CN113234647B - Aeromonas salmonicida from turbot and application thereof - Google Patents
Aeromonas salmonicida from turbot and application thereof Download PDFInfo
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Abstract
The invention discloses an aeromonas salmonicida BHAS-1 from turbot and application thereof, wherein the aeromonas salmonicida BHAS-1 is an aeromonas salmonicida subspecies, is separated from viscera of the turbot with red mouth, fin congestion and anus redness and swelling and evagination symptoms, carries 14 virulence genes, is obviously different from reported aeromonas salmonicida from different sources, is stored in a China typical culture storage center at 03 month and 24 days in 2021, and has the storage addresses of: the preservation number of the Wuhan in China is CCTCC NO: M2021264, and the classification is named as Aeromonas salmonicida BHAS-1Aeromonas salmonicidaBHAS-1. The aeromonas salmonicida BHAS-1 is used for preparing vaccines, can effectively prevent and control the turbot aeromonas disease, reduces the abuse of antibiotic drugs and ensures the quality safety of aquatic products.
Description
Technical Field
The invention relates to a bacterium and application thereof, in particular to an aeromonas salmonicida from turbot and application thereof, belonging to the technical field of pathogenic microorganism screening.
Background
Since the introduction of Scophthalmus in Scophthalmus of Pleuroideae of Scophthalmus in 1990, and the breakthrough of artificial breeding technique, the scale of cultivation is continuously increased, the formation of the fourth industrialized wave of marine cultivation in China is successfully promoted, and the Scophthalmus is an important kind of industrial cultivation in North China. However, with the development of high-density intensive culture mode, serious bacterial diseases appear in the culture process, which not only causes economic loss to culture units, but also more seriously restricts the green and healthy development of aquaculture industry. Generally speaking, the use of antibiotic fishery drugs can achieve good prevention and treatment effects on bacterial diseases, but frequent use of antibiotic fishery drugs not only can cause food safety hidden dangers, but also can cause pathogenic bacteria to gradually generate drug resistance or even multiple drug resistance, and the prevention and control difficulty is increased.
Scabies is a common and common bacterial disease of salmon and trout, and the pathogen is aeromonas salmonicida. Aeromonas salmonicida is a psychrophilic unpowered Aeromonas sobria, is widely distributed in seawater, and has been reported to infect fishes such as bass, cyprinidae and sole, besides salmon and trout. Aeromonas salmonicida (Aeromonas salmonicida) Can be divided into Salmon subspecies (A. salmonicida subsp. salmonicida) Colorless subspecies (A)A. salmonicida subsp. achromogenes) And Salmon subspecies Japan killing (A. salmonicida subsp. masoucida) Stachy subspecies (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B)A. salmonicida subsp.smithia) And pectolytic subspecies: (A. salmonicida subsp. pectinolytica) Different subspecies infect the object, the pathogenicity is slightly different. Generally, the pathogenicity of a pathogenic strain can be preliminarily judged by detecting the carrying condition of virulence genes of the pathogenic strain, the types and the quantity of virulence factors are different among different strains, and the comprehensive detection of the carrying condition of the virulence factors of the strain is an important means for judging the pathogenicity of the strain and establishing effective prevention and control measures.
The use of the vaccine can improve the specific immunity level of the fish, so that the fish body can generate immunity against the infection of specific pathogenic microorganisms, and the vaccine has no pollution and residue after injection, and is the main research direction for preventing and controlling fish diseases. However, in vaccine preparation, strains from different isolated sources have different protective ranges. Therefore, the development of a vaccine aiming at the multi-virulence factor carrying strain of the aeromonas salmonicida from the turbot has important significance.
Disclosure of Invention
The invention aims to provide an aeromonas salmonicida from turbot and application thereof.
In order to achieve the above object, the present invention adopts the following technical solutions:
an Aeromonas salmonicida BHAS-1 derived from Scophthalmus maximus is characterized in that the Aeromonas salmonicida BHAS-1 is Aeromonas salmonicida Salmonella salmonicida subspecies (b)Aeromonas salmonicida subsp. salmonicida) 14 virulence genes including aerosol gene, closed-banded toxin gene, DNase gene, serine protease gene, lipase gene, S-layer protein gene, heat-labile cell-exciting enterotoxin gene, cytotoxic enterotoxin gene, heat-stable cell-exciting enterotoxin gene, extracellular protease gene, flagellin gene, elastase gene, hemolysin gene and glycerophosphocholesterol acyltransferase gene are separated from the internal organs of turbot with red mouth, fin congestion and anus bulge symptoms, and the aeromonas salmonicida BHAS-1 is preserved in China center for type culture preservation, the preservation address is China Han, and the classification is named as aeromonas salmonicida BHAS-1Aeromonas salmonicidaBHAS-1, with a preservation date of: 24/03/2021, accession number: CCTCC NO: M2021264.
