An inactivated vaccine against Salmonella abortus equi

Abstract

The present invention belongs to the technical field of veterinary medicine preparation, and provides an inactivated vaccine against Salmonella abortus equi, using Salmonella abortus equi C.SDLCYANG.2021 as an antigen and nanoaluminum adjuvant and / or recombinant flagellin of Salmonella abortus equi as an adjuvant. After immunizing mice with this vaccine, they can completely resist the attack of strong strains, and adding recombinant flagellin can significantly reduce the number of bacteria carried by Salmonella abortus equi in the spleen; after immunizing donkey herds, it has good safety, no systemic and local adverse reactions after immunization, does not affect feeding, and has good protective efficacy, and can be used to prevent the abortion of pregnant female equines caused by this type of pathogenic bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drug preparation, and particularly relates to an inactivated equine Salmonella abortus vaccine. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be regarded as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Equine abortion Salmonella, also known as equine paratyphoid, is an infectious disease characterized by abortion in equine animals. The disease can occur throughout the year, mainly in early spring and late autumn. Under natural conditions, Salmonella abortus ( Salmonella abortus Equine (equi) only infects equines. Infected equines continuously excrete pathogens through semen, feces, and amniotic fluid of aborted fetuses, contaminating feed and drinking water, leading to persistent infection of the bacteria in farms, and female animals that have had abortions may be infected again.

[0004] With the expansion of large-scale donkey farming and the interregional transportation of donkey herds, miscarriages caused by Salmonella equi are becoming a common occurrence in major donkey-breeding areas in my country. A study showed that 94.3% of over 100 aborted donkey fetal samples tested were positive for Salmonella equi, indicating that Salmonella equi has become the most important cause of miscarriage in pregnant donkeys on large-scale breeding farms. Therefore, the development of a preventive and control agent for this disease is imperative.

[0005] my country successfully developed an attenuated Salmonella enterica equine abortion vaccine in the 1970s, which played a significant role in the prevention and control of the disease. However, it has the potential to spread the virus and interfere with clinical diagnosis. With the application of new adjuvants and the demand for industrial development, scholars at home and abroad have conducted research on whole-bacterial inactivated vaccines and subunit vaccines. For example, patent CN111100817A provides an inactivated vaccine prepared with the 20180316.H.AES.G strain and ISA35 adjuvant, which can affect the appetite of some horses. CN108220183A and CN111154678A provide inactivated vaccines prepared with the SMXJ-97 strain and Freund's incomplete adjuvant, and with the 20180422.D.DE.X strain and ISA35 adjuvant, respectively. However, these vaccines provide insufficient immune protection in mice, reaching only 80% and 87.5%, respectively. CN108218965A provides a recombinantly expressed flagellin protein, FliC, from Salmonella abortus equi. Using the recombinant FliC protein from this strain as an antigen for immunization, the researchers demonstrated a 75% protective immune response in experimental mice. The aforementioned inactivated or subunit vaccines have been associated with adverse reactions and poor protection rates. Therefore, further research into vaccines targeting Salmonella abortus equi is crucial for the prevention and treatment of the disease. Summary of the Invention

[0006] In response to the problems in the existing technology, the present invention provides a donkey-derived inactivated equine abortion Salmonella vaccine, which uses a newly isolated inactivated virulent strain as an antigen and nano-aluminum gel and recombinant self-flagellin as adjuvants. It has high safety and good epidemic prevention effect.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions.

[0008] A strain of Salmonella abortus ( Salmonella abortus ) C.SDLCYANG.2021, its accession number is CGMCC No. 31065.

[0009] The present invention provides a flagellin of Salmonella abortus equi, comprising a peptide segment with an amino acid sequence as shown in SEQ ID NO: 1.

[0010] Preferably, the nucleotide sequence of the gene encoding the equi abortus flagellin protein is shown as SEQ ID NO: 3.

[0011] To facilitate production, the present invention provides a recombinant flagellin of Salmonella abortus equi, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0012] In order to express the flagellin of Salmonella abortus equi, the present invention also provides a vector and a recombinant cell capable of expressing the above amino acid sequence. The cell is preferably Escherichia coli.

[0013] The present invention also provides a use of the recombinant flagellin of Salmonella equi abortus as an adjuvant for an inactivated Salmonella equi abortus vaccine.

[0014] When the above-mentioned recombinant flagellin of Salmonella abortus is used as an adjuvant, the dosage in the inactivated vaccine is 50 μg / mL-200 μg / mL.

