Actinobacillus pleuropneumoniae FJLYC01 and bivalent inactivated vaccine prepared from actinobacillus pleuropneumoniae FJLYC01

Abstract

The invention discloses actinobacillus pleuropneumoniae FJLYC01 and a bivalent inactivated vaccine prepared from the actinobacillus pleuropneumoniae FJLYC01, and relates to the technical field of microorganisms and immunity. The collection number of the actinobacillus pleuropneumoniae FJLYC01 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No.30270, and the actinobacillus pleuropneumoniae FJLYC01 is serum type 1 and is high in immunogenicity and good in immune effect. A bivalent vaccine prepared from the strain and haemophilus parasuis serum type 4 and type 5 strains has a very good immune protection effect on single or mixed infection of haemophilus parasuis and actinobacillus pleuropneumoniae, is safe and free of side effects, and reduces the number of immunization times and the stress of pigs compared with single-linked immunization; and the immune cost is reduced. The multi-prevention effect is achieved by one injection clinically, the hidden danger of poison dispersing does not exist, and safety and reliability are achieved.

Description

Technical Field

[0001] The present invention relates to the fields of microorganisms and immunology, and more particularly to an Actinobacillus pleuropneumoniae strain FJLYC01 and a bivalent inactivated vaccine prepared therefrom. Background Art

[0002] Clinically, Haemophilus parasuis often co-infects or secondary infects with Actinobacillus pleuropneumoniae. Their symptoms are similar and the drug treatment effect is not good. In addition, due to the abuse of antibiotics, drug-resistant strains have emerged, making drug treatment ineffective and wasteful; the residue of antibiotic drugs also makes meat animals face the problem of failing to pass when exported. In view of the current problem of co-infection of Haemophilus parasuis and Actinobacillus pleuropneumoniae and the lack of immune cross-protection between different serotypes, developing a bivalent inactivated vaccine that can simultaneously prevent Haemophilus parasuis and Actinobacillus pleuropneumoniae is the most economical and effective method at present.

[0003] Therefore, developing a bivalent inactivated vaccine that can simultaneously prevent Haemophilus parasuis and Actinobacillus pleuropneumoniae is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides an Actinobacillus pleuropneumoniae strain FJLYC01 and a bivalent inactivated vaccine prepared therefrom.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] An Actinobacillus pleuropneumoniae strain FJLYC01, with the preservation number of CGMCC No. 30270.

[0007] Another object of the present invention is to provide an immunogenic composition, which contains the above-mentioned Actinobacillus pleuropneumoniae strain FJLYC01.

[0008] Another object of the present invention is to provide the application of the above-mentioned Actinobacillus pleuropneumoniae strain FJLYC01 or the above-mentioned immunogenic composition, which is any one of the following:

[0009] a. Application in the preparation of drugs for preventing and / or treating Actinobacillus pleuropneumoniae infection or diseases caused by Actinobacillus pleuropneumoniae infection;

[0010] b. Application in the preparation of vaccines for preventing and / or treating Actinobacillus pleuropneumoniae infection or diseases caused by Actinobacillus pleuropneumoniae infection;

[0011] c. Use in the preparation of antibodies for preventing and / or treating Actinobacillus pleuropneumoniae infection or diseases caused by Actinobacillus pleuropneumoniae infection;

[0012] d. Use in the preparation of antiserum for preventing and / or treating Actinobacillus pleuropneumoniae infection or diseases caused by Actinobacillus pleuropneumoniae infection;

[0013] e. Use in the preparation of reagents or test kits for detecting Actinobacillus pleuropneumoniae;

[0014] f. Use in the immunogenicity evaluation of Actinobacillus pleuropneumoniae vaccines for non-diagnostic and non-therapeutic purposes;

[0015] g. Use in the protective evaluation of Actinobacillus pleuropneumoniae vaccines for non-diagnostic and non-therapeutic purposes;

[0016] h. Use in the preparation of Actinobacillus pleuropneumoniae-infected cell models;

[0017] i. Use in the preparation of Actinobacillus pleuropneumoniae-infected animal models;

[0018] j. Use in the screening or efficacy evaluation of drugs for preventing and / or treating Actinobacillus pleuropneumoniae infection or diseases caused by Actinobacillus pleuropneumoniae infection.

[0019] Another object of the present invention is to provide a vaccine, which contains an inactivated vaccine prepared from the above-mentioned Actinobacillus pleuropneumoniae FJLYC01.

[0020] Another object of the present invention is to provide a combined inactivated vaccine, which is prepared from the above-mentioned Actinobacillus pleuropneumoniae FJLYC01 and Haemophilus parasuis serotypes 4 and 5.

[0021] Preferably, it includes the following steps:

[0022] (1) Bacterial liquid culture

[0023] Respectively culture the bacterial liquids of Haemophilus parasuis serotype 4 strain LYH5, serotype 5 strain LY02 and Actinobacillus pleuropneumoniae serotype 1 strain FJLYC01 by liquid fermentation;

[0024] (2) Viable bacteria counting

[0025] Take the bacterial liquid cultured for 22 - 24 h for viable bacteria counting, and adjust the bacterial liquid concentration to 2.5×10 9 CFU / ml;

[0026] (3) Inactivation

[0027] Add formaldehyde solution to the bacterial liquid with adjusted concentration for inactivation;

[0028] (4) Adding a vaccine adjuvant

[0029] Mix the above-inactivated Haemophilus parasuis serotypes 4 and 5 and Actinobacillus pleuropneumoniae serotype 1 bacterial liquids in a volume ratio of 1:1:1, and prepare an oil-in-water emulsion inactivated vaccine with an oil phase:water phase ratio of 2:1, adding 1% aluminum stearate. Among them, the water phase is 4% Tween-80 and 96% bacterial liquid, and the oil phase is 94% white oil and 6% Span-80; the antigen content of both the Haemophilus parasuis and Actinobacillus pleuropneumoniae bivalent inactivated vaccines is 2.0×10 9 CFU / mL.

