CN102949714A - Swine Streptococcosis trivalent inactivated vaccine and preparation method thereof - Google Patents
Swine Streptococcosis trivalent inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of preparation of animal vaccines and particularly relates to a trivalent inactivated vaccine for Streptococcus equi subsp. zooepidemicus, Streptococcus suis serotype 2 and Streptococcus suis serotype 7, as well as a preparation method and application thereof. The inactivated vaccine disclosed by the invention is mainly applicable to prevention and treatment of swine Streptococcosis for sows, boars and piglets. The invention discloses separation, identification and application of three strains special for preparing the swine Streptococcosis trivalent inactivated vaccine, wherein the three strains for the vaccine are respectively a Streptococcus suis serotype 2-LT strain (with the collection number being CCTCC NO:M2011282), a Streptococcus suis serotype 7-YZ strain (with the collection number being CCTCC NO:M2011160) and a Streptococcus equi subsp. Zooepidemicus XS strain (with the collection number being CCTCC NO:M2011405). The inactivated vaccine disclosed by the invention can achieve multiple prevention effects with one injection, so that the stresses of swine are reduced, and the inactivated vaccine is safe and reliable and is free from the potential hazards of toxin spreading.
Description
Technical field
The invention belongs to the animal vaccine preparing technical field, be specifically related to tervalence inactivated vaccine and preparation method and the application of a kind of streptococcus equi epizootic disease subspecies+streptococcus suis 2-type+Streptococcus suis 7 types.Vaccine of the present invention mainly is applicable to sow, and boar and piglet are to the control of pig streptococcicosis.
Technical background
In recent years pig streptococcicosis at home and abroad infection rate and the sickness rate of Intensive Farm of Pig Raising day by day improve, the economic loss that causes is day by day serious, has become one of main epidemic disease of harm modernized pig raising industry.Known Streptococcus suis has 33 kinds of serotypes, 2 types wherein can cause pig acute sepsis, meningoencephalitis, arthritis and acute death, also can cause the specific crowd among the live pig practitioner to infect and death, be a kind of important infectious diseases common to human beings and animals (Wisselink etc., 2002).Abroad since nineteen sixty-eight, Holland reported first people infected the meningitis case, existing people more than 300 was dead because of the infected pigs streptococcus.In China, since the seventies, more than 20 the provinces and cities' generation or popular in the whole nation of this disease caused economic loss in various degree.6～August in 2005, it is sick that streptococcus suis 2-type has been broken out in the areas such as Ziyang City, Sichuan Province, Neijiang City, and cause 204 people to infect, 38 people dead (Zhao Zhanqin etc., journal of animal science and veterinary medicine, 2007).The streptococcus suis 2-type disease has become a kind of main epidemic disease of current serious harm China pig industry development, and has also caused serious harm for the mankind in extensively existence and seriously popular of China in recent years; Find simultaneously the pathogenic trend that continuous rising is arranged of Streptococcus suis 7 types, its sickness rate is high, incubation period long, cause price of deed rate to reduce, and has caused the tremendous economic loss to pig industry; And streptococcus equi epizootic disease subspecies (Lan Shi is the C group streptococcus in hiving off) still happen occasionally on China pig farm, and the pig industry of China in serious harm.The commercial goods vaccine only has streptococcus suis 2-type and streptococcus suis 2-type+streptococcus equi epizootic disease subspecies bivalent vaccine at present, but current Streptococcus suis 7 types are also more common, and existing vaccine can not play preventive effect to it, and the commercial goods vaccine needs repeatedly immunity, cost is higher, to pig stress be larger.
Therefore be badly in need of on the market that invention is a kind of new to prevent streptococcus suis 2-type sick simultaneously, the sick and three kinds of disease inactivated vaccines of streptococcus equi epizootic disease subspecies of Streptococcus suis 7 types are to address the above problem.
Summary of the invention
The objective of the invention is to be to provide a kind of Streptococcus suis tervalence inactivated vaccine, this inactivated vaccine is the tervalence inactivated vaccine of streptococcus equi epizootic disease subspecies+streptococcus suis 2-type+Streptococcus suis 7 types, this inactivated vaccine can effectively prevent streptococcus suis 2-type sick simultaneously, Streptococcus suis 7 types disease and three kinds of diseases of streptococcus equi epizootic disease subspecies, and only need pin immunity, reduced pig stress, reduced cost, reach the effect of " how anti-a pin is ", and do not have the hidden danger of loose poison.Inactivated vaccine of the present invention is safe and reliable.
Another object of the present invention is the preparation method that has been to provide a kind of Streptococcus suis tervalence inactivated vaccine.
In order to realize above-mentioned purpose, the present invention by the following technical solutions:
The applicant has prepared a kind of trivalent inactivated vaccine for swine streptococcosis, this inactivated vaccine contains the antigen that preserving number is the streptococcus equi epizootic disease subspecies XS of CCTCC NO:M2011405, and preserving number is the antigen of streptococcus suis 2-LT of CCTCC NO:M2011282 and antigen and the adjuvant of the Streptococcus suis 7-YZ that preserving number is CCTCC NO:M2011160; The deactivation of described antigen; Described adjuvant is white oil.The proportioning of the antigen of above-mentioned three kinds of bacterial strains in described inactivated vaccine is in equal proportions (1: 1: 1), and the cumulative volume of the antigen of described three kinds of bacterial strains accounts for 40%～45% of described vaccine cumulative volume.
The applicant provides a kind of preparation method of trivalent inactivated vaccine for swine streptococcosis, and its step comprises following content:
A. with streptococcus suis 2-LT (preserving number is CCTCC NO:M2011282), Streptococcus suis 7-YZ (preserving number is CCTCCNO:M2011160) and streptococcus equi epizootic disease subspecies XS (preserving number is CCTCC NO:M2011405) enrichment culture (this culture medium is available from U.S. company BD) on the TSB culture medium respectively, obtain respectively streptococcus suis 2-LT bacterium liquid, Streptococcus suis 7-YZ bacterium liquid and streptococcus equi epizootic disease subspecies XS bacterium liquid.
