CN112226379B - Erysipelothrix rhusiopathiae type 1a strain, swine erysipelas inactivated vaccine and application thereof - Google Patents
Erysipelothrix rhusiopathiae type 1a strain, swine erysipelas inactivated vaccine and application thereof Download PDFInfo
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Abstract
The invention discloses a swine erysipelas type 1a strain, a swine erysipelas inactivated vaccine and application thereof, and belongs to the technical field of biological products for livestock. The invention relates to a newly separated current strain erysipelothrix rhusiopathiae HG-1 strain, and the preservation number of the strain is CCTCC NO: M2019684. The strain is separated from heart blood of a fattened pig with acute septicemia, has strong toxicity, and has half lethal dose of 3.8 multiplied by 10 aiming at piglets with age of 8-9 weeks3And (4) CFU. Experiments prove that the inactivated vaccine prepared by the strain can effectively prevent swine erysipelas caused by type 1a and type 2 swine erysipelas, and meanwhile, the vaccine has good safety, and piglets immunized by the vaccine do not have symptoms such as red swelling, fever, death and the like of the inoculated part. The vaccine has the advantages of simple preparation process, good safety to pigs, capability of simultaneously protecting immune pigs against infection of erysipelothrix rhusiopathiae types 1a and 2, and the like.
Description
Technical Field
The invention relates to a swine erysipelas type 1a strain, a swine erysipelas inactivated vaccine prepared from the strain and application of the swine erysipelas inactivated vaccine, and belongs to the technical field of biological products for livestock.
Background
The Erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae) is a facultative anaerobic gram-positive gramnenobacter of the Erysipelothrix genus of the family Erysiridae, and has capsule, no flagellum and no spore production. The bacteria are widely distributed in nature and can cause erysipelas of pigs and other animals. Erysipelothrix rhusiopathiae is divided into 26 serotypes, namely type 1a, type 1b, type 2-24 and type N. The virulence of different serotype strains has obvious difference, wherein the strains of type 1a and type 1b have stronger virulence, and most of erysipelothrix rhusiopathiae separated from the body of the pig with acute septicemia is type 1 a. Virulence factors of erysipelothrix rhusiopathiae and their potential virulence factors that have been discovered so far are: neuraminidase, hyaluronidase, capsular and other surface proteins (e.g., SpaA, RspA, RspB), and the like.
In swine herd, erysipelas of swine caused by erysipelas is an acute and febrile infectious disease, clinically manifested as acute septicemia type, subacute rash type and chronic endocarditis type, which is widely present and prevalent worldwide. The clinical manifestations of erysipelothrix rhusiopathiae infected humans are highly similar to those of pigs, and the main manifestations are local skin damage (erysipelas-like), systemic skin damage and endocarditis-related septicemia. The swine erysipelas occurs in the world, causes serious economic loss to a plurality of countries such as Europe, Asia and the like, is proved to be sporadic in China, has obvious seasonality and frequent occurrence in summer, can affect pigs of various ages of days, has 80-90 percent of fatality rate of acute swine erysipelas, and causes serious economic loss to the pig industry in China.
Before the 80's of the 20 th century, swine erysipelas was one of three infectious diseases (swine fever, swine erysipelas and swine plague) which endanger the swine industry in China. The late 90 s in the 20 th century to the 21 st century have few reports on swine erysipelas cases, which were once thought to disappear in China. However, since 2010, swine erysipelas occurs in a plurality of provinces of China. At present, the swine erysipelas inactivated vaccine (C43-5 strain), the swine erysipelas live vaccine (GC42 strain or G4T10 strain) and the like which are developed in the 60 th of the 20 th century are still used in China. Therefore, the development of a novel swine erysipelas vaccine against the currently circulating strains and with greater immune potency is an inevitable requirement for the prevention and control of swine erysipelas.
Disclosure of Invention
The invention aims to provide a strain of erysipelothrix rhusiopathiae type 1 a.
Meanwhile, the invention also provides a swine erysipelas inactivated vaccine prepared from the swine erysipelas mycelium 1a type strain.
Finally, the invention also provides an application of the erysipelothrix rhusiopathiae type 1a strain in preparation of an inactivated erysipelothrix rhusiopathiae vaccine.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
erysipelothrix rhusiopathiae type 1a strain of swine, designated Erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae) HG-1, accession No.: CCTCC NO, M2019684, preservation date: 09/02/2019, depository: china Center for Type Culture Collection (CCTCC), collection address: wuhan university in Wuhan, Hubei (preservation center of Wuchang Lojia mountain Wuhan university, Wuhan city, Hubei province).
The Erysipelothrix rhusiopathiae 1a type strain HG-1(CCTCC NO: M2019684) is separated from heart blood of a fattened pig with acute septicemia, has strong toxicity, and has half lethal dose of 3.8 multiplied by 10 aiming at piglets with age of 8-9 weeks3And (4) CFU. Culturing the strain on a TSA culture medium containing 10% newborn calf serum at 37 ℃ for 24-48 hours to grow into small colonies which are smooth, neat in edge, bluish in color, colorless, transparent and large in dew-like tips; gram-positive, straight or slightly curved, elongated bacilli, blunt-rounded ends, no spores, no motility; the serous type is identified as type 1a, and can be prepared into inactivated vaccines for preventing the infection of currently popular type 1a and type 2 strains.
