CN102007941A - Application of Bdellovibrio nectophore in preparing bactericide for controlling red mouth disease of scophthalmus maximus - Google Patents
Application of Bdellovibrio nectophore in preparing bactericide for controlling red mouth disease of scophthalmus maximus Download PDFInfo
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Abstract
The invention discloses an application of Bdellovibrio nectophore in preparing a bactericide for controlling the red mouth disease of a scophthalmus maximus. Bdellovibrio is Bdellovibrio sp. BDM01. In the application method of the bactericide, the bactericide for controlling the scophthalmus maximus red mouth disease is splashed into a water body to cause the final concentration of the Bdellovibrio nectophore in the water body to be 10-107pfu/mL, or feeds are soaked by the bactericide for controlling the scophthalmus maximus red mouth disease for 15-45min to cause the concentration of the Bdellovibrio nectophore in the bactericide to be 10-107pfu/mL. The application of the Bdellovibrio nectophore in preparing the bactericide for controlling the scophthalmus maximus red mouth disease can effectively control and treat the red mouth disease of the scophthalmus maximus and has high safety, environmental protection and good application prospect and does not generate pathogenic bacteria resistance in the application of aquatic animal culture.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of application of mushroom, particularly Bdellovibrio leech plastid is prevented and treated application in the agent of the red mouth germ of turbot in preparation.
Background technology
Turbot (Scophthalmus maximus) is a kind of famous and precious flounder flounder class that originates in Europe, is under the jurisdiction of Pleuronectiformes, Bothidae, and brill belongs to.Mainly be distributed in the Atlantic coast, the North Sea, Black Sea and Mediterranean one band.Because having, it adapts to low water temperature life, fast growth, and delicious meat, acceptant artificial feed are easy to advantages such as intensive culture, become the main breed development object of many countries such as Europe and South America Chile in succession.Since turbot in 1992 was introduced into China, its breed had been radiated northern coastal provinces fast from Shandong, become a pillar industry of China's mariculture, has obtained good society and economic benefit.Yet along with the continuous expansion of the scale of breed, the disease problem of turbot is outstanding day by day, and the extensive phenomena of mortality frequently take place, and the medicament residue problem is also day by day serious, brings totally unfavorable influence for the sound development of turbot aquaculture.Red mouth disease is exactly wherein a kind of, has caused heavy economic loss for the breed dealer.The cardinal symptom of red mouth disease shows as: the sick fish kiss end and the outside of belly upper jaw are hemorrhage, the part fish gill cover has the hemorrhage little pothole that festers, the oral cavity internal haemorrhage, the enlargement of the sick flake eyeball of part is given prominence to, needled a large amount of festers in the adventitia, the congested no food of enteron aisle, Di has a small amount of thick liquid, and liver hyperemia, look dimness are the rotten shape that looses, and the spleen enlargement is rubescent.In therapeutic process, the raiser is the economic pursuit benefit, and phenomenon such as medication happens occasionally carelessly, causes the residue of pesticide problem to be on the rise.At present, prevent that the main method of disease from being the sterilizing medicine that adopts, but itself exist many shortcomings that as drug resistance and medicament residue that antibiotic use causes, the residual incident of turbot nitrofurans medicine that took place in 2006 is just very representative.Therefore, how to carry out healthy aquaculture, aquatic products circle that just becomes problem demanding prompt solution.
