A strain of Haemophilus somnus and its application

Abstract

The present invention proposes a strain of Haemophilus somnus and an application thereof. The strain of Haemophilus somnus is Haemophilus somnus HS / NMG / 2023, and its accession number is CGMCC No. 31702. The present invention provides a strain of Haemophilus somnus with stable passage, high bacterial count, strong pathogenicity and good immunogenicity. After being prepared into an inactivated vaccine for immunizing mice, it can produce an effective and stable immune protection effect against the prevalent pathogenic strains of Haemophilus somnus, and has good safety, providing important medical value for the prevention, control and treatment of Haemophilus somnus disease in pastoral areas, and has far-reaching and significant practical application significance.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to a strain of Haemophilus somnus and application thereof. Background Art

[0002] Haemophilus somnus disease in cattle is a multisystem disease caused by Haemophilus somnus, which infects systemic organs including nervous, respiratory, digestive, renal, muscular, skeletal and reproductive, and is sometimes called Haemophilus somnus syndrome. First of all, H. somnus is a common commensal bacteria in the upper respiratory tract of cattle and one of the four main bacteria that cause bovine respiratory disease (BRDC), which is most common in feedlot cattle. H. somnus can also cause other forms of systemic diseases in cattle, including sepsis, myocarditis, arthritis and thromboembolic meningoencephalitis (ITME). In addition, it is also a recognized pathogen that causes abortion and reproductive diseases in cattle, including infertility and endometritis. Haemophilus somniferum can cause bovine respiratory disease syndrome (BRDC) when infected alone or mixed with other pathogens. Upper respiratory tract infection causes suppurative laryngitis and tracheitis, and lower respiratory tract infection causes catarrhal pneumonia, suppurative pneumonia, thromboembolic meningoencephalitis (TEME), arthritis, fibrinous pleurisy, pericarditis, and sudden death caused by septic cardiovascular necrosis. Haemophilus somniferum causes BRDC. Sick cows and latent carrier cows are the main sources of infection. It mostly occurs in beef cattle, lactating cows, and grazing cows. There is no obvious seasonality, but it is more common in the damp, cold and condensed climates of winter and spring. It is a conditional pathogen. Feeding stress, transportation stress, and weaning stress can often cause the disease in cattle. At present, there are very few reports on bovine respiratory Haemophilus somniferum disease in China. In foreign cases, the positive reaction rate in cattle with respiratory symptoms was 59.6%, indicating that the respiratory infection rate of the disease is relatively high. Foreign research data show that 55% of male, female and calves in dairy cows have reproductive tract infections 27 weeks after calving, and the detection rate of this bacteria in the reproductive tract of cows is 33-76%. This bacteria is related to miscarriage and infertility. Pregnant cows infected with Haemophilus somniferum are likely to suffer miscarriage at any stage.

[0003] Haemophilus somnus belongs to the class Proteobacteria, class γ-Proteobacteria, order Pasteurelales, family Pasteurellaceae, and genus Haemophilus in bacterial taxonomy. Haemophilus somnus is a facultative anaerobic Gram-negative pleomorphic small coccobacillus, 1.0 μm or shorter in size, short chain or fibrous, non-motile, without spores and lacking flagella. On brain heart infusion blood solid medium, cultured at 37°C and 5% to 10% CO2 for 24 to 48 hours, yellow or off-white creamy round needle-shaped transparent raised small colonies are formed, which are moist and shiny, with a diameter of 1 to 2 mm. The strains show various forms of β-hemolytic, α-hemolytic, hemolytic or non-hemolytic, which only slightly turn the culture medium green. Its growth requires growth factors in animal tissues or bacterial extracts, but does not rely on X and V factors. Haemophilus somnus has a very short survival time in vitro. Data show that it can survive in nasal mucus or blood for more than 70 days at 23.5°C, in vaginal mucus for more than 5 days, and in urine for more than two hours (Frederick, W. et al., 1989).