[0015] The invention discloses an inactivated vaccine for equi abortions Salmonella, which takes equi abortions Salmonella C.SDLCYANG.2021 as an antigen and nano-aluminum gel and / or equi abortions recombinant flagellin as an adjuvant.

[0016] Preferably, the amount of nano-aluminum gel adjuvant added is 2.5 mg / mL, and the amount of recombinant protein added is 50 μg / mL-200 μg / mL.

[0017] The particle size of the nano-aluminum gel is 20 nm-100 nm.

[0018] The present invention has the following advantages:

[0019] The present invention provides a strain of equine abortion Salmonella C.SDLCYANG.2021, which is isolated from the tissue of a susceptible donkey, has strong virulence and can be used to produce an inactivated vaccine. The present invention prepares the recombinant flagellin B (FljB) of the C.SDLCYANG.2021 strain, which can be used as an adjuvant for inactivated vaccines. The inactivated Salmonella equi abortion vaccine provided by the present invention is safe for mice, has no adverse reactions after subcutaneous injection, can completely resist the attack of strong strains after immunizing mice, and can significantly reduce the number of Salmonella equi abortion bacteria in the spleen after adding the FljB recombinant protein adjuvant; it is safe after immunizing the donkey group, has no systemic and local adverse reactions after immunization, does not affect feeding, and has good protection, and can be used to prevent abortion in pregnant equine female livestock caused by such pathogens.

[0020] Biological deposit information

[0021] Salmonella abortus equi ( Salmonella abortus ) C.SDLCYANG.2021, deposited on June 24, 2024 in the General Microbiology Center of China Culture Collection Administration (CGMCC), the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No.31065. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The colony morphology of the isolated strain on SS medium (left) and MacConkey medium (right);

[0023] Figure 2 is the morphology of the isolated strain after Gram staining (1000×);

[0024] Figure 3 This is the SDS-PAGE electrophoresis of the recombinant FljB protein after expression and purification in recombinant BL21 (DE3); where M is the protein molecular weight standard, 1 is the uninduced recombinant bacteria, 2 is the expression product of the recombinant bacteria after induction, 3 is the supernatant after induction disruption, 4 is the precipitate after induction disruption, and 5 is the purified recombinant protein. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0026] Example 1 Isolation and identification of equine Salmonella abortus

[0027] 1. Isolation and biochemical identification

[0028] Collect the organ tissues of pregnant donkey fetuses suspected of being infected with Salmonella equine abortion, obtain the organ extract, streak inoculate on Salmonella chromogenic medium, incubate at 37℃ for 18-24h, pick the suspected single colony with lavender color, inoculate the isolated strain on MacConkey agar medium and SS medium for observation, and perform Gram staining. Figure 1-2 As shown: On MacConkey agar, the bacteria grows as smooth, colorless, translucent, round, raised colonies, while on SS agar, they appear as round, raised, colorless, transparent, round colonies. Gram staining reveals Gram-negative bacteria, consisting of scattered or individual short rods arranged in rows. Growth characteristics and Gram staining results are consistent with those of Salmonella.

[0029] Table 1 Biochemical identification of suspected strains

[0030]

[0031] Note: “+” indicates a positive reaction result; “-” indicates a negative reaction result.

[0032] The suspected single colony was subjected to biochemical identification, and the results are shown in Table 1: the strain produced gas during growth on triple sugar iron agar, and the culture medium showed a red slope and a yellow bottom; and the results of various biochemical tests were consistent with the biochemical characteristics of Salmonella.

[0033] The suspected single colony was subjected to serological characteristics determination and the strain was identified according to the identification instructions of Salmonella O4 factor serum and diphasic flagellar antigen He, n, x serum. Both showed agglutination, indicating that the strain was Salmonella equi abortus of Salmonella group B.

[0034] Place one drop of 1:500 acridinium yellow aqueous solution on a clean glass slide, then add one drop of 1×10 9 CFU / mL bacterial suspension, the two were mixed evenly, no agglutination occurred within 2-3 min, and the isolated bacteria had no SR mutation.

[0035] 2. Molecular Biological Identification

[0036] DNA of suspected single colonies was extracted and 16S rDNA identification was performed: 16S rDNA universal primers were used, upstream primer: 5'-AGAGTTTGATCMTGGCTCAG-3'; downstream primer: 5'-TACGGYTACCTTFTTACGACTT-3', and the genome of the isolated strain was used as a template for amplification. The strain information was obtained by sequencing and comparison. After identification, it was identified as Salmonella equi abortus. The strain was named C.SDLCYANG.2021 and deposited with CGMCC No. 31065.

