CN109880767A - One plant for preventing or treating the bacterial strain of vibriosis penaeus - Google Patents
One plant for preventing or treating the bacterial strain of vibriosis penaeus Download PDFInfo
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- CN109880767A CN109880767A CN201910220408.2A CN201910220408A CN109880767A CN 109880767 A CN109880767 A CN 109880767A CN 201910220408 A CN201910220408 A CN 201910220408A CN 109880767 A CN109880767 A CN 109880767A
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Abstract
The present invention provides one plant for preventing the bacillus of vibriosis penaeus, it is BHK201801 plants of bacillus, the China Committee for Culture Collection of Microorganisms's common micro-organisms center of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica is deposited on March 6th, 2019, deposit number is CGMCC NO:17307.Bacillus provided by the present invention can play the role of effective inhibiting effect to the vibrio frequens in litopenaeus vannamei breeding process, make it in prawn Colonization inside plants to play prevention litopenaeus vannamei vibriosis by the way that bacillus to be added in feed to and fed prawn.
Description
Technical field
The invention belongs to beneficial bacterium screening and applied technical fields, and in particular to one plant for preventing or treating prawn vibrios
The bacterial strain of disease.
Background technique
Litopenaeus vannamei (Litopenaeus vannamai) is because of its delicious meat, the spies such as the speed of growth is fast, resistance is strong
Point after introducing China from the 1980s, rapidly develops as the principal item of China's prawn culturing, its yield accounts for prawn at present
75% or more of cultured output.In recent years, with the rapid development of litopenaeus vannamei aquaculture, disease problem is increasingly prominent, sternly
The interests at prawn culturing family are damaged again, and shrimp culture industry is hindered further to develop.
Vibriosis is one of the Major Diseases during prawn culturing, has popular wide, disease incidence height, harm big and dead
The features such as rate is high.Currently, being determined that the cause of disease of vibriosis penaeus can be caused to have Vibrio harveyi (Vibrio harveyi), pair
Hemolysis vibrion (V.parahaemolyticus), vibrio alginolyticus (V.alginolyticus), Vibrio anguillarum (V.anguillarum),
Vibrio vulnificus (V.vulnificus), Kan Beishi vibrios (V.campbelli) and comma bacillus (V.cholerae) etc..Antibiotic
Equal drugs are the effective means for preventing and treating vibriosis penaeus, but equally can also cause the appearance of drug-resistant bacteria and cause a series of environment
Problem even endangers human health.Therefore, it needs to find a kind of new environmental-friendly effective means to support for litopenaeus vannamei
The prevention and treatment of vibriosis during growing.
Probiotics has been demonstrated to play a protective role to host by a variety of mechanism of action, it is considered to be antibiotic
Favorable substitutes.Currently, existing research shows that probiotics can play a positive role during prawn culturing, but probiotics
Selection it is most important, it is many commercialization probiotics practical application effect it is undesirable, this is close with the type of probiotics and source
It is related.Moreover, research discovery is relative to land source probiotics, the bacterium isolated from aquiculture animal or environment has more
Add significant probiotic effects.Therefore, screening obtains having the bacillus of vibrios antagonistic activity for right out of healthy prawn body
The prevention and treatment of vibriosis is most important during shrimp aquaculture.
Summary of the invention
It, can be effectively to prawn the purpose of the present invention is to provide one plant for preventing the bacillus of vibriosis penaeus
Vibrio frequens in breeding process play inhibiting effect, thus the high mortality of prawn caused by being effectively reduced because of vibriosis.
Bacillus provided by the present invention, be BHK201801 plants of bacillus (Bacillus sp.), in 2019 3
The moon is deposited in the China Microbiological bacterium of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica on the 6th
Kind preservation administration committee common micro-organisms center, deposit number are CGMCC NO:17307；
Bacillus of the invention is screened from healthy litopenaeus vannamei body, can be used for inhibiting during prawn culturing
Vibrio frequens；
Bacillus provided by the present invention is for being added in feed, and the feed containing bacillus is for preventing vannamei boone
Vibriosis penaeus.
