CN113018426B - Combined strain for preparing haemophilus parasuis vaccine, octavalent inactivated vaccine and application thereof - Google Patents
Combined strain for preparing haemophilus parasuis vaccine, octavalent inactivated vaccine and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological products for livestock, and particularly relates to preparation and application of a haemophilus parasuis serum type 1, type 2, type 4, type 5, type 7, type 12, type 13 and type 14 octavalent inactivated vaccine. The vaccine has 100% protection rate on infection of type 1, type 2, type 4, type 5, type 7, type 12, type 13 and type 14 strains, has better cross protection force on infection of type 3, type 6, type 8, type 9, type 11, type 15 and haemophilus parasuis which cannot be separated, and has simple preparation process, no toxic or side effect, good safety, long immunization period and good immunization effect.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a haemophilus parasuis serum type 1, type 2, type 4, type 5, type 7, type 12, type 13 and type 14 octavalent inactivated vaccine and application thereof.
Background
Haemophilus parasuisHaemophilus parasuisHPS) is a pathogenic bacterium of haemophilus parasuis disease in pigs, mainly causing serositis, arthritis and meningitis in pigs. The disease is widespread worldwide. Haemophilus parasuis can infect pigs of various ages, mainly occurs after weaning and in the nursery stage, mainly infects pigs of 2 to 4 months of age, and has a morbidity of 10 to 15 percent and a mortality rate in severe casesThe disease can reach 80%, becomes one of the main causes of death of weaned and nursing piglets in recent years, is one of the main pathogens of clinical mixed infection, causes great economic loss for pig industry, and seriously affects the healthy development of pig industry.
Haemophilus parasuis has more clinical serotypes, and the standard type has 15 serotypes, and more than 20% of non-parting strains clinically exist, the pathogen is distributed worldwide, the serotypes of epidemic strains are different from place to place, and the immunological cross-protection capability is lacked among the serotypes. Vaccine immunization is the best way for preventing and controlling haemophilus parasuis, most domestic commercial vaccines of haemophilus parasuis are bivalent vaccines aiming at serotype 4 and type 5, and the serotype difference between the commercial vaccines and main epidemic strains in various areas is large, so that the prevention and control effects are poor. There is therefore a need for a new vaccine that maximally contains the development of dominant strains that are prevalent in all different regions of the country, and that has an excellent protection against infection with all serotypes and non-typeable haemophilus parasuis.
Disclosure of Invention
Aiming at the defect and problem that the vaccine effect is poor due to the fact that the epidemic serotypes of haemophilus parasuis are different in all places and the lack of effective cross protection among the serotypes, the invention provides an octave inactivated vaccine of haemophilus parasuis serotypes 1, 2, 4, 5, 7, 12, 13 and 14.
The invention solves the technical problems by adopting the scheme that: provides a combined strain for preparing haemophilus parasuis vaccine, which comprises haemophilus parasuis @Haemophilus parasuis) Type 1 HN1397 strain with preservation number of CGMCC NO:16804; haemophilus parasuisHaemophilus parasuis) Type 2 HN1553 strain with preservation number of CGMCC NO:16801; haemophilus parasuisHaemophilus parasuis) 4 HNHPS1 strain with preservation number of CGMCC NO:13335; haemophilus parasuisHaemophilus parasuis) 5 HN1570 strain with preservation number of CGMCC NO:16802; haemophilus parasuisHaemophilus parasuis) 7 HN1565 strain with the preservation number of CGMCC NO:16803; haemophilus parasuisHaemophilus parasuis) 12 HN486 strain with preservation number of CGMCC NO: 16505; haemophilus parasuis（Haemophilus parasuis) 13 HN1601 strain with a preservation number of CGMCC NO:16806; haemophilus parasuisHaemophilus parasuis) 14 HN1513 strain with preservation number of CGMCC NO:16807.
the invention also provides an octavalent inactivated vaccine of haemophilus parasuis prepared by the combined strain, which comprises eight inactivated haemophilus parasuis bacterial liquids and an immune adjuvant.
