New use of radix cirsii japonici extract against rainbow trout iridovirus

Abstract

The present application relates to a new application of radix cirsii japonici extract in resisting largemouth bass iridovirus. The present application finds that the radix cirsii japonici extract has a significant effect in resisting largemouth bass iridovirus, can significantly reduce the copy number of largemouth bass iridovirus, inhibit the expression of largemouth bass iridovirus MCP protein, improve the phagocytic ability of largemouth bass kidney leukocytes, and effectively improve the survival rate of fish infected with largemouth bass iridovirus, and has good biological safety. Therefore, the radix cirsii japonici extract can be used for preparing products such as drugs, fish feed or fish feed additives resisting largemouth bass iridovirus, and provides an effective prevention and control means for largemouth bass and other fish breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the prevention and control of largemouth bass iridovirus, specifically to a new application of thistle extract against largemouth bass iridovirus. Background Technology

[0002] Largemouth bass is an important economically important aquaculture animal. According to data from the "2023 China Fisheries Statistical Yearbook," the national production of largemouth bass farming reached 802,500 tons. However, in recent years, the largemouth bass farming industry has faced serious threats from diseases. Outbreaks of diseases such as largemouth bass iridovirus disease have become one of the most significant factors restricting the success of largemouth bass farming. Largemouth bass virus (LMBV) belongs to the family Iridoviridae, genus Rhabdirivirus, and is an icosahedral cytoplasmic DNA virus with a genome similarity of >98% to that of mandarin frog iridovirus (MRV). Since its isolation and purification in 1991, LMBV has caused severe economic losses to my country's largemouth bass farming industry, with a mortality rate exceeding 90%. Symptoms include skin ulcers, ascites, and intestinal inflammation. However, to date, there are no commercially available vaccines or effective control drugs for LMBV, which is extremely detrimental to the control of the disease. Therefore, there is an urgent need to develop effective drugs against LMBV.

[0003] Traditional Chinese medicine (TCM) and its extracts are playing an increasingly important role in antiviral research. Their mechanism of action involves not only directly killing viruses, preventing viral adsorption and invasion of cells, and inhibiting viral adsorption, transcription, and replication in the body, but also improving the internal environment of animals, bidirectionally regulating the immune system, activating the phagocytic activity of immune cells to enhance immunity, and regulating various inflammatory factors to alleviate oxidative stress, thus exerting anti-inflammatory and antioxidant effects. *Cirsium japonicum*, the dried aerial part of the plant *Cirsium japonicum* (belonging to the Asteraceae family), contains various active ingredients such as glycosides, flavonoids, and sugars. Studies have found that it has significant hemostatic, hypotensive, antibacterial, and antiviral biological activities. Furthermore, *Cirsium japonicum* is listed in the "Natural Feed Plants Catalogue" by the Ministry of Agriculture and Rural Affairs of China, indicating low toxicity to animals. Summary of the Invention

[0004] Based on this, the purpose of this invention is to provide a new application of thistle extract against largemouth bass iridovirus, providing an effective prevention and control method for the aquaculture of largemouth bass and other fish.

[0005] The technical solutions for achieving the above objectives include the following.

[0006] The first aspect of the invention is to provide the use of thistle extract in the preparation of products resistant to largemouth bass iridovirus.

[0007] A second aspect of the invention is to provide the use of thistle extract in the preparation of products for the prevention and / or treatment of fish infected with largemouth bass iridovirus.

[0008] In some embodiments, the fish is at least one of the family Sunfishes in the order Perciformes; preferably, the fish is the largemouth bass.

[0009] In some embodiments, the thistle extract is a water extract of thistle, preferably a water extract of dried thistle flowers.

[0010] The method for preparing the water extract of thistle includes the following steps: the dried flower part of thistle is ground into powder, 100 grams of powder is soaked in 80-120 ml of water overnight, boiled for 4-8 hours, and then the supernatant is collected, filtered, and dried.

[0011] In some embodiments, the product is a medicine in which the active ingredient comprises thistle extract.

[0012] In some embodiments, the drug is an oral or injectable form.

