CN109609418A - One plant of erysipelothrix porci and its application - Google Patents

One plant of erysipelothrix porci and its application Download PDF

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CN109609418A
CN109609418A CN201910095766.5A CN201910095766A CN109609418A CN 109609418 A CN109609418 A CN 109609418A CN 201910095766 A CN201910095766 A CN 201910095766A CN 109609418 A CN109609418 A CN 109609418A
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erysipelothrix
porci
erysipelothrix porci
vaccine
bacterium
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CN109609418B (en
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徐引弟
王治方
张青娴
朱文豪
许峰
李海利
郎利敏
焦文强
张立宪
游
游一
王克领
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Institute of Animal Husbandry and Veterinary Medicine of Henan Academy of Agricultural Sciences
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Abstract

The invention discloses one plant of 1a type erysipelothrix porci and its applications.The deposit number of the erysipelothrix porci is CGMCC NO:16808, is signed as erysipelothrix porci HN1573.The strain isolation is from the painstaking effort for the growing and fattening pigs that acute sepsis death occurs, and when separating on TSA solid medium without other bacterial growths, only erysipelothrix porci is grown, and viral titer is high in TSB fluid nutrient medium, up to 109 CFU/mL or more；Using the vaccine for the bacterial strain HN1573 preparation that the present invention filters out, preparation process is simple, has good preventive effect to swine erysipelas.

Description

One plant of erysipelothrix porci and its application
Technical field:
The invention belongs to veterinary biological product technical fields, and in particular to one plant of erysipelothrix porci and its application.
Background technique:
Erysipelothrix porci (Erysipelothrix rhusiopathiae) is that a kind of small-sized, elongated, direct rod shape leather is blue Family name's positive bacteria can cause pig and many other animals erysipelas occur.Brickpox is usually expressed as acute or subacute septicemia With INVENTIONChronic proliferative lesion.Heart is main target organ, is usually expressed as significantly damaging.This disease of brickpox occurs All over the world, there is important economic significance in Europe, Asia, Australia and America.Erysipelothrix porci is also the one of the mankind Kind infecting both domestic animals and human encephalapthy agent, is skin disease-erysipeloid cause of disease.It is red that attenuated live vaccine and bacterium are already used to control pig Poison.However, the failure of attenuated vaccine is frequent occurrence, violent reaction or death are often caused after injection.Brickpox it is high-incidence still It is the problem of pork industry is paid close attention in world wide.In addition, the antibody that vaccine inoculation generates is difficult and wild strain Erysipelothrix sense Antibody caused by contaminating distinguishes.Therefore, it is necessary to develop new, more effective vaccine.
Erysipelothrix serotype is numerous, and what is had confirmed that so far has 26 serotypes (i.e. 1a, 1b, 2-24, N), different serum There are notable differences for the virulence of type Erysipelothrix bacterial strain, and the pathogenicity of 1a, 1b type is most strong, and the bacterial strain from septicemia case is mostly 1a type, the bacterial strain from rash block type case is mostly 2 types.Due to 1a type cause more in big pig acute sepsis it is dead, so once This disease, which occurs, would become hard to control, to cause great economy property loss to raiser, bring to the development of China's pig breeding industry huge Big difficulty.The many drugs formerly researched and developed can play good therapeutic effect to erysipelothrix porci disease, but with cultivation Scale development, the use of a large amount of antibacterials, the unreasonable use of off-drug period drug all cause the drug resistance phenomenon of pathogen more next It is more serious, it is increasingly detrimental to the prevention and treatment of clinical disease.Therefore needing to research and develop a kind of new has preferable immunogenicity Bacterial strain preparation vaccine.
Summary of the invention:
The treatment of current swine erysipelas there are aiming at the problem that, the present invention provides one plant of 1a type brickpox bacterium and its application.
The present invention solves scheme used by its technical problem: a kind of erysipelothrix porci, and the erysipelothrix porci is Erysipelothrix porci (Erysipelothrix rhusiopathiae) HN1573, deposit number: CGMCC NO:16808, preservation day Phase: on November 09th, 2018, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation Location: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
A kind of above-mentioned erysipelothrix porci, the erysipelothrix porci are 1a type erysipelothrix porci.
The present invention also provides a kind of application of erysipelothrix porci in the drug of preparation prevention and control swine erysipelas.
