CN103157100A - hemophilus parasuis disease, swine streptococcosis bivalent inactivated vaccine and preparation method thereof - Google Patents
hemophilus parasuis disease, swine streptococcosis bivalent inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a hemophilus parasuis disease, a swine streptococcosis bivalent inactivated vaccine and a preparation method thereof. The vaccine is prepared by adopting a hemophilus parasuis serotype 4 JS strain, a hemophilus parasuis serotype 5 ZJ strain and a streptococcus suis type 2 SC strain as antigens, and adopting a veterinarily acceptable adjuvant, concurrently provides immunoprophylaxis for hemophilus parasuis diseases caused by hemophilus parasuis, swine streptococcosis caused by streptococcus suis, and mixed infection of the hemophilus parasuis disease and the swine streptococcosis, has effects of low side effect, no endotoxin, no impurity protein in serum, good immunization safety, multi-prevention effect with one needle in the clinic, and cost reducing, and can meet different requirements of different users.
Description
Technical field
The present invention relates to the domestic animal biological pharmacy technical field, specifically, the present invention relates to a kind of Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis and preparation method thereof.
Background technology
Haemophilus parasuis and pig streptococcicosis are topmost two kinds of bacterial infectious diseases in present China swinery.Haemophilus parasuis is polyserositis and the arthritis of the pig that causes of haemophilus parasuis (Haemophilus parasuis, Hps), and this disease is called again the pig leather and draws Ze Shi sick (Glsser ' s Disease).Haemophilus parasuis can affect from the young pig at 2 age to 4 monthly ages in week, mainly falls ill with the child care stage after wean, usually sees the pig in 5～8 ages in week, and sickness rate can reach 40%, and mortality rate can reach 50%.Main clinic symptoms shows as cough, dyspnea, becomes thin, walks lamely and the thick unrest of quilt hair; Mainly cut open the inspection pathological changes and show as fibrinous pleurisy, pericarditis, peritonitis, arthritis and meningitis etc.In addition, haemophilus parasuis also can cause septicemia, and may stay sequela after actute infection, i.e. sow miscarriage, the chronic limping of boar.On the basis of a large amount of separation haemophilus parasuises, find that the serotype complexity of this bacterium is various, press Kieletein-Rapp-Gabriedson (KRG) agar diffusion serotype method, haemophilus parasuis can be divided into 15 serotypes at least; According to the evaluation of China's seroepidemiological survey and isolated strains, the most popular with 4,5 types.In recent years, carry out improperly due to the adjustment of the present breeding technology of China, and the new respiratory syndrome that happens suddenly makes this disease increasingly popular, endangers day by day serious.
Pig streptococcicosis is a kind of acute, hot infectious disease of infecting both domestic animals and human, show as acute hemorrhagic septicemia, endocarditis, meningitis, arthritis, suckling pig dysentery and the miscarriage of pregnant pig etc., under specific inducement effect, morbidity pig mortality rate can reach 80%, has a strong impact on the development of pig industry.Pig streptococcicosis is large tracts of land eruption and prevalence mistake once on China south China, southwest, East China, Jiangxi and Jiangsu and other places, and M ﹠ M is all higher, has caused serious economic loss, and main serotype is Lan Shi C group, is secondly Lan Shi D group, E group.In recent years, in the numerous serotypes of Streptococcus suis, streptococcus suis 2-type (R group) is the main cause of disease of pig, and is pathogenic the strongest, all Streptococcus suis that separate in characterizing healthy pig tonsil, 2 types reach 50.6%, and can infect the specific crowd morbidity, and can lethally die, harm is serious, wherein the Streptococcus suis of some areas, Jiangsu in 1998 outburst, cause 25 people to infect, and 14 people are dead; Part in Sichuan area outburst 2 type epidemic situations in 2005 cause 205 people to infect, and 37 people are dead.
adopting vaccine to carry out immunoprophylaxis is the fundamental means of controlling Haemophilus parasuis and pig streptococcicosis both at home and abroad, and the commercially available vaccine of market sale only has Haemophilus parasuis inactivated vaccine (serum 4 type MD0322+ Serotype 5 SH0165), Haemophilus parasuis inactivated vaccine (Z-1517 strain), Haemophilus parasuis inactivated vaccine (serum 1 type SV1+ serum 6 type SV6), Streptococcus suis C group live vaccine, Streptococcus suis bivalent inactivated vaccine (2 types+streptococcus equi epizootic disease subspecies), certain preventive effect has been played in the application of these vaccines, but along with having made existing vaccine, cause of disease epidemic strain and mixed infection is difficult to reach the immune effect of expection, and the commercial goods vaccine needs repeatedly immunity, cost is higher, to pig stress be larger, simultaneously for the vaccine of controlling Haemophilus parasuis and pig streptococcicosis, also still blank both at home and abroad in addition.Therefore, need a kind of Haemophilus parasuis of invention, inactivated combined vaccine against swine streptococcosis (JS strain+ZJ strain+SC strain) to address the above problem.
Patent CN101745105A discloses a kind of pig streptococcus illness, pasteurella multocida disease inactivated vaccine and preparation method thereof, this vaccine is that the Lan Shi C group horse with deactivation connects coccus epizootic disease subspecies C55138 strain culture fluid, concentrated solution is cultivated in streptococcus suis 2-type HA9801 strain, pasteurellosis bacillus capsular serotype A group bacterium is cultivated concentrated solution, pasteurellosis bacillus pod membrane B group bacterium is cultivated the concentrated solution mixed in equal amounts as antigen, make as adjuvant with gel aluminum hydroxide, its concentration process adopts the sedimentation method, removes the serum foreign protein that can not remove to greatest extent in the toxin process in endotoxin and culture medium.
