CN103898015B - A kind of vibrio alginolyticus phage and the application in sea cucumber disease prevention thereof - Google Patents
A kind of vibrio alginolyticus phage and the application in sea cucumber disease prevention thereof Download PDFInfo
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- CN103898015B CN103898015B CN201410108994.9A CN201410108994A CN103898015B CN 103898015 B CN103898015 B CN 103898015B CN 201410108994 A CN201410108994 A CN 201410108994A CN 103898015 B CN103898015 B CN 103898015B
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Abstract
The invention discloses a kind of vibrio alginolyticus phage and the application in sea cucumber disease prevention thereof, belong to biological technical field.This phage on March 11st, 2014 be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preserving number CGMCC? No.8792, does is Classification And Nomenclature Vibrio? alginolyticus? bacteriophage; Vibrio alginolyticus phage is Caudoviradles, Podoviridae, head diameter 50nm, the long 15nm of afterbody, and ungauged regions caudal sheath has 6 short-tail silks.Phage is applied to the infection preventing and treating corresponding vibrio alginolyticus in holothruian cultures process and pollution, tire>=10
5pFU/mL.Phage has restraining effect to vibrio alginolyticus, produces in 12h without bacteriophage resistant strain; Latent period 28min, burst size is 79, and it reduces two orders of magnitude in sea water culture environment in 14 days.
Description
Technical field
The invention belongs to biological technical field, relating to a strain can the phage isolates of Specific lytic vibrio alginolyticus (Vibrioalginolyticus) bacterial strain and the application in sea cucumber disease prevention area thereof.
Background technology
Sea cucumber (Holothurioidea) is one of maximum marine cultured animal of the Northern Coastal Region of China area output value.According to statistics, nearly 90,000 hectares of current China holothruian cultures area, nearly 100,000 tons of output, annual value of production more than 100 hundred million yuan (Chinese sea cucumber academic research and industry development forum, 2009).But in recent years along with the rapid increase of apostichopus japonicus culture scale and cultivation density, aquaculture water runs down, and disease problem is also on the rise.Vibrio alginolyticus (Vibrioalginolyticus) is a kind of common Marine Pathogenic Bacteria, and this bacterium causes adult sea cucumber to suffer from one of Main Pathogenic Bacteria of skin ulceration syndrome, and mortality ratio, up to 90%, seriously limits the sustainable and healthy development of holothruian cultures industry.Meanwhile, vibrio alginolyticus is also the encountered pathogenic bacteria causing coastland fishery products food poisoning and acute diarrhea clinically.
Phage is a bacterioid venereal disease poison, can in somatic cells quick copy propagation, and final cracking bacterium thus reach antibacterial effect.Phage therapy utilizes the characteristic of phage splitting bacterium to prevent and treat the bacterial infection of human or animal.In recent years, research finds that Phage therapy effectively can prevent and treat bacteriosis of aquatic livestock.The haliotis discus hannai Ino pustulosis that Yellowtail fish Lactococcus garvieae (Lactococcusgarvieae) infects, sweetfish cold water is sick, distortion pseudomonas (Pseudomonasplecoglossicida) is infected, prawn Vibrio harveyi (Vibrioharveyi) is sick and vibrio fluvialis (Vibriofluvialis) causes is prevented and treated as utilized phage.Although phage is confirmed in the lab in the effect of control terrestrial animal and aquatic animal (Yellowtail fish, sweetfish, prawn etc.) bacteriosis, but the research report had not yet to see about phage being used for sea cucumber disease control, and some key issues in Phage therapy process not yet solve, concrete mechanism of action is still not clear.
Phage isolates in the present invention is the virulent phage be separated from occurring in nature, through mouse safety experiment prove its safely, have no side effect, and to vibrio alginolyticus, there is strong splitting action.The sea cucumber disease be developed to as biological bactericide prevention and control are caused by vibrio alginolyticus is expected to after this phage is purified.
Summary of the invention
The invention provides a kind of disease applied lytic phage and prevent to be caused by vibrio alginolyticus in holothruian cultures, reduce the mortality ratio caused by this disease, the technological method of holothruian cultures Business Economic Benefit can be improved.
Technical scheme of the present invention is to provide a kind of vibrio alginolyticus phage, this vibrio alginolyticus phage is preserved on March 11st, 2014 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; preserving number CGMCCNo.8792, Classification And Nomenclature is vibrio alginolyticus phage Vibrioalginolyticusbacteriophage.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 vibrio alginolyticus phage is Caudoviradles, Podoviridae, head diameter 50nm, the long 15nm of afterbody, and ungauged regions caudal sheath has 6 short-tail silks.
