Method for gut microbiota transplantation between distantly related fish and application thereof

Abstract

This invention belongs to the field of aquaculture and microbial application technology, specifically relating to a method for intestinal flora transplantation between distantly related fish species and its application. The method includes: collecting intestinal contents from donor fish at the peak activity time of intestinal flora after feeding, and preparing a donor bacterial suspension through standardized processing; pretreating recipient fish after fasting with a mixed antibiotic solution containing vancomycin, metronidazole, neomycin, and ampicillin via combined gavage and enema for 24 hours; after pretreatment, transplanting the donor bacterial suspension into the recipient's intestine via gavage and enema; and feeding the recipient with a bacterial feed containing the donor bacterial suspension for 30 days after transplantation. This invention establishes a standardized transplantation process, overcomes the limitations of synspecific transplantation, and achieves efficient transplantation and long-term colonization of intestinal flora between distantly related fish species. It can be used for targeted regulation of the intestinal flora of recipient fish.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture and microbial application technology, specifically relating to a method for intestinal flora transplantation between distantly related fish species and its application. Background Technology

[0002] The gut microbiota plays a crucial role in host nutrient metabolism, immune regulation, and disease resistance. In aquaculture, regulating the gut microbiota through fecal microbiota transplantation (FMT) has become a potential strategy for improving the health, promoting growth, and enhancing the stress resistance of farmed fish.

[0003] However, existing fish fecal microbiota transplantation (FMT) techniques are primarily limited to interspecific strains or closely related species. While this homogeneous transplantation model is effective to some extent, its pool of beneficial microorganisms is relatively limited, making it difficult to overcome the inherent microbial structure of a species. Fish with different diets and evolutionary positions have developed gut microbiota highly compatible with their physiological characteristics through long-term adaptive evolution. For example, omnivorous fish have an abundance of fermentative microorganisms adapted to high-carbohydrate diets. These microbial resources are unavailable through homogeneous transplantation. Therefore, the homogeneous transplantation model significantly limits the application potential of FMT technology.

[0004] Currently, extending fecal microbiota transplantation (FMT) technology to distant fish species still faces multiple technical bottlenecks:

[0005] (1) Lack of standardized transplantation procedures. The key aspects such as the collection time of donor microbiota, processing methods, recipient pretreatment schemes, transplantation timing and transplantation methods lack unified and scientific operating standards, resulting in unstable transplantation effects, poor experimental repeatability, and difficulty in forming technical specifications that can be promoted.

[0006] (2) There are theoretical and technical obstacles to transplantation in distantly related fish species. When the donor and recipient fish are distantly related species (such as different families), their gut microbiota differ significantly in evolution, and their gut environments (such as pH, digestive enzyme composition, mucosal structure, and immune status) are quite different, making it difficult for exogenous microbiota to survive and colonize effectively in the xenobiotic gut. How to overcome host specificity and achieve stable implantation of cross-species microbiota is an unsolved technical problem in this field.

[0007] (3) Lack of guarantee of donor microbial activity. Current technology lacks scientific consideration of the timing of donor microbial collection. The metabolic activity of intestinal microbiota changes dynamically with the digestive process after food intake. Microbiota collected during the period of low activity have poor transplantation potential, which directly affects the transplantation success rate.

[0008] (4) Incomplete recipient pretreatment and unclear transplantation timing. Conventional antibiotic pretreatment methods may not be able to effectively eliminate or fully inhibit the recipient's original gut microbiota, resulting in insufficient ecological niches available for exogenous microbiota colonization. At the same time, if transplantation is performed too early after pretreatment, the residual antibiotics in the body will inhibit the activity of the transplanted microbiota. If transplantation is performed too late, the recipient's original microbiota will naturally rebound and reoccupy the ecological niche. Current technology has not provided a transplantation time window that takes both into account.

[0009] (5) Long-term colonization effect is not good. Existing methods mostly rely on single transplantation. The transplanted microbiota is easily affected by the rebound of the recipient's original microbiota or the influence of the breeding environment and gradually disappears, making it difficult to achieve long-term and stable functional modification of the recipient's intestinal microecology.

