CN103566364B - Porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine and preparation method thereof - Google Patents
Porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention provides 3 strain Actinobacillus pleuropneumoniaes and 2 strain Streptococcus suis, several antigen with the use of, can be near minimum by the mutual interference between antigen; Based on above-mentioned discovery, the invention provides and a kind of there is good immunogenic porcine contagious pleuropneumonia, inactivated combined vaccine against swine streptococcosis; This vaccine employs Actinobacillus pleuropneumoniae 1,5,7 type of deactivation and Streptococcus suis as antigen, it has virulent and immunogenicity, less antigen amount can be used in vaccine preparation process to reach good immune effect, thus reduce and produce and use cost, the application in suitable for mass production and plant.Porcine contagious pleuropneumonia of the present invention, Streptococcus suis bivalent inactivated vaccine, in the level of production cost identical with single Seedling and selling price, reach the effect that a pin two is anti-, single Seedling can be replaced to use, thus greatly reduce immune cost, there are coml success potentiality.
Description
Technical field
The present invention relates to a kind of porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine and preparation method thereof, belong to veterinary drug vaccines arts.
Background technology
Porcine contagious pleuropneumonia is a kind of respiratory system Important Infectious Diseases caused by Actinobacillus pleuropneumoniae (Actionobacilluspleuropneumoniae, APP).Primary disease mainly causes the one of pig with pleuritic cellulosic, hemorrhagic, necrotizing pneumonia, how dead rapidly in the most acute or acute course of disease, M & M is all more than 50%, it can betide the pig at any age, especially at Intensive Farm of Pig Raising, once generation can cause heavy economic losses.
Actinobacillus pleuropneumoniae is also called haemophilus parahaemolyticus (H.parahaemolyties), and this growth of pathogenic bacteria requires strict, is gram-negative coccobacillus, has pod membrane.This pathogen serotype is numerous, according to NAD dependency, biological I type and biological II type can be divided into, the actinobacillus pleuropneumoniae wherein relying on NAD divides biological I type (BiovarI) into, the actinobacillus pleuropneumoniae not relying on NAD divides biological II type (BiovarII) into, adopt traditional serotype method, 15 serotypes can be subdivided into again; Wherein biological I type comprises serum 1-12 type and 15 types, and biological II type comprises serum 13 type and 14 types.The cause of disease serotype of pig pleuropneumonia disease is numerous, brings difficulty to the Prevention and controls of pig pleuropneumonia disease.
Streptococcus suis (Streptococcussuis) is conditioned pathogen, be distributed widely in occurring in nature, be everlasting at the place such as respiratory tract, intestinal, reproductive tract of animal, pig meningitis, arthritis, endocarditis, septicemia, pneumonia and sudden death can be caused, the development of serious harm pig industry.Streptococcus suis is gram positive bacteria, can survive for a long time in external environment, particularly in feces and contaminated environment and the pig that carries disease germs be the important source of infection, there is not any symptom in the pig of super acute death.Pig, regardless of kind, age and sex, can infect primary disease throughout the year, and suckling and the rear piglet death rate of the onset of wean up to more than 80%, and have certain people and animals' common sense metachromia.Streptococcus suis is at least divided into 5 types: acute septic, meningitic, arthritis, suppurative and agalactia type.
Due to the numerous types of the sick serotype of pig pleuropneumonia and Streptococcus suis, to two kinds of disease coinfections, the plant of falling ill simultaneously, there is no effective counter-measure, usually bring massive losses.
Summary of the invention
In view of this, main purpose of the present invention is to provide a kind of Novel pig contagious pleuropneumonia, pig streptococcicosis bigeminy multivalent inactivated vaccine, comprise at least one Actinobacillus pleuropneumoniae antigen of deactivation and at least one Streptococcus suis antigen, the disease that sick and Streptococcus suis causes with prevention and therapy pig pleuropneumonia.
Actinobacillus pleuropneumoniae antigen refers to any compositions containing at least one Actinobacillus pleuropneumoniae antigen forms, and the immunne response that opposing Actinobacillus pleuropneumoniae infects can be induced, stimulates or be strengthened to described Actinobacillus pleuropneumoniae antigen inoculation pig.Actinobacillus pleuropneumoniae is the street strain comprising clinical separation well known to those skilled in the art, comprises 1 ~ 15 serotype identified at present.Preferably, described Actinobacillus pleuropneumoniae antigen is complete Actinobacillus pleuropneumoniae, be preferably deactivated form, more preferably, the hybrid antigen of described Actinobacillus pleuropneumoniae antigen to be the serotype of deactivation be 1 type, 5 types and 7 types; Most preferably be the Actinobacillus pleuropneumoniae serum 1 type LC strain of deactivation, and the Actinobacillus pleuropneumoniae Serotype 5 YC strain of deactivation, and the Actinobacillus pleuropneumoniae serum 7-type YS strain of deactivation.
Actinobacillus pleuropneumoniae antigen of the present invention can also comprise any one antigen of following compositions: as the trivalent inactivated vaccines for porcine infectious pleuropneumonia (APP serotype 1 type, 2 types and 7 types) of Wuhan Keqian Animal Biological Products Co., Ltd.; The trivalent inactivated vaccines for porcine infectious pleuropneumonia (APP serum 1 type, 2 types and 7 types) of Chengdu medical instruments factory of Zhongmu Industry Co., Ltd.
As is known by those skilled in the art, different microorganism field isolates has small variation in gene order, but, when make a variation do not affect its protein synthesis, structure or Main Function functional areas time, cannot be absolutely identical even if he of this microorganism plants field isolates gene order, its basic physiological function can't change to some extent.Therefore, often use 16SRibosomalRNA(16SrRNA in the world at present) carry out the resolution of bacteria type, therefore can compare with 16SrRNA relatively going up of similarity and obtain its homology.So have the prerequisite of homology at 16SrRNA under, Actinobacillus pleuropneumoniae antigen used in the present invention is not limited to field isolates used in the present invention.
16srRNA gene is the corresponding DNA sequence of rRNA that bacterial chromosome is encoded, and is present in the germy chromogene group of institute.16SrRNA has conservative and specificity and this gene order long enough (comprising about 50 functional domains) of height.
Therefore, the present invention includes and (the LC strain of Actinobacillus pleuropneumoniae three strain bacterial strain, YC strain and YS strain) homology of 16SrRNA on 80% more than bacterial strain, more preferably with LC strain or YC strain or YS strain 16SrRNA homology more than at least 90%, preferably more than at least 95%, the more preferably Actinobacillus pleuropneumoniae bacterial strain of more than at least 95% ~ 99%, namely (the LC strain of Actinobacillus pleuropneumoniae three strain bacterial strain is not changed, YC strain and YS strain) 16SrRNA prerequisite under, those skilled in the art can by simple screening or mutation Actinobacillus pleuropneumoniae of the present invention, obtain the bacterial strain with Actinobacillus pleuropneumoniae 16SrRNA very high homology of the present invention, and acquisition correspondingly has same or similar immunogenic antigen composition.