A kind of application of the above-mentioned erysipelothrix porci in the drug of preparation prevention and control swine erysipelas, the drug are vaccine.
A kind of erysipelothrix porci inactivated vaccine, which is made of erysipelothrix porci serotype antigen and adjuvant, described Erysipelothrix porci serotype antigen is the HN1573 plants of antigens of erysipelothrix porci inactivated.
A kind of above-mentioned erysipelothrix porci inactivated vaccine, the adjuvant are aluminum hydroxide adjuvant.
A kind of above-mentioned erysipelothrix porci inactivated vaccine, the erysipelothrix porci bacterium amount are 1 × 109CFU/mL。
Any of the above-described kind of erysipelothrix porci inactivated vaccine the preparation method comprises the following steps: erysipelothrix porci is inoculated into the training of TSB liquid Support base, be placed in 37 DEG C of shaking tables and cultivate, collect culture after 18h, measure concentration, adjust the bacterium number in culture be 2 × 109Formaldehyde is added to final concentration of 0.2%, in 37 DEG C of inactivation 12h in CFU/mL；Hydrogen is added in 9:1 to bacterium solution after inactivation by volume Aluminum adjuvant is mixed to prepare erysipelothrix porci inactivated vaccine.
Beneficial effects of the present invention:
1, the bacterial strain HN1573 in the present invention is isolated from the painstaking effort that the growing and fattening pigs of acute sepsis death occur, solid in TSA Without other bacterial growths when separating on body culture medium, only erysipelothrix porci is grown, the viral titer in TSB fluid nutrient medium Height, up to 109CFU/mL or more.
2, the bacterial strain HN1573 in the present invention has stronger pathogenicity to child care pig, causes the morbidity of child care pig dead, tool There is good immunogenicity.
3, according to the present invention in the vaccine of bacterial strain HN1573 preparation there is preferable protection to the erysipelothrix porci disease of pig Effect.
Detailed description of the invention:
Fig. 1 is bacterial strain HN1573 colonial morphology.
Fig. 2 is form under bacterial strain HN1573 thallus 100 × microscope of Gram's staining.
Fig. 3 is the PCR qualification result of bacterial strain HN1573, and the Marker in figure is DL 2000bp, is followed successively by from top to bottom 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp；Swimming lane 1 is that primer P1, P2 expand HN1573 plants of 16S rRNA bases Because of result；Swimming lane 2 is that primer P1, P2 expand vaccine strain G4T10 plants of 16S rRNA genetic results；Swimming lane 3 is primer P1, P2 amplification C500 plants of 16S rRNA genetic results of salmonella.
Fig. 4 is the serotype PCR qualification result of bacterial strain HN1573, and the Marker in figure is DL 2000bp, from top to bottom according to Secondary is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp；Swimming lane 1 is that primer P3, P4 expand HN1573 plants Glycerol-3-phosphatecytidylyltransferase genetic results；Swimming lane 2 is that primer P3, P4 expand vaccine strain G4T10 plants of glycerol-3-phosphate cytidylyltransferase genetic results；Swimming lane 3 is primer P3, P4 amplification C500 plants of results of salmonella.
Specific embodiment:
The present invention is described in more detail with reference to the accompanying drawings and examples.
The pathological material of disease of erysipelothrix porci of the present invention is adopted from August, 2018 on Henan Province, large-scale pig farm, Yuanyang County The painstaking effort of the 6 monthly ages growing and fattening pigs of the generation acute sepsis death of collection.
Embodiment 1: the separation and identification of bacterial strain HN1573
The preparation of 1.1 culture mediums
TSB fluid nutrient medium: TSB gravy powder (Tryptic Soy broth, pancreas peptone soybean broth) 30g is dissolved in In 1000mL ultrapure water, 115 DEG C of sterilizing 15min add fetal calf serum 50mL；
TSA solid medium: TSA agar powder (Tryptic Soy Agar, tryptose soya agar) 40g is dissolved in In 1000mL ultrapure water, 115 DEG C of sterilizing 15min add fetal calf serum 50mL；It is saved backup in 4 DEG C.