[0004] At present, for areas where Haemophilus somnus disease is prevalent, some preventive measures are mainly taken to reduce or lower the stress response of animals. After the disease occurs, only symptomatic treatment can be taken. The commonly used method is to use antibiotics on a large scale. Although there is a certain preventive effect, once the effect of antibiotics disappears, repeated infection may occur again, and it is also very easy to cause the emergence of strains with multiple drug resistance. The most effective strategy for the prevention and control of Haemophilus somnus disease in cattle is immune prevention. The immunization interval should be fully considered in advance, and vaccination should be used to reduce the infection of cattle to Haemophilus somnus. At present, there are reports abroad that the use of vaccine to reduce the incidence and mortality rate by immunizing twice at a certain interval, but there are few research reports on Haemophilus somnus disease in my country, especially in the isolation and identification of Haemophilus somnus and the development of vaccines. Therefore, the successful isolation and identification of Haemophilus somnus strains and the development of effective vaccines are of great significance for the prevention and control of Haemophilus somnus disease. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] The present invention provides a strain of Haemophilus somnus and its application. The strain is purified, separated and cultured for multiple generations in an optimal solid culture medium, is a strain currently prevalent in Haemophilus somnus, can be stably propagated in the culture medium, has a high bacterial count, strong pathogenicity and good immunogenicity. The strain is prepared into an inactivated vaccine, which can produce an effective and stable immune protection effect against the prevalent pathogenic strains of Haemophilus somnus, and provides a basis for effective vaccine preparation and prevention and control of Haemophilus somnus.

[0007] (II) Technical solution

[0008] In a first aspect, the present invention provides a strain of Haemophilus somnus, which has been deposited in the China General Microbiological Culture Collection Center on August 19, 2024. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Classification Name: Haemophilus somnus, Latin name: Haemophilus somnus, and the deposit number is CGMCC No. 31702.

[0009] In a second aspect, the present invention provides the use of the above-mentioned Haemophilus somnus strain in the preparation of a drug or reagent, wherein the drug or reagent is used to treat, prevent or diagnose a disease caused by Haemophilus somnus.

[0010] In a third aspect, the present invention provides a vaccine comprising the Haemophilus somnus or a culture thereof.

[0011] Based on the third aspect, further, the vaccine includes an inactivated antigen prepared from the Haemophilus somnus or a culture thereof and an adjuvant.

[0012] Preferably, the adjuvant is an aluminum hydroxide gel adjuvant, wherein the volume ratio of the adjuvant to the antigen is 1:3 to 1:7.

[0013] Preferably, the effective content of the antigen in the vaccine is not less than 5×10 9 CFU / ml.

[0014] In a fifth aspect, the present invention provides a method for preparing a vaccine, the preparation method comprising:

[0015] Step 1, fermenting and culturing the bacterial liquid of the Haemophilus somnus strain;

[0016] Step 2, concentrating and inactivating the bacterial solution in step 1 to obtain an inactivated antigen bacterial solution;

[0017] Step 3: Mix the obtained inactivated antigen bacterial solution with the adjuvant to prepare an inactivated vaccine.

[0018] Furthermore, the step 1 specifically includes:

[0019] 1) Primary seed propagation: Inoculate the bacterial liquid of Haemophilus somnus strain into a liquid culture medium of brain heart infusion yeast extract, and culture it at 37℃ and 5% CO2 for 8-12h. Then, streak it onto a brain heart infusion yeast extract blood plate, and culture it at 37℃ and 5% CO2 for 16-20h. Select typical colonies and inoculate them onto several brain heart infusion yeast extract blood slant plates, and culture them at 37℃ and 5% CO2 for 16-20h. Use them as primary seeds and store them at 2-8℃ for expanded culture. The usage period shall not exceed 7 days.

[0020] 2) Secondary seed propagation: Take one of the first-level seeds in step 1.1), wash off the bacterial moss on the blood slope of the brain heart infusion yeast extract liquid culture medium, inoculate it into the brain heart infusion yeast extract liquid culture medium, and culture it at 37℃ and 5% CO2 for 8-12h. After passing the purity test, store it at 2-8℃ as the second-level seed;

[0021] 3) Preparation of fermentation liquid: The bacterial liquid of Haemophilus somniferus was cultured in a shake flask, and the secondary seed liquid was added at a rate of 2% (v / v) to the liquid culture medium of brain heart infusion yeast extract. The mixture was evenly mixed and cultured at 37°C in an environment of 5% CO2 for 8 to 12 hours to obtain the bacterial liquid of Haemophilus somniferus strain.

