CN118086224A - Vibrio parahaemolyticus phage with cleavage spectrum multivalent and application thereof in sea cucumber culture - Google Patents
Vibrio parahaemolyticus phage with cleavage spectrum multivalent and application thereof in sea cucumber culture Download PDFInfo
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Abstract
The invention discloses a vibrio parahaemolyticus phage with a splitting spectrum multivalent property and application thereof in sea cucumber culture, belonging to the technical field of biology, wherein the phage is sent to China center for type culture Collection for preservation in 2024, and the preservation address is as follows: the preservation number of the Chinese university of Wuhan is CCTCC NO: m2024134. The phage VPP19 has better cracking effect on common pathogenic vibrio such as vibrio parahaemolyticus, vibrio lautus, vibrio alginolyticus, vibrio harveyi and vibrio robustus in the sea cucumber culturing process, and in addition, the vibrio parahaemolyticus phage VPP19 has high value-added titer, wide cracking spectrum, safety, high efficiency and better tolerance to temperature and pH value. The invention can effectively prevent diseases caused by common pathogenic vibrio in the sea cucumber culturing process, and provides excellent bacterial strain resources and biological preventive measures for large-scale fermentation production for phage therapy of the problem of the sea cucumber vibrio.
Description
Technical Field
The invention relates to the technical field of biology, in particular to vibrio parahaemolyticus phage with cleavage spectrum multivalent and application thereof in sea cucumber culture.
Background
At present, the scale and density of sea cucumber cultivation in China are larger and larger, but the cultivation is over-fast, the operation is not scientific, the importance degree of disease problems is insufficient, the cultivation technology level is uneven, the sea cucumber disease problems are inevitably caused to be serious, various obvious symptoms and the phenomenon of large-scale death of sea cucumbers occur, huge economic loss of farmers is caused, and the stable development of the industry is also seriously influenced. The skin rot syndrome of sea cucumber, also called skin disease and skin ulcer of sea cucumber, is the "first killer" in the sea cucumber cultivation process. Research on the beancurd sheet syndrome is mainly focused on etiology. Becker et al have originally reported skin ulcers, which are thought to occur as a result of co-infection with a variety of events and a variety of causative agents, including bacteria.
At present, the vibrio prevention and control of sea cucumber culture is mainly carried out by using antibiotics and probiotics, and according to various documents and related reports, the antibiotic resistance condition of the sea cucumber is more serious. The research on the skin formation of sea cucumber is carried out in Xiapu county of Fujian province 11 in 2023, the main pathogenic bacteria of the skin formation and enteritis of the sea cucumber are vibrio robustus, vibrio alginolyticus and vibrio harveyi, and the separated vibrio has high drug resistance to enrofloxacin. Therefore, it is necessary to develop a green and effective biological control method.
In the prior report, phage preparations for preventing and controlling one or more vibrio in the sea cucumber culturing process, such as vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi, vibrio robiosus and the like, are not available, and based on the phage preparations, the invention provides a vibrio parahaemolyticus phage with a cracking spectrum polyvalent and application of the phage in sea cucumber culturing.
Disclosure of Invention
In order to prevent the skin rot syndrome or other diseases caused by one or more vibrio such as vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi, vibrio robiosus and the like in the sea cucumber culture process and reduce the death rate caused by the diseases, one of the purposes of the invention is to provide a vibrio parahaemolyticus bacteriophage with a splitting spectrum multivalent;
the second object of the present invention is to provide the application of the vibrio parahaemolyticus phage VPP19 in preparing a medicament for treating or preventing diseases caused by one or more of vibrio parahaemolyticus, vibrio lautus, vibrio alginolyticus, vibrio harveyi, vibrio robustus and the like.
In order to achieve the above object, the present invention is realized by the following technical scheme: a vibrio parahaemolyticus phage with a splitting spectrum multivalent property comprises vibrio parahaemolyticus phage VPP19, the applicant separates a vibrio parahaemolyticus phage VPP19 from a water body in a sea area around a certain area of Jiangsu province, the phage form is shown in figure 1, plaque formed by the phage and host bacteria is transparent, round and 0.5mm in diameter, the phage is sent to China center for type culture collection for preservation at 2024, 1 month and 18 days, and the preservation address is: university of martial arts in chinese; the preservation number is CCTCC NO: m2024134, classification nomenclature: vibrio parahaemolyticus phage VPP19.
Further, a complex of the vibrio parahaemolyticus phage VPP19 is also included.
The application of the vibrio parahaemolyticus phage VPP19 or phage complex is applied to the sea cucumber culture industry to prepare bacteriostats of vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi and vibrio robustus.
