Method for evaluating hemorrhagic disease resistance of grass carp based on multi-dimensional indexes of intestinal mucosal barrier

Abstract

The invention provides a method for evaluating the hemorrhagic disease resistance of grass carp based on intestinal mucosal barrier multi-dimensional indexes, and relates to the field of aquaculture and molecular biology. According to the method, grass carps with different genetic backgrounds are taken as objects, and comparison shows that after GCRV infection for 48 hours, the intestinal mucosa permeability of gynogenetic grass carps and disease-resistant grass carps is remarkably lower than that of common grass carps, the intestinal mucosa villus length is remarkably higher than that of the common grass carps, and the expression of intestinal mucosa tight junction molecules is remarkably higher than that of the common grass carps; expression of intestinal mucosa immune related factors is obviously higher than that of common grass carp, diversity and abundance of intestinal flora are obviously higher than that of common grass carp, concentration of four key antioxidant enzymes in serum is obviously higher than that of common grass carp, and content of liver and intestinal VP56 is obviously lower than that of common grass carp. Therefore, after 15 days of GCRV infection, the survival rate of gynogenesis grass carps and disease-resistant grass carps is significantly higher than that of common grass carps. The invention provides a novel method for evaluating the hemorrhagic disease resistance of the grass carp.

Description

Technical Field

[0001] The invention relates to the fields of aquaculture and molecular biology, and in particular to a method for evaluating the anti-hemorrhagic disease ability of grass carp based on multidimensional indicators of intestinal mucosal barrier. Background Art

[0002] In patent authorization number CN107318719B, Hunan Normal University successfully created gynogenetic grass carp. It also successfully bred disease-resistant grass carp by hybridizing gynogenetic grass carp as the female parent and common grass carp as the male parent. After years of breeding experiments, it was found that this important strain of disease-resistant grass carp has the advantages of fast growth, good meat quality, and strong disease resistance. It has been promoted and demonstrated in many places such as Hunan, Sichuan, Chongqing, Guangxi, Hebei and Guangdong, and achieved good economic benefits.

[0003] However, in recent years, grass carp hemorrhagic disease is still the number one epidemic disease of grass carp, which has caused huge economic losses and seriously affected the healthy development of grass carp farming. Grass carp hemorrhagic disease is mainly caused by grass carp reovirus (GCRV). Its pathogenesis is complex and is closely related to the immune system, genetic background and environmental factors of grass carp. Studies have shown that the strength of the intestinal mucosal barrier function directly affects the grass carp's resistance to viruses and recovery speed. Therefore, it is particularly important to develop a method to effectively evaluate the intestinal mucosal barrier function of grass carp and then judge its ability to resist hemorrhagic disease. However, there is currently no report on the application of evaluating and comparing the intestinal mucosal barrier capacity after GCRV infection of common grass carp, gynogenetic grass carp and disease-resistant grass carp.

[0004] Traditional evaluation methods focus on one aspect of biomarkers, ignoring the complexity and dynamic changes of intestinal mucosal barrier function, reducing the accuracy and practicality of the evaluation. Therefore, the technical problem to be solved by the present invention is to more accurately predict and evaluate the ability of grass carp to resist hemorrhagic disease by innovatively establishing a comprehensive evaluation system based on multidimensional indicators of intestinal mucosal barrier.

[0005] The above-mentioned GCRV virus has been reported in the literature (Xu C, Qiao M, Huo X, Liao Z, Su J. An Oral Microencapsulated Vaccine Loaded by Sodium Alginate Effectively Enhances Protection Against GCRV Infection in Grass Carp (Ctenopharyngodon idella). Front. Immunol. 202213: 848958. doi: 10.3389 / fimmu.2022.848958). Summary of the invention

[0006] The present invention provides a method for evaluating the anti-hemorrhagic disease ability of grass carp based on multi-dimensional indices of intestinal mucosal barrier, and its purpose is to solve the above-mentioned problems existing in the background technology.

