In-vitro culture method of shrimp enterospora hepatopenaei

Abstract

The invention belongs to the technical field of aquatic pathogenic microorganism products, and particularly relates to an in-vitro culture method of enterospora hepatopenaei. The method comprises the following steps: obtaining purified spores of enterospora hepatopenaei; the method comprises the following steps: inoculating purified spores of enterospora hepatopenaei into a host cell being cultured for infection to obtain a host cell sample infected with enterospora hepatopenaei; and detecting the proliferation and maturation of the purified spores of the enterospora hepatopenaei in the host cell sample infected with the enterospora hepatopenaei by using a fluorescent staining method. The method provided by the invention provides a key technical platform for basic research, pathogenic mechanism analysis, drug screening and vaccine development of the shrimp enterospora hepatopenaei, has the advantages of convenience in acquisition of experimental materials, simplicity and convenience in operation, high repeatability and the like, and is suitable for in-vitro culture and proliferation of the pathogen in a laboratory.

Description

Technical Field

[0001] This invention belongs to the field of aquatic pathogenic microorganism products technology, specifically relating to an in vitro culture method for shrimp hepatic enterospora. Background Technology

[0002] Microsporidia are obligate intracellular parasites, with approximately 220 genera and over 1700 species reported globally, and new species are discovered every year. Microsporidia have an extremely wide host range and distribution, infecting almost all invertebrates and vertebrates, such as humans, rabbits, silkworms, bees, and crabs. Some microsporidia are important pathogens in humans, livestock, aquaculture, and economically important insects like silkworms and bees, causing serious losses to human health and economic development. However, to date, there are no effective means to control microsporidiasis.

[0003] Enterobius vermicularis, belonging to the same genus as Enterobius picori, which causes fatal diarrhea in humans, can infect major farmed shrimp species such as Litopenaeus vannamei. It can be directly transmitted through water, causing hepatopancreatic microsporidiasis in shrimp. Infected shrimp may still eat, but their growth is stunted, their immunity is weakened, and they are susceptible to infections by other pathogens such as Vibrio, resulting in significant economic losses for the shrimp farming industry.

[0004] Animal cell culture plays a crucial role in the study of human and animal pathogenic microorganisms. In recent years, research on the in vitro culture of microsporidia that cause human diseases has gradually increased, and culture systems for several species have been successfully established. However, compared with insect and mammalian microsporidia, progress in the in vitro culture of aquatic microsporidia remains relatively slow. Several studies have attempted to culture fish microsporidia in primary cells and cell lines. However, research on *Enterocera hepatispora* from shrimp remains limited, and it is currently impossible to observe its continuous proliferation and life cycle under in vitro conditions, thus greatly restricting the progress of research on its pathogenic mechanisms, drug screening, and vaccine development. Summary of the Invention

[0005] To address the problem that existing technologies cannot achieve continuous proliferation and life cycle observation of *Hepatocystis suis* under in vitro conditions, thus greatly limiting research on its pathogenic mechanism, drug screening, and vaccine development, this invention provides an in vitro culture method for *Hepatocystis suis*. To achieve the above objectives, this invention adopts the following technical solution.

[0006] This invention provides a method for in vitro culture of shrimp hepatic enterospora, comprising the following steps: Purified spores of shrimp hepatocystis were obtained.

[0007] The purified spores of the shrimp hepatic enterospora were inoculated into cultured host cells to obtain host cell samples infected with shrimp hepatic enterospora.

[0008] The proliferation and maturation of purified spores of *Hepatopanthera spp.* in host cell samples infected with *Hepatopanthera spp.* were detected using a fluorescence staining method.

[0009] The fluorescent staining method includes the following steps: The host cell samples infected with *Hepatocystis hepatis* were stained with fluorescent dyes, and the stained host cells were collected to observe whether mature spores formed within the stained host cells. The fluorescent dyes included DY96 and DAPI. DY96 can bind to chitin in the spore wall, thereby marking mature spores. DAPI can bind to the nuclear DNA of the host cell or mature spores, thereby marking the nucleus.

