Construction method and application of pseudo-sterile aquatic animal model

Abstract

The invention belongs to the technical field of biology, and discloses a construction method and application of a pseudo-sterile aquatic animal model, and the construction method is characterized by comprising the following steps: (1) fasting experimental aquatic animals; (2) feeding antibiotics to the fasting experimental aquatic animals in the step (1), wherein the mode of feeding the antibiotics to the fasting experimental aquatic animals is intragastric administration; and (3) carrying out a breeding experiment on the aquatic animals subjected to gavage of antibiotics in the step (2). According to the method, the mode of intragastric administration of the antibiotics once a week is adopted, the growth and health of the aquatic animals are not directly damaged while the effective content of the antibiotics is guaranteed and intestinal microorganisms are killed, the model construction method is easy to operate, the model stability is good, and the method is a better choice for establishing the pseudo-sterile aquatic animal model.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for constructing a pseudo-sterile aquatic animal model and application thereof. Background Art

[0002] Intestinal microorganisms are regarded as a "functional organ". They are huge in number and variety. They have been found to play an important role in the normal physiological functions of the body, such as development, evolution, nutritional metabolism, immunity and barrier protection, and the occurrence and development of diseases. In recent years, with the development of sequencing technology, the role of intestinal flora in the health of aquatic animals has received unprecedented attention. As the research continues to deepen, ordinary aquatic animals can no longer meet the needs of scientific research. Germ-free animal models, gnotobiotic animals and pseudo-germ-free animal models have become essential core tools for in-depth research on the intestinal microbiome.

[0003] At present, aseptic and gnotobiotic animal models, mainly based on model animals such as zebrafish (Danio rerio) and three-spined stickleback (Gasterosteus aculeatus), and economic fish such as rainbow trout (Oncorhynchus mykiss), Nile tilapia (Oreochromis niloticus) and European seabass (Dicentrarchus labrax), have been successfully established. They have been used to study the effects of microorganisms on host development, screen probiotic candidates, and host-pathogen interactions. However, the construction methods of the above animal models are only applicable to fry and juvenile fish, and it is difficult to apply them to fry and adult fish of aquaculture economic animals. In addition, due to the large differences in the physiological and metabolic functions of fish at different developmental stages, the development of a method that can effectively eliminate the intestinal flora of fry or adult aquatic animals will greatly promote the study of the interaction between aquatic animal hosts, microbiota, and environmental factors. Pseudo-aseptic animals refer to the use of antibiotics to make the intestines of experimental animals temporarily sterile. Its simple operation and low cost make it very popular in scientific research, but the current research on pseudo-sterile models for juvenile and adult economic fish in aquaculture is still very limited and needs further development and improvement.

[0004] In the context of global warming today, the health of aquaculture animals faces many challenges. As a key barrier connecting the body with the external environment, the intestine is one of the main target organs affected by high temperature stress. A large number of studies have found that high temperature can induce enteritis in fish and reduce their growth performance, but the mechanism is still unclear. The intestinal flora is a core component of the intestinal microecological system. Under steady-state conditions, intestinal symbiotic bacteria can not only enhance the host's resistance to pathogen colonization by competing for nutrients and adhesion sites, but also help maintain the integrity of the intestinal barrier and regulate the host's immune response. Previous studies have shown that high temperature can cause intestinal flora imbalance in aquatic animals, resulting in a significant increase in the abundance of pathogens and a significant decrease in the abundance of probiotics. However, it is still unclear whether the intestinal flora mediates high temperature-induced enteritis in fish. Therefore, this study intends to use a pseudo-sterile aquatic animal model to explore this issue. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a pseudo-sterile aquatic animal model and its application. By administering antibiotics once a week through oral administration, the impact on aquatic animals can be greatly reduced while killing intestinal microorganisms. The construction process is simple and the model has high stability.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for constructing a pseudo-sterile aquatic animal model, comprising the following steps:

[0008] (1) Fasting the experimental aquatic animals;

[0009] (2) administering antibiotics to the fasted aquatic animals in step (1), wherein the administration of antibiotics to the fasted aquatic animals is by intragastric administration;

[0010] (3) Conducting breeding experiments on aquatic animals that have been gavage-administered with antibiotics in step (2).