[0013] In some embodiments, the product is a fish feed or fish feed additive containing thistle extract.

[0014] In some embodiments, the application includes at least one of the following (1) to (4): (1) reducing the copy number of largemouth bass iridovirus; (2) inhibiting the expression or synthesis of largemouth bass iridovirus MCP protein; (3) activating the expression of Fc receptor-mediated phagocytic pathway-related genes, said gene-related genes including at least one of FcγRIa, Lyn, CFL, IL-10, IFN-α, Mx1, IFN-γ and ARPC5; (4) improving the phagocytic capacity of renal leukocytes.

[0015] A third aspect of the present invention is to provide a fish feed or fish feed additive that is resistant to largemouth bass iridovirus, said fish feed or fish feed additive comprising a. thistle extract and b. common fish feed ingredients or feed additive ingredients.

[0016] In some embodiments, the thistle extract is a water extract of thistle.

[0017] The present invention has the following beneficial effects:

[0018] This invention reveals that *Cirsium japonicum* extract has a significant antiviral effect against largemouth bass iridovirus (MLV). It can significantly reduce the copy number of MCP protein in largemouth bass MLV, inhibit the expression of MCP protein, and enhance the phagocytic capacity of largemouth bass kidney leukocytes. This effectively improves the survival rate of fish infected with MLV, and exhibits good biocompatibility. Therefore, *Cirsium japonicum* extract can be used to prepare drugs, fish feeds, or fish feed additives that combat MLV, providing an effective prevention and control measure for the aquaculture of largemouth bass and other fish species. Attached Figure Description

[0019] Figure 1 The results of safety analysis of thistle extract on SCB3 cells were analyzed using CCK8 assay.

[0020] Figure 2 The results show the in vitro anti-LMBV activity assay of thistle extract.

[0021] Figure 3 For the effect of thistle extract on LMBV EC 50 Value fitting analysis results.

[0022] Figure 4 This study presents the effects of Cirsium japonicum extract on the expression of LMBV MCP protein.

[0023] Figure 5 This study presents the results of an investigation into the inhibitory activity of Cirsium japonicum extract on LMBV at different life cycles.

[0024] Figure 6 Analysis of the survival curve of thistle extract against LMBV in largemouth bass.

[0025] Figure 7 The results show the analysis of the anti-LMBV tissue virus content of largemouth bass by thistle extract.

[0026] Figure 8 The results show the effect of thistle extract on the expression levels of immune-related genes in largemouth bass.

[0027] Figure 9 Flow cytometry analysis results of phagocytosis by leukocytes in the head kidney of largemouth bass treated with thistle extract. Red peak: leukocytes not phagocytosed; Blue peak: leukocyte phagocytosis fluorescence peak in the untreated group; Orange peak: leukocyte phagocytosis fluorescence peak in the thistle extract-treated group. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0030] Unless otherwise defined, all technical and scientific terms used in this invention are consistent with those belonging to this invention.

[0031] The meanings are generally understood to be the same by those skilled in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0033] To facilitate understanding of this technology, some terms and phrases are defined below.

[0034] TCID50: Half-maximal tissue culture infection dose, also known as 50% tissue cell infection dose, refers to the amount of virus that can cause half of the cells to become diseased or die in the wells of a culture plate or test tube, and is used to characterize the viral titer.

[0035] EC 50 The half-maximal effective concentration (MCC) represents the drug concentration at which 50% of the virus is inhibited. We will observe cytopathic effects at different drug concentrations. When significant cytopathic effects are observed in the virus control group, we will record the cytopathic effects in different drug concentration groups, and determine the viral copy number in each group using qPCR. Subsequently, we will determine the viral inhibition rate in each group and finally calculate the EC50 of the drug. 50 value.

[0036] This invention demonstrates through in vitro and in vivo experiments that the extract of thistle has a significant effect against largemouth bass iridovirus. Largemouth bass iridovirus can inhibit viral DNA replication, inhibit the expression of MCP protein, regulate the immune status of fish, and improve the phagocytic capacity of renal leukocytes, thereby effectively improving the survival rate of fish infected with largemouth bass iridovirus.

