CN103992990A - Vibrio cyclitrophicus phage and application thereof in prevention of diseases of holothurioidea - Google Patents
Vibrio cyclitrophicus phage and application thereof in prevention of diseases of holothurioidea Download PDFInfo
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- CN103992990A CN103992990A CN201410232545.5A CN201410232545A CN103992990A CN 103992990 A CN103992990 A CN 103992990A CN 201410232545 A CN201410232545 A CN 201410232545A CN 103992990 A CN103992990 A CN 103992990A
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Abstract
The invention belongs to the technical field of biology and relates to a vibrio cyclitrophicus phage and an application thereof in prevention of diseases of holothurioidea. The vibrio cyclitrophicus phage was collected in China General Microbiological Culture Collection Center on May 9, 2014, with the collection number of CGMCC No. 9101 and the category name of vibrio cyclitrophicus phage. Vibrio cyclitrophicus phage liquid is used for immersion bath of stichopus japonicus in disease outbreak period or the vibrio cyclitrophicus phage liquid after dilution is used for spraying in a holothurioidea pool, immersion bath or spraying is performed once every 5 days and continuously performed for 3 times and the application quantity every time is not less than 10<7>pfu/m<3> of aquaculture water body. The phage widely exists in nature as a natural antimicrobial microorganism, artificial screening, enrichment and use of a phage preparation can replace the use of traditional chemical medicines and antibiotics and the vibrio cyclitrophicus phage has the characteristics of no public hazard, no residues, low price, high efficiency, antibacterial effect and the like and is a natural bactericide with broad application prospect and huge potential.
Description
Technical scheme
The invention belongs to biological technical field, relating to a strain can specificity cracking have a liking for phage isolate and the application in sea cucumber disease prevention area thereof of ring vibrios (Vibrio cyclitrophicus) bacterial strain, thereby improves the economic benefit of holothruian cultures enterprise.
Background technology
Sea cucumber (Holothurioidea) is one of marine cultured animal of Northern Coastal Region of China area output value maximum.According to statistics, China's holothruian cultures area is nearly 90,000 hectares at present, nearly 100,000 tons of output, and more than 100 hundred million yuan of annual value of production (Chinese sea cucumber academic research and industry development forum, 2009), and in 2012, national annual value of production broke through 30,000,000,000.Yet along with the rapid increase of apostichopus japonicus culture scale and cultivation density, aquaculture water runs down in recent years, disease problem is also on the rise.Have a liking for a kind of bacterium that ring vibrios (Vibrio cyclitrophicus) Shi You Dalian University of Technology Life Science and Technology institute Animal Biotechnology and nutrition laboratory were found in ill sea cucumber in 2011, can cause the skin ulceration of sea cucumber childhood, together with other stichopus japonicus putrid skin disease pathogenic bacterium, seriously limit the sustainable and healthy development of holothruian cultures industry.
At present, the normal antibiotics that adopts is treated by bacterial stichopus japonicus putrid skin disease, result will cause the appearance of a large amount of Resistant strain, and destroy the balance of the micro-ecology of breeding environment, holothruian cultures are caused to further harm, therefore need a kind of novel antibacterial preparation to be used for preventing the generation of sea cucumber disease.
Phage is a kind of virus that can bacterial infection.Lytic phage (hereinafter to be referred as phage), after bacterial infection, can cause that host cell breaks, and utilizes this characteristic phage can be applied to antibacterial therapy.The 70-80 age in last century, the appearance of bacterial drug resistance problem, particularly superbacteria, application microbiotic carries out antibacterial therapy faces enormous challenge, and relevant phage is subject to common concern as the research of antibiotic preparation.At present, in the U.S. and some East European countries, phage has successfully been applied in animal food production process, control Salmonellas, Escherichia coli O 157: the control of the bacterial infections such as H7 listeria bacteria, streptococcus aureus, and have some products to emerge, aspect control animal bacteria disease, showing advantage.Therefore, aspect the prevention and control of aquatic animal disease, also in the urgent need to treating by phage.In recent years, research discovery Phage therapy can effectively be prevented and treated bacteriosis of aquatic livestock.The haliotis discus hannai Ino pustulosis that phage control Yellowtail fish Lactococcus garvieae (Lactococcus garvieae) infects as utilized, sweetfish cold water is sick, distortion pseudomonas (Pseudomonas plecoglossicida) infects, prawn Vibrio harveyi (Vibrio harveyi) disease and vibrio fluvialis (Vibrio fluvialis) cause etc.