[0011] In some embodiments, in step (1), the experimental aquatic animal is one of fish and crustacean, the weight of the experimental aquatic animal is 10g-2kg, and the fasting time is 24h-48h.

[0012] Preferably, the aquatic animal is a Lateolabrax having a body weight of 80-200 g.

[0013] In some embodiments, in step (2), the frequency of gavage is 1 day / time to 7 days / time.

[0014] In some embodiments, in step (2), the gavage method is: the fasted experimental aquatic animal is taken out from the breeding system and placed in a breeding water body containing anesthetics. After the experimental animal is anesthetized, it is taken out and placed in a basin containing water for weighing. The amount of antibiotics required for gavage is calculated based on the weight of the fasted experimental aquatic animal, and then the antibiotics are injected into the stomach of the experimental animal through the enema tube using a sterile syringe and a disposable enema tube.

[0015] Preferably, the anesthetic used for anesthesia is one of eugenol or ethyl m-aminobenzoate methanesulfonate.

[0016] In some embodiments, the antibiotic is a mixture of metronidazole, neomycin sulfate, and vancomycin hydrochloride;

[0017] The mixing ratio of the mixture is metronidazole: neomycin sulfate: vancomycin hydrochloride = 2:2:1, and the dosage of the antibiotic is 10-2000 mg of the antibiotic per kg of the fasted experimental aquatic animal.

[0018] In some embodiments, the antibiotic is a mixture of metronidazole, neomycin sulfate, and vancomycin hydrochloride dissolved in sterile saline or sterile PBS;

[0019] The mixing ratio of the mixture is metronidazole: neomycin sulfate: vancomycin hydrochloride = 2:2:1, and the dosage of the antibiotic is 10-2000 mg of the antibiotic per kg of the fasted experimental aquatic animal.

[0020] Preferably, the concentration of the antibiotic is 50 mg / mL-500 mg / mL.

[0021] In some embodiments, in step (3), the culture method is: carried out in an indoor recirculating aquaculture system, with a light cycle of 12h dark / light cycle, a temperature of 27±1°C, and feeding with feed that meets the nutritional needs of aquaculture animals, twice a day (8:00, 18:00), feeding when full.

[0022] In some embodiments, the method further comprises detecting the bacterial content in the hindgut tissue or hindgut contents by using a plate spreading method after the gavage.

[0023] The present invention also provides application of the constructed pseudo-sterile aquatic animal model in studying enteritis in fish induced by intestinal flora.

[0024] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0025] The present invention adopts the method of gavage of antibiotics once a week, which ensures the effective content of antibiotics and kills intestinal microorganisms without directly damaging the growth and health of aquatic animals. The model construction method is simple to operate, low in cost, and has good model stability. It is a better choice for establishing a pseudo-sterile aquatic animal model.

[0026] The constructed pseudo-sterile aquatic animal model confirmed that intestinal flora plays an important role in high temperature-induced enteritis in Lateolabrax japonicus. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 To illustrate the effect of adding 15g / kg antibiotics to feed on the number of intestinal microorganisms in the hindgut mucosa and hindgut contents of Lateolabrax japonicus, where A, B and C are the number of hindgut mucosa microorganisms of Lateolabrax japonicus after feeding feed containing 15g / kg antibiotics for 1, 3 and 12 days, respectively, and D, E and F are the number of intestinal microorganisms in the hindgut contents of Lateolabrax japonicus after feeding feed containing 15g / kg antibiotics for 1, 3 and 12 days, respectively;

[0028] Figure 2 To illustrate the effect of oral administration of 250-1000 mg / kg antibiotics on the number of microorganisms in the hind intestine mucosa of Lateolabrax after 1 day, where A: the number of microorganisms in the hind intestine mucosa of Lateolabrax after oral administration of 250 mg and 500 mg antibiotic mixture per kg body weight for 1 day, B: the number of microorganisms in the hind intestine mucosa of Lateolabrax after oral administration of 1000 mg antibiotic mixture per kg body weight for 1 day, "*" indicates P < 0.05;