Streptococcus suis antigen refers to any compositions containing at least one Streptococcus suis antigen forms, and the immunne response of opposing streptococcus suis infection can be induced, stimulates or be strengthened to described Streptococcus suis antigen inoculation pig.Streptococcus suis is the street strain comprising clinical separation well known to those skilled in the art, comprises the serotype of 1 ~ 9 pathogenic effects identified at present.Preferably, described Streptococcus suis antigen is complete Streptococcus suis, is preferably deactivated form, more preferably, and the hybrid antigen of described Streptococcus suis antigen to be the serotype of deactivation be 2 types and Streptococcus suis C group Malian drainage; Most preferably be the Streptococcus suis serum 2 type SC strain of deactivation, and the Streptococcus suis C group Malian drainage of deactivation.
Streptococcus suis antigen of the present invention can also comprise any one antigen of following compositions: as the Streptococcus suis inactivated vaccine (Malian drainage+streptococcus suis 2-type+Streptococcus suis 7 type) of Wuhan Keqian Animal Biological Products Co., Ltd.; The Streptococcus suis inactivated vaccine (Malian drainage+streptococcus suis 2-type) of Shanghai Hai Li Biotechnology Ltd.; The streptococcus suis 2-type inactivated vaccine of Guangdong Winsun Bio-Pharmaceutical Co., Ltd.; The Streptococcus suis inactivated vaccine (Malian drainage+streptococcus suis 2-type) of Binzhou, Shandong Hua Hong biological product Co., Ltd.
Also be included under 16SrRNA has the prerequisite of homology, Streptococcus suis antigen used in the present invention is not limited to field isolates used in the present invention.The present invention also to comprise with the homology of Streptococcus suis two strain bacterial strain (SC strain and Malian drainage) 16SrRNA the bacterial strain more than on 80%, more preferably with SC strain or Malian drainage 16SrRNA homology more than at least 90%, preferably more than at least 95%, the more preferably pig streptococcus bacterial strain of more than at least 95% ~ 99%, namely under not changing the prerequisite of Streptococcus suis two strain bacterial strain (SC strain and Malian drainage) 16SrRNA, those skilled in the art can by simple screening or mutation Streptococcus suis of the present invention, obtain the bacterial strain with Streptococcus suis 16SrRNA very high homology of the present invention, and acquisition correspondingly has same or similar immunogenic antigen composition.
Inventor is by long-term research and a large amount of tests, find current porcine contagious pleuropneumonia, pig streptococcicosis lacks appropriate therapeutic measure, the reason particularly lacking suitable bigeminy vaccine is, the serotype of Actinobacillus pleuropneumoniae is numerous, with Streptococcus suis hypotype and/or subspecies numerous, prepare in the single Seedling for porcine contagious pleuropneumonia or pig streptococcicosis and will mutually disturb between polyvalent antigen, thus affect the overall immune effect of its vaccine.But inventor is in long-term research and a large amount of tests, have unexpectedly discovered that at least three of Actinobacillus pleuropneumoniae kinds of serotypes, and Streptococcus suis at least two kinds of hypotypes or hypotype coupling time, influencing each other between antigen can be reduced; Particularly when use Actinobacillus pleuropneumoniae three kinds of serotypes 1,5,7 type time, the impact between antigen can be reduced significantly, while minimizing antigen consumption, obtain good immunogenicity, may be used for prevention and therapy porcine contagious pleuropneumonia and pig streptococcicosis simultaneously.
More unexpectedly, inventor have also obtained 3 strain Actinobacillus pleuropneumoniaes and 1 strain Streptococcus suis, with the Streptococcus suis of preserving number ATCC35246 with the use of, can be near minimum by the mutual interference between antigen, and obtain and a kind of there is good immunogenic porcine contagious pleuropneumonia, inactivated combined vaccine against swine streptococcosis
Based on above-mentioned discovery, a first aspect of the present invention is for providing porcine contagious pleuropneumonia, inactivated combined vaccine against swine streptococcosis, and it contains the antigen of Actinobacillus pleuropneumoniae serotype and the antigen of Streptococcus suis hypotype or subspecies.
Preferably, bivalent inactivated vaccine of the present invention is containing the antigen of at least three kinds of Actinobacillus pleuropneumoniae serotypes and the antigen of at least two kinds of Streptococcus suis hypotypes or subspecies.More preferably, in bivalent inactivated vaccine of the present invention, the antigen containing Actinobacillus pleuropneumoniae serum 1 type, Serotype 5 and serum 7-type; And the antigen of streptococcus suis 2-type.
Preferably, bivalent inactivated vaccine of the present invention, the antigen containing Actinobacillus pleuropneumoniae serum 1 type LC strain, Serotype 5 YC strain, serum 7-type YS strain, streptococcus suis 2-type (R group) SC strain and Streptococcus suis C group Malian drainage.
Preferably, bivalent inactivated vaccine of the present invention, in described bivalent inactivated vaccine, antigen ratio is: Actinobacillus pleuropneumoniae serum 1 type LC strain: Serotype 5 YC strain: serum 7-type YS strain: streptococcus suis 2-type SC strain: Streptococcus suis C group Malian drainage=2:2:2:1:1.
Preferably, bivalent inactivated vaccine of the present invention, according to bivalent inactivated vaccine according to claim 4, it is characterized in that, in described bivalent inactivated vaccine, the content of antigen is: before deactivation, Actinobacillus pleuropneumoniae serum 1 type, Serotype 5, serum 7-type viable count are all no less than 5.0 × 10 separately
7cFU/mL, streptococcus suis 2-type and Malian drainage viable count are all no less than 5 × 10 separately
9cFU/mL; Or above-mentioned antigen cumulative volume is 75% ~ 90% of described vaccine cumulative volume.
Secondly, at present in the vaccine for prevention and therapy porcine contagious pleuropneumonia or pig streptococcicosis, adjuvant used is oiliness vaccine, this is also because the serotype of Actinobacillus pleuropneumoniae is numerous, with Streptococcus suis hypotype and/or subspecies numerous, when using aqueous adjuvants, mutually will disturb between polyvalent antigen, thus affecting the overall immune effect of its vaccine; And inventor is by great many of experiments, is surprised to find that in above-mentioned bivalent inactivated vaccine of the present invention, adds the immunogenicity that aqueous adjuvants can't reduce vaccine.
Therefore, a second aspect of the present invention is to provide the bivalent inactivated vaccine aqueous adjuvants containing above-mentioned antigen; I.e. bivalent inactivated vaccine of the present invention, also containing adjuvant; More preferably, described adjuvant is aqueous adjuvants; Most preferably, described aqueous adjuvants is selected from one or more of MontanideGELPR polymer and homologue thereof.
In addition, vaccine of the present invention can also contain the known vaccine adjuvant such as antiseptic, immunostimulant; Preferably, bivalent inactivated vaccine of the present invention, the thimerosal also containing vaccine total amount 0.01%.
