Application of bacillus velezensis in biological prevention and treatment of aeromonas disease of aquatic animals

Abstract

The invention relates to the technical field of aquatic animal breeding, in particular to application of bacillus velezensis in biological prevention and treatment of aeromonas of aquatic animals. According to the application, the bacillus velezensis is found to have a relatively strong antagonistic effect on common aquatic pathogenic bacteria such as aeromonas hydrophila, aeromonas sobria, aeromonas cainea, aeromonas veronii, aeromonas salmonicida, pseudomonas fluorescens, shewanella putrefaciens, plesiomonas shigelloides and edwardsiella catfish, and the strain has no hemolytic activity; the strain does not carry common virulence genes of bacillus, is sensitive to most antibacterial drugs, and has no pathogenicity to grass carp. Therefore, the bacillus velezensis is an aeromonas antagonistic bacterium with good safety, can be used as a potential probiotic for preventing and treating aeromonas diseases of aquatic animals, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquaculture, and particularly relates to application of Bacillus velezensis in biological prevention and treatment of Aeromonas disease of aquatic animals. BACKGROUND

[0002] Aeromonas is a kind of gram-negative bacillus, which is widely distributed in soil, water environment and fish, shrimp and shellfish, etc. and is a common opportunistic pathogen in aquaculture, including Aeromonas hydrophila, Aeromonas sobria, Aeromonas veronii, Aeromonas caviae, Aeromonas schubertii and Aeromonas salmonicida. Aeromonas can produce enterotoxin, hemolysin, extracellular protease and other pathogenic factors during proliferation, and often causes large-scale disease and death of grass carp (Ctenopharyngodon idella), Hypophthalmichthys molitrix, crucian carp (Carassius auratus), Channel Catfish and other farmed fish, seriously affecting the healthy and sustainable development of aquaculture industry.

[0003] At present, the prevention and treatment of Aeromonas disease of aquatic animals mainly relies on antibiotics and chemical drugs, but the large use of antibiotics or chemical drugs will make the drug resistance of pathogenic bacteria continue to increase, the multiple drug resistance phenomenon become more serious and the quality and safety of aquatic products be affected. Biological prevention and control is to achieve the effect of "controlling bacteria with bacteria" under natural conditions, to inhibit the growth of pathogenic bacteria by using antagonistic bacteria, so as to reduce the occurrence of diseases.

[0004] Bacillus is a common probiotic bacteria in aquaculture, which has the advantages of improving the breeding environment, inhibiting the proliferation of pathogenic bacteria, promoting animal growth, and improving disease resistance, and is a potential biological prevention and control resource. At present, scholars have carried out screening and application research on Bacillus antagonistic to water-borne pathogenic bacteria. Kewcharoen et al. isolated a Bacillus subtilis (Bacillus subtilis) AQAHBS001 from the body of shrimp, found that it had antagonistic activity to many strains of pathogenic Vibrio Parahaemolyticus (Vibrio Parahaemolyticus), and could significantly improve the growth efficiency of Litopenaeus vannamei when it was mixed into feed and fed to Litopenaeus vannamei; Huang Xiaodong et al. screened a Baclicus lincheniformis (Baclicus lincheniformis) PNB3 from the breeding sediment, which could effectively inhibit the growth of Citrobacter freundii (Citrobacter freundii); Wang Jinyan et al. isolated a Bacillus velezensis (Bacillus velezensis) DY-6 from the breeding pond of Apostichopus japonicus, which had obvious antagonistic effect on Pseudoalteromonas nigrifaciens, Vibrio alginolyticus and other bacteria.

[0005] However, at present, there are few reports on Bacillus antagonistic to fish-borne pathogenic Aeromonas. SUMMARY

[0006] The application provides an application of Bacillus velezensis in biological prevention and treatment of Aeromonas disease of aquatic animals.