[0037] In some embodiments of the present invention, the use of thistle extract in the preparation of products resistant to largemouth bass iridovirus is provided.

[0038] In some embodiments of the present invention, the use of thistle extract in the preparation of products for the prevention and / or treatment of largemouth bass iridovirus infection in fish is provided.

[0039] In some preferred embodiments, the thistle extract is a water extract of thistle, which can be prepared according to conventional water extraction methods in the art.

[0040] For example, the preparation of the water extract of thistle according to the conventional methods described in the following examples is as follows:

[0041] Dried flowers of *Cirsium japonicum* purchased from the market were ground into powder. 100 g of powder was soaked in 100 mL of water at 4°C overnight to soften, then boiled for 6 hours. The supernatant was then collected, filtered three times through a 100 mm nylon mesh filter, and subsequently evaporated to dryness in a rotary evaporator. Prior to use in this study, the extract was dissolved in double-distilled water (ddH2O) and filtered through a 0.45 μm sterile filter.

[0042] The present invention will be further described in detail below with reference to specific embodiments.

[0043] 1. Materials and Methods

[0044] 1.1 Materials

[0045] LMBV was isolated and preserved by the Aquatic Disease Research Laboratory of the Animal Health Institute, Guangdong Academy of Agricultural Sciences. SCB3 cells were established and preserved by the Aquatic Disease Research Laboratory of the Animal Health Institute, Guangdong Academy of Agricultural Sciences. Nocardia seriolae was isolated and preserved by the Aquatic Disease Research Laboratory of the Animal Health Institute, Guangdong Academy of Agricultural Sciences, and labeled with fluorescein isothiocyanate (FITC). Largemouth bass (10g±2g) were purchased from Guangdong Liangshi Aquatic Seed Industry Co., Ltd. and temporarily housed in 40cm×30cm×40cm aquariums for 2 weeks with continuous aeration and water temperature maintained between 25-28℃. Before the experiment, 5 fish were randomly selected for dissection, and nucleic acid was extracted from brain, spleen, and kidney tissues. LMBV was detected using the LMBV qPCR method to ensure that the experimental fish did not carry LMBV.

[0046] 1.2 Safety determination of Cirsium japonicum extract on mandarin fish brain cells

[0047] The safety of *Cirsium japonicum* extract on mandarin fish brain cells was determined using the CCK8 assay. (5×10⁻⁶) 4SCB3 cells were seeded at 1 / mL into 96-well plates and cultured at 25°C until cell confluence reached over 80%. Thistle extract was fully dissolved in sterile ddH2O, filtered through a 0.45μm micromembrane, and added to SCB3 cells at final concentrations of 2000μg / mL, 1500μg / mL, 1000μg / mL, 500μg / mL, and 50μg / mL, respectively. Cells were incubated at 25°C for 48 h. Cells without thistle extract (0μg / mL) served as a control. Cell morphology differences were observed under a light microscope. Then, 20μL of CCK8 reagent was added to each well, and incubation continued for 4 h. OD was measured. 450 Changes in nm value.

[0048] 1.3 Determination of the in vitro anti-LMBV activity of Cirsium japonicum extract

[0049] 5×10 5 SCB3 cells per mL were seeded into 12-well plates and cultured in L15 medium containing 10% FBS at 25°C until cell confluence reached 80% or higher. LMBV cytotoxic supernatant (100 μL, 100 TCID50) was then added. 50 Incubate at 25°C for 1 hour with ( / mL) solution, discard the supernatant, wash three times with PBS, and then add *Cirsium japonicum* extract at final concentrations of 1000 μg / mL, 800 μg / mL, 600 μg / mL, 400 μg / mL, and 200 μg / mL, respectively. Simultaneously, set up a LMBV-only cytotoxic supernatant (100 μL, 100 TCID50) solution. 50 Cells inoculated with the virus ( / mL) and untreated cells were incubated at 25°C for 48 hours, and cytopathic effects were observed under an optical microscope. Subsequently, all wells were subjected to three freeze-thaw cycles, and 200 μL of the supernatant was collected from each well. DNA was extracted from each well using a viral nucleic acid extraction kit, and absolute quantification was performed using SYBR Green real-time quantitative PCR to determine the LMBV viral copy number. Based on this, the inhibition rate of *Cirsium japonicum* extract against LMBV was calculated using the following formula.