[0030] Preferably, the bacterial liquid culture in step (1) is specifically as follows: For Haemophilus parasuis serotypes 4 and 5, add to the semi-synthetic medium I according to a fermentation volume of 100 ml / 250 ml liquid loading amount, culture at 37 °C and 180 r / min for 22 - 24 hours, and harvest each type of bacterial liquid; for Actinobacillus pleuropneumoniae serotype 1, add to the semi-synthetic medium II according to a fermentation volume of 100 ml / 250 ml liquid loading amount, culture at 37 °C and 180 r / min for 22 - 24 hours, and harvest the bacterial liquid;

[0031] Among them, the preparation method of the semi-synthetic medium I is: Dissolve 30 g of tryptic soy broth in deionized water, make up the volume to 1000 mL, shake well and dissolve, then sterilize at 121 °C by high-pressure steam for 15 min, add 50 mL of filtered and sterilized newborn bovine serum, and 200 μL of filtered and sterilized 0.02% NAD; the preparation method of the semi-synthetic medium II is: Dissolve 30 g of tryptic soy broth in deionized water, make up the volume to 1000 mL, shake well and dissolve, then sterilize at 121 °C by high-pressure steam for 15 min, add 50 mL of filtered and sterilized newborn bovine serum, and 200 μL of filtered and sterilized 0.04% NAD.

[0032] Beneficial effects:

[0033] The present invention provides an Actinobacillus pleuropneumoniae strain FJLYC01, which is serotype 1, has high immunogenicity and good immune effect. The bivalent vaccine prepared with this strain and Haemophilus parasuis serotypes 4 and 5 strains has achieved good immune protection effects against single or mixed infections of Haemophilus parasuis and Actinobacillus pleuropneumoniae, is safe and has no side effects. Compared with single vaccination, it reduces the number of immunizations, reduces the stress of pigs, and reduces the immunization cost. Clinically, it achieves the effect of "preventing multiple diseases with one injection", and there is no hidden danger of spreading the virus, being safe and reliable. Description of the drawings

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0035] Figure 1 It is the phylogenetic tree of Actinobacillus pleuropneumoniae FJLYC01.

[0036] Figure 2 It is the genomic comparative analysis between Actinobacillus pleuropneumoniae FJLYC01 and strains AP76, JL03, KL16, L20, SAMN02469615.

[0037] Figure 3 It is the clinical lesion characteristics of piglets that died after being challenged with Actinobacillus pleuropneumoniae FJLYC01.

[0038] Figure 4 It is the histopathological changes of piglets that died after being challenged with Actinobacillus pleuropneumoniae FJLYC01.

[0039] Figure 5 It is the growth curve of Actinobacillus pleuropneumoniae FJLYC01.

[0040] Figure 6 It is the schematic diagram of the HPS+APP bivalent inactivated vaccine product prepared by the present invention.

[0041] Figure 7 It is the observation of clinical symptoms and pathological changes of piglets in each test group after immunization and challenge in Example 5; each row from top to bottom is a test group, namely groups 1-10.

[0042] Figure 8 It is the HPS antibody level of piglets after immunization and challenge in Example 5; note: * indicates P < 0.05 compared with the non-immunized control and blank control groups; ** indicates P < 0.01 compared with the non-immunized control and blank control groups; *** indicates P < 0.001 compared with the non-immunized control and blank control groups, the same below.

[0043] Figure 9 It is the APP antibody level of piglets after immunization and challenge in Example 5.

[0044] Figure 10 It is the T lymphocyte level of piglets after immunization and challenge in Example 5 (HPS control).

[0045] Figure 11 It is the T lymphocyte level of piglets after immunization and challenge in Example 5 (APP control).

[0046] Figure 12 The B lymphocyte levels of piglets after immunization and challenge in Example 5 (HPS control).

[0047] Figure 13 The B lymphocyte levels of piglets after immunization and challenge in Example 5 (APP control). Detailed implementation manners

[0048] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0049] Example 1 Isolation, identification and preservation of Actinobacillus pleuropneumoniae FJLYC01

[0050] 1. Trachea, lungs and other diseased materials of diseased pigs suspected of being infected with Actinobacillus pleuropneumoniae (APP) by autopsy were collected and inoculated on TSA (added with NAD, 4 μg / mL; calf serum, 5%; the addition concentration in TSB broth is the same) nutrient agar for isolation and culture, and incubated at 37 °C for 20 - 24 h. After picking smooth, round, needle-point-sized, colorless and translucent single colonies on the TSA medium for purification culture, a total of 31 APP strains were isolated. Through biochemical identification and 16S rRNA PCR analysis, a total of 3 serotypes were detected, with serotypes 1 and 7 being prevalent, followed by serotype 2. In cooperation with Zhao Fenghua Biotechnology (Fuzhou) Co., Ltd., 19 APP1-type strains were determined and screened.