[0010] Therefore, developing a standardized method that can overcome host barriers and achieve efficient, stable transplantation and long-term colonization of gut microbiota in distant fish species is of great scientific significance and application value for expanding probiotic resources, realizing the targeted delivery of beneficial functions from distant species, and innovating fish health management strategies. Summary of the Invention

[0011] To achieve the above objectives, the technical solution of this application discloses a method for intestinal flora transplantation between distantly related fish species, comprising the following steps:

[0012] (a) Collection and preparation of donor microbiota: At the time point of peak activity of intestinal microbiota after feeding, the intestinal contents of donor fish were collected and processed to prepare a donor microbiota suspension;

[0013] (b) Recipient pretreatment: For recipient fish that have been fasted, a mixed antibiotic solution containing vancomycin, metronidazole, neomycin and ampicillin was administered via a combination of gavage and enema for 24 hours.

[0014] (c) Microbial transplantation: After the pretreatment in step (b), the donor bacterial suspension prepared in step (a) was transplanted into the intestine of the recipient fish by a combination of gavage and enema.

[0015] (d) Feeding consolidation: After transplantation in step (c), the recipient fish are fed with a bacterial feed containing the donor bacterial suspension for 30 consecutive days.

[0016] Furthermore, the method for preparing the donor bacterial suspension in step (a) includes:

[0017] (a1) The intestines of donor fish were aseptically collected, longitudinally cut open, and the food residue and mucosal attachments in the intestinal lumen were scraped off.

[0018] (a2) Cut the scraped intestinal tissue into small pieces and place it in a sterile buffer solution with a weight of 5 times the total weight of the chyme, mucosal attachments and intestinal tissue.

[0019] (a3) Combine and mix the scraped material from step (a1) with the shaking liquid from step (a2), and filter it through a 300-mesh filter bag to obtain the filtrate;

[0020] (a4) Centrifuge the filtrate, discard the supernatant, and wash the resulting precipitate with sterile buffer to obtain bacterial sludge;

[0021] (a5) The bacterial sludge was resuspended in a sterile buffer containing 20% ​​glycerol by volume and stored at -80°C to obtain the donor bacterial suspension.

[0022] Furthermore, in step (a), the peak time point of intestinal flora activity is the time point at which the metabolic activity of intestinal live bacteria in the donor fish reaches its peak after feeding, as determined by the PMA-qPCR live bacteria detection pre-experiment; the peak time point is located 4 to 8 hours after feeding.

[0023] Furthermore, in step (b), the concentration of vancomycin in the mixed antibiotic solution is 0.02–0.04 g / L, the concentration of metronidazole is 1–2 g / L, the concentration of neomycin is 1–2 g / L, and the concentration of ampicillin is 1–2 g / L.

[0024] Furthermore, in step (b), the dosage of the combined gavage and enema administration is 0.01–0.02 mL / g of the recipient fish body weight.

[0025] Furthermore, in step (c), the transplantation is performed immediately after the pretreatment in step (b); the transplantation dose of the donor bacterial suspension is the body weight of the recipient fish.

[0026] Furthermore, in step (d), the bacterial cell concentration of the donor bacterial suspension in the bacterial bait is... CFU / g feed.

[0027] Furthermore, in step (d), the continuous feeding begins on the day after the transplantation in step (c) and is performed once a day.

[0028] Furthermore, the donor fish and the recipient fish belong to different species.

[0029] Furthermore, the above methods are applied to the targeted regulation of gut microbiota in recipient fish.

[0030] Beneficial effects:

[0031] (1) Breaking through species limitations and expanding the microbial community resource pool. This invention has for the first time achieved successful transplantation of gut microbiota between distantly related fish (different species, preferably different families), breaking through the limitation of existing technologies that are limited to transplantation within the same species. This allows beneficial microbial community resources from fish with different diets and different evolutionary positions to be utilized across species, providing a broader microbial community resource pool for screening beneficial microorganisms.

[0032] (2) High activity of donor microbiota. This invention uses a pre-experimental study of PMA-qPCR live bacteria detection to determine the peak time point of the metabolic activity of the intestinal microbiota after the donor ingests food. Microbiota are collected at the peak time point and, in conjunction with a standardized treatment process of anaerobic buffer system containing L-cysteine ​​and low-temperature preservation of glycerol, the activity and transplantation potential of the transplanted microbiota are guaranteed to the greatest extent.

