Novel ecological feed capable of preventing diseases of multiple parabramis pekinensis

Abstract

The invention provides a novel ecological feed capable of preventing multiple grass-bream diseases, the novel ecological feed comprises a feed matrix and an ecological feed leavening agent, and the ecological feed leavening agent comprises probiotics with surface expression antigens, the antigen is any one or more of a grass carp reovirus antigen, a flavobacterium johnsonii antigen, an aeromonas veronii antigen and a clonorchis sinensis antigen. The novel ecological feed with a disease-resistant function in a parabramis pekinensis breeding mode is prepared for the first time, the resistance of the parabramis pekinensis to various pathogens can be enhanced, and the survival rate of the parabramis pekinensis in the disease attack peak period can be increased by 50% or above after immunization. An oral immunization mode is adopted and can be completed in the feeding process, and the immunization and breeding cost is greatly saved.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to a novel multi-pathogen resistant ecological feed. Background Technology

[0002] Grass carp and bream often experience severe mortality in aquaculture. These fish are susceptible to various diseases, primarily hemorrhagic septicemia, septicemia, and parasitic diseases. Hemorrhagic septicemia caused by viruses is particularly serious. Grass carp reovirus GCRV has been confirmed as one of the most deadly reoviruses in aquaculture, and current treatments have limited effectiveness. Aeromonas verrucosa is a zoonotic bacterium widely present in the environment, especially in aquatic environments. It multiplies most rapidly in summer and autumn, and aquatic organisms with weakened immunity or wounds on their bodies are more susceptible to infection. Its main virulence factors include exotoxins, extracellular enzymes, and adhesion factors. This bacterium is highly pathogenic to poikilothermic aquatic animals, often causing serious complications such as hemorrhagic septicemia, and even endangering life.

[0003] Flavobacterium johnsonii can parasitize the gills of grass carp, causing severe bacterial infections. After infection, there is significant proliferation of gill lamellae and gill raker epithelium, along with vacuolation. Simultaneously, the central cartilage structure may also be damaged, leading to severe corrosive diseases. Clonorchis sinensis often causes hepatobiliary problems and ascites, with an infection rate exceeding 25% in freshwater fish in China. It is also one of the most widely transmitted zoonotic parasites. Overall, in polyculture systems, it is necessary to strengthen preventative measures against different types of parasites and bacteria to ensure smooth aquaculture and reduce the risk of losses. Summary of the Invention

[0004] The purpose of this invention is to provide a novel multi-functional ecological feed that can prevent and control various diseases such as bream virus disease, bacterial disease, and parasitic disease.

[0005] This invention provides a method for preparing a novel ecological feed for preventing various diseases in grass carp, comprising:

[0006] 1) Preparation of Bacillus compound fermentation agent: Bacillus subtilis glycerol bacteria with surface antigens and wild Bacillus subtilis WT55 were prepared into a bacterial solution with a final probiotic concentration of 10^10 CFU / mL.

[0007] 2) Preparation of Pichia pastoris compound fermentation agent: Pichia pastoris GS115 glycerol strain that secretes and expresses antigens and wild strain GS115 were prepared into a bacterial solution with a final concentration of 10^10 CFU / mL.

[0008] 3) Preparation of Lactococcus lactis compound fermentation agent: Lactococcus lactis zhy1 glycerol strain expressing antigen was prepared into a bacterial solution with a final concentration of Lactococcus lactis of 10^10 CFU / mL.

[0009] 4) Add at least two of the Bacillus compound fermentation agent, Pichia pastoris compound fermentation agent and Lactococcus lactis compound fermentation agent prepared in steps 1-3 to the feed matrix, as well as a mixed feed additive enzyme preparation, and ferment to obtain ecological feed.

[0010] The antigen is any one or more of grass carp reovirus GCRV, flagellin, Flavobacterium johnsonii, Aeromonas vesiculosus, and Clonorchis sinensis.

[0011] The antigen is in the state of a subunit antigen.

[0012] The effective bacteria content of the ecological feed prepared in step 4) is 4×10^7-4×10^9 CFU / g.

[0013] This invention also provides an ecological feed for preventing various diseases in grass carp and bream, comprising a feed matrix and an ecological feed fermentation agent. The ecological feed fermentation agent comprises probiotics expressing antigens on their surface, wherein the antigens are any one or more of grass carp reovirus antigen, Flavobacterium johnsonii antigen, Aeromonas verrucosa antigen, and Clonorchis sinensis antigen.

