CN101892175A - Bovine capsular serotype A Pasteurella mutocida, validation identification and application thereof - Google Patents
Bovine capsular serotype A Pasteurella mutocida, validation identification and application thereof Download PDFInfo
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Abstract
The invention discloses bovine capsular serotype A Pasteurella mutocida and application thereof. The bovine capsular serotype A Pasteurella mutocida is separated from disease samples with haemorrhagic septicomia of cattle in six provinces (cities) with the microbial preservation number of CGMCC No.3619. The bovine capsular serotype A Pasteurella mutocida is cultured by BHI, oil adjuvant is adopted to prepare inactivated immunogen, and a mouse model proves that the bovine capsular serotype A Pasteurella mutocida has complete protecting function on serotype A Pm velogenic attck, and does not have crossing protection with serotype B Pm. Testing results prove that vaccine can be prepared by the bovine capsular serotype A Pasteurella mutocida for preventing the haemorrhagic septicomia of cattle caused by the capsular serotype A Pasteurella mutocida. The strain cannot cause diseases when inoculated to chicks, which proves that the strain is chicken isolated capsular serotype A Pasteurella mutocida.
Description
Technical field
The present invention relates to a strain pasteurellosis bacillus, relate in particular to a strain ox source capsular serotype A type pasteurella multocida, the invention still further relates to the application of this ox source capsular serotype A type pasteurella multocida in control ox hueppe's disease, belong to pasteurellosis bacillus cause of disease and vaccine field.
Background technology
(Pasteurella mutocida is the pathogenic bacteria subpopulation with heterogeneous feature Pm) to pasteurella multocida, often causes domestic animals such as ox, pig, chicken, rabbit and the wildlife disease based on septicemia and diseases in respiratory system.Antigen-specific according to the capsular polysaccharide on thalline surface is divided into A, B, D, E and five pod membrane types of F (Capsulartype) with this bacterium.
Ox hueppe's disease (ox goes out to lose) causes by pasteurella multocida, is the ox transmissible disease of feature with sick damage of lung mainly, is generally sporadicly, and under the situation that acutely stress occur with new pod membrane type, to be region popular more.External ox goes out to lose main popular capsular serotype A, B or E type, and China mainly was the pod membrane Type B in the past, so vaccine strain also uses the Type B bacterium always.
Since two thousand six, determined to have taken place A type ox hueppe's disease epidemic situation the cows (comprising beef cattle and milk cow) of China 6 provinces (city).Because cows do not have immunizing power, it is popular that this disease is region in China, and sickness rate is up to 100% under the serious stressed condition, and sick ox lethality rate causes heavy economic losses up to 40%～60% to cattle-raising.Can estimate that this disease will be to one of the most serious transmissible disease of cattle-raising harm in the Future in China 5 to 10 years.
Summary of the invention
One of purpose of the present invention provides a strain ox source capsular serotype A type pasteurella multocida;
Two of purpose of the present invention is that above-mentioned ox source capsular serotype A type pasteurella multocida is applied to prevent and treat the ox hueppe's disease;
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
One strain ox source capsular serotype A type pasteurella multocida, its microbial preservation number are: CGMCC No.3619; Classification name: multocida (Pasteurellamultocida); The preservation time is: on January 29th, 2010; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The present invention is separated to pasteurella multocida (Pm) from the pathological material of disease of acute ox hueppe's disease takes place for Heilungkiang, Tianjin, Jilin, the Inner Mongol, Shandong and Ningxia.With reference to delivering document improvement PCR method, Pm kind conservative gene kmt, pod membrane biosynthesis gene and 16S rRNA gene are carried out pcr amplification and sequencing and analysis.