[0007] In order to achieve the above object, the embodiment of the present invention provides a method for evaluating the anti-hemorrhagic disease ability of grass carp based on multidimensional indicators of intestinal mucosal barrier, taking grass carp (common grass carp, gynogenetic grass carp and disease-resistant grass carp) of different genetic backgrounds as the object, and by comparison, it is found that 48 hours after GCRV infection, the intestinal mucosal permeability of gynogenetic grass carp and disease-resistant grass carp is significantly lower than that of common grass carp, the length of intestinal mucosal villi is significantly higher than that of common grass carp, the expression of intestinal mucosal ZO-1, Occludin and Claudin-1 is significantly higher than that of common grass carp, the expression of intestinal mucosal immune-related factors IL-22, MUC2, LEAP-2 and Hepcidin-1 is significantly higher than that of common grass carp, the diversity and abundance of intestinal flora are significantly higher than that of common grass carp, the concentrations of serum superoxide dismutase, catalase, acid phosphatase and alkaline phosphatase are significantly higher than those of common grass carp, and the content of liver and intestinal VP56 is significantly lower than that of common grass carp. Therefore, 15 days after GCRV infection, the survival rate of gynogenetic grass carp and disease-resistant grass carp is significantly higher than that of common grass carp. The method covers a multi-level and three-dimensional evaluation system including analyzing the mechanical barrier (integrity of physical structure), immune barrier (local immune response), microbial barrier (balance of intestinal flora) and antioxidant barrier (free radical scavenging ability) of the intestinal mucosa of grass carp, and can accurately evaluate the strength of grass carp's ability to resist hemorrhagic disease. The present invention can provide a new method for evaluating grass carp's ability to resist hemorrhagic disease.

[0008] The embodiment of the present invention provides a method for evaluating the anti-hemorrhagic disease ability of grass carp based on multi-dimensional indicators of intestinal mucosal barrier, comprising the following steps:

[0009] S1: Overall evaluation of intestinal mucosal permeability of grass carp

[0010] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, plasma was collected and the concentration changes of intestinal mucosal permeability markers in plasma were detected to analyze the changes in intestinal mucosal permeability of grass carp with different genetic backgrounds in response to GCRV infection;

[0011] S2: Evaluation of the mechanical barrier of the intestinal mucosa of grass carp

[0012] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the intestines were collected and intestinal mucosal tissue sections were made, and the changes in the length of the intestinal mucosal epithelial villi were examined by HE staining and microscopy, and the expression changes of intestinal mucosal tight junction molecules were observed to analyze the changes in the intestinal mucosal mechanical barrier of grass carp with different genetic backgrounds in response to GCRV infection;

[0013] S3: Evaluation of the intestinal mucosal immune barrier of grass carp

[0014] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the intestines were collected and the expression changes of intestinal mucosal immune-related factors were detected to analyze the changes in the intestinal mucosal immune barrier of grass carp with different genetic backgrounds in response to GCRV infection;

[0015] S4: Evaluation of the intestinal mucosal microbial barrier of grass carp

[0016] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the intestinal tract was collected and the diversity and abundance of intestinal flora were detected to analyze the changes in the intestinal microbial barrier of grass carp with different genetic backgrounds in response to GCRV infection;

[0017] S5: Evaluation of the antioxidant barrier of the intestinal mucosa of grass carp

[0018] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, serum was collected and the changes in the enzymatic activity of antioxidant enzymes in the serum were detected to analyze the changes in the intestinal mucosal antioxidant stress response capacity of grass carp with different genetic backgrounds in response to GCRV infection;

[0019] S6: Evaluation of viral load in grass carp

[0020] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the liver and intestine were collected and the expression changes of VP56 protein were detected to analyze the changes in viral load in grass carp with different genetic backgrounds when responding to GCRV infection;

[0021] S7: Assessment of grass carp survivability

[0022] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV. The observation was continued for 15 days, the specific time of death of each grass carp was recorded and the survival curve was drawn to analyze the differences in resistance of grass carp with different genetic backgrounds to GCRV infection.

[0023] According to one aspect of the embodiments of the present invention, the intestinal mucosal permeability markers include LPS and D-lactic acid.

[0024] According to one aspect of an embodiment of the present invention, the intestinal mucosal tight junction molecules include ZO-1, Occludin and Claudin-1.

[0025] According to one aspect of an embodiment of the present invention, the intestinal mucosal immunity-related factors include IL-22, MUC2, LEAP-2 and Hepcidin-1.