[0014] The probiotics include at least two of Bacillus subtilis, Pichia pastoris, and Lactococcus lactis.

[0015] The antigen is in the state of a subunit antigen.

[0016] Wherein, the lactococcus lactis is lactococcus ZHY1; the Bacillus subtilis is Bacillus subtilis WT55; and the Pichia pastoris is Pichia pastoris GS115.

[0017] The ecological feed fermentation agent includes at least two of the following (A1)-A3): A1) 1-2 parts by weight of Lactococcus lactis ZHY1, A2) 1-20 parts by weight of Bacillus subtilis WT55, and A3) 1-2 parts by weight of Pichia pastoris GS115.

[0018] The ecological feed also includes mixed feed additive enzyme preparations.

[0019] The present invention also provides a breeding method for improving the disease resistance of grass carp and / or bream, characterized in that grass carp and / or bream are fed with the aforementioned ecological feed.

[0020] The feeding method includes an immunization step for the fish.

[0021] 1) Initial immunization: Feed the fish with the ecological feed described above, feeding them until they are full, once a day in the evening for 7 consecutive days;

[0022] 2) Booster immunization: Start booster immunizations using the same method as the initial immunization, 15-21 days after the initial immunization.

[0023] 3) The initial immunization plus booster immunization is carried out for 2-3 cycles.

[0024] The feed bases mentioned above are all commercially available feeds for feeding grass carp and / or bream.

[0025] The advantages and effects of this invention are:

[0026] 1) This invention is the first to prepare a new type of ecological feed with disease resistance function in grass bream farming mode, which can enhance grass bream's resistance to a variety of pathogens and improve the survival rate of grass bream during the peak of disease outbreaks by more than 50% after immunization.

[0027] 2) This invention uses oral immunization, which can be completed during the feeding process, greatly saving immunization and breeding costs.

[0028] Preservation Instructions

[0030] Strain name: Lactococcus lactis ZHY1

[0031] Latin name: Lactococcus lactis

[0032] Strain number: ZHY1

[0033] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0034] Collection institution abbreviation: CGMCC

[0035] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0036] Date of preservation: March 17, 2021

[0037] CGMCC Registration Number: CGMCC No. 22024 Attached Figure Description

[0038] Figure 1 Antigen expression status.

[0039] Figure 2 Fermentation of novel ecological feeds with different formulations.

[0040] Figure 3 This represents the cumulative number of deaths during the later stages of the breeding process in the example.

[0041] Figure 4 This example compares the intestinal IgT levels of the immune group and the control group. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] The Bacillus subtilis wt55 used in the following examples is disclosed in the literature “Study about the combination strategy of Bacillus subtilis wt55 with AiiO-AIO6 to improve the resistance of zebraffsh to Aeromonas veronii infection, Fish and Shellffsh Immunology 128(2022)447–454”, and can be obtained by the public from the Feed Research Institute of the Chinese Academy of Agricultural Sciences for the purpose of repeating this experiment.

[0045] The commercial feed described in the following examples is grass carp extruded feed 2.0, produced by Jiangsu Huwei Agricultural and Animal Husbandry Development Co., Ltd.

[0046] Example 1: Preparation of strains carrying antigens

[0047] 1. Prepare Bacillus subtilis with surface-displayed antigens according to the method described in patent 202110838332.7, the specific steps of which are as follows:

[0048] 1. Preparation of bacterial culture

[0049] 1.1 Bacillus subtilis wt55 was cultured in LB medium at 37 degrees Celsius to obtain Bacillus subtilis wt55 bacterial suspension.

[0050] 1.2 Gene Synthesis and Recombinant Plasmid Construction

[0051] The DNA sequences of grass carp reovirus GCRV antigen (NCBI: 3K1Q_F), Clonorchis sinensis antigen (NCBI: AEQ59229), Aeromonas versicolor antigen (NCBI: WP_236322878), and Flavobacterium johnsonii antigen (NCBI: WP_014166251) were synthesized by Qingke Biotechnology Co., Ltd., and an 8×His tag was fused to the end of the sequence. The above gene sequences were used to replace the target gene AIO6 of plasmid pDG364-N-AIO6 disclosed in patent 202110838332.7, and the new recombinant plasmids were named pDG364-N-GCRV, pDG364-N-CsCP (clonorchis sinensis antigen plasmid), pDG364-N-FJ (flavobacterium johnsonii antigen plasmid), and pDG364-N-Aeromonasveronii (Aeromonas vesiculosus antigen plasmid).

