CN113278559A - Erizabeth milrina from rana nigromaculata and application thereof - Google Patents
Erizabeth milrina from rana nigromaculata and application thereof Download PDFInfo
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- CN113278559A CN113278559A CN202110656218.2A CN202110656218A CN113278559A CN 113278559 A CN113278559 A CN 113278559A CN 202110656218 A CN202110656218 A CN 202110656218A CN 113278559 A CN113278559 A CN 113278559A
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to an Erizabeth milpa sourced from Rana nigromaculata and application thereof. The invention separates and screens a strain of milrinia elizabeth from diseased frog brain and marrow tissues, and determines that the screened strain is the pathogenic bacteria of the rana nigromaculata white barrier disease through artificial infection of pathogenic bacteria. The application also further studies the sensitivity of the pathogenic bacteria to various antibiotics and plant extracts. The strain provided by the invention is a common disease of rana nigromaculata, namely 'wry head disease' and 'cataract' pathogen, and can be used for researching drug resistance and pathogenesis of the pathogen, or used as a strain material for tests such as vaccine development and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an Erizabeth milpa sourced from Rana nigromaculata and application thereof.
Background
In recent years, the rana nigromaculata breeding industry in China develops rapidly, but the rana nigromaculata has low immunity in a high-density mode, various diseases frequently occur and spread rapidly, and especially the 'head distortion disease' similar to meningitis. The disease has extremely strong infectivity and high mortality rate, and causes huge economic loss for the culture of rana nigromaculata. Researches show that the milelia elizakii is the main pathogen of the disease, the milelia elizakii (elizabethikingia miricola) is a gram-negative bacterium, and the cultured rana nigromaculata in many places such as Hunan province and Hubei province of China all erupts the 'head distortion disease' similar to meningitis since 2015. The disease has strong infectivity and high mortality, and the main symptoms are head distortion, cataract and loss of orientation ability, which causes huge economic loss for the culture of the rana nigromaculata.
The milrinia elizabetes is a conditionally pathogenic bacterium symbiotic in intestinal tracts of rana nigromaculata, can be widely spread among rana nigromaculata, and has extremely strong drug resistance, so that farmers can not recognize the pathogenic bacterium enough, and the phenomenon of indiscriminate medication in production is serious. Therefore, only by separating and identifying the pathogenic bacteria and carrying out sensitivity analysis on antibiotics, Chinese herbal medicines and the like, the proper medicine and reasonable concentration can be selected to prevent the disease.
In summary, the problems of the prior art are as follows:
at present, although the rana nigromaculata biogenetic phenomenon does not appear in the genus, the similarity of the rana nigromaculata biogenetic phenomenon with the milelizabethan which is a human source is high, the pathogenic mechanism and the like of the rana nigromaculata biogenetic phenomenon need to be emphasized, and a large number of strains are needed to support scientific research. The frog source milrinia elizabetha is studied later, the number of the frogs stored in each strain bank is small, the development of related scientific researches is not facilitated, for example, the research on drug sensitivity, pathogenic mechanism and the like is insufficient, so that the prevention means is lacked, and the human health and the healthy development of the frog breeding industry face important challenges.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an Erizabeth reevesii-derived rana nigromaculata and application thereof, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.
The invention is realized in such a way that a rana nigromaculata source Erizabeth, namely Erizabeth nigromaculata (Elizabethkingia miricola) JX27, has the preservation number: CCTCC NO: M2021473. The preservation unit: china center for type culture Collection, collection address: wuhan, Wuhan university, China; the preservation date is as follows: 2021, 4 and 27 months.
The invention also provides application of the rana nigromaculata-derived milrinia formosana strain in preparation of a reagent for inducing the wry head disease and/or the cataract of animals.
The invention also provides application of the rana nigromaculata-derived milrinia formosana strain in constructing an animal model of the wry head disease and/or cataract.
Further, the animal includes a frog.
Further, the frog comprises a Rana nigromaculata.
The invention also provides application of the antibiotic in preventing and treating milrinone and elizabeth, wherein the antibiotic comprises at least one of minocycline, doxycycline, cefoperazone, clindamycin, chloramphenicol, midecamycin, piperacillin, carbenicillin, erythromycin, vancomycin and ofloxacin.