[0007] In a first aspect, the application provides an application of Bacillus velezensis in biological prevention and treatment of Aeromonas disease of aquatic animals, and the preservation number of the Bacillus velezensis is CCTCC NO: M 2020811.

[0008] Optionally, the Aeromonas includes Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae, Aeromonas veronii, Aeromonas salmonicida, Pseudomonas fluorescens, Shewanella putrefaciens, Plesiomonas shigelloides, Edwardsiellaictaluri.

[0009] The application explores the antagonistic effect of Bacillus velezensis with preservation number CCTCC NO: M 2020811 on 9 common water-borne pathogenic bacteria, and the hemolytic activity, virulence gene and drug resistance of the strain. The safety of the Bacillus velezensis is analyzed and evaluated by infecting Aeromonas susceptible host grass carp with different concentrations of the Bacillus velezensis, so as to provide a reference for the application of the Bacillus velezensis in biological prevention and treatment of Aeromonas disease of aquatic animals.

[0010] The application detects the antagonism of Bacillus velezensis to 9 common aquatic pathogenic bacteria, and the results show that the Bacillus velezensis has good antagonistic activity to Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae, Aeromonas veronii, Aeromonas salmonicida, Pseudomonas fluorescens, Shewanella putrefaciens, Plesiomonas shigelloides and Edwardsiella tarda, and the inhibition zone diameter is 16.00mm-24.00mm. Among them, the inhibition zone diameters of the five strains of fish-derived Aeromonas are all more than 20.00mm.

[0011] The results show that the Bacillus velezensis has the biological characteristics of broad-spectrum antagonism to common aquatic pathogenic bacteria, and the antagonistic activity to Aeromonas pathogenic bacteria is stronger, which has potential application value in the biological control of Aeromonas disease of aquatic animals.

[0012] In the application process of Bacillus velezensis, its biological safety is also very important. Related studies show that Bacillus carries multiple virulence genes such as enterotoxin Nhe gene, enterotoxin T gene, enterotoxin FM gene, cytotoxin K gene, hemolysin BL gene and vomiting gene, which can cause skin ulceration of Cynoglossus semilaevis, hemorrhagic septicemia of Ictalurus punctatus, and has potential pathogenic risk to aquatic animals. Therefore, detecting the hemolytic activity and virulence genes of Bacillus velezensis has become an important link to evaluate its safety.

[0013] Optionally, the Bacillus velezensis has no hemolytic activity.

[0014] Optionally, the Bacillus velezensis does not carry the common virulence genes nheA, nheB, nheC, bceT, bceT, cytK, ces, hblA, hblC and hblD of Bacillus.

[0015] Optionally, the Bacillus velezensis is sensitive to 6 categories of antibacterial drugs, including aminoglycosides, sulfonamides, macrolides, tetracyclines, amphenicols and quinolones, and only resistant to penicillin in β-lactams.

[0016] The application detects the hemolytic activity and virulence genes of Bacillus velezensis, and the results show that the Bacillus velezensis has no hemolytic activity and does not carry the common virulence genes of Bacillus.

[0017] In addition, the application also detects the sensitivity of Bacillus velezensis to 7 categories of 22 kinds of antibacterial drugs and its pathogenicity test on grass carp. The results show that the Bacillus velezensis is sensitive to 6 categories of antibacterial drugs, including aminoglycosides, sulfonamides, macrolides and tetracyclines, and only resistant to penicillin in β-lactams, which is very weak. The Bacillus velezensis has no pathogenicity to grass carp, and has the biological safety for application in aquaculture.

[0018] In summary, the present application includes at least one of the following beneficial technical effects:

[0019] The present application found that Bacillus velezensis has strong antagonistic effect on 9 common aquatic pathogenic bacteria such as Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae, Aeromonas veronii, Aeromonas salmonicida, Pseudomonas fluorescens, Shewanella putrefaciens, Plesiomonas shigelloides and Edwardsiellaictaluri, and the strain has no hemolytic activity, does not carry common virulence genes of Bacillus, is sensitive to most antibacterial drugs, and has no pathogenicity to grass carp.