The application of the aeromonas salmonicida BHAS-1 from the turbot in preparing the vaccine is characterized in that the vaccine is an inactivated vaccine, is applied to vaccination of turbot cultivation, and is used for preventing bacterial diseases which take red mouth, fin congestion and anal swelling and evagination as main symptoms and are caused by the aeromonas salmonicida.
The invention has the advantages that: the invention discloses Aeromonas salmonicida BHAS-1, which is an Aeromonas salmonicida subspecies obtained by screening diseased turbot, the strain carries aerosol genes, closed-band toxin genes, DNA enzyme genes, serine protease genes, lipase genes, S-layer protein genes, heat-labile cell excitatory enterotoxin genes, cytotoxic enterotoxin genes, heat-stable cell excitatory enterotoxin genes, extracellular protease genes, flagellin genes, elastase genes, hemolysin genes and glycero-phosphate cholesterol acyltransferase genes, total 14 virulence genes are 14 virulence genes which are the first reported Aeromonas salmonicida from turbot sources carrying 14 virulence genes in China and are obviously different from reported Aeromonas salmonicida from different sources, the Aeromonas salmonicida BHAS-1 is used for preparing vaccines, can effectively prevent and control the Aeromonas salmonicida, reducing the abuse of antibiotic drugs and ensuring the quality safety of aquatic products.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
Strain separation, identification and virulence gene detection
1. Strain isolation
The diseased turbot sample is collected from a turbot farm on a cigarette bench in Shandong, and the individual mass is about 350 g. The manifestation of the diseased turbot is as follows: anorexia even without food intake, dislike of exercise, body color blackened, red mouth, redness of fin rays, and redness, swelling and swelling of anus. The autopsy finds that: liver congestion, swelling and congestion of the intestine or accumulation of fluid in the intestine, and swollen kidney with milky pus-like liquid.
Taking an individual endangered by a typical symptom of suffering turbot, carrying out aseptic dissection on internal organs, taking a liver, a kidney and a spleen, washing with aseptic water, grinding, streaking on a nutrient agar plate added with 2wt% of NaCl, carrying out inverted culture at 28 ℃ for 48h, and selecting dominant bacterial colonies with consistent forms for purification culture until a pure culture is obtained. Subjecting the pure culture to amplification culture on nutrient agar plate containing 2wt% NaCl, eluting bacterial colony with 0.9wt% sterile physiological saline to obtain bacterial suspension, and determining the strain concentration to 7.8 × 10 by turbidimetry7 CFU/mL。
Selecting healthy turbot with similar specification to diseased turbot as experimental fish, and artificially infecting (injecting the bacterial suspension into abdominal cavity). After temporarily rearing the experimental fish for one week, the experimental fish is randomly divided into 4 groups of 10 fish tails, wherein 3 groups are injected with 0.1mL of the bacterial suspension in a gradient manner, and 1 group is injected with 0.1mL of sterile normal saline. The water temperature of the culture is 18 +/-1 ℃, air is filled all day long, water is changed by 30 percent every day, the death number and disease symptoms are observed and recorded every day, and pathogenic bacteria are separated again from diseased individuals.
The infection result shows that the strain BHAS-1 shows stronger pathogenicity, can cause more than 90 percent of death of turbot in the experiment, and the death individual has clinical symptoms consistent with the diseased turbot. The strain BHAS-1 is proved to be a pathogen causing red mouth, redness and redness of fin ray and swelling and evagination of anus, and the strain can be isolated from an infected individual again.
2. Identification of strains
Use of bacteria 16SrDNAThe universal primers (27F, 1492R) are used for carrying out PCR amplification on the strain BHAS-1 to obtain a product with the length of 1452 bp, and the gene sequence of the product is shown in SEQ ID No. 1.