The haemophilus parasuis octavalent inactivated vaccine comprises serotype antigens of haemophilus parasuis type 1, type 2, type 4, type 5, type 7, type 12, type 13 and type 14 in a ratio of 1:1:1:1:1:1:1:1:1.
The haemophilus parasuis serum type 1, type 2, type 4, type 5, type 7, type 12, type 13 and type 14 antigens have a content of 1×10 10 CFU/mL。
The haemophilus parasuis octavalent inactivated vaccine is characterized in that: the immune adjuvant is Summit S550, a water-in-oil-in-water adjuvant for livestock and poultry.
The invention also provides a preparation method of the haemophilus parasuis octavalent inactivated vaccine, which comprises the following steps:
(1) Proliferation: the strain of HN1397, HN1553, HNHPS1, HN1570, HN1565, HN486, HN1601 and HN1513 are propagated and cultured respectively to obtain 8 haemophilus parasuis bacterial solutions, and viable counts are carried out respectively;
(2) And (3) inactivation: adding formaldehyde solution into bacterial solutions of haemophilus parasuis HN1397 strain, HN1553 strain, HNHPS1 strain, HN1570 strain, HN1565 strain, HN486 strain, HN1601 strain and HN1513 strain obtained in the step (1) according to 0.2% of the volume of the bacterial solutions, and inactivating 48 h at 37 ℃;
(3) Concentrating: concentrating the qualified bacterial solutions of haemophilus parasuis HN1397, HN1553, HNHPS1, HN1570, HN1565, HN486, HN1601 and HN1513 after inactivation by using an ultrafiltration device, and adjusting the concentration by using sterile physiological saline according to the viable bacteria count before inactivation;
(4) Preparing a vaccine: the haemophilus parasuis HN1397 strain, HN1553 strain, HNHPS1 strain, HN1570 strain, HN1565 strain, HN486 strain, HN1601 strain and HN1513 strain antigen liquid obtained after proliferation, inactivation and concentration are uniformly mixed according to a proportion, and an adjuvant is added, so that the haemophilus parasuis octavalent inactivated vaccine is prepared.
In the preparation method of the haemophilus parasuis octavalent inactivated vaccine, in the step (4), eight bacterial strain inactivated antigen solutions are uniformly mixed according to the proportion of 1:1:1:1:1:1:1:1, and water-in-oil-in-water adjuvant for livestock and poultry with the final volume of 30% is added for uniform mixing.
The invention has the beneficial effects that: according to the invention, dominant epidemic strains of haemophilus parasuis HN1553, HNHPS1, HN1570, HN1565, HN1397, HN486, HN1601 and HN1513 are selected from clinical isolates, wherein the strains HN1397, HN1553, HNHPS1, HN1570, HN486, HN1601 and HN1513 have strong pathogenicity on nursery pigs, and cause morbidity and mortality of the nursery pigs. The strain HN1565 in the invention has weak pathogenicity to the nursing piglets, but can cause respiratory symptoms of the nursing piglets, slow development and reduced feed return; the test shows that the eight haemophilus parasuis have good immunogenicity, the inactivated vaccine prepared by using the eight haemophilus parasuis as strain antigens has 100% protection rate on type 1, type 2, type 4, type 5, type 7, type 12, type 13 and type 14 infections, and has better cross protection force on type 3, type 6, type 8, type 9, type 11, type 15 and non-typing haemophilus parasuis infections, and the vaccine has the advantages of simple preparation process, no toxic or side effect, good safety, long immunization period and good immunization effect.
Drawings
FIG. 1 shows the PCR typing identification result of haemophilus parasuis, wherein the Marker is DL 2000 bp, and is 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp and 100 bp from top to bottom in sequence; lanes 1-15 are the amplification results of the H.parasuis serotype 1-15 standard strains, respectively.
Detailed Description
The invention will be further described with reference to the drawings and examples.