[0050] Inhibition rate (%) = (1-A / B)×100%.

[0051] Where A represents the number of virus copies in the pores of the LMBV cytotoxic supernatant when both thistle extract and LMBV cytotoxic supernatant are added; and B represents the number of virus copies in the pores of the LMBV cytotoxic supernatant when only LMBV cytotoxic supernatant is added.

[0052] 1.4 Effect of Cirsium japonicum extract on the expression of LMBV primary capsid protein (MCP)

[0053] To investigate the effect of thistle extract on LMBV MCP expression, SCB3 cells were seeded in a 25mm glass-bottom confocal culture dish and cultured to a monolayer. Then, 100 μL of LMBV cytotoxic supernatant (100 TCID50) was added.50 Incubate at 25°C for 1 hour ( / mL), discard the supernatant, wash 3 times with PBS, add 1000 μg / mL *Cirsium japonicum* extract, and continue culturing at 25°C. Collect cells at 48 and 72 hours, discard the culture medium, wash 3 times with PBS, add 1 mL of 4% paraformaldehyde to each dish for fixation for 20 min; wash 3 times with PBS, add 1 mL of 0.1% Triton 100 for permeabilization for 10 min; wash 3 times with PBS, add 1 mL of 1%... Block with BSA blocking solution for 30 min; remove the blocking solution, wash 5 times with PBS, add 1 mL of primary antibody dilution buffer (anti-MCP polyclonal antibody, 1:1000 dilution) and incubate overnight at 4°C; remove the primary antibody incubation buffer, wash 5 times with PBS, remove the primary antibody, add 1 mL of fluorescent secondary antibody (AF647-labeled goat anti-rabbit monoclonal antibody, 1:1000 dilution), incubate at 37°C for 1 h in the dark; remove the secondary antibody incubation buffer, wash 5 times with PBS, add 1 mL of DAPI dilution buffer (1:1000 dilution), incubate at 37°C for 10 min in the dark; remove the DAPI, wash 5 times with PBS, add 50 μL of anti-fluorescence quencher to the center ring of the glass dish, observe under a fluorescence microscope, and set up LMBV cytotoxic supernatant (100 μL, 100 TCID50). 50 / mL) Control group treated with the virus and control group without treatment.

[0054] 1.5 Determination of the effect of thistle extract on the activity of LMBV at different life stages

[0055] To further explore the potential mechanisms of drug antiviral activity, the viral copy number at three stages of the viral life cycle was measured.

[0056] (1) Adhesion stage: After cells were cultured in 48-well plates until a confluent monolayer was formed, the cells were pretreated with 1000 μg / mL thistle extract for 1 h, followed by inoculation with the virus at 4°C for 1 h, which enabled the virus to adhere to the cells and restricted its invasion. After washing three times with PBS, the cells were cultured in fresh medium for another 48 h. The cells were then collected for total DNA extraction and virus copy number detection to study the ability of the drug to prevent viral adhesion.

[0057] (2) Invasion stage: Cells were cultured in 48-well plates until a confluent monolayer was formed, and then inoculated with the virus. The virus and cells were co-incubated at 4°C for 30 min to induce invasion and limit its replication. After washing three times with PBS, the cells were treated with 1000 μg / mL thistle extract for 2 h, and then cultured in fresh medium for another 48 h. Total DNA was extracted and viral copy number was detected to study the drug's ability to prevent viral invasion.

[0058] (3) Replication stage: After the cells were cultured to a complete monolayer as described above, they were first co-incubated with the virus at 4°C for 30 min, and then transferred to 25°C for 4 h to allow the virus to enter the cells and begin replication. After that, the cells were treated with 1000 μg / mL thistle extract for 48 h, and total DNA was extracted and the viral copy number was detected to study the ability of the drug to prevent viral replication.