[0022] Furthermore, the step 2 specifically includes:

[0023] 1) Concentration of fermentation broth: The fermentation broth was concentrated by centrifugation at 3500 r / min, 4°C, for 20 min. The bacterial precipitate was then collected and resuspended in sterile PBS at pH 7.2 to 7.4 to prepare a 10-fold concentrated broth.

[0024] 2) Inactivation of fermented bacterial solution: Add formaldehyde solution at 0.1% to 0.2% (v / v) of the concentrated bacterial solution, inactivate at 37°C for 8 to 10 hours, shake several times during the process, and prepare an inactivated antigen bacterial solution;

[0025] Furthermore, the step 3 specifically includes: first adding the inactivated antigen-bacteria solution obtained in step 2 into an emulsifying tank, stirring at a low speed, and then slowly adding aluminum hydroxide gel adjuvant in a volume ratio of 1 part adjuvant to 5 parts antigen-bacteria solution, stirring to fully mix them, and preparing an inactivated vaccine.

[0026] (III) Beneficial effects

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a strain of Haemophilus somnus that is stable in passage, has a high bacterial count, is highly pathogenic, and has good immunogenicity. The results of the examples show that after immunizing mice with an inactivated Haemophilus somnus vaccine prepared using the strain as an antigen, it can produce an effective and stable immune protection effect against the prevalent pathogenic strains of Haemophilus somnus, and has good safety. Therefore, the inactivated vaccine provided by the present invention is expected to provide a good preventive effect on Haemophilus somnus disease in pastoral areas, and has no potential safety risks, providing important medical value for the prevention, control and treatment of Haemophilus somnus disease, and has far-reaching and significant practical application significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is a photo of the streak inoculation colony of the HS / NMG / 2023 strain.

[0029] Figure 2 This is a microscopic photograph of the HS / NMG / 2023 strain after Gram staining.

[0030] Figure 3 This is the S-shaped curve of HS / NMG / 2023 strain-specific PCR quantitative detection.

[0031] Figure 4 This is the gel electrophoresis diagram of 16SrDNA sequencing of HS / NMG / 2023 strain.

[0032] Figure 5 This is an information chart of the existing HS strains publicly available overseas.

[0033] Figure 6 This is the result of homology analysis of 16SrDNA sequencing of HS / NMG / 2023 strain.

[0034] Figure 7 This is the result of 16SrDNA sequencing evolutionary tree analysis of HS / NMG / 2023 strain.

[0035] Figure 8 This is the growth curve of the HS / NMG / 2023 strain P7.

[0036] The provided bovine Haemophilus somnus strain HS / NMG / 2023 was deposited in the China General Microbiological Culture Collection Center on August 19, 2024. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Classification Name: Haemophilus somnus, Latin name: Haemophilus somnus; The deposit number is CGMCC No.31702. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0039] The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0040] Example 1: Isolation and identification of Haemophilus somnus strains

[0041] 1.1 Culture medium and disease materials

[0042] Brain heart infusion yeast extract culture medium (brain heart infusion manufacturer: Guangdong Huankai Microbial Technology Co., Ltd., product number: 028360; yeast extract manufacturer: Guangdong Huankai Microbial Technology Co., Ltd., product number: 050070; horse serum manufacturer: Hohhot Caoyuan Lvye Bioengineering Materials Co., Ltd.) is prepared by obtaining different raw material components through commercial channels; the pathological materials are nasal swabs and diseased lung tissues collected from a cattle farm positive for Haemophilus somnifera in Inner Mongolia Autonomous Region in 2023.

[0043] It should be noted that the brain heart infusion yeast extract culture medium and brain heart infusion yeast extract blood agar plate mentioned in the present invention have the same composition and ratio unless otherwise specified.

