A method for constructing an aquatic animal model of co-infection

Abstract

The present invention discloses a method for constructing a mixed - infected aquatic animal model. A composition containing 2×10 6 TCID 50 ~3.33×10 6 TCID 50 of nervous necrosis virus and 1×10 6 cfu / mL~1×10 7 cfu / mL of Vibrio is used to infect Perciformes fish with a body length of 4 cm - 9 cm until infection symptoms appear, thus obtaining a mixed - infected aquatic animal model. The method provided by the present invention can quickly establish a multi - pathogen mixed infection of nervous necrosis virus and Vibrio in animals, showing typical mixed - infection symptoms, providing a new technical option for those skilled in the art to solve the problem of frequent multi - pathogen mixed infections in aquaculture, and contributing to the healthy development of the aquaculture industry.

Description

Technical Field

[0001] The present invention relates to the field of marine organisms, and specifically, to a method for constructing a mixed-infected aquatic animal model. Background Art

[0002] Trachinotus ovatus belongs to Osteichthyes, Perciformes, Carangidae, and Trachinotus; Epinephelus sp. belongs to Osteichthyes, Perciformes, Serranidae, and Epinephelinae. Due to their delicious meat, rich nutrition, strong stress resistance, fast growth rate, and high economic benefits, they are increasingly favored by consumers and breeders and have become important seawater fish farming varieties in southern China. At present, the vast majority of technicians in this field use high-density offshore cage farming of seawater fish. With the rapid development and disorderly expansion of this farming industry, the sea area load has become too large, the farming environment has been continuously deteriorating, and seawater fish diseases occur frequently and are becoming increasingly serious. Diseases have caused great economic losses to the farming production of seawater fish and seriously hindered the development of the aquaculture industry.

[0003] Most viral nervous necrosis diseases of marine fish in China are caused by Red-spotted grouper nervous necrosis virus (RGNNV). In severe cases, the mortality rate can reach 100%, which seriously threatens the healthy development of the seawater fish farming industry. Vibrio rotiferianus and Vibrio harveyi are both Gram-negative bacteria of the genus Vibrio and can cause diseases in various aquaculture animals and even trigger the outbreak of epidemic diseases. They have become important emerging pathogens in aquaculture diseases in China and have caused huge economic losses.

[0004] The aquaculture environment is complex, and the water body often contains a large number of viruses, bacteria, fungi, and parasites. The secondary and combined infections of multiple pathogens are common forms of the outbreak of aquatic animal diseases. Although mixed infections are very common under natural conditions, most of the existing technologies for the prevention and treatment of aquatic animal diseases remain at the level of single-pathogen infections, and it is not yet clear how to deal with and solve the conditions of mixed infections in aquatic animals. Summary of the Invention

[0005] To solve the problem of the lack of an aquatic animal model for mixed infection in the prior art, the present invention provides a method for constructing an aquatic animal model for mixed infection, targeting viral pathogens and bacterial pathogens with high incidence and strong pathogenicity in seawater aquaculture.

[0006] The first object of the present invention is to provide a composition for constructing an aquatic animal model for mixed infection.

[0007] The second object of the present invention is to provide the application of the above composition in constructing an aquatic animal model for mixed infection.

[0008] The third object of the present invention is to provide a method for constructing an aquatic animal model for mixed infection.

[0009] The fourth object of the present invention is to provide the application of the aquatic animal model for mixed infection constructed by the above method in evaluating methods for preventing and controlling aquaculture diseases.

[0010] To achieve the above objects, the present invention is realized through the following solutions:

[0011] Taking two seawater economic species, Trachinotus ovatus and Epinephelus spp., as objects, and using common viral pathogens (nervous necrosis virus) and bacterial pathogens (Vibrio rotiferianus and Vibrio harveyi) of seawater fish as infection pathogens to construct a mixed infection model. By designing various combinations of fry of different specifications and different infection concentrations of pathogens, and analyzing the disease incidence and mortality of fry samples among various combinations, the best combination suitable for studying the mechanism of mixed infection is finally explored. In addition, based on pathogen gene cloning, plasmid construction, and real-time fluorescence quantitative PCR technology, a pathogen detection standard curve is constructed to facilitate the detection of pathogen replication in various tissues of the host in the infection model.

[0012] A composition for constructing an aquatic animal model for mixed infection, comprising 1.8×10 6 TCID 50 ~3.5×10 6 TCID 50 nervous necrosis virus and 0.8×10 6 cfu / mL~1.2×10 7 cfu / mL vibrio.

[0013] Preferably, the nervous necrosis virus is nervous necrosis virus of Epinephelus akaara.

[0014] Preferably, the vibrio is Vibrio rotiferianus or Vibrio harveyi.

[0015] The application of any of the above compositions in constructing an aquatic animal model for mixed infection shall also be within the protection scope of the present invention.

[0016] A method for constructing a mixed-infected aquatic animal model, using a composition containing 1.8×10 6 TCID 50 ~3.5×10 6 TCID 50 nervous necrosis virus and 0.8×10 6 cfu / mL~1.2×10 7 cfu / mL vibrio to infect Perciformes fish with a body length of 4 cm to 9 cm until infection symptoms appear, thus obtaining a mixed-infected aquatic animal model; the infection symptoms are one or more of weakened swimming ability, loss of balance, fin congestion, swimming sideways, belly-up, body color darkening, and tail rot.

[0017] Preferably, 1.8×10 6 TCID 50 ~3.5×10 6 TCID 50 nervous necrosis virus and 0.8×10 6 cfu / mL~1.2×10 7 cfu / mL vibrio in the composition are used to infect Perciformes fish with a body length of 4 cm to 9 cm.

[0018] More preferably, the nervous necrosis virus is Epinephelus akaara nervous necrosis virus; the vibrio is one of Vibrio rotiferianus or Vibrio harveyi.

[0019] Further preferably, the Perciformes fish is Trachinotus ovatus with a body length of 4 cm to 6 cm, then 3.0×10 6 TCID 50 ~3.5×10 6 TCID 50 Epinephelus akaara nervous necrosis virus and 0.8×10 7 cfu / mL~1.2×10 7 cfu / mL Vibrio rotiferianus are used to infect the Trachinotus ovatus until infection symptoms appear.

[0020] Even more preferably, the Perciformes fish is Trachinotus ovatus with a body length of 4 cm to 6 cm, then 3.0×10 6 TCID 50 ~3.5×10 6 TCID 50 Epinephelus akaara nervous necrosis virus and 0.8×10 7 cfu / mL~1.2×10 7 cfu / mL Vibrio rotiferianus are used to infect the Trachinotus ovatus until infection symptoms appear.

[0021] More preferably, the Perciformes fish is Trachinotus ovatus with a body length of 4 cm to 6 cm, and then Trachinotus ovatus is infected with 3.33×10 6 TCID 50 Nervous necrosis virus of Epinephelus akaara and 1×10 7 cfu / mL Vibrio rotiferianus until infection symptoms appear.

[0022] Most preferably, first inject 3.33×10 6 TCID 50 Nervous necrosis virus of Epinephelus akaara into Trachinotus ovatus, and then inject 1×10 7 cfu / mL Vibrio rotiferianus into Trachinotus ovatus 12 h later. Infection symptoms appear 48 h later.

[0023] More preferably, the Perciformes fish is Epinephelus coioides with a body length of 7 cm to 9 cm, and then Epinephelus coioides is infected with 0.8×10 7 cfu / mL to 1.2×10 7 cfu / mL Nervous necrosis virus of Epinephelus akaara and 0.8×10 6 cfu / mL to 1.2×10 6 cfu / mL Vibrio harveyi until infection symptoms appear.

[0024] Even more preferably, the Perciformes fish is Epinephelus coioides with a body length of 7 cm to 9 cm, and then Epinephelus coioides is infected with 2×10 6 TCID 50 Nervous necrosis virus of Epinephelus akaara and 1×10 6 cfu / mL Vibrio harveyi until infection symptoms appear.