In the present invention, vibrio alginolyticus phage refined solution has good preventive effect (survival rate improves more than 50%) for the sea cucumber disease caused by vibrio alginolyticus.Vibrio alginolyticus phage refined solution is applied to the infection and pollution that prevent and treat corresponding vibrio alginolyticus in holothruian cultures process, wherein, tire>=10
5pFU/mL.This vibrio alginolyticus phage has good restraining effect to vibrio alginolyticus, produces in 12h without bacteriophage resistant strain; Latent period 28min, burst size is 79, and its phage refined solution reduces two orders of magnitude in sea water culture environment in 14 days.
Pnagus medius of the present invention is through mouse safety experiment, and abdominal injection gives phage experimental group 10
10the phage refined solution of PFU/kg, control group gives normal saline, and after giving 15 days continuously, mouse is taken off neck and put to death, postmortem, its lung, liver, kidney, abdominal cavity all do not find disease states, and this phage of preliminary identification is safe.
Beneficial effect of the present invention:
1. be separated acquisition one strain phage PVA1 and have good dissipation effect to pathogenic vibrio alginolyticus, and produce without bacteriophage resistant strain in 12h;
2. above-mentioned phage refined solution is in aquaculture water, tires and only have dropped two orders of magnitude in 14 days, and environmental resistance is better, has certain practicality basis;
3. the safety experiment display continuous 15 day administration no abnormality seen of above-mentioned phage refined solution in Mice Body, security in its body of preliminary identification;
4. above-mentioned phage refined solution (tire>=10
5pFU/mL) good preventive effect (survival rate improves more than 50%) is had for the sea cucumber disease caused by pathogenic vibrio alginolyticus;
5. phage is extensively present in occurring in nature as natural antibacterial microorganism, artificial screening, enrichment use 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 having a extensive future, have a high potential.
Accompanying drawing explanation
Fig. 1 is phage PVA1 transmission electron microscope picture.
Fig. 2 is phage PVA1 In Vitro Bacteriostasis result.
Fig. 3 is phage PVA1 one step growth.
Fig. 4 is the degraded situation of phage PVA1 refined solution in analog culture water body.
Fig. 5 is the preventive effect of various dose phage PVA1 refined solution for vibrio alginolyticus.
In figure:
doxycycline (5mg/L);
sulphuric acid kanamycin (10mg/L);
high dosage MOI=10;
middle dosage MOI=1;
low dosage MOI=0.1; blank.
Embodiment
Embodiment 1
The screening of phage and purifying
(1) water sampling and process
(1) preparation of Host Strains
Vibrio alginolyticus is inoculated in 2216E solid medium after bacteriological surveillance, is stored in 4 DEG C of refrigerators.Screening Host Strains is through 28 DEG C of incubated overnight, and the single bacterium colony of picking, is inoculated in 2216E liquid nutrient medium, after 28 DEG C of shaking culture 8h, is used for being separated phage and uses.
(2) process of water sample
Get and come from apostichopus japonicus culture field, Dalian and each 200mL of fish market, Daliang City sewage draining exit wastewater sample, pre-treatment is done to water sample: add CaCl2, MgCl
2, make its final concentration be 1mmol/L, act on about 10min.(2) enrichment of water sample phage
By above-mentioned through pretreated water sample, the centrifugal 5min of 10000g, collects supernatant; Supernatant is crossed 0.22 μm of filter membrane, except other compositions such as thalline, impurity, obtain phage stoste; Get 10mL filtrate add be in logarithmic phase (inoculation after about 5h, phage final concentration reaches 10
6-10
7during CFU/mL, i.e. logarithmic phase) 50mL bacterium liquid in, 28 DEG C of incubated overnight 12-18h, object is that phage quantity is increased, and observes and record; Collect bacterium liquid, the centrifugal 5min of 10000g, collect supernatant, after filter membrane (0.22 μm), obtain the phage supernatants after propagation.
(3) screening of phage
Adopt double-layer agar technique to carry out the qualification of phage, lower floor is the 2216E solid medium of 1.5% agar, be placed in 4 DEG C for subsequent use, with being front put in 28 DEG C, 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 DEG C stand-by, gets 8 10mL centrifuge tubes, adds 1mL Host Strains liquid respectively, draws phage suspension liquid and carries out gradient dilution to 10-
6the phage diluent of 7 kinds of different concns gradients is taken out 10 μ L respectively, be added in 10mL centrifuge tube, a control group is set, only adds 1mL Host Strains liquid and 10 μ L2216E substratum, at 28 DEG C, act on 10min, add after 5mL fully mixes containing the 2216E substratum of 0.5% agar, be added to upper strata at once, after to be solidified, be inverted after cultivating 24h and observe with or without Plaques assay.If form plaque on flat board, illustrate has the lytic phage for this Host Strains to exist in filtrate.
