Riemerella anatipestifer and application thereof in egg yolk antibody

Abstract

The invention discloses riemerella anatipestifer and application of the riemerella anatipestifer in an egg yolk antibody. The riemerella anatipestifer is named as BRA22A011, and the preservation number of the riemerella anatipestifer is CGMCC (China General Microbiological Culture Collection Center) No.45887. The invention also discloses an inactivated vaccine, wherein the antigen in the inactivated vaccine contains the inactivated riemerella anatipestifer BRA22A011. The invention further discloses an egg yolk antibody, and the egg yolk antibody is prepared by using the riemerella anatipestifer BRA22A011 as an antigen. The use mode of the egg yolk antibody is intramuscular injection inoculation, and the injection dosage is 0.5 mL. The riemerella anatipestifer BRA22A011 has high pathogenicity and strong immunogenicity, and the egg yolk antibody prepared from the riemerella anatipestifer BRA22A011 can replace antibiotics and vaccines, is safe, free of residues, free of drug resistance and environment-friendly, and has a good prospect in clinical application of preventing and treating infectious serositis of ducks. The riemerella anatipestifer BRA22A011 and the Escherichia coli BE-20261 are adopted for preparing the egg yolk antibody together, and the prevention and treatment effects of the egg yolk antibody are more obvious.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary biological products, in particular to Riemerella anatipestifer and application thereof in egg yolk antibodies. Background Art

[0002] Riemerella anatipestifer (RA) is currently the main pathogen that harms the waterfowl farming industry. It is widely distributed in duck farms around the world and is difficult to eliminate after infection. The infection rate of the disease can sometimes be as high as 90% or more, but due to the large differences in virulence between serotypes, the mortality rate ranges from 5% to 75%. The mortality rate of the disease can be as high as 90%. The acute type manifests as death from acute septicemia, while the chronic type generally has no obvious symptoms, and some may manifest as weight loss and decreased egg production.

[0003] The long-term and extensive use of antibiotics has greatly accelerated the evolution of drug resistance in pathogenic bacteria. New drug-resistant strains continue to emerge. Drug resistance causes the efficacy of drugs in the prevention and treatment of RA to continue to decline. At the same time, the problem of antibiotic residues caused by the extensive use of antibiotics is becoming increasingly serious. The problem of drug resistance and antibiotic residues caused by the abuse of antibiotics has become a global problem that needs to be solved urgently. Improving animal breeding conditions, vaccinating or using yolk antibodies to treat pathogens are effective ways to reduce the use of antibiotics.

[0004] Vaccination is currently the most effective means of preventing and treating Riemerella anatipestifer. At present, there are 21 recognized vaccine RA serotypes, and there is no cross-protection between the RA serotypes. Therefore, when developing vaccines and egg yolk antibodies, we must base ourselves on the serotype strains prevalent in the region and use them as vaccine strains. Although inactivated vaccines have been successfully used for preventive immunization of RA disease, there are some shortcomings in their production, such as inconvenient operation procedures, high costs, and greater stress on ducklings.

[0005] Therefore, the existing technology needs to be further improved. It is important to screen out Riemerella anatipestifer with strong pathogenicity and good immunogenicity and explore the preparation of its corresponding egg yolk antibodies. Summary of the invention

[0006] The present invention aims to solve the above problems and provides a Riemerella anatipestifer and its application in egg yolk antibodies.

[0007] Specifically, the technical solution provided by the present invention is as follows:

[0008] In a first aspect, the present invention provides a Riemerella anatipestifer, which is named as Riemerella anatipestifer BRA22A011, and the deposit number of Riemerella anatipestifer BRA22A011 is CGMCC No.45887.

[0009] In a second aspect, the present invention provides a use of Riemerella anatipestifer BRA22A011 as described in the first aspect in preparing an inactivated vaccine.

[0010] In a third aspect, the present invention provides an inactivated vaccine, wherein the antigen in the inactivated vaccine comprises the inactivated Riemerella anatipestifer BRA22A011 of claim 1.

[0011] In a fourth aspect, the present invention provides a use of Riemerella anatipestifer BRA22A011 as described in the first aspect in preparing egg yolk antibodies.

[0012] In a fifth aspect, the present invention provides an egg yolk antibody, wherein the egg yolk antibody is prepared using the Riemerella anatipestifer BRA22A011 described in the first aspect as an antigen.