1.2 bacterial strains are separately cultured
The painstaking effort of aseptic collection sick pig are inoculated on TSA solid medium, cultivate 24-36h in 37 DEG C of constant incubators, long Consistent tip-like size, colorless and transparent, round, smooth, neat in edge bacterium colony out (see Fig. 1).After purifying inoculation, it is blue to carry out leather Albert'stain Albert, the bacterium are gram-positive bacteria, straight or slightly bent microbacterium (microbacteria), catenation or Cheng Changsi (see Fig. 2).From bacterial strain Morphology and biochemical characteristic result preliminary judgement are erysipelothrix porci category, are named as bacterial strain HN1573.
The identification of 1.3 bacterial strains
1.3.1 design of primers
According to erysipelothrix porci according to Erysipelothrix 16S rRNA gene (NO.M23728) sequence, design a pair is general to draw Object, for the amplification of erysipelothrix porci, primer sequence is as follows:
P1:5 '-AGATGCCATAGAAACTGGTA-3 ' (SEQ ID NO.1)
P2:5 '-CTGTATCCGCCATAACTA-3 ' (SEQ ID NO.2)
Amplified fragments size is 407bp.
According to the sequence design of erysipelothrix porci glycerol-3-phosphate cytidylyltransferase gene The specific primer of a pair of of erysipelothrix porci 1a type, for the amplification of erysipelothrix porci 1a type, primer sequence is as follows:
P3:5 '-CTCCTAACGCTTTAGCACGC-3 ' (SEQ ID NO.3)
P4:5 '-TGATCCTTTGCCACTAATGC-3 ' (SEQ ID NO.4)
Amplified fragments size is 356bp.
1.3.2PCR identification
The DNA of bacterial strain HN1573 is extracted according to the operating procedure of DNA extraction kit, spectrophotometric determination concentration is 100ug/mL carries out PCR identification.
Pcr amplification reaction system is 25 μ L:10 × buffer, 2.5 μ L, 2.5mM dNTPs0.5 μ L, 10 μM/L universal primer DdH is added in each 1 μ L, 100ug/mLDNA template of 1 μ L, 5U/ μ L rTaq, 1 μ L of P1, P22O to 25 μ L.G4T10 plants of vaccine strain are Positive control, C500 plants of salmonella are negative control.
Reaction condition: 95 DEG C of initial denaturation 2min, into circulation: 94 DEG C of 30s, 60 DEG C of 30s, 68 DEG C of 1min, totally 35 recycle, Last 72 DEG C of extensions 10min, 4 DEG C of PCR product preservations.
Amplified production carries out electrophoresis respectively, and every hole is loaded 10 μ L, 1% agarose gel electrophoresis, observes result under ultraviolet light (see Fig. 3).As a result 407bp segment is amplified, is sequenced and erysipelothrix porci WH13013 plants (GenBank NO:CP017116) 100% is homologous, and sequencing result is as follows, it was demonstrated that bacterial strain HN1573 is erysipelothrix porci (Erysipelothrix rhusiopathiae)。
AGATGCCATAGAAACTGGTAGACTAGAGTGCAGGAGAGGTTAGTGGAATTCCATGTGTAGCGGTAAAA TGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTAACTGGCCTGTAACTGACGCTGAGGCTCGAAAGCGT GGGGAGCAAATAGGATTAGATACCCTAGTAGTCCACGCCGTAAACGATGGATACTAAGTGTTGGAGAAATTCAGTG CTGTAGTTAACGCAATAAGTATCCCGCCTGGGGAGTATGCGCGCAAGCGTAAAACTCAAAGGAATTGACGGGGGCC CGCACAAGCGGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATACCGCGCAAA AGCACAGAGATGTGTAATAGTTATGGCGGATACAG(SEQ ID NO.5)
1.3.3 Serotype Identification
Referring to 1.3.2PCR identification method, is expanded with specific primer P3, P4 of erysipelothrix porci 1a type, as a result amplified 356bp size segment (see Fig. 3), sequencing result is as follows, with WH13013 plants of erysipelothrix porci (GenBank NO:CP017116) 100% is homologous, it was demonstrated that bacterial strain HN1573 is 1a type erysipelothrix porci.