Patent CN101721696A discloses the secondary haemophilus serum of a boar 4 types, 5 types, 13 type trivalent oil emulsion inactivated vaccines.It is to use secondary three the main popular serotypes of haemophilus of China pig and separated strain as antigen, after cultivating expanding propagation 24h with trypticase soybean broth culture medium (TSB), through 0.3% formalin-inactivated 11～13h, the antigen of three serotypes mixes with the ratio of 1: 1: 1, then add the oil phase that equates with the antigen cumulative volume, then be prepared from through emulsifying.This vaccine adjuvant is white oil, and this Adjuvanted vaccines side reaction is larger, can form in the injection site inflammation or swelling; In addition, data shows according to the study, uses the mineral oil adjuvant can excite to a certain extent and improve the sickness rate of the swine diseases such as pig annulus.And in this vaccine preparation process, the antigen that obtains does not have judgement and the related check of inactivating efficacy after deactivation, can't learn whether the antigen deactivation is complete, and the antigen that obtains can not be as antigen for vaccine.
Therefore, now be badly in need of a kind of side effect of invention little, do not contain endotoxin and do not contain foreign protein, pig streptococcicosis and mixed infection both, vaccine and technology of preparing thereof safely and effectively that the Haemophilus parasuis that simultaneously immunoprophylaxis is caused by haemophilus parasuis and Streptococcus suis cause.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis and preparation method for above-mentioned the deficiencies in the prior art, this vaccine is by haemophilus parasuis serum 4 type JS strain (Haemophilus Parasuis Serotype 4, the Strain JS of deactivation; Be preserved in Chinese Typical Representative culture collection center, referred to as CCTCC; CCTCC NO:M 2011172), 5 type ZJ strain (Haemophilus Parasuis Serotype 5, Strain ZJ preservation date: on May 18th, 2011, preserving number:; Be preserved in Chinese Typical Representative culture collection center, referred to as CCTCC; CCTCC NO:M 2011173) and Streptococcus suis serum 2 type SC strain (Streptococcus suis Serotype 2, Strain SC preservation date: on May 18th, 2011, preserving number:; Be preserved in Chinese Typical Representative culture collection center, referred to as CCTCC; Preservation date: on October 12nd, 2011, preserving number: CCTCC NO:M2011351) as antigen, and the acceptable adjuvant of veterinary is made, the pig streptococcicosis that the Haemophilus parasuis that simultaneously immunoprophylaxis is caused by haemophilus parasuis and Streptococcus suis cause and mixed infection both, this vaccine side effect is little, do not contain and do not contain foreign protein in endotoxin and serum, have immunological safety preferably.
For this reason, the invention provides a kind of Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis, comprise: antigen and adjuvant, wherein, the inactivation antigen that described antigen comprises the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain concentrates-resuspended diluent.Described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen be concentrated-and the total content of resuspended diluent is 50%～90% (V/V) of vaccine total amount, and described adjuvant content is 10%～50% (V/V) of vaccine total amount.Described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen be concentrated-and the volume ratio of resuspended diluent is 2: 2: 1～4: 4: 1.
Above-mentioned term " vaccine total amount " refers to the total amount of antigen and adjuvant.
In a preferred embodiment of the invention, before concentrated-resuspended diluent deactivation that described vaccine contains haemophilus parasuis serum 4 type JS strain inactivation antigens, viable count is no less than 1.0 * 10
9CFU/mL; Described haemophilus parasuis Serotype 5 ZJ strain inactivation antigen is concentrated-resuspended diluent deactivation before viable count be no less than 1.0 * 10
9CFU/mL; Described streptococcus suis 2-type SC strain inactivation antigen is concentrated-resuspended diluent deactivation before viable count be no less than 5 * 10
8CFU/mL.
In other embodiment of the present invention, antigen of the present invention can also adopt other pathogenic haemophilus parasuis serological type strains and other Streptococcus suis serological type strains; Described other pathogenic haemophilus parasuis serological type strains comprise one or both in haemophilus parasuis serum 2 types, serum 12 types, serum 13 types etc.; Other Streptococcus suis serological type strains are the Streptococcus suis serum 7-type.
According to the present invention, described adjuvant is the acceptable aqueous adjuvant of veterinary, comprise aluminum salt series adjuvant, Montanide IMS series adjuvant or Montanide GEL series adjuvant, propolis, immunostimulating complex, two-way adjuvant 206, oil-in-water series adjuvant, the cytokine class adjuvant, nucleic acid and derivatives class adjuvant thereof, lecithin class adjuvant; Wherein, in the vaccine of aluminum salt series adjuvant, three kinds of inactivation antigens are concentrated-and the total content of resuspended diluent is 75%～85% (V/V), and adjuvant content is 15%～25% (V/V); In the vaccine of Montanide IMS series adjuvant, three kinds of inactivation antigens are concentrated-and the total content of resuspended diluent is 50%～60% (V/V), and adjuvant content is 40%～50% (V/V); In the vaccine of Montanide IMS series adjuvant, three kinds of inactivation antigens are concentrated-and the total content of resuspended diluent is 75%～90% (V/V), and adjuvant content is 10%～25% (V/V).