[0010] This invention provides a stable, reproducible, and highly sensitive in vitro culture method for *Enterocera hepatispora*. The method first obtains purified spores of *Enterocera hepatispora*, ensuring their purity and activity, effectively prolonging their survival and infectivity in vitro, thus solving the problem of maintaining microsporidia activity in existing technologies. Then, the purified spores are inoculated into cultured host cells for infection, obtaining host cell samples infected with *Enterocera hepatispora*. After inoculating the purified spores into the host cells, fluorescent staining is used to detect spore development within the cells, enabling continuous development and observation of the complete life cycle in the in vitro environment, overcoming the technical difficulty of tracking the life cycle of microsporidia in existing technologies. Specifically, DY96 dye combined with spore wall chitin is used to specifically label mature spores, while DAPI dye combined with DNA is used to label both the host cell nucleus and the spore cell nucleus, forming a dual fluorescent labeling system, significantly improving the clarity and reliability of developmental stage differentiation. This combined staining technique has high sensitivity and specificity, overcoming the problems of unclear labeling and large observational bias in traditional staining methods. In summary, this invention enables the continuous proliferation and complete life cycle development of shrimp hepatocystis under in vitro conditions, which can then be used for research on its pathogenic mechanisms, drug screening, and vaccine development, breaking through the technical bottlenecks that limit related research in the prior art.

[0011] Furthermore, the in vitro culture method for shrimp hepatocysts provided by this invention has been repeatedly verified and has good stability, making it suitable for different batches of spore samples.

[0012] Preferably, the purified spores of *Hepatospora sinensis* are mature microsporidian spores isolated from the hepatopancreatic tissue of shrimp infected with *Hepatospora sinensis*. The obtained purified spores of *Hepatospora sinensis* have high purity and strong viability, enabling them to efficiently infect host cells and complete their life cycle, thus ensuring the reliability and reproducibility of in vitro culture experiments.

[0013] In some embodiments of the present invention, the method for obtaining the mature microsporidia spores includes the following steps: Sequential sucrose solutions with weight-to-volume ratios of 90%, 60%, and 30% were prepared and sterilized. Simultaneously, grinding pestles, centrifuge tubes, and other equipment were also sterilized. Hepatopancreatic tissue from shrimp was dissected and homogenized with PBS solution, then filtered to obtain the stock solution. The solution underwent multi-stage differential centrifugation pretreatment, including centrifugation at 8000×g / 5min, discarding the supernatant, resuspending in 30mL PBS solution, centrifuging at 1000×g / 5min to collect the supernatant, and then concentrating at 8000×g / 5min. This washing process was repeated twice to obtain 2mL of concentrated suspension. A sucrose density gradient system was then constructed, and the sample suspension was stacked on top of the gradient solution. The solution was then ultracentrifuged at 40000×g / 4℃ for 40min to achieve stratification of the parasites. The target layer solution was collected, rapidly centrifuged at 8000×g / 1min, and purified by four washes with PBS solution. Finally, the purified product was diluted and microscopically counted in a clean bench.

[0014] Preferably, the staining time is 10 minutes.

[0015] Preferably, the method for inoculating purified spores of *Hepatocystis suis* into cultured host cells for infection includes the following steps: When the host cells being cultured reach a confluence of 80%, the purified spores of the shrimp hepatic enterospora are inoculated into the host cells being cultured and co-cultured.

[0016] The conditions for co-cultivation were 37℃, 5% CO2, and 95% humidity.

[0017] Preferably, the inoculation amount of purified spores of *Enterocera hepatica* is 10 per well. 6 In one preferred embodiment of the present invention, the culture vessel used for culturing the purified spores of the shrimp hepatocystis is a standard 24-well plate.