The present invention redesigns the primer of A type pod membrane biosynthesis gene and 16S rRNA gene.The Auele Specific Primer of the A type pod membrane synthetic gene that is redesigned is: capA1:5 ' TGC CAA AAT CGC AGT CAG TAT TTT TTA TCC3 ' (SEQ ID NO:1); CapA2:5 ' TGC CAT CAT TGT CAG TGA TTT ATT TTG TAA G 3 ' (SEQID NO:2); The special primer sequence of the 16S rRNA gene that is redesigned is: 16sU:5 ' TCA GAT TGA ACG CTG GCG GCA GGC TTA AC 3 ' (SEQ ID NO:3); 16sL:5 ' CAC CCC AGT CAT GAA TCA TAC CGT GGT AA 3 ' (SEQ IDNO:4).
When carrying out the multiplex PCR amplification, the present invention finds by a large amount of tests, with the bacterium liquid of ox source capsular serotype A type pasteurella multocida according to the resulting product of following processing as pcr template, has optimum expanding effect: the culture 250ul that pathogenic bacteria is cultivated 24h with BHI, centrifugal 2 minutes of 12000 * g inhales gently and abandons supernatant, suspends with the concussion of 50uL aseptic double-distilled water, boil 2min, as pcr template.
The multiplex PCR amplified production shows through agarose gel electrophoresis, from amplify two specific fragments with the identical size of positive control the capsular serotype A type pasteurella multocida of isolating ox source, be respectively the pasteurella multocida specificity kmt1 gene of 460bp and the capsular serotype A type biosynthesis gene hyaD-hyaC of 1044bp, and do not amplify any band in the negative control.
PCR product to Pm species specificity gene kmt1 carries out sequential analysis, and each the pod membrane type kmt1 dna homolog among itself and the GenBank among AF016259, AY225341, AY225342, AY225343, AY225344, DQ233648, DQ233649, the EU873317 is all more than 98%; With Clustal W methods analyst, homology has verified further that all more than 96.6% these strain isolateds are pasteurella multocida.The hyaD-hyaC gene of capsular serotype A type specificity is isometric among the capsular serotype A type biosynthesis gene fragment total length 1045bp, itself and AF067175, only at 562 C → T same sense mutations takes place, and homology is 99.9%; And with GenBank in to belong to the homology of AF169324 bcbD gene, AF302465 dcbF gene, AF302466 ecbJ gene, AF302467 fcbD gene of other pod membrane type very low, between 20.76%-35.78%.Pcr amplification and sequential analysis show that all the present invention is capsular serotype A type bacterium from the isolating Pm of above-mentioned six provinces (city).
16S rRNA genic system evolutionary analysis shows, strain isolated of the present invention (representative strains is Pm-TJ) is the highest with Britain's capsular serotype A type strain isolated PM338 homology, reach 99.93%, and with the homology of other ox source capsular serotype A type Pm decline is arranged slightly, but apparently higher than with the homology of other animal-origin capsular serotype A type Pm; And the homology of pod membrane B, D, E, F type Pm and the A type Pm in various different animals source all is starkly lower than the homology between the A type Pm.
In addition, the present invention finds that by a large amount of tests when the ox source capsular serotype A type pasteurella multocida of adopting the present invention of BHI culture medium culturing to be separated to, it is unattenuated to keep the bacterial strain virulence.
Adopt oily adjuvant to be prepared as the deactivation immunogen after BHI cultivates ox of the present invention source capsular serotype A type pasteurella multocida, proof has provide protection completely to A type Pm strong virus attack on mouse model, but does not have cross protection with Type B Pm.Test-results shows that ox of the present invention source capsular serotype A type pasteurella multocida can be prepared into vaccine and be applied to prevent the caused ox hueppe's disease of capsular serotype A type pasteurella multocida.
Ox of the present invention source capsular serotype A type pasteurella multocida inoculation chick is not pathogenic, and test-results shows that bacterial strain of the present invention is the capsular serotype A type pasteurella multocida of non-Ji Yuan.
Description of drawings
Fig. 1 China different zones ox source capsular serotype A type Pm strain isolated kmt1 gene and hyaD-hyaC gene multiplex PCR detect.A: Heilungkiang; B: Tianjin; C: Jilin; D: Ningxia; E: the Inner Mongol; F: Shandong; G: positive control; H: negative control; L:250bpDNA ladder.