Use of Aeromonas salmonicida housekeeping genesgyrBThe specific primer (F, R) is used for carrying out PCR amplification on the strain BHAS-1 to obtain a product with the length of 1073 bp, and the gene sequence of the product is shown in SEQ ID No. 2.
| 27F | 5＇-AGAGTTTGATCMTGGCTCAG-3＇ |
| 1492R | 5＇-GGTTACCTTGTTACGACTT-3＇ |
| F | 5＇-TCCGGCGGTCTGCACGGCGT-3＇ |
| R | 5＇-TTGTCCGGGTTGTACTCGTC-3＇ |
Through NCBI online BLAST comparison, MEGA 7.0 constructs a phylogenetic tree, and the result shows that: the strain BHAS-1 is Aeromonas salmonicida subspecies (Aeromonas salmonicida subsp. salmonicida）。
3. Strain virulence gene detection
Carrying out virulence gene detection on the strain BHAS-1. The detection result shows that the strain BHAS-1 carries: aerolysin gene (aer) Blocking band toxin groupDue to (zat) DNA enzyme gene (c)DNA) Serine protease gene (A), (B), (C)ser) Lipase gene (a)lip) S layer protein Gene: (sp) Heat-labile cell-exciting enterotoxin gene (a)alt) Cytotoxic enterotoxin gene (A)act) Heat stable cell excitable enterotoxin gene (a)ast) Extracellular protease gene (a)epa) Flagellin gene (a)fla) Elastase gene (a)ahyB) Hemolysin gene (b)hly) And glycerol phosphate cholesterol acyltransferase gene (gcat) There are 14 virulence genes in total.
This indicates that: the strain BHAS-1 is obviously different from the reported aeromonas salmonicida from different sources.
Secondly, preparing the inactivated vaccine
Activating purified Aeromonas salmonicida BHAS-1 stored in a refrigerator at-80 ℃ for later use, inoculating the activated Aeromonas salmonicida BHAS-1 into a nutrient agar plate added with 2wt% NaCl, and culturing at 28 ℃ for 48 h; picking single colony of exponential growth phase in nutrient broth, shake culturing at 28 deg.C for 24h, collecting culture solution, centrifuging the culture solution at 4 deg.C for 10 min at 5000 rpm, washing the centrifuged deposited bacteria with PBS buffer solution with pH =7.2 for 3 times, and preparing into 10% concentration with sterile physiological saline9 CFU/mL of a stock solution of the bacterial suspension; adding a formalin solution with the mass concentration of 37% into the bacterial suspension stock solution until the final concentration of formaldehyde is 0.2wt%, inactivating at 37 ℃ and 140 rpm, and detecting the inactivation condition by respectively taking the bacterial solutions which are inactivated for 12 h and 24 h. Centrifuging the inactivated bacteria solution at 4 deg.C and 5000 rpm for 10 min, washing the centrifuged deposited bacteria with PBS buffer solution with pH =7.2 for 3 times, and preparing into 10% concentration with sterile physiological saline9 CFU/mL aeromonas salmonicida BHAS-1 inactivated bacterial suspension; the inactivated vaccine is prepared by uniformly mixing Aeromonas salmonicida BHAS-1 inactivated bacteria suspension, cecropin (North China pharmaceutical division Co., Ltd.) and Freund's incomplete adjuvant (SIGMA Co., Ltd.) according to the volume ratio of 2:1: 3.
Thirdly, detecting the immune effect of the inactivated vaccine
Adjusting the concentration of the inactivated vaccine to OD600=0.1 +/-0.01, intraperitoneal injection immunization0.1mL of turbot is injected into each tail, and the sterile normal saline with the same dosage is injected into a control group. 20 animals were immunized per group, and 3 replicates, 1 control, were set. The water temperature of the cultivation is 18 +/-1 ℃, air is filled all day long, and the water is changed by 30% every day; 30 days after immunization, the vaccine is detoxified by Aeromonas salmonicida BHAS-1 and Aeromonas salmonicida RB0506 (see patent document CN110055194A, stored in China center for type culture Collection (CCTCC NO: 2018849) in 12 months and 03 days in 2018, and the injection concentration of each tail is 5.8 multiplied by 10100.1mL of CFU/mL bacterial liquid; the death number of each experimental group is observed and recorded every day, and the immune protection rate of the inactivated vaccine to the aeromonas salmonicida is calculated.
