A strain of Riemerella columbae from chicken and its application
By isolating and identifying RC TH22 of the chicken-derived romelli RC TH22 and confirming its pathogenicity, it was prepared into an inactivated vaccine, which solved the problem of insufficient etiology of serosal inflammatory disease in chickens and achieved effective prevention and control of the disease.
Patent Information
- Application Number
- CN202310584813.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-23
- Publication Date
- 2025-03-25
- Estimated Expiration
- 2043-05-23
AI Technical Summary
The prior art studies on the pathogenic etiology of chicken multiserose inflammation, and the pathogenic factors are complex, resulting in unsatisfactory prevention and treatment effects.
A chicken-derived RC TH22 strain was isolated and identified, and its pathogenicity was confirmed by artificial infection of chicks, and an inactivated vaccine was prepared to prevent and control chicken multiserose inflammation.
It enriches the etiology data of chicken multiserosus inflammation, provides materials for research on pathogenic mechanisms, and prepares vaccines that have a strong protective effect on pigeon Rimerci infection, providing an effective means for the prevention and control of chicken multiserosus inflammation.
Smart Images
Figure CN116515707B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and more specifically relates to a strain of chicken-derived Riemerella columbae and its application. Background Art
[0002] Riemerella belongs to the genus Riemerella sp. of the Flavobacteriaceae family, including Riemerella anatipestifer (RA), Riemerella columbina (RC), and Riemerella columbipharyngis, as well as some isolates whose taxonomic status has not yet been fully determined. Riemerella columbina (RC) is a type of non-spore-forming, non-motile, pleomorphic Gram-negative rod. At present, there are very few studies on the pathogenicity of RC. Occasionally, RC infection in domestic pigeons can cause pathological changes similar to those of Riemerella anatipestifer (RA), that is, polyserosal inflammation characterized by fibrinous exudates on the surface of the pericardium, liver capsule, air sac membrane, abdominal cavity and pleural cavity membrane, and severe sepsis.
[0003] Riemerilosis is mainly seen in waterfowl (ducks and geese) and is one of the most common bacterial infectious diseases in commercial meat ducks and geese. The isolation of Riemeril from chickens is relatively rare. Only Liu Fazhi et al. isolated a strain of Riemeril HB2013 from the trachea of chickens. The study confirmed that the HB2013 strain had no obvious pathogenicity to poultry (Liu Fazhi, Li Zhanhong, Yin Jinhua, Su Jingliang. Isolation and identification of Riemeril from chickens [J]. Chinese Journal of Veterinary Medicine, 2015, 51(05): 40-41.). Existing studies have shown that, except for one RC isolated from tree shrews, almost all RC isolates are derived from the upper respiratory mucosal surface of domestic pigeons, and there are no reports of isolation and identification from other animals.
[0004] At present, polyserosal inflammation in chickens occurs frequently, with complex pathogenic factors, and the phenomenon of mixed infection and cross infection of multiple pathogens is relatively common, which seriously threatens the health of chickens and brings great harm to the development of the poultry industry. Clinically, it is generally believed that the pathogenic microorganisms causing polyserosal inflammation in chickens are mainly H9 subtype avian influenza virus, Escherichia coli and mycoplasma, while Riemerella is less concerned. The limitations of clinical understanding of the pathogenic microorganisms of this disease may be an important reason for the unsatisfactory prevention and treatment of polyserosal inflammation in chickens. Summary of the invention
[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing problems, provide a strain of chicken-derived Riemerella columba RC TH22 and its application, enrich the etiological data of chicken polyserosal inflammation, and provide good material for in-depth research on the pathogenic mechanism of Riemerella columba that can cause polyserosal inflammation in chicks.
[0006] The first object of the present invention is to provide a chicken-derived Riemerella columbina RCTH22 strain.
[0007] The second object of the present invention is to provide the application of the strain.