The 16S rRNA genic system evolutionary tree of Fig. 2 Pm.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Separation and the evaluation of embodiment 1 ox of the present invention source capsular serotype A type pasteurella multocida
1.1 Pathogen Isolation and evaluation: the cause of disease material is respectively from lungs, liver, heart, kidney, respiratory tract and the intestinal tissue of Heilungkiang, Jilin, Ningxia, Shandong, the Inner Mongol, Tianjin and Guizhou morbidity ox.
After the pathological material of disease grinding, be made into 1: 10 suspension with aseptic PBS, 4 ℃ are spent the night, and get 3 of supernatant abdominal cavity inoculation small white mouses.With the centrifugal 10 minutes supernatant of suspension 12,000 * g, 3 of abdominal cavity inoculation small white mouses.After suspension was centrifugal, supernatant was through the bacteria-free filtrate of 0.2um membrane filtration, and 3 of small white mouses are inoculated in the abdominal cavity.2 of PBS contrasts.Every small white mouse inoculum size is 0.2ml, observes incidence day by day.Lungs, liver, heart and the blood of the dead mouse of aseptic collection 24～48h, the contact dyeing microscopic examination, and pathological material of disease is inoculated in the BHI substratum increases bacterium.The BHI culture inoculation blood agar of 24h, BHI agar, maconkey agar flat board, 37 ℃ of 24h cultivate the back and observe colonial morphology.Random choose list colony inoculation BHI, pure culture 24h, smear staining microscopy.With BHI 24h culture, shift being inoculated in the biochemical reaction pipe, according to sky, Hangzhou and microorganism reagent company limited " bacterium trace biochemical reaction pipe working instructions ", observing response result (Marvin's dagger-axe etc., Chinese Preventive Veterinary Medicine newspaper, 2008).
All can detect the Gram-negative bacillus pumilis in the pathological material of disease of various places, methylene blue and Wright's staining the two poles of the earth are dense dyes.With 3 mouse of pathological material of disease suspension abdominal cavity inoculation in 1: 10, dead in the 24～48h of inoculation back; The mouse that supernatant, 0.2um ultrafiltrated and PBS with the pathological material of disease suspension after centrifugal inoculates respectively, strong living in the 120h.Mouse lung, liver, heart tissue increase fungus in BHI, 24h on the blood agar plate grows the bacterium colony of rounded protuberance, smooth moistening, neat in edge, little blue flash of light, no haemolysis; On the Mai Kangkai flat board, do not grow; Riotous growth on the BHI agar plate forms the bacterium colony of circular flat, smooth moistening, neat in edge.The smear staining microscopy presents the Gram-negative tyrothricin, and methylene blue and Wright's staining can be seen the dense thalline that dyes in the two poles of the earth.Heilungkiang is identical with the cultural characters of Tianjin pathological material of disease isolate.Isolate glucose fermentation, fructose, N.F,USP MANNITOL and sorbyl alcohol; Maltose, lactose, rhamnosyl and galactitol test are negative; The hydrogen peroxide enzyme positive; The ornithine decarboxylase positive; The indole test is positive; Reduction nitrate; Produce hydrogen sulfide.
1.2 cause of disease is to the virulence of mouse: preliminary study shows, the single bacterium colony on the picking BHI agar plate, inoculation BHI are cultivated the 18-24h cultures in 37 ℃, and 3 mouse of every inoculation 100,000,000 bacterium are in 6～9h death; 3 mouse of every inoculation 0.1 hundred million bacterium are in 11～36h death; 3 mouse of every inoculation 0.01 hundred million bacterium are in 32～71h death.Strong the living of contrast mouse 120h.By optimizing culture condition, this bacterium strengthens significantly to the mouse virulence, and two groups of mouse that inoculate 100,10 bacterium respectively are all dead in 24～48 hours, and the mouse of 1 bacterium of inoculation had 1/3 generation dead in 48～72 hours.From dead mouse lungs, the liver bacterial isolate of falling ill, in BHI, inoculate mouse according to above-mentioned pathogen separation method after the pure culture once more, show same virulence.