[0029] Figure 3 To illustrate the effect of oral administration of 500 mg / kg antibiotics on the number of microorganisms in the hindgut mucosa of Leptospermum japonicum for 6 days. "*" indicates P < 0.05;

[0030] Figure 4 Effects of intragastric administration of antibiotics on food intake and survival rate of Lateolabrax japonicus for one week. A: Food intake of Lateolabrax japonicus after intragastric administration of 500 mg antibiotic mixture per kg body weight for one week. B: Survival rate of Lateolabrax japonicus after intragastric administration of 500 mg antibiotic mixture per kg body weight for one week. “*” indicates P<0.05, and “ns” indicates no significant difference.

[0031] Figure 5Effects of oral administration of antibiotics on gene expression levels of inflammatory cytokines in the intestine of Lateolabrax after one week of oral administration of 500 mg of antibiotic mixture per kg body weight. A: Gene expression level of tnf-α in the intestine of Lateolabrax after oral administration of 500 mg of antibiotic mixture per kg body weight for one week. B: Gene expression level of il-4 in the intestine of Lateolabrax after oral administration of 500 mg of antibiotic mixture per kg body weight for one week. B: Gene expression level of il-8 in the intestine of Lateolabrax after oral administration of 500 mg of antibiotic mixture per kg body weight for one week. "*" indicates P < 0.05, "**" indicates P < 0.01, and "ns" indicates no significant difference. DETAILED DESCRIPTION

[0032] The present invention is further described in detail in conjunction with specific embodiments. The following embodiments can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way.

[0033] The embodiment of the present invention provides a method for constructing a pseudo-sterile aquatic animal model, comprising the following steps:

[0034] (1) Fasting the experimental aquatic animals;

[0035] Specifically, an experimental aquatic animal of a fish or crustacean weighing 10 g to 2 kg is fasted for 24 hours.

[0036] (2) administering antibiotics to the fasted aquatic animals in step (1), wherein the administration of antibiotics to the fasted aquatic animals is by intragastric administration;

[0037] Specifically, after the fasted experimental aquatic animals are anesthetized with eugenol, they are gavaged with an antibiotic mixture of metronidazole: neomycin sulfate: vancomycin hydrochloride = 2:2:1 at a rate of 10-2000 mg per kg of the fasted experimental aquatic animals, with a gavage frequency of 1 day / time to 7 days / time.

[0038] The method also includes anesthetizing the fasted experimental aquatic animals with eugenol, and then gavaging 10-2000 mg of the antibiotic mixture per kg of the fasted experimental aquatic animals with a mixture of metronidazole, neomycin sulfate and vancomycin hydrochloride dissolved in sterile saline or sterile PBS, wherein the concentration of the antibiotics is 50 mg / mL-500 mg / mL and the gavage frequency is 1 day / time to 7 days / time.

[0039] The specific gavage operation is as follows: the fasted experimental aquatic animals are taken out from the breeding system and placed in a breeding water body containing eugenol, with a water temperature of 23-27°C, an ammonia nitrogen content of 0-0.1 mg / L, a nitrite content of 0.1-0.15 mg / L, and a dissolved oxygen of 10 mg / L. After the experimental animals are anesthetized, they are taken out one by one and placed in a basin containing water for weighing. The amount of antibiotics required for gavage is calculated based on the weight of each fish, and a 1 mL sterile syringe with a scale is used with a disposable enema tube (8 cm*2 mm) to absorb an appropriate amount of antibiotic suspension, and the antibiotic suspension is injected into the stomach of the experimental animal through the enema tube.

[0040] (3) Conduct breeding experiments on aquatic animals that have been fed with experimental antibiotics in step (2).