The invention also provides application of the plant extract in preventing and treating the milfoil elizabeth, wherein the plant extract comprises at least one of cinnamon polyphenol, radix curcumae extract, astragalus polysaccharide, dandelion extract, codonopsis pilosula extract, polygonum multiflorum extract, honeysuckle extract, polygonatum polysaccharide, chrysanthemum extract, radix bupleuri extract, gingerol, rhizoma alismatis extract and angelica extract.
Further, the elizasa millerii is deposited under the accession number: CCTCC NO: M2021473, JX27 of Isizabalaceae (Elizabethikingia miricola).
The frog source Elizabeth JX27 separated by the invention can be used as a scientific research standard strain, provides research materials for researching drug sensitivity characteristics, pathogenic mechanisms and the like, and also provides research materials for developing vaccines and diagnostic methods thereof.
In summary, the advantages and positive effects of the invention are:
the strain of the invention, namely the milrinia elizakii (E.miricola) JX27, is uniformly turbid in an LB liquid culture medium, the diameter of a bacterial colony on an LB agar culture medium plate is 2.5-3.0mm, the bacterial colony is circular, beige, smooth and opaque in surface, glossy, slightly protruded in the middle, neat in edge, free of halo and soft in texture.
Through detection of the resistance of the Mierlissajou Mireyi (E.miricola) JX27 to 30 antibiotics, the bacterium is only sensitive to antibiotics No. 12, 13, 19, 21, 22 and 30, is moderately sensitive to 5 antibiotics and is resistant to other 19 antibiotics.
Through detecting the bacteriostatic effect of 38 plant extracts on the JX27 of the milrinia elizasa (E.miricola), 13 plants are found to have bacteriostatic effects, wherein the No. 7 effect has the largest bacteriostatic circle.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.
In the following examples of the present invention, the temperature is not particularly limited, and all of the conditions are normal temperature conditions. The normal temperature refers to the natural room temperature condition in four seasons, no additional cooling or heating treatment is carried out, and the normal temperature is generally controlled to be 10-30 ℃, preferably 15-25 ℃.
The genes, proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.
The invention discloses an Erizabeth milrina from Rana nigromaculata and application thereof, and the specific embodiment of the Erizabeth milrina is shown in the following examples.
Example 1 screening and identification of strains
1. Isolation of the Strain
1) Preparation of a culture medium:
LB liquid medium: dissolving 10g of peptone, 5g of yeast extract and 10g of NaCl in 1000mL of ultrapure water, sterilizing at 121 ℃ for 15min, pouring into a disposable bacterial culture dish, and storing at 4 ℃ for later use after solidification;
LB solid medium: 10g of peptone, 5g of yeast extract, 10g of NaCl and 20g of agar powder, dissolving the peptone in 1000mL of ultrapure water, sterilizing the peptone for 15min at 121 ℃, pouring the peptone into a disposable bacterial culture dish, and storing the peptone for later use at 4 ℃ after solidification;
2) and (3) isolated culture of the strain:
aseptically collecting diseased frog brain and marrow tissues, clamping by using forceps, then grinding and inoculating on an LB solid culture medium, and culturing at constant temperature of 30 ℃ for 24h to grow single colonies; respectively inoculating the grown single colonies to a new LB solid culture medium, culturing at constant temperature of 30 ℃ for 24h, and observing whether the colony forms are consistent; repeating the above operations again; after single colony culture, the culture was inoculated into LB liquid medium and cultured at 30 ℃ and 180rpm for 24 hours in a shaker.
2. Identification of the strains:
1) morphological characteristics of
The medium is uniformly turbid in an LB liquid medium, the diameter of a colony on an LB agar medium plate is 2.5-3.0mm, the colony is circular, beige, smooth and opaque in surface, glossy, slightly raised in the middle, neat in edge, free of halo and soft in texture.