1.3, the PCR authentication method
(1) PCR design of primers: with reference to Pm specific specificity gene primer Kmt1, A, B, D, E, F pod membrane serotype specificity gene primer capA, capB, capD, capE, capF (Townsend etc., Journal of Clinical Microbiology, 2001), and 16S rRNA gene (Davies R L, Microbiology, 2004), the primer of A type pod membrane biosynthesis gene and 16S rRNA gene is redesigned.The A type pod membrane synthetic gene primer of redesign: capA1:5 ' TGC CAA AAT CGC AGT CAGTAT TTT TTA TCC3 '; CapA2:5 ' TGC CAT CAT TGT CAG TGA TTTATT TTG TAA G 3 '; The 16S rRNA gene primer sequence 16sU:5 ' TCA GAT TGA ACG CTG GCG GCA GGC TTA AC 3 ' of redesign; 16sL:5 ' CAC CCCAGT CAT GAA TCA TAC CGT GGT AA 3 '.
(2) sample preparation and PCR method: with pathogenic bacteria BHI culture, as each 250ul of ox oral secretion of negative control, centrifugal 2 minutes of 12000 * g abandons supernatant, suspends with the concussion of 50ul aseptic double-distilled water, as pcr template.50ul PCR reaction system is formed: distilled water 36ul, 5 times of Prime Star damping fluid 10ul, 10mmol/ldNTP 1.5ul, each 1.0ul of 10umol/l upstream and downstream primer, Prime Star TaqDNA polysaccharase 1.0ul, template 0.5ul.The pcr amplification reaction condition is: 95 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ of totally 30 circulations in 45 seconds; 72 ℃ of extensions in 10 minutes.The PCR product detects through 1% agarose gel electrophoresis.
1.4PCR somatotype and evaluation: the bacterium liquid of cultivating with BHI is template, adopts 1.3 described multiple PCR methods to increase; From the pathological material of disease of above-mentioned six provinces and cities, all amplify less than two specific bands about 500bp and 1000bp with above-mentioned multiple PCR method; Kmt1, kmt1+capA, kmt1+capB, kmt1+capD, kmt1+capE, kmt1+capF combination of primers, have only kmt1+capA to amplify simultaneously less than two specific bands about 500bp and 1000bp, other combinations only amplify the specific band (Fig. 1) less than 500bp.With from the pathological material of disease of above-mentioned six provinces and cities, the increase 16S rRNA gene fragment of 1468bp of PCR method.Sequence alignment shows that the 16S rRNA dna homolog of all strain isolateds is 100%.
1.5kmt1 gene sequencing and homology analysis: clone's kmt1 gene fragment is carried out two-way order-checking with the terminal cessation method of two deoxidations.Fragment total length less than 500bp is 460bp, the Blast Search Results shows, each the serotype kmt1 dna homolog among itself and the GenBank among AF016259, AY225341, AY225342, AY225343, AY225344, DQ233648, DQ233649, the EU873317 is all more than 98%; With Clustal W methods analyst, homology is all more than 96.6%.
1.6 the pod membrane synthetic gene sequence is measured and homology analysis
Capsular serotype A type biosynthesis gene fragment total length 1045bp, isometric with the hyaD-hyaC gene of capsular serotype A type specificity among the AF067175, at 562 C → T same sense mutation takes place only, homology is 99.9%; And with GenBank in to belong to AF169324 bcbD gene, AF302465 dcbF gene, AF302466 ecbJ gene, the AF302467 fcbD dna homolog of other serotypes very low, between 20.76%-35.78%.
