CN103157100B - 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 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 current 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 pig leather and draws Ze Shi sick (Glsser ' s Disease).Haemophilus parasuis can affect the young pig from 2 week age to 4 monthly ages, mainly after wean, falls ill with the child care stage, conventionally sees the pig in 5～8 week age, 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 inspection pathological changes and show as fibrinous pleurisy, pericarditis, peritonitis, arthritis and meningitis etc.In addition, haemophilus parasuis also can cause septicemia, and may leave sequela after actute infection, i.e. sow miscarriage, the chronic limping of boar.On the basis of a large amount of separated haemophilus parasuises, find that the serotype complexity of this bacterium is various, by Kieletein-Rapp-Gabriedson (KRG) agar diffusion serotype method, at least haemophilus parasuis can be divided into 15 serotypes; According to the evaluation of China's seroepidemiological survey and isolated strains, the most popular with 4,5 types.In recent years, due to the adjustment of the current breeding technology of China, carry out improperly, 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 area 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 serotype of Streptococcus suis, streptococcus suis 2-type (R group) is the main cause of disease of pig, pathogenic the strongest, in characterizing healthy pig tonsil separated all Streptococcus suis, 2 types reach 50.6%, and can infect specific crowd morbidity, and can lethally die, harm is serious, wherein the Streptococcus suis of some areas, Jiangsu in 1998 outburst, causes 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 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 to invent a kind of Haemophilus parasuis, 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 concentrated solution mixed in equal amounts as antigen, the gel aluminum hydroxide of usining is made as adjuvant, its concentration process adopts the sedimentation method, removes in toxin process and can not remove to greatest extent the serum foreign protein 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 that secondary three the main popular serotypes of haemophilus of application China pig and separated strain are as antigen, by cultivating after expanding propagation 24h by 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 equating with 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 injection site inflammation or swelling; In addition, data shows according to the study, uses mineral oil adjuvant can excite to a certain extent and improve the sickness rate of the swine diseasess such as pig annulus.And in this vaccine preparation process, the antigen obtaining does not have judgement and the related check of inactivating efficacy after deactivation, cannot learn that whether antigen deactivation is complete, the antigen obtaining can not be as antigen for vaccine.
Therefore, now be badly in need of a kind of side effect of invention little, not containing endotoxin with not containing foreign protein, pig streptococcicosis and the mixed infection of the two, safely and effectively vaccine and technology of preparing thereof that the Haemophilus parasuis that immunoprophylaxis is simultaneously 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 immunoprophylaxis is simultaneously caused by haemophilus parasuis and Streptococcus suis cause and the mixed infection of the two, this vaccine side effect is little,, containing or not foreign protein in endotoxin and serum, do not there is good immunological safety.
For this reason, the invention provides a kind of Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis, comprise: antigen and adjuvant, wherein, concentrate-resuspended diluent of 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.The total content of concentrate-resuspended diluent of described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen is 50%～90% (V/V) of vaccine total amount, and described adjuvant content is 10%～50% (V/V) of vaccine total amount.The volume ratio of concentrate-resuspended diluent of described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen 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, described vaccine is no less than 1.0 * 10 containing the front viable count of concentrate-resuspended diluent deactivation of haemophilus parasuis serum 4 type JS strain inactivation antigens
9cFU/mL; Before the concentrate-resuspended diluent deactivation of described haemophilus parasuis Serotype 5 ZJ strain inactivation antigen, viable count is no less than 1.0 * 10
9cFU/mL; Before the concentrate-resuspended diluent deactivation of described streptococcus suis 2-type SC strain inactivation antigen, viable count is 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 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, cytokine class adjuvant, nucleic acid and derivatives class adjuvant thereof, lecithin class adjuvant; Wherein, in the vaccine of aluminum salt series adjuvant, the total content of three kinds of concentrate-resuspended diluents of inactivation antigen is 75%～85% (V/V), and adjuvant content is 15%～25% (V/V); In the vaccine of Montanide IMS series adjuvant, the total content of three kinds of concentrate-resuspended diluents of inactivation antigen is 50%～60% (V/V), and adjuvant content is 40%～50% (V/V); In the vaccine of Montanide IMS series adjuvant, the total content of three kinds of concentrate-resuspended diluents of inactivation antigen 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, breeds respectively its production strain of preparation by the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain, and this product is checked purely;
Step B, is inoculated into respectively its seedling by 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 to 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 to Haemophilus parasuis, pig streptococcicosis bigeminy deactivation emulsion vaccine, and this product is carried out to steriling test, safety verification and efficacy test;
Step F, Haemophilus parasuis, pig streptococcicosis bigeminy deactivation emulsion vaccine subpackage, storage;
It is characterized in that: step e comprises with diluent the resuspended dilution of antigen concentrated solution of the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain; Concentrated-resuspended diluent of three kinds of obtained antigens is mixed, then add adjuvant mixing and emulsifying, and before emulsifying finishes, add antiseptic, 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 carries out first order seed breeding and secondary seed breeding makes the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain production strain to the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain.