[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:
[0009] The present invention is the first to isolate a strain of Riemerella columbina RC TH22 from the liver of a black-bone chicken suffering from severe pericarditis and perihepatitis. The strain has been deposited in the China Center for Type Culture Collection on July 11, 2022, with a deposit number of CCTCC NO: M 20221075. The RC TH22 strain was incubated at 37°C for 24 hours under aerobic conditions on a TSA plate containing 5% fetal bovine serum, and produced colonies with a diameter of 1 to 2 mm, grayish white, raised, smooth, and moist surfaces; the bacterial body was Gram-negative, and microscopic examination showed darkly stained pink rods at both poles; under an electron microscope with a resolution of 5 μm, it showed densely arranged rod-shaped bacteria with swollen poles.
[0010] The present invention artificially infects chicks with the isolated chicken-derived Riemerella RC TH22, returning to the original animal. The present invention first confirms that the chicken-derived Riemerella RC TH22 can cause polyserosal inflammation in chicks. After 24 hours of RC TH22 attack, clinical lesions similar to those of chicken-derived Escherichia coli EC101 attack appear, and both have lesions such as fibrinous pericarditis, hepatitis, and air sac inflammation, which are obviously pathogenic to chicks. At the same time, the present invention also determines the types of antibiotics sensitive to the RC TH22 strain. In the clinical treatment of Riemerella infection, florfenicol and roxithromycin are better choices for oral medication, and gentamicin, cefuroxime, and ceftazidime antibiotics can bring good therapeutic effects when injected, providing a clinical medication guide for the treatment of polyserosal inflammation in chickens. The present invention further enriches the host source of Riemerella RC TH22 and updates the understanding of pathogenic microorganisms of polyserosal inflammation in chickens in the veterinary field.
[0011] Furthermore, the present invention prepares the isolated bacteria RC TH22 into an inactivated vaccine. After immunizing chicks through an immune protection test, it is confirmed that the RC TH22 inactivated vaccine has a strong protective effect against the same strain, providing an effective means for the prevention and control of polyserosal inflammation in chickens caused by Riemerella infection.
[0012] Therefore, the present invention provides the following application of Riemerella columbina RC TH22 strain:
[0013] Application of Riemerella columbina RC TH22 strain in constructing an animal model of polyserosal inflammation in chickens.
[0014] Application of Riemerella columbina RC TH22 strain in the preparation of chicken polyserosal inflammation vaccine.
[0015] The invention provides a chicken polyserosal inflammation vaccine, which contains Riemerella columbina RCTH22 strain.
[0016] Preferably, the vaccine is an inactivated vaccine, wherein the concentration of RC TH22 strain is not less than 2×10 8 CFU / mL.
[0017] Preferably, the vaccine further contains a vaccine adjuvant, which may be a conventional commercial adjuvant in the art, such as the aluminum hydroxide / magnesium hydroxide alum adjuvant used in the present invention.
[0018] The invention provides a method for constructing an animal model of polyserosal inflammation in chickens infected with Riemerella columbina. The Riemerella columbina RC TH22 strain is used to infect chicks to obtain the animal model of polyserosal inflammation in chickens infected with Riemerella columbina.
[0019] Preferably, the infection method is subcutaneous injection in the neck.
[0020] Preferably, the injection concentration of the RC TH22 strain is not less than 1×10 9 CFU / ml.
[0021] The present invention also provides application of the chicken polyserosal inflammation animal model obtained by the above construction method in chicken polyserosal inflammation drug screening and vaccine effect evaluation.
[0022] The present invention also provides a method for screening drugs for chicken polyserosal inflammation, and the chicken polyserosal inflammation animal model obtained by the above-mentioned construction method is used as a pathogenic model for screening.
[0023] In addition, the present invention also provides a medicament for inhibiting Riemerella columbina, which contains one or more antibiotics selected from florfenicol, roxithromycin, neomycin, gentamicin, cefuroxime and ceftazidime.
