Application of enterococcus faecalis SZ07 in preparation of trachinotus ovatus nervous necrosis virus resisting fish feed and medicine

Abstract

The invention provides an application of enterococcus faecalis SZ07 in preparation of a trachinotus ovatus nervous necrosis virus resistant fish feed, the collection number of the enterococcus faecalis SZ07 is GDMCC No: 66159, and the collection center is Guangdong Microbial Culture Collection Center. Related experiments prove that the enterococcus faecalis SZ07 provided by the invention can effectively improve the intestinal morphology of the trachinotus ovatus, and more importantly, the enterococcus faecalis SZ07 can effectively regulate the immune response of a trachinotus ovatus body and improve the immunocompetence, antioxidant capacity and the like of the trachinotus ovatus, so that the antiviral capacity of the trachinotus ovatus is effectively improved, and the effect of preventing and treating the trachinotus ovatus is achieved. Particularly, the nervous necrosis virus resistance of the trachinotus ovatus is obviously improved. The enterococcus faecalis SZ07 provided by the invention can effectively prevent and control viral nervous necrosis of trachinotus ovatus, improve the survival rate, growth speed and disease resistance of trachinotus ovatus fries, and promote healthy development of trachinotus ovatus breeding industry.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to the application of Enterococcus faecalis SZ07 in the preparation of fish feed and drugs resistant to oval pomfret nerve necrosis virus. Background Technology

[0002] As a crucial species in the expansion of deep-sea aquaculture, the oval pomfret exhibits a pronounced gregarious swimming habit and rapid movement. It grows quickly, has delicious meat, and high economic value. Currently, the control of diseases such as viral nerve necrosis virus (VNN) in oval pomfret aquaculture mainly relies on traditional methods. Vaccination is commonly used for disease prevention, but because NNN virus has multiple genotypes and is prone to mutation, existing vaccines are insufficient to provide effective immune protection against all variants, leading to inconsistent control effects.

[0003] In aquaculture management, water quality control mainly relies on physical filtration and simple chemical adjustments, which are insufficient to precisely maintain the balance of the microbial community in the aquaculture water and effectively inhibit the growth of harmful microorganisms, thus creating favorable conditions for virus transmission. Regarding feed, while commercially available general-purpose feeds can meet the basic growth needs of oval pomfret, they lack functional components specifically designed to enhance the fish's immunity and resistance to viral infections.

[0004] In studies on the application of probiotics in pomfret farming, various common probiotic strains, such as lactic acid bacteria and Bacillus, have been introduced. However, these studies are mostly based on empirical screening and lack in-depth exploration of the mechanisms of action of probiotics in the pomfret's gut. Screening methods are limited to observing the effects of probiotics on fish growth rate and conventional water quality indicators, without analyzing the complex interactions between probiotics and the fish's immune system, gut microbiota, and viruses at the molecular level. Furthermore, existing studies have not comprehensively evaluated the functions of probiotics, often focusing only on single functions, such as growth promotion or water purification, while neglecting their potential to enhance the fish's antiviral capabilities. Summary of the Invention

[0005] To address the problems and shortcomings of existing technologies, this invention provides the application of Enterococcus faecalis SZ07 in the preparation of fish feed and drugs resistant to neuronecrosis virus in pomfret. Related experiments have demonstrated that the Enterococcus faecalis SZ07 provided by this invention can effectively improve the intestinal morphology of pomfret, and more importantly, it can effectively regulate the immune response of pomfret, improving their immunity and antioxidant capacity, thus effectively enhancing their antiviral ability, particularly significantly improving their resistance to neuronecrosis virus. In other words, the Enterococcus faecalis SZ07 provided by this invention can effectively control viral neuronecrosis disease in pomfret, improve the survival rate, growth rate, and disease resistance of pomfret fry, and promote the healthy development of the pomfret aquaculture industry.

[0006] According to a first aspect of the present invention, an application of Enterococcus faecalis SZ07 in the preparation of fish feed resistant to oval pomfret neuronecrosis virus is provided. The preservation number of Enterococcus faecalis SZ07 is GDMCC No: 66159, and the preservation center is the Guangdong Provincial Microbial Culture Collection Center. It should also be noted that the preservation date of Enterococcus faecalis SZ07 is April 18, 2025.

[0007] Enterococcus faecalis is part of the normal gut microbiota. In a healthy state, it, along with other gut microbes, constitutes a complex ecosystem. They maintain the stability of the gut microbiota by competing for nutrients and occupying the intestinal mucosal surface, thus inhibiting the overgrowth of harmful bacteria.

[0008] However, different strains of Enterococcus faecalis exhibit significant differences in function and effect. Some types of Enterococcus faecalis can be used as probiotics and clinically applied strains, while others are pathogenic strains. For example, Enterococcus faecalis Symbioflor-1 has been extensively studied and used clinically. Studies have shown that Symbioflor-1 can significantly reduce the recurrence frequency in patients with chronic relapsing bronchitis and reduce the frequency of antibiotic use. Enterococcus faecalis EF-2001 has shown significant potential in anti-aging and neuroprotection. Studies have also shown that some Enterococcus faecalis strains can express hemolysin, exhibiting strong pathogenicity and causing hospital-acquired infections such as endocarditis, sepsis, surgical wound infections, and urinary tract infections. Furthermore, the genome of Enterococcus faecalis contains a large number of mobile genetic elements that can transfer drug resistance genes and virulence factors through conjugation. For example, some multidrug-resistant Enterococcus faecalis strains can tolerate multiple antibiotics, leading to treatment difficulties.

[0009] Therefore, the rational selection and application of Enterococcus faecalis has become one of the challenges in its application. In particular, there are few reports on the application of Enterococcus faecalis against neuronecrosis virus, and there is virtually no research on the application of Enterococcus faecalis against ovoid pomfret neuronecrosis virus.

[0010] This invention, through long-term research, has discovered that Enterococcus faecalis SZ07 can effectively prevent and control the nerve necrosis virus in oval pomfret, thereby effectively improving the growth performance, disease resistance, and quality of oval pomfret. This is beneficial to promoting the healthy and sustainable development of the oval pomfret farming industry and increasing its economic benefits.

[0011] Specifically, Enterococcus faecalis can regulate the immune response of pomfret by enhancing bifunctional immune enzyme indicators (significantly increased ACP and ALP activities), improving antioxidant capacity (significantly increased SOD, GSH-Px, and CAT activities), and regulating the expression of immune-related factors (downregulating TNF-α and IL-8 expression and upregulating TGF-β and IL-10 levels). This enhances the pomfret's resistance to neuronecrosis virus during its growth process. It may do so by affecting the viral particle structure of pomfret neuronecrosis virus, interfering with the adsorption, invasion, and replication process of pomfret neuronecrosis virus during cell infection, or through other specific immune pathways. This provides a new and effective means for the prevention and control of viral neuronecrosis disease in pomfret.