[0033] (3) Thorough recipient pretreatment and scientific transplantation window. This invention uses four antibiotics with complementary action spectra in combination and achieves full intestinal coverage through combined gavage and enema administration, which can reduce the density of the recipient's intestinal flora by 5 to 6 orders of magnitude. At the same time, the transplantation time is established to be performed immediately after the pretreatment (24 hours after treatment). At this time, the recipient's original flora is at a very low level, the ecological niche is fully open, and the antibiotic residue in the body has dropped below the safety threshold, thus taking into account both the clearance effect and the safety of the transplanted flora.

[0034] (4) High colonization efficiency and long-term stable effect. This invention uses a single transplantation via both gavage and enema, supplemented by continuous feeding of a bacterial feed containing donor bacterial suspension for 30 days to consolidate the transplantation. This allows the donor's characteristic bacterial flora to stably colonize in the recipient's intestine and become the dominant flora, effectively overcoming the defect of easy disappearance of the flora after a single transplantation. Examples show that 30 days after transplantation, the relative abundance of the characteristic dominant bacterial genus Cetobacterium from the donor tilapia in the intestine of the recipient largemouth bass reached 49.55%, close to the donor level.

[0035] (5) Achieving cross-species transfer of beneficial functions. Examples show that the blood glucose level of the recipient largemouth bass after transplantation was significantly lower than that of the non-transplantation group (P<0.001), and the abundance of the potentially pathogenic bacteria Plesiomonas in the recipient's intestine was significantly reduced. This proves that the method of the present invention can directionally transfer the beneficial bacterial community function of the donor fish to the recipient, providing a new technical means for the targeted regulation of the intestinal health and productive traits of the recipient fish, and significantly increasing the probability of screening out specific beneficial functional strains or bacterial community combinations. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This shows the change in gut microbiota density over time of antibiotic treatment.

[0038] Figure 2 The changes in the abundance of gut microbiota at the genus level under different treatments. Detailed Implementation

[0039] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical terms involved in this invention are explained as follows:

[0041] The term "distantly related fish" refers to donor and recipient fish belonging to different species, preferably different families. For example, the donor tilapia (Oreochromis niloticus) belongs to the Cichlidae family of the order Perciformes, while the recipient largemouth bass (Micropterus salmoides, also known as the largemouth bass) belongs to the Sunfish family of the order Perciformes. Their divergence occurred approximately 112 million years ago, making them typical examples of distantly related fish. The method of this invention is also applicable to other combinations of fish from different families and genera, such as between Cyprinidae fish and carnivorous fish of the order Perciformes.

[0042] The term "intestinal contents" refers to the chyme and mucosal deposits in the intestinal lumen of fish. The chyme contains free bacteria, while the mucosal deposits contain colonizing bacteria attached to the intestinal wall. Together, they constitute a complete source of intestinal flora.

[0043] The term "peak time point of gut microbiota activity" refers to the time point at which the metabolic activity of the gut microbiota in donor fish rises to its peak as digestion progresses after feeding. This time point was determined through a preliminary experiment using PMA-qPCR viable bacteria detection: propidium azide bromide (PMA) can only penetrate the cell membrane of dead bacteria and covalently bind to their DNA, thereby inhibiting the amplification of dead bacterial DNA. Therefore, PMA-qPCR can selectively quantify viable bacteria. By tracking changes in the amount of viable bacteria at different time points after feeding, the peak activity time point can be determined. This peak time point is usually located within the range of 4 to 8 hours after feeding, but varies depending on the fish species, feed composition, and water temperature.

[0044] The term "gavage" refers to the administration of medication or bacterial suspension directly into the stomach of the recipient fish via a catheter through the mouth; the term "enema" refers to the administration of medication or bacterial suspension directly into the hindgut of the recipient fish via a catheter through the anus. Combining these two methods achieves complete intestinal coverage.

[0045] The term "bacterial bait" refers to a feed containing live bacteria, prepared by uniformly mixing a donor bacterial suspension with the recipient fish's regular feed.

[0046] The term "colonization" refers to the biological process by which exogenous donor microbiota survive, proliferate, and remain stable in the recipient's gut. This can be verified by comparing the similarity between the recipient's gut microbiota structure and the donor's microbiota structure before and after transplantation using 16S rDNA sequencing.

[0047] The first embodiment of this application discloses a method for intestinal microbiota transplantation between distantly related fish species, comprising the following steps in sequence: (a) donor microbiota collection and preparation, (b) recipient pretreatment, (c) microbiota transplantation, and (d) feeding consolidation. The technical means of each step will now be described in detail based on claim 1.