[0041] Specifically, it was carried out in an indoor recirculating aquaculture system at the Jimei University Fisheries Experimental Field, with a photoperiod of 12 h dark / light cycle, a temperature of 27±1°C, an ammonia nitrogen content of 0-0.1 mg / L, a nitrite content of 0.1-0.15 mg / L, and dissolved oxygen>10 mg / L.

[0042] The present invention also includes detecting the bacterial content of the hindgut tissue or hindgut contents by using a plate coating method after the intragastric administration is completed.

[0043] Specifically, the hindgut tissue or hindgut contents were weighed using a 1 / 10,000 analytical balance, 9 volumes of sterile saline were added according to the weight-to-volume ratio, homogenized at 70 Hz for 2 min, 100 μL of the homogenate was added to 900 μL of saline, and four consecutive 10-fold gradient dilutions were performed to form four concentration gradients. 100 μL of the diluted homogenate was spread on nutrient agar medium, cultured at 28°C for 48 h, the number of single colonies in the culture medium was counted, and the bacterial content in the sample was calculated according to the following formula:

[0044] Content of intestinal microorganisms (CFU / g) = number of single colonies * dilution factor / sample weight

[0045] The experimental data obtained were expressed as mean ± standard error, and one-way analysis of variance was performed using SPSS22.0 analysis software. The T-test method was used to compare the significance of differences among the groups. "*" indicates that the differences among the groups were significant (P<0.05).

[0046] According to the rodent standard, the pseudo-sterile animal model was successfully constructed when the bacterial removal rate reached more than 90%.

[0047] The calculation method of the antibacterial rate is: [1-(the content of intestinal microorganisms in the antibiotic treatment group / the content of intestinal microorganisms in the control group)]*100%.

[0048] The dosage of the anesthetic in the present invention is: the volume ratio of the anesthetic to water is 1:10000.

[0049] The present invention also provides an application of the constructed pseudo-sterile aquatic animal model in studying intestinal flora-induced fish enteritis. Specifically, the pseudo-sterile aquatic animal model constructed by the construction method is used to study whether intestinal flora mediates high temperature-induced fish enteritis.

[0050] Embodiment 1:

[0051] Metronidazole, neomycin sulfate and vancomycin hydrochloride were dissolved in sterile saline at a ratio of 2:2:1 to prepare an antibiotic mixture suspension with a concentration of 83.3 mg / mL. 45 healthy striped seabass were randomly distributed in three breeding barrels, with 15 in each breeding barrel, and fed with commercial feed (Zhuhai Haiwei Feed Co., Ltd., marine fish compound feed). Before the gavage experiment, the striped seabass was fasted for 24 hours and anesthetized with eugenol. During gavage, the striped seabass was fished out and weighed, and gavage was performed at a dose of 3 mL of antibiotic mixture suspension (i.e., containing 250 mg of antibiotic mixture, AB-250) per kg of body weight. After 24 hours of gavage, the hindgut tissue samples were collected for plate coating and counting.

[0052] Embodiment 2:

[0053] Metronidazole, neomycin sulfate and vancomycin hydrochloride were dissolved in sterile saline at a ratio of 2:2:1 to prepare an antibiotic mixture suspension with a concentration of 166.7 mg / mL. 45 healthy striped sea bass were randomly distributed in three breeding barrels, with 15 in each breeding barrel, and were fed with commercial feed (Zhuhai Haiwei Feed Co., Ltd., marine fish compound feed). Before the gavage experiment, the striped sea bass was fasted for 24 hours and anesthetized with eugenol. During gavage, the striped sea bass was fished out and weighed, and gavage was performed at a dose of 3 mL of antibiotic mixture suspension (i.e., containing 500 mg of antibiotic mixture, AB-500) per kg body weight. The hindgut tissue was collected for plate counts on the first and sixth days after gavage, and fasted for 24 hours before sampling.