A third aspect of the present invention is for providing Actinobacillus pleuropneumoniae in above-mentioned discovery and Streptococcus suis, it has virulent and immunogenicity, less antigen amount can be used in vaccine preparation process to reach good immune effect, thus reduce production and use cost, be specially:
Actinobacillus pleuropneumoniae serum 1 type LC strain, be separated by Pulaike Biological Engineering Co., Ltd., identify, carry out preservation at Chinese Typical Representative culture center (being called for short CCTCC), preservation date: on December 9th, 2011, preserving number is: CCTCCM2011458, and address is Wuhan, China Wuhan University; Actinobacillus pleuropneumoniae Serotype 5 YC strain, be separated by Pulaike Biological Engineering Co., Ltd., identify, carry out preservation, preservation date at Chinese Typical Representative culture center: on December 9th, 2011, preserving number is: CCTCCM2011459; Actinobacillus pleuropneumoniae serum 7-type YS strain, is separated by Pulaike Biological Engineering Co., Ltd., identifies, carry out preservation, preservation date at Chinese Typical Representative culture center: on December 9th, 2011, preserving number is: CCTCCM2011460.
Streptococcus suis 2-type SC strain, is separated by Pulaike Biological Engineering Co., Ltd., identifies, carry out preservation, preservation date at Chinese Typical Representative culture center: on October 12nd, 2011, preserving number is: CCTCCM2011351.
A third aspect of the present invention is the preparation method providing above-mentioned porcine contagious pleuropneumonia, inactivated combined vaccine against swine streptococcosis, comprises the following steps:
1) Actinobacillus pleuropneumoniae, Streptococcus suis are cultivated in breeding respectively;
2) deactivation mixing with certain proportion;
3) adjuvant and/or antiseptic is added; And/or immune improving agent, both obtain the finished product of porcine contagious pleuropneumonia, inactivated combined vaccine against swine streptococcosis.
Preferably, the preparation method of bivalent inactivated vaccine of the present invention, further comprising the steps:
1. bivalent inactivated vaccine according to claim 13, is characterized in that, described step is:
(1) respectively Actinobacillus pleuropneumoniae serum 1 type LC strain, Serotype 5 YC strain, serum 7-type YS strain, streptococcus suis 2-type (R group) SC strain and Streptococcus suis C group Malian drainage culture propagation are cultivated, obtain Actinobacillus pleuropneumoniae serum 1 type LC strain, Serotype 5 YC strain, serum 7-type YS strain, streptococcus suis 2-type (R group) SC strain and Streptococcus suis C group Malian drainage bacterium liquid;
(2) respectively (1) step is cultivated obtain Actinobacillus pleuropneumoniae serum 1 type LC strain, Serotype 5 YC strain, serum 7-type YS strain, add by respective total amount in streptococcus suis 2-type (R group) SC strain and Streptococcus suis C group Malian drainage bacterium liquid 0.2% formalin, place 37 DEG C of deactivation 24h, period stirs 3 ~ 5 times, concentrates bacterium liquid according to count results;
(3) by above-mentioned deactivation, concentrated Actinobacillus pleuropneumoniae serum 1 type LC strain, Serotype 5 YC strain, serum 7-type YS strain, streptococcus suis 2-type (R group) SC strain and Streptococcus suis C group Malian drainage bacterium liquid, mix in 2:2:2:1:1 ratio, and add the thimerosal of vaccine total amount 0.01%, then carry out emulsifying with adjuvant, adjuvant content is 10% ~ 25%; Finished product porcine contagious pleuropneumonia, inactivated combined vaccine against swine streptococcosis antigenic content are Actinobacillus pleuropneumoniae serum 1 type before deactivation, Serotype 5, serum 7-type viable count are all no less than 5.0 × 10
7cFU/mL, streptococcus suis 2-type and Malian drainage viable count are all no less than 5 × 10
9cFU/mL.
Therefore bivalent inactivated vaccine of the present invention may be used for prevention and therapy porcine contagious pleuropneumonia, pig streptococcicosis.
technique effect
Of the present invention have following advantage at least:
1. the present invention solves a difficult problem for prevention and therapy porcine contagious pleuropneumonia, pig streptococcicosis simultaneously first time, overcome Actinobacillus pleuropneumoniae and Streptococcus suis numerous types causes the difficulty cooperatively interacted, provide and can have good immunogenic antigen composition;
2. present invention finds 3 strain Actinobacillus pleuropneumoniaes and 1 strain Streptococcus suis, several antigen with the use of, can be near minimum by the mutual interference between antigen, and obtain a kind of have good immunogenic porcine contagious pleuropneumonia, inactivated combined vaccine against swine streptococcosis;
3. present invention uses Actinobacillus pleuropneumoniae 1,5,7 type of deactivation and Streptococcus suis as antigen, it has virulent and immunogenicity, less antigen amount can be used in vaccine preparation process to reach good immune effect, thus reduce and produce and use cost, the application in suitable for mass production and plant;
4. the present invention is in the preparation of vaccine, employs the Seedling bacterium liquid of joining that hollow fiber membrane ultrafiltration device concentrates, normal saline suspends and greatly reduces toxin, while Optimization Technology, improve the safety of vaccine.
5. first the present invention employs aqueous adjuvants at porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine, decrease and use oil adjuvant immunity pig and the stress that occurs, not only meet current pig vaccine aqueous adjuvants development trend, decrease and join red tape in Seedling process, and reduce the demand of manpower and materials.Represent the development trend that current pig vaccine is new, there is promotion potential.
6. porcine contagious pleuropneumonia of the present invention, Streptococcus suis bivalent inactivated vaccine, in the level of production cost identical with single Seedling and selling price, reach the effect that a pin two is anti-, single Seedling can be replaced to use, thus greatly reduce immune cost, there are coml success potentiality.
Detailed description of the invention
Specific experiment method in the embodiment of the present invention can see " People's Republic of China's veterinary drug allusion quotation (two 〇 mono-〇 versions) " and annex.Other not marked reagent or raw material all can be obtained according to prior art by those skilled in the art.
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation of embodiment 1 porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine
1. strain
Actinobacillus pleuropneumoniae serum 1 type LC strain, is separated by Pulaike Biological Engineering Co., Ltd., identifies, carry out preservation, preservation date at Chinese Typical Representative culture center: on December 9th, 2011, preserving number is: CCTCCM2011458.
Actinobacillus pleuropneumoniae serum 2 type XT strain, be disclosed in Chinese patent CN102391975A, preserving number is: CCTCCNOM2011410.
Actinobacillus pleuropneumoniae Serotype 5 YC strain, is separated by Pulaike Biological Engineering Co., Ltd., identifies, carry out preservation, preservation date at Chinese Typical Representative culture center: on December 9th, 2011, preserving number is: CCTCCM2011459.
Actinobacillus pleuropneumoniae serum 7-type YS strain, is separated by Pulaike Biological Engineering Co., Ltd., identifies, carry out preservation, preservation date at Chinese Typical Representative culture center: on December 9th, 2011, preserving number is: CCTCCM2011460.
Streptococcus suis 2-type SC strain, is separated by Pulaike Biological Engineering Co., Ltd., identifies, carry out preservation, preservation date at Chinese Typical Representative culture center: on October 12nd, 2011, preserving number is: CCTCCM2011351.
Streptococcus suis C group Malian drainage, from American Type Culture Collecti, preserving number ATCC35246.