In one embodiment of the invention, described aluminum salt series adjuvant comprises aluminium hydroxide gel, aluminum phosphate colloid etc.; Described Montanide IMS series adjuvant comprises 1312VG, 1313VG, 251C VG, 2215VG, 3012VG ST; Described Montanide GEL series adjuvant is GEL 01PR or Montanide PET GEL A; Described oil-in-water series adjuvant comprises MF59, Montanide ISA15A VG etc.; Described cytokine class adjuvant comprises interleukin (IL-1, IL-2, IL-4, IL-12), interferon (IFN-γ, IFN-α, IFN-β) etc.; Described nucleic acid and derivatives class adjuvant thereof comprise immunostimulatory sequence DNA (CpG DNA) or CpG oligodeoxynucleotide etc.
In a preferred embodiment of the invention, also contain antiseptic in described vaccine, comprise thimerosal, dimethylmercury or sodium azide, its content is 0～0.01% (V/V) of vaccine total amount.
The present invention also provides a kind of preparation method of vaccine of the present invention, comprising:
Steps A is bred respectively its production of preparation with the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain and is used strain, and this product is checked purely;
Step B is inoculated into respectively its seedling with the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain production with strain and makes antigen liquid with carrying out enrichment culture in culture medium, and this product is checked and count plate purely;
Step C, the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain antigen liquid make its inactivation antigen liquid through deactivation respectively, and this product is carried out deactivation check and safety examination;
Step D, the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen liquid are respectively through concentrated its inactivation antigen concentrated solution that makes;
Step e, the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen concentrated solution and adjuvant mixing and emulsifying are prepared Haemophilus parasuis, pig streptococcicosis bigeminy deactivation emulsion vaccine, and this product is carried out steriling test, safety verification and efficacy test;
Step F, Haemophilus parasuis, the packing of pig streptococcicosis bigeminy deactivation emulsion vaccine, storage;
It is characterized in that: step e comprises with the antigen concentrated solution resuspended dilution of diluent with the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain; Three kinds of antigens that obtain are concentrated-resuspended diluent mixing, then add the adjuvant mixing and emulsifying, and add antiseptic before emulsifying finishes, make Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis.
According to the present invention, diluent described in step e is normal saline; Described resuspended dilution comprises: it is 2.5 * 10 that the antigen concentrated solution of the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains is diluted to concentration
9CFU/mL～4.5 * 10
9CFU/mL, it is 5.0 * 10 that the antigen concentrated solution of streptococcus suis 2-type SC strain is diluted to concentration
9CFU/mL～5.5 * 10
9CFU/mL.
According in the inventive method, steps A comprises that first order seed breeding and secondary seed breeding are carried out in the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain to be made the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain and produce and use strain.
In a specific embodiment of the present invention, the first order seed breeding is carried out in the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain.With the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain freeze-drying lactobacillus, streak inoculation is on the TSA/NAD flat board respectively, under 37 ℃ of conditions, cultivate 18～24h, choose satisfactory bacterium colony, be inoculated in the TSB/NAD fluid medium, under 37 ℃ of conditions, cultivate 12～16h, as first order seed; Streptococcus suis 2-type SC strain freeze-drying lactobacillus, streak inoculation under 37 ℃ of conditions, are cultivated 18～24h as first order seed on Clumbia Sanguis caprae seu ovis flat board.
In another specific embodiment of the present invention, the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain first order seed are carried out the secondary seed breeding.With the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain first order seeds, the amount by 1% adds in the TSB/NAD fluid medium, cultivates 12～16h for 37 ℃, through purely after the assay was approved as secondary seed; Streptococcus suis 2-type SC selects good strains in the field for seed and gets satisfactory first order seed, be inoculated in the improvement martin's bouillon culture medium that contains the 15ml/L calf serum, under 37 ℃ of conditions, standing cultivation 12～16h, through the Sanguis caprae seu ovis flat board purely after the assay was approved as secondary seed; The secondary seed that obtains is used strain as producing.
According to the inventive method, seedling described in step B comprises with culture medium: haemophilus parasuis culture medium and cofactor thereof, Streptococcus suis culture medium and cofactor thereof.Wherein, described haemophilus parasuis culture medium is trypticase soybean broth culture medium (TSB), and its cofactor comprises calf serum, glucose.Described Streptococcus suis culture medium is improvement martin's bouillon culture medium, and its cofactor comprises calf serum, glucose.
In a specific embodiment of the present invention, it is commercially available haemophilus parasuis culture medium (TSB: tryptone 17.0g/L, soy peptone 3.0g/L, sodium chloride 5.0g/L, glucose 2.5g/L, dipotassium hydrogen phosphate 15.0g/L) that the seedling culture medium that adopts with strain is produced in the 4 type JS strains of inoculation haemophilus parasuis serum, 5 type ZJ strains; It is Streptococcus suis culture medium (improvement martin's bouillon: peptone 5.0g/L, magnesium sulfate 0.5g/L, glucose 20.0g/L, yeast powder 2.0g/L, dipotassium hydrogen phosphate 1.0g/L) that the seedling culture medium that adopts with strain is produced in inoculation streptococcus suis 2-type SC strain.
In an embodiment according to the inventive method, 0.2% (V/V) by total amount in haemophilus parasuis 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid and streptococcus suis 2-type SC strain antigen liquid that step C obtains to enrichment culture respectively adds respectively formalin, place 37 ℃ of deactivation 24h, during stir 3～5 times.