[0026] According to one aspect of an embodiment of the present invention, the antioxidant enzymes include superoxide dismutase, catalase, acid phosphatase and alkaline phosphatase.

[0027] According to one aspect of the embodiments of the present invention, the ELSIA method is used to detect changes in corresponding indicators in plasma and serum.

[0028] According to one aspect of an embodiment of the present invention, the qRT-PCR method is used to detect the expression of intestinal mucosal tight junction molecules, intestinal mucosal immune-related factors and VP56 protein.

[0029] According to one aspect of an embodiment of the present invention, 16S rRNA gene sequencing is used to detect changes in intestinal flora diversity and abundance.

[0030] According to one aspect of the embodiments of the present invention, intestinal mucosal tissue sections are prepared using paraffin sections.

[0031] The above scheme of the present invention has the following beneficial effects:

[0032] (1) The present invention deeply analyzes the anti-hemorrhagic disease ability of grass carp from multiple levels, such as the physical structure integrity of the intestinal mucosa of different grass carps, local immune response, intestinal flora homeostasis and free radical scavenging ability, to ensure the comprehensiveness and accuracy of the analysis results.

[0033] (2) The present invention has created a multi-dimensional evaluation system based on intestinal mucosal barrier function, which is used to accurately analyze the ability of grass carp to resist hemorrhagic disease. Through this multi-parameter, multi-level evaluation model, it can not only reveal the dynamic changes of the intestinal mucosal barrier of grass carp in resisting GCRV invasion, but also provide a powerful tool for grass carp variety selection and disease prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 is the test result of the effect of GCRV infection on the intestinal mucosal permeability markers LPS and D-lactic acid of three grass carps in Example 1 of the present invention; wherein A is plasma LPS and B is plasma D-lactic acid;

[0036] Figure 2 These are the test results of the changes in the intestinal mucosal morphology and structure of the three grass carps in Example 1 of the present invention when responding to GCRV infection; wherein A is the HE staining result of the intestinal mucosal tissue, and B is the result of the length of the intestinal mucosal villi;

[0037] Figure 3 is the test result of the changes in the intestinal mucosal mechanical barrier of the three grass carps in Example 1 of the present invention when responding to GCRV infection; wherein A is the expression result of the ZO-1 gene, B is the expression result of the Occludin gene, and C is the expression result of the Claudin-1 gene;

[0038] Figure 4 It is the test result of the key cytokines of the intestinal mucosal immune barrier of the three grass carps in Example 1 of the present invention when responding to GCRV infection; wherein A is the IL-22 gene, B is the MUC2 gene, C is the LEAP-2 gene, and D is the Hepcidin-1 gene;

[0039] Figure 5 is the test result of the changes in the intestinal microbial barrier of the three grass carps in Example 1 of the present invention when responding to GCRV infection; wherein A is the change in the Shannon diversity index of the intestinal flora, B is the change in the abundance of the intestinal flora at the phylum level, and C is the change in the β diversity index of the intestinal flora;

[0040] Figure 6 is the test result of the response capacity of the antioxidant enzymes of the three grass carps in response to GCRV infection in Example 1 of the present invention; wherein A is superoxide dismutase, B is catalase, C is acid phosphatase, and D is alkaline phosphatase;

[0041] Figure 7 It is the test result of the change of viral load of GCRV in three grass carp in Example 1 of the present invention; wherein A is the change of expression of liver VP56 protein, and B is the change of expression of intestinal VP56 protein;

[0042] Figure 8This is the test result of the difference in survival rate of three grass carps after being infected with GCRV in Example 2 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0044] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0045] The conventional experimental reagents, sequencing and experimental animals involved in the present invention are provided by the following companies:

[0046] 1. The total RNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0047] 2. Reverse transcription kit and qRT-PCR SYBR Mix were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0048] 3. Primer synthesis was completed by "Shanghai Bioengineering Co., Ltd."

