence.Get single bacterium colony smear gramstaining, be Gram-negative, be polymorphism, be single little coccobacillus or elongated thread bacillus.
Pure culture bacterium liquid is done to intersect line taking off on the fine sheep blood agar solid medium with streptococcus aureus, put 37 ℃, 5%CO
2After cultivating 24～48 hours under the condition, be little and transparent bacterium colony, more good the closer near the growth staphylococcus, present typical satellite growth phenomenon, and haemolysis does not appear in the haemophilus parasuis periphery of bacterial colonies on the blood agar plate.
Pure culture bacterium liquid is inoculated in the TSA/NAD solid medium, sticks the circular filter paper sheet that is impregnated with X, V, the X+V factor more respectively.Put 37 ℃, 5%CO
2Cultivated under the condition 24～48 hours, the result only has colony growth around the scraps of paper that contain V, the X+V factor, show that this bacterial strain only relies on the V factor, and do not rely on the X factor.
Pure culture bacterium liquid is inoculated micro-biochemical reaction pipe, replenish the NAD of final concentration 0.01% respectively, put 37 ℃ and leave standstill cultivation, judge the biochemical characteristic result behind the 48h.The 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 with 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 among the GenBank, primer sequence is: P1:5 '-gctccacaagctaacactttc-3 ', P2:5 '-catagaaacttcttttgaacc-3 ', give birth to worker's biotechnology company limited by Shanghai and synthesize, this primer amplification clip size is 1038bp.
2.3.2 FS0307 strain culture 1.5mL is got in the preparation of thallus DNA template, the centrifugal 5min of 10000r/min abandons supernatant, precipitate resuspendedly with 200 μ L distilled waters, behind 100 ℃ of water-bath 10min, the centrifugal 5min of 12000r/min collects supernatant, be pcr template ,-20 ℃ of preservations are standby.
2.3.3PCR amplification and order-checking use the thallus DNA that extracts as pcr template.The 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 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min eventually.Get 10 μ L pcr amplification products, sepharose with 1% electrophoresis under 120V voltage is identified, while, as positive control, the specific band of the 1038bp size identical with type strain appearred in the result, illustrates that the FS0307 strain that is separated to is haemophilus parasuis with type strain.
Transfer to the order-checking of Shanghai living worker's biotechnology company limited through reclaiming the pcr amplification product that is accredited as the positive.The result is shown in SEQ ID No.1 for haemophilus parasuis FS0307 strain P5 gene sequencing, and the sequence of announcing among this sequence and the GenBank is carried out BLAST comparison, reaches more than 95% with the homology of domestic and international existing haemophilus parasuis corresponding sequence.2.4 haemophilus parasuis FS0307 strain serological identification
Pressing Kleletein-Rapp-Gabriedson (KRG) agar diffusion serotype method identifies the serotype of haemophilus parasuis FS0307 strain.Haemophilus parasuis FS0307 strain is inoculated in the TSB substratum, 37 ℃, 5%CO in 5% ratio
2After cultivating 18～24 hours under the condition, 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 that supernatant is used for agar diffusion test.Prepare 1% agar plate, punching (6 on 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 rabbits (directly over the hole be labeled as 1, add 1 type hyper-immune serum, the serum of all the other each serotypes is pressed the 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, wet box is hatched for 37 ℃, observe and record result, the contrast of the accurate bacterial strain of bidding simultaneously at 24h, 48h, 72h respectively.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, do not have precipitation line with the serotype of other type type strain, illustrate that haemophilus parasuis FS0307 strain is serum 4 types.
2.5 animal challenge test
Serum 4 type haemophilus parasuis FS0307 strains are cultivated through the TSB/NAD liquid nutrient medium, carry out live bacterial count, after concentrating, make infection titer reach 1 * 10
9CFU/ml is through 5 of the healthy susceptible pigs of abdominal injection 50～60 ages in days, 5ml/ head.Other establishes 1 group of control group, and 5, abdominal injection TSB/NAD substratum, 5ml/ head.Raise antibiotic-free in the feed routinely after attacking poison.Observed 14, and surveyed body temperature every day, observe clinical symptom.Attack poison and cut open inspection after 14 days, according to clinical symptom with cut open the inspection pathology and calculate the morbidity number.Simultaneously, the pathological material of disease of organizing of getting the morbidity pig is done bacterium separation and evaluation.
