Method for constructing micropterus salmoides rhabdovirus activity system under water temperature cooperative control
By constructing a water temperature-coordinated largemouth bass rogue virus activity system and using a low-rate heating method, the difficulties existing in the actual application of existing prevention and control methods are solved, and the effect of inhibiting virus outbreaks and reducing fry mortality is achieved.
Patent Information
- Application Number
- CN202510168791.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-17
- Publication Date
- 2025-05-30
AI Technical Summary
The existing largemouth bass rhizovirus prevention and control methods have difficulties in actual application, including small specifications of fish fry that are not suitable for injection or oral immunity, incomplete development of the fry immune system, high production costs of subunit vaccines or DNA vaccines, and difficult to effectively add drug prevention and control to powder materials.
By constructing a water temperature-coordinated largemouth bass radix virus activity system, we explore the lethality rate of viruses under different living environment conditions, and use a low-rate heating method to inhibit the outbreak of largemouth bass fry and reduce the mortality rate of fry when the largemouth bass fry is out.
It has achieved the inhibition of the outbreak of rhodvirus disease during the production process and reduced the mortality rate of fry, which is operable, convenient and practical.
Smart Images
Figure CN120060162A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of prevention and control of Micropterus salmoides rhabdovirus, and particularly to a method for constructing a system for synergistically controlling the activity of Micropterus salmoides rhabdovirus with water temperature. Background Art
[0002] Micropterus salmoides, commonly known as the California bass, is a eurythermal fish species. It is characterized by fast growth, no intermuscular spines, and delicious meat, and is an important freshwater aquaculture variety in China. In recent years, the aquaculture of Micropterus salmoides has been growing steadily, and the total output has increased from 3.53 million tons in 2015 to 8.88 million tons in 2023. The aquaculture areas are mainly distributed in Guangdong, Zhejiang, Hubei and other places. However, with the expansion of the aquaculture scale, the disease problems of Micropterus salmoides have become increasingly prominent. In recent years, the prevalence trends of viral and bacterial diseases have gradually increased. Among them, rhabdovirus disease, iridovirus disease, Nocardia disease, etc. have seriously endangered the healthy development of the Micropterus salmoides aquaculture industry.
[0003] Among them, Micropterus salmoides rhabdovirus (MSRV) is a single-stranded RNA virus. This virus is characterized by strong infectivity and high lethality. The epidemic water temperature under natural conditions is 25 - 28 °C, and the mortality rate of infected fry is as high as 60% - 90%. In addition, another main characteristic of this virus is that it only infects fry with a length of 2 - 5 cm, and no deaths caused by this disease have been found in juvenile fish and adult fish under natural conditions (high-dose laboratory infections can cause death).
[0004] Regarding the diseases caused by this virus, the prevention and control methods reported so far include vaccines and drugs. Some research reports have shown that subunit vaccines or DNA vaccines can effectively reduce the mortality rate after rhabdovirus infection and improve the survival rate after being soaked, injected or orally gavaged. On the other hand, some research has found that adding arctigenin to the feed or directly injecting drugs (8-hydroxyquinoline, Magnolol derivatives, ribavirin) into the body can effectively improve the survival rate of bass after virus infection.
[0005] However, there are some problems with these methods in practical applications. Bass is a carnivorous fish, and during the fry cultivation process, it needs to go through different feeding conversions such as artemia - powder feed - pellet feed. The stage prone to rhabdovirus disease is the cultivation stage of artemia - powder feed. First of all, the fry are small in size at this stage and are not suitable for injection or oral immunization. In addition, the immune system of the fry is not fully developed, and the production cost of subunit vaccines or DNA vaccines is high, and they cannot be used in actual production before obtaining the new veterinary drug certificate recognized by the state. Secondly, the method of drug prevention and control can only be added to the powder feed, and the diameter of the powder feed is as small as 1 mm. Without a suitable preparation process, the liquid medicine cannot be directly sprayed onto the feed and mixed evenly like pellet feed. Therefore, these two methods are not feasible in practical applications and can only stay at the research level. Summary of the Invention
[0006] The present invention provides a method for constructing a temperature - coordinated control system for the activity of largemouth bass rhabdovirus. This construction method explores the lethality of largemouth bass rhabdovirus under different living environment conditions, and obtains the best prevention and control system for largemouth bass rhabdovirus disease, so as to be applied to the cultivation of largemouth bass, thereby improving the survival rate of largemouth bass.