In another embodiment according to the inventive method, also can adopt beta-propiolactone (BPL), glycidaldehyde (GDA),
60Other ablation methods such as Co irradiation and heating.
According to the inventive method, step D adopts the cross-flow ultrafiltration system to concentrate, and removes simultaneously toxin and foreign protein, and the molecular cut off of ultrafilter membrane used is 500KD～1000KD.
In other a embodiment of the present invention, step D can also adopt other isolation technics to concentrate, and removes simultaneously toxin and foreign protein.Affiliated other technologies comprise: centrifuging, Hollow Fiber Ultrafiltration method, asbestos and active carbon adsorption, gel chromatography, aluminium glue adsorption precipitation method etc.
In an embodiment according to the inventive method, step F covers bottle stopper with appropriate volume packing Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis, and the jewelling lid.
According to the inventive method, in the preparation process of described vaccine, steps A comprises to be checked with strain purely to the production that obtains; Step B comprises three kinds of antigen liquids that obtain is checked and count plate purely; The inactivation antigen liquid that step C comprises obtaining carries out deactivation check and safety examination; Step e comprises carries out physical behavior check, steriling test, safety verification and efficacy test to the Haemophilus parasuis that obtains, inactivated combined vaccine against swine streptococcosis.With safety, the reliability of guaranteeing vaccine.
According to the present invention, described pure check is after cultivating end, sampling, and according to " People's Republic of China's veterinary drug allusion quotation (version in 2010) " appendix, sample is checked purely, it is pure that result should be.Described count plate is after cultivating end, sampling, and haemophilus parasuis carries out count plate with dull and stereotyped cultivation of TSA/NAD; Streptococcus suis carries out count plate with blood agar plate, with the bacterium number of determining to cultivate.Described steriling test is according to " People's Republic of China's veterinary drug allusion quotation " appendix, testing sample to be carried out steriling test, and result should be asepsis growth.
In a specific embodiment according to the inventive method, step C carries out the deactivation check to the inactivation antigen liquid that obtains, that the haemophilus parasuis antigen liquid 5mL that gets deactivation is inoculated in the haemophilus parasuis TSB culture medium that 100mL contains 0.01%～0.05%NAD, 5%～10% serum, 0.1%～5% glucose, cultivate 24h for 37 ℃, then transplant 37 ℃ of cultivation 24h in above-mentioned 100mL culture medium; Get the haemophilus parasuis antigen liquid 0.5mL streak inoculation of deactivation in the TSA/NAD plating medium, cultivate 24h for 37 ℃.After two kinds of methods are cultivated, all should asepsis growth.
The Streptococcus suis antigen liquid 5mL that gets deactivation is inoculated in 100mL and adds 1.5% calf serum, 0.1%～5% glucose improvement martin's bouillon culture medium, cultivates 24h for 37 ℃, then transplants 37 ℃ of cultivation 24h in above-mentioned 100mL culture medium; Get the Streptococcus suis bacterium liquid 0.5mL streak inoculation of deactivation in the SBA plating medium, cultivate 24h for 37 ℃.After two kinds of methods are cultivated, all should asepsis growth.
In another specific embodiment according to the inventive method, step C carries out safety examination to inactivation antigen liquid, is bacterium liquid 5 of the Balb/C mices of intravenous injection 18～22g respectively of getting deactivation, every 0.2mL, 5/5 strong living in three days, the side is thorough for deactivation.
In the another specific embodiment according to the inventive method, step e is carried out safety verification to Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis.
Safety to white mice: get the Balb/C mice that three batches of Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis are inoculated respectively 18～22g, every subcutaneous injection 0.4ml observed 14, without local response and whole strong living as qualified.
Safety to piglet: get 5 of three batches of Haemophilus parasuis, the healthy susceptible pigs of each intramuscular injection 30 ages in days of inactivated combined vaccine against swine streptococcosis, every 4mL was qualified without local response and whole strong living in 14 days.
According to the inventive method, Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis are carried out efficacy test.
The Balb/C mice of Balb/C mice potency test: 18～22g, subcutaneous vaccination 0.1mL Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis, be divided into 3 groups on 21st～28 after exempting from, together with every group each 10 of matched groups, (bacteria containing amount is 4.0 * 10 to use respectively haemophilus parasuis serum 4 type JS strain lumbar injection 1ml
9CFU), 5 type ZJ strains are carried out lumbar injection 1ml (bacteria containing amount are 2.5 * 10
9CFU); (bacteria containing amount is 1.0 * 10 with streptococcus suis 2-type SC strain subcutaneous injection 1ml
2CFU), other 10 are made as blank, observe 14.
Piglet potency test: get Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis, every group of vaccine each with 15 of the healthy susceptible pigs of 28～35 ages in days, every intramuscular injection 2mL, contain 1 using dosage, after 21～28 days, the immunity test pig of having injected every kind of vaccine is divided into three groups at random, uses respectively described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain counteracting toxic substances.15 of contrast pigs are divided three groups, use respectively described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain counteracting toxic substances.
The 4 type JS strains of haemophilus parasuis serum: choose each 5 of immune swines, together with 5 of the identical contrast pigs of condition, the serum 4 type JS strain bacterium liquid of 1 morbidity amount of lumbar injection were observed 14, and immune swine is protected more than 4/5, contrasted pig and fell ill more than 4/5.