Attack malicious result and show, observe 14 continuously after, 5 experiment pig that the poison group is attacked in serum 4 type haemophilus parasuis FS0307 strains are first sequela all; The contrast pig is acted normally.Clinical symptom shows as, and experiment pig infection haemophilus parasuis strain isolated fervescence after 1 day engenders that subsequently appetite reduces, and thick random by hair, ear and whole skin are rubescent, and nose liquid increases, and expiratory dyspnea appears in the later stage, arthroncus, and ground for sleeping in does not rise.Cut open inspection and find, thoracic cavity, seroperitoneum appear in morbidity pig and the pig that dies of illness, and in various degree pleura, peritoneal adhesion are arranged, and arthroncus, joint cavity mucus increase, and lungs are hemorrhage, 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 the bacterium separation.The result is separated to the bacterium colony bacterium similar to the haemophilus parasuis type strain with thalli morphology from the sick pig of 4 hairs the poison group is attacked in the FS0307 strain.Simultaneously all bacteriums that are separated to are carried out PCR and identify that the result specific band occurs at the 1038bp place, proves that bacterial isolate is haemophilus parasuis.
The virulence test result of table 1 haemophilus parasuis FS0307 strain
Not immunity or inapplicable of "-" expression.
2.6 immunogenic mensuration
Cultivate serum 4 type haemophilus parasuis FS0307 strains with the TSB/NAD liquid nutrient medium, behind the live bacterial count, the viable bacteria culture is used final concentration 0.3% formaldehyde solution deactivation 12h respectively, and the bacterium number is condensed into 4 * 10
9CFU/ml through deactivation after the assay was approved, prepares vaccine by ratio 1 ︰ 3 emulsifications of water oil phase.Choose 25 of healthy susceptible pigs about 2 ages in week, be divided into 5 groups at random, 5 every group.The 1st～3 group every group respectively immune FS0307 strain vaccine 0.5ml, 1ml, 2ml, the 4th group for attacking malicious control group, and the 5th group is the normal healthy controls group.Each is organized vaccine immunity group first immunisation and uses Isodose vaccine booster immunization once after 3 weeks.Head exempted from back 42 days, and except 5 of normal healthy controls groups, all immune group forward to the pig of attacking malicious control group and attack malicious Animal House.The 1st～3 group and the 4th group every group respectively the FS0307 strain viable bacteria culture of intraperitoneal injection through concentrating (the bacterium number is (1 * 10
9CFU/ml), 5ml/ 4; The 5th group of intraperitoneal injection TSB/NAD substratum, the 5ml/ head.Attack the poison back and observed 14, cut open inspection.According to clinical symptom with cut open the inspection pathology and calculate the morbidity number and attack malicious protection ratio.Immunity is attacked malicious protection test result and is shown, attacks 5 pigs of malicious control group and all falls ill (wherein dead 2), the sick clinical symptom of typical haemophilus parasuis and significantly pathological change occur.The experiment pig of normal healthy controls group is not morbidity all.There are 2 clinical symptom and pathological change to occur in 5 pigs of FS0307 strain 0.5mL immune group, have only 1 the inspection pathology to occur cuing open in 5 pigs of 1mL immune group, attack malicious protection ratio and reach 3/5 and 4/5 respectively; And the experiment pig of 2mL immune group is attacked malicious protection ratio and is reached 5/5 all less than morbidity.Immunity is attacked malicious protection situation and is seen table 2 for details.
The immunogenicity determining test-results of table 2 haemophilus parasuis FS0307 strain
"-" expression is inapplicable.
By table 2 result as seen, the minimum immunoprotection dosage of haemophilus parasuis FS0307 strain is 1mL to contain viable count is 1 * 10
9CFU/mL.
Case study on implementation 2: serum 4 application of type haemophilus parasuis FS0307 strain in the haemophilus parasuis disease vaccine.
1 bacterial classification
The 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 stronger virulence, and good immunogenicity is arranged.