[0007] The object of the present invention is achieved through the following technical solutions: A method for constructing a temperature - coordinated control system for the activity of largemouth bass rhabdovirus, comprising the following steps: S01. Isolate largemouth bass rhabdovirus from diseased largemouth bass, infect grass carp ovary cells, and culture them in M199 medium at a temperature of 25°C. S02. When the cytopathic effect reaches 50%, lyse the infected cells by freeze - thawing, and centrifuge at 3000g for 30 minutes at 4°C; collect the supernatant containing virus particles and store it at - 80°C for virus challenge experiments. S03. Disinfect the pond water and centrifuge for 10 minutes to obtain disinfected pond water with suspended organic matter removed. S04. Inoculate largemouth bass rhabdovirus into 250 mL of pond water, stir evenly, incubate at a temperature of 10°C - 30°C, and perform virus enrichment and infectivity determination at 0, 1, 3, 5, 7, 10, and 14 days after inoculation.
[0008] Preferably, in step S04, the temperature of the pond water is selected as 30°C.
[0009] Preferably, in step S04, the heating rate is 0.5°C / h.
[0010] Preferably, the composition of the M199 medium is: Medium 199 basal medium containing 10% fetal bovine serum.
[0011] Preferably, in step S04, the virus enrichment and infectivity determination steps are as shown in S05 - S07: S05. Pass the above water sample through a negatively charged filter membrane with a pore size of 0.45 µm, filter it, cut the filter membrane into small pieces of 0.3 to 0.5 cm, and then put them into a 50 - mL centrifuge tube pre - filled with 5 mL of 3% beef extract and 6 - 8 zirconium beads, and vortex - shake to mix to obtain a supernatant; S06. Transfer the supernatant to a centrifuge tube containing 5 mL of 16% PEG8000, mix well, incubate at 4°C for 2 hours, and centrifuge at 13000 rpm for 5 min at 4°C, discard the supernatant; resuspend the precipitate with 300 µL of 0.2 mol / L Na 2 HPO 4 Collect the supernatant after centrifugation to obtain a virus - enriched solution; S07. Dilute the above virus - enriched solution 10 - fold and inoculate it into grass carp ovary cells, and calculate the median tissue culture infective dose.
[0012] Preferably, in step S06, the centrifugation conditions are: 13000 rpm, 5 min, 4°C.
[0013] Compared with the prior art, the advantages or beneficial effects of the technical solution of the present application include: The present invention discovers that by the way of low - rate temperature rise, when epizootic rhabdovirus disease breaks out in largemouth bass fry, it can inhibit the outbreak of rhabdovirus disease and reduce the fry mortality rate, and has operability, convenience and practicability in the production process. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 : Virus copy numbers of MSRV after incubation at water temperatures of 10°C, 20°C and 30°C for different times; Figure 2 : Virulence attenuation curves of MSRV after incubation at water temperatures of 10°C, 20°C and 30°C for different times; Figure 3 : Representative pictures of GCO cells infected by MSRV after incubation at water temperatures of 10°C, 20°C and 30°C for different times; Figure 4 : Cumulative mortality rates of largemouth bass after challenge with MSRV at different temperatures; Figure 5 : Histopathological changes of largemouth bass after MSRV infection analyzed by hematoxylin - eosin staining (A - C) and Prussian blue staining (D - F); Figure 6 : Cumulative mortality rates of largemouth bass in the control group and the experimental group; Figure 7 : Viral loads in the livers (A), spleens (B), and kidneys (C) of largemouth bass in the control group and the experimental group; Figure 8 : Activities of MDA (A), GSH-PX (B), SOD (C), CAT (D), LZM (E), and AKP (F) in the livers of largemouth bass in the control group; Figure 9 : Activities of MDA (A), GSH-PX (B), SOD (C), CAT (D), LZM (E), and AKP (F) in the livers of largemouth bass in the experimental group. Detailed implementation manners
[0015] The following will describe in detail the implementation manners of the present application in conjunction with the drawings and embodiments, so as to fully understand how the present application uses technical means to solve technical problems and the implementation process of achieving corresponding technical effects and implement accordingly. The embodiments of the present application and each feature in the embodiments can be combined with each other on the premise of not conflicting, and the formed technical solutions are all within the protection scope of the present application.