[0052] 1.3 Transformation of Bacillus subtilis with recombinant plasmids: The above four plasmids were transformed into Bacillus subtilis strain wt55.

[0053] 1.4 Screening of positive clones: The clones on the plate were verified by PCR and sequenced for analysis. Correct positive clones were used for protein expression analysis.

[0054] 1.5 Western Blot assay for target protein expression: Positive clones of Bacillus subtilis were cultured in LB medium for 48-72 hours. After observing spore formation under a microscope, the bacterial cells were collected, and the surface protein was extracted for Western Blot analysis. The results are as follows: Figure 1 As shown, only the Aeromonas vera antigen (NCBI: WP_236322878) was successfully expressed in Bacillus subtilis. For the antigen expression of other pathogens, attempts were made to use different probiotic expression vectors.

[0055] 2. Prepare Pichia pastoris strain that secretes and expresses antigens according to the method described in patent ZL202111241709.7. The specific steps are as follows:

[0056] 2.1 Culture of bacterial suspension: Pichia pastoris strain GS115 was cultured in YPDA liquid medium and then preserved as a 25% glycerol culture at -80℃.

[0057] 2.2 Construction of recombinant plasmids

[0058] 1) The DNA molecule encoding Clonorchis sinensis antigen (NCBI: AEQ59229) was ligated to linearized pGAP (ZL202111241709.7) to obtain the recombinant plasmid pGAP-CsCP.

[0059] 2) The DNA molecule encoding Flavobacterium johnsonii antigen (NCBI: WP_014166251) was ligated to linearized pGAP (ZL202111241709.7) to obtain the recombinant plasmid pGAP-FJ.

[0060] 2.3 Plasmid Transformation

[0061] The three plasmids were transformed into Pichia pastoris strain GS115.

[0062] 2.4 Screening of positive clones: The clones on the plate were verified by PCR and sequenced for analysis. Correct positive clones were used for protein expression analysis.

[0063] 2.5 testing

[0064] The positive clone of Pichia pastoris strain GS115 was cultured at 30°C for 48 hours. The supernatant of the culture medium was then further concentrated and analyzed by Western blotting. The results are shown in the table. Figure 1 As shown, the DNA molecule of Clonorchis sinensis antigen (NCBI: AEQ59229), which cannot be expressed in Bacillus subtilis, was successfully expressed in the yeast host, but Flavobacterium johnsonii antigen still could not be expressed in the yeast host.

[0065] 3. Prepare Lactococcus lactis with surface-displayed antigens according to the method described in patent 202110544436.7, the specific steps of which are as follows:

[0066] 2.1 Culture of bacterial suspension: Culture Lactococcus lactis zhy1 bacterial suspension for later use.

[0067] 2.2 Construction of recombinant plasmids

[0068] 1) Replace the DNA sequence (including small fragments including the SacI and XbaI recognition sites) between the SacI and XbaI recognition sites on plasmid pMG36e (Beijing Huayueyang Biotechnology, catalog number SWGH695) with the DNA molecule encoding the GCRV antigen (NCBI: 3K1Q_F), while keeping the other sequences of plasmid pMG36e unchanged, to obtain the recombinant plasmid pMG36e-GCRV.

[0069] 2) Replace the DNA sequence (including the small fragments including the SacI and XbaI recognition sites) between the SacI and XbaI recognition sites on plasmid pMG36e (Beijing Huayueyang Biotechnology, catalog number SWGH695) with the DNA molecule encoding Flavobacterium johnsonii antigen (NCBI: WP_014166251), while keeping the other sequences of plasmid pMG36e unchanged, to obtain the recombinant plasmid pMG36e-FJ.

[0070] 2.3 Plasmid Transformation

[0071] The three plasmids were transformed into Lactococcus lactis zhy1, respectively.

[0072] 2.4 Detection

[0073] The transformed positive lactococcus zhy1 was cultured to the late logarithmic growth phase, and the bacterial cells were collected and surface proteins were extracted for Western blotting analysis. The results are shown in the table. Figure 1 As shown: GCRV antigen, which cannot be expressed in Bacillus subtilis, was successfully expressed in Lactococcus lactis; Flavobacterium johnsonii antigen, which cannot be expressed in Bacillus subtilis and yeast, was successfully expressed in Lactococcus lactis.