Further, the method is also applied to the preparation of medicines for treating or preventing diseases caused by infection of vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi and vibrio robustus, wherein the diseases are sea cucumber beancurd skin syndrome or vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi and vibrio robustus and the like.
Further, it is also used for preparing feed or feed additive for antagonizing vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi and vibrio robustus.
The invention has the beneficial effects that:
1. The invention screens out the phage VPP19 of vibrio parahaemolyticus, vibrio lautus, vibrio alginolyticus, vibrio harveyi, vibrio robustum and other vibrio which can cause sea cucumber beancurd skin syndrome by multivalent lysis for the first time, the phage VPP19 has high cleavage rate to vibrio, the phage VPP19 has good effect on inhibiting different vibrio, and has great reference significance for the prevention, control and treatment of vibrio in sea cucumber culture or aquaculture in future.
2. The vibrio parahaemolyticus phage VPP19 with the cleavage spectrum multivalent property has high fermentation rate, and when the optimal multiplicity of infection MOI=0.01, the fermentation is carried out for 4 hours, the titer can reach 8.7X10 13 PFU/mL, and a theoretical basis is provided for industrial fermentation.
3. The vibrio parahaemolyticus phage VPP19 with the cleavage spectrum multivalent property has good environmental tolerance, and the titer of the vibrio parahaemolyticus phage VPP19 is not obviously changed by orders of magnitude when the vibrio parahaemolyticus phage VPP19 is processed for 2 to 24 hours within the pH range of 4 to 11; the vibrio parahaemolyticus phage VPP19 has longer survival time at the temperature of 45 ℃ and below, has no obvious order of magnitude difference in the phage titer after 24 hours at the temperature of 4 ℃, and has good stability at the temperature of 4-37 ℃.
Drawings
FIG. 1 is a plaque of the Vibrio parahaemolyticus phage VPP 19;
FIG. 2 shows the titers of the vibrio parahaemolyticus phage VPP19 under different infection complex conditions;
FIG. 3 is a test of the stability of the vibrio parahaemolyticus phage VPP19 at different pH values;
FIG. 4 is a test of the stability of the vibrio parahaemolyticus phage VPP19 at different temperatures;
FIG. 5 is a schematic diagram showing fermentation kinetics of Vibrio parahaemolyticus phage VPP 19;
FIG. 6 is a diagram showing annotation of DNA gene sequence information of the vibrio parahaemolyticus phage VPP 19.
Detailed Description
The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand.
Referring to fig. 1 to 6, the present invention provides the following technical solutions:
Example 1:
Separation and purification of vibrio parahaemolyticus phage VPP19
30 Parts of water sample is collected in a water body of a culture area in a certain eastern city of Shandong province, 30mL of water sample is centrifuged at 600 rpm for 10min, a supernatant is taken and sterilized by a 0.22 mu m filter, 5mL of filtrate is taken and evenly mixed with 0.5mL of vibrio parahaemolyticus (the concentration is about 5 multiplied by 108 CFU/mL), and the mixture is subjected to shaking culture in a shaking table at 200rpm at 37 ℃ for overnight fermentation. The fermentation broth was centrifuged at 6000rpm for 10min, the supernatant was sterilized with a 0.22 μm filter, 100. Mu.L of the filtrate was mixed with 300. Mu.L of Vibrio parahaemolyticus (concentration: about 5X 108 CFU/mL) uniformly, 2% sodium chloride TSB semisolid (containing 0.65% agar) was added, gently inverted and homogenized, poured onto a 2% sodium chloride TSA bottom plate, and after solidification, the petri dish was placed in a 37℃incubator for overnight culture. The clear and bright plaque spots are picked up, and after shaking and desorption in 1mL SM solution, the plaque spots are sterilized by a microporous filter membrane with the thickness of 0.22 mu m to obtain phage filtrate, the phage filtrate is inoculated into 5mL 2% sodium chloride TSB liquid culture medium, 500 mu L of corresponding host vibrio parahaemolyticus bacteria liquid is added and mixed uniformly, shaking culture is carried out at 200rpm at 37 ℃ for overnight, centrifugation is carried out at 6000rpm for 10min, the supernatant is taken out, and the microporous filter membrane with the thickness of 0.22 mu m is sterilized to obtain phage filtrate, and the plaque morphology is observed by adopting a double-layer flat plate method. And repeating the operation for 3-5 times to obtain the plaque with consistent shape and size.