2 production strain preparation
After 2.1 the one-level seeding is cultivated the secondary pig of 4 types of getting and has a liking for the seed lyophilized products dissolving of bacillus basis, be inoculated in the TSA/NAD solid medium, cultivated 24 hours down at 37 ℃, 5 above colonies typicals are selected in every strain, be inoculated in respectively in the TSB/NAD liquid nutrient medium, 37 ℃ of vibration 180r/min cultivated 18～24 hours.Gather in the crops culture then, carry out purely after the assay was approved, as the one-level seeding, put 2～8 ℃ and preserve down, preserve and be no more than 2.
2.2 one-level seeding culture is got in the breeding of secondary seeding, 1: 100 by volume inoculation TSB/NAD liquid nutrient medium, and 37 ℃ of vibration 180r/min cultivated 18～24 hours.Substratum, is put 2～8 ℃ and is preserved down as the secondary seeding through purely after the assay was approved, preserves and is no more than 2.
2.3 secondary seeding nutrient solution is got in the preparation of seedling bacterium liquid, be inoculated in the culture tank that fills the TSB/NAD liquid nutrient medium in 1: 100 by volume, 37 ℃ of shaking culture 24 hours, results and sampling are checked and live bacterial count purely according to existing " People's Republic of China's veterinary drug allusion quotation " appendix.Every batch of seedling bacterium liquid does not all have 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 the 500mL bottle, divide 3 bottles.Every bottle drips 10% formaldehyde solution, and limit edged mixing makes the formaldehyde final concentration of 3 bottles of cultures be respectively 0.1%, 0.2%, 0.3% and 0.4%, transfers to then in another bottle, puts 37 ℃ of deactivations, and every 2h jolting once.Reach 37 ℃ with the liquid in container temperature and pick up counting, 6h, 9h, 12h, 15h, 18h, 21h, 24h draw samples after beginning respectively at deactivation carry out pure and the 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% formaldehyde solution deactivation haemophilus parasuis FS0307 strain bacterium liquid does not all detect viable bacteria respectively when 18h and 12h, and is 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 for use, and every 2h jolting once.Inactivated bacterial liquid through the check asepsis growth be qualified, with this inactivated bacterial liquid as antigen for vaccine.
The result of table 3 different concns formalin-inactivated haemophilus parasuis FS0307 strain nutrient solution
"+" positive, expression have the haemophilus parasuis growth; "-" feminine gender is represented no haemophilus parasuis growth.
The preparation of 4 vaccines
4.1 the preparation of haemophilus parasuis oil adjuvant killed vaccine
4.1.1 94 parts of injection white oils are got in oil phase preparation, and Si Ben-806 part, stir, through 121 ℃ of sterilizations 30 minutes, it was standby to be cooled to room temperature.
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 standby.
4.1.3 seedling and packing are got 2.5 parts of oil phases and are joined in the aseptic beaker, start homogenizer, 10000r/min, behind homogeneous 30～60s, slowly add 1 part of water, continue emulsification, 12000～13000r/min, 5～8 minutes, the adding final concentration was 0.005% Thiomersalate solution before emulsification finishes, and makes water-in-oil emulsion.Get the 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 packing, seals, and labels, and puts 2～8 ℃ and preserves down.
4.2 the preparation of haemophilus parasuis water adjuvant inactivated vaccine
4.2.1 Seppic ISA201VG adjuvant is got in the adjuvant preparation, is heated to 31 ℃, and is standby.
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 ℃, and is standby.
4.2.3 the ISA201VG adjuvant is pressed in seedling and packing and the antigenic quality ratio is 1:1 proportioning (as haemophilus parasuis FS0307 strain bacterium liquid 10g, ISA201VG adjuvant 10g).Each all need be heated to 31 ℃ before mixing.This water antigen medium is added among the ISA201VG, maintain the temperature at 30 ° of C, mix with low-shearing power, obtain stabilization formulations.The qualified vaccine of emulsification is mixed quantitatively packing of back, seal, label, put 2～8 ℃ and preserve down.