[0048] Step (a): Donor microbial community collection and preparation

[0049] Intestinal contents of donor fish were collected at the peak time of intestinal flora activity after feeding and then processed to prepare a donor bacterial suspension.

[0050] The peak time point is chosen because the metabolic activity of the gut microbiota changes dynamically with the digestive process after feeding, and the microbiota collected at the peak activity point exhibits the best vitality and the highest transplantation potential. The peak time point is determined through a preliminary PMA-qPCR live bacteria detection experiment and is generally located within the range of 4–8 hours after feeding; in a specific embodiment of this invention, the peak time point for the donor tilapia is 6 hours after feeding.

[0051] In a further embodiment, the donor bacterial suspension is preferably prepared using the following standardized process:

[0052] (a1) Under aseptic conditions, the complete intestine from the pyloric caeca to the anus of the donor fish was collected, longitudinally cut open, and food residue and mucosal attachments in the intestinal lumen were scraped off using sterile instruments. Scraping off the mucosal attachments allows for the simultaneous acquisition of both free intestinal flora and colonized flora in the mucosa, ensuring the integrity of the source of the flora.

[0053] (a2) The scraped intestinal tissue is minced and placed in a sterile buffer solution with a weight of 5 times the total weight of the chyme, mucosal attachments, and intestinal tissue. The solution is then thoroughly shaken to release the microbial community deeply embedded within the tissue and to prevent its loss. The sterile buffer solution is preferably phosphate-buffered saline (PBS) containing 0.05% L-cysteine ​​by weight / volume. L-cysteine ​​acts as a reducing agent to maintain the anaerobic environment of the system and protect the activity of anaerobic bacteria (such as Cetobacterium). Alternatively, the sterile buffer solution can be sterile physiological saline or other commonly used anaerobic buffer solutions in the art; the amount of L-cysteine ​​added can be adjusted within the range of 0.03% to 0.1%.

[0054] (a3) Combine the scraped material from step (a1) with the shaken liquid from step (a2), shake to mix, and then quickly squeeze and filter using a filter bag to remove undigested feed residue and large particulate impurities, obtaining a filtrate containing bacteria. The filter bag is preferably 300 mesh; however, in cases where a small amount of fine residue is permissible, a filter bag in the range of 100 to 300 mesh may also be used.

[0055] (a4) Centrifuge the filtrate at a low temperature (preferably 4°C), discard the supernatant, resuspend the resulting precipitate in the above-mentioned sterile buffer solution, wash, and centrifuge again to obtain bacterial sludge. The washing step can remove digestive enzymes, bile acids, and other components in the chyme that may inhibit bacterial activity.

[0056] (a5) Resuspend the bacterial sludge in a sterile buffer solution containing 20% ​​glycerol (v / v), aliquot evenly, and store at -80°C to obtain a standardized donor bacterial suspension. Glycerol, as a cryoprotectant, prevents damage to the bacteria from ice crystals during cryopreservation; its v / v can be adjusted within the range of 15%–25%. Liquid nitrogen storage can also be used. Before transplantation, thaw the bacterial suspension and count the viable cells to control the transplantation dose.

[0057] Step (b): Receptor pretreatment

[0058] For the recipient fish after fasting, a mixed antibiotic solution containing vancomycin, metronidazole, neomycin and ampicillin was administered via a combination of gavage and enema for 24 hours as a pretreatment.

[0059] In this step, the four antibiotics have complementary spectrums of action: vancomycin mainly targets Gram-positive bacteria, metronidazole mainly targets anaerobic bacteria, neomycin mainly targets Gram-negative bacteria, and ampicillin is a broad-spectrum antibiotic. The combination of the four can achieve broad-spectrum and thorough clearance of various bacteria in the recipient's intestines, fully releasing the ecological niche; the combined administration of gavage and enema allows the antibiotic solution to cover both the foregut and hindgut of the recipient fish at the same time, avoiding the clearance dead zones caused by a single administration route.

[0060] The fasting period is 24–48 hours. Fasting empties the intestinal contents, reducing the dilution and blockage of antibiotics by food residue. The preferred concentrations of each component in the mixed antibiotic solution are: vancomycin 0.02–0.04 g / L, metronidazole 1–2 g / L, neomycin 1–2 g / L, and ampicillin 1–2 g / L. The preferred dosage for combined administration is 0.01–0.02 mL / g of recipient fish body weight. Before administration, the recipient fish can be anesthetized with a low-dose anesthetic (e.g., 20 mg / L MS-222) to reduce stress and operational damage.