[0054] Embodiment 3:

[0055] Metronidazole, neomycin sulfate and vancomycin hydrochloride were dissolved in sterile saline at a ratio of 2:2:1 to prepare an antibiotic mixture suspension with a concentration of 333.3 mg / mL. 45 healthy striped seabass were selected and randomly distributed in three breeding barrels, with 15 in each breeding barrel, and the striped seabass were fed with commercial feed. Before the gavage experiment, the striped seabass was fasted for 24 hours and anesthetized with eugenol. During gavage, the striped seabass was fished out and weighed, and gavage was performed at a dose of 3 mL of antibiotic mixture suspension (i.e., containing 1000 mg of antibiotic mixture, AB-1000) per kg of body weight. The hindgut tissue was collected 24 hours after the end of gavage for plate counting.

[0056] Embodiment 4:

[0057] 180 healthy japonica seabass (~140g) were randomly distributed in 12 culture barrels (450L), with 15 in each barrel, for a one-week feeding and growth experiment. Four treatment groups were set up in the experiment, namely the suitable temperature group (27℃, MAC), the high temperature group (33℃, HAC), the suitable temperature antibiotic treatment group (27℃, MA) and the high temperature antibiotic treatment group (33℃, HA), with 3 barrels in each group. The japonica seabass were fed with commercial feed (Zhuhai Haiwei Feed Co., Ltd., marine fish compound feed) twice a day (6:00, 18:00) until apparent satiation. Before the experiment, the MA group and the HA group were gavaged with an antibiotic mixture to remove intestinal flora, and the gavage dose was 500mg of antibiotics per kg of the fasted experimental aquatic animals; the MAC group and the HAC group were gavaged with an equal amount of physiological saline. During the breeding period, a natural light cycle was used, the ammonia nitrogen content was 0-0.1 mg / L, the nitrite content was 0.1-0.15 mg / L, and the dissolved oxygen was >10 mg / L. The specific preparation method of the antibiotic suspension was as follows: metronidazole, neomycin sulfate and vancomycin hydrochloride were dissolved in sterile saline at a ratio of 2:2:1 to prepare an antibiotic mixture suspension with a concentration of 166.7 mg / mL. The method of oral administration of antibiotics was as follows: fasting for 24 hours before the experiment, the fasted experimental aquatic animals were removed from the breeding system, and placed in a breeding water body containing eugenol, the water temperature was 23-27°C, the ammonia nitrogen content was 0-0.1 mg / L, the nitrite content was 0.1-0.15 mg / L, and the dissolved oxygen content was 10 mg / L. After the experimental animals were anesthetized, they were fished out one by one and placed in a basin containing water for weighing. The amount of antibiotics required for gavage was calculated according to the weight of each fish. A 1mL sterile syringe with a scale and a disposable enema tube (8cm*2mm) were used to draw an appropriate amount of antibiotic suspension, and the antibiotic suspension was injected into the stomach of the experimental animal through the enema tube. After gavage, the sea bass was returned to the original breeding barrel for a week of breeding experiments. After the experiment, the food intake and survival rate were counted, and samples were taken. Before sampling, the fish were anesthetized with eugenol, 3 fish were taken from each barrel, and intestinal tissue samples were collected in 2mL cryopreservation tubes with sterile ophthalmic scissors and ophthalmic forceps. After liquid nitrogen quick freezing, they were stored in a -80℃ ultra-low temperature refrigerator for RNA extraction.

[0058] Comparative Example 1:

[0059] Metronidazole, neomycin sulfate and vancomycin hydrochloride were mixed in a ratio of 2:2:1, and 15g of the above antibiotic mixture (AB-15g / kg) was added to each kg of commercial feed. After mixing evenly, the feed was refrigerated away from light. 45 healthy striped sea bass were randomly distributed in three breeding barrels, with 15 in each breeding barrel, and fed with the above feed for a 12-day breeding experiment. During the breeding period, they were fed twice a day (8:00, 18:00) until apparent satiation. Six fish were randomly selected on the 1st, 3rd and 12th days, and samples of hindgut tissue and hindgut contents were collected for plate coating and counting. Calculated at 2% feed intake, 20g of feed per kg of body weight of striped sea bass per day is equivalent to a daily intake of 300mg of antibiotic mixture.