The swine erysipelas inactivated vaccine has an antigen of an inactivated swine erysipelas rhusiopathia 1a type strain HG-1(CCTCC NO: M2019684).
Further, the swine erysipelas inactivated vaccine further comprises an adjuvant, such as a GEL water-soluble polymer adjuvant (e.g. commercially available Montanide)TMGEL 01PR water-soluble polymer adjuvant, MontanideTMGEL 02PR water-soluble polymer adjuvant, the content of the adjuvant is 10-20 percent by volume percentage), aluminum hydroxide GEL adjuvant (the content of the adjuvant is 20 percent by volume percentage) and the like.
Furthermore, the swine erysipelas inactivated vaccine also contains a preservative, such as 0.005% of thimerosal (volume percentage), 0.3% of phenol (volume percentage) and the like.
Further, the inactivation may be performed using a conventional inactivating agent, such as formaldehyde, beta-propiolactone (BPL), and the like.
Furthermore, in 1mL of the inactivated vaccine of erysipelas suis, the content of the inactivated thallus of the erysipelas suis type 1a strain HG-1(CCTCC NO: M2019684) is not less than 3.0 multiplied by 109CFU/mL。
The application method and the dosage of the swine erysipelas inactivated vaccine are as follows: intramuscular injection of neck (head indicated by bottle label) 1 head (2mL) each time regardless of pig size. The piglets are first immunized after 3-4 weeks of age and are immunized again after 3 weeks; the sow is first immunized 8-9 weeks before delivery, second immunized 3 weeks after delivery, and then immunized once every 5-6 weeks before delivery.
The erysipelothrix rhusiopathiae 1a type strain HG-1(CCTCC NO: M2019684) is inoculated into a suitable culture medium for purification and culture, then inoculated into a liquid culture medium (such as TSB) for proliferation and culture, the culture solution is inactivated by formaldehyde, and then the thalli are centrifugally collected and added with an adjuvant (such as Montanide)TMGEL 02PR water-soluble polymer adjuvant) to be emulsified into an inactivated vaccine which can effectively prevent the swine erysipelas caused by the erysipelas rhusiopathiae types 1a and 2. Meanwhile, the inactivated vaccine has good safety, and the piglet after the vaccine immunization does not have symptoms of red and swollen, fever, death and the like of the inoculated part.
The application of erysipelothrix rhusiopathiae type 1a strain in the preparation of erysipelothrix rhusiopathiae inactivated vaccine. Specifically, the preparation method of the swine erysipelas inactivated vaccine comprises the following steps:
1) inactivating the bacterial liquid of erysipelothrix rhusiopathiae type 1a bacterial strain HG-1(CCTCC NO: M2019684);
2) concentrating the inactivated bacterial liquid, removing supernatant, washing and diluting to obtain antigen liquid;
3) mixing the antigen solution with adjuvant and antiseptic uniformly.
In the step 1), the content of viable bacteria of erysipelothrix rhusiopathiae 1a type strain HG-1(CCTCC NO: M2019684) in the bacterial liquid is not less than 1.0 multiplied by 109CFU/mL。
In the step 2), a formaldehyde inactivating agent is adopted for inactivation, and the formaldehyde inactivating agent is added into a formaldehyde solution with the concentration of 37-40% according to 0.25-0.3% (volume fraction) of the total amount of the bacterial liquid; inactivating the cells at 37-39 ℃ for 24-48 hours.
In the step 2), sterile PBS solution (0.0l mol/L, pH value is 7.2) can be adopted for washing and diluting; washing, diluting with sterile PBS solution until the concentration of thallus antigen (inactivated erysipelothrix rhusiopathiae type 1a strain HG-1, CCTCC NO: M2019684) is at least 3.53 × 109CFU/mL。
In the step 3), the antigen liquid and the GEL adjuvant are mixed according to the volume ratio of 80-90% to 10-20%, or the antigen liquid and the aluminum hydroxide GEL adjuvant are mixed according to the volume ratio of 80-20%; stirring at room temperature at 500-800 r/min for 20-30 min until the mixture is uniformly mixed.
In the step 3), the preservative is added with thimerosal in an amount of 0.005% (volume percent) or added with phenol in an amount of 0.3% (volume percent).
In the step 3), the inactivated swine erysipelas vaccine contains inactivated erysipelothrix rhusiopathiae type 1a bacterial strain HG-1(CCTCC NO: M2019684) with the thallus content of not less than 3.0 multiplied by 109CFU/mL。
The swine erysipelas inactivated vaccine has simple preparation process and good safety to pigs, and can simultaneously protect immune pigs against the infection of erysipelas suis type 1a and type 2.
The invention has the beneficial effects that:
in the swinery of China, swine erysipelas is a typical bacterial infectious disease of 'new onset of old diseases', so that the research and development of a novel safe and efficient swine erysipelas vaccine is imperative. At present, the main popular serotype of the erysipelothrix rhusiopathiae in China is type 1a, and a small amount of type 2. The erysipelothrix rhusiopathiae type 1a strain HG-1(CCTCC NO: M2019684) is separated from Guangdong diseased swinery in 2018 at 6 months, and the menstrual blood serum type is identified as type 1 a. A large number of animal immune protection tests are carried out on the inactivated vaccine prepared from the erysipelothrix rhusiopathiae 1a type strain HG-1(CCTCC NO: M2019684), and the inactivated vaccine is proved to have good immune effect and can effectively prevent the infection of the currently popular type 1a and type 2 strains.