[0074] Example 2

[0075] I. Preparation of Ecological Feed Fermentation Agent

[0076] 1. Bacillus compound fermentation agent:

[0077] One strain of Bacillus subtilis displaying Aeromonas villus antigen on its surface and Bacillus subtilis WT55 were streaked onto LB agar plates and incubated overnight at 37°C. Single colonies from each strain were picked and inoculated into LB medium and incubated overnight at 37°C. OD 600 Approximately 0.8-1.5; adjust the OD of each strain. 600 ≈0.8, inoculate 1% of the sample into fresh LB medium, and co-culture at 37℃ for about 70 hours. Observe the spore conversion rate using an oil immersion microscope. When 100% of the sample is converted into spores, end the culture, centrifuge at 8000 r / min, remove the supernatant, and resuspend the bacterial cells with a bacterial strain protectant to make the final concentration of probiotics reach 10^10 CFU / mL.

[0078] 2. Pichia pastoris compound fermentation agent:

[0079] One strain of Pichia pastoris expressing Clonorchis sinensis antigen and two strains of Pichia pastoris GS115, stored at refrigeration, were streaked onto YPD anti-inflammatory plates and incubated at 30°C for 3-5 days. Single colonies of each strain were picked and inoculated into YPD medium and incubated overnight at 30°C. OD 600 Approximately 0.8-1.5; Adjust the OD of each vaccine strain. 600 ≈0.8, inoculate together with fresh YPD medium at an inoculation rate of 1%, co-culture at 30℃ for about 48 hours, centrifuge at 8000 r / min, remove the supernatant, and resuspend the cells with a strain protectant to achieve a final concentration of Pichia pastoris probiotics of 10^10 CFU / mL.

[0080] 3. Lactococcus lactis starter: One strain of Lactococcus lactis displaying GCRV antigen and one strain of Lactococcus lactis displaying Flavobacterium johnsonii antigen were streaked onto GM17 plates and incubated overnight at 30°C. Single colonies of each strain were picked and inoculated into GM17 medium and incubated overnight at 30°C. OD 600 Approximately 0.8-1.5; adjust the OD of the strain. 600 ≈0.8, inoculate together with 1% into fresh GM17 medium, incubate at 30℃ for about 40h, centrifuge at 8000r / min, remove the supernatant, resuspend the bacteria with a bacterial strain protectant, and the final concentration of Lactococcus lactis zhy1 reaches 10^10 CFU / mL.

[0081] In the above-mentioned method for preparing the starter culture, the strain protectant is a liquid prepared by dissolving trehalose and skim milk powder in distilled water in a certain proportion and sterilizing it at 115°C for 30 minutes, resulting in a trehalose weight percentage of 10% and a skim milk powder weight percentage of 10%.

[0082] Example 3: Preparation of a Novel Ecological Feed

[0083] I. Feed Preparation

[0084] 1. Add 20 mL of the prepared Bacillus compound fermentation agent, 2 mL of Pichia pastoris compound fermentation agent, and 2 mL of Lactococcus lactis fermentation agent to every 1 kg of commercial feed.

[0085] 2. Add 0.1% (equivalent to 1g) of mixed feed additive enzyme preparation (purchased from Hangzhou Baoankang Biotechnology Co., Ltd.) per 1kg of commercial feed.

[0086] 3. Add distilled water to bring the final moisture content of the feed to 35%, place it in a breathing bag, fully vent the air, and then place it in a 37℃ constant temperature incubator for fermentation for 120 hours to obtain a new type of ecological feed. (This is equivalent to adding 20mL of Bacillus compound fermentation agent, 2mL of Pichia pastoris compound fermentation agent, 2mL of Lactococcus lactis fermentation agent, and 0.1% of mixed feed additive enzyme preparation per 1kg of feed.)

[0087] II. Comparison of Fermentation Status

[0088] 1. Replace step one with one without adding enzyme preparation, keeping all other steps unchanged. Place in a 37℃ constant temperature incubator for 6 days. The fermentation progress is as follows: Figure 2 As shown;

[0089] 2. Replace the distilled water added in step one with 34%, halve the amount of fermentation starter, put it into a breathing bag, fully degas the bag, and place it in a 37℃ constant temperature incubator for fermentation for 2 days. The fermentation progress is as follows. Figure 2 As shown;

[0090] 3. Replace the amount of distilled water added in step one with 31%, keeping all other steps unchanged. Place the mixture in a breathing bag, fully degas it, and then place it in a 37℃ constant temperature incubator for fermentation for 2 days. The fermentation progress is as follows: Figure 2 As shown.