[0013] Further preferably, the egg yolk antibody is prepared by using Riemerella anatipestifer BRA22A011 and Escherichia coli BE-20261 as antigens as described in the first aspect.

[0014] Further preferably, the mass ratio of Riemerella anatipestifer BRA22A011 and Escherichia coli BE-20261 in the egg yolk antibody is 4:1.

[0015] Furthermore, the yolk antibody is administered by intramuscular injection.

[0016] Furthermore, the injection amount of the egg yolk antibody is 0.5 mL.

[0017] The present invention has the following advantages:

[0018] The Riemerella anatipestifer BRA22A011 of the present invention has high pathogenicity and strong immunogenicity. The egg yolk antibody prepared by the Riemerella anatipestifer BRA22A011 can replace antibiotics and vaccines, is safe and has no residue, will not produce drug resistance, is environmentally friendly, and has good prospects in clinical applications for preventing and treating duck infectious serositis. Escherichia coli is a mixed-sense bacterium. The present invention uses Riemerella anatipestifer BRA22A011 and Escherichia coli BE-20261 to jointly prepare the egg yolk antibody, and the prevention and treatment effects are more obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0020] Figure 1 : The morphology of Riemerella anatipestifer (BRA22A011) of the present invention on sheep blood agar plates;

[0021] Figure 2 : Gram staining result of Riemerella anatipestifer (BRA22A011) of the present invention;

[0022] Figure 3 : Electrophoresis detection diagram of ompA of Riemerella anatipestifer (BRA22A011) of the present invention;

[0023] Figure 4 : Electrophoresis detection diagram of 16S of Riemerella anatipestifer (BRA22A011) of the present invention. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0025] Example 1: Isolation, purification, identification and preservation of Riemerella anatipestifer

[0026] Sampling was conducted from a diseased farm in Weifang, Shandong. The liver or brain of the diseased ducks was collected aseptically (the liver was collected when there was typical fibrinous exudation, and the brain was collected when there were neurological symptoms). The inoculation loop was inserted into the tissue, and then streaked onto TSA blood agar plates and MacConkey agar plates. The plates were cultured in a 37°C incubator or candle jar for 24 to 48 hours. On the blood agar plate culture medium, moist, dew-drop-like, rounded, smooth-surfaced, neatly-margined colonies were observed. The colonies were 1 to 2 mm in diameter and off-white in color. No colonies grew on the MacConkey agar plate. The morphology of Riemerella anatipestifer on the sheep blood agar plate is as follows: Figure 1 shown.

[0027] Select a single typical colony for Gram staining and microscopic examination. Figure 2 As shown, Gram-negative, small rods without flagella, no movement, and no spore formation. Arranged singly, in pairs or in short chains, the bacterial morphology is rod-shaped, some are oval, and occasionally filamentous. The bacterial size is 0.2μm to 0.5μm wide and 1μm to 5μm long, and the filamentous bacteria can be as long as 11μm to 24μm. Pick a typical colony and continue to purify by streaking 3 to 5 times until the colony morphology is consistent, then pick a single colony and inoculate it in 5mL of TSB broth (2% fetal bovine serum) culture medium, 37℃, 200rpm shaking culture for 24h to obtain a uniform turbid bacterial suspension.

[0028] Then, through 16S rRNA molecular identification and serotype identification, it was determined to be pathogenic Riemerella anatipestifer, and one of the strains was named Riemerella anatipestifer BRA22A011. The identified strain was streaked on a TSA + 2% serum culture medium plate and cultured for 24-36 hours. All colonies were scraped into 60% glycerol-TSB liquid with an inoculation loop and stored in a -80°C refrigerator.

[0029] The isolated Riemerella anatipestifer BRA22A011 was preserved with the deposit number CGMCC No. 45887. The depositor was the General Microbiology Center of China Culture Collection Administration. The deposit address was No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was March 15, 2024. The classification name was Riemerella anatipestifer, and the test result was survival.

[0030] The laboratory isolated pathogenic Escherichia coli BE-20261, with the preservation number CGMCC No.28217, the preservation unit is the General Microbiology Center of China Culture Collection Administration, the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is August 23, 2023, and the classification name is: Escherichia coli, and the test result is survival.

[0031] The RA genome was extracted using a bacterial genome extraction kit, and PCR identification was performed using primers for the outer membrane protein A gene (ompA), a specific protein of Riemerella anatipestifer, as well as 16S identification. The electrophoresis detection of ompA is shown in the figure below. Figure 3 As shown (maker is D2000, from top to bottom are 2000, 1000, 750, 500, 250, 100 bp), the electrophoresis detection diagram of 16S is as follows Figure 4As shown (marker is D2000, from top to bottom are 2000, 1000, 750, 500, 250, 100 bp).