[0018] Preferably, the host cell is a rabbit kidney cell line. In a preferred embodiment of the present invention, shrimp hepatocystis and rabbit kidney cells are used as research objects. The rabbit kidney cell line is used as the host cell, and this cell line has been proven to support the proliferation of various microsporidia, exhibiting good versatility and stability. By selecting this cell line, the infection efficiency and in vitro proliferation capacity of shrimp hepatocystis can be significantly improved, thereby enabling the continuous development and complete observation of its life cycle process in the in vitro environment, solving the technical problem of difficulty in tracking the life cycle of microsporidia in the prior art. More preferably, the rabbit kidney cell line is RK13 cells.

[0019] Preferably, the host cell sample infected with *Enterocera hepatica* is a cell sample harvested 4 hours post-infection. That is, cell samples are collected 4 hours after infection and stained with DY96 and DAPI dyes. The specific steps are as follows:

[0020] (1) Cell fixation and permeabilization: Wash cells twice with PBS solution to remove culture medium. Add 4% paraformaldehyde (w / v) to fix at room temperature for 15 minutes. Wash three times with PBS solution for 5 minutes each time. Add 500 μL of 0.1% Triton X-100 (w / v) to treat for 1 hour. Wash three times with PBS solution for 5 minutes each time.

[0021] (2) DY96 staining: Under light-protected conditions, add 100 μL of diluted DY96 and treat at room temperature for 10 minutes. Wash three times with PBS solution for 5 minutes each time to remove unbound dye.

[0022] (3) DAPI counterstaining: Add 100 μL of DAPI working solution and incubate at room temperature in the dark for 10 minutes. Wash three times with PBS solution for 5 minutes each time.

[0023] (4) Mounting and observation: Add 5 μL of anti-quenching mounting medium to cover the coverslip, avoiding air bubbles. Apply nail polish to the edge of the coverslip to fix it in place. After air drying, observe under a fluorescence microscope.

[0024] Preferably, before staining the host cell samples infected with *Enterocera hepatispora* using fluorescent dye, the host cell samples are first washed with PBS solution, then fixed with 4% paraformaldehyde (w / v) for 15 minutes to obtain fixed host cells. The fixed host cells are then washed with PBS solution before staining. This process effectively maintains the integrity of the cell structure and preserves key spore components, improving staining quality and image clarity. This facilitates direct observation of the spore state within the host cells, overcoming problems such as uneven staining, fragile cell morphology, and difficulty in observation in existing technologies. It provides technical support for the accurate analysis of the in vitro proliferation process of microsporidia.

[0025] Preferably, the PBS solution has a concentration of 0.01M and a pH of 7.3.

[0026] In some embodiments of the present invention, host cell samples infected with *Enterocera hepatica* are collected at different time points after infection, total DNA is extracted using a marine animal tissue genomic DNA extraction kit, and the genome copy number of *Enterocera hepatica* in the samples is determined using qPCR.

[0027] The different time points are 1h, 3h, 5h, 7h, 9h and 12h.

[0028] Marine animal tissue genomic DNA extraction kit: TIANGEN.

[0029] For total DNA extraction, 0.1 mm silica beads were used for disruption. The disruption conditions were: shaking for 30 seconds every 5 minutes, repeated 5 times. Silica beads: Biospec, 11079101z.

[0030] The standard plasmid used to determine the genome copy number of *Hepatopanthera philoxeroides* in shrimp using qPCR was pET32-18s.