[0049] 4. HE staining kit and LPS kit were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0050] 5. D-lactic acid, superoxide dismutase, catalase, acid phosphatase and alkaline phosphatase kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0051] 6.16S rRNA gene sequencing was completed by Beijing Novogene Technology Co., Ltd.;

[0052] 7. Common grass carp, gynogenetic grass carp and disease-resistant grass carp were purchased from Hunan Yuelushan Aquatic Breeding Technology Co., Ltd.;

[0053] In view of the existing problems, the present invention provides a method for evaluating the anti-hemorrhagic disease ability of grass carp based on multi-dimensional indices of intestinal mucosal barrier, comprising the following steps:

[0054] S1: Overall evaluation of intestinal mucosal permeability of grass carp

[0055] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, plasma was collected and the concentration changes of intestinal mucosal permeability markers in plasma were detected to analyze the changes in intestinal mucosal permeability of grass carp with different genetic backgrounds in response to GCRV infection;

[0056] S2: Evaluation of the mechanical barrier of the intestinal mucosa of grass carp

[0057] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the intestines were collected and intestinal mucosal tissue sections were made, and the changes in the length of the intestinal mucosal epithelial villi were examined by HE staining and microscopy, and the expression changes of intestinal mucosal tight junction molecules were observed to analyze the changes in the intestinal mucosal mechanical barrier of grass carp with different genetic backgrounds in response to GCRV infection;

[0058] S3: Evaluation of the intestinal mucosal immune barrier of grass carp

[0059] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the intestines were collected and the expression changes of intestinal mucosal immune-related factors were detected to analyze the changes in the intestinal mucosal immune barrier of grass carp with different genetic backgrounds in response to GCRV infection;

[0060] S4: Evaluation of the intestinal mucosal microbial barrier of grass carp

[0061] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the intestinal tract was collected and the diversity and abundance of intestinal flora were detected to analyze the changes in the intestinal microbial barrier of grass carp with different genetic backgrounds in response to GCRV infection;

[0062] S5: Evaluation of the antioxidant barrier of the intestinal mucosa of grass carp

[0063] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, serum was collected and the changes in the enzymatic activity of antioxidant enzymes in the serum were detected to analyze the changes in the intestinal mucosal antioxidant stress response capacity of grass carp with different genetic backgrounds in response to GCRV infection;

[0064] S6: Evaluation of viral load in grass carp

[0065] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the liver and intestine were collected and the expression changes of VP56 protein were detected to analyze the changes in viral load in grass carp with different genetic backgrounds when responding to GCRV infection.

[0066] S7: Assessment of grass carp survivability

[0067] Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV. The observation was continued for 15 days, the specific time of death of each grass carp was recorded and the survival curve was drawn to analyze the differences in resistance of grass carp with different genetic backgrounds to GCRV infection.

[0068] According to one aspect of the embodiments of the present invention, the intestinal mucosal permeability markers include LPS and D-lactic acid.

[0069] According to one aspect of an embodiment of the present invention, the intestinal mucosal tight junction molecules include ZO-1, Occludin and Claudin-1.

[0070] According to one aspect of an embodiment of the present invention, the intestinal mucosal immunity-related factors include IL-22, MUC2, LEAP-2 and Hepcidin-1.

[0071] According to one aspect of an embodiment of the present invention, the antioxidant enzymes include superoxide dismutase, catalase, acid phosphatase and alkaline phosphatase.

[0072] According to one aspect of the embodiments of the present invention, the ELSIA method is used to detect changes in corresponding indicators in plasma and serum.

[0073] According to one aspect of an embodiment of the present invention, the qRT-PCR method is used to detect the expression of intestinal mucosal tight junction molecules, intestinal mucosal immune-related factors and VP56 protein.

[0074] According to one aspect of an embodiment of the present invention, 16S rRNA gene sequencing is used to detect changes in intestinal flora diversity and abundance.

[0075] The following is a detailed description through specific embodiments.

[0076] Example 1

[0077] The application test to evaluate the intestinal mucosal barrier capacity of three grass carp species after GCRV infection included the following steps:

[0078] Step (1): Preparation of experimental animals

[0079] Selected healthy grass carp, gynogenetic grass carp and disease-resistant grass carp, all weighing about 15g, and divided each grass carp into 2 groups, with 10 fish in each group. Before the formal experiment, the grass carp was allowed to adapt to the laboratory culture environment for more than 1 week to keep its physiological state stable. Grouping information is shown in Table 1 below:

[0080] Table 1 Experimental animal grouping information of this embodiment 1

[0081]

[0082] Step (2): GCRV infection

[0083] For each designated GCRV infection group, GCRV infection was performed by intraperitoneal injection with an injection volume of 100 μL and an injection concentration of 1.0×10 7 TCID 50 mL -1 The normal group consisted of normal grass carp without any treatment.