Example 1: isolation and identification of haemophilus parasuis
1.1 Source of disease material
In the period from 1 month 2016 to 12 months 2019, lung, heart blood, lymph node, spleen, brain, joint fluid and other diseases of pigs with clinical symptoms of suspected haemophilus parasuis disease in the provinces of Henan, shanxi, shandong, hubei, anhui, jiangsu, hebei and the like are collected together, and the ill pigs mostly show fever, dyspnea, joint swelling, lameness, skin and mucous membrane cyanosis, standing difficulty and even paralysis, stiff pigs or death.
1.2 Preparation of culture medium
TSB broth: TSB broth powder (Tryptic Soy broth, tryptone soy broth, BD company) 30g was dissolved in 1000mL ultrapure water and sterilized at 115 ℃ for 15min, and fetal bovine serum 50mL (Hychone company), sterile 1% nad (Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide, coenzyme i, roche company) 1mL was added.
TSA solid medium: 40g of TSA Agar powder (Tryptic Soy Agar, BD company) was dissolved in 1000mL of ultra pure water, sterilized at 115℃for 15min, and 50mL of fetal bovine serum and 1mL of sterile 1% NAD were added; preserving at 4 ℃ for standby.
1.3 Isolated culture of haemophilus parasuis
Collecting the lung, heart blood, lymph node, spleen, brain, joint fluid and other diseases of the sick or dead pig under the aseptic condition, inoculating the disease materials on a TSA solid culture medium containing NAD, culturing 36h in a constant temperature incubator at 37 ℃, selecting suspected colonies for subculture purification, culturing 24-48 h, observing the colony morphology of haemophilus parasuis, and growing uniform needle-tip-shaped, colorless, transparent, smooth and moist colonies with the diameter of 1-2 mm; purification and inoculation are carried out on a TSA solid culture medium without NAD, and growth cannot be carried out on the TSA solid culture medium without NAD; the purified individual colonies were picked for gram staining microscopy and the observed bacteria were characterized as gram negative bacteria, having a variety of different morphologies, ranging from individual cocci to elongate filamentous bacteria.
1.4 identification of haemophilus parasuis and identification of serotypes
The clinical isolated suspected strain is subjected to haemophilus parasuis identification and typing identification by Polymerase Chain Reaction (PCR) technology. The primer sequences and the amplified fragment lengths of the haemophilus parasuis types 1-15 are shown in Table 1.
And (3) PCR reaction: the PCR reaction system was 25. Mu.L, which included 10X Tap PCR master mix. Mu.L, 1.0. Mu.L each of the upstream and downstream primers, 5.0. Mu.L of sterile water, and 5. Mu.L of template. The reaction conditions are as follows: 94. after denaturation at 5min, 30 cycles of 94℃for 30 s,54℃for 30 s,72℃for 40 s were performed, and finally extension at 72℃for 10 min, and the PCR product was stored at 4 ℃. 10 mu L of PCR products are taken, electrophoresis is carried out for 45 min under the voltage of 1% agarose gel 100V, and then a gel imaging system is used for analyzing and observing results, and positive results are sequenced and compared.
The 60 haemophilus parasuis strains are co-separated by bacterial separation, purification and identification of the collected suspected samples, and the homology of the separated strains and the 16SrRNA sequence of the standard HPS is over 99 percent through gene comparison. The result of the PCR molecular serotyping identification of 60 haemophilus parasuis Henan isolates is shown in figure 1. The results show that: in 60 haemophilus parasuis, 2 strains exist in serotype 5, 12, 13, 10, 9, 8, 2, 6, 7, 1 and 14, and 4 strains exist in non-parting type. The results show that the type 5, type 13, type 4, type 12, type 2, type 7, type 1 and type 14 are the dominant epidemic serotypes in clinic.