CTCCTAACGCTTTAGCACGCTTTAGAATATTAATATGACCTTGATGTAATAAATCAAACGTACCGTAT GTTATAACTTTTTTCATTTGTTTACTCCTCCACTAATTAATTTGATGACTATTACACTTTATCCTCTCTACTAGAC TAAAATTTAACTATTTGCTAATAGTTCCAATATTTTCGAAAAAGTCGATAAATCTTTCAGAGGATTTATTGTCCAT ATATTTATTCACCTTATGATTGACTTCTTCTCTCTTACTTTTGAAACTATCGACTTCTTCTAATACATTTTCGAAG AATTTAATTAAACCATCTAAATCAGTTATCTTATAACCCGGCATTAGTGGCAAAGGATCA(SEQ ID NO.6)
1.4 virulence test
Experimental animal is piglet 10 of 30 ages in days wean health.Experimental animal is divided into two groups, and control group 5, test group 5 Only.Bacterial strain HN1573 is inoculated with TSB fluid nutrient medium, after 37 DEG C of shaking table culture 18h, measures cell concentration, continuous 10 times of dilutions Applying solid plate, count plate under low power lens calculate stoste cell concentration, adjust to 2.5 × 108CFU/mL, test group are dynamic Object passes through neck inoculated with subcutaneous injections by HN1573 plants of liquid cultures of neck inoculated with subcutaneous injections 2mL, control animals The TSB fluid nutrient medium of 32mL sterilizing.Situations such as being observed continuously after injection 2 weeks, observing and recording its morbidity number and death toll.
Observation discovery: test group animal shows high fever in inoculation HN1573 afterwards for 24 hours, and body temperature reaches 42-43 DEG C, delays not It moves back, sleeping ground, eat, the symptoms such as mucous membrane cyanosis, it is all dead in 3 days；Control animals body temperature between experimental period is normal and without bright Aobvious clinical symptoms.
It cuts open after the test and kills test group animal, while cuing open and killing 5 control animals；Pathological material of disease is acquired, brickpox silk is carried out Bacterium separation.Test group dissect changes in apparent systemic sepsis, and kidney extravasated blood, enlargement are in aubergine, and spleen hyperemia enlargement is in Cherry-red, lymph nodes of body as a whole hyperemia, bleeding, enlargement, Stomach duodenum are in acute hemorrhagic catarrhal inflammation.Control group dissect Apparent pathological change is not occurred, 5 parts of pathological material of diseases of control group are not separated to erysipelothrix porci, in 5 parts of pathological material of diseases of test group It is separated to erysipelothrix porci, PCR qualification result is consistent with HN1573.
Embodiment 2, the preparation of vaccine, safety and potency test
The preparation of 2.1 vaccines
Erysipelothrix porci bacterial strain HN1573 is inoculated into TSB fluid nutrient medium, is placed in 37 DEG C of shaking tables and cultivates, received after 18h Culture is taken, concentration is measured, adjusting the bacterium number in culture is 2 × 109CFU/mL, every 1000mL bacterium solution add 2mL formalin, In 37 DEG C of inactivation 12h.Aluminum hydroxide adjuvant is added in 9:1 to bacterium solution after inactivation by volume, is mixed to prepare erysipelothrix porci inactivation Vaccine.
The safety testing of 2.2 vaccines
30 age in days health child care pig 10 is chosen, is divided into 2 groups, every group 5.
Vaccine group: this vaccine of 4mL is subcutaneously injected in neck；
Control group: 4mL sterile saline is subcutaneously injected in neck.Animal heat is measured, clinical manifestation is observed, observes 2 altogether Week.
During observation, breathing, appetite, the state of mind be all just during entire observation for the child care pig of vaccine group and control group Often, illustrate inactivated vaccine safety of the invention.
The potency test of 2.3 vaccines
Choose 30 days sodium selenite 30, be divided into 6 groups, every group 5.Specifically: vaccine 1-4 group: every group is injected this respectively Vaccine 0.5ml, 1ml, 1.5ml and 2ml；5th group is non-immune group；6th group is healthy control group, injects 2mL sterile physiological salt Water.Vaccine group and two exempt to inject the same dose of vaccines or sterile saline after healthy control group 3 weeks.Two exempt from rear 14 days each groups With 109The HN1573 bacterium solution of CFU takes intravenous injection to attack poison.The clinical manifestation of piglet is observed, and measures body temperature daily, is observed altogether 2 weeks.Erysipelothrix porci is carried out to organs such as dead pig lungs to be separately cultured.