[0025] Most preferably, first inject 3.33×10 6 TCID 50 Nervous necrosis virus of Epinephelus akaara into Epinephelus coioides, and then inject 1×10 7 cfu / mL Vibrio rotiferianus into Epinephelus coioides 12 h later. Infection symptoms appear 48 h later.

[0026] Most preferably, first inject 1×10 7 cfu / mL Vibrio rotiferianus into Epinephelus coioides, and then inject 3.33×10 6 TCID 50 Nervous necrosis virus of Epinephelus akaara into Epinephelus coioides 12 h later. Infection symptoms appear 48 h later.

[0027] The application of any of the above - constructed aquatic animal models with mixed infection in evaluating methods for preventing and controlling diseases in aquaculture should also be within the protection scope of the present invention.

[0028] Use of the primer composition in preparing a kit for detecting an aquatic animal model with mixed infection constructed by any of the above methods, wherein the primer composition comprises any one or more of primer pairs 1 to 3; primer pair 1 is used for detecting nervous necrosis virus of Epinephelus akaara, and its nucleotide sequences are as shown in SEQ ID No.5 to 6; primer pair 2 is used for detecting Vibrio rotiferianus, and its nucleotide sequences are as shown in SEQ ID No.11 to 12; primer pair 3 is used for detecting Vibrio harveyi, and its nucleotide sequences are as shown in SEQ ID No.17 to 18.

[0029] Preferably, the kit further comprises any one or more of the plasmids with nucleotide sequences as shown in SEQ ID No.4, the plasmids with nucleotide sequences as shown in SEQ ID No.10, and the plasmids with nucleotide sequences as shown in SEQ ID No.16.

[0030] The plasmid with nucleotide sequence as shown in SEQ ID No.4 is a standard product of nervous necrosis virus of Epinephelus akaara; the plasmid with nucleotide sequence as shown in SEQ ID No.10 is a standard product of Vibrio rotiferianus; the plasmid with nucleotide sequence as shown in SEQ ID No.16 is a standard product of Vibrio harveyi.

[0031] Preferably, the kit further comprises real-time fluorescence quantitative PCR reaction reagents and an instruction manual, and the instruction manual records a formula for calculating the replication amount according to the Ct value, and the formula comprises the formula of the detection standard curve of nervous necrosis virus of Epinephelus akaara y=-0.2999x + 10.922 (R 2 =0.9995), the formula of the detection standard curve of Vibrio rotiferianus y=-0.293x + 11.78 (R 2 =0.9976), and the formula of the detection standard curve of Vibrio harveyi y=-1.348ln(x)+37.642 (R 2 =0.996), any one or more of them.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The method provided by the present invention can quickly establish a multi-pathogen mixed infection of nervous necrosis virus and vibrio in animals, showing typical mixed infection symptoms, providing a new technical option for those skilled in the art to solve the problem of frequent multi-pathogen mixed infection in aquaculture, and contributing to the healthy development of the aquaculture industry. Description of the Drawings

[0034] Figure 1For the case of the detection standard for nervous necrosis virus; A is the real-time fluorescence quantitative PCR amplification curve of the detection standard for nervous necrosis virus; B is the real-time fluorescence quantitative PCR melting curve of the detection standard for nervous necrosis virus; C is the detection standard curve for nervous necrosis virus.

[0035] Figure 2 For the case of the detection standard for Vibrio rotiferianus; A is the real-time fluorescence quantitative PCR amplification curve of the detection standard for Vibrio rotiferianus; B is the real-time fluorescence quantitative PCR melting curve of the detection standard for Vibrio rotiferianus; C is the detection standard curve for Vibrio rotiferianus.

[0036] Figure 3 For the case of the detection standard for Vibrio harveyi; A is the real-time fluorescence quantitative PCR amplification curve of the detection standard for Vibrio harveyi; B is the real-time fluorescence quantitative PCR melting curve of the detection standard for Vibrio harveyi; C is the detection standard curve for Vibrio harveyi.

[0037] Figure 4 For the mortality of Trachinotus ovatus infected by the single virus infection group.

[0038] Figure 5 For the mortality of Trachinotus ovatus infected by the single Vibrio infection group.

[0039] Figure 6 For the replication of nervous necrosis virus in the brain, eyes, liver and spleen of Trachinotus ovatus in the single virus infection group, mixed infection group 1 and mixed infection group 2 at 48 h post-infection; the vertical axis represents the copy number of the CP gene of nervous necrosis virus, and the horizontal axis represents the group; RGNNV represents the single virus infection group; Vibrio rotiferianus+RGNNV represents mixed infection group 1; RGNNV+Vibrio rotiferianus represents mixed infection group 2; ** represents significant P<0.01; *** represents significant P<0.001.

[0040] Figure 7 For the replication of Vibrio rotiferianus in the brain, eyes, liver and spleen of Trachinotus ovatus in the single Vibrio infection group, mixed infection group 1 and mixed infection group 2 at 48 h post-infection; the vertical axis represents the copy number of the Vr1 gene of Vibrio rotiferianus, and the horizontal axis represents the group; Vibrio rotiferianus represents the single Vibrio infection group; Vibrio rotiferianus+RGNNV represents mixed infection group 1; RGNNV+Vibrio rotiferianus represents mixed infection group 2; * represents significant P<0.05; ** represents significant P<0.01.

[0041] Figure 8HE staining results of the brain, eyes, liver and spleen of Trachinotus ovatus in the control group, single virus infection group, single Vibrio infection group, mixed infection group 1 and mixed infection group 2 at 48 h post-infection; PBS represents the control group; VR represents the single Vibrio infection group; RGNNV represents the single virus infection group; VR+RGNNV represents mixed infection group 1; RGNNV+VR represents mixed infection group 2.

[0042] Figure 9 Mortality of Epinephelus coioides infected with single virus infection group.

[0043] Figure 10 Mortality of Epinephelus coioides infected with single Vibrio infection group.

[0044] Figure 11 Pathological anatomy results of the brain, eyes, liver and spleen of Epinephelus coioides in the single virus infection group, single Vibrio infection group, mixed infection group 1 and mixed infection group 2 at 48 h post-infection; PBS represents the control group; Vibrio harveyi represents the single Vibrio infection group; Vibrio harveyi+RGNNV represents mixed infection group 1; RGNNV+Vibrio harveyi represents mixed infection group 2.

[0045] Figure 12 Replication of Vibrio harveyi in the liver tissue of Epinephelus coioides in the single Vibrio infection group, mixed infection group 1 and mixed infection group 2 at 48 h post-infection; the vertical axis represents the copy number of Vibrio harveyi Vhhp2, and the horizontal axis represents the group; Vibrio harveyi represents the single Vibrio infection group; Vibrio harveyi+RGNNV represents mixed infection group 1; RGNNV+Vibrio harveyi represents mixed infection group 2; * represents significant P<0.05.

[0046] Figure 13 Replication of nervous necrosis virus in the brain tissue of Trachinotus ovatus in the single virus infection group, mixed infection group 1 and mixed infection group 2 at 48 h post-infection; the vertical axis represents the copy number of the CP gene of nervous necrosis virus, and the horizontal axis represents the group; Vibrio harveyi represents the single Vibrio infection group; RGNNV+Vibrio harveyi represents mixed infection group 1; Vibrio harveyi+RGNNV represents mixed infection group 2; * represents significant P<0.05.