[0091] The results showed that the ratio in step one had the best fermentation effect.

[0092] Example 4

[0093] This experiment used 240 healthy adult grass carp. The experiment was divided into a control group and an immunized group, with four replicates per group and 50 fish per replicate, for a 6-week rearing trial. Each group used a net cage measuring 2 meters x 2 meters x 1 meter, with a water depth of approximately 2 meters. During the experiment, the fish were fed to their full capacity. The control group was fed regular feed throughout the trial. The immunized group was fed ecological feed as their last meal of the day during weeks 1, 3, and 5; otherwise, they were fed regular feed, the same as the control group.

[0094] After the rearing period, observe the disease and mortality of grass carp. Results are shown below. Figure 3 The morbidity and mortality rates in the control group were higher than those in the group immunized with the new ecological feed grass bream.

[0095] At the end of week 6, 10 fish from each group were collected as 10 independent samples, and hindgut mucus was collected. IgT levels in the hindgut mucus were measured using an ELISA kit. Results are shown below. Figure 4 The intestinal IgT level in the immune group was significantly higher than that in the control group.

[0096] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing a novel ecological feed for preventing various diseases in grass carp and bream, characterized in that, include: 1) Preparation of Bacillus compound fermentation agent: Bacillus subtilis glycerol bacteria with surface antigens and wild Bacillus subtilis WT55 were prepared into a bacterial solution with a final probiotic concentration of 10^10 CFU / mL. 2) Preparation of Pichia pastoris compound fermentation agent: Pichia pastoris GS115 glycerol strain that secretes and expresses antigens and engineered strain GS115 were prepared into a bacterial solution with a final concentration of Pichia pastoris probiotic reaching 10^10 CFU / mL. 3) Preparation of Lactococcus lactis compound fermentation agent: Lactococcus lactis zhy1 glycerol strain with surface display of antigen was prepared into a bacterial solution with a final concentration of Lactococcus lactis of 10^10 CFU / mL; 4) Add the Bacillus compound fermentation agent, Pichia pastoris compound fermentation agent and Lactococcus lactis compound fermentation agent prepared in steps 1-3 to the feed matrix, and ferment to obtain ecological feed. The antigen is any one or more of the following: grass carp reovirus GCRV antigen, Flavobacterium johnsonii antigen, Aeromonas versicolor antigen, and Clonorchis sinensis antigen.

2. The method for preparing a novel ecological feed for preventing various diseases in grass carp according to claim 1, characterized in that: The antigen is in the state of a subunit antigen.

3. The method for preparing a novel ecological feed for preventing various diseases in grass carp according to claim 2, characterized in that: The effective bacteria content of the ecological feed prepared in step 4) is 4×10^7-4×10^9 CFU / g.

4. An ecological feed for preventing various diseases in grass carp and bream, characterized in that, It includes a feed matrix and an ecological feed fermentation agent, wherein the ecological feed fermentation agent includes probiotics that express antigens on their surface, and the antigens are any one or more of grass carp reovirus antigen, Flavobacterium johnsonii antigen, Aeromonas verrucosa antigen, and Clonorchis sinensis antigen.

5. The ecological feed for preventing various diseases in grass carp and bream according to claim 4, characterized in that, The probiotics include at least two of Bacillus subtilis, Pichia pastoris, and Lactococcus lactis.

6. The ecological feed for preventing various diseases in grass carp and bream according to claim 4, characterized in that, The antigen is in the state of a subunit antigen.

7. The ecological feed for preventing various diseases in grass carp and bream according to claim 5, characterized in that, The lactococcus lactis is Lactococcus lactis ZHY1; the Bacillus subtilis is Bacillus subtilis WT55; and the Pichia pastoris is Pichia pastoris GS115.

8. The ecological feed for preventing various diseases in grass carp and bream according to claim 7, characterized in that, The ecological feed fermentation agent includes at least two of the following (A1)-A3): A1) 1-2 parts by weight of Lactococcus lactis ZHY1, A2) 1-20 parts by weight of Bacillus subtilis WT55, and A3) 1-2 parts by weight of Pichia pastoris GS115.

9. The ecological feed for preventing various diseases in grass carp and bream according to any one of claims 1-5, characterized in that, The ecological feed also includes mixed feed additives and enzyme preparations.

10. A breeding method for improving the disease resistance of grass carp and / or bream, characterized in that, Grass carp and / or bream are fed with the ecological feed described in any one of claims 1-6.

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