For the strain that acquired immunity is strong, the lyophilizing after the rejuvenation of responsive pig body is qualified of each strain is preserved and contains.
2. the preparation of porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine
2.1. the preparation of production seed
Actinobacillus pleuropneumoniae serum 1 type LC strain, 2 type XT strains, 5 type YC strains, 7 type YS strain freeze-drying lactobacillus, respectively streak inoculation in TSA/NAD(containing 5% calf serum) on flat board, put 37 DEG C to cultivate 16 ~ 24 hours, choose satisfactory bacterium colony, be inoculated in TSB/NAD(containing 5% calf serum) in fluid medium, cultivate 12 ~ 16 hours, as first order seed for 37 DEG C; Streptococcus suis 2-type SC strain, C group's Malian drainage ATCC35246 strain freeze-drying lactobacillus, streak inoculation is on Clumbia Sanguis caprae seu ovis flat board respectively, puts 37 DEG C and cultivates 18 ~ 24 hours as first order seed.
The culture of first order seed is got in Actinobacillus pleuropneumoniae serum 1 type LC strain, 2 type XT strains, 5 type YC strains, 7 type YS strains, amount by 0.1% adds TSB/NAD(containing 5% calf serum) in fluid medium, cultivate 12 ~ 16 hours for 37 DEG C, through checking purely as secondary seed; Satisfactory bacterium colony is chosen in streptococcus suis 2-type SC strain, C group's Malian drainage ATCC35246 strain respectively, be inoculated in the improvement martin's bouillon culture medium containing 15ml/L calf serum, 37 DEG C of quiescent culture 12 ~ 16 hours, dull and stereotyped pure after the assay was approved as secondary seed through Sanguis caprae seu ovis.
Adopt Actinobacillus pleuropneumoniae culture medium, Streptococcus suis culture medium commercially.
2.2. the cultivation of bacterium liquid
In Actinobacillus pleuropneumoniae culture medium (commercially TSB), add 0.01% ~ 0.05%NAD, 5% ~ 10% serum, Actinobacillus pleuropneumoniae serum 1 type LC strain, 2 type XT strains, 5 type YC strains, 7 type YS strain bacterium liquid are added in culture medium cultivate respectively by the amount of 0.1% respectively, mix rearmounted 37 DEG C to cultivate 16 ~ 18 hours, when the concentration OD600 of bacterium liquid reach more than 2.5, DO value starts to rise, pH value stops cultivation when being reduced to below 6.5.
In Streptococcus suis culture medium (commercially improveing martin's bouillon), add the L calf serum of 1.5%, the glucose of 0.1% ~ 5%, streptococcus suis 2-type SC strain, C group's Malian drainage ATCC35246 strain bacterium liquid are added in culture medium cultivate by the amount of 5% respectively, mix rearmounted 37 DEG C to cultivate 16 ~ 18 hours, when bacterial concentration OD600 reach more than 2.5, DO value starts to rise, pH value stops cultivation when being reduced to below 6.0.
2.3. deactivation
To in above-mentioned six kinds of cultured bacterium liquid, the 0.2%(V/V by total amount) add formalin respectively, be positioned over 37 DEG C of deactivation 24h, period stirs 3 ~ 5 times.
2.4. inspection
The 4 kinds of Actinobacillus pleuropneumoniae bacterium liquid getting deactivation get respectively 5mL be inoculated in 100mL containing 0.01% ~ 0.05%NAD, 5% ~ 10% serum Actinobacillus pleuropneumoniae TSB culture medium in, cultivate 24 hours for 37 DEG C, then to transplant in above-mentioned 100mL culture medium 37 DEG C and cultivate 24 hours; Get 3 kinds of Actinobacillus pleuropneumoniae bacterium liquid 0.5mL streak inoculation of deactivation in TSA/NAD plating medium, cultivate 24 hours for 37 DEG C.After two kinds of methods are cultivated, equal asepsis growth.
The 2 kinds of Streptococcus suis bacterium liquid getting deactivation respectively 5mL are inoculated in 100mL and add the L calf serum of 1.5%, the glucose improvement martin's bouillon culture medium of 0.1% ~ 5%, cultivate 24 hours for 37 DEG C, then to transplant in above-mentioned 100mL culture medium 37 DEG C and cultivate 24 hours; Get 2 kinds of Streptococcus suis bacterium liquid 0.5mL streak inoculation of deactivation in SBA plating medium, cultivate 24 hours for 37 DEG C.After two kinds of methods are cultivated, equal asepsis growth.
Get the Balb/C mice 5 of above-mentioned six kinds of bacterium liquid respectively intravenous injection 18 ~ 22g of deactivation, every 0.2mL, in three days, 5/5 strongly lives, side for deactivation thorough.
3. the preparation of porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine
3.1. the concrete composition proportion of vaccine
Existing 6 kinds of antigenic components (Actinobacillus pleuropneumoniae serum 1 type LC strain, 2 type XT strains, 5 type YC strains, 7 type YS strains, streptococcus suis 2-type SC strain and C group's Malian drainage ATCC35246 strain) can make following 45 kinds of porcine contagious pleuropneumonias, Streptococcus suis bivalent inactivated vaccine according to different collocation modes.Antigen total content percent by volume accounts for 10% ~ 25% at 75% ~ 90%, MontanideGELPR adjuvant, and we select the optimum percent by volume 10% of optimum percent by volume 90%, the MontanideGELPR adjuvant of antigen total content herein.Actinobacillus pleuropneumoniae serum 1 type LC strain, 2 type XT strains, 5 type YC strains and 7 type YS strains respectively account for cumulative volume 20%, streptococcus suis 2-type SC strain and C group's Malian drainage ATCC35246 strain respectively account for cumulative volume 10%, MontanideGELPR adjuvant accounts for cumulative volume 10%, and antigenic component is supplied with PBS less than 90% part.
Before this joins the antigens inactive of Seedling use, viable count is respectively Actinobacillus pleuropneumoniae serum 1 type LC strain 5.0 × 10
9cFU/ml, Actinobacillus pleuropneumoniae serum 2 type XT strain 3.0 × 10
9cFU/ml, Actinobacillus pleuropneumoniae Serotype 5 YC strain 2.0 × 10
9cFU/ml, Actinobacillus pleuropneumoniae serum 7-type YS strain 3.7 × 10
9cFU/ml, streptococcus suis 2-type SC strain 7.0 × 10
9cFU/ml and C group Malian drainage ATCC35246 strain 8.0 × 10
9cFU/ml.Join Actinobacillus pleuropneumoniae serum 1 type LC strain before contained deactivation, serum 2 type XT strain, Serotype 5 YC strain, serum 7-type YS strain viable count in Seedling time vaccines and be all adjusted to 5.0 × 10
7cFU/mL, streptococcus suis 2-type SC strain and Malian drainage ATCC35246 strain viable count are all adjusted to 5 × 10
9cFU/mL.