[0047] Figure 14HE staining results of the brain, eye, liver, and spleen tissues of orange-spotted groupers in the single-virus infection group, single-vibrio infection group, co-infection group 1, and co-infection group 2 at 48 h post-infection; PBS represents the control group; Vibrio harveyi represents the single-vibrio infection group; RGNNV+Vibrio harveyi represents co-infection group 1; Vibrio harveyi+RGNNV represents co-infection group 2. Detailed implementation manners

[0048] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] Example 1 Preparation of red-spotted grouper nervous necrosis virus (RGNNV) stock solution and its detection standard curve

[0050] I. Experimental method

[0051] (1) Preparation of RGNNV stock solution

[0052] 1. Strain isolation

[0053] In this example, RGNNV was isolated from diseased groupers infected with red-spotted grouper nervous necrosis virus using a conventional method. The specific isolation method is as follows:

[0054] Collect the brain tissues containing only RGNNV virus from diseased groupers and place them in a mortar; add an appropriate amount of PBS solution, grind with a grinding rod for 10 min, add liquid nitrogen for tissue cryopreservation, and then grind again; repeat the above grinding step 3 times to obtain a tissue suspension; let it stand overnight at 4°C; centrifuge the tissue suspension, collect the supernatant solution, and filter it through a 0.22 μm filter membrane to obtain the RGNNV virus suspension.

[0055] 2. Virus amplification and purification

[0056] The RGNNV virus suspension was amplified and purified using grouper spleen (GS) cells. The specific method is as follows:

[0057] GS cells were inoculated into Leibovitz's L-15 medium containing 10% v / v fetal bovine serum (FBS) and cultured in an incubator at 28°C. When the GS cells grew to monolayer covering the bottom of a 50 mL cell bottle and the density reached over 80%, 5 μL of the RGNNV virus suspension obtained in the previous step was added to infect the GS cells. After 2 h, the cells were washed 3 times with Leibovitz L15 medium containing 2% v / v fetal bovine serum to remove the excess RGNNV. Then, Leibovitz L15 medium containing 10% fetal bovine serum was added and the culture was continued. The cell supernatant containing the virus was collected 48 h - 72 h after infection, and the cell debris in the cell supernatant was removed by centrifugation. According to the above method, after continuous purification for 5 generations, the virus solution of RGNNV was obtained and stored at -80°C for standby.

[0058] 3. Titer determination

[0059] The titer of the RGNNV virus solution was determined by the TCID 50 method, and the titer was adjusted to 1×10 7 TCID 50 , thus obtaining the RGNNV stock solution.

[0060] (2) Construction of the RGNNV detection standard

[0061] The viral DNA in the RGNNV virus stock solution was extracted. Using the DNA of RGNNV as a template, PCR amplification was carried out with the upstream primer CP-F: 5’-ATGGTACGCAAAGGTGAGAAGA-3’ (SEQ ID No.1) and the downstream primer CP-R: 5’-TTAGTTTTCCGAGTCAACCCTG-3’ (SEQ ID No.2).

[0062] The PCR reaction system was as follows: ultrapure water, 6 μL; CP-F (SEQ ID No.1) (10 μmol / L), 1 μL; CP-R (SEQ ID No.2) (10 μmol / L), 1 μL; DNA template (about 100 ng / μL), 2 μL; TaKaRa Taq premix enzyme, 10 μL.

[0063] The PCR reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, for a total of 34 cycles; extension at 72°C for 10 min.

[0064] The recycled amplification product is the partial sequence (SEQ ID No.3) of the cp gene of RGNNV, named CP1. CP1 (SEQ ID No.3) was ligated to the pMD18-T vector, transformed into competent cells DH5α, and positive clones with correct sequencing results were selected to extract plasmids. The obtained pMD18-CP1 recombinant plasmid is the RGNNV detection standard (SEQ ID No.4).

[0065] (3) Construction of the RGNNV detection standard curve

[0066] The RGNNV detection standard (SEQ ID No.4) was diluted at a 10-fold titer for a total of 7 gradients, with 3 replicates for each gradient.

[0067] The mass concentration of the RGNNV detection standard (SEQ ID No.4) at each gradient was measured using a nucleic acid protein analyzer, and the plasmid copy number (i.e., the number of viral DNA copies per μL of plasmid) was calculated. The calculation formula used was: M represents the average molecular mass of each base, and X represents the number of bases in the recombinant plasmid.

[0068] Using the upstream quantitative primer CP-RT-F: 5’-CAACTGACAACGATCACACCTTC-3’ (SEQ ID No.5) and the downstream quantitative primer CP-RT-R: 5’-CAATCGAACACTCCAGCGACA-3’ (SEQ ID No.6), a real-time fluorescence quantitative PCR reaction was performed to measure the Ct value of the RGNNV detection standard (SEQ ID No.4) at each gradient. The real-time fluorescence quantitative PCR reaction system was: Green Realtime PCR Master Mix, 5 μL; CP-RT-F (SEQ ID No.5) (10 μmol / L), 0.3 μL; CP-RT-R (SEQ ID No.6) (10 μmol / L), 0.3 μL; the RGNNV detection standard (SEQ ID No.4) at each gradient, 1 μL; ultrapure water, made up to 10 μL. The real-time fluorescence quantitative PCR reaction conditions were: (1) Amplification curve (Ct value determination): 95°C, 1 min; 95°C, 15 s, 60°C, 15 s, 72°C, 45 s, for a total of 40 cycles; (2) Melting curve: 95°C, 15 s; 60°C, 15 s; 95°C, 15 s; 33°C, 15 s. During this process, fluorescence signals were continuously collected to form a melting curve.

[0069] After detection, the mass concentrations, corresponding plasmid copy numbers, and Ct values of RGNNV detection standards (SEQ ID No. 4) with different gradients are as follows: for gradient 1, the mass concentration is 5.1 ng / μL, the plasmid copy number is 7.63×10 8 copies / μL, and the Ct value is 6.726; for gradient 2, the mass concentration is 0.51 ng / μL, the plasmid copy number is 7.63×10 7 copies / μL, and the Ct value is 10.233; for gradient 3, the mass concentration is 0.051 ng / μL, the plasmid copy number is 7.63×10 6 copies / μL, and the Ct value is 13.550; for gradient 4, the mass concentration is 0.0051 ng / μL, the plasmid copy number is 7.63×10 5 copies / μL, and the Ct value is 16.894; for gradient 5, the mass concentration is 0.00051 ng / μL, the plasmid copy number is 7.63×10 4 copies / μL, and the Ct value is 20.039; for gradient 6, the mass concentration is 0.000051 ng / μL, the plasmid copy number is 7.63×10 3 copies / μL, and the Ct value is 23.680; for gradient 7, the mass concentration is 0.0000051 ng / μL, the plasmid copy number is 7.63×10 2 copies / μL, and the Ct value is 26.595.

[0070] According to the plasmid copy numbers and corresponding Ct values of the RGNNV detection standards (SEQ ID No. 4), an RGNNV detection standard curve is constructed.

[0071] II. Experimental Results

[0072] The real-time fluorescence quantitative PCR amplification curves of the RGNNV detection standards (SEQ ID No. 4) with 9 gradient concentrations are as shown in Figure 1 A, and the melting curves are as shown in Figure 1 B.

[0073] The RGNNV detection standard curve is as shown in Figure 1 C, and the formula of the standard curve is y = -0.2999x + 10.922 (R 2 = 0.9995), where x represents the Ct value, y represents the lg copy number, and R 2 represents the goodness of fit of the standard curve.

[0074] Example 2 Preparation of Vibrio rotiferianus Stock Solution and Its Detection Standard Curve

[0075] I. Experimental Method

[0076] (1) Preparation of Vibrio rotiferianus stock solution

[0077] Vibrio rotiferianus was isolated from diseased Trachinotus ovatus by the research group of Qin Qiwei from the College of Ocean Sciences, South China Agricultural University, and named VR-TO.

[0078] The Vibrio rotiferianus bacterial solution was evenly spread on brain heart infusion broth solid medium (BHI solid medium) and cultured at 28 °C for 18 h; single colonies were picked and inoculated into 5 mL of BHI liquid medium, and cultured at 28 °C for 18 - 20 h; the bacteria in the logarithmic phase were collected (the OD value of the bacterial solution was about 1.2, and the concentration was about 1×10 9 cfu / mL), and the Vibrio rotiferianus stock solution (i.e., VR-TO stock solution) was obtained and stored at -80 °C for later use.