[0044] 1.2 Isolation and culture

[0045] The nasal swabs and diseased lung tissues of sick cattle collected from Gelinger County, Hohhot City, Inner Mongolia were streaked and inoculated on the blood agar plate of brain heart infusion yeast extract, and cultured in a 5% CO2 constant temperature box at 37℃ for 16-20h. Among them, only one positive diseased material showed suspected Haemophilus somnus colonies on the blood agar plate. The suspicious colonies (off-white creamy round needle-shaped transparent protrusions, small colonies, moist and shiny, slightly hemolytic, after continuous subculture, the hemolytic property will weaken or disappear, making the culture medium slightly green) were picked. Figure 1As shown in the figure, the left picture is a colony cultured for 16 hours, and the right picture is an old colony cultured for 72 hours). The smear was performed aseptically and Gram staining was performed to observe the bacterial morphology and staining results. The results are shown in Figure 2. After Gram staining, polymorphic, Gram-negative spherical short rods were observed under the microscope, arranged in short chains or fibers.

[0046] 1.3 Identification of specific primers for quantitative PCR

[0047] 1) Take several single colonies of the suspected Haemophilus somnus examined under the microscope and inoculate them into appropriate amounts of brain heart infusion yeast extract medium (numbered 1-8, containing 0.5% yeast extract and 2% serum), and culture them at 37°C in a 5% CO2 incubator for 8-12 hours to obtain bacterial culture fluid;

[0048] 2) Take 200 μL of each of the above bacterial culture solutions as test samples (corresponding to brain heart infusion yeast extract culture medium numbers 1-8), extract genomic DNA respectively, and use it as a template to perform gene-specific PCR fluorescence quantitative detection of bovine Haemophilus somnus (Xu Liyuan et al., Fluorescence quantitative PCR detection kit for bovine Haemophilus somnus and its dedicated primers and TaqMan probe, Chinese invention patent, publication number: CN116219044A, publication date: 2023.06.06, main classification number: C12Q1 / 689), and the strain-specific PCR quantitative detection S-shaped curve is shown as follows Figure 3 As shown, except for samples 3 and 5 which did not show the characteristic S-shaped curve of fluorescence quantification, the other 6 samples all showed the characteristic S-shaped curve and were identified as positive for Haemophilus somniferus.

[0049] 1.4. 16S rDNA sequencing and identification

[0050] The Haemophilus somnus strains identified by PCR (corresponding to numbers 1-2, 4, 6-8) were streaked onto brain heart infusion yeast extract blood agar plates for purification, and inoculated into brain heart infusion yeast extract liquid medium (containing 0.5% yeast extract and 2% serum), purified continuously, and then PCR was performed using primers for 16S rDNA gene sequence (fragment size 1493 bp)

[0051] A small amount of PCR product was taken for electrophoresis in 1.5% agarose gel (a target band of about 1493 bp was visible, which was consistent with the expected size. The electrophoresis results were as follows Figure 4As shown, No. 1-6 correspond to No. 1, 2, 4, 6, 7, and 8 strains, respectively). The amplified PCR products were sent to Beijing Sangon Biotech Co., Ltd. for sequencing and the results were spliced. The sequencing results were analyzed by BLAST online comparison at NCBI, and 4 strains (corresponding to No. 2, 6, 7, and 8) were confirmed to be positive for Haemophilus somniferus.

[0052] The sequences of the four strains were compared with the reference sequences downloaded from the GenBank database for homology, and genetic evolution analysis was performed. The homology comparison results of their 16SrDNA gene sequences with the nucleotide sequences of the six reference genes of Haemophilus somnus were between 99.5% and 99.8%, and the homology with the nucleotide sequence of the reproductive tract strain HS / JS / 2024 isolated by our company in 2024 was 99.4% ( Figure 6 ), the homology of the four strains was 100%, which was very close to the homology of the current epidemic reference strains. This isolated strain can be used as a candidate vaccine strain for preventing the current clinical epidemic of Haemophilus lethargica disease.