[0012] Furthermore, the Enterococcus faecalis SZ07 provided in this invention possesses excellent stress resistance (resistance to high temperatures / bile salts / acids) and adhesion ability. Its excellent stress resistance allows it to stably exert its probiotic effects in complex aquaculture environments; its outstanding adhesion ability helps it colonize the fish intestines and continuously regulate the intestinal microecology. Therefore, Enterococcus faecalis SZ07 can better exert its corresponding effects in oval pomfret, achieving a more superior anti-neural necrosis virus effect.

[0013] Preferably, the fish feed includes a basal feed and Enterococcus faecalis SZ07; the inoculation amount of Enterococcus faecalis SZ07 in the basal feed is 1×10⁻⁶. 5 CFU / g ~ 1×10 9 CFU / g. Regarding the inoculation amount of Enterococcus faecalis SZ07, if the inoculation amount is too small, it will not effectively improve the anti-neurone necrosis virus effect of oval pomfret, nor will it effectively improve the immune performance of oval pomfret itself. Ultimately, this will lead to a higher probability of oval pomfret infection with neuronecrolysis virus during normal cultivation, and a decrease in the quality of oval pomfret. If the inoculation amount is too large, it will easily cause an imbalance in the intestinal flora homeostasis, which is not conducive to the function of the specific Enterococcus faecalis SZ07 screened in this invention. Therefore, it is also not conducive to effectively improving the anti-neurone necrosis virus effect of oval pomfret, and will also reduce the growth performance and quality of the cultivated oval pomfret.

[0014] Preferably, the inoculation amount of Enterococcus faecalis SZ07 in the basal feed is 1×10⁻⁶.7 CFU / g.

[0015] Preferably, the basic feed comprises the following components: 40-50 wt% crude protein, 2-6 wt% crude fiber, 4-10 wt% crude fat, and 10-20 wt% crude ash. Other unlisted components are conventional components in the art, such as vitamins, amino acids, and mineral additives, which can be added according to actual needs.

[0016] According to a second aspect of the present invention, there is an application of Enterococcus faecalis SZ07 in the preparation of a drug for treating ovoid pomfret neuronecrosis virus. The drug prepared using Enterococcus faecalis SZ07 also has a significant effect on the prevention and treatment of ovoid pomfret neuronecrosis virus.

[0017] According to a third aspect of the present invention, a method for cultivating oval pomfret resistant to nerve necrosis virus is provided. The cultivation method includes the following steps: Step 1, preparing a basic feed by mixing Enterococcus faecalis SZ07 with the basic feed and drying it naturally to obtain fish feed; the preservation number of Enterococcus faecalis SZ07 is GDMCC No: 66159, and the preservation center is Guangdong Provincial Microbial Culture Collection Center; Step 2, placing healthy oval pomfret juveniles in the following water environment for cultivation: the temperature is maintained at 26-28℃, the salinity is maintained at 28-30‰, the dissolved oxygen content is not less than 5mg / L, and the pH is maintained at 7.8-8.2; and during the cultivation period, the fish feed is fed twice a day.

[0018] The aquaculture ecosystem also significantly impacts the effectiveness of oval pomfret against neuronecrosis virus (NSV). Besides the aforementioned challenges in selecting and applying probiotics, current technologies often lack a comprehensive understanding of the complex relationships within the aquaculture ecosystem. In aquaculture management, factors such as feed, water quality, and probiotics are often viewed in isolation, failing to fully recognize their interrelationships and synergistic effects. For instance, feed nutrients affect fish metabolism and immunity, thus influencing the utilization of probiotics; water quality directly impacts probiotic activity, fish health, and virus transmission. This lack of systematic thinking prevents current methods from fundamentally addressing the problems of weak antiviral capabilities and poor quality improvement in oval pomfret fry.

[0019] Therefore, in the method for cultivating oval pomfret with resistance to neuronecrosis virus provided by this invention, during the cultivation process, not only is the feed inoculated with specifically screened Enterococcus faecalis SZ07, which is resistant to neuronecrosis virus in oval pomfret, but the temperature, salinity, dissolved oxygen, and pH of the water body during the cultivation process are also strictly controlled. The feed, water quality, and probiotics are considered as a whole, and the above factors are regulated from multiple aspects to ensure that the oval pomfret is in an excellent growth environment. This effectively enhances the oval pomfret's resistance to neuronecrosis virus and improves its immunity, thereby effectively preventing and treating neuronecrosis virus in oval pomfret and improving the quality of the cultivated oval pomfret.

[0020] Preferably, in step 1, the inoculation amount of Enterococcus faecalis SZ07 in the basal feed is 1×10⁻⁶. 5 CFU / g ~ 1×10 9 CFU / g.

[0021] Preferably, in step 1, the inoculation amount of Enterococcus faecalis SZ07 in the basal feed is 1×10⁻⁶. 7 CFU / g.

[0022] Preferably, before aquaculture in step 2, healthy oval pomfret juveniles are cultured for 1 to 3 weeks in advance.

[0023] Preferably, in step 2, a basic feed is given for 1 to 2 weeks before feeding the fish food.

[0024] Preferably, in step 2, during the aquaculture process, fish feed is given twice a day at 08:00 and 16:00.

[0025] According to a fourth aspect of the present invention, a method for screening probiotics resistant to neuronecrosis virus in oval pomfret is provided, comprising the following steps: S1. For an oval pomfret population naturally infected with neuronecrosis virus, oval pomfret exhibiting symptoms of viral neuronecrosis disease are used as the control group, and asymptomatic oval pomfret are used as the experimental group. Intestinal tissue samples, brain tissue samples, and spleen tissue samples are collected from oval pomfret in both the control and experimental groups; S2. RNA is extracted from the brain tissue samples and spleen tissue samples from both the control and experimental groups, and high-throughput sequencing is performed; S3. 3. Compare the gene expression of brain and spleen tissue samples from oval pomfret in the control and experimental groups to screen for differentially expressed genes; S4. Perform functional enrichment analysis on differentially expressed genes; S5. Perform microbial community analysis on intestinal tissue samples from oval pomfret in the control and experimental groups respectively; S6. Based on the analysis results of S4 and S5, determine the source of probiotic intestinal tissue samples and the type of probiotics, and isolate probiotics from the intestinal tissue sample source. Then, through further biological analysis, screen for probiotics with strong stress resistance and outstanding adhesion ability.

[0026] As described in the background section, existing technologies struggle to effectively control viral neuronecrosis in pomfret. Even in studies on the application of probiotics in pomfret farming, common probiotics are introduced, and screening is largely based on empirical methods. There is a lack of research into the molecular mechanisms by which probiotics colonize and multiply in the pomfret gut, and compete or synergize with other microorganisms. The failure to utilize multi-omics technologies to comprehensively analyze the probiotic functions at the gene, transcription, and protein levels leads to a blind screening process, making it difficult for selected probiotics to stably exert antiviral effects in actual farming environments. Furthermore, insufficient research into viral mutation characteristics and host interaction mechanisms results in delays in vaccine development and disease control measures, hindering effective responses to constantly evolving viral threats.