Phage isolate in the present invention is the virulent phage from occurring in nature separation, through mouse safety experiment prove its safely, have no side effect, and to have a liking for ring vibrios there is strong splitting action.After this phage is purified, be expected to be developed to for biological bactericide prevention and control are by having a liking for the microbial sea cucumber disease of ring arc.
Summary of the invention
The invention provides in a kind of application lytic phage prevention holothruian cultures and encircle the microbial disease of arc by having a liking for, reduce the mortality ratio being caused by this disease, can improve the technological method of holothruian cultures Business Economic Benefit.
Technical scheme of the present invention is to provide a kind of ring vibrios phage (Vibrio cyclitrophicus) of having a liking for, this phage is preserved on April 16th, 2014 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; preserving number CGMCC No.9101, Classification And Nomenclature is for having a liking for ring vibrios phage.China Committee for Culture Collection of Microorganisms's common micro-organisms centre address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101.This has a liking for ring vibrios phage (Vibrio Phage Vc1) for Caudoviradles, Stylovinidae, and head capsid diameter is 42 ± 3nm, afterbody long 146 ± 10nm, wide 8nm; One step growth shows about 35min in the latent period of this phage-infect Host Strains, the about 218PFU/ cells infected of burst size, and cracking performance is stronger.
Pnagus medius of the present invention is through mouse safety experiment, and abdominal injection gives phage experimental group 10
9the phagocytosis body fluid of pfu/kg, control group gives equivalent physiological saline, gives continuously after 20 days, the de-neck of mouse is put to death, postmortem, pathology symptom is not all found in its lung, liver, kidney, abdominal cavity, the security of this phage preparation of preliminary identification.Have a liking for the application of ring vibrios phage in sea cucumber disease prevention, with this, have a liking for ring vibrios phagocytosis body fluid the stichopus japonicus of illness outbreak phase is carried out to dipping bath maybe by after dilution, this is had a liking for ring vibrios phagocytosis body fluid and joins pond sprinkling, every 5 days dipping baths or spray 1 time, carry out continuously 3 times, use consumption>=10 at every turn
7pfu/m
3aquaculture water, to reach the effect for the treatment of or preventing corresponding disease.
Beneficial effect of the present invention:
1. sea cucumber putrid skin disease is bacteriosis serious in holothruian cultures process, after application of bacteriophage, can prevent and treat by the microbial sea cucumber putrid skin disease of respective fine, improves sea cucumber surviving rate, reduces financial loss.
2. use phage to control and have a liking for ring vibrios, substitute antibiotics control sea cucumber putrid skin disease, can reduce the usage quantity of microbiotic in holothruian cultures process, reduces the occurrence probability of Resistant strain, improves the action effect of antibiotic therapy other diseases.
3. phage is extensively present in occurring in nature as natural antibacterial microorganism, artificial screening, enrichment are also used phage preparation can replace traditional chemical drug, antibiotic use, have nuisanceless, noresidue, the features such as cheapness, high-efficiency antimicrobial are a kind of natural bactericidal agents that has a extensive future, has a high potential.
Accompanying drawing explanation
Fig. 1 is transmission electron microscope observing phage Vibrio Phage Vc1 form.
Fig. 2 is the one step growth of phage Vibrio Phage Vc1.
Embodiment
Below in conjunction with accompanying drawing and technical scheme, illustrate the specific embodiment of the present invention.
Embodiment 1
The screening of phage and purifying
(1) water sampling and processing
(1) preparation of Host Strains
Vibrio alginolyticus is inoculated in 2216E solid medium after bacteriology is identified, is stored in 4 ℃ of refrigerators.Screening is with Host Strains through 28 ℃ of incubated overnight, and the single bacterium colony of picking, is inoculated in 2216E liquid nutrient medium, after 28 ℃ of shaking culture 8h, is used for separated phage to use.
(2) processing of water sample
Get and come from fish market, apostichopus japonicus culture Chang Ji Daliang City, Dalian each 200mL of sewage draining exit wastewater sample, water sample is done to pre-treatment: add CaCl2, MgCl
2, making its final concentration is 1mmol/L, acts on about 10min.