Bdellovibrio is to parasitize other bacteriums, and can cause a bacterioid of host bacteria cracking.Littler than general bacterium, can pass through bacterial filter, the effect of similar phage is arranged.Unicellular, curved or funny point-like, shape in the shape of a spiral sometimes, big or small 0.2-0.6 * 0.8-1.2 micron, end is given birth to flagellum, mostly is one.Gram is negative, and cleavable causes a disease or potential pathogenic bacteria.Divide two stages the history of life of Bdellovibrio: free living, can move telotroch stage of not breeding and the leech plastid stage of in the periplasmic space of specific host bacterium, carrying out growth and breeding.Bdellovibrio is in the telotroch stage, and identification also with the host irreversible the combination is taken place, and invades the host then.Bdellovibrio leech plastid then is Bdellovibrio carries out growth and breeding in the periplasmic space of specific host bacterium a form.After Bdellovibrio entered host's bacterial cell periplasmic space, mainly the big molecule by the degraded host cell became monomer, took in then to be used for the synthetic of its large biological molecule.Along with cell proliferation and hydrolase produce, make the host cell wall destroyed, thereby cracking discharge Bdellovibrio.
The Bdellovibrio telotroch is meant the growth forms before Bdellovibrio is invaded host bacteria.It is very fast that the Bdellovibrio telotroch is invaded the process of host bacteria, can finish in general several seconds.For Bdellovibrio leech plastid, though exist the weakness that preparation is loaded down with trivial details, be difficult for preservation, the pathogenic bacteria of culturing in the pond with control of Bdellovibrio telotroch and elimination have rapid-action, energetic advantage.These advantages make the Bdellovibrio telotroch be suitable as the biological cleaning factor of restraining or removing pathogenic bacteria in organism and the environment thereof equally, thereby have overcome the side effect that the antibiotic abuse brings.
Summary of the invention
In order to overcome the weak point that abuse of antibiotics in the fish culture causes various food-safety problems, the invention provides a kind of Bdellovibrio telotroch and prevent and treat application in the agent of the red mouth germ of turbot in preparation.
The present invention is achieved through the following technical solutions:
The Bdellovibrio telotroch is prevented and treated application in the agent of the red mouth germ of turbot in preparation.
The preparation method of described Bdellovibrio telotroch is: the host is in the nutrient broth liquid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, culture fluid is through 4 ℃, behind the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB liquid nutrient medium, insert Bdellovibrio BDM01, under 25～35 ℃ of temperature, cultivate 36～48h with 150～300rpm shaking table, obtain containing the culture fluid of Bdellovibrio leech plastid and Bdellovibrio telotroch, culture fluid under 4 ℃ of temperature with 6000～8000rpm behind centrifugal 15～20min, supernatant is behind the centrifugal 15～20min of 16000～18000rpm, precipitation DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value are 7.2～7.6 phosphate buffer suspension, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL, this bacterium liquid are prevents and treats the agent of the red mouth germ of turbot.
Described Bdellovibrio is Bdellovibrio (Bdellovibrio sp.) BDM01, is preserved in Chinese typical culture collection center on April 28th, 2008, and deposit number is CCTCC NO:M208066.
Described Bdellovibrio telotroch is to splash to prevent and treat the agent of the red mouth germ of turbot in water body in the application that preparation prevents and treats in the agent of the red mouth germ of turbot, changes the microbial inoculum of evenly splashing in water body after the water at every turn, makes that Bdellovibrio telotroch final concentration is 10-10 in the water body
7Pfu/mL.
Described Bdellovibrio telotroch is that feed is soaked 15～45min with preventing and treating the agent of the red mouth germ of turbot in the application that preparation prevents and treats in the agent of the red mouth germ of turbot, and Bdellovibrio telotroch concentration is 10-10 in the microbial inoculum
7Pfu/mL throws feed every day three times, and early, middle and late respectively once (17:00), the process of at every turn throwing something and feeding continues 30min for 7:00,12:00.4% of the mass fraction of daily ration of feeding and fish average weight perfuses full pond.
Bdellovibrio BDM01 carries out ultraviolet mutagenesis by Bdellovibrio BDJ01 (CCTCC M 208011) and obtains, it is carried out morphologic observation under electron microscope after the negative staining: Bdellovibrio BDM01 is unicellular, arc, and size is 1.7 * 1.0 μ m, end is given birth to flagellum, and flagellum length is 3.5 μ m; It is cultivated the transparent circular plaque that can form diameter 3-4mm in three days with the double-layer plate method in 28 ℃.