[0020] Therefore, Bacillus velezensis is an Aeromonas antagonistic bacterium with good safety, which can be used as a potential probiotic for the prevention and treatment of Aeromonas disease in aquatic animals, and has good application prospect. The present application provides a new probiotic resource and a new idea for the biological prevention and control of bacterial diseases in aquatic animals. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 4 is the detection results of the antagonistic activity of Bacillus velezensis on 9 common aquatic pathogenic bacteria (A. The culture obtained the inhibition zone results; B. Statistical analysis results of the diameter of the inhibition zone). Note: Different letters on the column chart represent significant difference (P<0.05), and the same letter represents no significant difference (P>0.05).

[0022] Figure 2 Figure 5 is the PCR identification results of the virulence genes of Bacillus velezensis (A. Bacillus velezensis, 1-10 lanes represent the PCR amplification products of nheA, nheB, nheC, bceT, entFM, ces, cytK, hblA, hblC and hblD virulence genes of Bacillus velezensis; B. Control bacteria, 11-20 lanes represent the PCR amplification products of nheA, nheB, nheC, bceT, entFM, ces, cytK, hblA, hblC and hblD virulence genes of the control bacteria; M: DL2000 DNA Marker). DETAILED DESCRIPTION

[0023] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are for the purpose of describing specific embodiments only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term belongs.

[0024] It should be noted that the terms "first", "second" are used only for descriptive purpose, and cannot be construed as indicating or implying relative importance or implying a number of the technical features indicated. Thus, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0025] The endpoints of the ranges and any values disclosed in the present application are not limited to the precise values recited. The ranges or values should be construed as having a range beyond the endpoints to include values approximately equivalent to the recited values. For numeric values, the endpoints of each range, the endpoints of each range and individual point values, and individual point values can be combined with one another to generate one or more new numeric ranges, which should be considered as specifically disclosed herein.

[0026] In the present application, the term "comprising" or "including" is an open-ended expression, i.e. including the indicated content, but not excluding other aspects.

[0027] For the purpose of making the purpose, technical solutions and advantages of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative efforts fall within the scope of protection of the present application. The embodiments described below are exemplary, and are only used to explain the present application, and cannot be understood as limiting the present application.

[0028] Unless otherwise specified, the technical or conditions in the embodiments are carried out according to the technical or conditions described in the literature in the art or according to the product manual. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained by purchase.

[0029] The present application will be further described in detail below in combination with the embodiments and test results.

[0030] Embodiment 1

[0031] In this embodiment, the antagonistic activity of Bacillus velezensis with the preservation number of CCTCC NO: M 2020811 against 9 common aquatic pathogenic bacteria was determined.

[0032] The aquatic pathogens used include 9 strains of common aquatic pathogens, including Aeromonas hydrophila JS02 from grass carp, Aeromonas sobria LB13 from grass carp, Aeromonas caviae CD16 from crucian carp, Aeromonas veronii WT07 from crucian carp, Aeromonas salmonicida BZ01 from grass carp, Pseudomonas fluorescens B01 from mandarin fish, Shewanella putrefaciens JS09 ​​from yellow catfish, Plesiomonas shigelloides CJ14 from American shad, and Edwardsiella ictaluri DY08 from yellow catfish. The above strains were isolated from diseased fish and preserved in our laboratory.

[0033] 100 μL of each pathogen suspension (1.0×10 8 cfu / mL) was spread on LB solid medium (Beijing Luqiao Technology Co., Ltd.), and 80 μL of Bacillus velezensis suspension (1.0×10 8 cfu / mL) were added into the wells, repeated 3 times, allowed to stand for 10 min, and then cultured at 30°C for 24 h to observe the results and measure the diameter of the inhibition zone (d).

[0034] Test results such as Figure 1 shown.