[0051] 2. Based on the results of the virulence experiment, one Actinobacillus pleuropneumoniae serotype 1 strain with relatively strong virulence (FJLYC01) was selected and sent to BGI for gene sequencing analysis. Five representative Actinobacillus pleuropneumoniae strains (AP76, JL03, KL16, L20, SAMN02469615) were selected on Genebank and compared with the experimental strain for comparative genomic analysis, and the results were compared with the reference strains for sequence similarity. The sequences were analyzed by the CorePan1 analysis method. An evolutionary tree was constructed based on the CorePan1 analysis results among AP76, JL03, KL16, L20, SAMN02469615 and FJLYC01 strains. The results of the evolutionary tree showed that the strain FJLYC01 had the closest evolutionary relationship with JL03 and was in the same branch; followed by AP76; it was also relatively close to the L20 isolate in evolution; while it had a relatively distant evolutionary relationship with the APP isolates of KL16 and SAMN02469615. See attached Figure 1 .

[0052] 3. The Actinobacillus pleuropneumoniae serotype 1 strain FJLYC01 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on April 7, 2024. The deposit number is CGMCC No. 30270. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is taxonomically named Actinobacillus pleuropneumoniae.

[0053] Example 2 Gene Sequence Analysis of Actinobacillus pleuropneumoniae FJLYC01

[0054] 1. The full name of the VFDB database is Virulence Factors of Pathogenic Bacteria, which is a database dedicated to the study of virulence factors of pathogenic bacteria, chlamydia, and mycoplasma.

[0055] The APP FJLYC01 strain was compared with the VFDB database, and finally, the annotation results of 190 virulence factors were obtained, mainly including adhesins, immune regulation, nutritional factors, and exotoxins. Among them, the ID values of 4 virulence factors related to adhesins exceeded 99%. The virulence factors related to exotoxins are hlyA, hlyB, hlyC, hlyD, and argK. The related genes of adhesins, immune regulation, nutritional factors, and exotoxins are shown in Table 1.

[0056] Table 1

[0057]

[0058] 2. In order to explore the similarity between APP FJLYC01 and other genes, 5 representative strains of Actinobacillus pleuropneumoniae were selected on Genebank for comparative genomic analysis with the experimental strain. The comparative genomic analysis includes common genes, unique genes, gene families, and species evolution.

[0059] From the venn diagram of the genomic Pan gene set between APP FJLYC01 and AP76, JL03, KL16, L20, and SAMN02469615 strains, it can be seen that the number of their common genes is 1770, and the APP FJLYC01 strain has the most unique genes, with 107 (see Appendix Figure 2 ).

[0060] Example 3 Pathogenicity Study of Actinobacillus pleuropneumoniae FJLYC01

[0061] 1. Determination of the median lethal dose in mice

[0062] Sixty ICR mice were randomly divided into six groups, with 10 mice in each group. Among them, groups 1-5 were experimental groups, and group 6 was the control group. The mice were intraperitoneally injected with a concentration of 2.5×1010 , 2.5×10 9 , 2.5×10 8 , 2.5×10 7 , 2.5×10 6 CFU / mL of FJLYC01 bacterial solution, 0.5 ml each, and the control group was intraperitoneally injected with 0.5 ml of normal saline per mouse. On the second day after injection, observe and record the clinical symptoms of the inoculated mice, including whether they huddle together with listless spirit, whether the hair is rough and disordered, whether there is lameness, whether the breathing is normal, and whether there are other obvious neurological symptoms, etc. Record the number of dead mice in the experiment and calculate the LD 50 of this strain. At the same time, dissect the dead mice in the experiment and record the clinical pathological changes.

[0063] After inoculating with bacterial solutions of different concentration gradients, the mice in each group showed different situations of death. The group with a concentration of 2.5×10 10 CFU / mL was close to complete death, while the group with a concentration of 2.5×10 6 CFU / mL survived completely, as shown in Table 2. The median lethal dose of FJLYC01 for mice was calculated by the Karber method to be 8.0×10 8 CFU / mL.

[0064] Table 2

[0065]

[0066] Median lethal dose LD50 = 8.0×10 8 CFU.

[0067] 2. Determination of pathogenicity in piglets

[0068] Select 15 healthy piglets aged 20 - 21 days (negative for APP antibody, negative for circovirus and porcine reproductive and respiratory syndrome antibodies, negative for pseudorabies field virus antibody), divide them into 3 groups, and inoculate FJLYC01 bacterial solution by nasal inoculation (the inoculation method is shown in Table 3). At the same time, set up a control group of 5 pigs inoculated with normal saline by nasal cavity. Observe continuously for 2 weeks after inoculation. Criteria for judging illness: ① Listless spirit; ② Coughing and difficulty breathing; ③ Body temperature rising by more than 1℃; ④ Death; ⑤ For those without symptoms, hemorrhagic fibrinous pleuropneumonia or focal necrosis can be seen during autopsy. If any one of the above items appears, it is judged as ill.

[0069] The piglets challenged with FJLYC01 showed clinical symptoms 2 days after challenge. The body temperature of the piglets in the experimental group increased, and they showed clinical symptoms such as listlessness, huddling together, decreased appetite, and rough hair, accompanied by respiratory function disorders such as coughing and wheezing. In the first challenge group, 2 piglets died on the 5th day after challenge and 2 piglets died on the 9th day after challenge; 2 weeks after challenge, 4 piglets died in the first challenge group, 2 piglets died in the second challenge group, and 2 piglets in the third challenge group got sick without death.