[0084] Step (3): Overall evaluation of grass carp intestinal mucosal permeability

[0085] 48 hours after the above step (2), the plasma of the normal group and the GCRV infection group was collected. The concentration changes of LPS and D-lactic acid, markers of intestinal mucosal permeability, in the plasma were detected using LPS and D-lactic acid kits. The experimental results showed that there was no difference in the concentrations of plasma LPS and D-lactic acid in the normal group. However, after common grass carp, gynogenetic grass carp and disease-resistant grass carp were infected with GCRV, the concentrations of plasma LPS and D-lactic acid in common grass carp were significantly higher than those in gynogenetic grass carp and disease-resistant grass carp (Mann-Whitney test, P﹤0.01). Figure 1 These results showed that after GCRV infection, the intestinal mucosal permeability of gynogenetic grass carp and disease-resistant grass carp was significantly lower than that of common grass carp, indicating that the intestinal mucosal barrier capacity of gynogenetic grass carp and disease-resistant grass carp was higher than that of common grass carp.

[0086] Step (4): HE staining of grass carp intestinal mucosa tissue

[0087] 48 hours after the above step (2), the intestines of the normal group and the GCRV-infected group were collected and preserved in 4% paraformaldehyde. Afterwards, according to the method for making paraffin sections, the tissues were dehydrated, waxed, embedded, and sliced ​​to prepare tissue sections. According to the instructions of the HE staining kit, the intestinal sections were stained with HE. The staining results were photographed and the length of the intestinal villi was counted using an optical microscope. The experimental results showed that there was no difference in the length of the intestinal mucosal villi in the normal group. However, after common grass carp, gynogenetic grass carp and disease-resistant grass carp were infected with GCRV, the length of the intestinal mucosal villi of common grass carp was significantly lower than that of gynogenetic grass carp and disease-resistant grass carp (Mann-Whitney test, P﹤0.01). Figure 2 shown.

[0088] Step (5): Evaluation test of the mechanical barrier of the intestinal mucosa of grass carp

[0089] 48 hours after the above step (2), the intestines of the normal group and the GCRV infection group were collected and stored in liquid nitrogen. The intestinal cDNA was obtained using the instructions of the total RNA extraction kit and the reverse transcription reagent. Using the primers in Table 2 and the above-synthesized cDNA as the PCR template, the PCR reaction system for detecting the expression of intestinal tight junction molecules (ZO-1, Occludin and Claudin-1) was prepared according to the instructions of the qRT-PCR SYBRMix kit, and 18SrRNA was used as the internal reference gene. Afterwards, PCR was performed on the QuantStudio 5 real-time fluorescence quantitative PCR system (purchased from ABI, USA), and the qRT-PCR program was set as follows: 95°C, pre-denaturation for 30 seconds; 95°C denaturation for 10 seconds, 60°C reaction for 30 seconds, for a total of 40 cycles. After the reaction, 2 -△△t The experimental results showed that after common grass carp, gynogenetic grass carp and disease-resistant grass carp were infected with GCRV, the expression of ZO-1, Occludin and Claudin-1 in the intestinal mucosa of common grass carp was significantly lower than that of gynogenetic grass carp and disease-resistant grass carp (Mann-Whitney test, P﹤0.01). Figure 3 shown.

[0090] Step (6): Evaluation test of grass carp intestinal mucosal immune barrier

[0091] The cDNA obtained in the above step (5) was used as a PCR template, and the primers in Table 2 were used to detect the expression changes of intestinal mucosal immune-related factors IL-22, MUC2, LEAP-2 and Hepcidin-1 using qRT-PCR SYBRMix. The PCR reaction was the same as the above step (5), and 18SrRNA was used as the internal reference gene. The experimental results showed that after common grass carp, gynogenetic grass carp and disease-resistant grass carp were infected with GCRV, the expression of IL-22, MUC2, LEAP-2 and Hepcidin-1 in the intestinal mucosa of common grass carp was significantly lower than that of gynogenetic grass carp and disease-resistant grass carp (Mann-Whitney test, P﹤0.01). Figure 4 shown.