[0031] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides an in vitro culture method for *Enterocera hepatis*. This invention provides a stable, reproducible, and highly sensitive in vitro culture method for *Enterocera hepatis*. The method provided by this invention first obtains purified spores of *Enterocera hepatis* to ensure their purity and activity, effectively prolonging their survival and infectivity in vitro, thus solving the problem of maintaining microsporidia activity in existing technologies. Then, the purified spores of *Enterocera hepatis* are inoculated into cultured host cells for infection, obtaining host cell samples infected with *Enterocera hepatis*. By inoculating the purified spores of *Enterocera hepatis* into host cells, the development of the spores in the infected host cells is detected using fluorescence staining. This allows for continuous development and observation of the complete life cycle in the in vitro environment, solving the technical problem of difficulty in tracking the life cycle of microsporidia in existing technologies. This invention employs a dual fluorescent labeling system, utilizing DY96 dye combined with spore wall chitin to specifically label mature spores, while simultaneously using DAPI dye combined with DNA to label both host cell nuclei and spore cell nuclei. This significantly improves the clarity and reliability of distinguishing developmental stages. This combined staining technique exhibits high sensitivity and specificity, overcoming the problems of unclear labeling and large observational biases inherent in traditional staining methods. In summary, this invention enables the continuous proliferation and complete life cycle development of *Hepatocystis hepatis* under in vitro conditions, which can then be used for research on its pathogenic mechanisms, drug screening, and vaccine development. The in vitro culture method for *Hepatocystis hepatis* provided by this invention effectively overcomes the difficulties in achieving continuous proliferation and life cycle observation under in vitro conditions in existing technologies, breaking through the technical bottlenecks limiting research on its pathogenic mechanisms, drug screening, and vaccine development.

[0032] 2. The method provided by this invention can also be used for morphological observation, molecular biology research and drug intervention experiments of shrimp hepatic enterospora. It has good stability and reproducibility, and can provide a reliable experimental platform for subsequent research on pathogenic mechanisms, screening of new antimicrosporidian drugs and discovery of vaccine targets. Attached Figure Description

[0033] Figure 1These are images of the proliferation of *Enterocera hepatispora* in rabbit kidney cells, as described in this invention; wherein, Figure 1 Figure A in the figure is the standard curve of standard plasmid pET32a-18s-qPCR; Figure 1 Figure B in the figure shows the proliferation curve of mature spores of *Enterocera hepatispora* in rabbit kidney cells.

[0034] Figure 2 This is a stained image of rabbit kidney cells 4 hours after infection with *Enterocera hepatisporum*, as described in this invention; wherein, Figure 2 Image A in the image is a DY96 channel image; Figure 2 Image B in the image is the DAPI channel image; Figure 2 In the image, C represents the overlay of the DY96 channel and the DAPI channel. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0036] Example 1: Construction of standard plasmids 1. Materials and Methods 1.1 Sample The shrimp infected with enterospira spp. came from Guangzhou Double Helix Gene Technology Co., Ltd.

[0037] 1.2 Main Reagents 2×Prime STAR Max: TaKaRa; Agarose: Solarbio; 1×TAE buffer: Solarbio; Nucleic acid dye: Invitrogen; DNA Marker: TaKaRa; DNA loading buffer: Solarbio; EZNACycle-PureKit: Omega; ClonExpress II One Step Cloning Kit: Nanjing Novizan; Ampicillin: Solarbio; LB medium: Solarbio; Plasmid mini-extraction kit: TIANGEN.

[0038] 1.3. Bacterial strains and plasmids The pET32-a vector was purchased from Dalian Takara Bio Inc.; E. coli competent cells DH5α were preserved in our laboratory.

[0039] 1.4 Primer Synthesis The primers, including the upstream and downstream primers, were synthesized by Genewiz Biotechnology Co., Ltd.

[0040] The upstream primer, also known as Fw, is 18S-F. The nucleotide sequence of 18S-F is shown in SEQ ID NO.1:

[0041] 5'-gctgatatcggatccgaattcTCTATGCGGGAGATAACCACG-3'.

[0042] The downstream primer, also known as RV, is 18S-R. The nucleotide sequence of 18S-R is shown in SEQ ID NO.2:

[0043] 5'-ttgtcgacggagctcgaattcTTTACAAAAGCTAATCCTACTCATCTCA-3'.

[0044] 1.5 Sample Processing Hepatocellular and pancreatic tissues were collected under sterile conditions in a laminar flow hood, ground with liquid nitrogen, resuspended in 4 times the volume of PBS solution, and then filtered through a nylon membrane to obtain a tissue suspension.

[0045] The hepatopancreatic tissue comes from shrimp infected with enterocytozoonosis.