The invention takes the dominant epidemic strain erysipelothrix rhusiopathiae 1a type strain HG-1(CCTCC NO: M2019684) as a vaccine preparation strain, inoculates the strain in a TSB liquid culture medium (containing 10% newborn calf serum) for culture, obtains a culture, inactivates the culture by formaldehyde, and adds an adjuvant to mix to prepare the inactivated vaccine of erysipelothrix rhusiopathiae, the vaccine can be used for preventing erysipelothrix rhusiopathiae caused by the 1a type and the 2 type erysipelothrix rhusiopathiae, and has the advantages of good safety, high immune efficiency, long immune period and the like.
Deposit and survival evidence description
And (3) preserving the strain: erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae) HG-1, accession No.: CCTCC NO: M2019684, preservation date: 09/02/2019, depository: china Center for Type Culture Collection (CCTCC), collection address: wuhan university in Wuhan, Hubei (preservation center of Wuchang Lojia mountain Wuhan university, Wuhan city, Hubei province).
Drawings
FIG. 1 shows the colony morphology of strain HG-1;
FIG. 2 shows the gram-stained 100 Xmicroscopic morphology of HG-1 strain;
FIG. 3 shows the result of PCR identification of the strain HG-1;
FIG. 4 shows the result of serotype identification by means of molecular biology of the strain HG-1;
FIG. 5 shows the serotonergic method for serotype identification of the strain HG-1.
Detailed Description
The following examples are intended to illustrate the invention in further detail, but are not to be construed as limiting the invention in any way.
Example 1
Pig erysipelas killerA live vaccine, the antigen of which is inactivated erysipelothrix rhusiopathiae type 1a strain HG-1(CCTCC NO: M2019684); in the swine erysipelas inactivated vaccine, the content of the inactivated thallus of the swine erysipelas 1a type strain HG-1(CCTCC NO: M2019684) is not less than 3.0 multiplied by 109CFU/mL。
Example 2
The application of the erysipelothrix rhusiopathiae type 1a strain in the preparation of the erysipelothrix rhusiopathiae inactivated vaccine comprises the following steps:
1. seed preparation for production
1) First-order seed reproduction and identification
The production strain of erysipelothrix rhusiopathiae type 1a strain HG-1(CCTCC NO: M2019684) is inoculated on a TSA culture medium containing 10% newborn bovine serum, after the constant temperature culture at 37 ℃ for 36 hours, more than 5 typical colonies are selected, respectively inoculated on the TSA culture medium containing 10% newborn bovine serum, cultured at 37 ℃ for 24 hours, and taken as a first-grade seed after passing the pure inspection. The product is stored at the temperature of 2-8 ℃, the storage time is not more than 7 days, and the subculture is not more than 5 generations.
2) Two stage seed reproduction
Inoculating HG-1 strain primary seeds into a TSB liquid culture medium containing 10% newborn calf serum, placing the TSB liquid culture medium on a shaking bed for shaking culture at 37 ℃ and 180r/min for 14 hours, and using the HGB liquid culture medium as secondary seeds after passing inspection. The product is stored at 2-8 ℃, and the service life of the product is less than 7 days.
2. Culture of bacterial liquid
And (2) filling a proper amount of TSB liquid culture medium containing 10% newborn calf serum into a glass bottle according to the volume, then transferring the secondary seeds into the new TSB culture medium according to the proportion of 1:100, and performing shaking culture at 37 ℃ and 180r/min for 16 hours to obtain bacterial liquid.
3. Viable count
Viable bacteria count was carried out by using a TSA culture medium containing 10% newborn bovine serum suitable for growth of the present bacteria according to the method in the appendix of the Current "Chinese veterinary pharmacopoeia". Cultured in a bacteria bottle, the content of viable bacteria of erysipelothrix rhusiopathiae type 1a strain HG-1 is not less than 1.0 × 109CFU/mL。
4. Inactivation of bacterial liquid
The culture solution of erysipelothrix rhusiopathiae type 1a strain HG-1 is inactivated, formaldehyde solution (with the concentration of 37-40%) is added according to 0.3% (volume ratio) of the total amount of the bacterial solution, and the bacterial solution is inactivated at 37 ℃ for 48 hours.
5. Inactivation test
Inoculating 0.2mL of inactivated bacterial liquid into a TSA culture medium (containing 10% newborn calf serum) suitable for the growth of the bacteria, inoculating 0.2mL of inactivated bacterial liquid into a TSB liquid culture medium (containing 10% newborn calf serum) suitable for the growth of the bacteria according to the proportion of 1:100, and continuously culturing at 37 ℃ for 7 days until the bacteria grow aseptically, thus obtaining the qualified product.
6. Seedling preparation
1) Preparing an adjuvant
Montanide according to Sappoc specialty Chemicals (Shanghai) LtdTMGEL 02PR product instruction, the GEL adjuvant is autoclaved for 30min at 121 ℃, and cooled to room temperature for standby.