Haemophilus parasuis Serotype 5 ZJ strain: choose 5 of immune swines, together with 5 of the identical contrast pigs of condition, the Serotype 5 ZJ strain bacterium liquid of 1 morbidity amount of lumbar injection was observed 14, and immune swine is protected more than 4/5, and the contrast pig falls ill more than 4/5.
Streptococcus suis 2-type SC strain: choose 5 of immune swines, together with 5 of the identical contrast pigs of condition, the streptococcus suis 2-type SC strain bacterium liquid of 1 lethal dose of intravenous injection was observed 14, and immune swine is protected more than 4/5, the death 4/5 or more of contrast pig.
The present invention is based on prior art not enough and provide a kind of and can prevent simultaneously pig streptococcicosis that the Haemophilus parasuis that caused by haemophilus parasuis and Streptococcus suis cause and Haemophilus parasuis, the inactivated combined vaccine against swine streptococcosis (JS strain+ZJ strain+SC strain) of mixed infection both.This vaccine side effect is little, does not contain or not foreign protein in endotoxin and serum, has immunological safety preferably, reaches clinically the effect that a pin is prevented more, reduces costs, and can satisfy the requirement of different user.
Culture presevation
Haemophilus parasuis serum 4 type JS strains (Haemophilus Parasuis Serotype 4, Strain JS) are separated, are identified by Pulaike Biological Engineering Co., Ltd., (are called for short: CCTCC at Chinese Typical Representative culture collection center; Address: No. 16, Wuchang District Lopa Nationality an ancient woman's ornament hill path, Wuhan City, Hubei Province, Wuhan University) carry out preservation, preservation date: on May 18th, 2011, preserving number: CCTCC NO:M 2011172.
Haemophilus parasuis Serotype 5 ZJ strain (Haemophilus Parasuis Serotype 5, Strain ZJ) is separated, is identified by Pulaike Biological Engineering Co., Ltd., (is called for short: CCTCC at Chinese Typical Representative culture collection center; Address: No. 16, Wuchang District Lopa Nationality an ancient woman's ornament hill path, Wuhan City, Hubei Province, Wuhan University) carry out preservation, preservation date: on May 18th, 2011, preserving number: CCTCC NO:M 2011173.
Streptococcus suis serum 2 type SC strains (Streptococcus suis Serotype 2, Strain SC) are separated, are identified by Pulaike Biological Engineering Co., Ltd., (are called for short: CCTCC at Chinese Typical Representative culture collection center; Address: No. 16, Wuchang District Lopa Nationality an ancient woman's ornament hill path, Wuhan City, Hubei Province, Wuhan University) carry out preservation, preservation date: on October 12nd, 2011, preserving number: CCTCC NO:M 2011351.
Be the strong strain of acquired immunity, the lyophilizing after responsive pig body rejuvenation accreditation of each strain is preserved and preservation.
The specific embodiment
Describe the present invention in detail below in conjunction with embodiment, these embodiment only play the illustrative effect, are not limited to range of application of the present invention.
Concrete experimental technique of the present invention can be referring to " People's Republic of China's veterinary drug allusion quotation (in 2010 version) " and appendix.Other not marked reagent or raw material all can be obtained according to prior art by those skilled in the art.
Embodiment
Embodiment 1: preparation Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis (JS strain+ZJ strain+SC strain)
1. preparation is produced and is used strain
First order seed breeding and secondary seed breeding are carried out in the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain to be made the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain and produces and use strain.
(1) first order seed breeding
The first order seed breeding is carried out in the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain.With the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain freeze-drying lactobacillus, streak inoculation is on the TSA/NAD flat board, under 37 ℃ of conditions respectively, cultivate 18h, choose satisfactory bacterium colony, be inoculated in the TSB/NAD fluid medium, under 37 ℃ of conditions, cultivate 12h, as first order seed; Streptococcus suis 2-type SC strain freeze-drying lactobacillus, streak inoculation under 37 ℃ of conditions, are cultivated 18h as first order seed on Clumbia Sanguis caprae seu ovis flat board.
(2) secondary seed breeding
The 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain first order seed are carried out the secondary seed breeding.With the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain first order seeds, the amount by 1% adds in the TSB/NAD fluid medium, cultivates 12h for 37 ℃, through purely after the assay was approved as secondary seed; Streptococcus suis 2-type SC selects good strains in the field for seed and gets satisfactory first order seed, be inoculated in the improvement martin's bouillon culture medium that contains the 15ml/L calf serum, under 37 ℃ of conditions, standing cultivation 12h, purely after the assay was approved as secondary seed, the secondary seed that obtains is used strain as producing through the Sanguis caprae seu ovis flat board.
2. preparation antigen liquid
(1) preparation antigen liquid
In haemophilus parasuis culture medium (commercially available TSB: tryptone 17.0g/L, soy peptone 3.0g/L, sodium chloride 5.0g/L, glucose 2.5g/L, dipotassium hydrogen phosphate 15.0g/L), add 0.05%NAD, 10% serum, 5% glucose to be mixed with the seedling culture medium, the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains are produced and added in the seedling culture medium by 1% amount respectively with strain, after mix homogeneously, cultivate respectively separately.Condition of culture is: temperature is 37 ℃, and speed of agitator is 180rmp, and DO is 80～0, and pH value is 7.2～6.5, and incubation time is 18h.Concentration OD when culture fluid
600Reach more than 2.5, the DO value begins again after being down to 0 to rise, pH value is reduced to 6.5 and stops cultivation when following, gathers in the crops respectively antigen liquid.