[0037] 3. Pathogenicity

[0038] According to 1×106 CFU / 0.1 mL-1×10 9 20 g ± 1 g clean KM mice were challenged with the virus by intraperitoneal injection of CFU / 0.1 mL. The mortality rate is shown in Table 2.

[0039] Table 2 MLD of Salmonella abortus equi isolates 50 Determination

[0040]

[0041] MLD was calculated based on the improved Koch method. 50 for MLD 50 =10 5.7±0.4 CFU; 10 MLD 50 Five pregnant female mice were challenged with 0.1 mL of the solution, and all of them suffered miscarriages, indicating that the isolated strain was highly pathogenic.

[0042] 4. Immunogenicity

[0043] The bacteria were cultured and enriched using a common broth or other enrichment medium, and then 10-fold serial dilutions were performed with three replicates for each dilution. The cells were then counted using the plate spread method. The bacterial suspension was inactivated using a 0.8% formaldehyde solution at 37°C for 24 h. The bacterial suspension content was adjusted, and an alum adjuvant was added and emulsified to obtain an alum adjuvanted inactivated vaccine. Six 20 g ± 1 g clean KM mice were immunized subcutaneously in the back. The vaccine dose per mouse was 1.25 × 10 7 CFU / 0.1 mL, and a normal saline injection control group was set up. After 28 days, 1×10 standard strong strains were injected intraperitoneally. 8 After 14 days of challenge with CFU / 0.1mL, all the patients in the control group became ill and died, while the immunized group was 100% protected.

[0044] Example 2 Preparation of recombinant Salmonella abortus flagellin

[0045] The genome of donkey-derived equine abortion Salmonella C.SDLCYANG.2021 strain was extracted, and the FljB gene with the nucleotide sequence shown in SEQ ID NO: 3 was cloned by PCR using FljB-F: 5'-GGAGGATCCATGGCACAAGTAATCAACACTAACA-3' and FljB-R: 5'-CACAAGCTTAACGTAACAGAGACAGCACATTC-3' as the upstream and downstream primers, respectively. The PCR product was recovered by gel gel, and the PCR product and the pET-32a prokaryotic expression vector were digested with restriction endonucleases BamHI and HindIII, respectively. The target fragment and vector backbone were recovered, ligated with T4 DNA ligase, and transformed into DH5α Escherichia coli competent cells. Single clones were obtained by ampicillin (Amp) resistance screening, and plasmids were extracted after culture. The recombinant plasmids were obtained by correct sequencing. The recombinant plasmid was transformed into BL21 (DE3) competent cells, cultured on Amp-resistant plates, and single colonies were picked and placed in 5 mL of LB liquid medium containing Amp resistance. The cells were cultured in a 37°C shaker overnight. The next day, the colonies were inoculated into 100 mL of liquid LB medium containing Amp resistance at a ratio of 1:100, and cultured in a 37°C shaker. The OD 600 When the value was 0.6-0.8, IPTG with a working concentration of 0.1 mmol / L was added for induction, and cultured on a shaker at 37°C for 4 h to obtain the induced bacterial solution.

[0046] The recombinant BL21 (DE3) bacterial suspension was centrifuged at high speed to obtain bacterial slurry. The suspension was rinsed with PBS, resuspended, and then ultrasonically disrupted. The supernatant and precipitate were collected by centrifugation at 12,000 rpm for 10 minutes. The samples were subjected to SDS-PAGE electrophoresis to identify that the recombinant FljB protein was mainly expressed in a soluble form. The soluble expression product in the supernatant was collected, filtered through a 0.45 μm filter membrane, and purified by affinity chromatography using Ni Bestarose FF filler. Protein purification was performed according to the filler instructions. The SDS-PAGE electrophoresis of the supernatant and precipitated proteins and the purified protein is shown in Figure 2. Figure 3 The amino acid sequence of the obtained recombinant FljB protein is shown in SEQ ID NO: 2.

[0047] Example 3 Preparation of inactivated equine Salmonella abortus vaccine

[0048] 1. Vaccine Preparation

[0049] The equine abortion Salmonella C.SDLCYANG.2021 strain isolated in Example 1 was inoculated into Martin broth and cultured at 37°C for 24 h, with shaking 1-2 times in the middle to obtain seed liquid. The seed liquid was then inoculated into ordinary broth and fermented at 37°C for 24 h. The bacterial liquid was collected and counted by plate spreading method. 0.8% formaldehyde solution was added for inactivation for 48 h. The cells were collected by centrifugation, and the residual formaldehyde solution was washed with sterile saline and suspended. Thimerosal solution was added for preservation to obtain an inactivated bacterial liquid with a concentration of 2×10 10 CFU / mL, the concentration of the inactivated bacterial solution in the vaccine of the present invention is diluted appropriately with sterile physiological saline according to the animal immunization dose, and the vaccine is prepared in the following three ways:

[0050] (1) Aluminum gel adjuvant vaccine: Mix the inactivated bacterial solution and LV aluminum adjuvant in a mass ratio of 3:1 and emulsify;

[0051] (2) Nano-aluminum gel adjuvant vaccine: inactivated bacterial solution and nano-aluminum gel adjuvant (particle size range 20-100 nm) were mixed in a mass ratio of 3:1 and then emulsified;

[0052] (3) Nano-aluminum gel-FljB adjuvant vaccine: fully mix the bacterial solution with the recombinant FljB protein solution to obtain the recombinant protein bacterial solution. The dosage of recombinant protein for mice is 10 μg / mouse, and the dosage for this animal is 100 μg / head;

[0053] The recombinant protein bacterial solution and nano-aluminum gel adjuvant (particle size range 20-100 nm) were mixed in a mass ratio of 3:1 and then emulsified.

[0054] 2. Vaccine quality inspection

[0055] The three prepared vaccines were then tested for sterility, physical properties, and formaldehyde and thimerosal content in accordance with the current "Chinese Veterinary Pharmacopoeia", and all were found to be in compliance with the regulations for veterinary biological products.

[0056] 3. Vaccine safety testing

[0057] Clean KM mice (20 g ± 1 g) were injected subcutaneously on the back with 1 × 10 8 CFU / 0.1 mL of different vaccines were used, and the injection of an equal amount of normal saline was used as a control. The patients were observed for 14 consecutive days, and the food intake and body weight changes were recorded every day to observe local and systemic reactions.

[0058] Table 3 Safety tests of different vaccines

[0059]

[0060] The adverse reactions in mice after vaccine administration are shown in Table 3. Compared with the control group, mice in the nano-aluminum gel adjuvant vaccine group and the nano-aluminum gel-FljB adjuvant vaccine group were in good spirits, with no significant differences in food intake or body weight. Only a few mice had slight swelling at the injection site. However, mice in the LV aluminum gel adjuvant vaccine group experienced weight loss within 2 days and swelling at the injection site, which later formed a scab, indicating that the nano-aluminum gel adjuvant vaccine and the nano-aluminum gel-FljB adjuvant vaccine are safe.

[0061] 4. Vaccine shelf life

[0062] The shelf life of nano-aluminum gel adjuvant vaccine and nano-aluminum gel-FljB adjuvant vaccine was studied in KM mice. The vaccines were stored at 2-8 ° C for 1, 3, 6, 9, and 12 months, and samples were taken for immunization and challenge protection tests. 7 CFU / 0.1 mL of vaccine, 6 mice in the immunization group, and a normal saline injection control group were set up. 28 days later, 1×10 8 CFU / 0.1 mL was challenged with the virus and observed for 14 days. All the patients in the control group became ill and died, while the immunized group was 100% protected at each time point during the storage period.

[0063] Example 4 Immune Effect of Inactivated Equine Salmonella Abortus Vaccine

[0064] 1. Immune efficacy

[0065] The three vaccines prepared in Example 3 were tested for their immune efficacy: 6 clean KM mice (20 g ± 1 g) were immunized by subcutaneous injection at the back, containing 1.25×10 7 CFU / 0.1 mL, and a normal saline injection control group was set up. After 28 days, 1×10 standard strong strains were injected intraperitoneally. 8 The virus was challenged with 100 CFU / 0.1 mL, and the number of deaths was recorded after 14 days of observation.

[0066] Table 4 Immune efficacy test of aluminum gel adjuvant vaccine

[0067]

[0068] The results showed that all mice in the control group became ill and died, while the immunized group was 100% protected, indicating that the vaccine prepared from the equine abortion Salmonella C.SDLCYANG.2021 strain isolated and obtained in the present invention can provide good immune protection to mice.

[0069] 2. Immunoprotective dose

[0070] The nano-aluminum gel-FljB adjuvant vaccine was divided into different immunization doses and injected subcutaneously on the back of KM mice, with 6 mice in each group. A normal saline injection control group was set up. 28 days later, 1×10 standard strong strains were injected intraperitoneally.8 The virus was challenged with 100 CFU / 0.1 mL, and the number of deaths was recorded after 14 days of observation.