1.716S rRNA gene sequencing and phylogenetic analysis
The 16S rRNA gene fragment order comparison of amplification 1468bp shows that the 16S rRNA dna homolog of all strain isolateds is 100% from the pathological material of disease of above-mentioned six provinces and cities.Select the originate 16S rRNA gene order of various serotype Pm of different animals among the GenBank, together set up systematic evolution tree, the results are shown in Figure 2 with the 16S rRNA gene of we isolating capsular serotype A type Pm.
The immunogenicity test of test example 1 ox of the present invention source capsular serotype A type pasteurella multocida
1.1 immunogen preparing: 1 isolating Pm-TJ the 12nd of embodiment bacteria liquid of being commissioned to train is coated the BHI agar plate, cultivate about 24h for 37 ℃, be paved with whole flat board, collect lawn and make 10 until lawn
10CFU/mL bacterium liquid also adds 0.2% formaldehyde in 37 ℃ of deactivation 24h, with French adjuvant (the MONTANIDE ISA 15A VG) thorough mixing of 15% volume, and the preparation immunogen.
1.2 immunization protocol and attack poison protection result: get 15 SPF level mouse, be divided into three groups of A, B, C, 5 every group.The immunogen that A winding kind 1.1 is prepared, dosage is 2.5 * 10
8CFU/ only; B winding kind Type B ox goes out to lose deactivation vaccine, and dosage is 2.5 * 10
8CFU/ only; 5 mouse of C group are cooked blank.Attack poison with Pm-TJ the 12nd generation bacterium liquid after 21 days, observed 3, the statistics protection ratio.Pm-TJ and Type B ox with deactivation goes out to lose deactivation vaccine immune mouse respectively respectively, attacks A type Pm after 21 days, counts its protection ratio, and result's (table 1) shows that it is 100% that deactivation A type Pm group is attacked malicious protection ratio, and the Type B ox goes out to lose the deactivation vaccine group and the blank group is all dead.
Table 1 mouse immune protection test result
| ? | The A group | The B group | The C group |
| Mouse quantity | ?5? | ?5? | 5? |
| Immunogen | A type pm inactivated vaccine | Type B pm inactivated vaccine | Blank |
| Immunizing dose (CFU) | ?2.5×10 8 | ?2.5×10 8 | -? |
| Attack toxic agent amount (CFU) | ?50? | ?50? | 50? |
| Mortality ratio | ?0/5? | ?5/5? | 5/5? |
| Protection ratio | ?100％? | ?0％? | 0％? |
1.3 ox of the present invention source capsular serotype A type pasteurella multocida is pathogenic to chick:
Owing to cause that the pathogenic bacteria of fowl cholera also is A type Pm, so the present invention has carried out the pathogenic test of chick, 93 age in days SPF chick are divided into three groups of A, B, C, 3 every group, respectively 1,10,100 Pm-TJ of abdominal injection inoculation the 12nd generation viable bacteria, observe onset state day by day.
The chick pathogenicity test results of table 2 Pm-TJ
| ? | The A group | The B group | The C group |
| Attack toxic agent amount (CFU) | 1? | 10? | 100? |
| Mortality ratio | 0/3? | 0/3? | 0/3? |
Test-results shows (table 2), and A, B, three groups of chick of C inoculate 1,10,100 Pm-TJ viable bacteria respectively, and all healthy survival of all chick shows that China emerging ox source A type Pm and fowl source Pm are irrelevant after 3 days.
Sequence table
＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture
＜120〉ox source pod membrane serum A type pasteurella multocida and isolation identification and application
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Claims (7)
1. a strain ox source capsular serotype A type pasteurella multocida, its microbial preservation number are: CGMCC No.3619.
2. the described ox of claim 1 source capsular serotype A type pasteurella multocida is preparing the purposes of preventing and treating in the medicine that is caused the ox hueppe's disease by ox source capsular serotype A type pasteurella multocida.
3. a vaccine composition of preventing and treating the ox hueppe's disease is made up of the described ox of the claim 1 of significant quantity source capsular serotype A type pasteurella multocida and adjuvant.
4. according to the described vaccine of claim 3, it is characterized in that: described adjuvant is oily adjuvant.