[0070] Table 3

[0071]

[0072]

[0073] For the main clinical pathological features of the challenged and dead piglets, see Appendix Figure 3 , severe adhesion of the chest and lungs, and yellow fibrinous exudate in the thoracic cavity ( Figure 3 -a); the lungs were significantly swollen and congested, with nodules of different sizes on the lung surface, and the hilar lymph nodes of the lungs were congested and enlarged ( Figure 3 -b); the heart was enlarged, with fibrinous exudate on the surface, showing the typical pathological change of "hairy heart" ( Figure 3 -c); the trachea was filled with mucus, and the tracheal wall was congested and enlarged ( Figure 3 -d); the liver was brittle, and the edge was severely congested ( Figure 3 -e); the spleen was congested, and the kidneys were congested and enlarged ( Figure 3 -f).

[0074] For the histological and pathological changes of the challenged and dead piglets, see Appendix Figure 4 , Lungs: The alveolar septum was widened, the alveoli were filled with exfoliated epithelial cells, red blood cells and neutrophils, and the alveoli were squeezed and their shapes were blurred ( Figure 4 -A); Trachea: The goblet cells on the inner wall of the trachea were degenerated, lost their shapes and were arranged disorderly; the inner wall was filled with red blood cells and infiltrated with neutrophils ( Figure 4 -B); Liver: The hepatocytes were arranged irregularly, enlarged, with red blood cells between the cells and a small amount of inflammatory cells ( Figure 4 -C); Spleen: A large number of splenic endothelial cells were degenerated and necrotic, with a large number of red blood cells and neutrophil infiltration ( Figure 4 -D); Kidneys: The renal epithelial cells were swollen and necrotic, with neutrophil infiltration; the glomerular endothelial cells were swollen, hemorrhaged and infiltrated with neutrophils ( Figure 4 -E); Myocardium: The interstitial space of the muscle fibers was significantly widened, infiltrated with inflammatory cells, and the myocardial cells were enlarged ( Figure 4 -F).

[0075] Example 4 Preparation of Haemophilus parasuis vaccine and Actinobacillus pleuropneumoniae bivalent inactivated vaccine

[0076] 1. Growth curve of FJLYC01 strain

[0077] A single colony of FJLYC01 cultured on TSA medium at 30°C for 24 h was selected and inoculated into TSB medium (supplemented with 0.004% NAD) and cultured at 37°C with shaking at 180 rpm for 48 h. The OD was measured every 2 h. 600 The experiment was repeated three times to compare the growth of Actinobacillus pleuropneumoniae FJLYC01 at different times. The results showed that the FJLYC01 strain was in a flat growth period within 8 hours, entered a logarithmic growth period from 8 hours, and had an OD of 9h-20h. 600 The value continued to rise and at 20h OD 600 The value reaches a peak after 20 hours. 600 The value begins to decline and enters the decline period (see Appendix Figure 5 ).

[0078] 2. Preparation of a combined inactivated vaccine of Haemophilus parasuis and Actinobacillus pleuropneumoniae

[0079] (1) Bacterial culture

[0080] Liquid fermentation was used to culture the bacterial liquids of Haemophilus parasuis serotype 4 strain LYH5 (accession number: CGMCC NO.11144), Haemophilus parasuis serotype 5 strain LY02 (accession number: CGMCC NO.11145) and Bacillus pleuropneumoniae serotype 1 FJLYC01 (accession number: CGMCC NO.30270).

[0081] For Haemophilus parasuis serotypes 4 and 5, add semi-synthetic culture medium I at a fermentation volume of 100 ml / 250 ml, culture at 37°C, 180 r / min for 22 to 24 hours, and harvest the bacterial liquid of each type; for Bacillus pleuropneumoniae serotype 1, add semi-synthetic culture medium II at a fermentation volume of 100 ml / 250 ml, culture at 37°C, 180 r / min for 22 to 24 hours, and harvest the bacterial liquid.

[0082] Semi-synthetic medium I: Dissolve 30 g of tryptic soy broth (TSB) in deionized water and make up to 1000 mL. Shake well to dissolve and sterilize with high pressure steam at 121°C for 15 min. Add 50 mL of filter-sterilized newborn calf serum and 200 μL of filter-sterilized 0.02% NAD.

[0083] Semi-synthetic medium II: Dissolve 30 g of tryptic soy broth (TSB) in deionized water and make up to 1000 mL. Shake well to dissolve and sterilize with high pressure steam at 121°C for 15 min. Add 50 mL of filter-sterilized newborn calf serum and 200 μL of filter-sterilized 0.04% NAD.

[0084] (2) Viable bacteria count

[0085] Take 1 ml of the bacterial liquid cultured for 22 - 24 h, and conduct viable bacteria count according to the method in the appendix of the Pharmacopoeia of the People's Republic of China for Veterinary Drugs. Concentrate the viable bacteria content of each type to 2.5×10 9 CFU / ml according to the counting results.

[0086] (3) Inactivation

[0087] Add formaldehyde solution at 0.25% of the volume of the bacterial liquid respectively, stir and inactivate at 37℃ for 24 h. After sampling for inactivation inspection, store at 2 - 8℃ for no more than 7 days.