The present invention has following advantage and effect with respect to prior art:
1, Bdellovibrio telotroch of the present invention can effectively prevent and treat the red mouth disease of turbot in the application that preparation prevents and treats in the agent of the red mouth germ of turbot.
As show shown in the 1-2, Bdellovibrio telotroch described in the present invention can effectively reduce the probability that turbot is suffered from red mouth disease in the application that preparation prevents and treats in the agent of the red mouth germ of turbot, effect is remarkable on the red mouth disease of treatment, compares with the antibiotic method of the use of routine, and it is a kind of method of environmental protection.
The red mouth disease of turbot is a kind of bacteriosis, and bacteriosis is a The main pathogenic fungi with some bacterial classification in Aeromonas (Aeromonas), pseudomonas (Pseudoumonas), the vibrio (Vibrio).Bdellovibrio has the effect of class phage, and it has splitting action to the fish common pathogen, can play the effect of eliminating the water body pathogenic bacteria.In addition, Bdellovibrio also has certain degradation to the ammonia nitrogen in the water, can purifying water body, enhance the transparency, and the control fish disease there is certain effect.Therefore, Bdellovibrio can be used for the prevention and the treatment of the red mouth disease of turbot.
2, to prevent and treat in the application that is applied in aquaculture of aquatic animal in the agent of the red mouth germ of turbot safety in preparation good for Bdellovibrio telotroch of the present invention.
3, Bdellovibrio telotroch of the present invention preparation prevent and treat in the agent of the red mouth germ of turbot application effectively, environmental protection, do not produce the pathogen resistance, application prospect is good.
Concrete implementing measure
The present invention is described in further detail below in conjunction with embodiment, but embodiments of the present invention are not limited thereto.
The preparation of embodiment 1 microbial inoculum
The Bdellovibrio bacteriovorus bacterial strain that uses is Bdellovibrio BDM01.Described Bdellovibrio (Bdellovibriosp.) BDM01 is preserved in Chinese typical culture collection center on April 28th, 2008, and deposit number is CCTCC NO:M 208066; It is carried out morphologic observation under electron microscope after the negative staining: Bdellovibrio BDM01 is unicellular, arc, and size is 1.7 * 1.0 μ m, and end is given birth to flagellum, and flagellum length is 3.5 μ m; It is cultivated the transparent circular plaque that can form diameter 3-4mm in three days with the double-layer plate method in 28 ℃.
Be equipped with the common nutrient broth of 100mL (NB) medium (peptone 10g, beef extract 3g, sodium chloride 5g, distilled water 1000mL, inoculation 1mL concentration is 10 in conical flask pH7.4)
9The Aeromonas hydrophila of cfu/mL (Aeromonas hydrophila purchases in Guangdong Province DSMZ of Institute of Micro-biology, numbering GIM1.172), 200rpm, 28 ℃ of shaking tables were cultivated 18 hours, and culture fluid is abandoned supernatant respectively at 4 ℃, the centrifugal 15min of 5000rpm.Precipitation joined 100mL DNB (dilute nutrient broth) liquid nutrient medium (nutrient broth 0.8g is housed, caseinic acid hydrolysate 0.5g, yeast extract 0.1g is dissolved in the 1000mL distilled water, the pH value is 7.4) conical flask in, adding 1mL concentration again is 10
3The plaque of the Bdellovibrio BDM01 of pfu/mL.Constant temperature shaking table 250rpm, 28 ℃ of cultivation 36h.Culture fluid is got supernatant in 4 ℃ of centrifugal 20min of 6000rpm, again with supernatant respectively at 4 ℃ of centrifugal 20min of 16000rpm, keep precipitation, add 1mL physiological saline and suspend, can obtain concentration is 10
8The Bdellovibrio telotroch microbial inoculum of pfu/mL.