[0027] Furthermore, while there have been studies on some genes of the oval pomfret in the application of gene technology, there is a lack of comprehensive transcriptomic analysis of fish after natural infection with NNV and genomic studies related to probiotics.

[0028] This invention provides a probiotic screening system based on multi-omics technology. It verifies and systematically analyzes the immune response mechanism and gut microbiota of pomfret infected with neuronecrosis virus (NSV) at the gene, transcription, and protein levels, particularly the changes in probiotic gene function. This allows for the precise screening of probiotics associated with NSV immunity in pomfret. Furthermore, these probiotics can be utilized to effectively control NSV in pomfret, thereby improving the survival rate, growth rate, and disease resistance of pomfret seedlings and promoting the healthy development of the pomfret aquaculture industry.

[0029] In addition, the probiotics screened in this invention have strong stress resistance (resistant to high temperature, bile salts, and acid) and outstanding adhesion ability, which means that they have a strong ability to adapt to the intestinal environment, can settle in the intestine and play a stable role, help maintain the balance of intestinal flora, improve intestinal immunity, and thus help improve the overall intestinal flora balance and immunity of oval pomfret, thereby further improving the resistance of oval pomfret to nerve necrosis virus, optimizing the growth performance and disease resistance of oval pomfret.

[0030] Preferably, the probiotic is Enterococcus faecalis SZ07; the NCBI accession number for the annotated genome sequence of Enterococcus faecalis SZ07 is JBKOFI00000000.

[0031] Preferably, in S3, comparing brain tissue samples from oval pomfret in the control group and experimental group, there were 3507 differentially expressed genes, of which 1645 were downregulated and 1862 were upregulated; comparing spleen tissue samples from oval pomfret in the control group and experimental group, there were 24484 differentially expressed genes, of which 9097 were downregulated and 15387 were upregulated.

[0032] Preferably, in S4, the results of functional enrichment analysis of differentially expressed genes are as follows: significantly enriched immune-related pathways include the JAK-STAT signaling pathway, cytokine-cytokine receptor interaction, and MAPK signaling pathway; and in the experimental group, the cellular response of brain tissue samples from oval pomfret to cycloheximine was significantly upregulated.

[0033] Differentially expressed genes were significantly enriched in the aforementioned pathways, revealing an immune regulatory network against oval pomfret neuronecrosis virus. Among these, the JAK-STAT signaling pathway is a key pathway for cytokine signaling and is widely involved in the regulation of immune responses. During viral infection, cytokines such as interferon (IFN) activate the JAK-STAT pathway, inducing cells to produce antiviral proteins and inhibiting viral replication and spread. The interaction between cytokines and their receptors is an important mode of intercellular communication within the immune system. In anti-neuronenecrosis virus immunity, cytokines (such as interleukins and interferons) bind to receptors, activating downstream signaling pathways (such as JAK-STAT and MAPK), regulating the activity, proliferation, and differentiation of immune cells. The MAPK signaling pathway (including ERK, JNK, and p38) also plays an important role in the immune response. In the immune response against neuronecrosis virus (NNV), the MAPK pathway can function through the following mechanisms: regulating the production of cytokines, enhancing the activation and function of immune cells; regulating the expression of apoptosis-related genes, clearing virus-infected cells, and reducing viral replication and spread; and participating in the regulation of inflammatory responses by controlling the production of inflammatory factors and cellular stress responses to maintain the balance of the immune response.

[0034] Furthermore, in the experimental group, the cellular response to cycloheximine was significantly upregulated in the brain tissue samples of oval pomfret. Cycloheximine is a protein synthesis inhibitor, which is usually used to study the regulatory mechanisms of protein synthesis. This indicates that a special anti-oval pomfret neuronecrosis virus mechanism was initiated in the immunity against oval pomfret neuronecrosis virus, which enhances the antiviral ability of cells by regulating the synthesis or degradation of certain antiviral proteins.

[0035] Preferably, in S6, the isolated probiotics exhibit strong stress resistance and outstanding adhesion ability. Preferably, strong stress resistance includes strong resistance to high temperatures, strong resistance to bile salts, and strong resistance to acids.

[0036] Preferably, the above-mentioned method for screening probiotics against oval pomfret neuronecrosis virus further includes step S7, which involves: performing whole-genome sequencing on the obtained probiotics to analyze their gene composition and function. Specifically, the whole-genome sequencing of the isolated Enterococcus faecalis is performed to analyze its gene composition and function. Bioinformatics methods are used to identify genes related to stress resistance and adhesion ability. For example, the presence of specific adhesin genes explains the strain's outstanding adhesion ability; simultaneously, it may contain gene clusters encoding antimicrobial peptides, providing evidence for its pathogen-inhibiting activity.

[0037] In summary, the *Enterococcus faecalis* SZ07 strain obtained by screening using specific multi-omics technologies can effectively control nerve necrosis disease in oval pomfret. Furthermore, by combining it with specific aquaculture system conditions and optimizing feed formulation and aquaculture management, it further enhances the fish's own immunity and intestinal health, thereby effectively improving the oval pomfret's resistance to nerve necrosis virus. This significantly improves the survival rate and quality of oval pomfret seedlings, achieving sustainable, healthy, and ecological development of the aquaculture industry. Attached Figure Description

[0038] Figure 1 This is a branching diagram illustrating the differences between symptomatic and asymptomatic oval pomfret infected with spontaneous neuronecrosis in Example 1.

[0039] Figure 2 This is a heatmap showing the correlation between the microbial genus (left) and species (right) levels and the host transcriptome in Example 1.

[0040] Figure 3 This is a diagram showing the tissue results of predicting the bacteriocin gene cluster in the SZ07 genome using BAGEL4 in Example 1.

[0041] Figure 4 This is a linear comparison diagram of the genome of Enterococcus faecalis SZ07 in Example 1.

[0042] Figure 5The following are the results of the stress resistance test of Enterococcus faecalis SZ07 in Example 1: Temperature sensitivity test results (A); Bile salt concentration tolerance test results (B); Acid resistance test results (C).

[0043] Figure 6 Figure 1 shows the adhesion characteristics of Enterococcus faecalis SZ07 in Example 1: results of self-aggregation ability and hydrophobicity (A); results of co-aggregation ability with pathogens (B).

[0044] Figure 7 This is a diagram showing the observation results of an intestinal tissue section of an oval pomfret in Example 2.

[0045] Figure 8 This is a diagram showing the results of a section of the midgut tissue from an oval pomfret in Example 2.

[0046] Figure 9 This is a diagram showing the results of intestinal digestive enzyme activity detection in oval pomfret in Example 2.

[0047] Figure 10 This is a diagram showing the results of the detection of intestinal antioxidant indicators in oval pomfret in Example 2.

[0048] Figure 11 This is a diagram showing the results of the intestinal metabolic-immune dual-function enzyme detection in oval pomfret in Example 2.