B. respectively to the cultured streptococcus suis 2 of step a-LT strain bacterium liquid, Streptococcus suis 7-YZ bacterium liquid and streptococcus equi epizootic disease subspecies XS bacterium liquid, formalin by total amount (volume ratio) adding 0.3%～0.4%, place 37 ℃ of deactivations 48～72 hours, stirred 1 time every 3～4 hours during this time, collect and obtain each inactivated bacterial liquid.
The streptococcus suis 2 of the deactivation that c. step b collection is obtained-LT bacterium liquid, Streptococcus suis 7-YZ bacterium liquid and streptococcus equi epizootic disease subspecies XS bacterium liquid mixed in 1: 1: 1 by volume, obtain mixing inactivated bacterial liquid, again tween 80 and above-mentioned preparation mixed inactivated bacterial liquid by volume 4～6: 94～96, be preferably 4: 96, make water.Be 94: 6: 1～2 by volume with white oil (available from Exxon Mobil Corporation), Si Ben-80 and aluminium stearate, the ratio that is preferably 94: 6: 1 is made oil phase.With water and oil phase by 40%～45%: 55%～60% volume ratio is mixed, and is preferably 40%: 60%.Water is slowly added the oil phase homogenizing after 3～5 minutes, make even Emulsion, be oil emulsion inactivated vaccine.The preservation condition of trivalent oil emulsion inactivated vaccine of the present invention is: 2～8 ℃.
D, the oil emulsion inactivated vaccine that step c is obtained carry out packing and are product.
The isolation and identification method of above-mentioned antigen bacterial strain is as described below:
1. streptococcus suis 2-type bacterial strain LT strain (streptococcus suis 2-separation LT), screening and identification:
The applicant separates from the painstaking effort that nervous symptoms, acute death pig are arranged of in JIUYUE, 2004 Hubei Province pig farm, Luotian County censorship and obtains a strain and have pathogenic streptococcus suis 2-type bacterial strain (numbering LT strain).After identifying with this bacterial strain called after streptococcus suis 2-LT (Streptococcus suis 2-LT), deliver Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on August 9th, 2011, its deposit number is CCTCC NO:M2011282 (concrete Isolation and Identification method is seen embodiment 1).
2. the separation of Streptococcus suis 7-YZ, screening and identification:
The applicant separates from the pathological material of disease of Yongzhou City, Hubei Province pig farm censorship in August, 2005 and obtains a strain Streptococcus suis 7 type bacterial strains (being numbered the YZ strain), after identifying with this bacterial strain called after Streptococcus suis 7-YZ (Streptococcus suis 7-YZ).This bacterial strain is delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on May 5th, 2011, its deposit number is CCTCC NO:M2011160 (concrete Isolation and Identification method is seen embodiment 2).
3. separation, the screening and identification of streptococcus equi epizootic disease subspecies XS strain
The applicant separates from ill pig joint, pig farm, Xiaoshan, Zhejiang Province and obtains strain streptococcus equi epizootic disease subspecies (being numbered the XS strain), after identifying with these bacterial strain called after streptococcus equi epizootic disease subspecies XS (Streptococcus equi subsp.zooepidimicusXS).This bacterial strain is delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on November 22nd, 2011, its deposit number is CCTCC NO:M2011405 (concrete Isolation and Identification is seen embodiment 3).
The microbial characteristic of above-mentioned three antigen bacterial strains
Streptococcus suis 2-type, Streptococcus suis 7 types and streptococcus equi epizootic disease subspecies bacterial strain are Streptococcus, wherein streptococcus suis 2-type and Streptococcus suis 7 type antibacterials size are 0.1mm-1.0mm, canescence, translucent, smooth surface, circle, neat in edge, and Gram’s staining is positive, becomes two or short chain shape.Streptococcus equi epizootic disease subspecies strain size is about 0.5～1cm, and thalline is oval in the outmoded culture, and this bacterium exists with single or short chain form in the solid medium, and culture forms long-chain in the fluid medium, and the thalline surface energy forms pod membrane.
Streptococcus suis tervalence inactivated vaccine of the present invention is mainly used in that boar and piglet reduce the sickness rate of Pig Farm streptococcicosis to the prevention of pig streptococcicosis in the pig farm, improves the piglet survival rate.
Advantage of the present invention is to prevent simultaneously streptococcus suis 2-type; the disease that Streptococcus suis 7 types and streptococcus equi epizootic disease subspecies cause; immune protective effect for homologous serotype reaches more than 85%; with respect to present commercially available vaccine, can single needle the more serotype of immunity, reach the purpose of " pin is anti-how sick "; and easy to use; reduced pig stress, and do not have the hidden danger of loose poison, safe and reliable.
More detailed technical scheme sees that " specific embodiment " is described.
Description of drawings:
Sequence table SEQ ID NO:1 is the nucleotide sequence of amplification GDH gene.
Sequence table SEQ ID NO:2 is the nucleotide sequence of amplification CPS2J gene.
Sequence table SEQ ID NO:3 is the nucleotide sequence of amplification CPS7J gene.
Sequence table SEQ ID NO:4 is the nucleotide sequence of amplification SodA gene.
Sequence table SEQ ID NO:5 is the nucleotide sequence of amplification M-like protein gene.
Sequence table SEQ ID NO:6 is the nucleotide sequence of amplification Fnz gene.
Fig. 1: a kind of PCR schematic diagram of Streptococcus suis universal primer amplification.Among the figure:
M:DNA molecule Marker; 1～4: Streptococcus suis isolated strains and streptococcus suis 2-type reference culture; 5: Streptococcus suis LT separated strain.