[0088] (4) Vaccine preparation

[0089] a. Mix the inactivated Haemophilus parasuis serotype 4 and 5 and Actinobacillus pleuropneumoniae serotype 1 bacterial liquids in a volume ratio of 1:1:1, and prepare an oil-emulsion inactivated vaccine according to the conventional method (oil phase: white oil 94%, span - 80 6%; water phase: tween - 80 4%, bacterial liquid 96%; oil phase: water phase = 2:1), and add 1% aluminum stearate. Among them, the antigen content of the inactivated vaccine of Haemophilus parasuis and Actinobacillus pleuropneumoniae is 2.0×10 9 CFU / mL.

[0090] b. Emulsification: Take 2 parts of the oil phase and place it in the emulsification tank, start the motor and stir at low speed, while slowly adding 1 part of the water phase, and then emulsify at 4000 r / min for 30 min. After emulsification, take 10 ml of the sample and centrifuge at 3000 r / min for 15 min, and it should not be stratified. If there is stratification, re - emulsify once.

[0091] c. Sterility test of the vaccine

[0092] Take the above - mentioned vaccine and conduct sterility test according to the appendix of the Pharmacopoeia of the People's Republic of China for Veterinary Drugs, and the result shows no bacterial growth.

[0093] d. Sub - packaging: Quantitatively sub - package, cover and seal to obtain the inactivated vaccine of Haemophilus parasuis and Actinobacillus pleuropneumoniae (see attachment Figure 6 ).

[0094] 3. Physical property test of the vaccine

[0095] Viscosity determination: Take a pipette with an inner diameter of 1.2 mm, suck 1 ml of the vaccine emulsion at room temperature, and the time required to vertically release 0.4 ml is 6.5 s.

[0096] Emulsion stability determination: Store the vaccine at 37℃ for 21 days and observe whether it demulsifies. The result shows that the vaccine does not demulsify, indicating that the physical properties of the vaccine are good.

[0097] Immunization Efficacy of Haemophilus parasuis Vaccine and Actinobacillus pleuropneumoniae Bivalent Inactivated Vaccine

[0098] 1. Immunoprotection and Efficacy Evaluation of Piglets

[0099] (1) Fifty 21-day-old healthy piglets (negative for PRRSV, PCV, HPS, APP antigens and antibodies) were randomly divided into 10 groups (5 piglets / group). Each piglet was injected with 2 mL of each vaccine via intramuscular injection in the neck at 21 days of age. Piglets in groups 1-3 were injected with the HPS+APP bivalent vaccine prepared in Example 4 via intramuscular injection in the neck, piglets in groups 4-5 were injected with the HPS commercial vaccine via intramuscular injection in the neck, piglets in group 6 were injected with the APP commercial vaccine 2 mL / piglet via intramuscular injection in the neck. Piglets in groups 7-8 were control groups challenged with HPS type 4 and HPS type 5 without immunization, piglets in group 9 were the control group challenged with APP type 1 without immunization, and piglets in group 10 were the blank control group, immunized with PBS 2 mL / piglet. A second immunization was performed 21 days later with the same dose and route.

[0100] Blood samples were collected from piglets in each group on days 0, 21, 28, 35, 42 and 56 after immunization to detect the specific antibody levels of HPS and HNAPP in piglets and determine the levels of T and B lymphocytes.

[0101] Fourteen days after the second immunization, each group of piglets was challenged by intraperitoneal injection with a lethal dose LD 50 of the virus strain (see Table 4). The challenge method was as follows: 3 mL of the bacterial suspension of Haemophilus parasuis serotype 4 and serotype 5 was injected intraperitoneally per piglet, and 3 mL of Actinobacillus pleuropneumoniae type 1 was sprayed into the nasal cavity per piglet. All experimental piglets were weighed on an empty stomach in the morning on the day of immunization and after challenge, and the average daily weight gain of each piglet in each group was calculated. During the experiment, the clinical manifestations of piglets in each group were observed every day and the following indicators were recorded: ① Feed intake; ② Diarrhea, body surface hair color and appearance; ③ Allergic reaction; ④ Mortality rate of each group of pigs, etc.

[0102] The morbidity and mortality of piglets were observed and recorded 2 weeks after challenge, and the body temperature, clinical symptoms and clinical pathological autopsy changes of piglets after challenge were recorded.

[0103] Table 4 Piglet Immunization Test

[0104]

[0105]

[0106] (2) Challenge Protection Rate of Piglets

[0107] Fourteen days after the second immunization, the challenge protection results of each group of piglets were as follows:

[0108] (HPS+APP) Bivalent Vaccine Group Results:

[0109] After 18 h of challenge with Haemophilus parasuis serotype 4 of Group 1, symptoms such as huddling together, lying still, and listlessness appeared. After 21 h of challenge, the body temperature increased. The body temperature of piglets returned to normal 9 days after challenge. After 14 days of challenge, 5 piglets survived, and the protection rate was 100%;

[0110] After 18 h of challenge with Haemophilus suis serotype 5 of Group 2, lying still occurred. After 21 h of challenge, the body temperature increased. The body temperature of piglets returned to normal 9 days after challenge. After 14 days of challenge, 5 piglets survived, and the protection rate was 100%;

[0111] After 18 h of challenge with Actinobacillus pleuropneumoniae serotype 1 FJLYC01 of Group 3, lying still occurred. After 21 h of challenge, the body temperature increased. The body temperature of piglets returned to normal 9 days after challenge. After 14 days of challenge, 5 piglets survived, and the protection rate was 100%.