[0049] Figure 12 This is a diagram showing the results of the detection of antioxidant indicators in the liver of oval pomfret in Example 2.

[0050] Figure 13 This is a diagram showing the results of liver metabolism-immune dual-function enzyme detection in oval pomfret in Example 2.

[0051] Figure 14 This is a diagram showing the results of real-time fluorescence quantitative PCR of the spleen of an oval pomfret in Example 2.

[0052] Figure 15 This is a bar chart showing the community composition of the top 15 dominant taxa in the intestinal tissue of the oval pomfret in Example 2 (belonging to the horizontal community composition). Detailed Implementation

[0053] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0054] Example 1 The screening of intestinal probiotics resistant to oval pomfret nerve necrosis virus was carried out according to the following steps: S1. For a population of oval pomfret naturally infected with neuronecrosis virus (NNV), oval pomfret exhibiting symptoms of viral neuronecrosis (VNN) served as the control group, while asymptomatic oval pomfret served as the experimental group. Under sterile conditions, intestinal tissue, brain tissue, and spleen tissue samples were collected from both the control and experimental groups. Intestinal tissue samples were used for microbial analysis, while brain and spleen tissue samples were used for transcriptome analysis. The collected samples were immediately placed in a special preservation solution and stored at low temperature to prevent alterations in biomolecules and the structure of the microbial community. S2. RNA was extracted from brain tissue and spleen tissue samples of oval pomfret in the control group and experimental group, respectively, and high-throughput sequencing (i.e., transcriptome sequencing was performed using high-throughput sequencing technology). S3. Through bioinformatics analysis, the gene expression of brain tissue and spleen tissue samples from the control and experimental groups of pomfret was compared to screen for differentially expressed genes. In this study, it was found that pomfret naturally infected with neuronecrosis virus (control group) had 3,507 significantly differentially expressed genes in brain tissue samples (downregulated 1,645, upregulated 1,862) and 24,484 differentially expressed genes in spleen tissue samples (downregulated 9,097, upregulated 15,387). S4. Functional enrichment analysis was performed on differentially expressed genes; the results showed that immune-related pathways were significantly enriched in the JAK-STAT signaling pathway, cytokine-cytokine receptor interaction pathway, and MAPK signaling pathway; among them, the cellular response to cycloheximine was significantly upregulated in the brain tissue of the asymptomatic group (experimental group); S5. Microbial community analysis was performed on intestinal tissue samples from oval pomfret in both the control and experimental groups; specifically, 16S rRNA gene sequencing technology was used to analyze the intestinal flora structure. S6. Based on the analysis results of S4 and S5, determine the source of the intestinal tissue samples of intestinal probiotics and the type of intestinal probiotics, and isolate intestinal probiotics from the sample source. Then, through further biological analysis, screen out intestinal probiotics with strong stress resistance and outstanding adhesion ability.

[0055] In step S5 above, the test results of the gut microbiota structure are as follows: Figure 1 As shown, Figure 1 For analysis of branch example diagrams based on differential species annotation, from Figure 1It was found that the dominant bacterial groups in the asymptomatic group (experimental group) were Actinobacteria, Arcobacteraceae, Campylobacterales, Campylobacteria, Enterococci, Rhizobiaceae, Moraxellaceae, and Pseudomonadales; while the relatively abundant differentially abundant species in the symptomatic group (control group) were Streptococcaceae, Clostridia, and Hydrogenophilaceae. Furthermore, multivariate statistical analysis showed that the bacterial community in the asymptomatic group was species-specific, with significant enrichment of Enterococcus faecalis, while it was not detected in the symptomatic group.

[0056] Combining the analysis results of S4 and S5 (based on the results of multi-omics joint analysis), it can be concluded that the intestinal probiotic with resistance to oval pomfret nerve necrosis virus is Enterococcus faecalis (denoted as Enterococcus faecalis SZ07).

[0057] Specifically, firstly, such as Figure 2 As shown, Figure 2 Based on the correlation analysis results of the top 30 items with the most significant differences between microorganisms and transcriptomes, it is shown that Enterococcus spp. ( Enterococcus ) has a strong correlation with the transcriptome, and Enterococcus faecalis ( Enterococcus faecalis It showed significant correlation with multiple differentially expressed genes, suggesting that it may interact by regulating the expression of genes related to host immunity, metabolism, or barrier function, thereby achieving the effect of resisting oval pomfret nerve necrosis virus.

[0058] Secondly, as shown in Table 1, which presents the association analysis results between Enterococcus faecalis (a differentially expressed strain) and the expression of immune-related genes in the brain and spleen in the combined analysis of transcriptome and gut microbiota, it can be seen that the expression of immune-related genes in the brain and spleen that are significantly associated with Enterococcus faecalis as a differentially expressed strain is upregulated, showing tissue-specific differences. Upregulated records exist in the brain (such as transcript-associated genes such as TRINITY_DN20353_c0_g1_i2_3), indicating that some genes have an upregulated response in the brain. Most genes in the spleen are marked as upregulated, reflecting the general upregulation trend of associated genes in the spleen. This difference suggests: Enterococcus faecalis The selective regulation of genes in the host's brain and spleen may be related to the specialization of immune and physiological functions in different tissues. These results indicate that Enterococcus faecalis regulates the immune and physiological functions of different tissues by modulating immune-related genes in the brain and spleen, as well as spleen-related genes, thereby enhancing the anti-neurone necrosis virus activity of pomfret.

[0059] Thirdly, as shown in Table 2, which presents the functional classification results of Enterococcus faecalis-related genes in the combined transcriptome-gut microbiota analysis, it can be seen that, through NCBI homology comparison, the proteins encoded by the associated genes are involved in oxidative damage regulation (e.g., glutathione peroxidase 9), immune cell / signal regulation (e.g., GTPase IMAP family member 9, CMRF35-like molecule 3), cell cycle / development regulation (e.g., cyclin-dependent kinase 15, pituitary homeobox 3-like), and metabolism / transportation-related functions (e.g., apolipoprotein D-like, cytochrome P450 2J2-like). This truly reflects the selective regulation of multiple tissue genes by Enterococcus faecalis, exhibiting tissue-specific regulatory characteristics. This indicates that Enterococcus faecalis SZ07 effectively enhances the anti-neural necrosis virus effect in pomfret by regulating the above-mentioned multiple specific pathways.

[0060] Table 1. Combined analysis of transcriptome and gut microbiota

[0061] Table 2 Classification of related gene functions

[0062] Regarding whole-genome analysis, the sequencing results of Enterococcus faecalis SZ07 showed key genomic features. The genome of this bacterium is a circular chromosome, as shown in Table 3, with a size of 2,863,342 bp and a G+C content of 37.57 mol%. Functional gene annotation indicated that it contains 2,675 protein-coding sequences, 12 rRNA genes, and 64 tRNA genes, with a predicted total of 2,751 genes. Furthermore, the cylA gene encoding a cytolysin processing protein was not detected in the SZ07 genome, but it may exhibit an α-hemolytic phenotype due to non-cytotoxic metabolites.