The haemophilus parasuis of the 8 main epidemic serotypes are selected and named and preserved in the China general microbiological culture Collection center, namely haemophilus parasuis type 1 HN1397 strain with the preservation number of CGMCC NO:16804; haemophilus parasuis 2 HN1553 strain with a preservation number of CGMCC NO:16801; haemophilus parasuis 4 type HNHPS1 strain with a preservation number of CGMCC NO:13335; haemophilus parasuis 5 type HN1570 strain with a preservation number of CGMCC NO:16802; haemophilus parasuis 7 HN1565 strain with the preservation number of CGMCC NO:16803; haemophilus parasuis 12 type HN486 strain with a preservation number of CGMCC NO: 16505; haemophilus parasuis 13 HN1601 strain with a preservation number of CGMCC NO:16806; haemophilus parasuis 14 type HN1513 strain with a preservation number of CGMCC NO:16807. preservation date: haemophilus parasuis 4 type HNHPS1 strain is preserved in 2016, 11 and 22 days, and the other 7 strains are preserved in 2018, 11 and 9 days, and the preservation address is North Chen Xili No. 1, 3 in the Chaoyang area of Beijing city.
Example 2: toxicity test
And selecting 45 healthy nursery pigs at 9-10 weeks of weaning age as test animals. The test animals were randomly divided into 9 groups, 1 control group and 8 test groups, each group having 5 heads. Inoculating haemophilus parasuis strain HN1397, HN1553, HNHPS1, HN1570, HN1565, HN486, HN1601 and HN1513 respectively to TSB liquid culture medium, shake culturing at 37deg.C for 24h, measuring thallus concentration, continuously diluting 10 times, coating solid plate, counting viable bacteria, calculating thallus concentration of stock solution, and adjusting to 10 9 CFU/mL; animals of the 8 groups of test groups were inoculated with 3 mL of H.parasuis liquid cultures by intraperitoneal inoculation of HN1397, HN1553, HNHPS1, HN1570, HN1565, HN486, HN1601 and HN1513, respectively, and animals of the control group were inoculated with 3 mL sterilized TSB liquid cultures by intraperitoneal inoculation. The number of morbidity and mortality was observed and recorded by continuous observation for 2 weeks after injection.
Observations find that: the HN1397 strain, HN1553 strain, HNHPS1 strain, HN1570 strain, HN486 strain, HN1601 strain and HN1513 strain inoculated groups show symptoms such as wheezing, dyspnea and the like after 24 hours of inoculation of haemophilus parasuis, body temperature rises, ear, abdomen and buttocks cyanosis occurs, and death begins to occur after 2 days. The HN1565 strain inoculated group has respiratory symptoms such as wheezing, cough and the like at 36h after inoculation, gradually emaciation, retardation and reduced feed reward accompanied by body temperature rise, and does not have death. Animals in the control group were normothermic and had no apparent respiratory symptoms during the trial. The onset and mortality are shown in Table 2.
And after the test is finished, the animals of the test group are killed, 5 animals of the control group are killed at the same time, and the disease materials are collected for bacterial separation. The HN1397 strain, HN1553 strain, HNHPS1 strain, HN1570 strain, HN486 strain, HN1601 strain and HN1513 strain are inoculated to form a group, and the group is examined to obtain a large amount of cellulose-like exudation of the lung, and the lung is adhered with pleura, pericardial effusion, choriocarcinoma and cerebral hemorrhage; HN1565 strain inoculated group has small amount of cellulose exudation in thoracic cavity, lung hemorrhage and other pneumonia symptoms, pericardial effusion; no obvious pathological change occurs in the control group, no haemophilus parasuis is separated from 5 disease materials of the control group, haemophilus parasuis is separated from 5 disease materials of each of 8 test groups, and the PCR identification result is consistent with that of the inoculated strain.
By combining toxicity test results, it was confirmed that haemophilus parasuis strains HN1397, HN1570, HN486, HN1601 and HN1513 were more virulent, HN1553 and HNHPS1 were moderately virulent, and HN1565 was less virulent.