[0112] Results of the commercial vaccine group of Haemophilus parasuis (HPS):

[0113] After 18 h of challenge with Haemophilus parasuis serotype 4 of Group 4, symptoms such as huddling together, lying still, and listlessness appeared. One piglet died after 98 h of challenge, and then 4 piglets still survived. The protection rate was 80%;

[0114] After 18 h of challenge with Haemophilus suis serotype 5 of Group 5, lying still occurred. Two piglets died after 80 h of challenge. The spirit recovered after 98 h of challenge. After 14 days of challenge, 3 piglets survived, and the protection rate was 60%.

[0115] Results of the commercial vaccine group of APP:

[0116] After 18 h of challenge with Actinobacillus pleuropneumoniae serotype 1 FJLYC01 of Group 6, symptoms such as huddling together, lying still, and listlessness appeared. The spirit recovered after 48 h of challenge, and 5 piglets survived. The protection rate was 100%.

[0117] Results of the non-immunized control group:

[0118] After 18 h of challenge with Haemophilus parasuis serotype 4 of Group 7, symptoms such as huddling together, lying still, and listlessness appeared. One piglet died after 21 h of challenge, one piglet died after 48 h of challenge, one piglet died after 80 h of challenge, one piglet died 9 days after challenge, and one piglet was still alive 14 days after challenge, but was listless and had swollen joints. The protection rate was 20%.

[0119] After 18 h of challenge with Haemophilus parasuis serotype 5 of Group 8, symptoms such as huddling together, lying still, and listlessness appeared. Two piglets died after 21 h of challenge, one piglet died after 72 h of challenge, two piglets died after 80 h of challenge, and no piglets survived 14 days after challenge. The protection rate was 0%.

[0120] In Group 9, after 18 hours of challenge with Actinobacillus pleuropneumoniae serotype 1 FJLYC01, the piglets lay quietly. One piglet died 6 days after challenge. The mental state gradually recovered 7 days after challenge, but the 4 surviving piglets were all emaciated. Pathological examination of 3 surviving piglets showed typical pathological features of Actinobacillus pleuropneumoniae such as pleuro-pulmonary adhesion, swelling and congestion of the lungs, mucus filling the trachea, and congestion of the liver and spleen. The protection rate was 20%.

[0121] Results of Group 10 (blank control group): The piglets were intramuscularly injected with PBS and showed no symptoms and no deaths.

[0122] The experimental results showed that the protection rates of the HPS+APP combined vaccine against HPS serotypes 4 and 5 were both 100%; the protection rate against Actinobacillus pleuropneumoniae serotype 1 HNAPP1 was also 100%, and the effect was significant.

[0123] (3) Clinical symptom observation and pathological changes of piglets after challenge

[0124] For the piglets in the HPS+APP combined vaccine group challenged with HPS serotype 4 group (Group 1), the HPS+APP combined vaccine group challenged with HPS serotype 5 group (Group 2), the HPS+APP combined vaccine group challenged with APP serotype 1 group (Group 3), the HPS commercial vaccine group challenged with HPS serotype 4 group (Group 4), the HPS commercial vaccine group challenged with HPS serotype 5 group (Group 5), and the APP commercial vaccine group (Group 6), as well as the piglets in the unchallenged HPS serotype 4 control group (Group 7), the unchallenged HPS serotype 5 control group (Group 8), and the unchallenged APP serotype 1 control group (Group 9), the pathological changes after challenge are shown in the appendix Figure 7 For the piglets in the HPS+APP combined vaccine group (Groups 1, 2, and 3), the HPS commercial vaccine group (Groups 4 and 5), and the APP commercial vaccine group (Group 6), there were no obvious pathological changes. For the piglets in the unchallenged control groups (Groups 7 and 8), typical pathological changes of Haemophilus parasuis (HPS) and Actinobacillus pleuropneumoniae (APP) (Group 9) appeared after challenge. The lesions mainly showed that the thoracic and abdominal cavities were filled with effusion, pleuro-pulmonary adhesion, swelling of the lungs, with fibrinous exudation on the surface, fibrinous exudation in the abdominal cavity, enlargement of the liver and spleen, with fibrinous exudation on the surface, and fibrinous exudation in the pericardium.

[0125] (4) Average daily gain of piglets

[0126] Regarding the results of the average daily gain of the experimental piglets, the average daily gains of the piglets in the HPS+APP combined vaccine group, the HPS commercial vaccine group, and the APP commercial vaccine group were all higher than those in the unchallenged control group and the blank control group. Among them, the average daily gain of the piglets in the HPS+APP combined vaccine group was the highest, followed by the HPS commercial vaccine group. In the unchallenged control group (challenged with HPS), only 1 piglet survived 2 weeks after challenge, so only the average daily gain in 45 days was statistically analyzed (see Table 5).

[0127] Table 5 Statistical table of average daily gain of piglets

[0128]

[0129]

[0130] (5) Evaluation of specific immune response in piglets

[0131] Antibody level of HPS in piglets:

[0132] Detection results of antibody titer of Hps in piglets. Three weeks after the first immunization, the antibody value of Hps in piglet serum began to rise. The HPS+APP combined vaccine group was significantly higher than the non-immunized control group and the blank control group (P<0.05), while there was no significant difference between the HPS+APP combined vaccine group and the HPS commercial vaccine group (P>0.05). From 1 week to 2 weeks after the second immunization, the HPS+APP combined vaccine group was significantly higher than the HPS commercial vaccine group, the non-immunized control group and the blank control group (P<0.05). One week after challenge, the antibody level of the HPS+APP combined vaccine group was extremely significantly higher than that of the non-immunized control group and the normal saline group (P<0.001), and there was no significant difference between the HPS+APP combined vaccine group and the HPS commercial vaccine group (P>0.05). Two weeks after challenge, the HPS+APP combined vaccine group still maintained a relatively high antibody level (see attachment Figure 8 ).