The results of the calculations are shown in the following table.
Results of an immunoprotection test using a vaccine prepared from two Aeromonas salmonicida
| BHAS-1 vaccines | RB0506 vaccine | Physiological saline control group | |
| Aeromonas salmonicida BHAS-1 attacking toxin | 56/60 | 45/60 | 5/60 |
| Aeromonas salmonicida RB0506 challenge | 53/60 | 51/60 | 8/60 |
Note: indicates survival/challenge
The results show that:
(1) the aeromonas salmonicida BHAS-1 injected with the physiological saline solution starts to die in 3 days after challenge, a death peak appears in 5-8 days, and the death rate can reach as high as 91.67% in 10 days after challenge; after the challenge, one or more typical symptoms of red mouth, fin congestion, anus swelling and evagination, liver congestion, intestinal water filling, spleen swelling and necrosis and the like appear in the fish body, are similar to the body surface symptoms and the Caesarean lesion of the naturally infected fish with the hair dyeing disease, and can be separated from the infected dead individual to kill aeromonas salmonicida BHAS-1 again.
(2) The mortality rate of the 2 vaccine immune groups is obviously lower than that of the control group, the mortality rate of the inactivated vaccine immune group prepared by using the Aeromonas salmonicida BHAS-1 in the virus attack of the Aeromonas salmonicida BHAS-1 is obviously lower than that of the inactivated vaccine immune group prepared by using the Aeromonas salmonicida RB0506, namely, the immune protection rate of the former is obviously higher than that of the latter.
Therefore, the inactivated vaccine prepared based on the Aeromonas salmonicida BHAS-1 obtained by screening has higher immune protection rate, and can provide effective immune protection for diseases caused by the Aeromonas salmonicida.
It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.
Sequence listing
<110> ocean economic research institute of cigarette terrace city (Fishery technology promotion station of cigarette terrace city, ocean fishing and proliferation management station of cigarette terrace city)
<120> Scophthalmus maximus-derived aeromonas salmonicida and application thereof
<141> 2021-06-26
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1166
<212> RNA
<213> Aeromonas salmonicida (2 Ambystoma laterale x Ambystoma jeffersonia)
<400> 1
ggcaggccgg cagcacacag caagcgagcg gcagcgggaa agagcgcacg ccggcgagcg 60
gcggacgggg agaagccggg gacgcccagc gagggggaaa cagggaaacg acgcaaaccg 120
caacgcccac gggggaaagg aggggacccg ggcccgcgag gagaacccag ggggaagcag 180
ggggggaagg ccaccaaggc gacgacccag cggcgagagg agacagccac acggaacgag 240
acacggccag acccacggga ggcagcaggg ggaaagcaca agggggaaac ccgagcagcc 300
agccgcgggg aagaaggccc ggggaaagca ccagcgagga ggaaaggggc gccaaacggc 360
aacggacgac cgcagaagaa gcaccggcaa cccggccagc agccgcggaa acggaggggc 420
aagcgaacgg aaacgggcga aagcgcacgc aggcggggaa agagaggaaa gccccgggcc 480
aaccgggaag caaaaacgcc agcagagcga gaggggggag aaccagggag cgggaaagcg 540
agagacggag gaaaccgggg cgaaggcggc ccccggacaa agacgacgcc agggcgaaag 600
cgggggagca aacaggaaga acccggagcc acgccgaaac gagcgaggag gcggccgaga 660
cgggcccgga gcaacgcgaa acgaccgccg gggagacggc cgcaaggaaa accaaagaag 720
acgggggccc gcacaagcgg ggagcaggga acgagcaacg cgaagaacca ccggccgaca 780
gcggaaccgc agagagcggg aggcccggga acagaacaca gggcgcaggc gcgcagccgg 840
cggagagggg aagcccgcaa cgagcgcaac cccgccggcc agcacgaagg gggaaccaag 900
ggagacgccg ggaaaaccgg aggaaggggg gagacgcaag cacaggccca cggccagggc 960