2) Preparation of antigens
Concentrating the bacterial solution qualified for inactivation test by using a hollow fiber ultrafilter to remove the supernatant (or centrifuging the bacterial solution qualified for inactivation test by using a low-temperature high-speed centrifuge to remove the supernatant), washing with sterile PBS (0.0l mol/L, pH value of 7.2), diluting HG-1 strain bacterial antigen by using sterile PBS (0.0l mol/L, pH value of 7.2) according to the counting result of viable bacteria before inactivation, and adjusting the concentration to 3.53 multiplied by 109CFU/mL。
3) Seedling preparation
Mixing the antigen solution and GEL 02PR adjuvant at a volume ratio of 85:15, stirring at 500-800 r/min for 20-30 min at room temperature for full emulsification, adding thimerosal in an amount of 0.005% (volume percentage) before stopping stirring, and fully mixing. Finally, the content of the inactivated HG-1 strain in the vaccine is not less than 3.0 multiplied by 109CFU/mL。
4) Inspection of semi-finished product
The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the result shows that the bacteria-free growth is realized.
5) Dispensing
Quantitatively subpackaging, covering and sealing, and storing at 2-8 ℃.
Test example 1
Identification of erysipelothrix rhusiopathiae:
1. materials and methods
1.1 sources of pathological material
The pig feed is collected 6 months in 2018 from a pig with suspected swine erysipelas clinical symptoms at one end of a large-scale pig farm in Guangdong province. The sick pigs show symptoms of mental depression, high fever retention, anorexia, gait stiffness or stiff and even visual mucosa cyanosis, dyspnea and the like, and the time from morbidity to mortality is short and is between 2 and 5 days. Collecting heart blood and lung, and collecting tissues such as liver, kidney, lymph node, spleen, brain, and joint fluid for separation and identification.
1.2 preparation of the culture Medium
Preparation method of TSA (tryptone Soy Agar, Tryptic Soy Agar) culture medium: weighing 20g of TSA, weighing 500mL of distilled water, pouring into a conical flask, fully shaking for dissolving, sterilizing for 30min by high-pressure steam at 121 ℃, putting into a clean bench, cooling to about 60 ℃, adding 10% (50mL) of filter-sterilized newborn bovine serum into a culture medium, pouring into a flat plate to prepare the TSA culture medium, and cooling for later use.
The preparation method of the TSB (tryptone Soy Broth, Tryptic Soy Broth) culture medium comprises the following steps: weighing 3g of TSB and 100mL of distilled water, pouring into a conical flask, fully shaking up for dissolving, sterilizing for 30min by high-pressure steam at 121 ℃, putting into a super-clean workbench for cooling to about 60 ℃, adding 10 percent (10mL) of filter-sterilized newborn bovine serum into a culture medium, and cooling for later use.
The preparation method of the PBS buffer solution comprises the following steps: weighing 1.45g of disodium hydrogen phosphate (analytically pure), 0.15g of sodium dihydrogen phosphate (analytically pure) and 4.1g of sodium chloride (analytically pure), pouring into a 500mL conical flask, adding distilled water to dissolve and shake uniformly, and fixing the volume to 500 mL; sterilizing at 121 deg.C for 30min, and cooling.
1.3 methods
1.3.1 isolation and culture of bacteria
The collected tissues such as heart blood, lung and the like are streaked and inoculated in a TSA culture medium containing 10% newborn bovine serum, suspected colonies are picked out for subculture purification after 36 hours of culture at 37 ℃, and the colony morphology of the erysipelothrix rhusiopathiae is observed after 24 hours of culture. Meanwhile, 3-5 single colonies are picked for gram staining microscopy, and morphological characteristics of the thalli are observed. If the erysipelothrix rhusiopathiae is separated from different pathogenic tissues of the same case, only one isolated strain of the pathogenic tissue is selected for preservation, and the priority order of the isolated strain is heart blood, lung, liver, spleen, lymph node, joint fluid, kidney and brain.
1.3.2 microscopic examination
A single colony of the purified colonies was fixed on a spatula, and the morphology of the bacteria was observed by a microscope after gram staining.
1.3.3 PCR identification
1.3.3.1 primer design
Primers were synthesized based on the 16S rRNA gene sequences reported in the literature, and the PCR primer sequences were as follows:
upstream primer JD 01: 5'-AGAGTTTGATCCTGGCTCAG-3' (shown in SEQ ID NO. 1);
downstream primer JD 02: 5'-ACGGTTACCTTGTTACGACTT-3' (shown in SEQ ID NO. 2).
PCR amplification is carried out on the erysipelothrix rhusiopathiae. The size of the primer amplified fragment is expected to be about 1500 bp.
1.3.3.2PCR reaction system and reaction conditions
PCR reaction (25. mu.L): 12.5 μ L2 XTaq MasterMix (Dye) (2 XTaq MasterMix contains Taq DNA Polymerase, 3mM MgCl2And 400. mu.M each dNTP), 10.5. mu.L ddH2O, upstream and downstream primers were 1. mu. L, DNA template each.