Example 3: vaccine preparation, safety and efficacy testing
3.1 Preparation of vaccine
3.1.1 Strain and Strain propagation
First seed propagation
The haemophilus parasuis 1 type HN1397 strain, 2 type HN1553 strain, 4 type HNHPS1 strain, 5 type HN1570 strain, 7 type HN1565 strain, 12 type HN486 strain, 13 type HN1601 strain and 14 type HN1513 strain are respectively inoculated on TSA flat plates containing new born calf serum (final concentration 5%) and NAD (final concentration 0.01%), the flat plates are cultured for 18 to 24 hours at 37 ℃, typical colonies meeting the requirements are selected, the flat plates containing fetal calf serum (final concentration 5%) and NAD (final concentration 0.01%) are respectively inoculated in a passage way, and the flat plates are cultured for 24 hours at 37 ℃, and the flat plates are used as primary seeds after the test is passed.
Secondary seed propagation
The primary seeds of each strain are respectively inoculated into TSB liquid culture medium containing new born calf serum (final concentration 5%) and NAD (final concentration 0.01%), shake culture is carried out for 18-24 h at 37 ℃, and the primary seeds are used as secondary seeds after pure inspection is passed.
3.1.2 Culture of antigenic solution
Secondary seeds of the eight strains were inoculated at 1% respectively into TSB liquid medium containing neonatal bovine serum (final concentration 5%) and NAD (final concentration 0.01%), at 37 ℃. And (5) culturing for 18-24 hours by shaking and harvesting bacterial liquid.
3.1.3 Viable count
According to the current method of annex of Chinese animal pharmacopoeia, a TSA culture medium which is suitable for the growth of the strain and contains 5% of new born calf serum and 0.01% of NAD is used for counting the viable bacteria. Shake flask culture is used, viable bacteria count concentration is achieved, and the viable bacteria content of eight strain cultures is 1.0X10% 9 CFU/mL or more.
3.1.4 bacterial liquid inactivation
To each of the eight haemophilus parasuis strains of culture medium was added a total volume of 0.2% formaldehyde solution, and the mixture was inactivated at 37℃for 48 h.
3.1.5 Inactivation test
0.2mL of inactivated bacterial liquid of eight strains is respectively taken and inoculated with a TSA plate (containing 5% of new born calf serum and 0.01% of NAD), and the plate is cultured at 37 ℃ for 48 h, and no bacteria grow to be qualified.
3.1.6 Antigen concentration
And (5) respectively concentrating the eight antigen bacterial liquids which are qualified in the inactivation test through an ultrafiltration system device. Based on the result of counting live bacteria before inactivation, the concentration of the eight bacterial antigens was adjusted to 1.0X10 using sterile physiological saline 10 CFU/mL, and the eight antigen solutions were mixed uniformly in a ratio of 1:1:1:1:1:1:1.
3.1.7 Adjuvant preparation
Split charging beasts and birds with water-in-oil-in-water adjuvant Summit-S550 into bottles, sterilizing at 121deg.C for 30 min, and storing at room temperature for use.
3.1.8 Vaccine preparation
Adding 30% of water-in-oil-in-water adjuvant Summit-S550 for livestock and poultry according to the concentrated and uniformly mixed antigen liquid, stirring and fully and uniformly mixing, and subpackaging to obtain the haemophilus parasuis disease octave inactivated vaccine with the aluminium hydroxide gel adjuvant. The final vaccine contains eight strains with total viable count of 1.0X10 before inactivation 10 CFU/mL。
3.2 Safety test of vaccine
10 healthy piglets of 14 days of age are selected and divided into 2 groups of 5 piglets.
Vaccine group: neck intramuscular injection of 4 mL of the vaccine;
control group: neck intramuscular injection of 4 mL sterilized normal saline. Animal body temperature was measured and clinical manifestations were observed for a total of 2 weeks.
The observation shows that the nursery pigs of the vaccine group and the control group have normal respiration, appetite and mental state during the whole observation period, the average body temperature is raised by not more than 1 ℃, and no obvious local or systemic adverse reaction exists. The safety of the inactivated vaccine of the invention is demonstrated.