(2) enrichment of water sample phage
By the pretreated water sample of above-mentioned process, the centrifugal 5min of 10000g, collects supernatant; Supernatant is crossed to 0.22 μ m filter membrane, except other compositions such as thalline, impurity, obtain phage stoste; Get 10mL filtrate and add that (about 5h after inoculation, phage final concentration reaches 10 in logarithmic phase
6-10
7during CFU/mL, i.e. logarithmic phase) in 50mL bacterium liquid, 28 ℃ of incubated overnight 12-18h, object is to make the amplification of phage quantity, observes and record; Collect bacterium liquid, the centrifugal 5min of 10000g, collects supernatant, after filter membrane (0.22 μ m), obtains the phage supernatant liquor after propagation.
(3) screening of phage
Adopt double-layer plate method to carry out the evaluation of phage, lower floor is the 2216E solid medium of 1.5% agar, be placed in 4 ℃ standby, with being front put in 28 ℃, about 30min; Upper strata is 0.5% agar 2216E substratum, by upper strata substratum heating for dissolving, is positioned in the water-bath of 50 ℃ stand-byly, gets 8 10mL centrifuge tubes, adds respectively 1mL Host Strains liquid, draws phage suspension liquid and carry out gradient dilution to 10
-6the phage diluent of 7 kinds of different concns gradients is taken out respectively to 10 μ L, be added in 10mL centrifuge tube, a control group is set, only adds 1mL Host Strains liquid and 10 μ L2216E substratum, at 28 ℃, act on 10min, after the 2216E substratum that adds 5mL to contain 0.5% agar fully mixes, be added at once upper strata, after solidifying, after 24h is cultivated in inversion, observation has or not Plaques assay.If form plaque on flat board, illustrate that the lytic phage having for this Host Strains exists in filtrate.
(4) purifying of phage
Size, the shape of first separated plaque are inconsistent, to the further purifying of separated phage, make phage on flat board, form size and the consistent plaque of form.With aseptic 200 μ L rifle choicests, get each of the plaque that form size has notable difference, it is joined respectively in the centrifuge tube of the aseptic PBS of 1mL, vortex concussion 1min, is placed in 4 ℃, and 4h, makes phage fully be released in PBS; 4 ℃, the centrifugal 5min of 10000g, collect supernatant, crosses film (0.22 μ m), phage considered to liquid and carry out gradient dilution, and Double layer culture, repeats 3 times aforesaid operations, until the plaque form occurring, big or small in full accord obtains purifying phage.Phage electromicroscopic photograph is shown in accompanying drawing 1.
(5) enrichment of phage and amplification
Adopt liquid method of proliferating to breed.Concrete method is as follows: phagocytosis body fluid is joined by a certain percentage in the Host Strains liquid of cultivating 12h, in 28 ℃ of shaking tables, cultivate 12h again, by mixed solution in 4 ℃, the centrifugal 5min of 10000g, place to go bacterial debris, the membrane filtration of 0.22 μ m for supernatant liquor, can obtain the phage pregnant solution of high-titer.
Embodiment 2
Phage one step growth is measured
Phage-infect plural number (multiplicity of infection, MOI) adds the ratio of phage and Host Strains quantity while referring to primary infection.In Host Strains liquid, adding infection multiplicity is 0.1 phage, mixes, and 28 ℃ standing, absorption 15min; 4 ℃ of centrifugal 10min of 12000rpm, abandon supernatant, and precipitation is resuspended with 2216E liquid nutrient medium, repeats above-mentioned centrifugally operated, eliminate the not phage of absorption.Precipitation is resuspended in 100mL2216E liquid nutrient medium, and now timing is T
0=0; 28 ℃ of 120rpm cultivate, every 10min sampling and measuring phage titer.
Result shows, the about 35min in latent period of this phage-infect Host Strains, and the about 218PFU/cell of burst size, cracking performance is stronger, the results are shown in accompanying drawing 2.