Bacillus provided by the present invention can play effective suppression to the vibrio frequens in litopenaeus vannamei breeding process
Production is used, and so that it is prevented all receive in prawn Colonization inside plants to play by the way that bacillus to be added in feed to and fed prawn
The effect of shore vibriosis penaeus.
Specific embodiment
The present invention is described in detail below with reference to embodiment.
Embodiment 1: the screening and identification of the bacillus with fungistatic effect
1, sample acquires
Sample is that the litopenaeus vannamei for occurring to survive after vibriosis takes liver pancreas after litopenaeus vannamei is carried out surface sterilization
Gland and muscle grind in sterile saline and are diluted to suitable concentration, then draw appropriate homogenate with sterile pipette tips and are coated with
In on LB solid medium (pure water 1000mL, peptone 10g, yeast powder 5g, agar 20g, NaCl 30g, pH 7.2~7.4),
It is cultivated at 28 DEG C for 24 hours, prepares further screening.
2, Enrichment of bacteria
With aseptic inoculation ring, picking form single colonie of different sizes is placed in the LB liquid of 10mL on LB solid medium
In culture medium (pure water 1000mL, peptone 10g, yeast powder 5g, NaCl 30g, pH 7.2~7.4), be placed on shaking table (28 DEG C,
180r/min) enterprising oxygen culture of acting charitably obtains pure bacterial strain after culture for 24 hours.It finally simultaneously can success from picking on LB solid medium
The bacterium of enrichment has 37 plants, numbers respectively are as follows: BD1, BD2, BD3, BD4, BD5, BD6, BD7, BD8, BD9, BD10, BD11,
BD12、BD13、BD14、BD15、BD16、BD17、BD18、BD19、BD20、BD21、BD22、BD23、BD24、BD25、BD26、
BD27、BD28、BD29、BD30、BD31、BD32、BD33、BD34、BD35、BD36、BD37。
3, the separation of bacillus
Configuration stimulation gemma generate culture medium, formula are as follows: seawater 1000mL, peptone 10g, yeast extract 3g, starch 3g,
MgSO4 0.1g、KH2PO4 1.5g、Na2HPO4 2g、NaCl30g、pH 7.8。
By the single colonie isolated be inoculated in stimulation gemma generate culture medium in, in constant-temperature table (28 DEG C, 180r/
Min it) cultivates for 24 hours.Water-bath 20min is under the conditions of cultured bacterium solution is placed in 80 DEG C to screen the bacterial strain that can generate gemma.By water
Bacterium solution after bath is coated on LB solid medium after being diluted to suitable concentration with sterile saline, is cultivated under the conditions of 28 DEG C
24h。
By screening, isolate 15 plants of doubtful bacillus, respectively BD1, BD2, BD3, BD4, BD15, BD18,
BD19、BD22、BD23、BD24、BD27、BD28、BD31、BD36、BD37。
4, the screening of the bacillus with fungistatic effect
With C-L plants of Kan Beishi vibrios, LY-L plants of vibrio parahaemolytious, HAH plants of Vibrio harveyi, New Caledonia vibrios
(V.neocaledonicus) W-B plants, W-S plants of staphylococcus xylosus (Staptococcus xylosus), luminous Vibrio Y-1,
What the laboratories such as Y-2, Y-92 plants were screened early period can cause the bacterial strain of litopenaeus vannamei vibriosis as pathogen.
It screens to obtain the bacillus with fungistatic effect using filter paper enzyme.Pathogen and bacillus are inoculated in LB
In fluid nutrient medium, after (28 DEG C, 180r/min) of constant-temperature table cultures for 24 hours, 200 μ l pathogen bacterium solutions are taken to be evenly coated in LB solid
Body media surface.After bacterium solution is dry, aseptic filter paper piece (6mm) is affixed on top, and 10 μ L bacillus bacterium solutions are added to
On filter paper, blank control group adds the sterile LB liquid medium of same volume.After being cultivated for 24 hours under the conditions of 30 DEG C, observation
Whether inhibition zone is generated and with vernier caliper measurement antibacterial circle diameter.