[0060] Comparative Example 2:

[0061] 45 healthy japonica seabass were randomly distributed in three culture tanks, 15 in each tank, and fed with commercial feed for a 12-day culture experiment (CON-1). The feed was refrigerated. During the culture period, the japonica seabass were fed twice a day (8:00 and 18:00) until the fish were apparently full. The hindgut tissue and hindgut contents were collected on the 1st, 3rd and 12th days after the start of the experiment for plate smearing to calculate the bacterial content.

[0062] Comparative Example 3:

[0063] 45 healthy japonica seabass were randomly distributed in three breeding tanks, 15 in each tank, and fed with commercial feed. Before the gavage experiment, the japonica seabass were fasted for 24 hours and anesthetized with eugenol. During gavage, the japonica seabass were fished out and weighed, and gavage was performed at a dose of 3 mL of sterile saline (CON-2) per kg of body weight. The hindgut tissue was collected on the first and sixth days after gavage for plate counts, and fasted for 24 hours before sampling.

[0064] Depend on Figure 1 It can be seen that the consumption of feed containing 15 g / kg antibiotics (Comparative Example 1, AB-15 g / kg) for 1-12 days can reduce the content of microorganisms in the intestinal mucosa of Lateolabrax, but the difference is not significant (P>0.05). Similarly, compared with Comparative Example 2 (CON-1), the consumption of feed containing 15 g / kg antibiotics for 1-12 days has no significant effect on the content of microorganisms in the intestinal contents of Lateolabrax.

[0065] Depend on Figure 2It can be seen that oral administration of 250-1000 mg / kg of the antibiotic mixture (Examples 1-3) can significantly reduce the content of microorganisms in the intestinal mucosa of the Lateolabrax one day later. The results of this study show that compared with Comparative Example 3 (CON-2), the inhibition rate of the intestinal mucosal microorganisms of the Lateolabrax by 250 mg / kg antibiotic treatment (Example 1) is 91%, the inhibition rate of the intestinal mucosal microorganisms of the Lateolabrax by 500 mg / kg antibiotic treatment (Example 2) is 98%, and the inhibition rate of the intestinal mucosal microorganisms of the Lateolabrax by 1000 mg / kg antibiotic treatment (Example 3) is 95%. The above results show that the 500 mg / kg antibiotic treatment group has the best antibacterial effect. And by Figure 3 It can be seen that the antibacterial rate on the 6th day after treatment with 500 mg / kg antibiotics was 95%, and the intestinal mucosal microbial content was significantly lower than that of comparative example 3. The above study shows that compared with adding antibiotics directly to feed, oral administration of antibiotics can effectively eliminate intestinal microorganisms. The reasons may be multifaceted. First, part of the antibiotics added to the feed will be dissolved in the water during the feeding process, and the effective content of the antibiotics cannot be guaranteed; in addition, it is impossible to guarantee that each fish has ingested the same amount of feed, so the method of adding antibiotics to the feed may not guarantee a consistent effect.

[0066] In summary, oral administration of 250-1000 mg of antibiotic mixture (metronidazole, neomycin sulfate and vancomycin hydrochloride = 2:2:1) per kg body weight of Lateolabrax japonicus can significantly reduce the number of intestinal mucosal microorganisms. Among them, 500 mg / kg has a better effect, with an inhibition rate of up to 98% on microorganisms, and the antibacterial effect can be maintained for at least 6 days, which can be used to construct a pseudo-sterile Lateolabrax japonicus model.