[0133] Antibody level of APP in piglets:

[0134] Detection results of antibody titer of APP in piglets. Two weeks after the first immunization, the antibody value of APP in piglet serum began to rise. From 2 to 3 weeks after the first immunization, the HPS+APP combined vaccine group and the APP commercial vaccine group were significantly higher than the non-immunized control group and the blank control group (P<0.05), while there was no significant difference between the HPS+APP combined vaccine group and the HPS commercial vaccine group (P>0.05). One week after the second immunization, the HPS+APP combined vaccine group was significantly higher than the non-immunized control group and the blank control group (P<0.05), while there was no significant difference between the HPS+APP combined vaccine group and the HPS commercial vaccine group (P>0.05). Two weeks after the second immunization, the HPS+APP combined vaccine group was lower than the commercial vaccine group, but significantly higher than the non-immunized control group and the blank control group (P<0.05), while there was no significant difference between the HPS+APP combined vaccine group and the HPS commercial vaccine group (P>0.05). One week after challenge, the antibody levels of both the HPS+APP combined vaccine group and the APP commercial vaccine group decreased significantly (see attachment Figure 9 ).

[0135] Level of T lymphocytes in piglets:

[0136] Detection results of T lymphocyte stimulation index: Two weeks after the second immunization, the T lymphocyte stimulation index in the HPS+APP combined vaccine group was significantly higher than that in the non-immunized control group and the normal saline group (P<0.05). However, there was no significant difference between the HPS+APP combined vaccine group and the HPS commercial vaccine group and the APP commercial vaccine group (P>0.05). Two weeks after challenge, the T lymphocyte stimulation index in the HPS+APP combined vaccine group was extremely significantly higher than that in the non-immunized control group and the normal saline group (P<0.001), and the T lymphocyte stimulation index in the HPS+APP combined vaccine group was higher than that in the HPS commercial vaccine group and the APP commercial vaccine group (see Appendix Figure 10 and 11 ).

[0137] Level of B lymphocytes in piglets:

[0138] Detection results of B lymphocyte stimulation index: One week after the second immunization, the B lymphocyte stimulation index in the HPS+APP combined vaccine group was extremely significantly higher than that in the non-immunized control group and the normal saline group (P<0.001), and was also higher than that in the HPS commercial vaccine group and the APP commercial vaccine group, but the difference was not significant (P>0.05); Two weeks after challenge, the B lymphocyte stimulation index in the HPS+APP combined vaccine group was extremely significantly higher than that in the non-immunized control group and the normal saline group (P<0.001), and the B lymphocyte stimulation index in the HPS+APP combined vaccine group was significantly higher than that in the HPS commercial vaccine group and the APP commercial vaccine group (P<0.05) (see Appendix Figure 12 and 13 ).

[0139] Example 6 Safety evaluation of Haemophilus parasuis vaccine and Actinobacillus pleuropneumoniae combined inactivated vaccine

[0140] 1. Safety test of single-dose inoculation of the target animals once

[0141] Five 21-day-old piglets were respectively injected intramuscularly in the neck with 2 ml of the HPS+APP combined inactivated vaccine per head, and a normal saline immunization control group was set up. To observe whether the test pigs had allergies, whether there were abnormalities in spirit and diet, whether there was redness and swelling at the injection site, and whether the weight gain of piglets or the litter size of sows was normal within 2 weeks after the test pigs were vaccinated. The results showed that the piglets were in good spirits, there was no redness and swelling at the injection site after inoculation, and the spirit and feeding were normal; there was no significant difference from the control pigs, indicating that the trial-produced vaccine was safe (see Table 6).

[0142] Table 6

[0143]

[0144] 2. Safety test of super-dose inoculation of the target animals once

[0145] HPS+APP two-unit inactivated vaccine was injected into the neck muscle of 5 healthy susceptible piglets at 21 days old, 4 ml / pig, to observe the safety of the experimental pigs after vaccination, and a normal saline immunization control group was set up. The results showed that no redness or swelling reaction occurred at the injection site of the 21-day-old piglets after vaccination, and their spirits and feeding were normal, with no significant difference from the control pigs, indicating that the trial vaccine is safe for one overdose vaccination (see Table 7).

[0146] Table 7

[0147]

[0148] 3. Single-dose repeated vaccination safety test

[0149] HPS+APP two-unit inactivated vaccine was used to vaccinate healthy susceptible piglets aged 10 to 15 days, and the vaccination was repeated once after 14 days to observe the safety of repeated vaccination of single doses of vaccine on vaccinated pigs, and a normal saline immunization control group was set up. The results showed that the vaccine had no adverse effects on the vaccinated pigs, no allergic reactions, no adverse reactions such as redness and swelling at the vaccination site, the spirit and feed intake of the vaccinated pigs were normal, and the weight gain of the piglets was not significantly different from that of the control pigs, indicating that repeated vaccination of single doses of the trial vaccine is safe (see Table 8).