The experimental results showed that there is 3 bacillus (BD4, BD22, BD28) that can have antibacterial effect at least one kind of pathogen
Fruit, wherein BD28 effect is best, there is apparent inhibitory effect, antibacterial circle diameter point to C-L, HAH, W-S, Y-1, Y-2, Y-92
It Wei not 6.51mm, 13.76mm, 10.29mm, 6.71mm, 8.77mm, 7.60mm.Followed by BD22 has Y-2 and Y-92 bright
Aobvious inhibitory effect, antibacterial circle diameter are respectively 7.96mm and 7.02mm；And BD4 only has inhibitory effect to W-S, inhibition zone is straight
Diameter is 8.07mm.
5. Bacteria Identification
It is white on LB solid medium to choose the best bacterial strain BD28 of fungistatic effect, opaque, surface is smooth and side
Edge is neat.Gram's staining result is the positive, and direct rod shape has gemma without pod membrane.
The gene sequencing of 16S rDNA is carried out to bacterial strain BD28, and carries out homologous sequence ratio with BLAST in NCBI
It is right, Phylogenetic Analysis then is carried out to determine bacterial strain type to sequencing result with MEGA 5.0.
Identified, bacterial strain BD28 and Sha Fu bacillus have highest similitude, are named as bacillus (Bacillus
Sp.) BHK201801 plants, it is micro- that Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences are deposited on March 6th, 2019
Biological study China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number be CGMCC NO:
17307。
Embodiment 2: not homologous bacillus to prawn grow, survival rate and stress influence
With from intracorporal bacillus BHK201801 plants of healthy prawn and land source bacillus LD05 as experiment
Bacterial strain.BHK201801 plants of bacillus and LD05 are inoculated in after being cultivated for 24 hours in LB liquid medium, centrifugation (2392 × g,
10min) and with sterile saline be diluted to suitable concentration, be stirred homogeneously into prawn mixed feed, make its final concentration of 1
×107cfu/g.It is spare under the conditions of feed is dried under the conditions of 35 DEG C and is stored in 4 DEG C.Only to add equivalent sterile physiological
The feed of salt water is as control group feed.With corresponding feed feeding prawn totally 50 days, the state of prawn is observed at any time, records prawn
Growing state and survival rate.Daily management during prawn culturing includes artificial bait throwing in, changes water suction bottom, daily fishing pond, growth
Monitoring etc..
26-28 DEG C of temperature, salinity 26-30, pH 7.8-8.3, dissolved oxygen > 5mg/L are maintained in breeding process, feed 4 daily
Meal, is satiated with food and feeds, and residual bait and excreta are sucked out after feeding 1h and supplements fresh seawater.The operation of fishing pond is carried out daily, pulls dead shrimp out.
Prawn culturing is first stopped eating 1 day in circulation, starts to feed after prawn adapts to environment.First feed
Prawn mixed feed feeds 4 meal daily, and the time is respectively daily 7:00,11:00,14:00,18:00.Feed mixed feed 7
Start to feed the mixed feed containing not homologous bacillus to experimental group prawn after it.Prawn immunological stress is observed in breeding process
Reaction measures body length, weight, survival rate and the immune enzyme activity etc. of each group prawn after 50 days.
Culture efficiency is analyzed as follows:
1. bacillus BHK201801 plants of feeding significantly improves prawn growth: measurement prawn body length and body after cultivation 50 days
It weighs and calculates specific growth rate, the results showed that, relative to control group, bacillus BHK201801 plants of feeding makes prawn body is long to increase
Add 3.1%, weight gain 15.5%, specific growth rate increases by 28.5%.Feed the body of the prawn of land source bacillus LD05
Length, weight etc. have different degrees of reduction compared with control group.
2. BHK201801 plants of bacillus are conducive to surviving for prawn: bacillus BHK201801 plants of feeding survives prawn
Rate improves 25% compared with control group, and the bacillus LD05 for feeding land source makes prawn survival rate improve 5% compared with control group.