[0063] Table 3 Summary of the genomic characteristics of Enterococcus faecalis SZ07

[0064] Furthermore, such as Figure 3 As shown, Figure 3The results of bacterial gene cluster structure analysis using the BAGEL tool are shown, with different color modules indicating gene function categories (such as modification, immunity / transport, etc.). The results indicate that the UviB gene (potentially involved in metabolic regulation or stress response) and the Fss2 gene (encoding fibrinogen-binding MSCRAMM adhesin) were annotated in the genome.

[0065] Furthermore, such as Figure 4 As shown, Figure 4 The results show the genome alignment of Enterococcus faecalis SZ07, Symbioflor 1, and V583 based on the Mauve program. The results indicate that the collinear regions of the genomes of Enterococcus faecalis SZ07 and Enterococcus faecalis Symbioflor 1 are contiguous, and the gene sequence is well consistent; however, compared with Enterococcus faecalis V583, the collinear regions are fragmented, and there is genomic rearrangement.

[0066] In summary, compared to traditional random screening methods for probiotics, this invention, based on a deep understanding of the immune response of fish under viral infection, changes in intestinal flora, and strain gene function, has identified a *Enterococcus faecalis* strain with clear antiviral potential. Furthermore, experimental data show that this strain exhibits inhibitory activity against common fish pathogens, and strain SZ07 demonstrates antibacterial activity against *Photobacterium damselae*, with an inhibition zone diameter of 11.35 ± 0.37 mm.

[0067] Meanwhile, the strain was tested for stress resistance (resistance to high temperatures / bile salts / acids) and adhesion ability (self-polymerization, hydrophobicity, and co-aggregation ability with pathogenic microorganisms). The test methods are as follows: Temperature sensitivity experiment: The strain was inoculated into sterile MRS liquid medium, and the inoculated medium was placed in a water bath at 30℃, 40℃, 50℃, 60℃, and 70℃ for 15 min, respectively, followed by incubation at 37℃ for 12 hours. After incubation, the OD600 value of each experimental group was measured. The group treated in the water bath at 30℃ served as the control group. Three replicates were set for each treatment group, and the survival rate of the strain in each experimental group was calculated. The survival rate was calculated using the following formula (where A is the OD600 value of the experimental group and A0 is the OD600 value of the control group): Survival rate (%) = A / A0 × 100.

[0068] Bile salt tolerance test: A mixed culture system was prepared by mixing porcine bile salt MRS culture medium (270 mL) and overnight bacterial culture (30 mL) at a volume ratio of 9:1. 5 mL of each system was taken and incubated at 37 ℃ for 4 h. After incubation, the culture was collected using a pipette, and the absorbance (OD600) at 600 nm was measured spectrophotometrically. This was used to evaluate the survival rate of strain SZ07 under simulated bile conditions. The specific calculation formula (where A1 is the OD600 at 4 h, and A2 is the OD600 at 0 h) is: Survival rate (%) = A1 / A2 × 100.

[0069] Acid tolerance test (to determine the survival ability of isolated strains in low pH environment): Fresh overnight bacterial culture was inoculated into MRS liquid medium with pH values ​​of 2, 3, and 4, and cultured at 37 ℃ (150 rpm) for 24 h. Bacterial culture under normal growth conditions was used as a control. The OD600 of the bacterial culture was measured using a microplate reader. The survival rate was calculated using the following formula (where A5 is the OD600 value of the experimental group and A6 is the OD600 value of the control group): Survival rate (%) = 100 × A5 / A6.

[0070] Self-polymerization ability determination experiment: Take fresh overnight bacterial culture, centrifuge at 10,000 × g for 5 minutes (4℃) to collect bacterial cells, suspend the cells in phosphate-buffered saline (PBS) and correct OD600 to 0.5. Take 2 mL of bacterial suspension and incubate at 37℃ for 2 hours, then take 1 mL of the supernatant to detect the OD600 value (recorded as A7). The formula for calculating self-polymerization ability is: Self-polymerization ability (%) = (1 – A7 / 0.5) × 100. Grading criteria: 16%-35% is low self-polymerization ability, 36%-50% is moderate, and more than 50% is high self-polymerization ability.

[0071] Hydrophobicity determination experiment: Fresh overnight bacterial culture was centrifuged at 10,000 × g for 5 minutes (4 ℃), the supernatant was discarded, the bacterial cells were washed 3 times with PBS, resuspended in 0.1 mol / L potassium nitrate solution, and the OD600 was corrected to 0.5. 3 mL of the bacterial suspension was mixed with 1 mL of xylene, allowed to stand for 10 minutes, shaken for 1 minute, and then allowed to stand for another 30 minutes. The OD600 value of the aqueous phase was then measured (recorded as A8). The formula for calculating hydrophobicity is: Hydrophobicity (%) = (1 – A8 / 0.5) × 100. The criteria for classifying the hydrophobicity of bacteria are: less than 20% is non-hydrophobic, 20%–50% is moderately hydrophobic, and more than 50% is highly hydrophobic.

[0072] Coagulation ability assay for pathogenic microorganisms: After correcting the OD600 of fresh overnight culture of isolated strains to 0.5, equal amounts of Escherichia coli, Staphylococcus aureus, and Salmonella suspensions were mixed and incubated at room temperature. Samples were taken at 0 hours and 18 hours, and the OD600 value of the supernatant was measured (denoted as A9). The coagulation ability was calculated using the formula: Coagulation ability (%) = (0.5 – A9) / 0.5 × 100.

[0073] Test results of the above experiments Figure 5 , 6 As shown. Among them, Figure 5 A presents the results of the temperature stress resistance experiment of strain SZ07. Using 30 ℃ as the baseline control, the survival rate of the strain was measured at different temperature gradients: when the temperature was set at 40 ℃, 50 ℃, and 60 ℃, the survival rate of the strain remained at 77.72%, 80.68%, and 77.85%, respectively; when the temperature was increased to 70 ℃, the survival rate decreased to 31.77%. These experimental results indicate that the strain can still maintain some survival ability under the extreme temperature condition of 70 ℃, showing significant high-temperature tolerance. One-way ANOVA revealed the effect of temperature on strain survival. The results showed that the survival rate of the strain in the 70 ℃ experimental group was significantly different from that in other temperature groups (p<0.05), while the survival rate of the strain in the 30 ℃–60 ℃ range did not show significant fluctuations, indicating that this temperature range is the suitable growth temperature range for strain SZ07. Furthermore, from… Figure 5 As shown in B, the survival rate of strain SZ07 in MRS culture media with added 0.1%, 0.2%, 0.3%, and 0.4% (m / V) porcine bile salts all exceeded 100%, indicating that strain SZ07 has strong bile salt tolerance. Furthermore, from... Figure 5 As shown in C, the survival rate of this probiotic strain varies significantly under different pH conditions. The growth of strain SZ07 shows an increasing trend as the pH increases from 2 to 4. At pH 2 or 3, the survival rate reaches over 25%, while at pH 4, the survival rate is as high as 85%.