[0079] (2) Construction of Vibrio rotiferianus detection standard

[0080] Extract the Vibrio rotiferianus DNA from the Vibrio rotiferianus stock solution. Using the Vibrio rotiferianus DNA as a template, PCR amplification was performed with the upstream primer VR-TO-F: 5’-GAAAGCGTGGGGAGCAAACAGGATT-3’ (SEQ ID No.7) and the downstream primer VR-TO-R: 5’-CTGCCCTCTGTATGCGCCATTGTAG-3’ (SEQ ID No.8).

[0081] The PCR reaction system was as follows: ultrapure water, 6 μL; VR-TO-F (SEQ ID No.7) (10 μmol / L), 1 μL; VR-TO-R (SEQ ID No.8) (10 μmol / L), 1 μL; DNA template (about 100 ng / μL), 2 μL; TaKaRar Taq premix enzyme, 10 μL.

[0082] The PCR reaction conditions were: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, for a total of 34 cycles; extension at 72 °C for 10 min.

[0083] The amplified product was recovered to obtain a partial sequence of the 16S gene of VR-TO (SEQ ID No.9), named Vr1.

[0084] Vr1 (SEQ ID No.9) was ligated to the pMD18-T vector, transformed into DH5α cells, and positive clones with correct sequencing results were selected to extract plasmids. The pMD18-Vr1 recombinant plasmid was the Vibrio rotiferianus detection standard (SEQ ID No.10).

[0085] (3) Construction of Vibrio rotiferianus detection standard curve

[0086] The Vibrio rotiferianus detection standard (SEQ ID No.10) was serially diluted 10-fold to a total of 7 dilutions, with 3 replicates for each dilution.

[0087] The mass concentration of the Vibrio rotiferianus detection standard (SEQ ID No.10) at each dilution was measured using a nucleic acid protein analyzer, and the plasmid copy number was calculated according to the formula in Example 1.

[0088] Real-time fluorescence quantitative PCR was performed using the upstream quantitative primer VR-RT-F: 5’-GGGAGTACGGTCGCAAGATT-3’ (SEQ ID No.11) and the downstream quantitative primer VR-RT-R: 5’-GCTGGCAAACAAGGATAAGG-3’ (SEQ ID No.12) to measure the Ct value of the Vibrio rotiferianus detection standard (SEQ ID No.10) at each dilution. The reaction system for real-time fluorescence quantitative PCR was as follows: Green Realtime PCR Master Mix, 5 μL; VR-RT-F (SEQ ID No.11) (10 μmol / L), 0.3 μL; VR-RT-R (SEQ ID No.12) (10 μmol / L), 0.3 μL; the Vibrio rotiferianus detection standard (SEQ ID No.10) at each dilution, 1 μL; ultrapure water was added to make up to 10 μL. The reaction conditions for real-time fluorescence quantitative PCR were: (1) Amplification curve (Ct value measurement): 95 °C, 1 min; 95 °C, 15 s, 60 °C, 15 s, 72 °C, 45 s, for a total of 40 cycles; (2) Melting curve: 95 °C, 15 s; 60 °C, 15 s; 95 °C, 15 s; 33 °C, 15 s. Fluorescence signals were continuously collected during this process to form a melting curve.

[0089] After detection, the mass concentration, corresponding plasmid copy number, and Ct value of the Vibrio rotiferianus detection standard (SEQ ID No.10) at different dilutions were as follows: the mass concentration of dilution 1 was 60 ng / μL, the plasmid copy number was 2.17×10 9 copies / μL, and the Ct value was 8.379; the mass concentration of dilution 2 was 0.6 ng / μL, the plasmid copy number was 2.17×10 8 copies / μL, and the Ct value was 11.472; the mass concentration of dilution 3 was 0.06 ng / μL, the plasmid copy number was 2.17×10 7 copies / μL, and the Ct value was 15.152; the mass concentration of dilution 4 was 0.006 ng / μL, the plasmid copy number was 2.17×10 6copies / μL, with a Ct value of 18.732; the mass concentration of gradient 5 is 0.0006 ng / μL, and the plasmid copy number is 2.17×10 5 copies / μL, with a Ct value of 22.254; the mass concentration of gradient 6 is 0.00006 ng / μL, and the plasmid copy number is 2.17×10 4 copies / μL, with a Ct value of 25.873; the mass concentration of gradient 7 is 0.000006 ng / μL, and the plasmid copy number is 2.17×10 3 copies / μL, with a Ct value of 28.188.

[0090] According to the plasmid copy numbers and corresponding Ct values of the Vibrio rotiferianus detection standard (SEQ ID No.10), a detection standard curve for Vibrio rotiferianus was constructed.

[0091] II. Experimental Results

[0092] The real-time fluorescence quantitative PCR amplification curves of the 9-gradient concentration Vibrio rotiferianus detection standard (SEQ ID No.10) are as shown in Figure 2 A in the figure, and the melting curves are as shown in Figure 2 B in the figure.

[0093] The detection standard curve for Vibrio rotiferianus is as shown in Figure 2 C in the figure, and the formula of the standard curve is y = -0.293x + 11.78 (R 2 = 0.9976), where x represents the sample CT value obtained by fluorescence quantitative PCR, y represents the lg copy number, and R 2 represents the goodness of fit of the standard curve.

[0094] Example 3 Preparation of the original solution of Vibrio harveyi and its detection standard curve

[0095] I. Experimental Method

[0096] (1) Preparation of the original solution of Vibrio harveyi

[0097] Vibrio harveyi was isolated from diseased groupers by the research group of Qin Qiwei from the College of Ocean Sciences, South China Agricultural University, and named VH-EC.

[0098] The Vibrio harveyi bacterial solution was evenly spread on the lysogeny broth solid medium (LB solid medium) and cultured at 28°C for 18 h; single colonies were picked and inoculated into 5 mL of LB liquid medium, and then cultured at 28°C for 14 - 16 h; the bacteria in the logarithmic phase were collected (the OD value of the bacterial solution was about 1.2, and the concentration was about 1×10 9 cfu / mL), thus obtaining the original solution of Vibrio harveyi (i.e., the original solution of VH-EC), which was stored at -80°C for later use.

[0099] (2) Construction of Vibrio harveyi detection standard

[0100] Extract the Vibrio harveyi DNA from the stock solution of Vibrio harveyi. Using the Vibrio harveyi DNA as a template, perform PCR amplification with the upstream primer VH-01F: 5’-GTGGTGGCGAACTGGATGTAA-3’ (SEQ ID No.13) and the downstream primer VH-01R: 5’-GAGTATTCGTTGCCGAGTAAAGC-3’ (SEQ ID No.14).

[0101] The PCR reaction system is as follows: ultrapure water, 6 μL; VH-01F (SEQ ID No.13) (10 μmol / L), 1 μL; VH-01R (SEQ ID No.14) (10 μmol / L), 1 μL; DNA template (about 100 ng / μL), 2 μL; TaKaRar Taq premix enzyme, 10 μL.

[0102] The PCR reaction conditions are as follows: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, for a total of 34 cycles; extension at 72 °C for 10 min.

[0103] Recover the amplification product to obtain a partial sequence of the Vhhp2 gene of VH-EC, named VHH1 (SEQ ID No.15), and its homology with other Vibrio harveyi Vhhp2 gene sequences in the public database is more than 95%.

[0104] Ligate VHH1 (SEQ ID No.15) to the pMD18-T vector, transform it into DH5α cells, select the positive clones with correct sequencing results and extract the plasmids. The obtained pMD18-Vr1 recombinant plasmid is the Vibrio harveyi detection standard (SEQ ID No.16).

[0105] (3) Construction of Vibrio harveyi detection standard curve

[0106] Perform 10-fold serial dilution on the Vibrio harveyi detection standard (SEQ ID No.16), with a total of 9 dilution gradients, and set 3 replicates for each gradient.

[0107] Use a nucleic acid and protein analyzer to measure the mass concentration of the Vibrio harveyi detection standard (SEQ ID No.16) at each gradient, and calculate the plasmid copy number according to the formula in Example 1.