[0024] The present invention has the following beneficial effects:
[0025] The present invention isolates a strain of Riemerella columbina RC TH22 from chickens for the first time. It is very rare to isolate Riemerella columbina from chicks with certain pathogenicity, which has extremely important research value. The present invention isolates a strain of chicken-derived Riemerella columbina RCTH22 from the liver of a black chicken suffering from severe pericarditis and perihepatitis for the first time, further enriching the host source of Riemerella columbina and updating the understanding of pathogenic microorganisms of polyserosal inflammation in chickens in the veterinary field. The inventor artificially infects chicks with the isolated Riemerella columbina and returns to the original animal. It is confirmed for the first time that the pathogen can cause polyserosal inflammation in chicks. The present invention uses Riemerella columbina as a vaccine strain for pigeon Riemerella disease for the first time to prepare an inactivated vaccine, and achieves an ideal immune protection effect, which has very important practical significance for effectively preventing and treating polyserosal inflammation in chicks. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the Gram staining microscopic examination image of the strain in the pathogen identification experiment;
[0027] Figure 2 The morphology of the strain under electron microscope in the pathogen identification experiment;
[0028] Figure 3 This is a picture of the anatomical lesions of different treatment groups in the animal regression experiment;
[0029] Figure 4 This is a survival curve diagram of different treatment groups after virus attack in the animal regression experiment;
[0030] Figure 5 This is a survival curve diagram of different treatment groups in the immune protection experiment. DETAILED DESCRIPTION
[0031] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
[0032] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.
[0033] The solid culture medium preparation method used was as follows: 40 g of tryptone soy agar TSA was dissolved in 950 mL of deionized water, sterilized at 121° C. for 15 minutes, 50 mL of fetal bovine serum was added, and the culture medium was dispensed into sterile dishes to make plates.
[0034] The liquid culture medium preparation method adopted by the present invention is as follows: 30g of peptone soy broth TSB is dissolved in 950mL of deionized water, sterilized at 121°C for 15 minutes, 50mL of sterile fetal bovine serum is added, and stored at 4°C.
[0035] The chicken-derived Escherichia coli EC101 used in the present invention is separated, identified and preserved by the laboratory of the inventor's unit.
[0036] Example 1 Isolation and Identification of Strain
[0037] 1. Isolation and culture
[0038] The samples in this example were obtained from 21-day-old reserve hens suffering from pericarditis, perihepatitis, and air sac inflammation in a chicken farm in Jiangxi Province. The liver tissue of the diseased animal was cut and transported to the laboratory at 4°C. The liver tissue block was soaked in a 75% ethanol solution for 1 min; it was washed twice with 1.5 mL of sterile saline to remove the ethanol residue on the surface; the liver tissue block was grinded and spread on a TSA plate containing 5% fetal bovine serum, and cultured at a constant temperature of 37°C for 24 hours; a single colony plate was streaked for subculture, and the colony morphology was observed.
[0039] On TSA plates containing 5% fetal bovine serum, incubated at 37°C for 24 hours under aerobic conditions, the resulting colonies were 1 to 2 mm in diameter, off-white in color, with raised, smooth, and moist surfaces.
[0040] 2. Pathogen identification
[0041] Use a bacterial loop to scrape multiple purified colonies for Gram staining microscopy. Use a sterile toothpick to pick up a purified single colony and place it in an EP tube. After resuspending it with 0.5 mL of saline, apply it to a TSA plate containing 5% fetal bovine serum, and gently cover it with a cover glass on the plate agar. After culturing at 37°C for 24 hours, send the bacteria transferred to the cover glass for electron microscopy.
[0042] The results are as follows Figure 1 As shown in the figure, pink bacilli with deep staining at both poles can be seen under the oil microscope at 1000 times magnification, and Gram staining is negative; under the electron microscope with a resolution of 5 μm, as shown in the figure, Figure 2 As shown, densely arranged rod-shaped bacteria with swollen poles can be seen.
[0043] 3. Biological identification
[0044] A single colony after purification was picked and inoculated into TSB liquid culture medium containing 5% fetal bovine serum. After culturing at 37°C and 180 r / min for 24 h, 2 mL of bacterial solution was centrifuged at 5000 r / min for 5 min, the supernatant was removed, and the culture was frozen in liquid nitrogen for 2-4 h. It was then transported and stored on dry ice and sent to Guangzhou Kidio Biotechnology Co., Ltd. for whole genome sequencing analysis. The 16S rRNA sequence annotated in the sequencing file was subjected to BLAST homology comparison analysis.
[0045] The nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID NO: 1. The BLAST comparison analysis of the NCBI database found that the isolated bacteria had the highest homology with Riemerella columbina strain RCAD0985 (Sequence ID: MT804608.1): 99.21%. Combined with its colony morphology and molecular biological characteristics, the strain was determined to be Riemerella columbina, with an NCBI accession number of CP103950. The strain was identified and named Riemerella columbina RC TH22, and was deposited in the China Center for Type Culture Collection on July 11, 2022, with a deposit number of CCTCC NO: M 20221075, and the deposit address is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province.