[0074] Figure 6 A shows that the strain exhibits significant self-aggregation ability (75.43%) and strong hydrophobicity (79.31%). Figure 6 Data from sample B showed that its co-aggregation activities with *Escherichia coli*, *Staphylococcus aureus*, and *Salmonella* were 68.53%, 68.62%, and 47.91%, respectively. One-way ANOVA revealed that the strain's co-aggregation ability with the first two Gram-positive bacteria was significantly higher than that with *Salmonella* (p<0.05). Comprehensive analysis indicates that this strain possesses high self-aggregation, strong hydrophobicity, and co-aggregation activity with intestinal pathogens, characteristics that provide important prerequisites for its probiotic effects in the intestinal environment.

[0075] In conclusion, Figure 5 as well as Figure 6 The results indicate that this strain exhibits strong stress resistance (tolerance to high temperatures, bile salts, and acids) and outstanding adhesion ability. Further experimental data also show that this strain is sensitive to vancomycin, providing a new and effective strain resource for the development of antiviral probiotics for oval pomfret. Simultaneously, the strain's phenotypic characteristics, such as strong stress resistance, enable it to stably exert its probiotic effects in complex aquaculture environments; its outstanding adhesion ability facilitates its colonization in the fish's intestines, continuously regulating the intestinal microecology.

[0076] It should also be noted that the specific isolation and identification steps for Enterococcus faecalis are as follows: Intestinal tissue was collected from asymptomatic, healthy oval pomfret. The collected samples underwent a series of pretreatment steps to ensure the quality of the samples used for isolation. Subsequently, the intestinal tissue was streaked onto YPD agar plates using the streak plating method. The plates were incubated at 37°C for 18 hours. After incubation, well-isolated colonies were carefully picked and inoculated into MRS liquid medium. The inoculated MRS liquid medium was incubated at 37°C and 150 rpm on a shaker to promote bacterial growth. After enrichment culture in MRS liquid medium, genomic DNA was extracted from the cultured bacteria. Using specific primers targeting the 16S ribosomal RNA gene, the 16S ribosomal RNA gene was amplified by polymerase chain reaction (PCR). The PCR product was then purified and sequenced. The obtained sequence was compared with a reference sequence in a public database, identifying the isolated bacteria as Enterococcus faecalis. Finally, the identified Enterococcus faecalis isolates were preserved in MRS broth with 50% glycerol and stored at -80°C for long-term preservation.

[0077] Colonies of isolated strain SZ07 on YPD agar medium are pale grayish-white, round, flat, slightly raised, with neat edges and a moist, smooth surface. After incubation at 37°C for 20–24 h, the diameter of a single colony is approximately 1 mm. The isolated strain is Gram-positive, and microscopic examination reveals that it consists of round or oval cocci, arranged singly or in pairs.

[0078] Example 2 This embodiment sets up 5 experimental groups and one control group, and prepares 6 experimental feeds with different inoculation doses. The specific inoculation doses of Enterococcus faecalis SZ07 are as follows: (Control group, labeled C0) (Group E1) (Group E2) (Group E3) (Group E4) (Group E5) The control group was fed with feed containing Enterococcus faecalis but without inoculation. Groups E1, E2, E3, E4, and E5 were experimental groups.

[0079] Based on the above variables, the oval pomfret was bred according to the following steps: Step 1: Prepare the feed. The control group's feed is a basal feed, whose main nutritional components include 45 wt% crude protein, 4 wt% crude fiber, 7 wt% crude fat, and 15 wt% crude ash. Other unlisted components are conventional components in this field, such as vitamins, amino acids, and mineral additives, which can be added according to actual needs. The experimental groups' feed consists of the basal feed plus different inoculation amounts of Enterococcus faecalis SZ07. The prepared feed is placed in a ventilated environment to dry naturally, then packaged into sealed bags, labeled with the group, and stored in a refrigerator at 4°C. A fresh batch of feed is prepared every 7 days. Step 2: Culture of oval pomfret. The culture experiment used an indoor factory-style seawater culture system. Healthy oval pomfret juveniles were temporarily raised in the factory-style culture pond for two weeks. Then, 360 fish were randomly selected and evenly distributed into 6 large net cages, with 60 fish per cage, and 3 parallel groups per group. Before the formal experiment, the fish were fed a basic feed for one week, and the experimental period was 42 days. During the culture period, the water temperature was maintained at 26-28℃, the salinity at 28-30‰, the dissolved oxygen content at no less than 5mg / L, and the pH at 7.8-8.2. During the culture period, the fish were fed twice daily at 08:00 and 16:00, and their growth and feeding were observed and recorded. At the same time, uneaten feed and feces in the culture system were regularly cleaned to maintain water quality.

[0080] After the experiment, liver, spleen, and whole intestinal tissue samples were collected from oval pomfret in both the experimental and control groups. These samples were flash-frozen in liquid nitrogen and then stored at -80°C for molecular biological analysis, including biochemical index detection, spleen immune-related gene expression detection, and gut microbiota sequencing analysis. The midgut tissue was fixed with fresh 4% paraformaldehyde tissue fixative for tissue section preparation and measurement.

[0081] Histological examination revealed significant differences in intestinal structure among the treatment groups. Figure 7 , 8 Compared with the control group (C0), the villus length of the midgut in groups E3, E4, and E5 (p<0.05) and the villus width in groups E4 and E5 (p<0.05) were significantly increased; while the villus width in groups E1, E2, and E3 was not statistically different from that in the control group, and the muscle layer thickness in groups E2, E3, and E5 was significantly lower than that in the control group (p<0.05).

[0082] Microscopic image analysis of tissue sections revealed that the intestinal villi in the experimental group exhibited a slender and dense morphology, while the muscle layer thickness was significantly thinner. No pathological damage was observed in the intestinal tissue, indicating that the addition of Enterococcus faecalis SZ07 not only did not cause structural damage to the intestine of the oval pomfret, but also significantly improved the intestinal absorptive surface area and functional activity by optimizing villus morphological parameters and reducing muscle layer thickness. Furthermore, the improvement in villus length and width was particularly pronounced in the E5 group; the villus length increased, and its width also significantly expanded, while the muscle layer thickness was significantly reduced compared to the control group (p<0.05). A healthier intestinal morphology helps improve the absorption efficiency of nutrients, providing sufficient energy and nutritional support for the immune system, and can help regulate the expression of immune-related genes, enhance anti-inflammatory responses, reduce inflammation levels, and thus improve the oval pomfret's ability to resist neuronecrosis virus, thereby improving the survival rate and quality of the oval pomfret.