2) 16S rDNA sequence analysis:
16S rDNA identification was performed on the isolated JX 27. The primer sequences were universal 16S rDNA primers 27f (SEQ ID NO. 1: 5 '-AGAGTTTGATCCTGGCTCAG-3') and 1492r (SEQ ID NO. 2: 5 '-GGTTACCTTGTTACGACTT-3'). Synthesized by Shanghai Co Ltd in bioengineering. PCR amplification was performed using total DNA of the strains obtained by screening as a template, and the amplification reaction system (50. mu.L) was: 10 XBuffer 5 uL, dNTP 4.0 uL, upstream and downstream primers 1.0 uL, taq enzyme 0.5 uL, template 1.0 uL, ddH2O37.5. mu.L. Reaction procedure: 5min at 95 ℃; 30s at 94 ℃, 30s at 54 ℃, 30s at 72 ℃ and 32 cycles; extension at 72 ℃ for 10 min. The target fragment is sent to Huahua big gene for sequencing, the sequencing result is compared and analyzed with 16S rDNA sequences (CP040516.1, MF800858.1, MK333252.1, MF375904.1 and the like) registered in GeneBank, and the homology reaches 100%. Strain JX27 was determined to be elizabeth (elizabethhkingia) by morphological characterization and 16S rDNA sequence analysis.
3) Analysis of gyrB sequence:
gyrB assay was performed on the isolated JX 27. The primer sequences are general gyrB primers UP1f (SEQ ID NO. 3: 5' -GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3 ') and UP2r (SEQ ID NO. 4: 5' -AGCAGGGTACGGATGTGCGA)GCCR-3'). Synthesized by Shanghai Co Ltd in bioengineering. PCR amplification was performed using total DNA of the strains obtained by screening as a template, and the amplification reaction system (50. mu.L) was: 10 XBuffer 5 uL, dNTP 4.0 uL, upstream and downstream primers 1.0 uL, taq enzyme 0.5 uL, template 1.0 uL, ddH2O37.5. mu.L. Reaction procedure: 5min at 95 ℃; 30s at 94 ℃, 30s at 54 ℃, 30s at 72 ℃ and 32 cycles; extension at 72 ℃ for 10 min. The target fragment is sent to Huahua big gene for sequencing, and the sequencing result is compared with 16S rDNA sequences (CP040516.1, AY468477.1, CP023746.1, LN995715.1 and the like) registered in GeneBank, and the homology reaches 100%. The strain was identified as elizasa milrina (e.miricola) by morphological characteristics and gyrB sequence analysis.
It was deposited under the name of Elizabeth Miricola (Elizabethikingia miricola) JX27, deposited in the following units: china center for type culture Collection, collection address: wuhan, Wuhan university, China; the preservation date is as follows: 2021, 4 month, 27 day, accession no: CCTCC NO: M2021473.
(3) Artificial infection with pathogenic bacteria
Separating the isolated bacterium JX27 at 1X 108The concentration of each/mL was challenged with 0.1mL intramuscular injection of healthy frogs, using sterile Phosphate Buffered Saline (PBS) pH7.2 as a control. The disease death condition is observed and recorded after 15 days of routine management, bacteria are separated again from infected frogs which produce typical cataract symptoms, and the homology of the separated bacteria and the milrinia elizabetha (E.miricola) reaches 100 percent through the identification of 16S rDNA and gyrB molecules, so that the pathogenic bacterium JX27 is confirmed to be the pathogenic bacterium of the rana nigromaculata cataract.
Example 2 drug susceptibility test of Elizabeth Mierfecti JX27
1. Study on antibiotic drug susceptibility
The drug sensitivity of pathogenic bacteria is measured by adopting a drug sensitive paper diffusion method: 0.1mL of overnight culture liquid is uniformly coated in an agar culture medium, 30 drug sensitive tablets are attached to the culture medium, 1 drug sensitive tablet is placed in each culture medium, the result is observed and recorded after 24 hours, and the experimental result is judged according to the size of the inhibition zone.
The sensitivity of the pathogenic bacteria JX27 to 30 antibacterial drugs is shown in Table 1 (because the bacteriostatic pictures are too many, the relevant experimental results are only provided in a table form, and no effect graph is provided any more). It can be seen from the table that among the 30 antibiotic drugs, there are only 6 types of sensitivity (minocycline, doxycycline, cefoperazone, clindamycin, chloramphenicol, and midecamycin, respectively), 5 types of moderate sensitivity (piperacillin, carbenicillin, erythromycin, vancomycin, and ofloxacin, respectively), and 19 types of resistance. Therefore, the effect of preventing and treating diseases may not be achieved at all because the drugs are not applied according to the drug sensitivity results in the actual production.