[0016] It should be clear that the following described embodiments are only a part of the embodiments of the present application, rather than all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative efforts belong to the protection scope of the present application. Embodiment
[0017] This embodiment provides a method for constructing a temperature-controlled largemouth bass rhabdovirus activity system, and the method includes the following steps: S01. Isolate largemouth bass rhabdovirus from diseased largemouth bass, infect grass carp ovarian cells, and culture them in M199 medium under the temperature condition of 25°C.
[0018] S02. After the cytopathic effect forms to 50%, disrupt the infected cells by freeze-thawing, and centrifuge at 3000g for 30 minutes at 4°C; collect the supernatant containing virus particles and store it at -80°C for the challenge experiment.
[0019] S03. Disinfect the pond water and centrifuge for 10 minutes to obtain disinfected pond water with suspended organic matter removed.
[0020] S04. Inoculate largemouth bass rhabdovirus into 250 mL of pond water, stir evenly, incubate under the temperature condition of 10 - 30°C, and perform virus enrichment and infectivity determination at 0, 1, 3, 5, 7, 10, and 14 days after inoculation. Among them, the basic water temperature is 25°C, and the heating rate is 0.5°C / h.
[0021] In this study, compared with 10 °C and 20 °C, the viral copy number at 30 °C decreased significantly, indicating that the persistence of MRSV was negatively correlated with water temperature. The infectivity of MSRV at 20 °C was higher than that at 10 °C and 30 °C, which might be related to the fact that the epidemic water temperature of MSRV was 20 °C - 25 °C.
[0022] S05. Filter the above water sample through a negatively charged filter membrane with a pore size of 0.45 µm. After filtration, cut the filter membrane into small pieces of 0.3 to 0.5 cm, and put them into a 50 mL centrifuge tube pre-filled with 5 mL of 3% beef extract and 6 - 8 zirconium beads, and vortex and mix to obtain the supernatant.
[0023] Filtration through a 0.45 µm negatively charged filter membrane enriches MSRV in water. Filter membranes are commonly used to capture viruses in water and are used for the isolation of infectious salmon anemia virus (ISAV), salmon alphavirus (SAV), and TiLV.
[0024] S06. Transfer the supernatant to a centrifuge tube containing 5 mL of 16% PEG8000, mix well, incubate at 4 °C for 2 hours, and centrifuge at 13000 rpm for 5 min at 4 °C, then discard the supernatant; resuspend the precipitate with 300 µL of 0.2 mol / L Na 2 HPO 4 Collect the supernatant by centrifugation to obtain the virus enrichment solution. Among them, the centrifugation conditions are: 13000 rpm, 5 min, 4 °C.
[0025] S07. Dilute the above virus enrichment solution 10 - fold and inoculate it into grass carp ovary cells, and calculate the median tissue culture infective dose.
[0026] The composition of the above M199 medium is: Medium 199 basal medium containing 10% fetal bovine serum.