(4) purifying of phage
Size, the shape of the plaque of first separation are inconsistent, to the further purifying of phage be separated, make phage on flat board, form size and the consistent plaque of form.Get form size with aseptic 200 μ L rifle choicests and have each one of the plaque of notable difference, it joined in the centrifuge tube of the aseptic PBS of 1mL respectively, vortex concussion 1min, is placed in 4 DEG C, 4h, phage is fully released in PBS; 4 DEG C, the centrifugal 5min of 10000g, collect supernatant, crosses film (0.22 μm), phage is considered liquid and carry out gradient dilution, Double layer culture, repeats 3 aforesaid operations, till plaque morphology, the size of appearance are completely the same, namely obtains purified phage.Phage electromicroscopic photograph is shown in accompanying drawing 1.
(5) enrichment of phage and amplification
Liquid method of proliferating is adopted to breed.Concrete method is as follows: joined by phagocytosis body fluid in the Host Strains liquid cultivating 12h by a certain percentage, 12h is cultivated again in 28 DEG C of shaking tables, by mixed solution in 4 DEG C, the centrifugal 5min of 10000g, place to go bacterial debris, the supernatant liquor membrane filtration of 0.22 μm, can obtain the phage pregnant solution of high-titer.
Embodiment 2
The In Vitro Bacteriostasis effect of phage
(1) antibacterial experiment in vitro of phage
Setup Experiments is 4 groups, gets 4 50mL centrifuge tubes, is labeled as A, B, C, D respectively; Wherein A is negative control group, and B, C are positive controls, and D is experimental group.Add 30mL2216E liquid nutrient medium in A, (concentration is 10 to 1mL bacterium liquid
8cFU/ml), 1mLPBS; Add 30mL2216E liquid nutrient medium in B, 1mL bacterium liquid, 1mL sulphuric acid kanamycin (SulfateKanamycin) concentration is 10mg/L; Add 30mL2216E liquid nutrient medium in C, 1mL bacterium liquid, 1mL doxycycline (Doxycycline) concentration is 5mg/L; Add 30mL2216E liquid nutrient medium, 1mL bacterium liquid in D equally, add 1mLPVA1 phage pregnant solution, tiring is 10
9pFU/mL; After vibration 10min, put into the value that microplate reader surveys OD600nm, record; Put into 28 DEG C of shaking tables to cultivate, sample the value of a survey OD600nm per half an hour, change in record 12h.
Result shows, and phage experimental group can suppress the growth of Host Strains in 12h, and produces without corresponding resistant bacterial strain, the results are shown in accompanying drawing 2.
(2) one step growth
One step growth experiment is utilized to detect the latent period of lytic phage infection host bacterium and the granule number of final release phage.
(1) in the 2216E liquid nutrient medium of 100mL, add 200 μ L Host Strains incubated overnight, make cell concentration about 1 × 10
9cFU/mL;
(2) get in 20mL2216E fresh culture and add 2mL and to spend the night bacterium liquid and phage conserving liquid, adjustment infection multiplicity is that 0.1,28 DEG C of standing 15min make phage fully be adsorbed on after on Host Strains to get 1mL aforesaid liquid;
(3) 4 DEG C, the centrifugal 10min of 10000g;
(4) supernatant is removed, with 1mL2216E liquid nutrient medium suspension bacterial sediment;
(5) repeating step 3 and 4 liang to three times, by the bacteriophage elution that do not adsorb in supernatant;
(6) above-mentioned adsorption liquid is added in the fresh 2216E substratum of 19mL, fully mix, 28 DEG C, 120rpm cultivation;
(8) draw 100 μ L nutrient solutions every 5min and detect phage titer.
Result shows, and phage PVA1 latent period is 28min, and burst size is 79, the results are shown in accompanying drawing 3.
Embodiment 3
The environmental resistance of phage
(tire is 4.2 × 10 to the high-titer pregnant solution of acquisition phage PVA1
10pFU/ml).100mL breeding seawater, 1 healthy sea cucumber of 9g and phagocytosis body fluid are put into 500mL beaker, and initial potency is 1.5 × 10
7pFU/ml, cultivation room temperature is placed, and every 24h uses double-layer agar technique to survey and tires once, record phage degradation situation in 14 days.