Fig. 2: a kind of PCR schematic diagram of streptococcus suis 2-type primer amplification.Among the figure:
1: streptococcus suis 2-LT separated strain; 2:DNA molecule Marker3～5: Streptococcus suis separated strain; 6: negative control (H2O.
Fig. 3: a kind of PCR schematic diagram of Streptococcus suis universal primer amplification.Among the figure:
1～4: the Streptococcus suis to be identified of picking; 5: Streptococcus suis 7 type reference cultures, 6: Streptococcus suis YZ separated strain.
Fig. 4: the PCR schematic diagram of Streptococcus suis 7 type primer amplifications.Among the figure
1: Streptococcus suis 7-YZ; 2～4: the Streptococcus suis to be identified 5 of picking: negative control.
Fig. 5: streptococcus equi epizootic disease subspecies latex agglutination schematic diagram.Among the figure:
1,2 and 4～6 are respectively A, B, and D, F, G group's standard latex, 3 is C group's standard latex.
Fig. 6: the PCR schematic diagram of streptococcus equi epizootic disease subspecies primer expansion.Among the figure:
M:DNA molecule marker; 1-3: streptococcus equi epizootic disease subspecies bacterial strain to be identified; 4: the streptococcus equi epizootic disease subspecies XS strain of separation
The specific embodiment
In order to make the present invention easier to understand, the below will further set forth embodiments of the invention.The present invention will be further described and demonstration in conjunction with implementing.But the present embodiment is not limitation of the present invention.
Embodiment 1: separation and the evaluation of streptococcus suis 2-LT strain.
Pathological material of disease to be separated---respectively organizing pathological material of disease is that applicant's inventor separates in JIUYUE, 2004 is located away from the Luotian County of Hubei pig farm and has the painstaking effort of nervous symptoms, acute death pig and obtains.Its concrete separation method is: the aseptic collection painstaking effort are inoculated on TSA (available from the U.S. company BD) plate, cultivate after 12-24 hour for 37 ℃ and observe.The picking diameter is that the petite of 0.1mm-1.0mm, canescence, translucent, smooth surface, circle, neat in edge is inoculated in 37 ℃ of shaking table overnight incubation in the TSB fluid medium.Antibacterial after the pure culture is carried out gram stain microscopy.Then use observation by light microscope, Gram’s staining is positive, becomes two or short chain shape coccus, to its purification that goes down to posterity.
The classification of streptococcus suis 2-type is identified, adopts PCR method, biochemical identification and slide agglutination test to identify, concrete steps are as follows:
(1) PCR method is identified: according to the synthetic Streptococcus suis universal primer of list of references (Zhao Zhanqin, 2007) and streptococcus suis 2-type Auele Specific Primer.Wherein Streptococcus suis universal primer JP4 and JP5 be according to the glutamate, Glu dehydrogenase gene (gdh, the gene accession number:
Gb|EF539838.1|) design:
Forward primer is JP4 (5 '-CCATGGACAGATAAAGATGG-3 ');
Downstream primer is JP5 (5 '-GCAGCGTATTCTGTCAAACG-3 '), but amplification length is the target DNA fragment (seeing SEQ ID NO:1) (its PCR picture is seen accompanying drawing 1) of 689bp.
The Auele Specific Primer of streptococcus suis 2-type be according to Streptococcus suis have type specificity capsular polysaccharide antigen gene cps2J (the gene accession number: JN024705.1) design:
Forward primer is cps2J-F (5 '-TGATAGTGATTTGTCGGGAGGG-3 '),
Downstream primer is cps2J-R (5 '-GAGTATCTAAAGAATGCCTATTG-3 '), but amplification length is 557bp target DNA fragment (seeing SEQ ID NO:2) (its PCR picture is seen accompanying drawing 2).
The preparation of pcr template:
Get the bacterium liquid of 1ml cultivation in aseptic 1.5ml centrifuge tube, centrifugal 5 minutes of 12000r/min removes supernatant, with 200 μ l sterilized water suspension mixings, boiled 10 minutes in 100 ℃ of water-baths, then placed rapidly cooled on ice 5 minutes, centrifugal 2 minutes of 12000r/min, supernatant is pcr template.
PCR reaction system (cumulative volume 25 μ l):
10 * Taq Buffer2.5 μ l, 2 μ M dNTPs, 1.5 μ l, each 1 μ l of 20 μ M upstream and downstream primers, TaqDNA polymerase 1 μ l, sterilized water 14 μ l, template 4 μ l.
Annealing temperature is: 94 ℃ 30 seconds, 58 ℃ 60 seconds, 72 ℃ 40 seconds, 30 the circulation.
PCR result observes:
Get pcr amplification reaction product 10 μ l and Mark2000plus 5 μ l, be added in 0.8% (V/V) agarose gel that contains EB, electrophoresis is 30 minutes under 80 volts of voltages, then observes under Burdick lamp.
(2) biochemical identification: from the TSA flat board, be accredited as positive single bacterium colony the picking step (1), carry out conventional biochemical identification test.The micro biochemical assessor operates according to the said firm's product description available from Hangzhou microorganism reagent company limited.Every biochemical indicator of streptococcus suis 2-LT strain all meets (seeing Table 1) as a result.