[0108] Using the upstream quantitative primer VHRT-F: 5’-GATGGATATGGTGGTGGCGAACTG-3’ (SEQ ID No.17) and the downstream quantitative primer VHRT-R: 5’-CACACGGCAATACAACCAGCATTC-3’ (SEQ ID No.18), a real-time fluorescence quantitative PCR reaction was carried out to measure the Ct values of the Vibrio harveyi detection standards (SEQ ID No.16) at each gradient. The real-time fluorescence quantitative PCR reaction system was as follows: Green Realtime PCR Master Mix, 5 μL; VHRT-F (SEQ ID No.17) (10 μmol / L), 0.3 μL; VHRT-R (SEQ ID No.18) (10 μmol / L), 0.3 μL; the Vibrio harveyi detection standards (SEQ ID No.16) at each gradient, 1 μL; ultrapure water, made up to 10 μL. The real-time fluorescence quantitative PCR reaction conditions were: (1) Amplification curve (Ct value determination): 95°C, 1 min; 95°C, 15 s, 60°C, 15 s, 72°C, 45 s, for a total of 40 cycles; (2) Melting curve: 95°C, 15 s; 60°C, 15 s; 95°C, 15 s; 33°C, 15 s. During this process, fluorescence signals were continuously collected to form a melting curve.

[0109] After detection, the mass concentrations, corresponding plasmid copy numbers, and Ct values of the Vibrio harveyi detection standards (SEQ ID No.16) at different gradients were as follows: the mass concentration of gradient 1 was 22 ng / μL, the plasmid copy number was 7×10 9 copies / μL, and the Ct value was 7.683; the mass concentration of gradient 2 was 2.2 ng / μL, the plasmid copy number was 7×10 8 copies / μL, and the Ct value was 9.985; the mass concentration of gradient 3 was 0.22 ng / μL, the plasmid copy number was 7×10 7 copies / μL, and the Ct value was 13.016; the mass concentration of gradient 4 was 0.022 ng / μL, the plasmid copy number was 7×10 6 copies / μL, and the Ct value was 16.03; the mass concentration of gradient 5 was 0.0022 ng / μL, the plasmid copy number was 7×10 5 copies / μL, and the Ct value was 19.114; the mass concentration of gradient 6 was 0.00022 ng / μL, the plasmid copy number was 7×10 4 copies / μL, and the Ct value was 23.747; the mass concentration of gradient 7 was 0.000022 ng / μL, the plasmid copy number was 7×10 3copies / μL, and the Ct value was 25.837.

[0110] According to the plasmid copy number and the corresponding Ct value of the Vibrio harveyi detection standard (SEQ ID No. 16), a detection standard curve for Vibrio harveyi was constructed.

[0111] II. Experimental Results

[0112] The real-time fluorescence quantitative PCR amplification curves of the Vibrio harveyi detection standard (SEQ ID No. 16) at 9 gradient concentrations are shown as A in Figure 3 and the melting curves are shown as B in Figure 3 .

[0113] The detection standard curve for Vibrio harveyi is shown as C in Figure 3 , and the formula of the standard curve is y = -1.348ln(x) + 37.642 (R 2 = 0.996), where x represents the plasmid copy number, y represents the Ct value, and R 2 represents the goodness of fit of the standard curve.

[0114] Example 4 Pathogenicity of RGNNV and Vibrio rotiferianus to Trachinotus ovatus of Different Sizes

[0115] I. Experimental Method

[0116] (1) Domestication of Trachinotus ovatus

[0117] Trachinotus ovatus were temporarily cultured in a seawater circulation system at 25 - 30 °C for two weeks. The seawater salinity was about 30‰, the pH was about 8, and the dissolved oxygen was above 5.6 mg / L.

[0118] After the temporary culture, 30 Trachinotus ovatus with a body length of 5.0 ± 1.0 cm were randomly divided into 3 groups, with 10 in each group; another 30 Trachinotus ovatus with a body length of 10.0 ± 1.0 cm were randomly divided into 3 groups, with 10 in each group.

[0119] (2) Infection Experiment

[0120] The 6 groups of Trachinotus ovatus were intraperitoneally injected according to the information shown in Table 1.

[0121] Table 1 Infection of Trachinotus ovatus of Different Sizes with RGNNV and Vibrio rotiferianus

[0122]

[0123] After injection, they were continuously cultured in a seawater circulation system at 25 - 30 °C for one week, and the morbidity of Trachinotus ovatus was observed every day, and the mortality was counted.

[0124] The symptoms of RGNNV infection are one or more of the following: weakened swimming ability, loss of balance, and blackening of body color.

[0125] The symptoms of Vibrio rotiferianus infection are one or more of the following: weakened swimming ability, loss of balance, fin congestion, and skin ulceration.

[0126] II. Experimental Results

[0127] The results showed that for Trachinotus ovatus fry with a body length of 5.0 ± 1.0 cm, when stimulated with the original RGNNV solution with a concentration of 1×10 7 TCID 50 (i.e., the R-5 experimental group) for 60 h, the mortality rate was 60% and obvious RGNNV infection symptoms appeared. When injected with 100 μL of the original Vibrio rotiferianus solution with a concentration of 1×10 9 cfu / mL (i.e., the V-5 experimental group) for 60 h, the mortality rate was 100% and obvious Vibrio rotiferianus infection symptoms appeared. For Trachinotus ovatus fry with a body length of 10.0 ± 1.0 cm, when injected with 100 μL of the original RGNNV solution with a concentration of 1×10 7 TCID 50 (i.e., the R-10 experimental group) and the original Vibrio rotiferianus solution with a concentration of 1×10 9 cfu / mL (i.e., the V-10 experimental group) for 60 h, the mortality rate was 0% and no obvious disease symptoms appeared.

[0128] In summary, for Trachinotus ovatus fry with a body length of 10.0 ± 1.0 cm, within 60 h of high-concentration infection with the two pathogens, there were no obvious disease symptoms, so it was not suitable for constructing a multi-pathogen mixed infection model. However, for Trachinotus ovatus fry with a body length of 5.0 ± 1.0 cm, within 60 h of high-concentration infection with the two pathogens, there were obvious disease symptoms and a high lethality rate, and it was more sensitive to virus infection, so it was more suitable for constructing a multi-pathogen mixed infection model.

[0129] Example 5 Pathogenicity of Different Concentrations of RGNNV and Vibrio rotiferianus to Trachinotus ovatus

[0130] I. Experimental Method

[0131] (1) Domestication of Trachinotus ovatus

[0132] Trachinotus ovatus were temporarily cultured in a seawater circulation system at 25 - 30 °C for two weeks. The seawater salinity was about 30‰, the pH was about 8, and the dissolved oxygen was above 5.6 mg / L.

[0133] After the temporary culture ended, 110 Trachinotus ovatus with a body length of 5.0 ± 1.0 cm were randomly divided into 11 groups, with 10 tails in each group.

[0134] (2) Infection Experiment

[0135] Inject 11 groups of Trachinotus ovatus intraperitoneally according to the information shown in Table 2.

[0136] Table 2 Infection of Trachinotus ovatus with RGNNV and Vibrio rotiferianus at different concentrations

[0137]

[0138] After 12 h, inject 100 μL of PBS buffer into each Trachinotus ovatus.

[0139] Keep them in a seawater circulation system at 25-30 °C for another week, observe the morbidity of Trachinotus ovatus every day, and count the mortality rate.

[0140] The infection symptoms of RGNNV are one or more of the following: weakened swimming ability, loss of balance, and blackening of body color.

[0141] The infection symptoms of Vibrio rotiferianus are one or more of the following: weakened swimming ability, loss of balance, fin congestion, and skin ulceration.

[0142] II. Experimental results

[0143] As Figure 4 shown, within 60 h after injecting 100 μL of the original RGNNV solution with a concentration of 3.33×10 6 TCID 50 into Trachinotus ovatus with a body length of 5.0±1.0 cm, typical RGNNV infection symptoms appeared, the mortality rate was 0%, and the mortality rate was 10% at 72 h.