3. BHK201801 plants of bacillus can reduce prawn stress reaction: in breeding process, feeding the prawn of bacillus
Apparent stress reaction is not shown, and the part prawn of control group and land source bacillus LD05 group is before culture experiment
Phase, show obvious stress reaction, including antenna and tail fan it is rubescent, jejunum sky stomach etc., in cultivation mid-term stress reaction
It fades away.
Embodiment 3: bacillus is for preventing the vibriosis penaeus as caused by Vibrio harveyi
Prawn (bacillus group) is fed with mixed feed of the method preparation containing bacillus in embodiment 2, with not
Mixed feed containing bacillus is to control, after two groups of prawns feed 15 days, is infused with HAH plants of Vibrio harveyi for pathogen
Challenge viral dosage is penetrated, which is proved to be able to cause prawn hepatopancrease lesion.After pathogen is cultivated for 24 hours in LB culture medium, from
The heart (2392 × g, 10min) and with sterile saline be diluted to concentration be 1 × 107The bacteria suspension of cfu/mL.Injection site is
Between prawn abdomen the 4th and the 5th uromere, injection dosage is 50 μ L.Daily management during prawn culturing include artificial bait throwing in,
Water suction bottom, daily fishing pond, growth monitoring etc. are changed, injection attacks poison and observes prawn incidence in the process and record prawn accumulation death
Rate.
24-26 DEG C of temperature, salinity 26-30, pH 7.7-8.1, dissolved oxygen > 5mg/L are maintained in breeding process, feed 4 daily
Meal, is satiated with food and feeds, and residual bait and excreta are sucked out after feeding 1h and supplements fresh seawater.The operation of fishing pond is carried out daily, pulls dead shrimp out.
It attacks during poison, it is consistent in condition of water quality and breeding process, it is lasting to inflate, it does not feed.
It is as follows to attack malicious interpretation of result:
1. bacillus BHK201801 plants of reduction hepatopancrease lesion degree of feeding: attacking discovery feeding gemma bar during poison
BHK201801 plants of bacterium of prawn hepatopancrease lesion degree is considerably lighter, hepatopancrease and shrimp body light discolouration, and control group prawn liver
Pancreas and shrimp body obviously turn yellow, and hepatopancrease erosion is fuzzy, jejunum sky stomach.
2. feeding the bacillus BHK201801 plant reduction prawn death rate: after injecting pathogen HAH, two groups pairs when preceding 6h
Shrimp death condition is without significant difference, and control group prawn cumulative mortality is apparently higher than the prawn of feeding bacillus when 9h, arrives
When 12h, the prawn cumulative mortality for feeding bacillus is substantially less than control group.Compared to control group, bacillus is fed
BHK201801 plants make prawn cumulative mortality have dropped 36.8%.
It is above-mentioned the result shows that bacillus provided by the present invention can be to the common arc in litopenaeus vannamei breeding process
Bacterium plays effective inhibiting effect, by bacillus is added in feed and is fed prawn make its in prawn Colonization inside plants from
And play the role of preventing litopenaeus vannamei vibriosis.
Claims (6)
1. a kind of bacillus, which is characterized in that the bacillus is bacillus (Bacillus sp.) BHK201801
Strain is deposited on March 6th, 2019 Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica
China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are CGMCC NO:17307.
2. bacillus described in claim 1 is screened from healthy litopenaeus vannamei body.
3. the application of vibrio frequens disease of the bacillus described in claim 1 during inhibiting prawn culturing.
4. application of the bacillus described in claim 1 as feed addictive.
5. a kind of feed addictive of litopenaeus vannamei, which is characterized in that the feed addictive is using claim 1 institute
The bacillus preparation stated.
6. a kind of feed of litopenaeus vannamei, which is characterized in that be added to feed described in claim 5 in the feed and add
Add agent.
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| CN112646786A (en) * | 2021-01-21 | 2021-04-13 | 海南海壹水产种苗有限公司 | Rapid preliminary separation method for vibrio kammaticus phage |
| CN112725217A (en) * | 2020-11-19 | 2021-04-30 | 中国海洋大学 | Composite flora for prawn culture water regulation |
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