[0046] The PBS solution was purchased from Solarbio Science & Technology Co., Ltd., with a concentration of 0.01M and a pH of 7.3.

[0047] 1.6 DNA Extraction Total DNA was extracted according to the instructions for the Marine Animal Tissue Genomic DNA Extraction Kit. The Marine Animal Tissue Genomic DNA Extraction Kit used was TIANGEN. The specific method is as follows:

[0048] (1) Centrifuge the tissue suspension at 12,000 rpm for 5 minutes, take the precipitate, add 400 μL of GA buffer to resuspend, shake for 30 seconds every 5 minutes, and repeat 5 times.

[0049] (2) Add 20 μL of Proteinase K solution with a concentration of 20 mg / mL, vortex to mix, and centrifuge briefly to remove water droplets from the inner wall of the tube cap. Incubate in a water bath at 56°C for 2 hours until the tissue is completely dissolved.

[0050] (3) Add 200 μL of buffer GB, mix thoroughly by inverting, and incubate at 70°C for 10 min.

[0051] (4) Add 200 μL of anhydrous ethanol and mix thoroughly by inverting.

[0052] (5) Add the solution and flocculent precipitate obtained in the previous step into the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

[0053] (6) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0054] (7) Add 600 μL of washing solution PW to the adsorption column CB3, centrifuge at 2,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0055] (8) Repeat step (7) once.

[0056] (9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for 5 minutes to thoroughly dry any residual rinsing liquid in the adsorption material.

[0057] (10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50 μL of elution buffer TE to the middle of the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0058] (11) Store the eluted DNA at -20°C.

[0059] 1.7 Amplification of the target gene PCR amplification was performed using a reaction system consisting of 25 µL of 2×Master Mix, 2 µL each of 10 pmol / µL upstream and downstream primers, 5 µL of template DNA, and nuclease-free water to a final volume of 50 µL. Reaction conditions are shown in Table 1. PCR products were subjected to 1.5% agarose gel electrophoresis and stained with nucleic acid dyes. Electrophoresis was performed at 150 V for 30 min, using standard molecular weight controls. Results were processed using a gel imaging system.

[0060] Table 1. Reaction conditions and reaction system for high-fidelity enzyme PCR in this experiment. .

[0061] Note: Fw is the upstream primer, and RV is the downstream primer.

[0062] 1.8 Purification and gel recovery of PCR amplification products The PCR products obtained above were purified using a gel extraction and purification kit, following the instructions. The procedure is as follows:

[0063] (1) Transfer the gel to the UV lamp and cut out the desired target band.

[0064] (2) Weigh the centrifuge tube, then transfer the gel block containing the target fragment to a 1.5 mL EP tube, weigh it again to obtain the total weight, and calculate the gel weight and volume. Add the same volume of XP2 Binding Buffer as the gel, place it in a 60℃ water bath and heat until the gel melts, vortexing every 3 minutes. The target fragment is the 18S gene amplification product.

[0065] (3) Insert the HiBind® DNA Mini binding column into the 2mL collection tube.

[0066] (4) Transfer the molten DNA melt to the HiBind® DNA Mini binding column, centrifuge at 10,000×g for 1 min at room temperature, discard the filtrate, and put the binding column back into the 2mL collection tube.

[0067] (5) Add 300µL Binding Buffer to the binding column, centrifuge at room temperature for 13,000×g for 1min, and discard the filtrate.

[0068] (6) Insert the binding column into a 2 mL collection tube, add 700 µL SPW Buffer, centrifuge at 10,000 × g for 1 min at room temperature, and discard the filtrate.

[0069] (7) Put the binding column back into the 2mL collection tube, centrifuge at 13,000×g for 2min at room temperature, and dry the remaining liquid in the binding column.

[0070] (8) Place the binding column in a new 1.5 mL EP tube, add 30 µL of preheated Elution Buffer to the matrix of the binding column, let stand at room temperature for 1 min, centrifuge at 13,000 × g for 1 min, and elute the DNA.