3.3 Efficacy test of vaccine
160 healthy piglets of 15 days old are selected, and 16 vaccine immunization groups and 16 control groups are established, wherein each group is provided with 5 piglets. Each vaccine group was injected with 2ml of the vaccine, and 3 weeks later, the vaccine group was given an equivalent dose of the vaccine without vaccination. The vaccine immunization group of 16 groups and the control group of 16 groups are respectively used for 10 days after the secondary immunization 9 The strain of CFU is H.parasuis type 1 HN1397, type 2 HN1553, type 4 HNHPS1, type 5 HN1570, type 7 HN1565, type 12 HN486, type 13 HN1601 and type 14 HN1513, type 3, type 6, type 8, type 9, type 11, type 15 and type 15 strains, and the strain liquid which is not able to be typed is used for intraperitoneal toxicity attack. The clinical manifestations of each group of piglets were observed, the body temperature was measured daily, and the total observation was 2 weeks.
Calculating morbidity and mortality of experimental piglets in the whole observation period, measuring body temperature, and carrying out bacterial isolated culture on organs such as the lungs of dead pigs.
Observations find that: the vaccine group and the control group have obvious differences, the next day after the virus attack, the piglets of the control group begin to have clinical symptoms, the body temperature rises, the breathing is difficult, the piglets begin to die after 2 days, and the pigs of different strain virus attack groups have different degrees of morbidity and death. The control group was examined for lesions of varying degrees, with effusion in the chest and abdominal cavities, and with cellulosic exudates, pericardial effusion, cellulosic exudates in the pericardial cavity, and pericardial adhesions. The lung is swollen, the two sides of the lung are blood stasis or bleeding, and the surface is cellulose exudates. Meningeal hyperemia and cerebrospinal fluid increase. In the 16 vaccine groups, pigs did not experience morbidity and mortality. The results of immune challenge for the different serotypes are shown in Table 3.
As can be seen from the immune challenge test results in Table 3, the vaccine 2ml has 100% protection rate on the infection of the clinical haemophilus parasuis serum type 1, type 2, type 4, type 5, type 7, type 12, type 13 and type 14, and has excellent cross protection on the infection of the standard strains type 3, type 6, type 8, type 9, type 11 and type 15 and haemophilus parasuis which cannot be typed, thus indicating that the inactivated vaccine has excellent protection effect on all haemophilus parasuis infection.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Sequence listing
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<213> Artificial sequence (Artificial Sequence)
<400> 16
ctccatagaa cctgctgctt gag 23
<210> 17
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
agccacatca attttagcct catca 25
<210> 18
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
ccttaaatag cctatgtctg tacc 24
<210> 19
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
ggtgacattt atgggcgagt aagtc 25
<210> 20
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
gcactgtcat caataacaat cttaagacg 29
<210> 21
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
ccatctcttt aactaatggg actg 24
<210> 22
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
ggacgccaag gagtattatc aaatg 25
<210> 23
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
atggctcacg atccgaaag 19
<210> 24
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
atttcccttt cctaaacgc 19
<210> 25
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ccatctcttt aactaatggg actg 24
<210> 26
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ggacgccaag gagtattatc aaatg 25
<210> 27
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
gctggttatg actatttctt tcgcg 25
<210> 28
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
gctcccaaga ttaaaccaca agcaag 26
<210> 29
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
caagttcgga ttgggagcat atatc 25
<210> 30
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
cctatatcat ttgttggatg tacg 24
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
gtgatgagga agggtggtgt 20
<210> 32
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
ggcttcgtca ccctctgt 18
Claims (7)
1. A combination strain for preparing haemophilus parasuis vaccine, characterized in that: comprises haemophilus parasuis 1 type HN1397 strain with the preservation number of CGMCC NO:16804; haemophilus parasuis 2 HN1553 strain with a preservation number of CGMCC NO:16801; haemophilus parasuis 4 type HNHPS1 strain with a preservation number of CGMCC NO:13335; haemophilus parasuis 5 type HN1570 strain with a preservation number of CGMCC NO:16802; haemophilus parasuis 7 HN1565 strain with the preservation number of CGMCC NO:16803; haemophilus parasuis 12 type HN486 strain with a preservation number of CGMCC NO: 16505; haemophilus parasuis 13 HN1601 strain with a preservation number of CGMCC NO:16806; haemophilus parasuis 14 type HN1513 strain with a preservation number of CGMCC NO:16807.