A vibrio parahaemolyticus phage VPP19 is separated from the collected water sample, and is transparent, round and 0.5mm in diameter with plaque formed by the host bacteria as shown in figure 1. The phage was sent to China center for type culture Collection, and named after classification, at 2024, 1 month, 18 days: vibrio parahaemolyticus phage (Vibrio parahaemolyticusphage) VPP19, address: chinese, university of martial arts, deposit number: cctccc NO: m2024134.
Example 2:
Determination of the potency of Vibrio parahaemolyticus phage VPP19 under different infection Complex conditions
Host bacteria vibrio parahaemolyticus VPFJ of vibrio parahaemolyticus phage VPP19 is inoculated in an EP tube with 3mL of 2% sodium chloride TSB, and shake culture is carried out for 5-8h at 37 ℃ to obtain VPFJ bacterial liquid. Bacterial solutions were inoculated at 2% inoculum size into 100mL of TSB medium, phage VPP19 was inoculated until VPGX grew to log initiation, and 3 replicates per group were run according to different multiplicity of infection (multiplicity of infection, MOI; MOI = phage number/bacterial number, see figure 2 in particular). Shaking culture was performed at 200rpm in a shaking table at 37℃for 12 hours.
The results are shown in fig. 2, where the fermentation titer is up to 8.7x10 13 PFU/mL when moi=0.01.
Example 3:
PH stability test of Vibrio parahaemolyticus phage VPP19
Adding 0.1mL of vibrio parahaemolyticus phage VPP19 fermentation broth into 0.9mL of TSB liquid culture medium (with the titer of 5 x10 8 pfu/mL after dilution) with the pH of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 respectively, placing into a 25 ℃ incubator for co-incubation for 0h, 1h, 2h and 24h, sampling, adjusting the pH to the original culture medium Ph=7.4 by using hydrochloric acid and sodium hydroxide solution respectively, performing multiple ratio dilution, and measuring phage titer by using a double-layer plate method; each point was averaged in duplicate and the experiment was repeated 3 times.
As a result, as shown in FIG. 3, the potency of the vibrio parahaemolyticus phage VPP19 was not significantly changed by an order of magnitude when it was treated for 1 to 24 hours in the pH range of 4 to 11.
Example 4:
temperature stability test of Vibrio parahaemolyticus phage VPP19
The vibrio parahaemolyticus phage VPP19 fermentation broth was filtered by centrifugation, and 1mL was dispensed into 1.5mL centrifuge tubes (initial titer 3.5 x 10 8 pfu/mL). Centrifuge tubes containing phage were placed at constant temperature of 4, 25, 37, 45, 55, 65, 75℃for 0h, 1h, 2h, 24h, 48h, 92h, 1w, 2w, 4w, 8w, 12w, 16w, 20w, 24w, 28w, 32w, 36w, 40w, 44w, 48w, 52w, respectively. After the action time is over, the sample tube is taken out and immediately placed in an ice bath for cooling, and phage titer is measured by adopting a double-layer flat plate method after proper dilution. Each point was averaged in duplicate and the experiment was repeated 3 times.
As shown in FIG. 4, the vibrio parahaemolyticus phage VPP19 has longer survival time at 45 ℃ and below, has no obvious magnitude order difference of the phage titer after 48 hours at 4 ℃, has good stability at 4-37 ℃ and can be effectively stored.
Example 5:
host range of phage VPP19
The vibrio used in the experiment is parahaemolyticus, splendidus, alginolyticus, harveyi and tonic.
Respectively picking 20 strains of vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi and vibrio robustum, respectively inoculating into 3mL of EP tube of 2% sodium chloride TSB, and oscillating at a constant temperature of 200rpm for 8 hours at 37 ℃ to obtain vibrio bacterial liquid; preparing enough TSA bottom plate, uniformly mixing 0.5mL of vibrio bacterial liquid with 6mL of 2% sodium chloride TSB semisolid culture medium (containing 0.65% agar) at about 50 ℃, and slowly spreading on the 2% sodium chloride TSA bottom plate; after the plate culture medium is solidified to normal temperature, 5 mu L of phage VPP19 with the titer of 5 multiplied by 10 8 PFU/ml is respectively dripped on the plate, after the air drying, the plate is placed in a biochemical incubator with constant temperature of 37 ℃ for culturing for about 8 hours, and the result is observed. Each set of experiments was repeated 3 times.