(3) slide agglutination test: with the streptococcus suis 2 that separates-LT strain inoculation TSB culture medium (available from U.S. company BD), put 37 ℃ of shaking table 170rpm/min, cultivated 8 hours, and made OD600 reach 1.3～1.5, adopt the method for slide coagulation that this bacterium liquid is carried out the serological typing evaluation.Concrete operation method is as follows:
Get 1 of clean slide, drawing 3 μ l Streptococcus suis (SS) standard positive serums (available from Copenhagen, Denmark Statens Seruminstitut) with micropipettor drips on microscope slide, other draws 3 μ l normal saline and does contrast, then draw 3 μ l bacterium liquid and add respectively in serum and the normal saline, fully mixing (annotate: each application of sample all will be changed the TIP head of pipettor).Shake gently microscope slide, observed result behind 1～2min.Obvious visible coagulation piece occurs after serum mixes with bacterium liquid to be checked, liquid becomes transparent, and saline control is dripped and still is even cloudy state namely to be judged to be the agglutination positive.If for even cloudy state, obvious visible coagulation piece do not occur and be judged to be feminine gender after serum mixes with bacterium liquid.Slide agglutination test shows, the streptococcus suis 2 that the present invention separates-LT strain only with streptococcus suis 2-type standard positive serum generation agglutination, not with other serotype coagulations.
The test of table 1 streptococcus suis 2-type biochemical identification
Annotate: 1, VP: glucose phosphate salt peptone water
2, reference culture is the streptococcus suis 2-type reference culture, comes from China Veterinery Drug Inspection Office.
Embodiment 2: the separation of Streptococcus suis 7-YZ and evaluation.
The separation of Streptococcus suis 7 type YZ
Pathological material of disease to be separated---respectively organizing pathological material of disease is that applicant's inventor separates from the pathological material of disease of Yongzhou City, Hubei Province pig farm censorship in August, 2005 and obtains.Its concrete separation method is: aseptic collection painstaking effort, lung tissue, spleen tissue, joint fluid are inoculated on the TSA plate, cultivate after 12-24 hour for 37 ℃ and observe.The picking diameter is that the petite of 0.1mm-1.0mm, canescence, translucent, smooth surface, circle, neat in edge is inoculated in 37 ℃ of shaking table overnight incubation in the TSB fluid medium.Antibacterial after the pure culture is carried out gram stain microscopy.Then use observation by light microscope, Gram’s staining is positive, becomes two or short chain shape coccus, to its purification that goes down to posterity, and called after Streptococcus suis 7-YZ.
The classification of Streptococcus suis 7-YZ is identified
Adopt PCR method, biochemical identification and slide agglutination test to identify, concrete steps are as follows:
(1) PCR method is identified: according to the synthetic Streptococcus suis universal primer of list of references (Zhao Zhanqin, 2007) and Streptococcus suis 7 type specificity primers.Wherein Streptococcus suis universal primer JP4 and JP5 be according to the glutamate, Glu dehydrogenase gene (gdh, the gene accession number:
Gb|EF539838.1|) design:
Forward primer JP4:(5 '-GCAGCGTATTCTGTCAAACG-3 ');
Downstream primer JP5:(5 '-CCATGGACAGATAAAGATGG-3 '), amplification obtains the target DNA fragment that length is 689bp (seeing SEQ ID NO:1) (its PCR picture is seen accompanying drawing 3).
The Auele Specific Primer of Streptococcus suis 7 types be according to Streptococcus suis have type specificity capsular polysaccharide antigen gene cps7 (the gene accession number:
Gb|FJ572226.1|) design:
Forward primer cps7-F:(5 '-AGCTCTAACACGAAATAAGGC-3 '),
Downstream primer cps7-R:(5 '-GTCAAACACCCTGGATAGCCG-3 '), to obtain length be 252bp target DNA fragment (seeing SEQ ID NO:3) (its PCR picture is seen Fig. 4) in amplification.
The preparation of pcr template:
Get 1ml bacterium liquid in aseptic 1.5ml centrifuge tube, centrifugal 5 minutes of 12000r/min removes supernatant, with 200 μ l sterilized water suspension mixings, boiled 10 minutes in 100 ℃ of water-baths, then placed rapidly cooled on ice 5 minutes, centrifugal 2 minutes of 12000r/min, supernatant is pcr template.
PCR reaction system (cumulative volume 25 μ l):
10 * Taq Buffer2.5 μ l, 2 μ M dNTPs, 1.5 μ l, each 1 μ l of 20 μ M upstream and downstream primers, TaqDNA polymerase 1 μ l, sterilized water 14 μ l, template 4 μ l.
Annealing temperature is: 94 ℃ 30 seconds, 56 ℃ 60 seconds, 72 ℃ 40 seconds, 30 the circulation.
PCR result observes:
Get pcr amplification reaction product 10 μ l and Mark2000plus 5 μ l, be added in 0.8% agarose gel that contains EB, electrophoresis is 30 minutes under 80 volts of voltages, then observes under Burdick lamp.
(2) biochemical identification: from the TSA flat board, be accredited as positive single bacterium colony the picking step (1), carry out conventional biochemical identification test.The micro biochemical assessor operates to specifications available from Hangzhou microorganism reagent company limited.Every biochemical indicator of Streptococcus suis YZ all meets (seeing Table 2) as a result.
(3) slide agglutination test: with the TSB culture medium that the Streptococcus suis YZ inoculation that separates is purchased, put 37 ℃ of shaking table 170rpm/min, cultivated 6 hours, make OD600 reach 1.3～1.5, adopt the method for slide coagulation that this bacterium liquid is carried out the serological typing evaluation.Operational approach is as follows:
Get 1 of clean slide, drawing 3 μ l Streptococcus suis (SS) standard positive serums (available from Copenhagen, Denmark Statens Seruminstitut) with micropipettor drips on microscope slide, other draws 3 μ l normal saline and does contrast, then draw 3 μ l bacterium liquid and add respectively in serum and the normal saline, fully mixing (annotate: each application of sample all will be changed the TIP head of pipettor).Shake gently microscope slide, observed result behind 1～2min.Obvious visible coagulation piece occurs after serum mixes with bacterium liquid to be checked, liquid becomes transparent, and saline control is dripped and still is even cloudy state namely to be judged to be the agglutination positive.If for even cloudy state, obvious visible coagulation piece do not occur and be judged to be feminine gender after serum mixes with bacterium liquid.Slide agglutination test shows, Streptococcus suis YZ strain only with Streptococcus suis 7 type standard positive serum generation agglutination, not with other serotype coagulations.