And (3) PCR reaction conditions: first 95 deg.C (pre-denaturation) for 4 min; then circulating for 30 times at 94 deg.C (denaturation) for 30s, at 56 deg.C (annealing) for 30s, and at 72 deg.C (extension) for 2 min; then 72 ℃ (re-extension) for 10 min; finally, the mixture is stored temporarily at 4 ℃.
And (3) respectively carrying out 1% agarose gel electrophoresis on the PCR products, adding 5 mu L of sample into each hole, adding 10 mu L of marker, observing the result by using a gel imaging system after 30min, sending the PCR products to a biological engineering (Shanghai) corporation for sequence determination, and carrying out NCBI database search and comparison analysis on the sequencing result by applying online biology software Standard Nucleotide BLAST (http:// BLAST.
1.3.4 serotype identification method for isolates
1.3.4.1 molecular biology methods for identifying serotypes
A pair of specific primers of erysipelothrix rhusiopathiae type 1a is designed according to the sequence of the GCT (glycerol-3-phosphate cytylyltransferase) gene of erysipelothrix rhusiopathiae, and is used for identifying the serotype of the erysipelothrix rhusiopathiae type 1a, and the primer sequences are as follows:
upstream primer P3: 5'-CTCCTAACGCTTTAGCACGC-3' (shown in SEQ ID NO. 3);
the downstream primer P4: 5'-TGATCCTTTGCCACTAATGC-3' (shown in SEQ ID NO. 4).
PCR amplification is carried out on the erysipelothrix rhusiopathiae. The size of the primer amplified fragment is expected to be 356 bp.
1.3.4.2 PCR reaction System and reaction conditions
PCR amplification reaction (25. mu.L): 12.5 μ L Mix, 10.5 μ L ddH2O, upstream and downstream primers were 1. mu. L, DNA template each. The swine erysipelothrix rhusiopathiae type 1a reference strain CVCC43008 is used as a positive control.
And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 2min, entering circulation: at 94 ℃ for 30s, at 60 ℃ for 30s, at 68 ℃ for 1min, for 35 cycles, and finally at 72 ℃ for 10min, the PCR product is stored at 4 ℃.
And (3) respectively carrying out electrophoresis on the amplification products, adding 5 mu L of sample into each hole, adding 10 mu L of sample into marker, carrying out electrophoresis on 1% agarose gel, and observing the result by using a gel imaging system after 30 min.
1.3.4.3 serological factor method for identifying serotypes
Inoculating erysipelothrix rhusiopathiae HG-1 strain to a TSA solid culture medium containing 10% newborn bovine serum, culturing at 37 ℃ for 36 hours, selecting a single colony, performing subculture purification for 24 hours, eluting the lawn with sterile PBS, centrifuging at 7000r/min for 5min, discarding the supernatant, concentrating and uniformly mixing the sterile PBS, treating with high-pressure steam at 121 ℃ for 2 hours, centrifuging at 7000r/min for 10min, and obtaining the supernatant as a typing antigen for agar diffusion test, which is also called a heat stability antigen.
The isolated erysipelothrix rhusiopathiae strain was identified by Kieletein-Rapp-Gabridson (KRG) agar diffusion serotyping using standard typing sera as follows: dissolving 1g of Noble agar and 8.5g of sodium chloride in 100mL of PBS, heating in a microwave oven to completely dissolve, cooling to about 56 ℃, adding 0.1g of preservative (thimerosal), preparing a gel plate with the thickness of about 3-4 mm, and storing at 4 ℃ for later use. Punching holes (the aperture is 3mm and the hole distance is 4mm) on the flat plate according to the requirements by using a puncher, and heating and sealing the bottom by using an alcohol lamp; a certain type of standard positive serum is added into the central hole, and the antigen to be detected is added into each peripheral hole, wherein each hole is about 15 mu L. Negative sera were also used as controls. The mixture was placed in a wet box and left at 37 ℃ for 24 hours, and the results were observed for three consecutive days. If a clear white precipitation line appears between the antigen and the antibody hole, the antibody is judged to be positive, otherwise, the antibody is judged to be negative.
1.3.5 virulence test
Inoculating erysipelothrix rhusiopathiae 1a type HG-1 strain to a TSA culture medium containing 10% of newborn bovine serum, recovering and culturing at 37 ℃ for 36 hours, selecting a single colony, performing subculture purification for 24 hours, selecting the purified single colony to a TSB liquid culture medium containing 10% of newborn bovine serum, placing the single colony in a constant temperature shaking table, performing shake culture at 37 ℃ for 14 hours to obtain a seed solution, transferring the seed solution to a new TSB liquid culture medium containing 10% of newborn bovine serum according to the proportion of 1:100, and performing shake culture at 37 ℃ for 12 hours to obtain the seed solution. The prepared bacterial liquid was diluted 10-fold to 4 th, 5 th, 6 th and 7 th gradients (10 th, respectively)5、104、103、102). Dividing 25 healthy piglets of 8-9 weeks into 5 groups (5 piglets/group) at random, and inoculating the ear vein (1 mL/head) of the first 4 groups of healthy piglets with the 4 gradient diluted bacterial liquid. And simultaneously, diluting and counting the original bacteria liquid, and determining the actual bacteria amount of inoculation. The fifth group was inoculated as a blank with sterile PBS (0.0l mol/L, pH value 7.2). Continuously observing and recording the morbidity and mortality for 15 days, performing autopsy immediately for the dead, collecting tissues and organs of each part of the body, performing bacteria isolation and identification, and calculating half lethal dose (50% lethase, LD) of 1a type HG-1 strain of Erysipelothrix rhusiopathiae for piglets according to Reed-Muench method50)。
1.3.6 creation of Primary seed lots of Erysipelothrix rhusiopathiae
The separated erysipelothrix rhusiopathiae type 1a HG-1 strain is used as a vaccine strain for developing the erysipelothrix rhusiopathiae inactivated vaccine. The strain of bacteria is preserved in 25-30 bottles as an original seed batch.