[0053] 1.5 Biochemical identification

[0054] Referring to the biochemical characteristics of Pasteurellaceae and Haemophilus recorded in the Bergey's Manual of Bacterial Identification, the four strains of Haemophilus somnus identified by quantitative PCR specific primers and 16SrDNA sequencing were subjected to sugar fermentation test (muscarinic acid, glucose, fructose, inositol, raffinose, sorbitol, sucrose, mannitol, xylose, lactose), VP test, MR test, and nitrate reduction test. The methods and results were determined according to the instructions of the purchased biochemical identification tube. The biochemical identification results of the four isolated strains were consistent, which was consistent with the biochemical characteristics of the genus in the Bergey's Manual of Bacterial Identification and literature records.

[0055] Note: “+” indicates positive; “-” indicates negative

[0056] 1.6 Colony count and growth curve determination

[0057] 1) Dilute the Haemophilus somnus bacterial suspension into 10-fold gradients using brain heart infusion yeast extract liquid culture medium. Select 3 appropriate serial dilutions (10 -6 , 10 -7 , 10 -8 ), use a pipette to draw 100µl drop of brain heart infusion yeast extract blood agar plate (different components purchased from the market are prepared by yourself, the ingredients are brain heart infusion and 0.5% yeast extract, 1.5% agar, 5% sterile defatted and defibrillated sheep blood, mix thoroughly and spread on the plate and store at 4℃ for later use), each dilution is parallel to 3 plates, inverted in a 5% CO2 incubator at 37℃ for incubation for 16-20h, observe the growth of colonies on each culture plate, and count the colonies on each culture plate.

[0058] 2) Take the culture of Haemophilus somniferus to draw the growth curve of the strain. Take the culture once every 2 hours and drop it on the brain heart extract yeast extract blood agar plate. Continue to take it until the end of 24 hours. Put the plate after each sampling into a 5% CO2 constant temperature box at 37℃ for 16 to 20 hours to form colonies. Summarize the colony count results of each time period (take the average of the colony counts of two culture plates) to draw a growth curve, see Figure 8 .

[0059] 1.7 Virulence screening in mice

[0060] 1) Strain culture: The four strains identified as positive for Haemophilus somnus after purification were inoculated with 2% (v / v) in a brain heart infusion yeast extract plus 2% serum liquid culture medium, and cultured at 37°C, 5% CO2 for 8-12 hours. The culture liquid was streaked and inoculated on a brain heart infusion yeast extract blood plate, and cultured at 37°C, 5% CO2 for 16-20 hours. Typical colonies were selected under a microscope and inoculated on several brain heart infusion yeast extract blood slants, and cultured at 37°C, 5% CO2 for 16-20 hours. The bacterial moss on one blood slants was rinsed with brain heart infusion yeast extract culture medium, and then inoculated into 200 ml brain heart infusion yeast extract culture medium, and cultured at 37°C in a 5% CO2 incubator for 8-12 hours.

[0061] 2) Toxicity test: 10 healthy susceptible mice weighing 18-22 g were selected and divided into 5 groups. Two mice in each group were intraperitoneally injected with 4 fresh culture solutions and liquid culture medium control (0.2 ml / mouse). The mice were observed for 3 days and the mortality was recorded.

[0062]

[0063] Note: "G-" indicates negative Gram staining, and "+" indicates positive PCR identification.

[0064] As can be seen from Table 2, the purified strains numbered 2 and 6 have strong virulence, and all 2 mice were killed. Combined with the growth of the strains in the previous strain growth curve determination, strain No. 6 was selected as a candidate strain for vaccine development and named HS / NMG / 2023. In the process of strain purification, separation and culture, the present invention screened a large number of nasal swabs and lung tissues of positive samples of bovine Haemophilus somnus for flat blood streak subculture. Only one positive sample was purified and cultured by continuous picking and streaking to obtain typical colony growth. During the subculture process, a single typical colony derived from a positive sample was continuously picked and streaked to obtain multiple purified clone strains. After liquid culture medium amplification culture and Gram staining microscopy, specific primer quantitative detection, 16SrDNA sequencing, growth curve determination, mouse pathogenicity determination, etc., it was found that some strains were difficult to purify and could not be subcultured continuously, and some strains had low bacterial counts and weak virulence. After a large number of screenings, only two strains with both good passage stability and high virulence were found. Combined with the growth conditions in the growth curve assay, the strain with the highest colony count was selected. After verification by the immunogenicity test, the preserved strain HS / NMG / 2023 with strong pathogenicity and good immunogenicity of the present invention was obtained.