The concrete composition proportion of table 1 vaccine
3.2. vaccine formulation process
Specific as follows: bacterium liquid hollow fiber membrane ultrafiltration device is concentrated, elimination supernatant, and with the resuspended dilution of normal saline, with reference to colony counting result, adjustment bacterial concentration is Actinobacillus pleuropneumoniae serum 1 type LC strain, 2 type XT strains, 5 type YC strains, 7 type YS strains each 5.0 × 10
7cFU/ml and streptococcus suis 2-type SC strain, C group's Malian drainage ATCC35246 strain each 5.0 × 10
9cFU/ml.Often kind of a vaccine is prepared 100ml(vaccine 45 and is prepared 111ml), wherein MontanideGELPR gets 10ml, in each vaccine, antigenic content is according to proportions in above form, vaccine 1 ~ vaccine 44 all needs to add PBS and adjusts antigen cumulative volume to 90ml, containing 6 kinds of antigenic components in vaccine 45, prepare about 110ml vaccine (LC strain 20ml+XT strain 20ml+YC strain 20ml+YS strain 20ml+SC strain 10ml+C group streptococcus equi veterinary subspecies 10ml+MontanideGELPR11ml) altogether according to each antigen ratio.Each vaccine composition is in joining after Seedling ratio adds, and stirring at low speed emulsifying, adds the thimerosal of 0.01% of vaccine total amount before stopping stirring.Often kind of formula vaccine prepares 3 batches.
4. safety testing
4.1. to the safety of white mice
The vaccine getting preparation inoculates the Balb/C mice 5 of 18 ~ 22g, every subcutaneous injection 0.2ml respectively, observes 14d, without local response and all strong work.
4.2. to the safety of piglet
Get vaccine respectively healthy each 5 of the susceptible pig of intramuscular injection 30 age in days of preparation, every 4mL, all strong alive without local response in 14 days.
5. potency test
Often organize the Balb/C mice that 18 ~ 22g used by vaccine, subcutaneous vaccination 0.1mL dyad inactivated vaccine, exempts from rear 28d and is divided into 6 groups, often organizes each 10 together with matched group, uses Actinobacillus pleuropneumoniae serum 1 type LC strain lumbar injection 5LD respectively
50(bacteria containing amount is 4.0 × 10
5cFU), 2 type XT strain lumbar injection 5LD
50(bacteria containing amount is 4.0 × 10
5cFU), lumbar injection 5LD is carried out in 5 type YC strains
50(bacteria containing amount is 5.0 × 10
5cFU); Lumbar injection 5LD is carried out in 7 type YS strains
50(bacteria containing amount is 5.0 × 10
6cFU); Streptococcus suis 2-type SC strain subcutaneous injection 2LD
50(bacteria containing amount is 1.0 × 10
2cFU), Streptococcus suis C group Malian drainage ATCC35246 strain subcutaneous injection 5LD
50(bacteria containing amount is 4.0 × 10
2cFU) other 10 are set to blank, observe 14d.
Often organize the healthy susceptible pig 30 that 28 ~ 35 ages in days used by vaccine, every intramuscular injection 2mL, containing 1 using dosage, after 28d days, the immunity test pig of having injected often kind of vaccine is divided into 6 groups at random, respectively with described Actinobacillus pleuropneumoniae serum 1 type LC strain, 2 type XT strains, 5 type YC strains, 7 type YS strains, streptococcus suis 2-type SC strain and Malian drainage ATCC35246 strain counteracting toxic substances separately.Contrast pig 30, divides 6 groups, respectively with described Actinobacillus pleuropneumoniae serum 1 type LC strain, 2 type XT strains, 5 type YC strains, 7 type YS strains, streptococcus suis 2-type SC strain and Malian drainage ATCC35246 strain counteracting toxic substances separately.Observe 14 days.Efficacy test result, sees the following form 2.
Table 2 vaccine potency assay
Note: porcine contagious pleuropneumonia morbidity standard: heating (body temperature more than 40.5 DEG C, continue 1 ~ 3 day), lethargy appear in morbidity pig, poly-heap, dyspnea, diarrhoea, vomiting, by the thick clinical symptoms such as random of hair.Cut open inspection to dead pig and dying pig, be full of cystose blood sample viscosity exudate, pulmonary congestion, edema in visible pig trachea and bronchus, pneumonia focus is dimmed, hardening, fragile, and fibrinous pleurisy district presents kermesinus or blackout.
Known by potency test; vaccine 43 and vaccine 45 formula effectively can be protected porcine contagious pleuropneumonia, Streptococcus suis two kinds of diseases; by the known vaccine of formula 43 a kind of antigen component fewer than vaccine 45; but can reach the effect of the general immune of vaccine 45, these two kinds of porcine contagious pleuropneumonias, pig streptococcicosis are provided comprehensive and effectively protected!
Embodiment 2 porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine contrast test
Current commercially available porcine contagious pleuropneumonia inactivated vaccine primarily of before the section of Wuhan and in herd 2 enterprises of Chengdu medical instruments factory and produce, Streptococcus suis vaccine is primarily of before the section of Wuhan, and Shanghai Hai Li, grand 4 enterprises in Yongshun, Guangdong and Shandong China produce.For the vaccine that above Ji Jia enterprise produces, we adopt this to study identical preparation method to prepare bivalent inactivated vaccine that is several and commercially available prod phase homologous serotype, and compare this several bivalent inactivated vaccine.A series of comparison is carried out subsequently by obtaining the product of optimum bigeminy vaccine with above-mentioned a few family of market sale more afterwards.Wherein commercially available relevant porcine contagious pleuropneumonia vaccine and streptococcus vaccine details are as following table 3, table 4:
The commercially available trivalent inactivated vaccines for porcine infectious pleuropneumonia of table 3.
The commercially available Streptococcus suis vaccine of table 4.
1. the preparation of porcine contagious pleuropneumonia of the present invention, Streptococcus suis bivalent inactivated vaccine
The serotype different with Streptococcus suis inactivated vaccine according to the commercially available trivalent inactivated vaccines for porcine infectious pleuropneumonia cited by above table and collocation mode; we have carried out porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine preparation according to this research preparation method to above several serotype to this several compound mode; and the porcine contagious pleuropneumonia under this several compound mode, Streptococcus suis bivalent inactivated vaccine are carried out to the comparison of protective efficacy, to drawing optimum combination.Detailed composition is as follows:
The preparation of 1.1 porcine contagious pleuropneumonias (serum 1 type LC strain+serum 2 type XT strain+serum 7-type YS strain), pig streptococcicosis (Malian drainage+streptococcus suis 2-type SC strain) bivalent inactivated vaccine (V1)
The preparation of 1.2 porcine contagious pleuropneumonias (serum 1 type LC strain+Serotype 5 YC strain+serum 7-type YS strain), pig streptococcicosis (Malian drainage+streptococcus suis 2-type SC strain) bivalent inactivated vaccine (V2)
Note: join above before Seedling bacterium liquid joins Seedling and concentrate with hollow fiber membrane ultrafiltration device, elimination supernatant, and with the resuspended dilution of normal saline, with reference to colony counting result, adjustment Actinobacillus pleuropneumoniae serum 1 type LC strain, 5 type YC strains, 7 type YS strain bacterial concentrations are respectively 5.0 × 10
7cFU/ml, adjustment Actinobacillus pleuropneumoniae serum 2 type XT strain bacterial concentration is 3.0 × 10
9cFU/ml, adjustment streptococcus suis 2-type SC strain and C group's Malian drainage ATCC35246 strain bacterial concentration are respectively 5.0 × 10
9cFU/ml, according to joining Seedling concrete composition proportion Standard entertion adjuvant, stirring at low speed emulsifying, adds the thimerosal of 0.01% of vaccine total amount before stopping stirring.