2. Results of the experiment
2.1 culture characteristics of the strains
The erysipelothrix rhusiopathiae isolate can grow into small colonies which are smooth, have neat edges, have bluish colors, are colorless and transparent, and have needle tips like macrobeads after being cultured for 36 hours at 37 ℃ on a TSA culture medium containing 10 percent newborn bovine serum, and are shown in figure 1. The strain can also grow on a common culture medium, but the strain grows better on a culture medium containing serum.
2.2 morphology of the strains
The bacteria were gram-positive, straight or slightly curved, elongated bacilli, blunt-rounded at both ends, non-sporulating, non-motile, as shown in fig. 2, when observed under a microscope after gram staining. Can react with beta-galactoside, acetamide, glucose, lactose, mannose, mannitol, sorbitol, hydrogen sulfide, urea, etc. positively, and react with sucrose, ribose, xylose, bile bacteriolysis control, bile esculin, ornithine, arabitol, nitrate (reduction), etc. negatively.
2.3 PCR identification of Erysipelothrix rhusiopathiae
Respectively carrying out electrophoresis on the amplification products, observing in a gel imaging system, and amplifying to obtain a fragment with the size of 1500bp as shown in figure 3, wherein a Marker in the figure is DL 2000, and the size is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom; lanes 1-6 are amplification results of HG-1 strain 16S rRNA genes; lane 7 is a negative control. Then sending to sequencing, and the sequencing result is shown as SEQ ID NO. 5. The sequence determination result is compared and analyzed by online biological analysis software BLAST (https:// blast.ncbi.nlm.nih.gov/blast.cgi), and the result shows that the genetic relationship between the strain and other erysipelothrix rhusiopathiae is more than 99%.
2.4 identification of serotype by the method of molecular biology of Erysipelothrix rhusiopathiae
Respectively carrying out electrophoresis on the amplification products, observing in a gel imaging system, and amplifying to obtain a 356bp fragment as shown in figure 4, wherein a Marker in the figure is DL 2000, and the Marker is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom; lane 1 is strain HG-1, lane 2 is a positive control, type 1a reference strain CVCC43008, lane 3 is a negative control. The results demonstrate that the serotype of strain HG-1 is type 1 a.
2.5 serological factor method for identifying serotype of Erysipelothrix rhusiopathiae
Identifying the separated erysipelothrix rhusiopathiae strain by using standard parting serum according to a Kieletein-Rapp-Gabridson (KRG) agar diffusion serum parting method, wherein standard positive serum is added into a middle hole, heat stable antigens of the identified strain are respectively added into peripheral holes, and a precipitation line is judged to be positive when the precipitation line appears, as shown in figure 5, the middle hole in the figure is standard 1a type serum, 1 is added with HG-1 heat stable antigen, 2 is added with 1a type reference strain CVCC43008 antigen as a positive control, and 3 is a negative control. The isolate identified by this method was erysipelothrix rhusiopathiae type 1a strain.
2.6 virulence of the Strain
Subjecting Erysipelothrix rhusiopathiae HG-1 strain to pig half Lethal Dose (LD)50) The test result shows that the erysipelothrix rhusiopathiae HG-1 strain shows strong toxicity and LD thereof50Equal to 3.8 × 103CFU (see table 1). According to the results of virulence test, the erysipelothrix rhusiopathiae type 1a HG-1 strain is determined to be used as a vaccine strain of the erysipelothrix rhusiopathiae inactivated vaccine.
TABLE 1 Swine erysipelothrix rhusiopathiae HG-1 strain piglet virulence test results
The observation shows that: animals began to develop diseases 36 hours after inoculating HG-1 strain, and showed clinical symptoms of mental depression, anorexia or complete abolition, joint swelling, lameness or lying difficulty, cyanosis of visible mucous membrane and the like, and all died within 4 days after toxin attack. The pigs in the control group had normal indexes and no obvious clinical symptoms in the observation period.
Dissecting the pigs which die from diseases, collecting the pathological materials, and carrying out separation and identification. The autopsy result shows that the change of the systemic septicemia is shown; the spleen is remarkably congested and swollen; pulmonary congestion, edema; the lymph nodes of the whole body are swollen and purple red; small bleeding spots are visible on the epicardium; the kidneys swollen and red with diffuse bleeding spots. After the observation period, 5 pigs in the control group were also dissected and the morbid material was collected, and no obvious pathological changes were found by the autopsy. The erysipelothrix rhusiopathiae can be separated from the diseased material of the diseased and dead pigs, is actually HG-1 strain through identification, and is not separated from the diseased material of a control group.