Observation discovery: vaccine group and non-immune group have apparent difference, attack after poison second day, non-immune group starts for second day There are clinical symptoms, body temperature increases, and sleeping ground is not eaten, mucous membrane cyanosis, starts within the 2nd day to occur dead, 5 all death in 1 week.
Dissect death Ren sus domestica, spleen, lymph nodes of body as a whole enlargement extravasated blood or bleeding.The piglet of healthy control group does not send out Disease.And in vaccine group, there are 3 clinical symptoms and case variation occur in 5 piglets of 0.5ml vaccine group；The 5 of 1ml vaccine group There are 2 clinical symptoms and case variation occur in head piglet；Have in 5 piglets of 1.5ml vaccine group 1 occur clinical symptoms and Case variation；5 piglets of 2ml vaccine group do not fall ill.Immunization protection situation is shown in Table 1.
Table HN1573 plants of Immunization test results of 1 erysipelothrix porci
Note: " --- " indicates not applicable.
As can be seen from the above table, it is 1 × 10 that the minimum immunoprotection dosage of this vaccine, which is 0.5ml bacteria containing amount,9CFU/mL.It says Bright inactivated vaccine of the invention has good protecting effect.
The foregoing is merely the optimal embodiments of the present invention, and for those skilled in the art, the present invention can have Various modifications and variations.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on, should all It is included within protection scope of the present invention.
Sequence table
<110>Animal Busbandry &. Veterinary Medicine Inst., Henan Prov. Academy of Agriculture
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Claims (8)

1. one plant of erysipelothrix porci, it is characterised in that: the erysipelothrix porci be erysipelothrix porci (Erysipelothrix rhusiopathiae) HN1573, deposit number: CGMCC NO:16808, preservation date: on November 09th, 2018, preservation list Position: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: BeiChen West Road, Chaoyang District, BeiJing City 1 Number institute 3.
2. one plant of erysipelothrix porci according to claim 1, which is characterized in that the erysipelothrix porci is 1a type brickpox Silk bacterium.
3. application of one plant of erysipelothrix porci in the drug of preparation prevention and control swine erysipelas.
4. application of the one plant of erysipelothrix porci according to claim 3 in the drug of preparation prevention and control swine erysipelas, special Sign is that the drug is vaccine.
5. a kind of erysipelothrix porci inactivated vaccine, which is characterized in that the vaccine is by erysipelothrix porci serotype antigen and adjuvant It is made, the erysipelothrix porci serotype antigen is the HN1573 plants of antigens of erysipelothrix porci inactivated.
6. a kind of erysipelothrix porci inactivated vaccine according to claim 5, which is characterized in that the adjuvant is aluminium hydroxide Adjuvant.
7. a kind of erysipelothrix porci inactivated vaccine according to claim 5 or 6, which is characterized in that the erysipelothrix porci Bacterium amount is 1 × 109 CFU/mL。
8. according to erysipelothrix porci inactivated vaccine described in claim 4-7 any claim the preparation method comprises the following steps: by pig pellet Aeontium bacterium is inoculated into TSB fluid nutrient medium, is placed in 37 DEG C of shaking tables and cultivates, and culture is collected after 18h, measures concentration, adjustment training Supporting the bacterium number in object is 2 × 109Formaldehyde is added to final concentration of 0.2%, in 37 DEG C of 12 h of inactivation in CFU/mL；Bacterium after inactivation Aluminum hydroxide adjuvant is added in 9:1 to liquid by volume, is mixed to prepare erysipelothrix porci inactivated vaccine.
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CN113897315A (en) * 2021-10-26 2022-01-07 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) Erysipelothrix rhusiopathiae and application thereof

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Publication number Priority date Publication date Assignee Title
CN112226379A (en) * 2019-12-26 2021-01-15 河南科技大学 Erysipelothrix rhusiopathiae type 1a strain, swine erysipelas inactivated vaccine and application thereof
CN112226379B (en) * 2019-12-26 2022-06-14 河南科技大学 Erysipelothrix rhusiopathiae type 1a strain, swine erysipelas inactivated vaccine and application thereof
CN113897315A (en) * 2021-10-26 2022-01-07 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) Erysipelothrix rhusiopathiae and application thereof

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