[0092] Step (7): Evaluation test of grass carp intestinal mucosal microbial barrier

[0093] 48 hours after the above step (2), the intestines of the normal group and the GCRV infection group were collected and stored in liquid nitrogen. Afterwards, the 16S rRNA gene sequencing technology was used to analyze the changes in the microbiota of each sample. The experimental results showed that after common grass carp, gynogenetic grass carp and disease-resistant grass carp were infected with GCRV, the diversity and abundance of the intestinal flora of common grass carp were significantly lower than those of gynogenetic grass carp and disease-resistant grass carp (Mann-Whitney test, P﹤0.01). Figure 5 shown.

[0094] Step (8): Evaluation test of the antioxidant barrier of grass carp intestinal mucosa

[0095] 48 hours after the above step (2), the serum of the normal group and the GCRV infection group was collected. The concentration changes of the four key antioxidant enzymes in the serum were detected using superoxide dismutase, catalase, acid phosphatase and alkaline phosphatase kits. The experimental results showed that there was no difference in the concentrations of serum superoxide dismutase, catalase, acid phosphatase and alkaline phosphatase in the normal group. However, after common grass carp, gynogenetic grass carp and disease-resistant grass carp were infected with GCRV, the concentrations of superoxide dismutase, catalase, acid phosphatase and alkaline phosphatase in the serum of common grass carp were significantly lower than those in the serum of gynogenetic grass carp and disease-resistant grass carp (Mann-Whitney test, P﹤0.01). Figure 6 shown.

[0096] Step (9): Application test to analyze the differences in viral loads in three grass carp species infected with GCRV

[0097] 48 hours after the above step (2), the livers and intestines of the normal group and the GCRV infection group were collected and stored in liquid nitrogen. The cDNA of the liver and intestine was obtained using the instructions of the total RNA extraction kit and the reverse transcription reagent. The primers in Table 2 were used to detect the expression changes of VP56, an important gene of GCRV, using qRT-PCR SYBR Mix. The PCR reaction was the same as the above step (5), with 18SrRNA as the internal reference gene. The experimental results showed that after common grass carp, gynogenetic grass carp and disease-resistant grass carp were infected with GCRV, the content of VP56 in the liver and intestine of common grass carp was significantly higher than that in the liver and intestine of gynogenetic grass carp and disease-resistant grass carp (Mann-Whitney test, P﹤0.01). Figure 7 shown.

[0098] Table 2 Primers used in Example 1

[0099]

[0100]

[0101] Example 2

[0102] The application test to analyze the differences in survival rates of three grass carp infected with GCRV specifically includes the following steps:

[0103] Step (1): Preparation of experimental animals

[0104] Select healthy common grass carp, gynogenetic grass carp and disease-resistant grass carp, all weighing about 15g, and divide each grass carp into 2 groups, with 20 fish in each group. Before the formal experiment, let the grass carp adapt to the laboratory breeding environment for more than 1 week to keep its physiological state stable. Grouping information is shown in Table 3 below:

[0105] Table 3 Experimental animal grouping information of this embodiment 2

[0106]

[0107] Step (2): GCRV infection

[0108] For each designated GCRV infection group, GCRV infection was performed by intraperitoneal injection with an injection volume of 100 μL and an injection concentration of 1.0×10 7 TCID 50 mL -1 The normal group consisted of normal grass carp without any treatment.

[0109] Step (3): Analyze survival rate

[0110] After the above step (2), the observation was continued for 15 days, the number of grass carp deaths was counted every day, and the survival rate of grass carp infected with GCRV was analyzed. The experimental results showed that after 15 days, the survival rates of gynogenetic grass carp and disease-resistant grass carp (45% and 40%, respectively) were significantly higher than the survival rate of ordinary grass carp (10%). Figure 8 (Log-rank test, P﹤0.01) as shown.