[0032] Table 1: Primer sequences

[0033]

[0034] The 16S sequence is shown in SEQ ID NO: 1:

[0035]

[0036] Example 2: Riemerella anatipestifer toxicity assay

[0037] A single colony of pure culture of the isolated bacteria was streaked onto a TSA plate (added with 2% calf serum before use) and cultured at 37°C for 24-36 hours.

[0038] All colonies on the plate were scraped and mixed with sterile saline to make a bacterial suspension, and 0.5 mL / bird (1.0 × 10^7 CFU / mL) was injected intramuscularly into 10 healthy susceptible ducks at 18 days of age. At the same time, a sterile saline injection control group of 10 ducks was set up. After the challenge, the ducks were observed for 10 consecutive days, and the spirit, appetite, drinking water, movement and death of the ducks were recorded daily. The dead ducks were subjected to pathological autopsy, bacterial isolation and identification.

[0039] The results showed that 36 hours after infection with BRA22A011 strain, ducks began to show symptoms such as lethargy, slow movements or prone position, eye and nasal discharge, and ataxia. The symptoms of the other 7 strains occurred in some cases at 96-120 hours, and some showed no clinical symptoms. All ducks became ill after infection with BRA22A011 strain, and most of the infected ducks died within 10 days, with a mortality rate of 8 / 10. Typical pericarditis, perihepatitis, and air sac inflammation were found in autopsy. 04, BRA22A005, BRA22A006, BRA22A008, BRA22A009, and BRA22A0010 had an incidence rate of 0 / 10-6 / 10 and a mortality rate of 0 / 10-2 / 10, so the BRA22A011 strain was determined to be the most virulent; the inoculum was isolated from brain tissue on TSA culture medium, and its biochemical and culture characteristics were consistent with the identification results of the BRA22A011 strain, so this study selected the BRA22A011 strain as the highly virulent strain for vaccine.

[0040] Table 2: Results of the virulence test of Riemerella anatipestifer

[0041]

[0042] Example 3: Immunogenicity determination of the virulent isolate of Riemerella anatipestifer BRA22A011

[0043] The virulent isolate BRA22A011 was inoculated into TSB medium (2% calf serum was added before use) and then the viable bacteria were counted by plate culture counting method. The bacteria were collected and the concentration was adjusted to 3.0×10 6 , 3.0×10 7 , 3.0×10 8 , 3.0×10 9 , 3.0×10 10 CFU / mL, and then the inactivated vaccines were made into oil-adjuvanted inactivated vaccines according to the oil-water ratio of 2:1, so that the bacterial count per milliliter of antigen liquid was 1.0×10 6 , 1.0×10 7 , 1.0×10 8 , 1.0×10 9 , 1.0×10 10 CFU.

[0044] Sixty healthy susceptible ducklings aged 60 days were selected, 10 ducks / group, and inactivated vaccines containing different concentrations of antigen were injected subcutaneously in the neck, 0.5 mL / duck. Ten ducks were used as non-immune controls. On the 14th day after vaccination, each duck was injected intramuscularly with 0.5 mL (1.0×10 7 CFU / mL) BRA22A011 strain bacterial liquid was observed for 10 days to determine the immune protection effect of the vaccine.

[0045] The experimental results are shown in Table 3. Analysis shows that when the antigen content reaches 1.0×10 7 CFU / mL and above, the immunization dose was 0.5mL, the protection reached 8 / 10, and there was no death; when the antigen content reached 1.0×10 8 CFU / mL, the immunization dose was 0.5mL, the protection reached 9 / 10, and there was no death; when the antigen content reached 1.0×10 9 CFU / mL and above, the immunization dose was 0.5mL, the protection reached 10 / 10, and there was no death; 10 / 10 of the control group became ill and 6 of them died. The results showed that when the antigen content reached 1.0×10 8 When the CFU / mL was above, the protection against virus infection reached more than 9 / 10, with no death and good immunogenicity.

[0046] Table 3: Results of immunogenicity tests of BRA22A011 isolates

[0047]

[0048] Example 4: Preparation of inactivated vaccines of Riemerella anatipestifer, Escherichia coli and Riemerella anatipestifer

[0049] (1) Cultivation of Riemerella anatipestifer: The highly virulent isolate BRA22A011 was inoculated into TSB medium (with 2% calf serum added before use) and then the viable bacteria were counted by plate culture counting method. The bacteria were collected and their concentration was adjusted to 3.0×10^9 CFU / mL.