[0150] Table 8

[0151]

[0152] 4. Safety test for pregnant sows

[0153] HPS+APP two-inactivated vaccine was used to inject 5 pregnant sows 15 days before delivery into the neck muscle, 2ml / head, and a normal saline immune control group was set up. The test pigs were observed to see if they had allergies, abnormal spirits and diets within 2 weeks after vaccination, whether there was redness and swelling at the injection site, and whether the sows gave birth normally. Results No redness or swelling appeared at the injection site of pregnant sows 15 days before delivery. On the second day of vaccination, the body temperature of some sows rose slightly. On the third day, the body temperature returned to normal, and the spirit and feeding were normal. The healthy piglet rate of sows in the immunization group was 85.25%; the healthy piglet rate of sows in the control group was 57.74%.

[0154] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0155] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Actinobacillus pleuropneumoniae FJLYC01, characterized in that: The deposit number is CGMCC No.30270.

2. An immunogenic composition, characterized in that The immunogenic composition contains the Actinobacillus pleuropneumoniae FJLYC01 according to claim 1.

3. Use of Actinobacillus pleuropneumoniae FJLYC01 according to claim 1 or the immunogenic composition according to claim 2, characterized in that: Any of the following: a. Use in the preparation of a drug for the prevention and / or treatment of Actinobacillus pleuropneumoniae infection or a disease caused by Actinobacillus pleuropneumoniae infection; b. Use in the preparation of vaccines for the prevention and / or treatment of Actinobacillus pleuropneumoniae infection or diseases caused by Actinobacillus pleuropneumoniae infection; c. Use in the preparation of antibodies for the prevention and / or treatment of Actinobacillus pleuropneumoniae infection or diseases caused by Actinobacillus pleuropneumoniae infection; d. Use in the preparation of antiserum for the prevention and / or treatment of Actinobacillus pleuropneumoniae infection or diseases caused by Actinobacillus pleuropneumoniae infection; e. Application in the preparation of reagents or kits for detecting Actinobacillus pleuropneumoniae; f. Application in the evaluation of immunogenicity of Actinobacillus pleuropneumoniae vaccines for non-diagnostic and therapeutic purposes; g. Application in the evaluation of protective efficacy of Actinobacillus pleuropneumoniae vaccines for non-diagnostic and therapeutic purposes; h. Application in the preparation of cell models infected with Actinobacillus pleuropneumoniae; i. Application in the preparation of an animal model of Actinobacillus pleuropneumoniae infection; j. Application in drug screening or efficacy evaluation for preventing and / or treating Actinobacillus pleuropneumoniae infection or diseases caused by Actinobacillus pleuropneumoniae infection.

4. A vaccine, characterized in that The vaccine contains the inactivated vaccine prepared from Actinobacillus pleuropneumoniae FJLYC01 according to claim 1.

5. A two-combination inactivated vaccine, characterized in that: The dual inactivated vaccine is prepared from Actinobacillus pleuropneumoniae FJLYC01 and Haemophilus parasuis serotypes 4 and 5 as described in claim 1.

6. The method for preparing the dual inactivated vaccine according to claim 5, characterized in that: The following steps are involved: (1) Bacterial culture Liquid fermentation was used to culture Haemophilus parasuis serotype 4 strain LYH5, 5 strain LY02 and Actinobacillus pleuropneumoniae serotype 1 strain FJLYC01 respectively. (2) Live bacteria count Take the culture solution after 22-24 hours of culture to count the viable bacteria and adjust the concentration of the culture solution to 2.5×10 9 CFU / ml; (3) Inactivation Add formaldehyde solution to the bacterial solution with adjusted concentration to inactivate it; (4) Adding vaccine adjuvant The inactivated Haemophilus parasuis serotypes 4 and 5 and Actinobacillus pleuropneumoniae serotype 1 bacterial liquids were mixed in a volume ratio of 1:1:1, and the oil phase and the water phase were prepared into an oil emulsion inactivated vaccine in a ratio of oil phase:water phase = 2:1, and 1% aluminum stearate was added, wherein the water phase was 4% Tween-80 and 96% bacterial liquid, and the oil phase was 94% white oil and 6% Siben-80; the antigen content of the combined inactivated vaccine of Haemophilus parasuis and Actinobacillus pleuropneumoniae was 2.0×10 9 CFU / mL.

7. The method for preparing the bivalent inactivated vaccine according to claim 5, characterized in that: Step (1) bacterial liquid culture specifically comprises: adding semi-synthetic culture medium I to serotypes 4 and 5 of Haemophilus parasuis according to a fermentation volume of 100 ml / 250 ml, culturing at 37° C., 180 r / min for 22 to 24 hours, and harvesting the bacterial liquid of each type; adding semi-synthetic culture medium II to serotype 1 of Actinobacillus pleuropneumoniae according to a fermentation volume of 100 ml / 250 ml, culturing at 37° C., 180 r / min for 22 to 24 hours, and harvesting the bacterial liquid; The preparation method of the semi-synthetic culture medium I is as follows: 30 g of tryptic soy broth is dissolved in deionized water, the volume is adjusted to 1000 mL, after being fully shaken and dissolved, the volume is sterilized at 121°C with high pressure steam for 15 minutes, and 50 mL of filtered sterilized newborn calf serum and 200 μL of filtered sterilized 0.02% NAD are added; the preparation method of the semi-synthetic culture medium II is as follows: 30 g of tryptic soy broth is dissolved in deionized water, the volume is adjusted to 1000 mL, after being fully shaken and dissolved, the volume is sterilized at 121°C with high pressure steam for 15 minutes, and 50 mL of filtered sterilized newborn calf serum and 200 μL of filtered sterilized 0.04% NAD are added.

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