The test of table 2 Streptococcus suis 7-YZ biochemical identification
Annotate: 1, VP: glucose phosphate salt peptone water
2, reference culture is Streptococcus suis 7 type reference cultures, comes from China Veterinery Drug Inspection Office.
Embodiment 3: separation and the evaluation of streptococcus equi epizootic disease subspecies XS
Pathological material of disease to be separated---respectively organizing pathological material of disease is that applicant's inventor is located away to separate in the ill pig joint, Xiaoshan, Zhejiang pig farm April calendar year 2001 and obtains.Its concrete separation method is: take a sample from the pathological material of diseases such as the painstaking effort of doubtful sick pig, lung tissue, spleen tissue, joint fluid in the sterile working, and at first streak inoculation on the TSA solid medium is cultivated the single bacterium colony of picking after 24～48 hours, carried out pure culture again for 37 ℃.The above-mentioned pure culture bacterium colony of picking smear, visible Gram’s staining becomes positive, becomes two or short chain shape coccus.With its called after streptococcus equi epizootic disease subspecies XS.
The classification of streptococcus equi epizootic disease subspecies XS is identified
Adopt latex agglutination test, biochemical identification and PCR method to identify, concrete steps are as follows:
(1) latex agglutination test: with the culture of purification, the single bacterium colony of picking goes down to posterity, cultivated 16-18 hour for 37 ℃, a picking 4-5 colonies typical, with streptococcus latex agglutination standard diagnostics test kit (the enzyme 200 μ l in (Streptococcus Grouping kit is available from OXIOD company product), 37 ℃ of water bath processing 10 minutes, behind the mixing at room temperature with reactant emulsion, and establish blank.The result shows, C group streptococcus separated strain can specificly produce agglutination with latex, bacterium colony only with C group's standard latex generation coagulation, with A, B, D, F, agglutination (seeing Fig. 5) does not all occur in G group's standard latex.
(2) biochemical identification: from the TSA flat board, be accredited as positive single bacterium colony the picking step (1), carry out conventional biochemical identification test.The micro biochemical assessor operates according to the said firm's product description available from Hangzhou microorganism reagent company limited.Every biochemical indicator of streptococcus equi epizootic disease subspecies XS strain all meets (seeing Table 3) as a result.
(3) PCR identifies: according to the synthetic primer SodA gene of C group streptococcus and class M gene and the Fnz gene of evaluation streptococcus equi epizootic disease subspecies identified of list of references (Li Fang, 2009).The primer SodA1 and the SodA2 that wherein identify the C group streptococcus are synthetic according to superoxide dismutase A gene (SodA):
Forward primer SodA1:CAG CAT TCC TGC TGA CAT TCG TCA GG,
Downstream primer SodA2:CTG ACC AGC CTT ATT CAC AAC CAG C, but amplification length is the purpose band (seeing SEQ ID NO:4) of 235bp;
The primer M1 and the M2 that further identify streptococcus equi epizootic disease subspecies are according to the M-like protein gene design,
M1：CTA?TCG?GTG?GTC?GTA?ATG?GAG?A，
M2:ACC TGC CAT AAC TGC AAG AGC T, the purpose that can increase fragment 704bp (seeing SEQ ID NO:5);
Primers F nz1 and Fnz2 are according to the fibronectin binding protein gene design,
Fnz1：GAG?GTG?GAC?CAG?CTT?CAC?CT，
Fnz2:GAG GAA AGA CGG TCC AGA CG, the purpose that can increase fragment 539bp (seeing SEQ IDNO:6).
Amplification is seen Fig. 6.
The preparation of pcr template
With inoculating loop picking list bacterium colony, be inoculated in the 5ml TSB fluid medium 8-10 hour results of 37 ℃ of static cultivations bacterium liquid.Get 1ml bacterium liquid in aseptic 1.5ml centrifuge tube, centrifugal 5 minutes of 12000r/min removes supernatant, with 200 μ l sterilized water suspension mixings, boiled 10 minutes in 100 ℃ of water-baths, then placed rapidly cooled on ice 5 minutes, centrifugal 2 minutes of 12000r/min, supernatant is pcr template.
PCR reaction system (25 μ l)
10 * Taq Buffer2.5 μ l, 2 μ M dNTPs, 4 μ l, TaqDNA polymerase 1 μ l, template 5 μ l are to add primer at 2: 2: 1 with three pairs of primers of above-mentioned design according to concentration ratio M: Fnz: SodA, remainingly supply with distilled water.
Annealing temperature is: 94 ℃ 30 seconds, 56 ℃ 40 seconds, 72 ℃ 40 seconds, 30 the circulation
PCR result observes
Get pcr amplification reaction product 10 μ l and Mark2000plus 5 μ l, be added in 0.8% agarose gel that contains EB, electrophoresis is 30 minutes under 80 volts of voltages, then observes under Burdick lamp.See Fig. 6.
The biochemical test of table 3 streptococcus equi epizootic disease subspecies XS
Annotate: 1, VP: glucose phosphate salt peptone water;
2, reference culture is streptococcus equi epizootic disease subspecies, comes from China Veterinery Drug Inspection Office.
