CN103191421A - 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 disease vaccine field in the veterinary biological product, particularly relate to the application of a strain serum 5 type haemophilus parasuis strains.
Background technology
Haemophilus parasuis (Haemophilus parasuis, HPs) be a kind of gram-negative coccobacillus in the pasteurellosis bacillus section haemophilus, it is a kind of conditionality pathogen of pig, be worldwide distribution, infect to cause with polyserositis, arthritis 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 rate 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) culture medium and tryptone Semen sojae atricolor (TSB) culture medium are to cultivate the optimum medium of haemophilus parasuis.Cultivate the visible needle point size of 24h canescence bacterium colony at the TSA plating 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, HPs cultivations of can not going down to posterity is not suitable for the subculture in vitro separately cultivation on the chocolate flat board, but the chocolate agar flat board can be for separating of this bacterium, supplies with 5%～10% carbon dioxide (CO during first separation and Culture
2) can promote growth.Need in meat soup or TSB culture medium, 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 metabolite V factor can be discharged in the culture medium in the aureus growth process, so HPs and staphylococcus aureus co-inoculation are cultivated in the blood agar plate, 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 the environment resistance extremely a little less than, In 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% separated strain 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 antibacterial under medicine pressure, to select the drug 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 antibiotic 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 pathogenicity 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 the application of a strain serum 5 type haemophilus parasuis vaccine strains (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 serum, 5 type haemophilus parasuis vaccine strains, its called after haemophilus parasuis (Haemophilus parasuis) of classifying, bacterial strain number is XX0306, bacterial strain number is the XX0306 strain, be preserved in Chinese typical 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 the inventor 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 5 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 W/O/W type vaccine.
Wherein, serum 5 type haemophilus parasuis vaccine strains and pharmaceutically acceptable carrier or adjuvant constitute medicine, as unit price Seedling, multivalence Seedling or connection Seedling etc.
Wherein, described serum 5 type haemophilus parasuis vaccine strains bacteria containing amount in medicine (preferred vaccine) is 3.5 * 10
9More than the CFU/mL.
Beneficial effect: serum 5 type haemophilus parasuis XX0306 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; duration is long; and the pig to homology strain counteracting toxic substances 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.
The specific 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 5 type haemophilus parasuis XX0306 strains.
1. produce and use culture medium
TSA solid medium preparation: accurately take by weighing TSA powder 40g, be dissolved in the 940mL distilled water, fully shake up back 121 ℃ of high pressure steam sterilization 15min, when being cooled to 50～60 ℃, add Ox blood serum 50mL, the 0.01%NAD of 10mL filtration sterilization of filtration sterilization, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices is standby behind the mix homogeneously.
TSB fluid medium preparation: accurately take by weighing TSB powder 30g, be dissolved in the 940mL distilled water, fully shake up back 121 ℃ of high pressure steam sterilization 15min, face the Ox blood 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 Jiangsu; be feature with persistent fever, pleuritis, peritonitis, arthritis etc.; choose lung, hydrothorax, pericardial fluid, the joint fluid inoculation TSA/NAD solid medium of disease pig; put under 37 ℃, 5%CO2 condition and cultivated 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, carry out twice TSA/NAD solid medium sub-clone again and cultivate.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 petite of the translucent protuberance of canescence.Get single bacterium colony smear Gram, be Gram-negative, be pleomorphism, be the 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 staphylococcus 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, galactose, xylose and fructose azymic, 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 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 among the GenBank, primer sequence is: P1:5 '-ctccacaagctaacactttc-3 ', P2:5 '-catagaaacttcttttgaacc-3 ', give birth to worker's biological engineering company limited by Shanghai and synthesize, this primer amplification clip size is 1038bp.
2.3.2 XX0306 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 condition is: 95 ℃ of pre-degeneration 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, while, as positive control, the specific band of the 1038bp size identical with type strain appearred in the result, illustrates that the XX0306 strain that is separated to is haemophilus parasuis with type strain.