2. porcine contagious pleuropneumonia, Streptococcus suis bivalent inactivated vaccine animal experiment compare
2.1 piglet contrast tests
Select 35 ~ 40 age in days ablactational baby pig 85, be divided into 4 groups, the 1st group 25, the 2nd group 25, the 3rd group 30, the 4th group 5.1st group of every pig musculi colli vaccinate (V1) 2ml respectively, after immunity, 28d inoculates 2ml again; 2nd group of every pig musculi colli vaccinate (V2) 2ml respectively, after immunity, 28d inoculates 2ml again; 3rd group as counteracting toxic substances matched group, the 4th group as blank group.42d after first immunisation, the 1st, 2,3 group of each random choose 5 piglets carry out Actinobacillus pleuropneumoniae 1 type LC strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head; 1st, 2,3 groups of each random chooses, 5 piglets carry out Actinobacillus pleuropneumoniae 7 type YS strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head; 1st, 2,3 groups of each random chooses, 5 piglets carry out C group's Malian drainage ATCC35246 strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head; 1st, 2,3 groups of each random chooses, 5 piglets carry out streptococcus suis 2-type SC strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head; 1st, 3 groups of each random chooses, 5 piglets carry out Actinobacillus pleuropneumoniae 2 type XT strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
9cFU/ head; 2nd, 3 groups of each random chooses, 5 piglets carry out Actinobacillus pleuropneumoniae 5 type YC strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head;
Continuous Observation 10 days after counteracting toxic substances, and slaughter after within the 10th day after counteracting toxic substances, weighing, cut open inspection.Judge according to correlated results such as body temperature, tissue injury and antibody horizontals.Result of the test is as table 5-table 8.
Table 5. tests grouping and process
The natural law that after table 6. counteracting toxic substances, each group test pig body temperature surpasses 40.5 DEG C compares
Can be found out by above-mentioned body temperature production table, the pig after Combined vaccine V2 immunity only has 2 test pig to occur that body temperature raises after counteracting toxic substances, and only maintains the time of 1 day; Pig after Combined vaccine V1 immunity has 5 test pig to occur that body temperature raises after counteracting toxic substances, also only maintains the time of 1 day, and the Vaccine effectiveness that after visible Combined vaccine V1 and V2 immunity piglet, counteracting toxic substances produces is all relatively good, and Combined vaccine V2 is more excellent.
Table 7. counteracting toxic substances Hou Gezu test pig tissue injury and bacterium picking out rate
Can be found out by above-mentioned tissue injury and bacterium picking out rate information slip, there is not any tissue injury in the test pig after Combined vaccine V2 immunity, do not detect any antibacterial after counteracting toxic substances yet; Test pig after Combined vaccine V1 immunity has 3 test pig to occur that individual tissues damages after counteracting toxic substances, and indivedual pig detects antibacterial simultaneously, and the Vaccine effectiveness Combined vaccine V2 that after visible Combined vaccine V1 and V2 immunity piglet, counteracting toxic substances produces is more excellent.
Latter 14 days antibody horizontals exempted from by table 8. respectively group test pig two
Note: "-" represents that antibody is negative
Exempt from latter 14 days antibody horizontal information slips by above-mentioned each group of test pig two can find out, antibody horizontal (comprising APP-1 type, APP-5 type, APP-7 type, SS-2 type and the Malian drainage) majority that test pig two after Combined vaccine V2 immunity is exempted from latter 14 days is 1:800, and indivedual pig antibody horizontal reaches 1:1600; Antibody horizontal (comprising APP-1 type, APP-2 type, APP-7 type, SS-2 type and the Malian drainage) majority that test pig two after Combined vaccine V1 immunity is exempted from latter 14 days is 1:800; there is 1:400 in indivedual pig antibody horizontal, the Vaccine effectiveness Combined vaccine V2 that after visible Combined vaccine V1 and V2 immunity piglet, counteracting toxic substances produces is more excellent.
2.2 farrowing sow contrast tests
Select antenatal 6 weeks farrowing sows 12, be divided into 3 groups, often organize 4.1st group of every pig musculi colli vaccinate (V1) 2ml respectively, inoculates 2ml in antenatal 2 weeks again; 2nd group of every pig musculi colli vaccinate (V2) 2ml respectively, inoculates 2ml in antenatal 2 weeks again; 3rd group as blank group.Observe sow spirit after inoculation, whether state of searching for food, inoculation position occur red and swollenly reacts, farrowing situation and pig maternal antibody continued case of farrowing.Result of the test is as table 9-table 15.
Table 9. divides into groups immunity and observed after immunization information slip
Can be found out by above-mentioned each group of farrowing sow observed after immunization information slip; farrowing sow after Combined vaccine V1 and V2 immunity after vaccination spirit and situation of searching for food all normal; there is not red and swollen phenomenon in inoculation position yet; the pig that farrows also 100% strong to live, but there is indivedual weak son in blank pig that group is farrowed, and death in 2 days after birth; result shows; Combined vaccine V1 and V2 all without any side effect, and all can produce effective protection to farrowing sow, significant for farrowed pig.
Table 10. farrowing sow farrows pig APP-1 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 11. farrowing sow farrows pig APP-2 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 12. farrowing sow farrows pig APP-5 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 13. farrowing sow farrows pig APP-7 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 14. farrowing sow farrows pig SS-2 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 15. farrowing sow farrows pig Malian drainage maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
This result of study shows: Combined vaccine V1 and V2 all can produce effective protection after immune piglet, but from clinical and cut open inspection symptom, Combined vaccine V2 is slightly better than Combined vaccine V1; Combined vaccine V1 and V2 does not all produce side reaction to sow after immune farrowing sow, all can produce long-time effective protection to produced piglet simultaneously.Therefore can reach a conclusion: apply the porcine contagious pleuropneumonia prepared by this research method, Streptococcus suis bivalent inactivated vaccine can produce effective protection to piglet and farrowing sow.
Embodiment 3 bigeminy vaccine and single Seedling conbined usage contrast test
2 result of the tests in conjunction with the embodiments, the porcine contagious pleuropneumonia prepared by known this research method of application, Streptococcus suis bivalent inactivated vaccine can produce effective protection to piglet and farrowing sow.The present embodiment is chosen optimum combination porcine contagious pleuropneumonia (serum 1 type LC strain+Serotype 5 YC strain+serum 7-type YS strain), Streptococcus suis (Malian drainage+streptococcus suis 2-type SC strain) bivalent inactivated vaccine and commercially available single Seedling conbined usage and is carried out effect Comparability test.