Embodiment 4:
The preparation method of a kind of trivalent inactivated vaccine for swine streptococcosis (streptococcus equi epizootic disease subspecies+streptococcus suis 2-type+Streptococcus suis 7 types), its step is as follows:
This product is to select streptococcus suis 2-LT, Streptococcus suis 7-YZ and streptococcus equi epizootic disease subspecies XS to be inoculated in the upper cultivation of TSB culture medium (the TSB culture medium is available from U.S. company BD), with culture through the formalin deactivation, remove residual toxin, add the white-oil adjuvant configuration and form (seeing following concrete steps), be used for prevention by streptococcus suis 2-type, caused various antibacterials infect for Streptococcus suis 7 types and streptococcus equi epizootic disease subspecies XS strain.This has comprised present popular several most important bacterial disease.Making the strain that product of the present invention uses is streptococcus suis 2-LT (preserving number CCTCC NO:M2011282), Streptococcus suis 7-YZ (CCTCC NO:M2011160) and streptococcus equi epizootic disease subspecies XS (CCTCC NO:M2011405).
Above-mentioned streptococcus suis 2-LT, Streptococcus suis 7-YZ and streptococcus equi epizootic disease subspecies XS are conventional lyophilizing and preserve.
Vaccine is made and the inspection of semifinished product
1. produce the preparation with seed
1.1 the breeding of first order seed
Freeze-drying lactobacillus (streptococcus suis 2-LT, Streptococcus suis 7-YZ and streptococcus equi epizootic disease subspecies XS) is inoculated in the TSA solid medium, putting 37 ℃ cultivated 24 hours, then choose well-grown bacterium colony, inoculation TSA solid medium several, put 37 ℃ and cultivated 12～16 hours, as first order seed.2～8 ℃ of preservations, storage life is no more than 5.Go down to posterity in the TSA culture medium, be no more than for 5 generations.
1.2 the secondary seed breeding is got first order seed and is inoculated in the TSB fluid medium, puts 37 ℃ and cultivates 12～16 hours, by " Chinese veterinary pharmacopoeia " (Chinese agriculture publishing house, version in 2010), appendix is checked purely, and is qualified rear as secondary seed.2～8 ℃ of preservations, storage life is no more than 5.
1.3 the seedling culture medium is the TSB fluid medium.
2 seedling bacterium solution preparations (the antigen preparation of inactivated vaccine)
2.1 antibacterial culturing is cultivated respectively preparation with the bacterium liquid (streptococcus suis 2-LT, Streptococcus suis 7-Y strain and streptococcus equi epizootic disease subspecies XS) of above-mentioned three kinds of serotypes.With the streptococcus suis 2-type of accreditation, Streptococcus suis 7 types and streptococcus equi epizootic disease subspecies secondary seed solution are inoculated in the TSB fluid medium by 1% (volume ratio) respectively, put 37 ℃ and cultivate 12 hours;
2.2 count plate is carried out in check and count of bacteria sampling purely, and purely checks by above-mentioned " Chinese veterinary pharmacopoeia " appendix, should be pure.
2.3 the bacterium liquid that deactivation is up to the standards adds formalin by 0.4% of bacterium liquid total amount (volume ratio), 37 ℃ of deactivations 48 hours, during stirred 1 time every 4 hours, then sampling is carried out deactivation by above-mentioned " Chinese veterinary pharmacopoeia " and is checked, should be without bacterial growth.
2.4 the concentrated centrifugal 10000rpm/min of bacterium liquid that deactivation is up to the standards, centrifugal 5 minutes; Regulate respectively streptococcus suis 2-type bacterium liquid by the count plate result before the deactivation with normal saline again, Streptococcus suis 7 type bacterium liquid and streptococcus equi epizootic disease subspecies XS strain bacterial concentration to 1.125 * 10
10CFU/ml.Steriling test is done in sampling, should be without bacterial growth.
3 join Seedling
3.1 94 parts of Exxon Mobil import white oils (available from Exxon Mobil Corporation) (take milliliter as unit) are got in the preparation of oil phase, add 1 part of aluminium stearate (in grams), the limit edged stirs, until transparent, add again Si Ben-80 6 part (take milliliter as unit), fully mixing in 130 ℃ of sterilizations 30 minutes, is cooled to room temperature for subsequent use.
Mix 3.2 above-mentioned three kinds of bacterium liquid that the preparation of water will concentrate are 1: 1: 1 ratio by volume, add the tween 80 after sterilizing, the limit edged stirs, and to fully dissolving, making its final concentration is 4.0% (volume ratio).
3.3 the volume ratio of emulsifying water and oil phase is 1: 1.5.Water is slowly added the oil phase homogenizing after 3～5 minutes, shear, make even Emulsion.
3.4 the packing quantitative separating, 2～8 ℃ of preservations are put in the sealing of jumping a queue.
1 testing program
Laboratory animal: the healthy ablactational baby pig of 28～35 ages in days; Healthy pregnant 70 age in days sows.
1.1 the safety testing to a single dose inoculation of ablactational baby pig
Respectively by the healthy ablactational baby pig of musculi colli inoculation 28～35 ages in days, 5 of the every batch of vaccine injections were observed 14 with 1 single dose 2ml inoculation for every, and mensuration body temperature with 5 batches of inactivated vaccines of the present invention of preparation.
1.2 the safety testing to ablactational baby pig single dose repeated inoculation
With 5 batches of inactivated vaccines of the present invention respectively by the healthy ablactational baby pig of musculi colli inoculation 28～35 ages in days, 5 of the every batch of vaccine injections, interval every single dose repeated inoculation 2ml on the 21st observed 14, and mensuration body temperature.
1.3 the safety to an overdose inoculation of ablactational baby pig
With 5 batches of inactivated vaccines of the present invention respectively by the healthy ablactational baby pig of musculi colli inoculation 28～35 ages in days, 5 of the every batch of vaccine injections, every overdose inoculation 4ml observed 14.
1.4 the safety testing to a single dose inoculation of in-pig
With 5 batches of inactivated vaccines of the present invention respectively by 70 days healthy sow of musculi colli inoculation gestation, 5 of the every batch of vaccine injections, every with 1 single dose 2ml inoculation, observes its clinical manifestation and farrowing situation.