[0061] The pretreatment time was set at 24 hours, which was sufficient to reduce the density of the recipient's gut microbiota by 5 to 6 orders of magnitude without damaging the recipient's condition due to excessive treatment.

[0062] Step (c): Microbial transplantation

[0063] After the pretreatment in step (b) is completed, the donor bacterial suspension prepared in step (a) is transplanted into the intestine of the recipient fish via gavage and enema.

[0064] Preferably, the transplantation is performed immediately after pretreatment, and the transplantation dose of the donor bacterial suspension is [missing value]. CFU / g recipient fish body weight. The timing of transplantation is based on the systematic study in Example 1 of this invention: 24 hours after antibiotic treatment, the recipient's intestinal flora density is at an extremely low level, the ecological niche is fully open, and antibiotic residues in the body have basically disappeared (the plasma inhibition zone disappears), so they will not inhibit the transplanted flora. If transplantation is delayed (e.g., until 48 hours), the recipient's original flora will naturally recover and reoccupy the ecological niche, leading to transplantation failure. Therefore, "immediate transplantation after pretreatment" is the key window to ensure transplantation success rate. Transplantation via both gavage and enema allows the donor flora to reach both the foregut and hindgut of the recipient simultaneously, increasing the contact area between the flora and the intestinal mucosa and improving colonization efficiency.

[0065] Step (d): Feeding Consolidation

[0066] After transplantation in step (c), the recipient fish are fed with a bacterial feed containing the donor bacterial suspension for 30 consecutive days.

[0067] Preferably, the final concentration of the donor bacterial suspension in the bacterial bait is [missing value]. The feed concentration is CFU / g, and the microbial feed should be prepared and used immediately. Continuous feeding begins the day after transplantation and is administered once daily. This step, by continuously and at low doses supplementing the recipient's gut with donor microbiota, helps the already colonized donor microbiota maintain its dominance in competition with the recipient's rebounding microbiota and the microbiota of the aquaculture environment. This is a key means to achieve long-term stable colonization and overcome the short-lived effect of single transplantation in existing technologies. The feeding cycle can be appropriately extended according to the feeding cycle of the recipient fish species, but should not be shorter than the time required for stable microbial colonization.

[0068] It should be noted that the types and dosages of anesthetics, centrifugation conditions, feed particle size, and specific daily feeding times in the above steps are all conventional choices in the field. Those skilled in the art can make conventional adjustments according to the size of the fish and the breeding conditions, all of which fall within the protection scope of this invention.

[0069] Overall technical effect: The above four steps and their key parameters (peak time of donor collection, quadruple antibiotic formulation and concentration, dual-route administration, 24-hour pretreatment duration, immediate transplantation window after pretreatment, transplantation dose, bacterial feed concentration and 30-day feeding cycle) work together to form a complete, repeatable and standardized intestinal flora transplantation process for distant fish, which together achieves a technical effect chain of "efficient clearance - precise transplantation - long-term colonization".

[0070] The technical means and effects of this application will be described in detail below through specific embodiments.

[0071] The following examples further illustrate the present invention using tilapia as the donor and largemouth bass as the recipient. The donor, tilapia, belongs to the family Cichlidae in the order Perciformes, while the recipient, largemouth bass, belongs to the family Sunfishes in the order Perciformes. They diverged approximately 112 million years ago and are typical distantly related species. Their glucose metabolism physiology differs significantly: tilapia are omnivorous and can tolerate up to 33% starch in their diet; their gut microbiota is adapted to processing high-carbohydrate feeds. Largemouth bass, on the other hand, are typical carnivorous fish with extremely limited carbohydrate utilization; long-term consumption of feed containing only 13%–15% starch results in pathological reactions such as slowed growth, lipid metabolism disorders, and impaired intestinal barrier. The following examples aim to verify the feasibility of the method of the present invention among distantly related fish species, with the functional endpoint of improving recipient glucose tolerance and identifying functional strains that enhance glucose tolerance.