Embodiment 2
Select physical health, individual juvenile fish of uniform size, average weight is 55 ± 2.5g, according to 300 tails/m
2Cultivation density drop in the pond, be 4 months each experimental period of culturing the pond.
Embodiment 2.1
Normal flowing water (adopt outer draining, whenever quantity of exchanged water is 8 streamflows round the clock) is thrown in conventional turbot pellet (breeding way of plant produced), not with microbial inoculum.
Embodiment 2.2
Changed water one time in per 40 days, and threw in conventional turbot pellet, not with microbial inoculum.
Embodiment 2.3
The embodiment 1 that splashes in water obtains microbial inoculum, makes that Bdellovibrio telotroch concentration reaches 10pfu/mL in the water body, and the conventional turbot pellet of feeding adds bacterium after changing a water in per 40 days.
Embodiment 2.4
The microbial inoculum that the embodiment 1 that splashes in water obtains makes that Bdellovibrio telotroch concentration reaches 10 in the water body
2Pfu/mL, the conventional turbot pellet of feeding adds bacterium after changing a water in per 40 days.
Embodiment 2.5
The microbial inoculum that feeding obtains through embodiment 1 soaks the conventional turbot pellet of 30min, and Bdellovibrio telotroch concentration reaches 10pfu/mL in the used microbial inoculum, bait throwing in every day three times, early, middle and late each (7:00 once, 12:00,17:00), the process of at every turn throwing something and feeding continues 30min.The mass fraction of daily ration of feeding and fish average weight is 4%, perfuses full pond, changes one time water in per 40 days.
Embodiment 2.6
The microbial inoculum that feeding obtains through embodiment 1 soaks the feed of 30min, and Bdellovibrio telotroch concentration reaches 10 in the used microbial inoculum
2Pfu/mL, bait throwing in every day three times, it is early, middle and late that respectively once (17:00), the process of at every turn throwing something and feeding continues 30min for 7:00,12:00.The mass fraction of daily ration of feeding and fish average weight is 4%, perfuses full pond, changes one time water in per 40 days.
Embodiment 2.7
The microbial inoculum that the embodiment 1 that splashes in water obtains, make that Bdellovibrio telotroch concentration reaches 10pfu/mL in the water body, changed in per 40 days behind water with microbial inoculum, the microbial inoculum that the while feeding obtains through embodiment 1 soaks the conventional turbot pellet of 30min, bait throwing in every day three times, early, middle and late each once (7:00,12:00,17:00), the process of at every turn throwing something and feeding continues 30min.The mass fraction of daily ration of feeding and fish average weight is 4%, perfuses full pond.Bdellovibrio telotroch concentration reaches 10pfu/mL in the used microbial inoculum.
Embodiment 2.8
The microbial inoculum that the embodiment 1 that splashes in water obtains makes that Bdellovibrio telotroch concentration reaches 10 in the water body
2Pfu/mL adds bacterium after changing a water in per 40 days, and feeding soaks the conventional turbot pellet of 30min through the Bdellovibrio telotroch simultaneously, bait throwing in every day three times, early, middle and late each once (17:00), the process of at every turn throwing something and feeding continues 30min for 7:00,12:00.The mass fraction of daily ration of feeding and fish average weight is 4%, perfuses full pond.Bdellovibrio telotroch concentration reaches 10 in the used microbial inoculum
2Pfu/mL.
Experimental result
The sick incidence of the red mouth of table 1.
Result of the test shows, under normal factory breeding way, the incidence of red mouth disease is about 10%, adopts behind the Bdellovibrio telotroch the sick incidence of red mouth to have significantly and descends, and minimum is 0.85%.Changing under the regimen condition of same frequency, the sick incidence of the red mouth of blank nearly 20%, and red mouth disease do not occur after having used the Bdellovibrio telotroch illustrate that Bdellovibrio swimming physical efficiency effectively prevents the generation of red mouth disease.In addition, in independent oral or dipping, or both combine, and its effect is all very remarkable.