[0059] In all the above experiments, a control group that was only exposed to the virus and a control group of untreated cells were set up.

[0060] 1.6 In vivo anti-LMBV activity assay of Cirsium japonicum extract

[0061] To investigate the in vivo inhibitory activity of *Cirsium japonicum* extract against LMBV, largemouth bass (body weight 10.0±2.0 g) were randomly divided into four groups of 100 fish each. 5.0 g, 10.0 g, and 15.0 g of *Cirsium japonicum* extract were weighed and added to 20 times their volume of sterile ddH2O. After thorough dissolution, the solution was filtered through a 0.45 μm micromembrane and mixed into 1000 g of basal diet. The mixtures were thoroughly dried and then fed to the groups twice daily for 15 consecutive days. The control group was fed the basal diet. Thirty fish from each of the experimental and control groups were randomly selected and injected intraperitoneally with 100 μL of 1×10... 7 TCID 50 The fish were placed in 40cm×30cm×40cm aquariums with LMBV cytotoxic supernatant at a constant volume (mL). Continuous aeration was maintained, and the water temperature was kept between 25-28℃. Daily mortality was observed and recorded. Dead largemouth bass were dissected, and tissue DNA was extracted from the spleen, kidneys, liver, brain, and intestines. Viral copy numbers in each tissue were determined. Survival rate curves were plotted using GraphPadPrism version 8.0 software, and significance analysis was performed.

[0062] 1.7 Effects of thistle extract on mRNA expression of immune-related genes in largemouth bass

[0063] Five fish were randomly selected from each of the experimental and control groups. Spleen and head / kidney tissues were harvested. RNA was extracted using a tissue RNA extraction kit and then reverse transcribed into cDNA using a reverse transcription kit. The previously established SYBR green qPCR method for immune-related genes was used. -△△CTThe effects of thistle extract on the mRNA expression levels of interleukin-1β (IL-1β), IL-10, tumor necrosis factor-α (TNF-α), Fcγ receptor Ia (FcγRIa), FcγRIIb, tyrosine-protein kinase (Lyn), actin-related protein 2 / 3 complex (subunit 5, ARPC5), cofilin (CFL), neutrophil cytosolic factor 1 (NCF1), interferon-α (IFN-α), IFN-γ, and Mx1 genes in largemouth bass were determined using a method. The primers used for detection are shown in Table 1.

[0064] Table 1. Immune-related genes of largemouth bass and primers for LMBV detection

[0065]

[0066]

[0067] 1.8 Effect of Cirsium japonicum extract on phagocytic capacity of leukocytes in the head kidney of largemouth bass

[0068] Five fish were randomly selected from each of the experimental and control groups. Head kidney tissue was collected, and white blood cells were extracted and counted using a fish tissue leukocyte extraction kit. (2 × 10⁻⁶) 6 100 / mL head kidney white blood cells and 2×10 7 Nocardia seriolae (CFU / mL) of fish was co-incubated at 25°C for 4 h. Subsequently, the cells were centrifuged at 250 g for 30 min, the supernatant was discarded, and the precipitated cells were washed three times with PBS. The cells were then completely resuspended in 1 mL of PBS, and flow cytometry was used to analyze the effect of thistle extract on the phagocytic capacity of leukocytes in the head kidney of largemouth bass.

[0069] 1.9 Data Statistical Analysis

[0070] The safety of *Cirsium japonicum* extract against mandarin fish brain cells and its in vitro anti-LMBV activity were determined using SPSS 23.0 software via one-way ANOVA. The effect of *Cirsium japonicum* extract on changes in the mRNA expression of immune-related genes in largemouth bass was analyzed using SPSS 23.0 software via independent samples t-test. Survival rate curves were analyzed using GraphPadprism version 8.0 software, and statistical differences were analyzed using Log-rank (Mantel-Cox). *P<0.05 indicates a significant difference. **P<0.01 indicates a highly significant difference.