1.5 the safety testing to in-pig single dose repeated inoculation
With 5 batches of inactivated vaccines of the present invention respectively by 70 days healthy sow of musculi colli inoculation gestation, 5 of the every batch of vaccine injections, interval every single dose repeated inoculation 2ml on the 21st carried out repeated inoculation the 2nd time after 14 days, observed its clinical manifestation and farrowing situation.
1.6 the safety testing to an overdose inoculation of in-pig
Respectively through 70 days healthy sow of musculi colli 5 gestation of inoculation, every pig inoculation twice immunizing dose (4ml) is observed its clinical manifestation and farrowing situation with 5 batches of inactivated vaccines of the present invention.
2. result of the test
2.1 inactivated vaccine of the present invention is to the safety of 1 single dose inoculation of ablactational baby pig
After 5 batches of inactivated vaccines of the present invention are inoculated respectively the healthy ablactational baby pig of 28～35 ages in days with 2ml, 5 of the every batch of vaccine injections, piglet body temperature, breathing situation, mental status, appetite etc. are all normal, have no abnormal changes.Illustrate that this inactivated vaccine is to ablactational baby pig safety fine (table 4).
Mean body temperature behind table 45 batches of inactivated vaccine single doses inoculation of the present invention 28～35 age in days ablactational baby pig
2.2 vaccine is to the safety of ablactational baby pig single dose repeated inoculation
With 5 batches of vaccines respectively by the healthy ablactational baby pig of musculi colli inoculation 28～35 ages in days, 5 of the every batch of vaccine injections, every inoculation 2ml, carry out repeated inoculation after 21 days the 2nd time, the 2ml/ head is observed the clinical manifestation of target animals, observed altogether 14 after each inoculation, and measure body temperature.The result is at whole viewing duration body temperature, breathing, appetite, the mental status normal (table 5).
Table 55 batches of inactivated vaccines of the present invention are to the safety of the healthy ablactational baby pig single dose of 28～35 ages in days repeated inoculation
2.3 inactivated vaccine vaccine of the present invention is to the safety testing of 1 overdose inoculation of ablactational baby pig
Get 5 batches of inactivated vaccines of the present invention respectively by musculi colli with the healthy ablactational baby pig of 4ml dose inoculation 28～35 ages in days, all pigs at whole viewing duration body temperature without obvious rising, breathing, appetite, the mental status be normal (table 6) all, shows that inactivated vaccine vaccine of the present invention is safe to 1 overdose inoculation of ablactational baby pig.
Mean body temperature behind the healthy ablactational baby pig of table 6 inactivated vaccine overdose inoculation of the present invention 28～35 ages in days changes
2.4 inactivated vaccine of the present invention is to the safety (product subcase) of a single dose inoculation of pregnant animal (in-pig)
Respectively by the healthy gestation of musculi colli inoculation sow on the 70th, every batch of inactivated vaccine is injected 5 with 5 batches of inactivated vaccine vaccines of the present invention, and every inoculation 2ml observes clinical manifestation, and measures body temperature.The healthy gestation of as a result 5 batches of inactivated vaccine of the present invention inoculations sows on the 70th are at whole viewing duration body temperature, breathing, appetite, the mental status all normal (table 7), and farrowing is normal, the situations (table 8) such as miscarriage, stillborn fetus occur.
The mean body temperature that single dose of 5 batches of inactivated vaccines of the present invention of table 7 is inoculated behind the healthy in-pig changes
Single dose of the healthy in-pig of table 8 is inoculated the farrowing situation behind the inactivated vaccine of the present invention
2.5 inactivated vaccine of the present invention is to the safety of pregnant animal (in-pig) single dose repeated inoculation
5 batches of inactivated vaccines of the present invention are inoculated healthy in-pig by musculi colli respectively, 5 pigs of every batch of inactivated vaccine injection, every inoculation 2ml, carry out repeated inoculation after 21 days the 1st time, the 2ml/ head carried out the 2nd time repeated inoculation after 14 days, observe the clinical manifestation of target animals (pig), observed altogether 14 after each inoculation, and measure body temperature.The 5 batches of inactivated vaccines of the present invention are respectively by the healthy in-pig of musculi colli with 2ml dose inoculation 3 times as a result, and at whole viewing duration body temperature, breathing, appetite, the mental status all normal (table 9), the institute pig that farrow also all is good for work (table 10).
5 batches of inactivated vaccines of the present invention of table 9 are to the safety of healthy in-pig single dose repeated inoculation
Farrowing situation behind the healthy in-pig single dose of the table 10 repeated inoculation inactivated vaccine of the present invention
2.6 inactivated vaccine of the present invention is to the safety of an overdose inoculation of pregnant animal (in-pig)
The ANOMALOUS VARIATIONS (table 11) that does not occur the body temperature aspect after 1 overdose inoculation of in-pig (4ml), untoward reaction did not all appear within the whole observation period, and last farrowing achievement miscarriage, stillborn fetus and mummy tire do not occur without significant difference yet.5 batches of inactivated vaccines of the present invention of the above results explanation without impact, are safe (tables 12) to in-pig on the reproductive performance of sow.
Overdose of 5 batches of inactivated vaccines of the present invention of table 11 is inoculated the healthy pregnant 70 days mean body temperatures behind the sow and is changed
Overdose of the healthy in-pig of table 12 is inoculated the farrowing situation behind the inactivated vaccine of the present invention
The potency test of embodiment 6 inactivated vaccines of the present invention
1 testing program:
With 42 of the healthy ablactational baby pig of 28～35 ages in days, wherein 21 inactivated vaccine 2ml that each intramuscular injection embodiment 1 makes contain 1 using dosage, and head exempts to carry out in rear 21 days the immunity second time; Remaining 21 not immune matched groups of conduct.Measure animal heat after the immunity, observe clinical manifestation.