Embodiment 3
Select the turbot (it is hemorrhage to be characterized as the fish kiss end and the outside of belly upper jaw, and the fish gill cover has the hemorrhage little pothole that festers, and the eyes enlargement is outstanding) of just suffering from red mouth disease, culture in capacity is 1 ton breed pond, each cultures pond 150 tails, and test period is 7 days, writes down disease fish quantity every day.
Embodiment 3.1
For normal flowing water (adopt outer draining, whenever quantity of exchanged water is 8 streamflows round the clock), throw in conventional turbot pellet (breeding way of plant produced)
Embodiment 3.2
For throwing in conventional turbot pellet, do not add the Bdellovibrio telotroch, duration of test does not change water.
Embodiment 3.3
Clean for adding sharp bacterium, sharp bacterium mixed only add low amounts of water after in conventional turbot pellet, stirring evenly and make it bonding, directly throw something and feed.The each medication 12g of every 500kg fish.
Embodiment 3.4
The microbial inoculum that the embodiment 1 that splashes in water obtains makes that Bdellovibrio telotroch concentration reaches 10 in the water body
3Pfu/mL, the conventional turbot pellet of feeding, duration of test does not change water.
Embodiment 3.5
The microbial inoculum that the embodiment 1 that splashes in water obtains makes that Bdellovibrio telotroch concentration reaches 10 in the water body
5Pfu/mL, the conventional turbot pellet of feeding, duration of test does not change water.
Embodiment 3.6
The microbial inoculum that the embodiment 1 that splashes in water obtains makes that Bdellovibrio telotroch concentration reaches 10 in the water body
7Pfu/mL, the conventional turbot pellet of feeding, duration of test does not change water.
Embodiment 3.7
The microbial inoculum that feeding obtains through embodiment 1 soaks the conventional turbot pellet of 30min, and used Bdellovibrio telotroch concentration reaches 10
3Pfu/mL, bait throwing in every day three times, early, middle and late each once, (17:00), the process of at every turn throwing something and feeding continues 30min for 7:00,12:00.The mass fraction of daily ration of feeding and fish average weight is 4%, perfuses full pond, and duration of test does not change water.
Embodiment 3.8
The microbial inoculum that feeding obtains through embodiment 1 soaks the conventional turbot pellet of 30min, and used Bdellovibrio telotroch concentration reaches 10
5Pfu/mL, bait throwing in every day three times, early, middle and late each once, (17:00), the process of at every turn throwing something and feeding continues 30min for 7:00,12:00.The mass fraction 4% of daily ration of feeding and fish body weight perfuses full pond, and duration of test does not change water.
Embodiment 3.9
The microbial inoculum that feeding obtains through embodiment 1 soaks the feed of 30min, and used Bdellovibrio telotroch concentration reaches 10
7Pfu/mL, bait throwing in every day three times, early, middle and late each once, bait throwing in every day three times, early, middle and late each once (17:00), the process of at every turn throwing something and feeding continues 30min for 7:00,12:00.
The mass fraction of daily ration of feeding and fish average weight is 4%, perfuses full pond, and duration of test does not change water.
Embodiment 3.10
The microbial inoculum that the embodiment 1 that splashes in water obtains makes that Bdellovibrio telotroch concentration reaches 10 in the water body
3Pfu/mL, duration of test do not change water, and simultaneously feeding soaks the conventional turbot pellet of 30min through the Bdellovibrio telotroch, bait throwing in every day three times, early, middle and late each once (17:00), the process of at every turn throwing something and feeding continues 30min for 7:00,12:00.The mass fraction of daily ration of feeding and fish average weight is 4%, perfuses full pond.Used Bdellovibrio telotroch concentration reaches 10
3Pfu/mL.