[0065] Example 2: Preparation of inactivated vaccine of Haemophilus somnus

[0066] In this embodiment, an inactivated Haemophilus somnus vaccine is prepared using the HS / NMG / 2023 strain of Haemophilus somnus isolated in the above Example 1, which specifically includes the following steps.

[0067] 2.1 Fermentation culture of Haemophilus somnus HS / NMG / 2023 strain was performed to obtain fermentation liquid, specifically:

[0068] 1) Primary seed propagation: 200 μL of Haemophilus somniferus HS / NMG / 2023 strain was inoculated into 10 mL of brain heart infusion yeast extract liquid culture medium (brain heart infusion containing 0.5% yeast extract and 2% horse serum, stored at 4°C for standby use), cultured at 37°C, 5% CO2 for 8-12 h, then streaked and inoculated onto brain heart infusion yeast extract blood plates (different components purchased from the market were prepared by themselves, the components were brain heart infusion and containing 0.5% yeast extract, 1.5% agar, 5% sterile defatted and defibrated sheep blood, fully mixed and spread on plates and stored at 4°C for standby use), cultured at 37°C, 5% CO2 for 16-20 h, typical colonies were selected, and several brain heart infusion yeast extract blood slants were inoculated respectively, cultured at 37°C, 5% CO2 for 16-20 h, as primary seeds, stored at 2-8°C for expanded culture, and the usable period shall not exceed 7 days.

[0069] 2) Secondary seed propagation: Take one primary seed, wash off the bacterial moss on the blood slope of brain heart infusion yeast extract with 4 mL of brain heart infusion yeast extract liquid culture medium (same as above), inoculate into 200 mL of brain heart infusion yeast extract liquid culture medium, incubate at 37°C, 5% CO2 environment for 8-12 hours, and after passing the purity test, store at 2-8°C as secondary seed;

[0070] 3) Preparation of fermentation liquid: The liquid culture of Haemophilus somnus was cultured in a shake flask. The secondary seed liquid was added to the liquid culture medium of brain heart extract yeast extract (same as above) at a rate of 2% (v / v), mixed evenly, and cultured at 37°C in a 5% CO2 environment for 8 to 12 hours.

[0071] After the bacterial liquid culture is completed, samples are taken for purity inspection according to the appendix of the 2015 edition of the "Chinese Pharmacopoeia of Veterinary Medicine", and they should be pure; at the same time, samples are taken for live bacteria count.

[0072] 2.2 The fermented bacterial solution obtained in step 2.1 is concentrated and inactivated to obtain an inactivated antigen bacterial solution, specifically:

[0073] 1) Concentration of fermentation broth: The fermentation broth was concentrated by centrifugation at 3500 r / min, 4°C, for 20 min. The bacterial precipitate was then collected and resuspended in sterile PBS at pH 7.2-7.4 to prepare a 10-fold concentrated broth.

[0074] 2) Inactivation of fermented bacterial solution: Add formaldehyde solution at 0.1% to 0.2% (v / v) of the concentrated bacterial solution, inactivate at 37°C for 8 to 10 hours, shake several times during the period, and prepare the inactivated antigenic bacterial solution. Inactivation test is carried out according to the appendix of the 2015 edition of the Chinese Veterinary Pharmacopoeia, and there should be no sterile growth.