[0071] Table 5 Immune protection dose of nano-aluminum gel-FljB adjuvant vaccine

[0072]

[0073] The results showed that 6.25×10 6 The groups with CFU / 0.1 mL immunization dose and above were all 100% protected, while all the patients in the control group became ill and died, indicating that the prepared vaccine can stimulate the body to produce good immune protection at a lower immunization dose.

[0074] 3. Bacterial load in the spleen of mice after challenge

[0075] The three vaccines prepared in Example 3 (1.25×10 7 CFU / 0.1 mL), nano-aluminum gel and FljB (20 μg / mouse) adjuvant were injected subcutaneously on the back of 20 g±1 g clean KM mice, and a normal saline injection control group was set up at the same time. 28 days later, 1×10 standard strong strains were injected intraperitoneally 8 The mice were challenged with the virus using a concentration of CFU / 0.1 mL. Four days after challenge, mice were sacrificed and their spleens were collected under a sterile environment. If mice in the control group became ill and died within four days, their spleens were immediately counted upon death. The bacterial load in mouse spleens was enumerated using the SS plate spread method. The spleens were ground and mixed with sterile saline at a ratio of 1:10, then serially diluted 10-fold. The plates were spread and incubated overnight at 37°C before enumeration. Statistical comparisons and analyses were performed using SPSS software (Duncan model).

[0076] Table 6 Effects of adjuvants on bacterial load in mouse spleen

[0077]

[0078] Note: Compared with the control P <0.01, ## indicates comparison with LV aluminum gel adjuvant vaccine P <0.01.

[0079] The results are shown in Table 6. There was no significant difference in the bacterial load in the spleen between the saline control group and the mixed adjuvant group, indicating that the use of 2 times the amount of FljB adjuvant combined with nano-aluminum gel adjuvant could not reduce the bacterial load. The bacterial load in the LV aluminum gel adjuvant vaccine group, nano-aluminum gel adjuvant vaccine group, and nano-aluminum gel-FljB adjuvant vaccine group were all lower than that in the saline control group and the mixed adjuvant group. The statistical analysis showed that the difference was extremely significant ( P<0.01), and the bacterial load in the spleen decreased by one order of magnitude, indicating that the immunopotency of the nano-aluminum gel adjuvant was better than that of the LV aluminum gel adjuvant. The adjuvant dosage of 10 μg / animal could further enhance the immune efficacy of the vaccine, and the immunopotency was significant.

[0080] 4. Safety of Immunized Animals

[0081] Ten non-pregnant female donkeys, 10 pregnant female donkeys, and 10 breeding male donkeys were randomly selected and divided into two groups according to the donkey herd. The nano-aluminum gel adjuvant vaccine and the nano-aluminum gel-FljB adjuvant vaccine were injected into the neck muscle respectively, with a dose of 7.5×10 9 CFU / 3 mL of vaccine, and observe for 14 consecutive days, record food intake every day, and observe local and systemic reactions.

[0082] The results showed that there was no significant change in the feed intake of the test donkeys, their mental state was good, no local swelling was observed, and there were no other local adverse reactions such as ulceration. The pregnant donkeys did not suffer from miscarriage, indicating that the nano-aluminum gel adjuvant vaccine and the nano-aluminum gel-FljB adjuvant vaccine are safe for these animals.

[0083] 5. Immune effect

[0084] A total of 102 female donkeys were immunized, including 24 non-pregnant female donkeys and 78 late pregnant female donkeys. They were randomly divided into two groups and injected intramuscularly with nano-aluminum gel adjuvant vaccine or nano-aluminum gel-FljB adjuvant vaccine in the neck, respectively. The immunization was carried out twice on day 0 and day 30, with a dose of 2.5×10 9 CFU / 1 mL of vaccine, and after 6 months of observation, no outbreak of pathogenic abortion caused by Salmonella equi was observed in pregnant donkeys.

[0085] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. An inactivated vaccine against Salmonella abortus equi, characterized in that, Using Salmonella abortus equi C.SDLCYANG.2021 as an antigen and nano-aluminum hydroxide gel and recombinant flagellin of Salmonella abortus equi as adjuvants; The preservation number of the Salmonella abortus equi C.SDLCYANG.2021 is CGMCC No. 31065; The amino acid sequence of the recombinant flagellin of Salmonella abortus equi is shown in SEQ ID NO:

2.

2. The inactivated vaccine of Salmonella abortus equi according to claim 1, characterized in that, The addition amount of the nano-aluminum hydroxide gel adjuvant is 2.5 mg / mL, and the addition amount of the recombinant protein is 50 μg / mL - 200 μg / mL; The particle size of the nano-aluminum hydroxide gel is 20 nm - 100 nm.

Images