In a 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 are carried out to first order seed breeding.By the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain freeze-drying lactobacillus, streak inoculation is on TSA/NAD flat board respectively, under 37 ℃ of conditions, cultivate 18～24h, choose satisfactory bacterium colony, be inoculated in 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, on Clumbia Sanguis caprae seu ovis flat board, under 37 ℃ of conditions, is cultivated 18～24h as first order seed.
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 to secondary seed breeding.By the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain first order seeds, the amount by 1% adds in 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 15ml/L calf serum, under 37 ℃ of conditions, standing cultivation 12～16h, through Sanguis caprae seu ovis flat board purely after the assay was approved as secondary seed; Obtained secondary seed is used as to production strain.
According to the inventive method, seedling described in step B comprises by 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 adopting 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 adopting with strain is produced in inoculation streptococcus suis 2-type SC strain.
According in the inventive method embodiment, 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, by 0.2% (V/V) of total amount, add respectively formalin, place 37 ℃ of deactivation 24h, during stir 3～5 times.
According in another embodiment of 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 cross-flow ultrafiltration system to concentrate, and removes toxin and foreign protein simultaneously, 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 toxin and foreign protein simultaneously.Affiliated other technologies comprise: centrifuging, Hollow Fiber Ultrafiltration method, asbestos and active carbon adsorption, gel chromatography, aluminium glue adsorption precipitation method etc.
According in the inventive method embodiment, step F, with appropriate volume subpackage Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis, covers bottle stopper, and jewelling lid.
According to the inventive method, in the preparation process of described vaccine, steps A comprises to be checked with strain purely to obtained production; Step B comprises three kinds of obtained antigen liquids is checked and count plate purely; Step C comprises obtained inactivation antigen liquid is carried out to deactivation check and safety examination; Step e comprises carries out physical behavior check, steriling test, safety verification and efficacy test to obtained Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis.To guarantee safety, the reliability of vaccine.
According to the present invention, described pure check is after cultivation finishes, sampling, and according to < < People's Republic of China (PRC) veterinary drug allusion quotation (version in 2010) > > appendix, sample is checked purely, it is pure that result should be.Described count plate is after cultivation finishes, 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, to determine the bacterium number of cultivation.Described steriling test is, according to < < People's Republic of China (PRC) veterinary drug allusion quotation > > appendix, testing sample is carried out to steriling test, and result should be asepsis growth.
According in the inventive method specific embodiment, step C carries out deactivation check to obtained inactivation antigen liquid, that the haemophilus parasuis antigen liquid 5mL that gets deactivation is inoculated in the haemophilus parasuis TSB culture medium of 100mL containing 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 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 SBA plating medium, cultivate 24h for 37 ℃.After two kinds of methods are cultivated, all should asepsis growth.
According in another specific embodiment of 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, side is that deactivation is thorough.
According in the another specific embodiment of 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, observes 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 to 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, after exempting from, within 21st～28, be divided into 3 groups, 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, containing 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, divide 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, the serum 4 type JS strain bacterium liquid of 1 morbidity amount of lumbar injection, observe 14, and immune swine more than 4/5 is protected, contrast pig and more than 4/5 fall ill.