Embodiment 3.11
The microbial inoculum that the embodiment 1 that splashes in water obtains makes that Bdellovibrio telotroch concentration reaches 10 in the water body
5Pfu/mL, duration of test do not change water, and simultaneously feeding soaks the conventional turbot pellet of 30min through the Bdellovibrio telotroch, bait throwing in every day three times, early, middle and late each once (17:00), the process of at every turn throwing something and feeding continues 30min for 7:00,12:00.The mass fraction of daily ration of feeding and fish average weight is 4%, perfuses full pond.Used Bdellovibrio telotroch concentration reaches 10
5Pfu/mL.
Embodiment 3.12
The microbial inoculum that the embodiment 1 that splashes in water obtains makes that Bdellovibrio telotroch concentration reaches 10 in the water body
7Pfu/mL, duration of test do not change water, and simultaneously feeding soaks the conventional turbot pellet of 30min through the Bdellovibrio telotroch, bait throwing in every day three times, early, middle and late each once (17:00), the process of at every turn throwing something and feeding continues 30min for 7:00,12:00.The mass fraction of daily ration of feeding and fish average weight is 4%, perfuses full pond.Used Bdellovibrio telotroch concentration reaches 10
7Pfu/mL.
Experimental result
The sick cure rate (%) of the red mouth of table 2. turbot
The result shows, uses the Bdellovibrio telotroch can 100% to cure red mouth disease, and Bdellovibrio telotroch concentration is high more, and the sick recovery from illness of red mouth is fast more.Dipping and oral independent use all can reach the effect of the red mouth disease of treatment, but required time is longer, and both use simultaneously then can reach with lower concentration and cure red mouth disease faster.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spiritual essence of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. the Bdellovibrio telotroch is prevented and treated application in the agent of the red mouth germ of turbot in preparation.
2. prevent and treat application in the agent of the red mouth germ of turbot according to the described Bdellovibrio telotroch of claim 1 in preparation, it is characterized in that: described Bdellovibrio telotroch preparation method is:
The host is in the nutrient broth liquid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, culture fluid is through 4 ℃, behind the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB liquid nutrient medium, insert Bdellovibrio BDM01, under 25～35 ℃ of temperature, cultivate 36～48h with 150～300rpm shaking table, obtain containing the culture fluid of Bdellovibrio leech plastid and Bdellovibrio telotroch, culture fluid under 4 ℃ of temperature with 6000～8000rpm behind centrifugal 15～20min, supernatant is behind the centrifugal 15～20min of 16000～18000rpm, precipitation DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value are 7.2～7.6 phosphate buffer suspension, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL, this bacterium liquid are prevents and treats the agent of the red mouth germ of turbot.
3. Bdellovibrio telotroch according to claim 1 is prevented and treated application in the agent of the red mouth germ of turbot in preparation, it is characterized in that: described Bdellovibrio is Bdellovibrio (Bdellovibrio sp.) BDM01, be preserved in Chinese typical culture collection center on April 28th, 2008, deposit number is CCTCC NO:M 208066.
4. prevent and treat application in the agent of the red mouth germ of turbot according to the described Bdellovibrio telotroch of claim 1 in preparation, it is characterized in that: described Bdellovibrio telotroch is to splash to prevent and treat the agent of the red mouth germ of turbot in water body in the application that preparation prevents and treats in the agent of the red mouth germ of turbot, makes that Bdellovibrio telotroch final concentration is 10-10 in the water body
7Pfu/mL.
5. prevent and treat application in the agent of the red mouth germ of turbot according to the described Bdellovibrio telotroch of claim 1 in preparation, it is characterized in that: described Bdellovibrio telotroch is that feed is soaked 15～45min with preventing and treating the agent of the red mouth germ of turbot in the application that preparation prevents and treats in the agent of the red mouth germ of turbot, and Bdellovibrio telotroch concentration is 10-10 in the microbial inoculum
7Pfu/mL.
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