2.7 preservation of Primary seed of Erysipelothrix rhusiopathiae
HG-1 plants were stored in 25 to 30 bottles as a stock seed lot. The freeze-dried erysipelothrix rhusiopathiae is in a solid but fluffy honeycomb structure by taking skim milk as a protective agent, and is immediately dissolved by adding water. The original seeds are batched, are stuck with labels containing information such as strain names, storage time, storage persons and the like, and are stored in a refrigerator at the temperature of minus 80 ℃. And 4, performing recovery inspection on the preserved strains after three days, wherein the preserved strains have no mixed bacteria and good activity, and the results show that the original seed batches meet the requirements.
The erysipelothrix rhusiopathiae type 1a HG-1 strain is sent to a China center for type culture collection to be preserved, wherein the preservation information is as follows: and (3) preserving the strain: erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae) HG-1, accession No.: CCTCC NO, M2019684, preservation date: 09/02/2019, depository: china Center for Type Culture Collection (CCTCC), preservation address: wuhan university in Wuhan, Hubei (preservation center of Wuchang Lojia mountain Wuhan university, Wuhan city, Hubei province).
Test example 2
Example 2 testing of finished product of swine erysipelas inactivated vaccine:
1) traits
The product is an off-white suspension, a small amount of precipitate is formed at the bottom of the product after long-term storage, and the product is uniform suspension after shaking.
2) Load check
The inspection is carried out according to the appendix of the current Chinese beast pharmacopoeia, and the regulations are met.
3) Sterility testing
The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the sterile growth is carried out.
4) Safety inspection
Safety test for piglet vaccination: 3 batches of swine erysipelas inactivated vaccines (batch numbers: 201901, 201902 and 201903) trial-produced according to the method of example 2 are adopted to respectively inoculate healthy piglets of 3-4 weeks old with 2 times of dose (4 mL/head), meanwhile, a control group is set to inoculate sterile PBS (0.0lmol/L, pH value 7.2) (4 mL/head), the immune part is neck muscle, and clinical symptoms and pathological changes of the piglets are closely observed after immunization.
The results show that the 3 batches of the vaccinated piglets have no local or systemic adverse reactions such as mental state, appetite abnormality, vomit, diarrhea and even death and the like caused by vaccination. The results of the temperature measurement before and one week after the inoculation show that the piglets in the immune group have transient temperature rise, but the temperature rise averagely does not exceed 1 ℃ at 24 hours after the inoculation, and the piglets are recovered to be normal within 48 hours. After the observation period is finished, the immune parts of all groups of experimental piglets are observed in an anatomical mode, the immune parts of the piglets of 3 vaccine groups have no obvious phenomena of vaccine residues, local inflammation, tissue lesion and the like, granuloma is not formed, and the piglet vaccine has no obvious difference compared with a control group. The test results show that the swine erysipelas inactivated vaccine in the example 2 is safe to be inoculated by target animals (piglets at 3-4 weeks old) with the minimum day age.
5) Efficacy test
A piglet immunity counteracting method: 30 healthy piglets of 4-5 weeks old are randomly divided into 6 groups (5 piglets/group), and the specific grouping conditions are shown in table 2. Groups 1 and 4 are vaccine immunization groups, each piglet is injected with 2mL of vaccine through neck muscle, and after 3 weeks, the piglet is inoculated with 2mL of vaccine according to the same method. Piglets in groups 2 and 5 were not vaccinated and were vaccinated with 2mL sterile PBS as a blank control. Piglets in group 3 and 6 were not inoculated as healthy control group, and were kept in isolation without toxicity. Groups 1 and 2 were injected intra-auricular intravenously with about 40LD 14 days after the 2 nd inoculation501mL of bacterial liquid of 1a type parent strain HG-1 of erysipelothrix rhusiopathiae (viable count is 1.6 multiplied by 10)5CFU), group 4 and group 5 were injected with about 4LD by ear margin intravenous injection501mL of erysipelothrix rhusiopathiae type 2 reference strain CVCC43006 bacterial liquid (the number of viable bacteria contained is about 6.8 multiplied by 10)6CFU), all piglets were observed daily for morbidity to day 14.
The results of immune tests (see table 2) show that the swine erysipelas inactivated vaccine prepared from the swine erysipelas 1a type strain HG-1 has better resistance to the 1a type parent strain HG-1 and the 2 type reference strain CVCC43006, the protection rate to the 1a type parent strain HG-1 is 100%, and no obvious clinical symptoms appear on the piglets after challenge; the strain has good protection effect (80%) on a type 2 reference strain CVCC43006, and only 1 piglet after challenge has slight symptoms of mental depression, inappetence and the like.
TABLE 2 Erysipelothrix rhusiopathiae HG-1 strain piglet immunoprotection test results
Note: "-" indicates inapplicable.
As can be seen from the table above, the inactivated swine erysipelas vaccine prepared from the type 1a strain HG-1 of the swine erysipelas has good immune protection effect on both the type 1a and the type 2 swine erysipelas.
6) Measurement of residual Formaldehyde and Thimerosal
The determination is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the regulations are met.
Experiments prove that the swine erysipelas inactivated vaccine can effectively resist the infection of the original strains of the swine erysipelas 1a and 2 and the current epidemic strains.