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, observes 14, and immune swine more than 4/5 is protected, contrast pig more than 4/5 falls ill.
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, observes 14, and immune swine more than 4/5 is protected, more than 4/5 death of contrast pig.
The present invention is based on prior art not enough and provide a kind of and can prevent 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 the mixed infection of the two simultaneously.This vaccine side effect is little,, containing or not foreign protein in endotoxin and serum, does not have good immunological safety, reaches clinically the how anti-effect of a pin, reduces costs, and can meet the requirement of different user.
culture presevation
Haemophilus parasuis serum 4 type JS strains (Haemophilus Parasuis Serotype 4, Strain JS),, evaluation separated by Pulaike Biological Engineering Co., Ltd., (be called for short: CCTCC at Chinese Typical Representative culture collection center; Address: No. 16, Luo Jia Shan road, Wuchang District, 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),, evaluation separated by Pulaike Biological Engineering Co., Ltd., (be called for short: CCTCC at Chinese Typical Representative culture collection center; Address: No. 16, Luo Jia Shan road, Wuchang District, 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),, evaluation separated by Pulaike Biological Engineering Co., Ltd., (be called for short: CCTCC at Chinese Typical Representative culture collection center; Address: No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province, Wuhan University) carry out preservation, preservation date: on October 12nd, 2011, preserving number: CCTCC NO:M 2011351.
For the strong strain of acquired immunity, the lyophilizing after responsive pig body rejuvenation accreditation of each strain is preserved and preservation.
The specific embodiment
Below in conjunction with embodiment, describe the present invention in detail, these embodiment only play illustrative effect, are not limited to range of application of the present invention.
Specific experiment method of the present invention can be referring to < < People's Republic of China (PRC) 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: prepare Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis (JS strain+ZJ strain+SC strain)
1. prepare production strain
The 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain are carried out to the breeding of first order seed breeding and secondary seed and make the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain production strain.
(1) first order seed breeding
The 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain are carried out to first order seed breeding.By the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain freeze-drying lactobacillus, streak inoculation is on TSA/NAD flat board, under 37 ℃ of conditions respectively, cultivate 18h, choose satisfactory bacterium colony, be inoculated in 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, on Clumbia Sanguis caprae seu ovis flat board, under 37 ℃ of conditions, is cultivated 18h as first order seed.
(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 to secondary seed breeding.By the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain first order seeds, the amount by 1% adds in 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 15ml/L calf serum, under 37 ℃ of conditions, standing cultivation 12h, through Sanguis caprae seu ovis flat board, purely after the assay was approved as secondary seed, obtained secondary seed is used as to production strain.
2. prepare antigen liquid
(1) prepare 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 seedling culture medium, the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains are produced and by 1% amount, added in seedling culture medium 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, DO value starts 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 seedling culture medium, streptococcus suis 2-type SC strain is produced and added in seedling culture medium by 5% amount with strain, after mix homogeneously, cultivate.Condition of culture is: temperature is 37 ℃, and speed of agitator is 200rmp, and DO is 80～0, pH value 7.4～6.5, and incubation time is 18h.Concentration OD when culture fluid
600reach more than 2.5, DO value starts 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) check purely
After cultivation finishes, from three kinds of obtained antigen liquids, sample respectively, according to < < People's Republic of China (PRC) veterinary drug allusion quotation (version in 2010) > > appendix, purely check, should be pure.
(3) count plate
From three kinds of obtained antigen liquids, sample 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, to determine the bacterium number of cultivation.
3. antigen liquid deactivation
(1) antigen liquid deactivation
The antigen liquid being up to the standards carries out respectively deactivation.In above-mentioned three kinds of cultured antigen liquids, by 0.2% (V/V) of total amount, add formalin, 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, under 37 ℃ of conditions, cultivate 24h, 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 TSA/NAD plating medium, under 37 ℃ of conditions, cultivate 24h.After two kinds of methods are cultivated, all should asepsis growth.