5 vaccine inspection after constructions
5.1 outward appearance haemophilus parasuis oil adjuvant killed vaccine outward appearance is the oyster white homogeneous latex emulsion.Haemophilus parasuis water adjuvant inactivated vaccine outward appearance is milky emulsion, and the bottom does not have tangible water liquation and goes out.
5.2 formulation is water-in-oil-type.Get a cleaning suction pipe, draw a small amount of vaccine and drip in cold water, be the cloud diffusion except first, each equal indiffusion later on illustrates that formulation stablizes.Haemophilus parasuis water adjuvant inactivated vaccine is the water-in-oil-in-water type; Get a cleaning 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 the water-in-oil-in-water type.
Add centrifuge tube 5.3 stability is drawn vaccine 10ml, with 3000r/min centrifugal 15 minutes, the pipe end water of separating out surpassed the regulation of 0.5ml.
5.4 viscosity existing " Chinese veterinary drug allusion quotation " appendix is tested, should be up to specification.
5.5 steriling test is tested the equal asepsis growth of the vaccine of preparation by existing " Chinese veterinary drug allusion quotation " appendix.
5.6 the Thiomersalate residual quantity is tested by existing " Chinese veterinary drug allusion quotation " appendix, the Thiomersalate residual quantity is 0.006～0.007% in the vaccine, meets the regulation of veterinary biologics general rule.
5.7 formaldehyde content is measured and tested by existing " Chinese veterinary drug allusion quotation " appendix, formaldehyde content is 0.03～0.11% in the vaccine, meets the regulation of veterinary biologics general rule.
5.8 safety examination repeats (2mL/ head with haemophilus parasuis inactivated vaccine single dose (2mL/ head), the single dose of prepared in laboratory, 2 times) and heavy dose of repetition (4mL/ head) musculi colli injection healthy susceptible piglet in 2 ages in week, and 90 days sow of heavy dose of (4mL/ head) musculi colli injection gestation, each group is 6.The clinical manifestation of piglet and sow, part, the systemic reaction of record piglet and sow, and the farrowing situation of sow are observed in the immunity back; Respectively before inoculation, in back 14 days of the inoculation, survey rectal temperature every day, heavy dose of inoculation proof test group after immunity 30,60 and 90 days extracts 2 pigs respectively, cuts open and kills, and the injection site is carried out the local organization cut sections for microscopic examination.The result shows that experiment pig appetite is normal, mental status is good; Sows farrowing 100% strong living, and do not have stillborn foetus and miscarriage; 24-48 hour piglet and sow fervescence all are no more than 1 ℃ behind the vaccine immunity, but be obvious one cross property, time of occurrence is of short duration, and pig is not only produced detrimentally affect.The vaccine safety that preparation is described is reliable.
5.9 potency test is chosen 5 of healthy susceptible piglets in 2 ages in week, each 1 part of neck intramuscular injection vaccine (2mL), after 21 days with the Isodose booster immunization once.Set up the identical pig of condition to attack each 5 of malicious control groups, normal healthy controls group simultaneously, head exempts to attack poison in back 42 days, attacks the poison back and observes 14, cuts open inspection, according to clinical symptom with cut open the inspection pathology and calculate the morbidity number and attack malicious protection ratio.The result shows that two attack malicious control group test pig all falls ill, and 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. a strain serum 4 type haemophilus parasuis vaccine strains, its called after haemophilus parasuis (Haemophilus parasuis) of classifying, bacterial strain number is FS0307, be preserved in Chinese typical culture collection center, deposit number: CCTCC NO:M2013094, preservation date: on March 21st, 2013.
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| WO2015051371A3 (en) * | 2013-10-04 | 2015-07-09 | Merial Limited | Haemophilus parasuis vaccine serovar type four |
| JP2016533353A (en) * | 2013-10-04 | 2016-10-27 | メリアル インコーポレイテッド | Hemophilus paraswiss vaccine serotype 4 |
| US9504740B2 (en) | 2013-10-04 | 2016-11-29 | Merial, Inc. | Haemophilus parasuis vaccine serovar type four |
| CN107245459A (en) * | 2017-04-11 | 2017-10-13 | 河南省农业科学院畜牧兽医研究所 | One plant of haemophilus parasuis and its application |
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| CN103194413B (en) | 2014-12-03 |
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