5. primer that is used to identify the A type pod membrane synthetic gene of the described ox of claim 1 source capsular serotype A type pasteurella multocida, it is characterized in that: its base sequence is shown in SEQ IDNO:1 and the SEQ ID NO:2.
6. primer sequence that is used to identify the 16S rRNA gene of the described ox of claim 1 source capsular serotype A type pasteurella multocida, it is characterized in that: its base sequence is shown in SEQ IDNO:3 and the SEQ ID NO:4.
7. a method of cultivating the described ox of claim 1 source capsular serotype A type pasteurella multocida comprises: adopt the BHI substratum to cultivate.
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| CN103513031A (en) * | 2012-06-21 | 2014-01-15 | 普莱柯生物工程股份有限公司 | Establishing method and application of pasteurella multocida indirect haemagglutination assay |
| CN106755462A (en) * | 2017-01-11 | 2017-05-31 | 南阳师范学院 | A kind of ring mediated isothermal amplification detection primer composition of ox pasteurella multocida, detection box and its application |
| CN107569681A (en) * | 2017-08-30 | 2018-01-12 | 中国农业科学院哈尔滨兽医研究所 | A kind of ox pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof |
| CN111621450A (en) * | 2020-07-10 | 2020-09-04 | 青岛易邦生物工程有限公司 | Duck-source gallibacterium and application thereof |
| CN112760394A (en) * | 2021-02-23 | 2021-05-07 | 福建省农业科学院畜牧兽医研究所 | Multiplex PCR primer for identifying avian pasteurella multocida and serotypes thereof |
| CN116059333A (en) * | 2021-10-19 | 2023-05-05 | 西南大学 | Bovine origin A type Pasteurella multocida gene deletion strain and application thereof |
| WO2023231250A1 (en) * | 2022-06-02 | 2023-12-07 | 金宇保灵生物药品有限公司 | Bovine pasteurella multocida capsular type a capsular polysaccharide vaccine and preparation method therefor |
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2010
- 2010-02-26 CN CN2010101155529A patent/CN101892175B/en not_active Expired - Fee Related
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| CN103513031A (en) * | 2012-06-21 | 2014-01-15 | 普莱柯生物工程股份有限公司 | Establishing method and application of pasteurella multocida indirect haemagglutination assay |
| CN103513031B (en) * | 2012-06-21 | 2016-08-31 | 普莱柯生物工程股份有限公司 | The method for building up of a kind of pasteurella multocida indirect hemagglutination test method and application thereof |
| CN106755462A (en) * | 2017-01-11 | 2017-05-31 | 南阳师范学院 | A kind of ring mediated isothermal amplification detection primer composition of ox pasteurella multocida, detection box and its application |
| CN107569681A (en) * | 2017-08-30 | 2018-01-12 | 中国农业科学院哈尔滨兽医研究所 | A kind of ox pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof |
| CN111621450A (en) * | 2020-07-10 | 2020-09-04 | 青岛易邦生物工程有限公司 | Duck-source gallibacterium and application thereof |
| CN111621450B (en) * | 2020-07-10 | 2022-07-05 | 青岛易邦生物工程有限公司 | Duck-source gallibacterium and application thereof |
| CN112760394A (en) * | 2021-02-23 | 2021-05-07 | 福建省农业科学院畜牧兽医研究所 | Multiplex PCR primer for identifying avian pasteurella multocida and serotypes thereof |
| CN116059333A (en) * | 2021-10-19 | 2023-05-05 | 西南大学 | Bovine origin A type Pasteurella multocida gene deletion strain and application thereof |
| CN116059333B (en) * | 2021-10-19 | 2024-01-09 | 西南大学 | Bovine origin A type Pasteurella multocida gene deletion strain and application thereof |
| WO2023231250A1 (en) * | 2022-06-02 | 2023-12-07 | 金宇保灵生物药品有限公司 | Bovine pasteurella multocida capsular type a capsular polysaccharide vaccine and preparation method therefor |
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