The Streptococcus suis antigen liquid 5mL that gets deactivation is inoculated in 100mL, in the improvement martin's bouillon culture medium that contains 1.5% calf serum, 5% glucose, under 37 ℃ of conditions, cultivate 24h, then transplant in above-mentioned 100mL culture medium, under 37 ℃ of conditions, cultivate 24h; Get the Streptococcus suis antigen liquid 0.5mL streak inoculation of deactivation in SBA plating medium, under 37 ℃ of conditions, cultivate 24h.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, side for deactivation thorough.
4. ultrafiltration and concentration
The Pellicon cross-flow ultrafiltration system of above-mentioned three kinds of inactivation antigen Ye Yong U.S. Millipore companies is concentrated, 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 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.Concentrate-resuspended diluent of three kinds of antigens is mixed by the volume ratio of 3.5: 3.5: 1.According to 75%～85% (V/V) measure three kinds of antigens concentrated-resuspended diluent mixture, according to 15%～25% (V/V) amount, add adjuvant mixing and emulsifying again, and before emulsifying finishes, add the thimerosal of 0.01% (V/V) of vaccine total amount, 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 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 is all diluted to concentration
9cFU/mL.Concentrate-resuspended diluent of three kinds of antigens is mixed by the volume ratio of 2: 2: 1.According to 50%～60% (V/V) measure three kinds of antigens concentrated-resuspended diluent mixture, according to 40%～50% (V/V) amount, add adjuvant mixing and emulsifying again, and before emulsifying finishes, add the dimethylmercury of 0.01% (V/V) of vaccine total amount, 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 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 is all 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.Concentrate-resuspended diluent of three kinds of antigens is mixed by the volume ratio of 4: 4: 1.According to 75%～90% (V/V) measure three kinds of antigens concentrated-resuspended diluent mixture, then add 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, according to < < People's Republic of China (PRC) veterinary drug allusion quotation > > appendix, carry out steriling test, result asepsis growth (in Table 1).
Table 1 Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis steriling test
Note: T.G represents sulphur glycollate culture medium, G.A represents peptone from casein agar culture medium, G.P dextrose peptone medium; "-" represents asepsis growth.
7. subpackage, storage
With 20mL/ bottle, 50mL/ bottle, 100mL/ bottle subpackage, cover bottle stopper, and jewelling lid, in 2～8 ℃ of storages.
Embodiment 2: safety research
(1) safety testing of vaccine to 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, observes 14, all without local response and all strong alive, in 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 is in Table 3.
The safety testing result of table 3 vaccine to piglet
From safety testing result, can find out, 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, is divided into 3 groups on the 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 is in 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, containing 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, divide 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, observe 14 days, and immune swine more than 4/5 is protected, and contrast pig more than 4/5 falls ill.
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, observes 14 days, and immune swine more than 4/5 is protected, and contrast pig more than 4/5 falls ill.
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, observes 14 days, and immune swine more than 4/5 is protected, and contrast pig is more than 4/5 dead.Assay is in Table 5.
Table 5 vaccine potency assay
Note: Haemophilus parasuis morbidity standard: heating (body temperature more than 40.5 ℃, continues 1～5), lethargy appear in morbidity pig, cough, dyspnea, becomes thin, walks lamely and by the thick clinical symptoms such as disorderly of hair.Dying pig is cutd open to inspection, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, there is serosity or fibrinous exudate in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
From above-described embodiment, can find out, 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, piglet protective rate is reached more than 80%, all to haemophilus parasuis, streptococcus suis infection has good protection, comparatively speaking, Haemophilus parasuis prepared by Montanide GEL PR adjuvant, inactivated combined vaccine against swine streptococcosis immune effect is best.
Claims (10)
1. Haemophilus parasuis, an inactivated combined vaccine against swine streptococcosis, comprising: antigen and adjuvant, and wherein, 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 is concentrated-resuspended diluent; The preserving number of described haemophilus parasuis serum 4 type JS strains is CCTCC NO:M 2011172; The preserving number of described haemophilus parasuis Serotype 5 ZJ strain is CCTCC NO:M 2011173; The preserving number of described streptococcus suis 2-type SC strain is CCTCC NO:M 2011351.