Transfer to Shanghai living worker's biological engineering company limited order-checking through reclaiming the pcr amplification product that is accredited as the positive.Haemophilus parasuis XX0306 strain OmpA gene sequencing the results are shown in shown in the SEQ ID No.1, 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 XX0306 strain serological Identification
Pressing Kleletein-Rapp-Gabriedson (KRG) agar diffusion serotype method identifies the serotype of haemophilus parasuis XX0306 strain.Haemophilus parasuis XX0306 strain is inoculated in the TSB culture medium, 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 typing antigen that supernatant is used for agar gel 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 heat treatment typing 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 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, do not have precipitation line with the serotype of other type type strain, illustrate that haemophilus parasuis XX0306 strain is serum 5 types.
2.5 animal challenge test
Serum 5 type haemophilus parasuis XX0306 strains are cultivated through the TSB/NAD fluid medium, carry out count plate, after concentrating, make infection titer reach 1 * 10
9CFU/ml is through 5 of the healthy susceptible pigs of lumbar injection 50～60 ages in days, 5ml/ head.Other establishes 1 group of matched group, and 5, lumbar injection TSB/NAD culture medium, 5ml/ head.Raise antibiotic-free in the feedstuff behind the counteracting toxic substances routinely.Observed 14, and surveyed body temperature every day, observe clinical symptoms.Counteracting toxic substances cutd open inspection after 14 days, according to clinical symptoms with cut open the inspection pathological changes and calculate the morbidity number.Simultaneously, the pathological material of disease of organizing of getting the morbidity pig is done antibacterial separation and evaluation.
The counteracting toxic substances result shows, observe 14 continuously after, 5 experiment pig of serum 5 type haemophilus parasuis XX0306 strain counteracting toxic substances groups are first sequela all; The 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 for sleeping inly.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, articular cavity mucus increase, and lungs are hemorrhage, 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 the antibacterial separation.Be separated to the bacterium colony antibacterial similar to the haemophilus parasuis type strain with thalli morphology in the sick pig of 4 hairs of result from XX0306 strain counteracting toxic substances group.Simultaneously all antibacterials 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 XX0306 strain
Not immunity or inapplicable of "-" expression.
2.6 immunogenic mensuration
Cultivate serum 5 type haemophilus parasuis XX0306 strains with the TSB/NAD fluid medium, behind the count plate, the viable bacteria culture is used final concentration 0.3% formalin 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 emulsifyings 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.Distinguish immune XX0306 strain vaccine 0.5ml, 1ml, 2ml for the 1st～3 group every group, the 4th group is the counteracting toxic substances matched 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, the pig of all immune group and counteracting toxic substances matched group forwards the counteracting toxic substances Animal House to.The 1st～3 group and the 4th group every group respectively the XX0306 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 culture medium, the 5ml/ head.Observed 14 behind the counteracting toxic substances, cut open inspection.According to clinical symptoms with cut open the inspection pathological changes and calculate morbidity number and counteracting toxic substances protective rate.Immunity counteracting toxic substances protection test result shows that 5 pigs of counteracting toxic substances matched group all fall ill (wherein dead 2), the sick clinical symptoms of typical haemophilus parasuis and significantly pathological change occur.The experiment pig of normal healthy controls group is not morbidity all.Have 2 clinical symptoms and pathological change to occur in 5 pigs of XX0306 strain 0.5mL immune group, have only 1 the inspection pathological changes to occur cuing open in 5 pigs of 1mL immune group, the counteracting toxic substances protective rate reaches 3/5 and 4/5 respectively; And the experiment pig of 2mL immune group all less than the morbidity, the counteracting toxic substances protective rate reaches 5/5.Immunity counteracting toxic substances protection situation sees table 2 for details.
The immunogenicity determining result of the test of table 2 haemophilus parasuis XX0306 strain
"-" expression is inapplicable.
By table 2 result as seen, the minimum immunoprotection dosage of haemophilus parasuis XX0306 strain is 1mL to contain viable count is 1 * 10
9CFU/mL.
Embodiment 2: serum 5 application of type haemophilus parasuis XX0306 strain in the haemophilus parasuis disease vaccine.
1 strain
The seedling strain is serum 5 type haemophilus parasuis XX0306 strains, 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 stronger virulence, and good immunogenicity is arranged.