1. the preparation of vaccine and acquisition
1.1 porcine contagious pleuropneumonias (serum 1 type LC strain+Serotype 5 YC strain+serum 7-type YS strain), Streptococcus suis (Malian drainage+streptococcus suis 2-type SC strain) bivalent inactivated vaccine
Prepare Combined vaccine according to the preparation method of V2 in embodiment 2, see the following form 16.
1.2 trivalent inactivated vaccines for porcine infectious pleuropneumonias are purchased from Wuhan Keqian Animal Biological Products Co., Ltd., and batch number is 111007; Streptococcus suis inactivated vaccine is purchased from Shanghai Hai Li Biotechnology Ltd., and batch number is 20111022.
2. animal experiment design
2.1 piglet animal experiments
Select 35 ~ 40 age in days ablactational baby pig 85, be divided into 4 groups, the 1st group 25, the 2nd group 25, the 3rd group 30, the 4th group 5.1st group of every piglet musculi colli vaccinate (V2) 2ml respectively, after immunity, 28d inoculates 2ml again; 2nd group of every piglet carries out head according to Streptococcus suis inactivated vaccine 20111022 and trivalent inactivated vaccines for porcine infectious pleuropneumonia 111007 operation instructions respectively and exempts to exempt from two; 3rd group as counteracting toxic substances matched group, the 4th group as blank group.Two exempt from rear 14d, and the 1st, 2,3 group of each random choose 5 piglets carry out Actinobacillus pleuropneumoniae 1 type LC strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head; 1st, 2,3 groups of each random chooses, 5 piglets carry out Actinobacillus pleuropneumoniae 7 type YS strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head; 1st, 2,3 groups of each random chooses, 5 piglets carry out C group's Malian drainage ATCC35246 strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head; 1st, 2,3 groups of each random chooses, 5 piglets carry out streptococcus suis 2-type SC strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head; 2nd, 3 groups of each random chooses, 5 piglets carry out Actinobacillus pleuropneumoniae 2 type XT strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
9cFU/ head; 1st, 3 groups of each random chooses, 5 piglets carry out Actinobacillus pleuropneumoniae 5 type YC strain counteracting toxic substances, and every collunarium bacterium liquid 3ml, viable bacteria content is 1 × 10
8cFU/ head; Continuous Observation 10 days after counteracting toxic substances, and slaughter after within the 10th day after counteracting toxic substances, weighing, cut open inspection.Judge according to correlated results such as body temperature, tissue injury and antibody horizontals.
2.3 farrowing sow animal experiments
Select antenatal 6 weeks farrowing sows 12, be divided into 3 groups, often organize 4.1st group of every farrowing sow musculi colli vaccinate (V2) 2ml respectively, inoculates 2ml in antenatal 2 weeks again; 2nd group of every farrowing sow carries out head according to Streptococcus suis inactivated vaccine 20111022 and trivalent inactivated vaccines for porcine infectious pleuropneumonia 111007 operation instructions respectively and exempts to exempt from two; ; 3rd group as blank group.Observe sow spirit after inoculation, whether state of searching for food, inoculation position occur red and swollenly reacts, farrowing situation and pig maternal antibody continued case of farrowing.
3. animal test results
3.1 piglet animal test results
The natural law that after table 17. counteracting toxic substances, each group test pig body temperature surpasses 40.5 DEG C compares
Can be found out by above-mentioned body temperature production table, the pig after Combined vaccine V2 immunity only has 1 test pig to occur that body temperature raises after counteracting toxic substances, and only maintains the time of 1 day; Pig after Streptococcus suis inactivated vaccine 20111022 and trivalent inactivated vaccines for porcine infectious pleuropneumonia 111007 combined immunization has 6 test pig to occur that body temperature raises after counteracting toxic substances; hold time about 1 ~ 2 day, to be used alone as seen after Combined vaccine V2 immunity piglet the Vaccine effectiveness that produces of counteracting toxic substances than conbined usage Streptococcus suis and porcine contagious pleuropneumonia trivalent deactivation list Seedling effect good.
Table 18. counteracting toxic substances Hou Gezu test pig tissue injury and bacterium picking out rate
Can be found out by above-mentioned tissue injury and bacterium picking out rate information slip, there is not any tissue injury in the test pig after Combined vaccine V2 immunity, do not detect any antibacterial after counteracting toxic substances yet; Tissue injury is there is individually in the test pig after Streptococcus suis inactivated vaccine 20111022 and trivalent inactivated vaccines for porcine infectious pleuropneumonia 111007 combined immunization after counteracting toxic substances; detect antibacterial simultaneously, to be used alone as seen after Combined vaccine V2 immunity piglet the Vaccine effectiveness that produces of counteracting toxic substances than conbined usage Streptococcus suis and porcine contagious pleuropneumonia trivalent deactivation list Seedling effect good.
Latter 14 days antibody horizontals exempted from by table 18. respectively group test pig two
Note: "-" represents that antibody is negative
Exempt from latter 14 days antibody horizontal information slips by above-mentioned each group of test pig two can find out, antibody horizontal (comprising APP-1 type, APP-5 type, APP-7 type, SS-2 type and the Malian drainage) majority that the test pig two after Combined vaccine V2 immunity is exempted from latter 14 days is 1:800; Antibody horizontal (comprising APP-1 type, APP-2 type, APP-7 type, SS-2 type and the Malian drainage) majority that test pig two after Streptococcus suis inactivated vaccine 20111022 and trivalent inactivated vaccines for porcine infectious pleuropneumonia 111007 combined immunization is exempted from latter 14 days is 1:400; there is 1:800 in minority pig antibody horizontal, to be used alone as seen after Combined vaccine V2 immunity piglet the Vaccine effectiveness that produces of counteracting toxic substances than conbined usage Streptococcus suis and porcine contagious pleuropneumonia trivalent deactivation list Seedling effect good.
3.2 farrowing sow animal test results
Table 19. divides into groups immunity and observed after immunization information slip
Can be found out by above-mentioned each group of farrowing sow observed after immunization information slip, the farrowing sow after Combined vaccine V2 immunity after vaccination spirit and situation of searching for food all normal, there is not red and swollen phenomenon in inoculation position yet, institute farrow pig also 100% be good for alive; Although farrowed pig 100% is strong after inoculation lives for the farrowing sow after Streptococcus suis inactivated vaccine 20111022 and trivalent inactivated vaccines for porcine infectious pleuropneumonia 111007 combined immunization, but occur lethargy, feed intake decline phenomenon, and there is trickle redness in vaccine injection site.Analyzing reason may be that single Seedling conbined usage wants vaccinate 4 times, and injects cumulative volume also than using Combined vaccine will exceed one times, therefore may bring corresponding side reaction.Result shows, and Combined vaccine V2 immunity without any side effect, and all can produce effective protection to farrowing sow, significant for farrowed pig; And single Seedling conbined usage can serve side reaction to farrowing sow band, therefore use Combined vaccine more effective than single Seedling conbined usage, meaning is large.