Two exempted from rear 14 days, streptococcus suis 2-type, and Streptococcus suis 7 types and streptococcus equi epizootic disease subspecies all adopt the vein counteracting toxic substances, use respectively 2.0 * 10
6CFU streptococcus suis 2-LT strain, 4.0 * 10
9The Streptococcus suis 7 type YZ strains and 5 * 10 of CFU
4The streptococcus equi epizootic disease subspecies XS strain counteracting toxic substances of CFU is observed its clinical symptoms and death condition behind the counteracting toxic substances, calculate the protective rate of inactivated vaccine.
2 result of the tests:
2.1 body temperature situation after the inactivated vaccine inoculation of the present invention
Inactivated vaccine Pigs Inoculated of the present invention is without obviously body temperature reaction, and appetite, spirit normally occur without other visible clinical responses, and the vaccine injection part is without inflammatory reaction (table 13) such as swelling.
Mean body temperature behind the table 13 inactivated vaccine inoculation piglet of the present invention (28～35 age in days) changes
As shown in table 13, the observed result of vaccine after to the healthy ablactational baby pig immunity inoculation of 28～35 ages in days 2ml show, vaccinated pig of the present invention only shows a fervescence of crossing property, and mean body temperature raises and is no more than 1 ℃.In addition, appetite, spirit are all acted normally after all piglet inoculations, without other visible clinical response.
2.2 inactivated vaccine immune efficacy of the present invention is measured
Piglet two was exempted from rear 14 days, used respectively 2.0 * 10
6CFU streptococcus suis 2-LT strain, 7.0 * 10
9The Streptococcus suis 7-YZ strain and 5.0 * 10 of CFU
4The streptococcus equi epizootic disease subspecies XS strain counteracting toxic substances of CFU, the result is as shown in table 14.
Table 14 inactivated vaccine epidemic disease of the present invention immune protective efficiency result
List of references:
[1]Wisselink?H?J，Joosten?J?J，Smith?H?E.Multiplex?PCR?assays?for?simultaneous?detection?of?sixmajor?serotypes?and?two?virulence?associated?phenotypes?of?Streptococcus?suisin?tonsillarspecimens?from?pigs[J].J?Clin?Microbiol，2002，40(8)：2922～2929.
[2] Zhao Zhanqin etc., the streptococcic isolation identification in pig source and biological characteristic research. journal of animal science and veterinary medicine, 2007,38 (4): 367～381.
[3] Li Fang etc., streptococcus equi epizootic disease subspecies multi-PCR detection method set up and use. Agricultural University Of Nanjing's Master's thesis, 2009 (middle National IP Networks: http://epub.cnki.net/grid2008/detail.aspx? QueryID=106﹠amp; CurRec=1).
Claims (4)
1. trivalent inactivated vaccine for swine streptococcosis, it is characterized in that, this inactivated vaccine contains the antigen that preserving number is the streptococcus equi epizootic disease subspecies XS of CCTCC NO:M2011405, and preserving number is the antigen of streptococcus suis 2-LT of CCTCC NO:M2011282 and antigen and the adjuvant of the Streptococcus suis 7-YZ that preserving number is CCTCC NO:M2011160; The deactivation of described antigen; Described adjuvant is white oil.
2. trivalent inactivated vaccine for swine streptococcosis as claimed in claim 1 is characterized in that, the volume ratio of described three kinds of antigens is 1: 1: 1, and the cumulative volume of described antigen is 40%～45% of described inactivated vaccine cumulative volume.
3. the preparation method of a trivalent inactivated vaccine for swine streptococcosis is characterized in that, it comprises the steps:
A. be the streptococcus suis 2-LT bacterial strain of CCTCC NO:M2011282 with preserving number, preserving number is that CCTCC NO:M2011160 Streptococcus suis 7-YZ bacterial strain and preserving number are the streptococcus equi epizootic disease subspecies XS bacterial strain difference enrichment culture of CCTCC NO:M2011405, obtain streptococcus suis 2-LT bacterium liquid, Streptococcus suis 7-YZ bacterium liquid and streptococcus equi epizootic disease subspecies XS bacterium liquid;
B. respectively to above-mentioned cultured streptococcus suis 2-LT bacterium liquid, press the formalin of bacterium liquid cumulative volume adding 0.3%～0.4% in Streptococcus suis 7-YZ bacterium liquid and the streptococcus equi epizootic disease subspecies XS strain bacterium liquid, place 37 ℃ of deactivations 48～72 hours, stirred 1 time every 4 hours during this time, be the bacterium liquid of deactivation;
C. with the streptococcus suis 2 of the deactivation of above-mentioned collection-LT bacterium liquid, Streptococcus suis 7-YZ bacterium liquid and streptococcus equi epizootic disease subspecies XS bacterium liquid by volume are that 1: 1: 1 ratio is mixed, be to mix at 4～6: 94～96 more by volume with tween 80 and above-mentioned mixed bacteria liquid, make water; Be that 94: 6: 1～2 ratio is made oil phase by volume with white oil, Si Ben-80 and aluminium stearate; Be that 1: 1.5 ratio is mixed by volume with described water and oil phase, water was slowly added the oil phase homogenizing after 3～5 minutes, make even Emulsion, be inactivated vaccine;
D. the inactivated vaccine packing that step c is obtained gets product.
4. one kind is exclusively used in the bacterial strain of producing trivalent inactivated vaccine for swine streptococcosis claimed in claim 1, it is characterized in that this bacterial strain is respectively streptococcus suis 2-LT, is deposited in Chinese Typical Representative culture collection center, and its preserving number is CCTCC NO:M2011282; Streptococcus suis 7-YZ, be deposited in Chinese Typical Representative culture collection center, its preserving number is CCTCC NO:M2011160 and streptococcus equi epizootic disease subspecies-XS, is deposited in Chinese Typical Representative culture collection center (CCTCC), and its preserving number is CCTCC NO:M2011405.
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