[0075] 2.3 The inactivated antigenic bacterial solution obtained in step 2.2 is mixed with the aluminum hydroxide gel adjuvant in a volume ratio of 3:1 to 7:1 (preferably 5:1) to prepare an inactivated vaccine, specifically:

[0076] First add the inactivated antigen-bacteria solution obtained in step 2.2 to the emulsification tank, stir at low speed for 5 minutes, then slowly add aluminum hydroxide gel adjuvant in a volume ratio of 1 part adjuvant to 5 parts antigen-bacteria solution, stir thoroughly for 15 to 20 minutes to mix thoroughly. It has been verified that the final vaccine contains no less than 5×10 9 CFU / ml. According to the Appendix of the 2015 edition of the Chinese Veterinary Pharmacopoeia, sterility test should be carried out, and there should be no sterile growth.

[0077] 2.4 Packaging

[0078] After passing the above sterility test, quantitative packaging should be carried out. Stirring should be done at any time during packaging to ensure uniform mixing. Seal with a stopper and label. Store at 2-8℃.

[0079] According to the above method steps, this embodiment uses HS / NMG / 2023 strain to prepare three batches of inactivated Haemophilus somnus vaccines (vaccine batch numbers are: S24001, S24002, and S24003).

[0080] 2.5 Vaccine product testing

[0081] 1) Properties: After standing, the upper layer is a clear liquid and the lower layer is a grayish white precipitate. After shaking, it becomes a uniform suspension.

[0082] 2) Filling quantity inspection: Inspection shall be carried out in accordance with the appendix of Part III of the current Chinese Pharmacopoeia of Veterinary Medicine and comply with the regulations.

[0083] 3) Sterility test: Test according to the third appendix of the current "Chinese Pharmacopoeia of Veterinary Medicine", sterile growth.

[0084] 4) Safety test: Randomly select vaccines from each batch of vaccine products (vaccine batch numbers: S24001, S24002, S24003) for testing, and conduct safety tests on each batch of vaccines. The test method is: use 2 guinea pigs weighing 350-450g, each subcutaneously inject 2ml of vaccine; use 5 mice weighing 18-22g, each subcutaneously inject 0.5ml of vaccine, and observe daily for 7 days. After testing, no guinea pigs or mice injected with vaccines from each batch showed death or obvious local reactions or systemic adverse reactions caused by the injection of vaccines.

[0085] 2.6 Evaluation of the immune efficacy of inactivated Haemophilus somnus vaccine in mice

[0086] 1) Determination of the minimum lethal dose in mice A batch of fresh bacterial solution was prepared according to the steps in Example 2, with the batch number being JY23001. Twenty mice weighing 18-22 g were selected and randomly divided into four groups, with 5 mice in each group. Three of the groups were intraperitoneally injected with 0.2 ml of bacterial solution (containing 2×10 live bacteria) 9 CFU / pc, 4×10 8 CFU / pc, 8×10 7 CFU / mouse), and the remaining 5 mice were used as controls and injected with the same volume of brain heart infusion yeast extract liquid culture medium and observed for 5 days. 9 CFU / pc, 4×10 8 All mice with 8×10 CFU / mouse died within 5 days. 7Two mice with CFU / mouse died, while all control mice were alive. The results are shown in Table 3, indicating that the minimum lethal dose of Haemophilus somnus HS / NMG / 2023 strain for 18-22 g mice by intraperitoneal injection is 4×10 8 CFU / piece.

[0087]

[0088] 2) Immunization and challenge Fifteen healthy mice were randomly divided into three groups, each with five mice. The three batches of vaccines (S23001, S23002, and S23003) prepared in Example 2 were subcutaneously inoculated with 0.2 ml of each. Five control mice with the same conditions were inoculated with sterilized PBS in the same way as controls. 21 days after the inoculation, the second immunization was performed with the same dose and the same inoculation method. 14 days after the second immunization, the 15 immunized mice and the five control mice were challenged with Haemophilus somnus (HS / NMG / 2023 strains, the challenge dose was 4×10 8 CFU), and then observed for 10 consecutive days to observe the death of mice and determine the protection status. The test results are shown in Table 4 below.