[0072] Example 1: Determination of the optimal transplantation time for recipient fish

[0073] 1. Materials and Methods

[0074] 1.1 Pretreatment of Recipient Fish: Healthy largemouth bass were fasted for 24 hours and then anesthetized with a low dose (20 mg / L) of MS-222. A mixed antibiotic solution was administered via gavage and enema at a dose of 0.01 mL / g of fish body weight. The mixed antibiotic solution consisted of: vancomycin 0.04 g / L, metronidazole 2 g / L, neomycin 2 g / L, and ampicillin 2 g / L.

[0075] 1.2 Sample Collection and Testing:

[0076] Intestinal flora density determination: Recipient fish were sacrificed before antibiotic treatment (0 h) and at 6 h, 24 h, and 48 h after treatment, and intestinal contents were aseptically collected. Samples were added to sterile PBS containing 0.05% L-cysteine ​​at a ratio of 1:9 (w / v), homogenized, serially diluted, and plated on anaerobic agar plates. After anaerobic incubation at 28°C for 24 hours, bacterial counts were performed. Intestinal flora density was calculated using the formula: CFU / g = average colony count × dilution factor / sample wet weight.

[0077] Plasma antibiotic residue detection: Blood was collected from recipient fish simultaneously, and plasma was separated. 100 μL of plasma was added to a pre-coated E. coli (…). In Mueller-Hinton plates containing CFU / mL, the diameter of the inhibition zone was measured after 24 hours of incubation at 37°C to assess the residual antibiotic activity in vivo.

[0078] 2 Results and Analysis

[0079] 2.1 Intestinal flora clearance effect: such as Figure 1 As shown, within 6 to 24 hours after antibiotic treatment, the intestinal flora density of the recipient fish decreased by approximately 5 to 6 orders of magnitude compared to before treatment (0 hours), reaching its lowest point at 6 hours; by 48 hours, the intestinal flora showed natural recovery, with the density returning to near its initial level. The results indicate that the quadruple antibiotic combined with pretreatment regimen of this invention has a thorough eradication effect, but the eradication window is temporary.

[0080] 2.2 Antibiotic Residue Dynamics: As shown in Table 1, a clear inhibition zone was still observed in the plasma collected 6 h after treatment, indicating that an effective concentration of antibiotic residues existed in the body at this time; the inhibition zone of the plasma collected 24 h after treatment had basically disappeared, indicating that the antibiotics had been effectively metabolized and cleared, and the residual concentration in the body had dropped to a level that had no significant inhibitory effect on microorganisms.

[0081] Table 1. Antibacterial efficacy test of residual antibiotics in plasma

[0082] .

[0083] 3. Conclusion

[0084] Based on the combined data of bacterial clearance effectiveness and antibiotic residues, 24 hours post-treatment was determined to be the optimal time for bacterial transplantation, with the following advantages:

[0085] (1) Niche openness: The original microbial community has been effectively suppressed and its density is at an extremely low level, providing sufficient niche space for the colonization of exogenous donor microbial community;

[0086] (2) Safe window period: The antibiotic residue in the body has been reduced to below the safe threshold, which can minimize its inhibition of the activity of transplanted microbiota;

[0087] (3) Operational feasibility: It avoids the natural recovery of the microbial community caused by excessively long treatment time (such as 48 h) and ensures the effectiveness of the pretreatment window.

[0088] Therefore, this invention establishes transplantation immediately after recipient pretreatment (i.e., 24 hours after treatment) as a fixed operation time point in the standardized process.

[0089] Example 2: Validation experiment of tilapia gut microbiota transplantation into largemouth bass

[0090] 1. Materials and Methods

[0091] 1.1 Preparation of donor bacterial suspension: Healthy tilapia were selected as donors. Six hours after feeding (the time point of peak activity determined by PMA-qPCR preliminary experiments), the tilapia were anesthetized and aseptically dissected.

[0092] (a1) Cut off the intestine from the pylorus to the anus, cut it longitudinally, and scrape off all the food residue and mucosal attachments;

[0093] (a2) Cut the intestinal tissue into small pieces, add 5 times its weight of sterile PBS containing 0.05% L-cysteine ​​and shake.

[0094] (a3) Combine the scraped material with the shaking liquid, mix well, and then filter by rapid squeezing through a filter bag; (a4) Centrifuge and wash the filtrate to obtain bacterial sludge;

[0095] (a5) Resuspend the bacterial sludge in a sterile buffer solution containing 20% ​​glycerol, aliquot, and store at -80°C. Perform viable cell counts before transplantation to ensure the concentration meets the requirements.