[0050] (2) Culture of Escherichia coli: Escherichia coli BE-20261 was inoculated into TSB medium for culture, and then live bacteria were counted by plate culture counting method and the bacteria were collected.

[0051] (3) Bacterial liquid test

[0052] Live bacteria count: Live bacteria count shall be carried out in accordance with the appendix of the current "Pharmacopoeia of Veterinary Medicine of the People's Republic of China".

[0053] (4) Inactivation of Riemerella anatipestifer and Escherichia coli: add formaldehyde solution at a ratio of 0.1 of the total bacterial solution, mix thoroughly, and inactivate by shaking at 37°C for 48 hours.

[0054] (5) Inactivation test of Riemerella anatipestifer and Escherichia coli: Take the inactivated bacterial solution and inoculate it with TSB (add 2% calf serum before use), incubate at 37°C for 36 hours, and check for sterile growth.

[0055] (6) Seedling matching

[0056] A. Preparation of oil phase: Take 94 parts of white oil for injection and 6 parts of Span 80, mix them and heat to 90°C while stirring. Sterilize them under high pressure at 121°C for 30 minutes. This is the oil phase.

[0057] B. Preparation of aqueous phase: Add 4 parts of sterilized Tween-80 to 96 parts of antigens (Riemerella anatipestifer: Escherichia coli = 4:1) and (Riemerella anatipestifer: Escherichia coli = 10:0) that have passed the test, shake thoroughly until the Tween-80 is completely dissolved to obtain the aqueous phase.

[0058] C. Emulsification: The ratio of water phase to oil phase is 1:2 (V / V). Inject the oil phase into the premixing tank, stir at 3500r / min for 15min, slowly add the water phase, and then inject into the emulsification tank for emulsification.

[0059] D. Packaging: Aseptically pack in quantitative quantities, seal the bottle mouth, and store at 2-8℃.

[0060] Example 5 Immunization of laying hens

[0061] Immunization group 1: Riemerella anatipestifer: Escherichia coli = 4:1; Immunization group 2: Riemerella anatipestifer: Escherichia coli = 10:0. The two groups were immunized for laying hens respectively. The immunization procedure is as follows

[0062] (1) For the first immunization, 0.5 mL / hen was injected intramuscularly with the prepared immunogen into commercial laying hens about 150 days old;

[0063] (2) Second vaccination: 15 days after the first vaccination, intramuscular injection of the prepared immunogen, 1.0 mL / animal;

[0064] (3) For the third immunization, 1.5 mL / mouse was injected intramuscularly 15 days after the second immunization.

[0065] (4) For the fourth vaccination, 2 mL / mouse was injected intramuscularly 30 days after the third vaccination.

[0066] (5) 14 days after the fourth vaccination, start collecting high-immunity eggs and store them in a 4°C refrigerator for later use.

[0067] Example 6: Yolk antibody preparation process

[0068] (Separate extraction of Riemerella anatipestifer and Escherichia coli, Riemerella anatipestifer egg yolk antibodies)

[0069] S1. Detect egg titer: Use ELISA test method to detect egg yolk antibody titer; the ELISA result of Riemerella anatipestifer egg yolk antibody ELISA in the immunized group should not be less than 3.0.

[0070] S2. Cleaning and disinfection: Soak the eggs in 0.1% 42°C Chlorhexidine solution for 15 minutes, remove them from the water, rinse them with running water to remove surface dirt, spray fumigate the eggs with 0.1% Weikang (glutaraldehyde) for 30 minutes, blow dry the eggs and then crack them.

[0071] S3. Egg yolk pretreatment: Use an egg beater to separate egg white and yolk; mix and stir evenly according to 1:1 (v / v, egg yolk: 0.02mol / L disodium hydrogen phosphate solution); filter with a 60-mesh wire sieve to remove impurities in the egg yolk liquid; transfer the above egg yolk liquid to a sedimentation tank, and add the remaining 2 times volume of disodium hydrogen phosphate solution (0.02mol / L); stir at 1000rpm for 60min. At this time, the pH value of the mixed solution is about 7.2, the color is milky yellow, turbid and uniform. Note: At this time, it must be slow and uniform, and the time is sufficient to prevent the egg yolk from denaturing.