[0071] 2 Results

[0072] 2.1 Safety determination of Cirsium japonicum extract on mandarin fish brain cells

[0073] CCK8 analysis showed that after incubation of SCB3 cells with 1000 μg / mL, 500 μg / mL, and 250 μg / mL *Cirsium japonicum* extract for 48 h, the OD450 values ​​were not significantly different from the control group. However, after incubation of SCB3 cells with 2000 μg / mL and 1500 μg / mL *Cirsium japonicum* extract for 48 h, the OD450 values ​​were significantly lower than those of the control group (P<0.05). Figure 1 The results showed that 1000 μg / mL or <1000 μg / mL of thistle extract was a safe concentration for SCB3 cells.

[0074] 2.2 Determination of the in vitro anti-LMBV activity of Cirsium japonicum extract

[0075] The viral copy number in each group was detected using real-time quantitative PCR technology, and the results are as follows: Figure 2 As shown in the SPSS one-way ANOVA, the LMBV viral copy numbers in the 1000 μg / mL, 800 μg / mL, 600 μg / mL, and 400 μg / mL *Cirsium japonicum* extract groups were significantly lower than those in the control group (P<0.05). Only the 200 μg / mL *Cirsium japonicum* extract group showed no significant difference in LMBV viral copy number compared to the control group. Figure 2 The relative inhibition rates of each group were calculated using the inhibition rate formula. The relative inhibition rates of *Cirsium japonicum* extract groups of 1000 μg / mL, 800 μg / mL, 600 μg / mL, 400 μg / mL, and 200 μg / mL against LMBV were 98.79%, 96.12%, 87.52%, 56.16%, and 35.89%, respectively. The inhibitory effect of *Cirsium japonicum* extract on LMBV was analyzed using GraphPad Prism version 8.0 software. 50 The value was 457.3 μg / mL. Figure 3 ).

[0076] 2.3 Thistle extract inhibits the expression of LMBV MCP protein.

[0077] To further verify the antiviral effect of the drug, the expression level of LMBV MCP protein was detected by immunofluorescence. The results showed that the addition of thistle extract to SCB3 cells significantly reduced the fluorescence intensity, proving that thistle extract inhibited the expression of LMBV MCP protein, further verifying the antiviral effect of thistle extract. Figure 4 ).

[0078] 2.3 Effects of Cirsium japonicum extract on the inhibitory activity of LMBV at different life cycles

[0079] To further investigate the mechanism of thistle extract's anti-LMBV effect, this study used SCB3 as a cell model and evaluated the differences in its inhibitory effect on LMBV during the viral adhesion, invasion, and replication stages using qPCR. The results showed that thistle extract did not reduce LMBV copy number during the LMBV adhesion stage, but it significantly reduced LMBV copy number during the invasion and replication stages. Specifically, the 1000 μg / ml extract reduced LMBV copy number by 98.46% and 99.31% during the invasion and replication stages, respectively. Figure 5 ).

[0080] 2.4 Results of in vivo anti-LMBV activity study of thistle extract

[0081] The in vitro anti-LMBV activity assay of *Cirsium japonicum* extract showed that the control group of largemouth bass died 3 days after infection, with a mortality rate of 100% by day 6. In contrast, the mortality rates of the 0.5%, 1.0%, and 1.5% *Cirsium japonicum* extract groups were 50%, 40%, and 60% respectively by day 20 post-infection. Compared with the control group, the relative protection rates (RPS) of the 0.5%, 1.0%, and 1.5% *Cirsium japonicum* extract groups were 50%, 60%, and 40%, respectively. Log-rank (Mantel-Cox) analysis showed that, compared with the control group, feeding with 0.5%, 1.0%, and 1.5% *Cirsium japonicum* extract significantly improved the survival rate of largemouth bass (P<0.01). Figure 6 ).