As shown in Table 1, the vibrio parahaemolyticus phage VPP19 can lyse 19 strains of 20 strains of vibrio parahaemolyticus with a lysis rate of 95%; 17 strains in 20 vibrio robustus can be cracked, and the cracking rate is 85%; can crack 18 strains in 20 strains of vibrio splendidus with a cracking rate of 90 percent; 18 strains in 20 strains of vibrio alginolyticus can be cracked, and the cracking rate is 90%; can crack 18 strains in 20 strains of Vibrio harveyi with a cracking rate of 90 percent. The vibrio parahaemolyticus phage VPP19 has great application value in vibrio prevention and control of the beancurd sheet syndrome in the sea cucumber culture process. In the lysis experiments on all 100 strains of Vibrio, VPP19 was able to lyse 90 of them with a lysis rate of 90%. Experimental results prove that the phage has very outstanding effects of resisting the inhibition of vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi and vibrio robustus, and has great value for the prevention and control of vibrio in sea cucumber and related aquaculture industry in future.
TABLE 1 lytic Effect of phage VPP19 on Vibrio 100 strains
Note that: "-" indicates no lysis, "+" indicates lysis, and plaque is clear; "++" indicates lysis and plaque is clear.
Example 6:
Fermentation preparation of vibrio parahaemolyticus phage VPP19
The single colony of vibrio parahaemolyticus VPFJ is picked up on a plate, inoculated in 3mL of 2% sodium chloride TSB culture medium, and cultured for 8 hours at 200rpm at 37 ℃ to obtain host bacterial liquid. The bacterial suspension was inoculated in an inoculum size of 2% into 120mL of 2% sodium chloride TSB medium, and cultured at 37℃and 200rpm until the early logarithmic phase, and the bacterial liquid OD 600 was 0.1.
The industrial fermentation is a 10L fermentation tank, the fermentation system is 6L, the culture medium is TSB, the initial pH value is 7.4, the vibrio parahaemolyticus is inoculated according to the inoculation amount of 1% by adopting a flame method, bacteriophage VPP19 is added into the fermentation system under the optimal infection condition MOI=0.01, sterile air is introduced in the fermentation process, and 3 per mill of defoamer is added, so that the fermentation preparation time is 12h. From the beginning of fermentation, 20ml of fermentation broth was taken from the sampling port at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, centrifuged at 6000rpm for 10min, and the supernatant was sterilized with a microporous filter membrane of 0.22 μm to obtain a phage-containing filtrate, and the titer thereof was measured, as described in example 2.
After fermentation, taking out all the mixed liquid of phage and host bacteria from the sampling port, inoculating into a sterile container, centrifuging at 6000rpm for 10min, taking supernatant, filtering into a sterile filter device by a vacuum pump, obtaining phage fermentation liquid, and preserving at 4 ℃.
As can be seen from FIG. 6, the titer of the vibrio parahaemolyticus phage VPP19 reaches the maximum of 7.4X10 13 PFU/mL in 4h of fermentation preparation, and the titer is stabilized at 10 13 PFU/mL in the period of 5h to 12 h; from the results, it is clear that the industrial production of phage by fermentation method is feasible, and provides effective reference data for phage production.
Example 7:
experiment for preventing vibrio composite infection in apostichopus japonicus by vibrio parahaemolyticus phage VPP19
In this section, experiments were carried out with reference to the toxicity attack and clinical reference basis of Vibrio lautus in Apostichopus japonicus (Jing Kailin, research on the protective effect of specific egg yolk antibody on Vibrio lautus infected apostichopus japonicus [ D ]. University of continuous company, 2016.) with respect to the toxicity attack and clinical reference basis of Vibrio lautus in Apostichopus japonicus, the sea cucumber infected with Vibrio lautus would have shaking head and vomiting of intestines, large-area epidermis ulceration would appear in late stage, and finally autolysis death would occur.
The stichopus japonicus in the experiment is purchased in a farm of Fujian Xiapu, the appearance of the purchased stichopus japonicus is normal, the feeding condition is normal, the average weight is 7.8+/-0.33 g, 250 stichopus japonicus are placed in an aquarium to be suitable for feeding for 7d before the experiment, the salinity of a water body is 2.5%, the pH value is 7.6+/-0.1, the temperature is 17 ℃, the dissolved oxygen is 6.6+/-0.1 mg/L, and the death rate is stabilized after the death rate is stabilized (extremely low value), and the experiment is carried out. Vibrio parahaemolyticus, vibrio robustus, vibrio splendidus, vibrio alginolyticus and vibrio harveyi used in this example are VPFJ47, VFFJ10, VSFJ1, VAJS178 and VHFJ25, respectively, and the phage selected is vibrio parahaemolyticus phage VPP19 of the present invention.