Embodiment 3
Phage host range is analyzed
The cracking ability of the phage of investigation screening to different bacterium, the host range of structure phage.The bacterium of selecting comprises:
Separated pathogenic bacteria is had a liking for ring vibrios (Vibrio cyclitrophicus) and Vibrio splindidus (Vibrio splendidus) ABTNL01, Vibrio vulnificus (Vibrio vulnificus) 1H00047, Vibrio harveyi (Vibrio harveyi) 1H00040, tower vibrios (Vibrio tubiashii) 1H00065, Ye Shi Pseudoalteromonas (Pseudoalteromonas elyakovii) 1B00018, produce black Pseudoalteromonas (Pseudoalteromonas nigrifaciens) 1B00017, Shigellae (Shigella sp.) 1B00019, seabed Psychrobacter (Psychrobacter submarinus) 1B00013.Result is as shown in the table: this phage can only be had a liking for ring vibrios by its Host Strains of cracking, and host specificity is strong.
The host range of table 1 phage Vc1
Note 1): cracking is designated as "+"; Can not cracking be designated as "-"
Embodiment 4
Phage control is by having a liking for the microbial sea cucumber disease analyzing of applying effects of ring arc
Get purchased from 40 of the healthy sea cucumbers of Dalian holothruian cultures factory, be divided at random four groups, 10 every group, adopt dipping bath mode to process each group.
First group: negative control group.Adding bacterium liquid to final concentration is 2 * 10
6cFU/mL.
Second group: positive controls.Adding bacterium liquid to final concentration is 2 * 10
6cFU/mL, adds sulphuric acid kanamycin to final concentration 10mg/L after 6h.
The 3rd group: phage preventive experiment group.Add phage to final concentration 2 * 10
7pFU/mL, after 6h, adding bacterium liquid to final concentration is 2 * 10
6cFU/mL.
The 4th group: Phage therapy experimental group.Adding bacterium liquid to final concentration is 2 * 10
6cFU/mL, adds phage to final concentration 2 * 10 after 6h
7pFU/mL (MOI=10).
Observe the survival condition of respectively organizing stichopus japonicus, statistics survival rate, result is as shown in the table:
The stichopus japonicus that table 2 phage Vibrio Phage Vc1 treatment is infected by VDL-1
Claims (2)
1. have a liking for a ring vibrios phage, it is characterized in that, this phage is preserved on April 16th, 2014 that " Classification And Nomenclature encircles vibrios phage for having a liking for for China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preserving number CGMCC No.9101.
2. have a liking for the application of ring vibrios phage in sea cucumber disease prevention for one kind, it is characterized in that, with having a liking for ring vibrios phagocytosis body fluid, the stichopus japonicus of illness outbreak phase is carried out to dipping bath maybe the ring vibrios phagocytosis body fluid had a liking for after dilution is joined to pond and sprayed, every 5 days dipping baths or spray 1 time, carry out continuously 3 times, use consumption>=10 at every turn
7pfu/m
3aquaculture water.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391358A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio mediterrae virulent phage vB _ Vmem-Yong and application thereof |
| CN112391355A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong3 and application thereof |
| CN116790516A (en) * | 2023-05-22 | 2023-09-22 | 中国海洋大学 | Phage for lysing algicidal bacteria and application thereof |
Citations (1)
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| CN102550458A (en) * | 2012-01-19 | 2012-07-11 | 广东海洋大学 | Technique for allowing aquatic invertebrate larvae and parents to harmlessly carry Vibrio phages |
Non-Patent Citations (1)
| Title |
|---|
| 杨扬: ""应用噬菌体防治弧菌引发的刺参腐皮综合征"", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391358A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio mediterrae virulent phage vB _ Vmem-Yong and application thereof |
| CN112391355A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong3 and application thereof |
| CN112391355B (en) * | 2019-08-14 | 2023-12-01 | 宁波大学 | Vibrio harveyi efficient lytic phage vB_VhaS-yong3 and application thereof |
| CN112391358B (en) * | 2019-08-14 | 2023-12-01 | 宁波大学 | Vibrio mediterranean virulent phage vB_VmeM-Yong and application thereof |
| CN116790516A (en) * | 2023-05-22 | 2023-09-22 | 中国海洋大学 | Phage for lysing algicidal bacteria and application thereof |
| CN116790516B (en) * | 2023-05-22 | 2023-11-24 | 中国海洋大学 | Phage for lysing algicidal bacteria and application thereof |
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Application publication date: 20140820 |