[0035] Depend on Figure 1 (A) It can be seen that the diameters of the inhibition zones of Bacillus Velez against 9 common aquatic pathogens ranged from 16.00 mm to 24.00 mm. It had the strongest antagonistic activity against Aeromonas salmonicida BZ01, with an inhibition zone diameter of (23.23±0.41) mm, which was significantly higher than the inhibition zone diameters against other pathogens (P<0.05). Bacillus Velez also had good antagonistic activity against Aeromonas hydrophila JS02, Aeromonas welchii WT07, Aeromonas caviae CD16, and Aeromonas sobria LB13, with inhibition zone diameters of 20.00 mm to 23.00 mm. Bacillus Velez had the weakest antagonistic activity against Shewanella putrefaciens JS09 ​​(P<0.05), but the inhibition zone diameter against it was still (16.10±0.38) mm.

[0036] Depend on Figure 1(B)It can be seen that the inhibition effect of B. velezensis on 9 strains of pathogenic bacteria is Aeromonas salmonicida BZ01>Aeromonas hydrophila JS02>Aeromonas veronii WT07>Aeromonas caviae CD16>Aeromonas sobria LB13>Aeromonas ichthiosigma DY08>Pseudomonas fluorescens B01>Leptococcus jellisoni CJ14>Shewanella putrefaciens JS09. Thus, it is shown that B. velezensis has antagonistic effect on 9 common aquatic pathogenic bacteria, and has strong antagonistic activity on Aeromonas pathogenic bacteria.

[0037] Example 2

[0038] In this example, the hemolytic activity of B. velezensis with accession number CCTCC NO: M 2020811 was determined.

[0039] The B. velezensis was streak inoculated on a Columbia blood agar plate, and after 24 h incubation at 30°C, whether a hemolytic ring was produced around the colony was observed. If no hemolytic ring was produced, it was γ-hemolytic; if a green hemolytic ring was produced, it was α-hemolytic; and if a transparent hemolytic ring was produced, it was β-hemolytic.

[0040] From the culture results, it can be seen that no hemolytic ring was produced around the colony of B. velezensis. It is shown that B. velezensis is γ-hemolytic and has no hemolytic activity.

[0041] Example 3

[0042] In this example, the virulence genes of B. velezensis with accession number CCTCC NO: M 2020811 were determined.

[0043] Referring to the prior art, 10 common Bacillus virulence genes in B. velezensis, including enterotoxin Nhe genes (nheA, nheB and nheC), enterotoxin T gene bceT, enterotoxin FM gene entFM, cytotoxin K gene cytK, emetic gene ccs and hemolysin BL gene (hblA, hblC and hblD), were detected by PCR, and grass carp-derived B. cereus BFK20206 (isolated from diseased fish and preserved by the laboratory) was used as a control strain. The PCR amplification primers (as shown in Table 1) were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd., and the PCR products were subjected to 1% agarose gel electrophoresis and photographed under a gel imaging system to record the results.

[0044] Table 1 Virulence gene primer sequences

[0045]

[0046]

[0047] The electrophoresis results of the PCR products are as follows:Figure 2 As shown.

[0048] By Figure 2 It can be seen that the amplification results of 10 kinds of virulence genes of Bacillus velezensis (A) including enterotoxin Nhe gene (nheA, nheB and nheC), enterotoxin T gene bceT, enterotoxin FM gene entFM, cytotoxin K gene cytK, vomiting gene ces and hemolysin BL gene (hblA, hblC and hblD) are all negative. The amplification results of 5 kinds of virulence genes of the control strain BFK20206 (B) including nheA, ces and hemolysin hblA, hblC and hblD are all negative, and the amplification results of 5 kinds of virulence genes including nheB, nheC, bceT, entFM and cytK are positive, and the sizes of the virulence gene amplification products are consistent with the literature reports.