The experimental design was as follows, the apostichopus japonicus was equally divided into 5 groups:
1) Control group: each apostichopus japonicus is injected with an equal volume of physiological saline (the same toxin attacking mode is adopted below);
2) Attack the poison group: each apostichopus japonicus is injected with 100 mu L of bacterial liquid with the concentration of 1X 10 6 CFU/mL. (VPFJ, VFFJ, 10, VSFJ, VAJS, 178, VHFJ25, mixing, supra);
3) Treatment group: each apostichopus japonicus is injected with 100 mu L of bacterial liquid with the concentration of 1X 10 6 CFU/mL; after 6h, phage VPP 191X 10 6 PFU/treatment alone were injected;
4) Preventive group: 1 day before the beginning of the experiment, phage VPP191×10 6 PFU/each stichopus japonicus is injected, and then 100 μl of bacterial liquid of 1×10 6 CFU/mL is injected into each stichopus japonicus;
5) Antibiotic group: each apostichopus japonicus is injected with 100 mu L of bacterial liquid with the final concentration of 1X 10 6 CFU/mL. After 12h, doxycycline was added in the indicated amounts for treatment.
The experiment lasts for 7 days, the symptom and death number of the apostichopus japonicus are recorded, after the observation date is over, the tissues of each group of apostichopus japonicus are respectively taken and mixed and ground, and the total counting of vibrio is carried out by using a TCBS plate. The survival rate of apostichopus japonicus was calculated for each group in the following manner.
Total A: the total number of apostichopus japonicus in each group is tested;
Death of A: the total number of stichopus japonicus death and panning is tested in each group;
C: the survival rate of apostichopus japonicus in each group is tested,%.
The experimental results are shown in table 3, the survival rate of the blank group is 98%, and the survival rate of the apostichopus japonicus in the virus attack group is 0%; the survival rate of the treatment group is 88%, the survival rate of the prevention group is 92%, and the survival rate of the antibiotic group is 90%. Experimental results show that vibrio parahaemolyticus phage VPP19 can effectively kill vibrio, the content of vibrio is effectively reduced in prevention and treatment groups, the survival rate of apostichopus japonicus is improved, the embodiment provides an important reference value for preventing and treating the skin rot syndrome of the sea cucumber in the future, and the application of the bacterial strain to preventing and treating the vibrio parahaemolyticus diseases of apostichopus japonicus and aquatic animals thereof can be used as a biological antibacterial agent.
TABLE 2 control effect of phage VPP19 on Vibrio in Apostichopus japonicus
Example 8:
genome functional annotation of Vibrio parahaemolyticus phage VPP19 whole genome
Phage VPP19 showed a genome length of 46284bp, a GC content of 49.2%, a total of 83 ORFs (Open READING FRAME, ORFS) and 64 CDS (coding sequences), the longest ORFs being 2730bp in length and the shortest ORFs being 303bp in length, using the RAST online annotation tool. By BLAST alignment, 55 out of 64 CDSs detected are CDSs of the determined functional protein, e.g., 26 are phage protein (phage protein) CDSs and 13 putative protein CDSs; the gene genealogy is shown in FIG. 6.
While the fundamental and principal features of the invention and advantages of the invention have been shown and described, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (5)
1. A vibrio parahaemolyticus bacteriophage having a lytic spectrum multivalent nature, characterized in that: the method comprises the steps that the vibrio parahaemolyticus phage VPP19 is separated from a water body of a certain surrounding sea area in Jiangsu province by the applicant, and the vibrio parahaemolyticus phage VPP19 is sent to a China center for type culture collection for preservation at the 1 st month of 2024, wherein the preservation address is as follows: university of martial arts in chinese; the preservation number is CCTCC NO: m2024134, classification nomenclature: vibrio parahaemolyticus phage VPP19.
2. Phage VPP19 according to claim 1, characterized in that: also included are complexes of vibrio parahaemolyticus phage VPP 19.
3. Use of the vibrio parahaemolyticus phage VPP19 or phage complex according to claim 2, characterized in that: is applied to the sea cucumber culture industry to prepare bacteriostats of vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi and vibrio robustus.
4. The use of a vibrio parahaemolyticus phage VPP19 or phage complex according to claim 3, characterized in that: and also can be used for preparing medicines for treating or preventing diseases caused by infection of vibrio parahaemolyticus, vibrio lautus, vibrio alginolyticus, vibrio harveyi and vibrio robustus, wherein the diseases are sea cucumber beancurd skin syndrome or vibrio parahaemolyticus, vibrio lautus, vibrio alginolyticus, vibrio harveyi and vibrio robustus.
5. The use of a vibrio parahaemolyticus phage VPP19 or phage complex according to claim 3, characterized in that: but also can be used for preparing feed or feed additive for antagonizing vibrio parahaemolyticus, vibrio splendidus, vibrio alginolyticus, vibrio harveyi and vibrio robustus.
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