[0083] The detection results of key enzyme indicators in the intestinal tissue of oval pomfret are as follows: Figures 9-11 As shown in Figure 9, firstly, regarding intestinal lipase activity, the values ​​of experimental groups E1, E3, and E4 were significantly higher than those of the control group C0 (p<0.05). Similarly, the values ​​of intestinal trypsin activity in experimental groups E1, E3, and E4 were also significantly higher than those of the control group C0 (p<0.05). The amylase activity in the experimental groups was also significantly higher than that of the control group C0 (p<0.05). The lipase, trypsin, and amylase activities in group E3 were all significantly higher than those in group C0 (p<0.05). This phenomenon suggests that adding [the following text is incomplete and requires further context:] to the feed... Enterococcus faecalis SZ07 can effectively enhance the digestive enzyme activity of pomfret, thereby strengthening its ability to digest and absorb nutrients. Secondly, such as Figure 10 As shown, regarding intestinal superoxide dismutase (SOD) activity, the values ​​in groups E1, E4, and E5 were significantly higher than those in the control group C0 (p<0.05). Regarding intestinal glutathione peroxidase (GSH-PX) and catalase (CAT) activities, the activities in groups E1, E2, E3, E4, and E5 were significantly higher than those in the control group C0 (p<0.05). Thirdly, as... Figure 11 As shown, the results of the intestinal metabolic-immune dual-function enzyme activity analysis showed that, compared with the control group C0, the intestinal acid phosphatase activity (ACP) in groups E3, E4, and E5 was significantly increased; the intestinal alkaline phosphatase activity (ALP) in groups E3, E4, and E5 was significantly higher than that in the control group C0 (p<0.05). Among them, the intestinal GSH-Px, liver SOD, and intestinal CAT activities in group E3 were significantly increased compared with the control group (p<0.05). Compared with group C0, the ACP and ALP activities in each experimental group (E1-E5) showed a significant increasing trend (p<0.05).

[0084] The detection results of key enzyme indicators in the liver tissue of oval pomfret are as follows: Figure 12 , 13 As shown in the figure. Compared with group C0, the activities of SOD, GSH-Px, and CAT in the liver and intestines of groups E4 and E5 were significantly enhanced. p <0.05%, compared with the control group, the activities of liver and intestine GSH-Px, liver SOD, and intestine CAT were significantly increased in the E3 group. p <0.05), with no significant differences in other indicators. SOD converts superoxide radicals into hydrogen peroxide, while GSH-Px further decomposes hydrogen peroxide into water, thus forming a complete antioxidant chain. CAT effectively mediates intracellular metabolites through its redox active site. The decomposition reaction converts it into water molecules. and oxygen As a key antioxidant enzyme, it can effectively scavenge excess reactive oxygen free radicals in the body, thereby inhibiting cellular oxidative damage caused by abnormal accumulation of hydrogen peroxide. Experimental results showed that, compared with the C0 group, the activities of ACP and ALP in each experimental group (E1-E5) showed a significant increasing trend. p <0.05%. Experimental results showed that, compared with group C0, the activities of ACP and ALP in each experimental group (E1-E5) exhibited a significant increasing trend ( p <0.05), high levels of ACP and ALP activity can enhance humoral immunity and improve disease resistance. The test methods for the content of various enzymes are as follows: same as above.

[0085] The detection results of key enzyme indicators in the spleen tissue of oval pomfret are as follows: Figure 14 It can be seen that adding the screened Enterococcus faecalis SZ07 to the feed can downregulate the expression of TNF-α and IL-8, upregulate the levels of TGF-β and IL-10, regulate the immune response of fish, and enhance antiviral ability, providing a new and effective means for the prevention and control of viral neuronecrosis in oval pomfret. Specifically, the transcriptional level of the TNF-α gene showed a dose-dependent downregulation trend. Compared with the control group, the expression level of the NF-κB gene in group E2 was significantly increased, while the expression trends of the NF-κB gene in other experimental groups were unstable compared with the control group. Compared with the control group, the expression level of the IL-8 gene in all experimental groups showed a significant downregulation trend (p<0.05), with the expression levels of IL-10 and TGF-β genes in group E5 being significantly higher than those in the control group (p<0.05). It is noteworthy that although the expression levels of IL-10 and TGF-β in groups E1-E4 did not reach statistical significance, they all showed varying degrees of upregulation.

[0086] The methods for testing the content of various enzymes are as follows: Intestinal and liver tissue samples were collected from each group of oval pomfret. After homogenization, the tissue supernatant was obtained for multi-index enzyme activity detection. This study selected superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), acid phosphatase (ACP), alkaline phosphatase (ALP), as well as trypsin, amylase, and lipase as detection indicators. Quantitative analysis of all indicators was performed using a professional enzyme activity detection kit provided by Jinshaoyuan (Shanghai) Biotechnology Co., Ltd., and the experimental procedures were strictly followed according to the standard operating procedures included in the kit.

[0087] Meanwhile, regarding the key enzyme indicators in the intestinal, liver, and spleen tissues of the oval pomfret, it's important to note the following: First, the intestine is the primary site for nutrient digestion and absorption in fish, and also a crucial line of defense for the immune system. The activity of digestive enzymes in the intestine (such as amylase and protease) not only affects nutrient absorption but also relates to the balance of the intestinal microbiota, thus influencing immune function. Second, the liver is a vital metabolic and immune organ in fish, and changes in its enzyme activity can reflect its physiological function and immune status. The activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in the liver are closely related to immune capacity. These enzymes can scavenge free radicals in the body, reduce oxidative damage, and thus enhance immune function. Third, the spleen is an important immune organ in fish, and changes in its enzyme activity can reflect the activation status of the immune system. The expression levels of some immune-related factors have a significant impact on the immune capacity of the oval pomfret. Therefore, key enzyme indicators in intestinal tissue, liver tissue, and spleen tissue are important indicators for assessing the immune capacity of oval pomfret, and the levels of these indicators are closely related to antiviral capacity.