2. an octavalent inactivated vaccine of haemophilus parasuis prepared from the combination strain of claim 1, wherein: comprises eight inactivated haemophilus parasuis bacterial liquids and an immunoadjuvant.
3. The haemophilus parasuis octavalent inactivated vaccine according to claim 2, wherein: haemophilus parasuis type 1, type 2, type 4, type 5, type 7, type 12, type 13, type 14 serotype antigen ratio is 1:1:1:1:1:1:1:1.
4. The haemophilus parasuis octavalent inactivated vaccine according to claim 2, wherein: the content of the type 1, type 2, type 4, type 5, type 7, type 12, type 13 and type 14 antigens of haemophilus parasuis is 1×10 10 CFU/mL。
5. The haemophilus parasuis octavalent inactivated vaccine according to claim 2, wherein: the immune adjuvant is Summit S550, a water-in-oil-in-water adjuvant for livestock and poultry.
6. A method for preparing the haemophilus parasuis octavalent inactivated vaccine according to any one of claims 3 to 5, wherein: the method comprises the following steps:
(1) Proliferation: the strain of HN1397, HN1553, HNHPS1, HN1570, HN1565, HN486, HN1601 and HN1513 are propagated and cultured respectively to obtain 8 haemophilus parasuis bacterial solutions, and viable counts are carried out respectively;
(2) And (3) inactivation: adding formaldehyde solution into bacterial solutions of haemophilus parasuis HN1397 strain, HN1553 strain, HNHPS1 strain, HN1570 strain, HN1565 strain, HN486 strain, HN1601 strain and HN1513 strain obtained in the step (1) according to 0.2% of the volume of the bacterial solutions, and inactivating 48 h at 37 ℃;
(3) Concentrating: concentrating the qualified bacterial solutions of haemophilus parasuis HN1397, HN1553, HNHPS1, HN1570, HN1565, HN486, HN1601 and HN1513 after inactivation by using an ultrafiltration device, and adjusting the concentration by using sterile physiological saline according to the viable bacteria count before inactivation;
(4) Preparing a vaccine: the haemophilus parasuis HN1397 strain, HN1553 strain, HNHPS1 strain, HN1570 strain, HN1565 strain, HN486 strain, HN1601 strain and HN1513 strain antigen liquid obtained after proliferation, inactivation and concentration are uniformly mixed according to a proportion, and an adjuvant is added, so that the haemophilus parasuis octavalent inactivated vaccine is prepared.
7. The method for preparing the haemophilus parasuis octavalent inactivated vaccine according to claim 6, wherein in the step (4), eight bacterial strain inactivated antigen solutions are uniformly mixed according to the ratio of 1:1:1:1:1:1:1:1, and added with 30% of water-in-oil-in-water adjuvant for livestock and poultry in the final volume for uniform mixing.
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| CN104611274A (en) * | 2015-02-11 | 2015-05-13 | 青岛农业大学 | Haemophilus parasuis and application thereof |
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| CN108441446A (en) * | 2018-03-27 | 2018-08-24 | 河南科技大学 | A kind of trivalent inactivated vaccine against Haemophilus parasuis infection and its production method and application |
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| RU2673715C2 (en) * | 2013-10-04 | 2018-11-29 | Мериал, Инк. | Haemophilus parasuis vaccine serovar type 4 |
| WO2016119078A1 (en) * | 2015-01-29 | 2016-08-04 | 山东省农业科学院畜牧兽医研究所 | Combined use of haemophilus parasuis lc strain and lz-20100109 strain |
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| CN104611274A (en) * | 2015-02-11 | 2015-05-13 | 青岛农业大学 | Haemophilus parasuis and application thereof |
| CN107245459A (en) * | 2017-04-11 | 2017-10-13 | 河南省农业科学院畜牧兽医研究所 | One plant of haemophilus parasuis and its application |
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