[0144] As Figure 5 shown, within 60 h after injecting 100 μL of the original Vibrio rotiferianus solution with a concentration of 1×10 7 cfu / mL into Trachinotus ovatus with a body length of 5.0±1.0 cm, the swimming ability was significantly weakened, and the mortality rate was 0%.

[0145] The above results show that the infection concentration of RGNNV is 3.33×10 6 TCID 50 , and the infection concentration of Vibrio rotiferianus is 1×10 7 cfu / mL, which can not only cause typical morbidity symptoms in Trachinotus ovatus but also be suitable for sample collection at different infection time points within 60 h. The above concentrations are suitable as the infection concentrations in the construction of the mixed infection model.

[0146] Example 6 Construction of a Trachinotus ovatus model with mixed infection of RGNNV and Vibrio rotiferianus

[0147] I. Experimental method

[0148] (1) Domestication of Trachinotus ovatus

[0149] The Trachinotus ovatus were temporarily cultured in a seawater circulation system at 25-30 °C for two weeks. The seawater salinity was about 30‰, the pH was about 8, and the dissolved oxygen content was above 5.6 mg / L.

[0150] After the temporary culture, 50 Trachinotus ovatus with a body length of 5.0±1.0 cm were randomly divided into 5 groups, with 10 fish in each group.

[0151] (2) Infection experiment

[0152] The 5 groups of Trachinotus ovatus were intraperitoneally injected according to the information shown in Table 3.

[0153] Table 3 Mixed infection of Trachinotus ovatus with RGNNV and Vibrio rotiferianus

[0154]

[0155] They were continuously cultured in a seawater circulation system at 25-30 °C, and the mortality rate was counted 14 days after infection.

[0156] (3) Sample collection and preservation

[0157] Taking the time of the second injection in the infection experiment as 0 h of infection, the status of each group of Trachinotus ovatus was observed every 6 h. Samples of four tissues, namely the brain, eye, liver, and spleen, of each group of Trachinotus ovatus were collected at 48 h after infection. Three fish were collected from each group each time and stored in liquid nitrogen for later use.

[0158] (4) Detection of pathogen replication

[0159] RNA was extracted from the samples of the four tissues collected in the previous step and reverse-transcribed into cDNA.

[0160] Using the cDNA of the samples as a template, the upstream quantitative primer CP-RT-F (SEQ ID No.5) and downstream quantitative primer CP-RT-R (SEQ ID No.6) for detecting RGNNV in Example 1, and the upstream quantitative primer VR-RT-F (SEQ ID No.11) and downstream quantitative primer VR-RT-R (SEQ ID No.12) for detecting Vibrio rotiferianus in Example 2 were used for qRT-PCR detection.

[0161] The qRT-PCR reaction system (10 μL) was: 2×SYBR Green Realtime PCR Mix, 5 μL; upstream quantitative primer, 0.3 μL; downstream quantitative primer, 0.3 μL; cDNA, 1 μL; ddH2O, 3.4 μL. The qRT-PCR reaction conditions were: reaction at 95 °C for 1 min; reaction at 95 °C for 5 s, annealing at 60 °C for 15 s, extension at 72 °C for 45 s, and repeated for 30 cycles.

[0162] Using the RGNNV detection standard curve constructed in Example 1 and the Vibrio rotiferianus detection standard curve constructed in Example 2, calculate the copy number of pathogens in the sample.

[0163] (5) Pathological detection

[0164] Perform HE staining and pathological observation on the tissues of the brain, eyes, liver, and spleen of the groupers in each group collected 48 h after infection.

[0165] II. Experimental results

[0166] Continuously observe the groupers in each group and find that the infection symptoms of the single Vibrio infection group are: weakened swimming ability, fin congestion, and caudal fin ulceration; the infection symptoms of the single virus infection group are: lying on the side or swimming in circles, and the body color turns black; the infection symptoms of the mixed infection group 1 are: lying on the side or swimming in circles, significantly decreased swimming ability, black body and rotten tail phenomena, and a small number of deaths within 36 h after infection; the infection symptoms of the mixed infection group 2 are: darker body color, spinning on the water surface, occasionally swimming wildly, and the number of deaths within 36 h after infection is more than that of the mixed infection group 1. It shows that the mixed infection mode has stronger pathogenicity than the single infection mode.

[0167] The statistical results of the mortality rate 14 days after infection show that the survival rates of each group are as follows: the mortality rate of the control group is 4%; the mortality rate of the single virus infection group is 20%; the mortality rate of the single Vibrio infection group is 4%; the mortality rate of the mixed infection group 1 is 12%; the mortality rate of the mixed infection group 2 is 32%. It shows that mixed infection will cause stronger lethality than single infection, and when mixed infected, the lethality of being infected with RGNNV first and then Vibrio rotiferianus is stronger.

[0168] As Figure 6As shown, at 48 h post-infection, the replication levels of RGNNV in the brain, eyes, liver, and spleen of the co-infection group 1 (Vibrio rotiferianus + NNV) (27,438,270.55 copies / μL; 2,991,854.923 copies / μL; 7,694.923655 copies / μL; 24,356.54015 copies / μL) were all lower than those in the single-virus infection group (RGNNV) (148,058,586.5 copies / μL; 10,157,066.14 copies / μL; 3,538,723.187 copies / μL; 3,724,993.605 copies / μL). However, the replication levels of the RGNNV virus in the brain, eyes, liver, and spleen of the co-infection group 2 (NNV + Vibrio rotiferianus) (940,407,295.4 copies / μL; 111,946,522.2 copies / μL; 25,820.82438 copies / μL; 3,946,695.084 copies / μL) were significantly higher than those in the single-virus infection group (RGNNV) (148,058,586.5 copies / μL; 10,157,066.14 copies / μL; 3,538,723.187 copies / μL; 3,724,993.605 copies / μL) and the co-infection group 1 (27,438,270.55 copies / μL; 2,991,854.923 copies / μL; 7,694.923655 copies / μL; 24,356.54015 copies / μL). This indicates that the RGNNV infection level in the co-infection group 2 was the highest.

[0169] As Figure 7As shown, at 48 h post-infection, the replication levels of Vibrio rotiferianus in the brain, eyes, liver, and spleen of the co-infection group 1 (Vibrio rotiferianus + NNV) (18,530.54337 copies / μL; 39,786.99961 copies / μL; 84,909.97128 copies / μL; 14,862.94926 copies / μL) were all higher than those in the single-bacterial infection group (Vibrio rotiferianus) (9,206.090329 copies / μL; 16,106.71867 copies / μL; 21,819.3549 copies / μL; 7,420.351859 copies / μL), while the replication levels of Vibrio rotiferianus in the brain, eyes, liver, and spleen of the co-infection group 2 (NNV + Vibrio rotiferianus) (48,955.73948 copies / μL; 136,962.2036 copies / μL; 135,298.4831 copies / μL; 41,738.78444 copies / μL) were significantly higher than those in the single-bacterial infection group (Vibrio rotiferianus) (9,206.090329 copies / μL; 16,106.71867 copies / μL; 21,819.3549 copies / μL; 7,420.351859 copies / μL) and the co-infection group 1 (18,530.54337 copies / μL; 39,786.99961 copies / μL; 84,909.97128 copies / μL; 14,862.94926 copies / μL) (18,530.54337 copies / μL; 39,786.99961 copies / μL; 84,909.97128 copies / μL; 14,862.94926 copies / μL). This indicates that the Vibrio rotiferianus infection level in the co-infection group 2 was the highest.