[0049] It can be seen that Bacillus velezensis does not carry 10 kinds of common virulence genes of Bacillus, which suggests that the virulence of the strain is small and the pathogenicity is weak.

[0050] Example 4

[0051] In this example, the drug sensitivity test of Bacillus velezensis with preservation number CCTCC NO: M 2020811 was carried out.

[0052] The sensitivity of Bacillus velezensis to 7 categories of 22 kinds of antibacterial drugs (purchased from the United Kingdom Oxoid Company, all stored at 4℃ for standby) including aminoglycosides, β-lactams, quinolones and the like was determined by K-B paper disc diffusion method, and the test and judgment results were referred to the United States Clinical and Laboratory Standardization Association (CLSL) “M100 Antimicrobial Drug Sensitivity Test Execution Standard”.

[0053] The detection results are shown in Table 2.

[0054] Table 2 Drug sensitivity test results of Bacillus velezensis

[0055]

[0056]

[0057] As shown in Table 2, the B. velezensis is sensitive to 21 kinds of antibacterial drugs such as streptomycin, neomycin, tobramycin, kanamycin, gentamicin, amikacin, enrofloxacin, norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin, florfenicol, doxycycline, erythromycin, azithromycin, sulfamethoxazole, cotrimoxazole, ampicillin, ceftazidime, cephalothin, cephalexin, etc., and is only resistant to penicillin. It is shown that the B. velezensis is sensitive to 6 kinds of antibacterial drugs such as aminoglycosides, sulfonamides, macrolides, tetracyclines, amphenicols, and quinolones, and is only resistant to penicillin in the β-lactam class, and it can be considered that it is not resistant in principle.

[0058] Example 5

[0059] In this example, the pathogenicity test of the B. velezensis with the preservation number of CCTCC NO: M 2020811 on grass carp was carried out.

[0060] The experimental grass carp was about 10.0 cm-15.0 cm in length, which was provided by a fish farm in Jinshan District, Shanghai.

[0061] 150 grass carps were divided into 4 experimental groups and 1 control group, 15 grass carps per group, and 2 parallel groups per group. The grass carps in the experimental groups were respectively injected with 100 μL of B. velezensis bacterial suspension with the concentrations of 1.5×10 6 cfu / mL, 1.5×10 7 cfu / mL, 1.5×10 8 cfu / mL, and 1.5×10 9 cfu / mL through abdominal cavity injection, and the grass carps in the control group were injected with the same amount of sterile normal saline through abdominal cavity injection. The experimental period was 7 days, and the swimming ability and reaction ability of the grass carps were observed every day, and the number of deaths of the grass carps in each group was recorded.

[0062] The test results show that the grass carps in each test group feed normally and have good vitality, and no abnormal reaction and death occurs, and the grass carps in the control group are also healthy and alive. It is shown that the B. velezensis has good biological safety on the grass carp.

[0063] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0064] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same. Although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent replacements for some of the technical features therein. Such modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Application of Bacillus Velezii in biological control of Aeromonas disease in aquatic animals, characterized in that: The deposit number of the Bacillus Velez is CCTCC NO: M 2020811.

2. The use according to claim 1, characterized in that The Aeromonas include Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae, Aeromonas vernix, Aeromonas salmonicida, Pseudomonas fluorescens, Shewanella putrefaciens, Pseudomonas shigelloides, and Edwardsiella catfish.

3. The use according to claim 1, characterized in that The Bacillus Velezii has no hemolytic activity.

4. The use according to claim 1, characterized in that The Velez Bacillus does not carry virulence genes commonly found in Bacillus species. nheA、nheB 、 nhC 、 Bt 、 Bt 、 cytK 、 ces 、 hblA, hblC, and hblD .

5. The use according to claim 1, characterized in that The Bacillus Velezii is sensitive to six major types of antibiotics, including aminoglycosides, sulfonamides, macrolides, tetracyclines, amides, and quinolones, and is only resistant to penicillin among the β-lactams.

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