[0096] 1.2 Recipient pretreatment and transplantation: Healthy largemouth bass were selected as recipients and randomly divided into transplantation group and non-transplantation group.

[0097] Pretreatment: After fasting for 24 hours, both groups of recipient fish were pretreated for 24 hours according to the protocol determined in Example 1 (combined administration of mixed antibiotics by gavage and enema, 0.01 mL / g body weight).

[0098] Transplantation procedure: Immediately after pretreatment, a single transplantation was performed on the transplantation group: the resuscitated and washed donor bacterial suspension was transferred according to... The dose of CFU / g fish body weight was injected into the recipient's intestine via gavage and enema; the non-transplant group was perfused with an equal volume of sterile buffer containing 0.05% L-cysteine.

[0099] "Starting the day after transplantation, the transplant group was fed a bacterial feed mixed with donor bacterial suspension (final bacterial concentration)." The non-transplanted group was fed the same amount of ordinary feed once a day for 30 consecutive days, with normal feeding and management during the period.

[0100] 1.3 Sample collection and 16S rDNA sequencing analysis: Blood samples were collected from the transplant group and the non-transplant group 30 days after treatment, and blood glucose was measured using a portable blood glucose meter; intestinal contents and donor bacterial suspension were collected from the recipient group before antibiotic treatment, after antibiotic treatment, and 30 days after treatment in the transplant group and the non-transplant group, and sent to the biotechnology company for 16S rDNA sequencing. Data analysis and comparison were performed using a cloud platform.

[0101] 2 Results and Analysis

[0102] 2.1 Characteristics of the gut microbiota of donor tilapia: such as Figure 2 As shown, the gut microbiota of tilapia exhibits highly specific enrichment at the genus level: *Cetobacterium* is the most dominant genus, with a relative abundance of approximately 51.09%, followed by *Barnesiellaceae_unclassified*, *Akkermansia*, and *Romboutsia*. This community structure, dominated by anaerobic fermenting bacteria, reflects the gut microecological characteristics of tilapia as an omnivorous fish, and differs significantly from the original microbiota composition of carnivorous largemouth bass, providing a clear donor marker for verifying distant transplantation.

[0103] 2.2 Evaluation of transplantation effect: The blood glucose test results (Table 2) showed that the blood glucose level in the transplantation group was significantly lower than that in the non-transplantation group 30 days after transplantation (P<0.001), indicating that the transplantation of tilapia gut microbiota improved the glucose metabolism status of California bass.

[0104] Table 2. Effects of gut microbiota transplantation on blood glucose levels in largemouth bass.

[0105] .

[0106] Sequencing analysis ( Figure 2The results showed that the gut microbiota of the recipient group showed a significant trend toward the structure of the donor microbiota 30 days after transplantation: (1) Successful colonization of donor characteristic bacteria - the donor-specific dominant bacteria genus Cetobacterium was detected in the gut of the recipient group, with a relative abundance of 49.55%, close to the donor level, while it was almost not detected in the non-transplantation group and the groups before and after antibiotic treatment (<1%); Romboutsia from the donor was also stably present in the transplantation group, with a relative abundance of 6.10%; (2) The original gut microbiota of the recipient was reconstructed - the abundance of the original dominant bacteria genus of the recipient (such as Plesiomonas) in the transplantation group was significantly reduced and replaced by the donor microbiota, indicating that the exogenous microbiota has a strong competitive advantage and colonization ability in the recipient gut.

[0107] 2.3 Natural recovery characteristics of the non-transplantation group: 30 days after antibiotic treatment, the intestinal flora of the non-transplantation group showed a trend of recovery to the pre-treatment state, but failed to fully recover to the original level. At the genus level, the conditionally pathogenic bacteria Plesiomonas still dominated, indicating that it is difficult to rebuild a healthy intestinal microecology in a short period of time by relying solely on natural recovery. This highlights the advantages of the transplantation method of the present invention in rapidly and directionally reconstructing the intestinal flora.

[0108] 2.4 Functional analysis of key microbiota: Cetobacterium and Romboutsia, which were successfully colonized in the transplantation group, are important potential probiotics in the fish gut and are related to beneficial functions such as short-chain fatty acid production and sugar metabolism. At the same time, the abundance of Plesiomonas, a potential pathogenic bacterium, was significantly lower in the transplantation group than in the non-transplantation group, suggesting that the gut microbiota of tilapia has a probiotic protective effect on largemouth bass.