[0170] As Figure 8As shown, in the brain and eyes, compared with the control group (PBS), there were no obvious changes in the Vibrio anguillarum single-infection group (VR); in the RGNNV single-infection group, phenomena such as tissue loosening and vacuolization occurred; although obvious tissue lesions such as blurred tissue contours and tissue looseness also appeared in the mixed-infection group 1 (VR+RGNNV), the degree of its lesions did not show an obvious aggravation compared with the RGNNV single-infection group (RGNNV) and the Vibrio anguillarum single-infection group (VR); while in the mixed-infection group 2 (RGNNV+VR), compared with the RGNNV single-infection group, its lesions were more severe, vacuolization was more obvious, and necrosis occurred in the brain tissue. In the liver and spleen, compared with the control group (PBS), the cell arrangement in the Vibrio anguillarum single-infection group (VR) was irregular, the cell contours were not obvious, and a small amount of vacuoles were produced; in the RGNNV single-infection group, nuclear condensation, blurred tissue contours, and obvious vacuolization occurred; although obvious tissue lesions such as blurred tissue contours and tissue looseness also appeared in the mixed-infection group 1 (VR+RGNNV), the degree of its lesions did not show an obvious aggravation compared with the RGNNV single-infection group (RGNNV) and the Vibrio anguillarum single-infection group (VR); while the lesions in the mixed-infection group 2 (RGNNV+VR) were the most severe, with blurred tissue contours, tissue looseness, irregular cell morphology, and obvious vacuolization.

[0171] The above results indicate that the condition of the mixed-infection group 2 is the most serious, the infection levels of RGNNV and Vibrio rotiferianus are the highest, showing typical symptoms of dual infection of RGNNV and Vibrio rotiferianus, and the model of Trachinotus ovatus with mixed infection of RGNNV and Vibrio rotiferianus is successfully constructed.

[0172] Example 7 Pathogenicity of RGNNV and Vibrio harveyi to Epinephelus coioides of different body sizes

[0173] I. Experimental methods

[0174] (1) Domestication of Epinephelus coioides

[0175] Epinephelus coioides were temporarily cultured in a seawater circulation system at 25-30 °C for two weeks. The seawater salinity was about 30‰, the pH was about 8, and the dissolved oxygen was above 5.6 mg / L.

[0176] After the temporary culture, 30 Epinephelus coioides with a body length of 4.0±1.0 cm were randomly divided into 3 groups, with 10 fish in each group; another 30 Epinephelus coioides with a body length of 8.0±1.0 cm were randomly divided into 3 groups, with 10 fish in each group.

[0177] (2) Infection experiment

[0178] Six groups of Epinephelus coioides were intraperitoneally injected according to the information shown in Table 4.

[0179] Table 4 RGNNV and Vibrio harveyi infections in Epinephelus coioides of different body sizes

[0180]

[0181] After injection, the fish were continuously reared in a seawater circulation system at 25 - 30 °C for one week, and the morbidity of Epinephelus coioides was observed daily and the mortality rate was counted.

[0182] The symptoms of RGNNV infection were: weakened swimming ability, blackened body color, and circular swimming.

[0183] The symptoms of Vibrio harveyi infection were: weakened swimming ability, fin congestion, lying on the bottom, immobility, and skin ulceration.

[0184] II. Experimental Results

[0185] The results showed that for Epinephelus coioides fry with a body size of 4.0 ± 1.0 cm, after injection with the original RGNNV solution at a concentration of 1×10 7 TCID 50 (i.e., the R - 4 experimental group) and the original Vibrio harveyi solution at a concentration of 1×10 9 cfu / mL (i.e., the V - 4 experimental group), the mortality rate was 100% after 60 h; for Epinephelus coioides fry with a body size of 8.0 ± 1.0 cm, after injection of 100 μL of the original RGNNV solution at a concentration of 1×10 7 TCID 50 (i.e., the R - 8 experimental group), the mortality rate was 50% after 60 h and obvious RGNNV infection symptoms appeared, and after injection of 100 μL of the original Vibrio harveyi solution at a concentration of 1×10 9 cfu / mL (i.e., the V - 8 experimental group), the mortality rate was 100% after 60 h and obvious Vibrio harveyi infection symptoms appeared.

[0186] In summary, for Epinephelus coioides fry with a body size of 4.0 ± 1.0 cm, within 60 h of high - concentration infection with the two pathogens, they have weak tolerance to the two pathogens and are not suitable for constructing a multi - pathogen mixed - infection model; while for Epinephelus coioides fry with a body size of 8.0 ± 1.0 cm, obvious infection symptoms appear within 60 h of high - concentration infection with the two pathogens, and there is a high lethality rate. They are more sensitive to virus infection and are more suitable for constructing a multi - pathogen mixed - infection model.

[0187] Example 8 Pathogenicity of Different Concentrations of RGNNV and Vibrio harveyi to Epinephelus coioides

[0188] I. Experimental Method

[0189] (1) Domestication of Epinephelus coioides

[0190] The Epinephelus coioides were temporarily cultured in a seawater circulation system at 25-30 °C for two weeks. The seawater salinity was about 30‰, the pH was about 8, and the dissolved oxygen content was above 5.6 mg / L.

[0191] After the temporary culture, 110 Epinephelus coioides with a body length of 8.0 ± 1.0 cm were randomly divided into 11 groups, with 10 fish in each group.

[0192] (2) Infection experiment

[0193] The 11 groups of Epinephelus coioides were intraperitoneally injected according to the information shown in Table 5.

[0194] Table 5 Infection of Epinephelus coioides with different concentrations of RGNNV and Vibrio harveyi

[0195]

[0196] They were continuously cultured in a seawater circulation system at 25-30 °C for one week. The morbidity of the Epinephelus coioides was observed every day, and the mortality rate was counted.

[0197] The infection symptoms of RGNNV were: weakened swimming ability, blackened body color, and swimming in circles.

[0198] The infection symptoms of Vibrio harveyi were: weakened swimming ability, fin congestion, lying on the bottom, immobility, and skin ulceration.

[0199] II. Experimental results

[0200] As Figure 9 shown, within 60 h after injecting 100 μL of the original RGNNV solution with a concentration of 2×10 6 TCID 50 into the Epinephelus coioides with a body length of 8.0 ± 1.0 cm, typical RGNNV infection symptoms appeared, and the mortality rate was 0%. At 72 h, the mortality rate was 10%.

[0201] As Figure 10 shown, within 60 h after injecting 100 μL of the original Vibrio harveyi solution with a concentration of 1×10 6 cfu / mL into the Epinephelus coioides with a body length of 8.0 ± 1.0 cm, the swimming ability was significantly weakened, and the mortality rate was 0%.

[0202] The above results indicate that the infection concentration of RGNNV is 2×10 6 TCID 50 , and the infection concentration of Vibrio harveyi is 1×10 6 cfu / mL, which can not only cause typical morbidity symptoms in Epinephelus coioides but also be suitable for sample collection at different infection time points within 60 h. The above concentrations are suitable as the infection concentrations for constructing a mixed infection model.

[0203] Construction of an Aquatic Animal Model with Mixed Infection of RGNNV and Vibrio harveyi

[0204] I. Experimental Methods

[0205] (1) Domestication of Epinephelus coioides

[0206] The Epinephelus coioides were temporarily cultured in a seawater circulation system at 25 - 30°C for two weeks. The seawater salinity was about 30‰, the pH was about 8, and the dissolved oxygen content was above 5.6 mg / L.

[0207] After the temporary culture, 50 Epinephelus coioides with a body length of 8.0 ± 1.0 cm were randomly divided into 5 groups, with 10 fish in each group.

[0208] (2) Infection Experiment

[0209] The 5 groups of Epinephelus coioides were intraperitoneally injected according to the information shown in Table 6.

[0210] Table 6 Mixed Infection of Epinephelus coioides with RGNNV and Vibrio harveyi

[0211]

[0212] They were continuously cultured in a seawater circulation system at 25 - 30°C, and the mortality rate was counted 14 days after infection.

[0213] (3) Sample Collection and Preservation

[0214] Taking the time of the second injection in the infection experiment as the 0 h of infection, the status of each group of Epinephelus coioides was observed every 6 h. Samples of four tissues, namely the brain, eye, liver, and spleen of each group of Epinephelus coioides, were collected at 48 h after infection. Three fish were collected from each group each time and stored in liquid nitrogen for later use.