In Streptococcus suis culture medium (commercially available improvement martin's bouillon: peptone 5.0g/L, magnesium sulfate 0.5g/L, glucose 20.0g/L, yeast powder 2.0g/L, dipotassium hydrogen phosphate 1.0g/L), add 1.5% calf serum, 5% glucose to be mixed with the seedling culture medium, streptococcus suis 2-type SC strain is produced add in the seedling culture medium by 5% amount with strain, cultivate after mix homogeneously.Condition of culture is: temperature is 37 ℃, and speed of agitator is 200rmp, and DO is 80～0, and pH value 7.4～6.5, incubation time are 18h.Concentration OD when culture fluid
600Reach more than 2.5, the DO value begins again after being down to 0 to rise, pH value is reduced to 6.0 and stops cultivation when following, gathers in the crops respectively antigen liquid.
(2) pure check
After cultivating end, take a sample from three kinds of antigen liquids that obtain respectively, purely check according to " People's Republic of China's veterinary drug allusion quotation (version in 2010) " appendix, should be pure.
(3) count plate
Take a sample from three kinds of antigen liquids that obtain respectively, haemophilus parasuis carries out count plate with dull and stereotyped cultivation of TSA/NAD; Streptococcus suis carries out count plate with blood agar plate, with the bacterium number of determining to cultivate.
3. antigen liquid deactivation
(1) antigen liquid deactivation
The antigen liquid that is up to the standards carries out respectively deactivation.In above-mentioned three kinds of cultured antigen liquids, add formalin by 0.2% (V/V) of total amount, and under 37 ℃ of conditions deactivation 24h, during stir 5 times.
(2) deactivation check
2 kinds of haemophilus parasuis antigen liquid 5mL that get respectively deactivation are inoculated in the haemophilus parasuis TSB culture medium that 100mL contains 0.05%NAD, 10% calf serum, 5% glucose, cultivate 24h under 37 ℃ of conditions, transplant again in above-mentioned 100mL culture medium, and cultivate 24h under 37 ℃ of conditions; Get 2 kinds of haemophilus parasuis bacterium liquid 0.5mL streak inoculation of deactivation in the TSA/NAD plating medium, cultivate 24h under 37 ℃ of conditions.After two kinds of methods are cultivated, all should asepsis growth.
The Streptococcus suis antigen liquid 5mL that gets deactivation is inoculated in 100mL, contain in the improvement martin's bouillon culture medium of 1.5% calf serum, 5% glucose, cultivate 24h under 37 ℃ of conditions, then transplant in above-mentioned 100mL culture medium, cultivate 24h under 37 ℃ of conditions; Get the Streptococcus suis antigen liquid 0.5mL streak inoculation of deactivation in the SBA plating medium, cultivate 24h under 37 ℃ of conditions.After two kinds of methods are cultivated, all should asepsis growth.
(3) safety examination
Get above-mentioned three kinds of inactivation antigen liquid 5 of Balb/C mices of intravenous injection 18～22g respectively, every 0.2mL, 5/5 strong living in three days, the side is thorough for deactivation.
4. ultrafiltration and concentration
Above-mentioned three kinds of inactivation antigen liquid are concentrated with the Pellicon cross-flow ultrafiltration system of U.S. Millipore company, and supernatant discarded makes its inactivation antigen concentrated solution.
5. prepare Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis (JS strain+ZJ strain+SC strain)
(1) preparation Haemophilus parasuis, pig streptococcicosis bigeminy aluminium glue adjuvant inactivated vaccine
With the above-mentioned three kinds of inactivation antigen concentrated solutions of the resuspended dilution of normal saline, with reference to the colony counting result, it is 2.85 * 10 that the antigen concentrated solution of the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains is diluted to concentration
9CFU/mL, it is 5.0 * 10 that the antigen concentrated solution of streptococcus suis 2-type SC strain is diluted to concentration
9CFU/mL.With three kinds of antigens concentrated-resuspended diluent mixes by the volume ratio of 3.5: 3.5: 1.The three kinds of antigens that measure according to 75%～85% (V/V) concentrate-resuspended diluent mixture, add the adjuvant mixing and emulsifying according to 15%～25% (V/V) amount again, and add the thimerosal of 0.01% (V/V) of vaccine total amount before emulsifying finishes, make Haemophilus parasuis, pig streptococcicosis bigeminy aluminium glue adjuvant inactivated vaccine.
(2) preparation Haemophilus parasuis, pig streptococcicosis bigeminy IMS1313 adjuvant inactivated vaccine
With the above-mentioned three kinds of inactivation antigen concentrated solutions of the resuspended dilution of normal saline, with reference to the colony counting result, it is 5.0 * 10 that the antigen concentrated solution of the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains, streptococcus suis 2-type SC strain all is diluted to concentration
9CFU/mL.With three kinds of antigens concentrated-resuspended diluent mixes by the volume ratio of 2: 2: 1.The three kinds of antigens that measure according to 50%～60% (V/V) concentrate-resuspended diluent mixture, add the adjuvant mixing and emulsifying according to 40%～50% (V/V) amount again, and add the dimethylmercury of 0.01% (V/V) of vaccine total amount before emulsifying finishes, make Haemophilus parasuis, pig streptococcicosis bigeminy IMS1313 adjuvant inactivated vaccine.