[0046] Example 2 Animal regression experiment
[0047] The purified RC TH22 strain and chicken Escherichia coli EC101 were inoculated into TSB medium supplemented with 5% fetal bovine serum and cultured at 37°C and 180 r / min for 24 h. The culture solution was centrifuged at 5000 r / min for 5 min, the supernatant was removed, and the cells were resuspended in sterile saline to adjust the concentration of the culture solution to 1×10 9 CFU / mL. 60 7-day-old SPF chicks were randomly divided into three groups. Each chick in the experimental group (RC) was challenged with 1 mL of RC TH22 bacterial solution, each chick in the experimental group (EC) was challenged with 1 mL of chicken-derived Escherichia coli EC101 bacterial solution, and each chick in the control group (C) was injected with 1 mL of normal saline. All three groups were challenged with subcutaneous injection in the neck. After challenge, continuous observation and recording were performed for 7 days.
[0048] like Figure 3 As shown in Figure 2, 24 hours after the infection, both the EC and RC groups developed fibrinous pericarditis, fibrinous hepatitis, turbidity and thickening of the air sac wall, and increased exudate. The heart, liver, and air sac of the control group were normal. The statistical results of the survival rate are shown in Figure 2. Figure 4 As shown in the figure, compared with the control group, the EC group and the RC group both showed more serious mortality after the challenge. RC TH22 and EC101 could be identified from the livers of the dead chicks in the EC group and the RC group, respectively.
[0049] Example 3 Antibiotic sensitivity test of isolated bacteria
[0050] The drug sensitivity of bacteria was detected according to the Kirby-Bauer disc diffusion method and judgment criteria recommended by the American Clinical Laboratory Standards Institute (CLSI). The isolated bacteria RC TH22 identified in Example 1 was inoculated on a TSA plate containing 5% fetal bovine serum and cultured at 37°C for 24 hours; 3-5 single colonies were dipped with a sterile cotton swab, diluted in 1 mL of normal saline, and the concentration of the bacterial solution was adjusted to 0.5 McFarland turbidimetric; another sterile cotton swab was soaked in the bacterial suspension and evenly applied to the surface of the fresh TSA culture medium; the plate was dried at room temperature for 5 minutes, and the drug sensitivity sheet was taken with tweezers and pasted on the agar surface; within 15 minutes after the drug sensitivity sheet was pasted, the plate was inverted and placed in an incubator, and cultured at 37°C for 24 hours; the plate was taken out, the diameter of the inhibition zone was measured, and the result was judged to be limited to the bacteria that could not be seen by the naked eye at the edge of the inhibition zone, and reported as sensitive (S), intermediate (I), and resistant (R).
[0051] The results of antibiotic sensitivity analysis are shown in Table 1 below, which show that the isolate RC TH22 is sensitive to florfenicol, roxithromycin, neomycin, gentamicin, cefuroxime, and ceftazidime, moderately sensitive to streptomycin, and resistant to polymyxin B, enrofloxacin, lincomycin, spectinomycin, doxycycline, ciprofloxacin, amoxicillin, ampicillin, kanamycin, and cephradine. It can be seen that in the clinical treatment of pigeon Riemerella infection, florfenicol and roxithromycin are better choices for oral medication; neomycin, gentamicin, cefuroxime, and ceftazidime can bring good therapeutic effects when injected.
[0052] Table 1 Antibiotic sensitivity analysis results
[0053]
[0054]
[0055] Note: R means resistant; I means intermediately sensitive; S means sensitive.
[0056] Example 4 Immunoprotective Test
[0057] The isolated and purified RC TH22 strain was inoculated into TSB medium supplemented with 5% fetal bovine serum and cultured at 37°C and 180 r / min for 24 h. The culture solution was centrifuged at 5000 r / min for 5 min, the supernatant was removed, and the cells were resuspended to 3×10 8 CFU / mL, add formaldehyde to a final concentration of 0.4%, and inactivate at 37°C for 48 hours, stirring once every 4 hours.