[0089]

[0090] From the results in Table 4 above, it can be seen that the mice were challenged with Haemophilus somnus 14 days after the second immunization with the corresponding inactivated vaccine. Among the three batches of vaccines, batches S23001 and S23002 showed an immune protection effect of 5 / 5, and batch S230032 showed an immune protection effect of 4 / 5. However, all 5 control mice died after the challenge. This shows that the inactivated Haemophilus somnus vaccine prepared by the present invention can enable the immunized mice to obtain relatively stable and effective protection against the challenge strain after inoculation.

[0091] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Haemophilus somnus, characterized in that The Haemophilus somnus strain ( Haemophilus somnus )'s accession number is CGMCC No.31702.

2. Use of the Haemophilus somnus strain as claimed in claim 1 in the preparation of a drug for treating or preventing Haemophilus somnus disease.

3. A vaccine, characterized in that The vaccine contains the Haemophilus somnus according to claim 1, or a culture thereof.

4. The vaccine according to claim 3, characterized in that The vaccine comprises an inactivated antigen prepared from the Haemophilus somnus or its culture and an adjuvant.

5. The vaccine according to claim 4, characterized in that The adjuvant is aluminum hydroxide gel adjuvant, wherein the volume ratio of the adjuvant to the antigen is 1:3 to 1:

7.

6. The vaccine according to claim 4, characterized in that The content of the antigen in the vaccine is not less than 5×10 9 CFU / ml.

7. A method for preparing a vaccine according to any one of claims 3 to 6, characterized in that: The preparation method comprises: Step 1, fermenting and culturing the bacterial liquid of the Haemophilus somnus strain according to claim 1; Step 2, concentrating and inactivating the bacterial solution in step 1 to obtain an inactivated antigen bacterial solution; Step 3: Mix the obtained inactivated antigen bacterial solution with the adjuvant to prepare an inactivated vaccine.

8. The preparation method according to claim 7, characterized in that: The step 1 specifically includes: 1) Primary seed propagation: Inoculate the bacterial liquid of Haemophilus somnus strain into a liquid culture medium of brain heart infusion yeast extract, incubate it at 37℃, 5% CO2 for 8-12h, then streak it onto a brain heart infusion yeast extract blood plate, incubate it at 37℃, 5% CO2 for 16-20h, select typical colonies, inoculate several brain heart infusion yeast extract blood slants, incubate them at 37℃, 5% CO2 for 16-20h, use them as primary seeds, and store them at 2-8℃ for expanded culture; 2) Secondary seed propagation: Take one of the first-level seeds in step 1), wash off the bacterial moss on the blood slope of the brain heart infusion yeast extract liquid culture medium, inoculate it into the brain heart infusion yeast extract liquid culture medium, and culture it at 37℃ and 5% CO2 for 8-12h. After passing the purity test, store it at 2-8℃ as the second-level seed; 3) Preparation of fermentation liquid: The bacterial liquid of Haemophilus somnus was cultured in a shake flask, and the secondary seed liquid was added at a rate of 2% v / v to the liquid culture medium of brain heart infusion yeast extract, the mixture was evenly mixed, and the culture was carried out at 37°C in an environment of 5% CO2 for 8-12 hours to obtain the bacterial liquid of Haemophilus somnus strain.

9. The preparation method according to claim 8, characterized in that: The step 2 specifically includes: 1) Concentration of fermentation broth: The fermentation broth was concentrated by centrifugation at 3500 r / min, 4°C, for 20 min. The bacterial precipitate was then collected and resuspended in sterile PBS at a pH of 7.2 to 7.4 to prepare a 10-fold concentrated broth. 2) Inactivation of fermented bacterial solution: add formaldehyde solution at 0.1% to 0.2% v / v of the concentrated bacterial solution, inactivate at 37° C. for 8 to 10 hours, and shake well to prepare an inactivated antigen bacterial solution.

10. The preparation method according to claim 9, characterized in that: The step 3 specifically comprises: first adding the inactivated antigen-bacteria solution obtained in step 2 into an emulsifying tank, stirring at a low speed, then adding aluminum hydroxide gel adjuvant in a volume ratio of 1 part adjuvant to 5 parts antigen-bacteria solution, stirring to fully mix, and preparing an inactivated vaccine.

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