(3) preparation Haemophilus parasuis, pig streptococcicosis bigeminy GEL adjuvant inactivated vaccine
With the above-mentioned three kinds of inactivation antigen concentrated solutions of the resuspended dilution of normal saline, with reference to the colony counting result, it is 2.5 * 10 that the antigen concentrated solution of the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains all is diluted to concentration
9CFU/mL, it is 5.0 * 10 that the antigen concentrated solution of streptococcus suis 2-type SC strain is diluted to concentration
9CFU/mL.With three kinds of antigens concentrated-resuspended diluent mixes by the volume ratio of 4: 4: 1.The three kinds of antigens that measure according to 75%～90% (V/V) concentrate-resuspended diluent mixture, then add the adjuvant mixing and emulsifying according to 10%～25% (V/V) amount, make Haemophilus parasuis, pig streptococcicosis bigeminy GEL adjuvant inactivated vaccine.
6. the steriling test of vaccine
Get above-mentioned vaccine, carry out steriling test according to " People's Republic of China's veterinary drug allusion quotation " appendix, as a result asepsis growth (seeing Table 1).
Table 1 Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis steriling test
Annotate: T.G represents sulphur glycollate culture medium, and G.A represents the peptone from casein agar culture medium, the G.P dextrose peptone medium; "-" expression asepsis growth.
7. packing, storage
With 20mL/ bottle, 50mL/ bottle, the packing of 100mL/ bottle, cover bottle stopper, and the jewelling lid, in 2～8 ℃ of storages.
Embodiment 2: safety research
(1) safety testing of vaccine to the Balb/C mice
The vaccine of getting embodiment 1 preparation is inoculated respectively each 10 of the Balb/C mices of 18～22g, and every subcutaneous injection 0.4ml observed 14, all without local response and all strong alive, saw Table 2.
Table 2 vaccine is to Balb/C mice safety testing result
(2) safety testing of vaccine to piglet
Get 5 of the healthy susceptible pigs of each intramuscular injection 30 ages in days of the prepared vaccine of embodiment 1, every 4mL lived without local response and whole being good in 14 days.Assay sees Table 3.
The safety testing result of table 3 vaccine to piglet
Can find out from the safety testing result, Haemophilus parasuis, pig streptococcicosis bigeminy aluminium hydroxide gel adjuvant inactivated vaccine (JS strain+ZJ strain+SC strain), Haemophilus parasuis, pig streptococcicosis bigeminy Montanide IMS1313VG adjuvant inactivated vaccine (JS strain+ZJ strain+SC strain), Haemophilus parasuis, pig streptococcicosis bigeminy Montanide GEL PR adjuvant inactivated vaccine (JS strain+ZJ strain+SC strain) inactivated vaccine are to Balb/C mice, piglet safety.
Embodiment 3: potency test (testing in our company's experimental animal room)
(1) Balb/C mice potency test
The Balb/C mice of 18～22g, subcutaneous vaccination 0.1mL dyad inactivated vaccine was divided into 3 groups on 21st～28 after exempting from, and together with every group each 10 of matched groups, (bacteria containing amount is 4.0 * 10 to use respectively haemophilus parasuis serum 4 type JS strain lumbar injection 1ml
9CFU), 5 type ZJ strains are carried out lumbar injection 1ml (bacteria containing amount are 2.5 * 10
9CFU); (bacteria containing amount is 1.0 * 10 with streptococcus suis 2-type SC strain subcutaneous injection 1ml
2CFU), other 10 are made as blank, observe 14, and assay sees Table 4.
Table 4Balb/C mice potency test result
(2) piglet potency test
Get prepared vaccine, every group of vaccine each with 15 of the healthy susceptible pigs of 28～35 ages in days, every intramuscular injection 2mL, contain 1 using dosage, after 21～28 days, the immunity test pig of having injected every kind of vaccine is divided into three groups at random, uses respectively described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain counteracting toxic substances.15 of contrast pigs are divided three groups, use respectively described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain counteracting toxic substances.
Haemophilus parasuis serum 4 type JS strains: choose each 5 of immune swines, together with 5 of the identical contrast pigs of condition, 1 morbidity amount (2.0 * 10 of lumbar injection
10CFU) serum 4 type JS strain bacterium liquid were observed 14 days, and immune swine is protected more than 4/5, and the contrast pig falls ill more than 4/5.
Haemophilus parasuis Serotype 5 ZJ strain: choose 5 of immune swines, together with 5 of the identical contrast pigs of condition, 1 morbidity amount (1.0 * 10 of lumbar injection
10CFU) Serotype 5 ZJ strain bacterium liquid was observed 14 days, and immune swine is protected more than 4/5, and the contrast pig falls ill more than 4/5.
Streptococcus suis 2-type SC strain: choose 5 of immune swines, together with 5 of the identical contrast pigs of condition, 1 lethal dose (5.0 * 10 of intravenous injection
8CFU) streptococcus suis 2-type SC strain bacterium liquid was observed 14 days, and immune swine is protected more than 4/5, and the contrast pig is dead more than 4/5.Assay sees Table 5.