TABLE 1 antibacterial Activity of different antibacterial drugs against JX27
Note: s, sensitivity; i, moderate sensitivity; r, drug resistance
2. Plant extract sensitivity study
38 plant extracts (American ginseng saponin, curculigo orchioides extract, cinnamon polyphenol, curcuma aromatica extract, astragalus polysaccharides, dandelion extract, codonopsis pilosula extract, polygonum multiflorum extract, ginkgo biloba extract, honeysuckle extract, tribulus terrestris saponin, cnidium fruit extract, dried orange peel extract, ampelopsis japonica extract, aloe extract, polygonatum polysaccharides, chrysanthemum extract, bupleurum root extract, gingerol, alisma orientale extract, sennoside, prepared rhizome of rehmannia extract, glycyrrhizic acid, bighead atractylodes rhizome extract, asparagus extract, dried rehmannia root extract, ligusticum chuanxiong hort extract, artemisia leaf extract, silymarin, angelica sinensis extract, chicoric acid, bacopa monniera herb, gypenoside, lotus seed extract, dried ginger extract, eucommia bark extract, schisandra chinensis extract, salidroside; saint-bioscience company) were purchased, diluted with sterile water to 3 concentration gradients, the sensitivity of the plant extract of pathogenic bacteria is determined by a drug sensitive paper diffusion method. And uniformly coating 0.1mL of overnight culture solution in concentration into an agar culture medium, dripping 0.1mL of 38 plant extract solution with different concentrations onto a sterile filter paper sheet in the center of the culture medium, and observing and recording the size of a bacteriostatic circle after 24 h. The 13 plant extracts are found to have bacteriostatic effects on pathogenic bacteria JX27, wherein the codonopsis pilosula extract has the largest bacteriostatic circle, as shown in Table 2.
TABLE 2 plant extracts with antibacterial activity against JX27
The strain JX27 provided by the invention is a pathogen of Rana nigromaculata cataract and head strait disease, and can be used as a standard test strain for researchers.
The invention provides a drug sensitive result aiming at the strain JX27 antibiotic and the plant extract, which can provide a foundation for developing the preventive drugs for cataract and strait head.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> institute of livestock and veterinary sciences of agricultural science institute of Jiangxi province
<120> Erizabeth milbefaciens derived from rana nigromaculata and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agagtttgat cctggctcag 20
<210> 2
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggttaccttg ttacgactt 19
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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agcagggtac ggatgtgcga gccr 24
Claims (8)
1. An Erichloropectinatus miiliensis strain from Rana nigromaculata, which is called the Erichryopectinatus miricola (Elizabethkingia miricola) JX27 and has the preservation number: CCTCC NO: M2021473.
2. Use of an Erichloropectia miehei strain derived from Rana nigromaculata according to claim 1 for the preparation of an agent for inducing wry head and/or cataracts in an animal.
3. The use of an Erichrya nigromaculata-derived Miehelizasa strain of claim 1 in constructing an animal model of facial paralysis and/or cataract.
4. Use according to claim 2 or 3, characterized in that: the animal comprises a frog.
5. Use according to claim 4, characterized in that: the frog comprises a Rana nigromaculata.
6. The application of the antibiotic in preventing and treating the milelia mirabilis is characterized in that: the antibiotic comprises at least one of minocycline, doxycycline, cefoperazone, clindamycin, chloramphenicol, midecamycin, piperacillin, carbenicillin, erythromycin, vancomycin, and ofloxacin.
7. The application of the plant extract in preventing and treating the Elizabeth milrinia is characterized in that: the plant extract comprises at least one of cinnamon polyphenol, radix curcumae extract, astragalus polysaccharide, dandelion extract, codonopsis pilosula extract, polygonum multiflorum extract, honeysuckle extract, polygonatum polysaccharide, chrysanthemum extract, radix bupleuri extract, gingerol, rhizoma alismatis extract and angelica sinensis extract.
8. Use according to claim 6 or 7, characterized in that: the milelia elizasa is a preservation number: CCTCC NO: M2021473, JX27 of Isizabalaceae (Elizabethikingia miricola).
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