While specific embodiments of the present invention have been described above, the embodiments of the present invention are not limited to the specific embodiments described above. All changes, modifications, substitutions, combinations, and simplifications which may be made by those skilled in the art and which are within the scope of the claims are to be considered as being equivalent substitutions within the scope of the invention.
Sequence listing
SEQUENCE LISTING
<110> university of Henan science and technology
<120> Erysipelothrix rhusiopathiae type 1a strain, swine erysipelas inactivated vaccine and application thereof
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<213> Erysipelothrix rhusiopathiae type 1a strain HG-1
<221> 16S rRNA of Strain HG-1
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gcagtcgaac gaagtgaaga ggagcttgct ccttggaact tagtggcgaa cgggtgagta 60
atacataagc aacctgcctc gatgcctggg ataacagagg gaaacttctg ctaataccgg 120
atacgttaat ctaagacatc ttagattaat taaagatggg atacatcaca acgagatggg 180
cttatggcgc attagttagt tggtaaggta acggcttacc aagacgatga tgcgtagccg 240
acctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc tacgggaggc 300
agcagtaggg aattttcggc aatgggggaa accctgaccg agcaacgccg cgtgagtgaa 360
gacggccttc gggttgtaaa gctctgttgt aagggaagaa cgataggaag agggaatgct 420
tcttatatga cggtacctta ccagaaagcc acggctaact acgtgccagc agccgcggta 480
atacgtaggt ggcaagcgtt atccggaatt attgggcgta aagggagcgc aggcggttta 540
tcaagtttat ggttaaagtt cggggcttaa ccccgtgatg ccatagaaac tggtagacta 600
gagtgcagga gaggttagtg gaattccatg tgtagcggta aaatgcgtag atatatggag 660
gaacaccagt ggcgaaggcg gctaactggc ctgtaactga cgctgaggct cgaaagcgtg 720
gggagcaaat aggattagat accctagtag tccacgccgt aaacgatgga tactaagtgt 780
tggagaaatt cagtgctgta gttaacgcaa taagtatccc gcctggggag tatgcgcgca 840
agcgtaaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagtat gtggtttaat 900
tcgaagcaac gcgaagaacc ttaccaggtc ttgacatacc gcgcaaaagc acagagatgt 960
gtaatagtta tggcggatac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020
tgggttaagt cccgcaacga gcgcaaccct tgtctttagt taccagcatt aagttgggga 1080
ctctaaagag actgccggtg ataaaccgga ggaaggtggg gatgacgtca aatcatcatg 1140
ccccttatga tctgggctac acacgtacta caatggcgta tacagagggc agcgaagcag 1200
cgatgcggag cgaatctcag aaagtacgtc tcagttcgga ttggagtctg caactcgact 1260
ccatgaagtc ggaatcgcta gtaatcgcgg atcagaatgc cgcggtgaat acgttctcgg 1320
gccttgtaca caccgcccgt caaaccatga gagttggtaa tacccgaagc cggtggccta 1380
acctagttac taggagggag ccgtcgaa 1408
Claims (10)
1. Erysipelothrix rhusiopathiae type 1a strain, which is characterized in that: the erysipelothrix rhusiopathiae type 1a strain is erysipelothrix rhusiopathiae ((R))Erysipelothrix rhusiopathiae) HG-1, accession number: CCTCC NO: M2019684.
2. The swine erysipelas inactivated vaccine is characterized in that: the antigen of the inactivated swine erysipelas vaccine is an inactivated swine erysipelas 1a type strain, the swine erysipelas 1a type strain is erysipelas erythraea (Erysipelothrix rhusiopathiae) HG-1, and the preservation number is as follows: CCTCC NO: M2019684.
3. The swine erysipelas inactivated vaccine of claim 2, characterized in that: the swine erysipelas inactivated vaccine also contains an adjuvant and a preservative.
4. The swine erysipelas inactivated vaccine of claim 3, characterized in that: the adjuvant is one of GEL water-soluble polymer adjuvant and aluminum hydroxide GEL adjuvant.
5. The swine erysipelas inactivated vaccine of claim 3, characterized in that: the preservative is one of thimerosal and phenol.
6. The swine erysipelas inactivated vaccine of any one of claims 2 to 5, characterized in that: in the swine erysipelas inactivated vaccine, the content of the inactivated thallus of the swine erysipelas mycelium 1a type strain is not less than 3.0 multiplied by 109CFU/mL。
7. The use of the erysipelothrix rhusiopathiae type 1a strain of claim 1 in the preparation of an inactivated erysipelas suis vaccine.
8. Use according to claim 7, characterized in that: the preparation method of the swine erysipelas inactivated vaccine comprises the following steps:
1) inactivating the bacterial liquid of erysipelothrix rhusiopathiae type 1a bacterial strain;
2) concentrating the inactivated bacterial liquid to remove supernatant, washing and diluting to obtain antigen liquid;
3) mixing the antigen solution with adjuvant and antiseptic uniformly.
9. Use according to claim 8, characterized in that: in the step 1), the content of viable bacteria of erysipelothrix rhusiopathiae type 1a bacterial strain in the bacterial liquid is not less than 1.0 multiplied by 109CFU/mL。
10. Use according to claim 8, characterized in that: in the step 2), a formaldehyde inactivating agent is used for inactivation.
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