[0072] S4. Acid extraction: Slowly add 1 times sedimentation liquid (PVP and trehalose are 6.66g / L and 4.16g / L respectively, fully dissolved in purified water of equal mass with egg yolk) into the sedimentation tank at a ratio of 1:1 (v / v, egg yolk: sedimentation agent). At this time, the solution is milky yellow, uniform and turbid. Carefully adjust the pH value of the sedimentation liquid to 6.0 with 30% citric acid solution, and sediment at 4°C for 12h. After the sedimentation is completed and the titer of the supernatant is not less than 2.0, all the supernatant can be transferred out to the next step.

[0073] S5. Caprylic acid degreasing: Add caprylic acid to the extracted supernatant at a final concentration of 0.1% to remove fat, mix thoroughly and place at room temperature for 2 to 6 hours, remove impurities through filter cloth, and the antibody stock solution is obtained.

[0074] S6. Sterile filtration: Sterilize by filtering with a 0.22um microporous filter.

[0075] S7. Ultrafiltration to remove viruses: Use an ultrafiltration membrane with a molecular weight cutoff of 1000 kDa to remove viruses.

[0076] S8. Ultrafiltration concentration: Use an ultrafiltration membrane with a molecular weight cutoff of 100 kDa to concentrate the egg yolk antibody twice.

[0077] S9. Antibody titer determination: The ELISA result of Riemerella anatipestifer egg yolk antibody should not be less than 2.0.

[0078] S10. Packaging: Pack in quantitative quantities, seal with a lid, and store at 2-8℃.

[0079] Example 7: Riemerella anatipestifer and Escherichia coli challenge and protection experiment

[0080] 120 susceptible ducklings aged 18 days were divided into 4 groups, 30 ducks in each group, and the concentration of Riemerella anatipestifer was adjusted to 1.0×10 7 CFU / mL. The specific grouping and treatment are as follows:

[0081]

[0082] The ducks were observed for 10 consecutive days, and their spirit, appetite, drinking water, exercise and death were recorded daily. The dead ducks were pathologically examined and bacteria were isolated and identified.

[0083] The morbidity and mortality rates of the control group A were both 0%.

[0084] 36 hours after the infection, ducklings in the non-treated group B began to show symptoms such as lethargy, slow movements or prone position, eye and nasal discharge, and ataxia. All ducks became ill after infection with the BRA22A011 strain, and most of the infected ducks died within 10 days, with a mortality rate of 8 / 10. Typical pericarditis, perihepatitis, and air sac inflammation were found in autopsy. Brain tissue was taken to isolate the inoculum on tryptone soy agar medium.

[0085] The protection rate of duck plague antibody treatment group C was 8 / 10;

[0086] The protection rate of duck plague + large intestine antibody treatment group D was 10 / 10; the autopsy showed no typical pericarditis, perihepatitis, and air sacculitis, and the inoculated bacteria could not be isolated from the brain tissue.

[0087] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A Riemerella anatipestifer, characterized in that The Riemerella anatipestifer is named as Riemerella anatipestifer BRA22A011, and the deposit number of the Riemerella anatipestifer BRA22A011 is CGMCC No.45887.

2. Use of the Riemerella anatipestifer BRA22A011 as claimed in claim 1 in the preparation of an inactivated vaccine.

3. An inactivated vaccine, characterized in that: The antigen in the inactivated vaccine comprises the inactivated Riemerella anatipestifer BRA22A011 according to claim 1.

4. Use of the Riemerella anatipestifer BRA22A011 as claimed in claim 1 in the preparation of egg yolk antibodies.

5. An egg yolk antibody, characterized in that: The egg yolk antibody is prepared by using the Riemerella anatipestifer BRA22A011 described in claim 1 as an antigen.

6. The egg yolk antibody according to claim 5, characterized in that: The egg yolk antibody is prepared by using Riemerella anatipestifer BRA22A011 and Escherichia coli BE-20261 as antigens as described in claim 1.

7. The egg yolk antibody according to claim 6, characterized in that: The mass ratio of Riemerella anatipestifer BRA22A011 and Escherichia coli BE-20261 in the egg yolk antibody is 4:

1.

8. The egg yolk antibody according to any one of claims 5 or 6, characterized in that: The yolk antibody is administered by intramuscular injection.

9. An egg yolk antibody according to claim 8, characterized in that: The injection amount of the egg yolk antibody is 0.5 mL.

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