2 production strain preparation
After 2.1 the one-level seeding is cultivated the secondary pig of 5 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 fluid 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 fluid medium, and 37 ℃ of vibration 180r/min cultivated 18～24 hours.Culture medium, 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 culture fluid is got in the preparation of seedling bacterium liquid, be inoculated in the culture tank that fills the TSB/NAD fluid medium in 1: 100 by volume, 37 ℃ of shaken cultivation 24 hours, results and sampling are checked and count plate 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% formalin, 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% formalin deactivation haemophilus parasuis XX0306 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% formalin 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 variable concentrations formalin-inactivated haemophilus parasuis XX0306 strain culture fluid
"+" 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 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 packing are got 2.5 parts of oil phases and are joined in the aseptic beaker, start homogenizer, 10000r/min, behind homogenizing 30～60s, slowly add 1 part of water, continue emulsifying, 12000～13000r/min, 5～8 minutes, the adding final concentration was 0.005% thimerosal solution before emulsifying 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 emulsifying is qualified.The vaccine qualified to emulsifying 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 XX0306 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 XX0306 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 force, obtain stabilization formulations.To quantitatively packing behind the qualified vaccine mix homogeneously of emulsifying, seal, label, put 2～8 ℃ and preserve down.
5 vaccine product inspections
5.1 outward appearance haemophilus parasuis oil adjuvant killed vaccine outward appearance is the milky 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 dosage form 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 dosage form stablizes.Haemophilus parasuis water adjuvant inactivated vaccine is the W/O/W 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 milky foreign country, be illustrated as the W/O/W 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 thimerosal residual quantity is tested by existing " Chinese veterinary drug allusion quotation " appendix, the thimerosal residual quantity is 0.006～0.007% in the vaccine, meets the regulation of veterinary biologics general rule.
5.7 content of formaldehyde is measured and tested by existing " Chinese veterinary drug allusion quotation " appendix, content of formaldehyde 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 general 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 the anus temperature every day, heavy dose of inoculation safety 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 fetus 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 harmful effect.Head exempts from the back 30 days sick bivalent inactivated vaccine immune group of haemophilus parasuis and observes obvious inflammatory reaction, and head exempts from back tissue slice on the 60th, 90 and all do not observe tangible inflammatory reaction.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 cervical region intramuscular injection vaccine (2mL), after 21 days with the Isodose booster immunization once.Set up the identical pig of condition to make each 5 of counteracting toxic substances matched groups, normal healthy controls group simultaneously, head exempts from back 42 days counteracting toxic substances, observes 14 behind the counteracting toxic substances, cuts open inspection, according to clinical symptoms with cut open the inspection pathological changes and calculate morbidity number and counteracting toxic substances protective rate.The result shows that two counteracting toxic substances matched group test pig are all fallen ill, and 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 (5)
1. the application of serum 5 type haemophilus parasuis vaccine strains in preparation treatment pig haemophilus parasuis medicine;
Wherein, described serum 5 type haemophilus parasuis vaccine strains, its called after haemophilus parasuis (Haemophilus parasuis) of classifying, bacterial strain number is XX0306, be preserved in Chinese typical culture collection center, deposit number: CCTCC NO:M2013095, preservation date: on March 21st, 2013.
2. application according to claim 1 is characterized in that, described medicine is vaccine.
3. application according to claim 2 is characterized in that, described vaccine is water-in-oil type vaccine or oil in water vaccine or W/O/W type vaccine.
4. application according to claim 1 is characterized in that, serum 5 type haemophilus parasuis vaccine strains and pharmaceutically acceptable carrier or adjuvant constitute medicine.
5. according to any described application in the claim 1 to 4, it is characterized in that described serum 5 type haemophilus parasuis vaccine strains bacteria containing amount in medicine is 3.5 * 10
9More than the CFU/mL.
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| 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 |
| 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 |
| CN115414350A (en) * | 2022-09-22 | 2022-12-02 | 湖北省农业科学院畜牧兽医研究所 | Application of mangiferin in preparation of medicament for inhibiting haemophilus parasuis |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN115414350A (en) * | 2022-09-22 | 2022-12-02 | 湖北省农业科学院畜牧兽医研究所 | Application of mangiferin in preparation of medicament for inhibiting haemophilus parasuis |
| 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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| CN103191421B (en) | 2014-08-06 |
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