Table 20. farrowing sow farrows pig APP-1 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 21. farrowing sow farrows pig APP-2 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 22. farrowing sow farrows pig APP-5 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 23. farrowing sow farrows pig APP-7 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 24. farrowing sow farrows pig SS-2 type bacterium maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
Table 25. farrowing sow farrows pig Malian drainage maternal antibody information slip
Note: "-" represents that antibody horizontal is negative
This result of study shows: Combined vaccine V2 not only can not produce any side reaction after immune farrowing sow, and can produce strong protection to farrowing sow, its maternal antibody level of pig that farrowing sow farrows not only the persistent period long, and antibody horizontal is very homogeneous; Although also can protection be produced to farrowing sow after Streptococcus suis inactivated vaccine 20111022 and trivalent inactivated vaccines for porcine infectious pleuropneumonia 111007 combined immunization; but some side reactions can be produced to farrowing sow after immunity; affect farrowing sow production status; simultaneously there is uneven, that the persistent period is short phenomenon in farrowing sow institute pig antibody horizontal of farrowing, and therefore Combined vaccine use is more excellent than single Seedling conbined usage effect.
Can find out according to above result of the test, homologous series adjuvant is comprised with MontanideGELPR() the Novel pig contagious pleuropneumonia made for adjuvant, inactivated combined vaccine against swine streptococcosis (LC strain+YC strain+YS strain+SC strain+Malian drainage) is not only easy to use, safer, immune effect is compared with two kinds of single Seedling connection and use and will get well, piglet and farrowing sow side reaction can not be caused after injection, and effectively can prevent Actinobacillus pleuropneumoniae and the microbial porcine contagious pleuropneumonia of hammer and pig streptococcicosis, there is good market prospect.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (11)
1. a bivalent inactivated vaccine, is characterized in that, described bivalent inactivated vaccine contains the antigen of Actinobacillus pleuropneumoniae serum 1 type, Serotype 5 and serum 7-type; And the antigen of streptococcus suis 2-type and Streptococcus suis C group Malian drainage; Wherein, in described bivalent inactivated vaccine, the content of antigen is: the Actinobacillus pleuropneumoniae serum 1 type before deactivation, Serotype 5, serum 7-type viable count are all no less than 5.0 × 10 separately
7cFU/mL, streptococcus suis 2-type and Malian drainage viable count are all no less than 5 × 10 separately
9cFU/mL; Or above-mentioned antigen cumulative volume is 75% ~ 90% of described vaccine cumulative volume.
2. bivalent inactivated vaccine according to claim 1, it is characterized in that, described bivalent inactivated vaccine contains the antigen of Actinobacillus pleuropneumoniae serum 1 type LC strain CCTCCM2011458, Serotype 5 YC strain CCTCCM2011459, serum 7-type YS strain CCTCCM2011460, streptococcus suis 2-type SC strain CCTCCM2011351 and Streptococcus suis C group Malian drainage ATCC35246 strain.
3. bivalent inactivated vaccine according to claim 2, it is characterized in that, in described bivalent inactivated vaccine, antigen ratio is: Actinobacillus pleuropneumoniae serum 1 type LC strain: Serotype 5 YC strain: serum 7-type YS strain: streptococcus suis 2-type SC strain: Streptococcus suis C group Malian drainage=2:2:2:1:1.
4. the bivalent inactivated vaccine according to claims 1 to 3 any one, is characterized in that, containing adjuvant in described bivalent inactivated vaccine.
5. bivalent inactivated vaccine according to claim 4, is characterized in that, described adjuvant is aqueous adjuvants.
6. bivalent inactivated vaccine according to claim 5, is characterized in that, described aqueous adjuvants is selected from one or more of MontanideGELPR polymer and homologue thereof.
7. an Actinobacillus pleuropneumoniae, is characterized in that, described Actinobacillus pleuropneumoniae is respectively Actinobacillus pleuropneumoniae serum 1 type LC strain, and preserving number is CCTCCM2011458; Actinobacillus pleuropneumoniae Serotype 5 YC strain, preserving number is: CCTCCM2011459; Actinobacillus pleuropneumoniae serum 7-type YS strain, preserving number is: CCTCCM2011460.
8. a Streptococcus suis, is characterized in that, described Streptococcus suis is respectively streptococcus suis 2-type SC strain, and preserving number is: CCTCCM2011351.
9. a preparation method for the bivalent inactivated vaccine of porcine contagious pleuropneumonia, pig streptococcicosis, comprises the following steps:
1) Actinobacillus pleuropneumoniae serum 1 type, Serotype 5 and serum 7-type, streptococcus suis 2-type and Streptococcus suis C group Malian drainage are cultivated in breeding respectively;
2) deactivation and with certain proportion mixing, wherein, in described bivalent inactivated vaccine, the content of antigen is: the Actinobacillus pleuropneumoniae serum 1 type before deactivation, Serotype 5, serum 7-type viable count are all no less than 5.0 × 10 separately
7cFU/mL, streptococcus suis 2-type and Malian drainage viable count are all no less than 5 × 10 separately
9cFU/mL; Or above-mentioned antigen cumulative volume is 75% ~ 90% of described vaccine cumulative volume;
3) adjuvant and/or antiseptic is added; And/or immune improving agent, obtain the finished product of bivalent inactivated vaccine of porcine contagious pleuropneumonia, pig streptococcicosis.
10. preparation method according to claim 9, comprises the following steps:
(1) respectively Actinobacillus pleuropneumoniae serum 1 type LC strain, Serotype 5 YC strain, serum 7-type YS strain, streptococcus suis 2-type (R group) SC strain and Streptococcus suis C group Malian drainage culture propagation are cultivated, obtain Actinobacillus pleuropneumoniae serum 1 type LC strain, Serotype 5 YC strain, serum 7-type YS strain, streptococcus suis 2-type (R group) SC strain and Streptococcus suis C group Malian drainage bacterium liquid;
(2) respectively (1) step is cultivated obtain Actinobacillus pleuropneumoniae serum 1 type LC strain, Serotype 5 YC strain, serum 7-type YS strain, add by respective total amount in streptococcus suis 2-type (R group) SC strain and Streptococcus suis C group Malian drainage bacterium liquid 0.2% formalin, place 37 DEG C of deactivation 24h, period stirs 3 ~ 5 times, concentrates bacterium liquid according to count results;
(3) by above-mentioned deactivation, concentrated Actinobacillus pleuropneumoniae serum 1 type LC strain, Serotype 5 YC strain, serum 7-type YS strain, streptococcus suis 2-type (R group) SC strain and Streptococcus suis C group Malian drainage bacterium liquid, mix in 2:2:2:1:1 ratio, and add the thimerosal of vaccine total amount 0.01%, then carry out emulsifying with adjuvant, adjuvant content is 10% ~ 25%; Finished product porcine contagious pleuropneumonia, inactivated combined vaccine against swine streptococcosis antigenic content are Actinobacillus pleuropneumoniae serum 1 type before deactivation, Serotype 5, serum 7-type viable count are all no less than 5.0 × 10
7cFU/mL, streptococcus suis 2-type and Malian drainage viable count are all no less than 5 × 10
9cFU/mL.
11. bivalent inactivated vaccines as described in claim 1 ~ 6 any one are in the application preparing prevention and therapy porcine contagious pleuropneumonia, pig streptococcicosis Chinese medicine.
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