[0082] The viral load in the liver, spleen, kidney, foregut, midgut, and hindgut tissues of largemouth bass was quantitatively analyzed using a real-time fluorescence quantitative PCR method for LMBV at 6 days and 20 days post-infection. Results are as follows: Figure 6 As shown, compared with the control group, 0.5% ( Figure 7 a) 1.0% Figure 7 b) and 1.5% Figure 7c) In the group fed with thistle extract, the viral copy numbers in the liver, spleen, kidney, foregut, midgut, and hindgut tissues of largemouth bass were significantly reduced at 6 days and 20 days post-infection (P<0.01), with reductions of 0.5%, 1.0%, and 1.0% respectively.

[0083] The 1.5% thistle extract feeding group showed a highly significant decrease in viral copy number in all tissues at 20 days post-infection compared to 6 days post-infection (P<0.01). Figure 7 ).

[0084] 2.5 Effects of thistle extract on immune-related levels in largemouth bass

[0085] 2.5.1 Effects of thistle extract on mRNA expression of immune-related genes in largemouth bass

[0086] To investigate the in vivo anti-LMBV mechanism of *Cirsium japonicum* extract, the expression levels of immune-related genes in largemouth bass fed with *Cirsium japonicum* extract were analyzed. Real-time quantitative PCR results showed that, compared to the untreated group, the expression of FcγRIa, Lyn, CFL, IL-10, IFN-α, and Mx1 genes in the spleen and kidney tissues of largemouth bass treated with *Cirsium japonicum* extract was significantly higher than that in the untreated group (*P<0.05, **P<0.01). The expression of IFN-γ and ARPC5 genes in the head kidney tissue of largemouth bass treated with *Cirsium japonicum* water extract was also significantly higher than that in the untreated group. Compared to the untreated group, the expression levels of IL-1β and TNF-α genes in the spleen and kidney tissues of largemouth bass treated with *Cirsium japonicum* extract were significantly reduced (*P<0.05, **P<0.01). Figure 8 The results showed that thistle extract significantly activated the Fc receptor-mediated phagocytic pathway in largemouth bass.

[0087] 2.5.2 Effect of Cirsium japonicum extract on phagocytic capacity of leukocytes in the head kidney of largemouth bass

[0088] To investigate the effect of thistle extract on the phagocytic capacity of largemouth bass phagocytes, we isolated head kidney leukocytes from the thistle extract-fed group and the control group. Flow cytometry was used to detect the phagocytic capacity of the leukocytes for Nocardia bacteria. The results are as follows: Figure 9 As shown, compared to the untreated group (20.4% phagocytosis rate), the addition of thistle extract significantly improved the phagocytic capacity of largemouth bass head kidney leukocytes (72.3% phagocytosis rate). Figure 9 ).

[0089] In conclusion, thistle extract can effectively combat largemouth bass iridovirus, improve the survival rate of infected fish, and provide an effective prevention and control measure for the aquaculture of largemouth bass and other fish species.

[0090] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Application of dried flower water extract of thistle in the preparation of drugs against largemouth bass iridovirus.

2. Application of the aqueous extract of dried thistle flowers in the preparation of drugs for the prevention and / or treatment of fish infected with largemouth bass iridovirus.

3. The application as described in claim 2, characterized in that, The fish in question is at least one species belonging to the family Sunfishes in the order Perciformes.

4. The application as described in claim 3, characterized in that, The fish in question is the largemouth bass.

5. The application as described in claim 1 or 2, characterized in that, The drug is available in oral or injectable form.

6. The application as described in any one of claims 1 to 5, characterized in that, The application includes at least one of the following (1) to (4): (1) reducing the copy number of largemouth bass iridovirus; (2) inhibiting the expression or synthesis of largemouth bass iridovirus MCP protein; (3) activating the expression of Fc receptor-mediated phagocytic pathway-related genes, wherein the related genes include FcγRIa , Lyn , CFL , IL-10 , IFN-α , Mx1, IFN-γ and ARPC5 At least one of them; (4) enhance the phagocytic capacity of renal leukocytes.

7. A fish feed or fish feed additive, characterized in that, The fish feed or fish feed additive includes a. a water extract of dried thistle flowers as the sole active ingredient, and b. a daily feed ingredient or feed additive ingredient for fish.

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