2. vaccine according to claim 1, it is characterized in that: the total content of concentrate-resuspended diluent of described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen 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: the volume ratio of concentrate-resuspended diluent of described haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains and streptococcus suis 2-type SC strain inactivation antigen is 2:2:1～4:4:1.
4. vaccine according to claim 3, is characterized in that: described vaccine is no less than 1.0 * 10 containing the front viable count of concentrate-resuspended diluent deactivation of haemophilus parasuis serum 4 type JS strain inactivation antigens
9cFU/mL; Before the concentrate-resuspended diluent deactivation of described haemophilus parasuis Serotype 5 ZJ strain inactivation antigen, viable count is no less than 1.0 * 10
9cFU/mL; Before the concentrate-resuspended diluent deactivation of described streptococcus suis 2-type SC strain inactivation antigen, viable count is no less than 5 * 10
8cFU/mL.
5. vaccine according to claim 1, is characterized in that: described adjuvant comprises aluminum salt series adjuvant, Montanide IMS series adjuvant or Montanide GEL series adjuvant; Wherein, in the vaccine of aluminum salt series adjuvant, the total content of three kinds of concentrate-resuspended diluents of inactivation antigen is 75%～85% (V/V), and adjuvant content is 15%～25% (V/V); In the vaccine of Montanide IMS series adjuvant, the total content of three kinds of concentrate-resuspended diluents of inactivation antigen is 50%～60% (V/V), and adjuvant content is 40%～50% (V/V); In the vaccine of Montanide GEL series adjuvant, the total content of three kinds of concentrate-resuspended diluents of inactivation antigen is 75%～90% (V/V), and adjuvant content is 10%～25% (V/V).
6. vaccine according to claim 1, is characterized in that: in described vaccine, also contain antiseptic, comprise thimerosal or dimethylmercury or sodium azide, its content is 0.01% (V/V) of vaccine total amount.
7. a preparation method for vaccine according to claim 6, comprising:
Steps A, breeds respectively its production strain of preparation by the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain, and this product is checked purely;
Step B, is inoculated into respectively its seedling by 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 to 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 to Haemophilus parasuis, pig streptococcicosis bigeminy deactivation emulsion vaccine, and this product is carried out to steriling test, safety verification and efficacy test;
Step F, Haemophilus parasuis, pig streptococcicosis bigeminy deactivation emulsion vaccine subpackage, storage;
It is characterized in that: step e comprises with diluent the resuspended dilution of antigen concentrated solution of the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains and streptococcus suis 2-type SC strain; Concentrated-resuspended diluent of three kinds of obtained antigens is mixed, then add adjuvant mixing and emulsifying, and before emulsifying finishes, add antiseptic, make Haemophilus parasuis, inactivated combined vaccine against swine streptococcosis.
8. method according to claim 7, is characterized in that: in step e, diluent is normal saline; 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, is characterized in that: seedling described in step B comprises by 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 cross-flow ultrafiltration system to concentrate, and removes toxin and foreign protein simultaneously, and the molecular cut off of ultrafilter membrane used is 500KD～1000KD.
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| CN104248758A (en) * | 2013-11-08 | 2014-12-31 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and application thereof |
| CN107589256A (en) * | 2017-09-30 | 2018-01-16 | 天津瑞普生物技术股份有限公司 | The method of inspection of the type of Haemophilus parasuis 4,5 type bivalent inactivated vaccine effect |
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| CN110327460A (en) * | 2019-06-05 | 2019-10-15 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccine and preparation method |
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| CN113730563A (en) * | 2021-09-15 | 2021-12-03 | 龙岩学院 | Haemophilus parasuis adjuvant vaccine and preparation method thereof |
| CN113832068B (en) * | 2021-10-14 | 2023-07-25 | 河南兴华生物技术有限公司 | Streptococcus strain and vaccine for preventing and treating swine streptococcosis |
| CN114805554B (en) * | 2022-04-13 | 2023-08-18 | 西南民族大学 | Refined egg yolk antibody injection for resisting streptococcus suis and haemophilus parasuis and preparation method thereof |
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