[0067] In addition, by Figure 4 A shows that, under 27℃, intragastric administration of antibiotics has no significant effect on the food intake of Lateolabrax, while under 33℃, intragastric administration of antibiotics significantly reduces the food intake of Lateolabrax. Figure 4 B shows that intragastric administration of antibiotics had no significant effect on the survival rate of Lateolabrax japonicus. Figure 5 It can be seen that compared with the moderate temperature group (MAC), the high temperature group (HAC) treatment for one week significantly upregulated the gene expression levels of inflammatory cytokines tnf-α, il-4 and il-8 in the intestinal inflammatory cytokines of the japonicus in the Lateolabrax japonicus. Under high temperature conditions, after the intestinal flora was cleared with antibiotics, the gene expression levels of inflammatory cytokines tnf-α, il-4 and il-8 in the intestinal inflammatory cytokines of the japonicus in the HA group were significantly downregulated compared with the HAC group. Under moderate temperature conditions, the use of antibiotics to clear the intestinal flora did not affect the gene expression levels of the above cytokines, that is, there was no significant difference between the MAC group and the MA group. The above results show that clearing the intestinal flora can significantly inhibit the gene expression levels of inflammatory cytokines in the japonicus intestinal inflammatory cytokines induced by high temperature, indicating that the intestinal flora plays an important role in high temperature-induced enteritis in japonicus.

[0068] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for constructing a pseudo-sterile aquatic animal model, characterized in that: The following steps are involved: (1) Fasting the experimental aquatic animals; (2) administering antibiotics to the fasted aquatic animals in step (1), wherein the administration of antibiotics to the fasted aquatic animals is by intragastric administration; (3) Conducting breeding experiments on aquatic animals that have been gavage-administered with antibiotics in step (2).

2. The method for constructing a pseudo-sterile aquatic animal model according to claim 1, characterized in that: The experimental aquatic animal in step (1) is one of fish and crustacean, the weight of the experimental aquatic animal is 10g-2kg, and the fasting time is 24h-48h.

3. The method for constructing a pseudo-sterile aquatic animal model according to claim 1, characterized in that: The gavage method in step (2) is as follows: the fasted experimental aquatic animal is taken out from the breeding system and placed in a breeding water body containing anesthetics. After the experimental animal is anesthetized, it is taken out and placed in a basin containing water for weighing. The amount of antibiotics required for gavage is calculated based on the weight of the fasted experimental aquatic animal, and then the antibiotics are injected into the stomach of the experimental animal through the enema tube using a sterile syringe and a disposable enema tube.

4. The method for constructing a pseudo-sterile aquatic animal model according to claim 1, characterized in that: The frequency of gavage in step (2) is 1 day / time to 7 days / time.

5. The method for constructing a pseudo-sterile aquatic animal model according to claim 1 or 3, characterized in that: The antibiotic is a mixture of metronidazole, neomycin sulfate and vancomycin hydrochloride; The mixing ratio of the mixture is metronidazole: neomycin sulfate: vancomycin hydrochloride = 2:2:1, and the dosage of the antibiotic is 10-2000 mg of the antibiotic per kg of the fasted experimental aquatic animal.

6. The method for constructing a pseudo-sterile aquatic animal model according to claim 1, characterized in that: The antibiotic is a mixture of metronidazole, neomycin sulfate and vancomycin hydrochloride dissolved in sterile saline or sterile PBS; The mixing ratio of the mixture is metronidazole: neomycin sulfate: vancomycin hydrochloride = 2:2:1, and the dosage of the antibiotic is 10-2000 mg of the antibiotic per kg of the fasted experimental aquatic animal.

7. The method for constructing a pseudo-sterile aquatic animal model according to claim 6, characterized in that: The concentration of the antibiotic is 50 mg / mL-500 mg / mL.

8. The method for constructing a pseudo-sterile aquatic animal model according to claim 1, characterized in that: The culture method of step (3) is: carried out in an indoor recirculating aquaculture system, with a light cycle of 12h dark / light cycle, a temperature of 27±1°C, and feeding with feed that meets the nutritional needs of aquaculture animals twice a day (8:00, 18:00), feeding when full.

9. The method for constructing a pseudo-sterile aquatic animal model according to claim 1, characterized in that: It also includes detecting the bacterial content in the hindgut tissue or hindgut contents by using a plate spreading method after the gavage.

10. Use of the pseudo-sterile aquatic animal model constructed according to the method according to any one of claims 1 to 9 in studying the role of intestinal flora in high temperature-induced enteritis in fish.

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