[0027] A total of 720 largemouth bass were evenly divided into 12 groups, with 3 replicates in each group (20 fish / replicate), and they were reared at 15 °C, 20 °C, 25 °C, and 30 °C for 15 days respectively. Anesthetize the fish with MS - 222 and intraperitoneally inject MSRV (8.5×10 5 copies / mL and 8.5×10 4 copies / mL) or PBS (control group), and the injection dose is 0.1 mL / fish. After injection, transfer the fish to the corresponding rearing temperature of 15 °C, 20 °C, 25 °C, or 30 °C, and observe continuously for 14 days. Observe and remove the dead fish every day, and take liver, spleen, and kidney samples to detect MSRV.
[0028] Three hundred and sixty Micropterus salmoides that had been acclimated for 15 days at 25 °C were evenly divided into six groups, with three replicates in each group (20 fish per replicate). The fish were anesthetized with MS-222 and intraperitoneally injected with MSRV (three experimental groups) or PBS (three control groups) at a dose of 0.1 mL per fish. In experimental group 1 (T1) and control group 1 (C1), the fish were transferred to aquariums with a constant temperature of 25 °C ± 0.5 °C; in experimental group 2 (T2) and control group 2 (C2), the fish were transferred to aquariums with a slowly rising water temperature, from 25 °C to 30 °C at a rate of 0.5 °C / h; in experimental group 3 (T3) and control group 3 (C3), the fish were transferred to aquariums with a rapidly rising water temperature, from 25 °C to 30 °C at a rate of 2 °C / h. Under sterile conditions, at 2.5, 10, 24, 48, 72, and 120 hours post-challenge (hpc), three fish from each group were sampled for their livers (divided into two parts), spleens, and kidneys, and immediately used for determining virus loads. In addition, the second liver sample was used for enzyme activity determination. Brain, liver, spleen, and kidney samples from randomly selected dead fish were subjected to histopathological analysis.
[0029] Conclusion: 1. Virus loads of MSRV in water after incubation at different temperatures The virus in pond water was concentrated through a filter membrane, and RT-qPCR was used to quantitatively detect the virus copy number. The virus copy number in water at 10 °C decreased by 5.899 log within 11 days 10 , the virus copy number at 20 °C decreased by 5.397 log within 7 days 10 , and the virus copy number at 30 °C decreased significantly by 4.62 log within 1 day 10 . At 10 °C, 20 °C, and 30 °C, the virus copy number in pond water decreased below the detection threshold within 11 days, 7 days, and 3 days, respectively.
[0030] 2. Infectivity of MSRV in water after incubation at different temperatures The infectivity of the virus in pond water decreased the fastest at 30 °C, and the TCID 50 of the virus decreased by 4.025 log within 1 day 10 , and the virus lost its infectivity. At 10 °C, the TCID 50 of the virus decreased by 50% within 1 day, and at 20 °C, the TCID 50 of the virus decreased by 50% within 3 days, and the virus lost its infectivity within 5 days ( Figure 2 ). The morphological changes of infected GCO cells were as shown in Figure 3 , including cell vacuolization, syncytium formation, clumping, and cell detachment, etc.
[0031] 3. Resistance of Micropterus salmoides to MSRV at different water temperatures Fish died within 5 days after virus challenge. When challenged with low concentration (8.5×10 4 copies / mL), the mortality rates of bass at 15℃, 20℃, 25℃, and 30℃ were 56.67%, 73.33%, 60%, and 5% respectively ( Figure 4 A). When challenged with high concentration (8.5×10 5 copies / mL), the mortality rates of bass at 15℃, 20℃, 25℃, and 30℃ reached 80%, 100%, 100%, and 40% respectively ( Figure 4 B). No death occurred in the control group (injected with PBS). The results showed that the mortality rate of largemouth bass infected at 30℃ was significantly lower than that at the other three temperatures.
[0032] 4. Histopathological analysis Results of hematoxylin-eosin (HE) staining and Prussian blue (PB) staining showed that a large amount of hemosiderin appeared in the liver ( Figure 5 A) and spleen ( Figure 5 B), and cell necrosis, infiltration of inflammatory cells, fibrous hyperplasia, and granulomas were found in the kidney ( Figure 5 C).