acacacggca caaggcgcga cagagggcgc aagcagcgaa ggagcgaacc caaaaagcgc 1020
gcgagccgga cggagcgcaa ccgacccgga agcggaacgc agaacgcgaa cagaagcgcg 1080
ggaaacgccc gggccgacac accgcccgca caccagggag ggggcaccag aagagaagca 1140
acccgggagg gcgaccacgg gaagcc 1166
<210> 2
<211> 878
<212> RNA
<213> Aeromonas salmonicida (2 Ambystoma laterale x Ambystoma jeffersonia)
<400> 2
ggcccacgcc ccgacagcgg cgaccaacgc cgcaaggcca gcacgagcag accacaccgg 60
ggagccgcag gcgccaccaa gcagaggcga cagcagcagc ggcacgggac cgaaggcgcc 120
ggccgagccc gaccacaccg aacccgacac acgagaccgg ccaagcgcgc gcgagccccc 180
ccaacccggg cccaccgcgc aagagagcgg aggccgcgag ggcacgcaga gggcggacaa 240
ggcgcggaaa ccgaaccaga acaagacccc gaccacccaa gggccacccc acagagcagg 300
acggcacggg cgaagcgcca gcagggaacg agccacagga aggggacgcc accaacaaca 360
cccgcagcgg gaggcggacc accgggggcc gacgcgcgac ccgaccccaa caccaaggac 420
aaagaggaac agcaagaagg ccaagcggcc gccagcggcg acgaggcgga aggcgagcgg 480
caccggcaag gaccggaccc aagcccccca gaccaaggac aaacggcccc gaaggaagac 540
cgccggaaca ggcgagggga gaagccggga gccgcggaaa acccgggcga gccaagaagg 600
gcaacaagac acgagcggcc cggcccggaa gcggcccgca aggcccgcga gcgaccgccg 660
caaggggcgc ggaacgccgg cgcccggcaa gcggcagacg ccaggagaaa gacccggccc 720
cccgaaccac aagggaaggg gaccgcggcg gccgccaagc agggcgcaac cggaagaacc 780
aggccaacgc cgccaagggc aagaccgaac gagagaaggc ccgcgacaag agaccccgca 840
agagggggca cccgacacag acccggggcc aaggccgc 878
Claims (4)
1. Aeromonas salmonicida from turbotWherein the Aeromonas salmonicida BHAS-1 is Aeromonas salmonicida Salmonella subspecies (Aeromonas salmonicida subsp. salmonicida) The Aeromonas salmonicida BHAS-1 is preserved in China center for type culture Collection, the preservation address is Wuhan in China, and the preservation date is as follows: 24/03/2021, accession number: CCTCC NO: M2021264, and is named as Aeromonas salmonicida BHAS-1 by classification.
2. Use of Aeromonas salmonicida derived from turbot in the preparation of a vaccine according to claim 1, wherein the vaccine is used for vaccination in the breeding of turbot.
3. The use according to claim 2, wherein the vaccine is an inactivated vaccine.
4. Use according to claim 2, wherein the vaccine is for use in the prevention of bacterial disease caused by aeromonas salmonicida.
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| CN104560851A (en) * | 2014-12-29 | 2015-04-29 | 江苏海健前沿生物科技有限公司 | Aeromonas salmonicida live vaccine preparation as well as preparation method and application of lyophilized vaccine product |
| CN109999192A (en) * | 2019-04-23 | 2019-07-12 | 山东省海洋生物研究院 | A kind of aeromonas salmonicida inactivated vaccine prepared using Nano-Zinc |
| CN110055194A (en) * | 2019-04-23 | 2019-07-26 | 山东省海洋生物研究院 | A kind of aeromonas salmonicida in cold water rainbow trout source |
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| CN108939062A (en) * | 2018-05-04 | 2018-12-07 | 中国水产科学研究院黄海水产研究所 | A kind of aeromonas salmonicida vaccine and its application |
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| CN104560851A (en) * | 2014-12-29 | 2015-04-29 | 江苏海健前沿生物科技有限公司 | Aeromonas salmonicida live vaccine preparation as well as preparation method and application of lyophilized vaccine product |
| CN109999192A (en) * | 2019-04-23 | 2019-07-12 | 山东省海洋生物研究院 | A kind of aeromonas salmonicida inactivated vaccine prepared using Nano-Zinc |
| CN110055194A (en) * | 2019-04-23 | 2019-07-26 | 山东省海洋生物研究院 | A kind of aeromonas salmonicida in cold water rainbow trout source |
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