[0111] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for evaluating the ability of grass carp to resist hemorrhagic disease based on multidimensional indices of intestinal mucosal barrier, characterized in that: The steps include: S1: Overall evaluation of intestinal mucosal permeability of grass carp Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, plasma was collected and the concentration changes of intestinal mucosal permeability markers in plasma were detected to analyze the changes in intestinal mucosal permeability of grass carp with different genetic backgrounds in response to GCRV infection; S2: Evaluation of the mechanical barrier of the intestinal mucosa of grass carp Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the intestines were collected and intestinal mucosal tissue sections were made, and the changes in the length of the intestinal mucosal epithelial villi were examined by HE staining and microscopy, and the expression changes of intestinal mucosal tight junction molecules were observed to analyze the changes in the intestinal mucosal mechanical barrier of grass carp with different genetic backgrounds in response to GCRV infection; S3: Evaluation of the intestinal mucosal immune barrier of grass carp Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the intestines were collected and the expression changes of intestinal mucosal immune-related factors were detected to analyze the changes in the intestinal mucosal immune barrier of grass carp with different genetic backgrounds in response to GCRV infection; S4: Evaluation of the intestinal mucosal microbial barrier of grass carp Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the intestinal tract was collected and the diversity and abundance of intestinal flora were detected to analyze the changes in the intestinal microbial barrier of grass carp with different genetic backgrounds in response to GCRV infection; S5: Evaluation of the antioxidant barrier of the intestinal mucosa of grass carp Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, serum was collected and the changes in the enzymatic activity of antioxidant enzymes in the serum were detected to analyze the changes in the intestinal mucosal antioxidant stress response capacity of grass carp with different genetic backgrounds in response to GCRV infection; S6: Evaluation of viral load in grass carp Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV; after 48 hours, the liver and intestine were collected and the expression changes of VP56 protein were detected to analyze the changes in viral load in grass carp with different genetic backgrounds when responding to GCRV infection; S7: Assessment of grass carp survivability Two groups of common grass carp, gynogenetic grass carp and disease-resistant grass carp were set up, one group was infected with GCRV and the other was not infected with GCRV. The observation was continued for 15 days, the specific time of death of each grass carp was recorded and the survival curve was drawn to analyze the differences in resistance of grass carp with different genetic backgrounds to GCRV infection.

2. The method for evaluating the anti-hemorrhagic disease ability of grass carp based on multidimensional indices of intestinal mucosal barrier according to claim 1 is characterized in that: The intestinal mucosal permeability markers include LPS and D-lactic acid.

3. The method for evaluating the anti-hemorrhagic disease ability of grass carp based on multidimensional indices of intestinal mucosal barrier according to claim 1 is characterized in that: The intestinal mucosal tight junction molecules include ZO-1, Occludin and Claudin-1.

4. The method for evaluating the anti-hemorrhagic disease ability of grass carp based on multidimensional indices of intestinal mucosal barrier according to claim 1, characterized in that: The intestinal mucosal immunity-related factors include IL-22, MUC2, LEAP-2 and Hepcidin-1.

5. The method for evaluating the anti-hemorrhagic disease ability of grass carp based on multidimensional indices of intestinal mucosal barrier according to claim 1, characterized in that: The antioxidant enzymes include superoxide dismutase, catalase, acid phosphatase and alkaline phosphatase.

6. The method for evaluating the anti-hemorrhagic disease ability of grass carp based on multidimensional indices of intestinal mucosal barrier according to claim 1, characterized in that: The ELSIA method was used to detect the changes of corresponding indicators in plasma and serum.

7. The method for evaluating the anti-hemorrhagic disease ability of grass carp based on multidimensional indices of intestinal mucosal barrier according to claim 1, characterized in that: The qRT-PCR method was used to detect the expression of intestinal mucosal tight junction molecules, intestinal mucosal immune-related factors and VP56 protein.

8. The method for evaluating the anti-hemorrhagic disease ability of grass carp based on multi-dimensional indices of intestinal mucosal barrier according to claim 1, characterized in that: 16S rRNA gene sequencing was used to detect changes in intestinal flora diversity and abundance.

9. The method for evaluating the anti-hemorrhagic disease ability of grass carp based on multidimensional indices of intestinal mucosal barrier according to claim 1, characterized in that: Use paraffin sections to prepare intestinal mucosal tissue sections.

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