Table 5 vaccine potency assay
Annotate: Haemophilus parasuis morbidity standard: heating (body temperature continues 1～5 more than 40.5 ℃), lethargy appear in the morbidity pig, cough, dyspnea, become thin, walk lamely and by the thick clinical symptoms such as disorderly of hair.Dying pig is cutd open inspection, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, serosity or fibrinous exudate appear in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
can find out from above-described embodiment, Haemophilus parasuis, pig streptococcicosis bigeminy aluminium hydroxide gel adjuvant inactivated vaccine (JS strain+ZJ strain+SC strain), Haemophilus parasuis, pig streptococcicosis bigeminy Montanide IMS1313VG adjuvant inactivated vaccine (JS strain+ZJ strain+SC strain), Haemophilus parasuis, pig streptococcicosis bigeminy Montanide GEL PR adjuvant inactivated vaccine (JS strain+ZJ strain+SC strain) inactivated vaccine can reach more than 90% to the protective rate of Balb/C mice, the piglet protective rate is reached more than 80%, all to haemophilus parasuis, streptococcus suis infection has good protection, comparatively speaking, the Haemophilus parasuis of Montanide GEL PR adjuvant preparation, the inactivated combined vaccine against swine streptococcosis immune effect is best.
Claims (10)
1. a Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis comprise: antigen and adjuvant, wherein, described antigen comprises the inactivation antigen of the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain concentrated-resuspended diluent.
2. vaccine according to claim 1, it is characterized in that: described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen be concentrated-and the total content of resuspended diluent is 50%～90% (V/V) of vaccine total amount, and described adjuvant content is 10%～50% (V/V) of vaccine total amount.
3. vaccine according to claim 2 is characterized in that: and described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen be concentrated-and the volume ratio of resuspended diluent is 2: 2: 1～4: 4: 1.
4. vaccine according to claim 3 is characterized in that: before concentrated-resuspended diluent deactivation that described vaccine contains haemophilus parasuis serum 4 type JS strain inactivation antigens, viable count is no less than 1.0 * 10
9CFU/mL; Described haemophilus parasuis Serotype 5 ZJ strain inactivation antigen is concentrated-resuspended diluent deactivation before viable count be no less than 1.0 * 10
9CFU/mL; Described streptococcus suis 2-type SC strain inactivation antigen is concentrated-resuspended diluent deactivation before viable count be no less than 5 * 10
8CFU/mL.
5. vaccine according to claim 1, it is characterized in that: described adjuvant is the acceptable aqueous adjuvant of veterinary, comprise aluminum salt series adjuvant, Montanide IMS series adjuvant or Montanide GEL series adjuvant, propolis, immunostimulating complex, two-way adjuvant 206, oil-in-water series adjuvant, the cytokine class adjuvant, nucleic acid and derivatives class adjuvant thereof, lecithin class adjuvant; Wherein, in the vaccine of aluminum salt series adjuvant, three kinds of inactivation antigens are concentrated-and the total content of resuspended diluent is 75%～85% (V/V), and adjuvant content is 15%～25% (V/V); In the vaccine of Montanide IMS series adjuvant, three kinds of inactivation antigens are concentrated-and the total content of resuspended diluent is 50%～60% (V/V), and adjuvant content is 40%～50% (V/V); In the vaccine of MontanideIMS series adjuvant, three kinds of inactivation antigens are concentrated-and the total content of resuspended diluent is 75%～90% (V/V), and adjuvant content is 10%～25% (V/V).
6. vaccine according to claim 1, it is characterized in that: also contain antiseptic in described vaccine, comprise thimerosal or dimethylmercury or sodium azide, its content is 0～0.01% (V/V) of vaccine total amount.
7. the preparation method of the described vaccine of any one in a claim 1～6 comprises:
Steps A is bred respectively its production of preparation with the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain and is used strain, and this product is checked purely;
Step B is inoculated into respectively its seedling with the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain production with strain and makes antigen liquid with carrying out enrichment culture in culture medium, and this product is checked and count plate purely;
Step C, the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain antigen liquid make its inactivation antigen liquid through deactivation respectively, and this product is carried out deactivation check and safety examination;
Step D, the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen liquid are respectively through concentrated its inactivation antigen concentrated solution that makes;
Step e, the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen concentrated solution and adjuvant mixing and emulsifying are prepared Haemophilus parasuis, pig streptococcicosis bigeminy deactivation emulsion vaccine, and this product is carried out steriling test, safety verification and efficacy test;
Step F, Haemophilus parasuis, the packing of pig streptococcicosis bigeminy deactivation emulsion vaccine, storage;
It is characterized in that: step e comprises with the antigen concentrated solution resuspended dilution of diluent with the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain; Three kinds of antigens that obtain are concentrated-resuspended diluent mixing, then add the adjuvant mixing and emulsifying, and add antiseptic before emulsifying finishes, make Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis.
8. method according to claim 7, it is characterized in that: diluent described in step e is normal saline; Described resuspended dilution comprises: it is 2.5 * 10 that the antigen concentrated solution of the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains is diluted to concentration
9CFU/mL～4.5 * 10
9CFU/mL, it is 5.0 * 10 that the antigen concentrated solution of streptococcus suis 2-type SC strain is diluted to concentration
9CFU/mL～5.5 * 10
9CFU/mL.
9. method according to claim 7, it is characterized in that: seedling described in step B comprises with culture medium: haemophilus parasuis culture medium and cofactor thereof, Streptococcus suis culture medium and cofactor thereof; Wherein, described haemophilus parasuis culture medium is trypticase soybean broth culture medium, and its cofactor comprises calf serum, glucose; Described Streptococcus suis culture medium is improvement martin's bouillon culture medium, and its cofactor comprises calf serum, glucose.
10. method according to claim 7 is characterized in that: step D adopts the cross-flow ultrafiltration system to concentrate, and removes simultaneously toxin and foreign protein, and the molecular cut off of ultrafilter membrane used is 500KD～1000KD.
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