[0109] 3. Conclusion

[0110] In this embodiment, the intestinal flora of tilapia was successfully transplanted to the distantly related largemouth bass in strict accordance with the method described in the claims: (1) 30 days after transplantation, the dominant bacterial genera (Cetobacterium, Romboutsia) of the donor successfully colonized in the recipient's intestine and became the dominant flora, confirming that the method of the present invention can overcome species limitations to achieve distant flora transplantation; (2) compared with the control group that relied solely on natural recovery, the present method can reconstruct the recipient's intestinal flora more quickly and in a more targeted manner; (3) transplantation significantly inhibited the abundance of the recipient's original potential pathogenic bacteria and significantly reduced the recipient's blood glucose level, realizing the cross-species transfer of the donor's beneficial functions, and providing direct evidence for the invention's objective of "obtaining flora with specific beneficial functions for the recipient fish species".

[0111] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions 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 invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for intestinal flora transplantation between distantly related fish species, characterized in that, Includes the following steps: (a) Collection and preparation of donor microbiota: At the time point of peak activity of intestinal microbiota after feeding, the intestinal contents of donor fish were collected and processed to prepare a donor microbiota suspension; (b) Recipient pretreatment: For recipient fish that have been fasted, a mixed antibiotic solution containing vancomycin, metronidazole, neomycin and ampicillin was administered via a combination of gavage and enema for 24 hours. (c) Microbial transplantation: After the pretreatment in step (b), the donor bacterial suspension prepared in step (a) was transplanted into the intestine of the recipient fish by a combination of gavage and enema. (d) Feeding consolidation: After transplantation in step (c), the recipient fish are fed with a bacterial feed containing the donor bacterial suspension for 30 consecutive days.

2. The method according to claim 1, characterized in that, The method for preparing the donor bacterial suspension in step (a) includes: (a1) The intestines of donor fish were aseptically collected, longitudinally cut open, and the food residue and mucosal attachments in the intestinal lumen were scraped off. (a2) Cut the scraped intestinal tissue into small pieces and place it in a sterile buffer solution with a weight of 5 times the total weight of the chyme, mucosal attachments and intestinal tissue. (a3) Combine and mix the scraped material from step (a1) with the shaking liquid from step (a2), and filter it through a 300-mesh filter bag to obtain the filtrate; (a4) Centrifuge the filtrate, discard the supernatant, and wash the resulting precipitate with sterile buffer to obtain bacterial sludge; (a5) The bacterial sludge was resuspended in a sterile buffer containing 20% ​​glycerol by volume and stored at -80°C to obtain the donor bacterial suspension.

3. The method according to claim 1 or 2, characterized in that, In step (a), the peak time point of intestinal flora activity is the time point at which the metabolic activity of intestinal live bacteria in donor fish reaches its peak after feeding, as determined by the PMA-qPCR live bacteria detection pre-experiment; the peak time point is located 4 to 8 hours after feeding.

4. The method according to claim 1, characterized in that, In step (b), the concentration of vancomycin in the mixed antibiotic solution is 0.02–0.04 g / L, the concentration of metronidazole is 1–2 g / L, the concentration of neomycin is 1–2 g / L, and the concentration of ampicillin is 1–2 g / L.

5. The method according to claim 1 or 4, characterized in that, In step (b), the dosage of the combined gavage and enema administration is 0.01 to 0.02 mL / g of the recipient fish body weight.

6. The method according to claim 1, characterized in that, In step (c), the transplantation is performed immediately after the pretreatment in step (b); the transplantation dose of the donor bacterial suspension is... CFU / g of recipient fish body weight.

7. The method according to claim 1, characterized in that, In step (d), the bacterial cell concentration of the donor bacterial suspension in the bacterial bait is [missing information]. CFU / g feed.

8. The method according to claim 1 or 7, characterized in that, In step (d), the continuous feeding begins on the day after the transplantation in step (c) and is performed once a day.

9. The method according to claim 1, characterized in that, The donor fish and the recipient fish belong to different species.

10. The application of the method according to any one of claims 1-9 in the targeted regulation of the intestinal flora of recipient fish.

Images