Result shows: under the condition of breeding seawater, cultivation room temperature, and phagocytosis body fluid have dropped two orders of magnitude in 14 days, and phage environmental resistance is better, for the environment water application of phage provides possibility, the results are shown in accompanying drawing 4.
Embodiment 4
Mouse safety experiment
Laboratory animal is cleaning grade kunming mice, body weight 18 ± 4g, and 2 monthly ages, (conformity certification number: 00001317) was provided by Dalian Medical Univ's Experimental Animal Center.Experimental animal feeding in cleaning grade Animal House, room temperature 18 DEG C, relative humidity 40%-80%, 12h sunshine and dim circulation.Experiment mice totally 10, male, after adaptability raises 3, random one-tenth two groups (control group, phage group), often organize 5, phage experimental group abdominal injection gives 10
10pFU/kg phage refined solution, control group gavage gives normal saline, and mouse, after 15 days, is taken off neck and puts to death by successive administration, dissects internal organ and observes pathology situation.
Result shows, 10
10under PFU/kg dosage, phage refined solution does not make significant difference to experiment mice daily behavior, and anatomic observation lung, liver, kidney, abdominal cavity all do not find disease states.
Embodiment 5
Various dose phage PVA1 refined solution is to the preventive effect of vibrio alginolyticus
Sea cucumber is provided by Dalian Bay culturing marine products field in experiment, individual heavy 5 ~ 8g, and before experiment, sea cucumber adaptability cultivates 7 days, fasting 1 day, water temperature 18 DEG C, logical oxygen lucifuge.Experiment grouping: (1) positive controls one (5mg/L doxycycline); (2) positive controls two (10mg/L sulphuric acid kanamycin); (3) high dosage phage group (final concentration 10
7pFU/mL); (4) dosage phage group (final concentration 10 in
6pFU/mL); (5) low dosage phage group (final concentration 10
5pFU/mL); (6) blank group (equivalent sterilizing seawater).Totally six groups, often organize 10 healthy sea cucumbers and establish three parallel laboratory tests simultaneously.Experimental technique: set up each experimental group by above-mentioned condition, uses 5.7 × 10 after 1h
6the every 24h dipping bath of CFU/mL vibrio alginolyticus attacks poison once, records each group of sea cucumber death condition.
Experimental result shows, and after 5 days, blank group sea cucumber is all dead, and the survival rate of doxycycline group is 80% simultaneously, and the survival rate of sulphuric acid kanamycin group is 47%, and the phage group survival rate of high, normal, basic dosage is respectively 73%, 50%, 50%, sees accompanying drawing 5.As can be seen here, phage PVA1 has certain preventive effect to vibrio alginolyticus, can be applied to the prevention of vibrio alginolyticus in holothruian cultures as a kind of biological antibiosis agent.
Claims (2)
1. a vibrio alginolyticus phage, it is characterized in that: this vibrio alginolyticus phage is preserved on March 11st, 2014 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; preserving number CGMCCNo.8792, Classification And Nomenclature is Vibrioalginolyticusbacteriophage.
2. the application of vibrio alginolyticus phage according to claim 1, is characterized in that: vibrio alginolyticus phage is applied to the infection and pollution that prevent and treat vibrio alginolyticus in holothruian cultures, wherein, tire>=10
5pFU/mL; Vibrio alginolyticus phage is inhibited to vibrio alginolyticus, produces in 12h without bacteriophage resistant strain.
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| CN112391354A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio alginolyticus efficient lytic phage vB _ ValM-Yong3 and application thereof |
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| CN110468110B (en) * | 2019-09-11 | 2022-08-19 | 大连理工大学 | Vibrio parahaemolyticus bacteriophage and application thereof in disease prevention of stichopus japonicus |
| CN111855984A (en) * | 2020-08-03 | 2020-10-30 | 浙江大学 | Method for evaluating biological safety by using intestinal flora of mice |
| CN112852750B (en) * | 2020-10-26 | 2023-04-21 | 河海大学 | Marine-derived vibrio phage, microecological preparation, and preparation methods and applications thereof |
| CN112553171B (en) * | 2020-12-27 | 2022-06-21 | 威海长青海洋科技股份有限公司 | Vibrio phage preparation and application thereof |
| CN113512539B (en) * | 2021-04-25 | 2023-06-27 | 中国海洋大学 | Phage and application thereof |
| CN115161290A (en) * | 2022-03-31 | 2022-10-11 | 青岛百奥安泰生物科技有限公司 | Vibrio alginolyticus bacteriophage with wide lysis spectrum and application thereof |
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