[0215] (4) Detection of Pathogen Replication

[0216] RNA was extracted from the samples of the four tissues collected in the previous step and reverse transcribed into cDNA.

[0217] Using the cDNA of the samples as a template, qRT-PCR detection was performed with the upstream quantitative primer CP-RT-F (SEQ ID No.5) and downstream quantitative primer CP-RT-R (SEQ ID No.6) for detecting RGNNV in Example 1, and the upstream quantitative primer VHRT-F (SEQ ID No.17) and downstream quantitative primer VHRT-R (SEQ ID No.18) for detecting Vibrio harveyi in Example 3.

[0218] The qRT-PCR reaction system (10 μL) is as follows: 2×SYBR Green Realtime PCR Mix, 5 μL; upstream quantitative primer, 0.3 μL; downstream quantitative primer, 0.3 μL; cDNA, 1 μL; ddH2O, 3.4 μL. The qRT-PCR reaction conditions are: reaction at 95°C for 1 min; reaction at 95°C for 5 s, annealing at 60°C for 15 s, extension at 72°C for 45 s, repeated for 30 cycles.

[0219] Using the RGNNV detection standard curve constructed in Example 1 and the Vibrio harveyi detection standard curve constructed in Example 3, calculate the copy number of pathogens in the sample.

[0220] (5) Pathological detection

[0221] Perform HE staining and pathological observation on the tissues of the brain, eyes, liver, and spleen of each group of orange-spotted groupers collected 48 h after infection.

[0222] II. Experimental results

[0223] Continuously observing each group of orange-spotted groupers, it was found that the infection symptoms of the single Vibrio infection group were: weakened swimming ability, fin congestion, lying on the bottom, immobility, and skin ulceration; the infection symptoms of the single virus infection group were: weakened swimming ability, blackening of body color, and circular swimming; the infection symptoms of the co-infection group 1 were: weakened swimming ability, blackening of body color, and fin congestion; the infection symptoms of the co-infection group 2 were: weakened swimming ability, fin congestion, lying on the bottom, immobility, and skin ulceration. It shows that the co-infection mode has stronger pathogenicity than the single infection mode.

[0224] The statistical results of the mortality rate 14 days after infection showed that the mortality rate of the control group was 0%; the mortality rate of the single virus infection group was 0%; the mortality rate of the single Vibrio infection group was 3%; the mortality rate of the co-infection group 1 was 10%, and the mortality rate of the co-infection group 2 was 10%. It shows that the co-infection mode has stronger lethality than the single infection mode.

[0225] As Figure 11 shown, there were no obvious disease symptoms in the internal organs of the orange-spotted groupers in the control group (PBS), the spleen in the single Vibrio infection group (Vibrio harveyi) was significantly enlarged, there were no obvious disease symptoms in the internal organs of the single virus infection group (RGNNV), and liver enlargement and congestion were found in both the co-infection group 1 (RGNNV + V. harveyi) and the co-infection group 2 (V. harveyi + RGNNV), and a large amount of ascites was deposited.

[0226] As Figure 12As shown, at 24 h post-infection, the RGNNV replication levels in the brain, eye, liver, and spleen of co-infection group 1 (Vibrio harveyi + NNV) ((2,104,706.75 copies / μL; 76,384.42 copies / μL; 704.55 copies / μL; 33,349.19 copies / μL) and those in the brain, eye, liver, and spleen of co-infection group 2 (RGNNV + Vibrio harveyi) (18,815,318.95 copies / μL; 84,193.66 copies / μL; 361.08 copies / μL; 24,848.58 copies / μL) were significantly higher than those in the single-virus infection group (RGNNV) (327,340.14 copies / μL; 359.42 copies / μL; 259.96 copies / μL; 16,746.06 copies / μL).

[0227] As Figure 13 shown, at 24 h post-infection, the RGNNV replication levels in the brain, eye, liver, and spleen of co-infection group 1 (Vibrio harveyi + NNV) (415.59 copies / μL; 373.13 copies / μL; 920.46 copies / μL; 308.23 copies / μL) and the Vibrio harveyi replication levels in the brain, eye, liver, and spleen of co-infection group 2 (RGNNV + Vibrio harveyi) (303.35 copies / μL; 285.83 copies / μL; 658.92 copies / μL; 286.99 copies / μL) were significantly higher than those in the single Vibrio infection group (Vibrio harveyi) (143.31 copies / μL; 175.98 copies / μL; 385.17 copies / μL; 166.75 copies / μL).

[0228] As Figure 14As shown, in the brain and eyes, compared with the control group (PBS), the pathological vacuolization in the co-infection group 1 (RGNNV+V. harveyi) and co-infection group 2 (V. harveyi+RGNNV) was more severe than that in the single Vibrio infection group (V. harveyi) and single virus infection group (RGNNV), and cell shedding was obvious. In the liver, hyaline degeneration in the co-infection group 1 (RGNNV+V. harveyi) and co-infection group 2 (V. harveyi+RGNNV) was obvious and higher than that in the single Vibrio infection group (V. harveyi) and single virus infection group (RGNNV); in the spleen, the congestion, hemorrhage and inflammatory reaction in the co-infection group 1 (RGNNV+V. harveyi) were stronger than those in the single virus infection group (RGNNV), and the congestion, hemorrhage and cell death in the co-infection group 2 (V. harveyi+RGNNV) were more than those in the co-infection group 1 (RGNNV+V. harveyi). It is indicated that both co-infection modes can cause more severe histopathological changes compared with single pathogen infection.

[0229] The above results show that both co-infection group 1 and co-infection group 2 presented typical symptoms of dual infection with RGNNV and V. harveyi, the infection levels of RGNNV and V. harveyi were both high, and the model of orange-spotted grouper with co-infection of RGNNV and V. harveyi was successfully constructed.

[0230] The above results show that the model of orange-spotted grouper with co-infection of RGNNV and V. harveyi was successfully constructed.

[0231] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description and ideas. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for constructing a mixed-infected aquatic animal model, characterized in that, Use 3.0×10 6 TCID 50 ~3.5×10 6 TCID 50 Epinephelus akaara nervous necrosis virus and 0.8×10 7 cfu / mL~1.2×10 7 cfu / mL Vibrio rotiferianus to infect Trachinotus ovatus with a body length of 4 cm - 6 cm until infection symptoms appear, thus obtaining a mixed-infected aquatic animal model; the infection symptoms include one or more of weakened swimming ability, loss of balance, fin congestion, swimming sideways, belly turning, body color darkening, and tail rot.

2. The method according to claim 1, wherein Use 3.33×10 6 TCID 50 Epinephelus akaara nervous necrosis virus and 1×10 7 cfu / mL Vibrio rotiferii to infect the Trachinotus ovatus until infection symptoms appear.

3. The method according to claim 2, wherein First, inject the Trachinotus ovatus with 3.33×10 6 TCID 50 nervous necrosis virus of Epinephelus akaara. After 12 h, inject the Trachinotus ovatus with 1×10 7 cfu / mL Vibrio rotiferianus. Infection symptoms appear after 48 h.

4. A method for constructing a mixed-infected aquatic animal model, characterized in that, Use 1.8×10 6 TCID 50 ~2.2×10 6 TCID 50 Epinephelus akaara nervous necrosis virus and 0.8×10 6 cfu / mL~1.2×10 6 cfu / mL Vibrio harveyi are used to infect Epinephelus coioides with a body length of 7 cm - 9 cm until infection symptoms appear, thus obtaining a mixed-infected aquatic animal model; the infection symptoms include one or more of weakened swimming ability, loss of balance, fin congestion, swimming sideways, belly-up, body color darkening, and tail rot.

5. The method according to claim 4, wherein Use 2×10 6 TCID 50 of nervous necrosis virus of Epinephelus akaara and 1×10 6 cfu / mL of Vibrio harveyi to infect the orange-spotted grouper until infection symptoms appear.

6. Use of the mixed-infected aquatic animal model constructed by the method according to any one of claims 1 to 5 in evaluating a method for preventing and treating diseases in aquaculture.

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