[0088] Therefore, the test results regarding key enzyme indicators in the intestinal, liver, and spleen tissues of *Enterococcus faecalis* all indicate that *Enterococcus faecalis* SZ07 effectively enhances the autoimmune capacity of *Enterococcus faecalis* by regulating the expression of these key enzymes, thereby effectively improving its antiviral ability. In particular, it has a significant effect on the resistance to *Enterococcus faecalis* neuronecrosis virus, demonstrating that *Enterococcus faecalis* SZ07 can effectively control viral neuronecrosis disease in *Enterococcus faecalis* through the regulation of these key enzyme indicators. This can effectively improve the survival rate, growth rate, and disease resistance of *Enterococcus faecalis* seedlings, promoting the healthy development of the *Enterococcus faecalis* aquaculture industry. In addition, the intestinal microbiota structure was sequenced, and the specific testing was entrusted to Shanghai Ouyi Biotechnology Co., Ltd. The specific intestinal microbiota sequencing analysis method was as follows: (1) DNA extraction and PCR amplification: Genomic DNA of intestinal samples was extracted according to the MagPure Soil DNA LQKit kit operation guide. The DNA quality was double-verified by agarose gel electrophoresis combined with NanoDrop2000 (USA). Barcode-specific primers were selected to amplify the target region of 16S rRNA gene of bacteria (Takara Bio) with genomic DNA as template. Universal primers were used to target and amplify the V3-V4 hypervariable region with 343F (5'-TACGGRAGGCAGCAG3') / 798R (5'-AGGGTATCTAATCCT-3'), and the amplified products were used for subsequent analysis. (2) Library preparation and high-throughput sequencing: After verifying the integrity of the PCR amplification products by agarose gel electrophoresis, the first round of PCR products were purified by magnetic bead purification system (AMPure XP) and used as templates for exponential amplification required for library construction. The final product was precisely quantified by a fluorescence quantitative analyzer (Qubit), and the concentration was homogenized before constructing a sequencing library. The final library was sequenced by paired ends using an Illumina NovaSeq 6000 sequencing system (EasyBio, China) to obtain the raw sequence data. (3) Bioinformatics analysis: The raw sequencing data was preprocessed by trimming adapter sequences using Cutadapt software; the DADA2 algorithm was used to select qualified paired-end sequences based on the default parameters of the QIIME 2 analysis framework, and representative sequences and ASV abundance tables were generated. All ASV representative sequences were then aligned to the Silva database (version 138) to complete the alignment annotation; this process was completed by the preset parameters of the q2-feature-classifier module to complete the taxonomic annotation. The α-diversity of the gut microbiome of *Siniperca ovalis* was assessed using QIIME 2 software and the Ace, Chao1, Shannon, and Simpson indices. The Binary-Jaccard distance matrix was calculated using R, and β-diversity (beta diversity) was analyzed based on principal coordinates analysis (PCoA). Simultaneously, linear discriminant analysis (LEfSe) was used to analyze the differences in species abundance spectra. DC42 represented the control group (C0); DL42 represented the supplementary group. Enterococcus faecalis group (E1); DF42: added Enterococcus faecalis group (E2); DM42: added Enterococcus faecalis group (E3); DT42: Added Enterococcus faecalis group (E4); DH42: added Enterococcus faecalis group (E5).

[0089] Gut microbiota sequencing results as follows Figure 15 As shown, compared to the control group, the Shannon and Simpson diversity indices were increased and the Ace index was decreased in groups E3 and E4, and the Chao1 abundance index decreased in all experimental groups (E1-E5). Except for group E4, the relative abundance of Lactobacillus increased in all experimental groups, the abundance of Cetacea increased in groups E1-E3, and the abundance of Pseudomonas decreased in groups E1-E4. These results also indicate that the Enterococcus faecalis SZ07 strain provided in this invention can optimize the intestinal flora structure, thereby further enhancing the immunity of pomfret and effectively improving its resistance to neuronecrosis virus, demonstrating its potential as a probiotic for aquaculture.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. The application of Enterococcus faecalis SZ07 in the preparation of fish feed resistant to oval pomfret nerve necrosis virus, characterized in that: The accession number of the Enterococcus faecalis SZ07 is GDMCC No: 66159, and the accession center is Guangdong Provincial Microbial Culture Collection Center.

2. The application of Enterococcus faecalis SZ07 as described in claim 1 in the preparation of fish feed resistant to oval pomfret nerve necrosis virus, characterized in that: Fish feed includes basal feed and the aforementioned Enterococcus faecalis SZ07; The inoculation amount of Enterococcus faecalis SZ07 in the basal feed was 1×10⁻⁶. 5 CFU / g ~ 1×10 9 CFU / g.

3. The application of Enterococcus faecalis SZ07 as described in claim 2 in the preparation of fish feed resistant to oval pomfret nerve necrosis virus, characterized in that: The inoculation amount of Enterococcus faecalis SZ07 in the basal feed was 1×10⁻⁶. 7 CFU / g.

4. The application of Enterococcus faecalis SZ07 as described in claim 2 in the preparation of fish feed resistant to oval pomfret nerve necrosis virus, characterized in that: The basic feed comprises the following components: 40-50 wt% crude protein, 2-6 wt% crude fiber, 4-10 wt% crude fat, and 10-20 wt% crude ash.

5. Application of Enterococcus faecalis SZ07 in the preparation of drugs against oval pomfret nerve necrosis virus.

6. A method for cultivating oval pomfret resistant to nerve necrosis virus, characterized in that, The cultivation method includes the following steps: Step 1: Prepare basic feed. Mix Enterococcus faecalis SZ07 with basic feed and allow it to dry naturally to obtain fish feed. The preservation number of Enterococcus faecalis SZ07 is GDMCC No: 66159, and the preservation center is Guangdong Provincial Microbial Culture Collection Center. Step 2: Place healthy oval pomfret juveniles in the following water environment for rearing: maintain the temperature at 26-28℃, the salinity at 28-30‰, the dissolved oxygen content at no less than 5mg / L, and the pH at 7.8-8.2; and feed them the fish feed twice a day during the rearing period.

7. The method for cultivating oval pomfret resistant to nerve necrosis virus as described in claim 6, characterized in that: In step 1, the inoculation amount of Enterococcus faecalis SZ07 in the basal feed is 1×10⁻⁶. 5 CFU / g ~ 1×10 9 CFU / g.

8. The method for cultivating oval pomfret resistant to nerve necrosis virus as described in claim 7, characterized in that: In step 1, the inoculation amount of Enterococcus faecalis SZ07 in the basal feed is 1×10⁻⁶. 7 CFU / g.

9. A method for screening probiotics resistant to oval pomfret nerve necrosis virus, characterized in that, Includes the following steps: S1. For a population of oval pomfret naturally infected with neuronecrosis virus, oval pomfret with symptoms of viral neuronecrosis were used as the control group, and asymptomatic oval pomfret were used as the experimental group. Intestinal tissue samples, brain tissue samples and spleen tissue samples were collected from oval pomfret in the control group and the experimental group, respectively. S2. RNA was extracted from brain tissue samples and spleen tissue samples from oval pomfret in the control group and experimental group, respectively, and high-throughput sequencing was performed. S3. Compare the gene expression of brain tissue samples and spleen tissue samples from oval pomfret in the control group and experimental group to screen out differentially expressed genes; S4. Perform functional enrichment analysis on the differentially expressed genes; S5. Microbial community analysis was performed on intestinal tissue samples from oval pomfret in both the control and experimental groups; S6. Based on the analysis results of S4 and S5, determine the source of the probiotic intestinal tissue sample and the type of probiotic, and isolate the probiotic from the source of the intestinal tissue sample.

10. The method for screening probiotics against oval pomfret nerve necrosis virus as described in claim 9, characterized in that, The probiotic is Enterococcus faecalis SZ07; The accession number of the Enterococcus faecalis SZ07 is GDMCC No: 66159, and the accession center is Guangdong Provincial Microbial Culture Collection Center.

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