[0033] 5. Water temperature regulation test Results showed that no fish death and clinical symptoms were found in groups C1, C2, and C3. The cumulative mortality rates of groups T1, T2, and T3 were 59.52%, 14.28%, and 88.09% respectively ( Figure 6 ). Detection of viral loads in the liver ( Figure 7 A), spleen ( Figure 7 B), and kidney ( Figure 7 C) by RT-qPCR found that the viral loads in the liver, spleen, and kidney of group T2 were the lowest compared with those of groups T1 and T3.
[0034] 6. Enzyme activities in liver tissue The activities of CAT ( Figure 8 D) and AKP ( Figure 8 F) in group C3 were significantly higher than those in group C2 at 10 - 24 h after infection (p<0.05); the activities of SOD ( Figure 8 C) and LZM ( Figure 8 E) were significantly higher than those in group C2 at 2.5 - 10 h after infection (p<0.05). The SOD activity in group T2 was significantly higher than those in groups T1 and T3 at 48 - 72 h after infection (p<0.05) ( Figure 9 C). The CAT activity in group T2 was significantly higher than those in groups T1 and T3 at 24 - 72 h after infection ( Figure 9 D).
[0035] High water temperature (30 °C) can reduce the infectivity of MSRV and increase the resistance of largemouth bass to MSRV. Increasing the water temperature at a low rate (0.5 °C / h) can significantly reduce mortality by decreasing the viral load in the liver, spleen, and kidney, while increasing the SOD and CAT activities in the liver. Increasing the temperature at a low rate is an effective strategy for preventing the outbreak of MSRV disease. These findings can improve the biosecurity of largemouth bass farms to control the outbreak of MSRV disease.
Claims
1. A method for constructing a water temperature-controlled largemouth bass rhabdovirus activity system, characterized in that: The steps include: S01, isolating the largemouth bass rhabdovirus from the infected largemouth bass, infecting the grass carp ovary cells, and culturing them in M199 medium at 25°C; S02. When the cytopathic effect reached 50%, the infected cells were destroyed by freeze-thaw and centrifuged at 3000 g for 30 min at 4°C; the supernatant containing viral particles was collected and stored at -80°C for the challenge experiment; S03, disinfecting the pond water and centrifuging for 10 minutes to obtain the pond water that has been disinfected and has had suspended organic matter removed; S04. Inoculate the largemouth bass rhabdovirus into 250 mL of pond water, stir evenly, incubate at 10℃-30℃, and perform virus enrichment and infectivity assays at 0, 1, 3, 5, 7, 10 and 14 days after inoculation.
2. The construction method according to claim 1, characterized in that: In step S04, the pond water temperature is selected to be 30°C.
3. The construction method according to claim 2, characterized in that: In step S04, the heating rate is 0.5°C / h.
4. The construction method according to claim 1, characterized in that: The composition of M199 medium is: Medium 199 basal medium containing 10% fetal bovine serum.
5. The construction method according to claim 1, characterized in that: In step S04, virus enrichment and infectivity determination steps are shown in S05-S07: S05, filtering the water sample through a negatively charged filter membrane with a pore size of 0.45 µm, cutting the filter membrane into small pieces of 0.3 to 0.5 cm, placing the pieces into a 50-ml centrifuge tube pre-filled with 5 ml of 3% beef extract and 6-8 zirconium beads, vortexing and mixing to obtain a supernatant; S06. Transfer the supernatant to a centrifuge tube containing 5 mL 16% PEG8000, mix thoroughly, incubate at 4°C for 2 hours, centrifuge at 13000 rpm for 5 min at 4°C, discard the supernatant; resuspend the precipitate with 300 µL 0.2 mol / L Na2HPO4; collect the supernatant after centrifugation to obtain a virus enrichment solution; S07. Dilute the above virus enrichment solution 10 times and inoculate it into grass carp ovary cells, and calculate the half tissue cell infection dose.
6. The construction method according to claim 5, characterized in that: In step S06, the centrifugation conditions are: 13000 rpm, 5 min, 4°C.