[0058] The inactivated bacterial solution was spread on a TSA plate containing 5% fetal bovine serum and cultured at 37°C for 24 hours. The live bacteria test was performed to ensure that the inactivation was complete and no colonies grew on the plate. This shows that there is no biosafety hazard in vaccine immunization. Then aluminum hydroxide / magnesium hydroxide alum adjuvant (trade name: imject Alum, manufacturer: Thermo Scientific) was added and mixed thoroughly to make the final concentration of RC TH22 strain 2×10 8 CFU / mL, and the RC TH22 strain inactivated vaccine was obtained.
[0059] One-week-old chicks were randomly divided into two groups, with 20 chicks in each group. 0.5 mL of RC TH22 inactivated vaccine and saline were injected subcutaneously in the neck. 14 days after the first immunization, the second immunization was performed, and the immunization dose for the second immunization was 0.8 mL / bird. 15 days after the second immunization, the live bacteria of RC TH22 strain were used for the challenge, and the challenge dose was 2×10 7 CFU. The mortality of chicks was counted for 7 consecutive days after infection.
[0060] The results are as follows Figure 5 As shown, compared with the control group, the mortality rate of chickens in the vaccine immunization group was significantly reduced after challenge with RC TH22 strain, indicating that the prepared RC TH22 strain inactivated vaccine has good immune protection and provides an effective means for the prevention and control of polyserosal inflammation in chickens caused by Riemerella infection in pigeons.
[0061] In summary, the present invention isolated a strain of Riemerella columbina from the liver of a black-bone chicken suffering from severe pericarditis and perihepatitis for the first time, and confirmed for the first time that the pathogen can cause polyserosal inflammation in chicks, enriched the host source of Riemerella columbina, updated the understanding of the pathogenic microorganisms of avian polyserosal inflammation in the veterinary field, and provided good material for basic research on avian polyserosal inflammation. The present invention also tested a variety of antibiotics that are sensitive to the isolated bacteria through drug sensitivity tests, providing a medication guide for the treatment of polyserosal inflammation in chickens. An inactivated vaccine effective for pigeon Riemerella disease was successfully created, and an effective method for clinical prevention and control of polyserosal inflammation in chickens was explored, providing an effective means for the prevention and control of polyserosal inflammation in chickens caused by Riemerella columbina infection.
[0062] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A strain of Riemerella columbae from chicken ( Riemerella columbina ) RC TH22 strain, characterized in that The strain was deposited in the China Center for Type Culture Collection on July 11, 2022, with the deposit number: CCTCC NO:M20221075.
2. Use of the Riemerella columbae RC TH22 strain described in claim 1 in the preparation of a chicken polyserosal inflammation vaccine.
3. Use of the Riemerella columbae RC TH22 strain described in claim 1 in constructing an animal model of chicken polyserosal inflammation.
4. A chicken polyserosal inflammation vaccine, characterized in that: Contains the Riemerella pigeon RC TH22 bacteria as described in claim 1; the vaccine is an inactivated vaccine.
5. The vaccine according to claim 4, characterized in that The concentration of RC TH22 strain in the vaccine is not less than 2×10 8 CFU / mL.
6. A method for constructing an animal model of polyserosal inflammation in chickens infected with Riemerella pigeonii, characterized in that: The Riemerella pigeonii RC TH22 strain described in claim 1 is used to infect chicks to obtain an animal model of chicken polyserosal inflammation infected with Riemerella pigeonii.
7. The method according to claim 6, characterized in that The infection method is subcutaneous injection in the neck, and the injection concentration of RC TH22 strain is not less than 1×10 9 CFU / ml.
8. Application of the chicken polyserosal inflammation animal model obtained by the method of claim 6 or 7 in the screening of therapeutic drugs for chicken polyserosal inflammation and the evaluation of vaccine effects.
9. A method for screening a drug for treating chicken polyserosal inflammation, characterized in that: The method is to use the chicken polyserosal inflammation animal model obtained by any one of the methods described in claims 6 to 7 as a pathogenic model for screening.
Citation Information
Patent Citations
Riemerella anatipestifer infection attenuated live virus vaccine and preparation method thereof
CN107513510A