[0071] 1.9 Single enzyme digestion of plasmid pET-32a(+) The pET-32a(+) plasmid was extracted according to the plasmid extraction instructions of the small plasmid extraction kit, and an endonuclease was used. EcoR I was subjected to single enzyme digestion, and the enzyme digestion reaction system is shown in Table 2: Table 2 Enzyme digestion reaction system .

[0072] Mix thoroughly and incubate at 37°C for 2 hours. Add 2 μL of 10× loading buffer to the enzyme digestion product and recover the target band using 1% agarose gel electrophoresis.

[0073] 1.10 Recombination reaction The target gene product and the recovered linearized vector pET-32a(+) were ligated using a one-step cloning kit. The reaction system is shown in Table 3. The ligation product was transformed according to the instructions for use with DH5α competent cells. The cloning kit used was the ClonExpress MultiS One Step Cloning Kit.

[0074] Table 3 Recombination Reaction System .

[0075] 1.11. Screening and Sequencing of Positive Clones The colony PCR reaction system and reaction conditions are shown in Table 1. Then, the positive strains were sent for sequencing. The correctly sequenced strains were amplified and cultured, and the plasmids were extracted for later use, resulting in the recombinant plasmid pET-32a-18s.

[0076] Example 2: Purification of shrimp hepatic enterospora, obtaining purified spores of shrimp hepatic enterospora. 1. Preparations: Solution preparation: Prepare sucrose solutions with mass / volume percentage concentrations of 90%, 60%, and 30% in 15 mL centrifuge tubes and autoclave them.

[0077] Equipment preparation: grinding pestle, grinding bowl, 15mL centrifuge tubes, 50mL centrifuge tubes, PBS solution, scissors, tweezers and nylon cloth. All the above equipment shall be sterilized by high temperature and high pressure.

[0078] 2. Dissect the diseased shrimp, remove their hepatopancreas tissue in a mortar, add PBS solution, filter through nylon cloth, and collect the filtrate in a 50mL centrifuge tube for later use. The diseased shrimp refer to those infected with enterocytozoonosis, sourced from Guangzhou Double Helix Gene Technology Co., Ltd.

[0079] 3. Centrifuge the solution at 8000×g and 4℃ for 5 min, and discard the supernatant.

[0080] 4. Add 30 mL of PBS solution to the precipitate, vortex, and centrifuge at 1000 × g and 4 °C for 5 min.

[0081] 5. Collect the supernatant solution into a new 50mL centrifuge tube, centrifuge at 8000×g and 4℃ for 5min, and discard the supernatant solution.

[0082] 6. Repeat step 4 once.

[0083] 7. Collect the supernatant solution into a new 50mL centrifuge tube and centrifuge at 8000×g and 4℃ for 5min. Discard the supernatant solution, suspend the precipitate in 2mL of PBS solution to obtain a microspore suspension after washing with PBS solution, and store at 4℃ for later use.

[0084] 8. Slowly add 2 mL of 90% sucrose solution, 2 mL of 60% sucrose solution, 2 mL of 30% sucrose solution, and 2 mL of shrimp hepatic enterospora stock solution to the centrifuge tube in sequence, adhering to the wall. Centrifuge at 40,000 × g and 4 °C for 40 min using an ultra-high-speed refrigerated centrifuge to achieve microspore separation.

[0085] 9. Collect the solution between each layer, centrifuge at 8000×g and 4℃ for 1 min, discard the supernatant, resuspend in 1 mL of PBS solution, and then centrifuge at 8000×g and 4℃ for 2 min to obtain purified shrimp hepatic enterospora.

[0086] 10. Repeat step 9 four times. Dilute and count the purified shrimp hepatic enterospora in a clean bench, and store at 4°C for later use.

[0087] Example 3: In vitro culture of shrimp hepatic enterospora. Purified spores of *Enterobacter hepatisporus* were inoculated into a cultured rabbit kidney cell line to obtain host cell samples infected with *Enterobacter hepatisporus*. Fluorescent staining was used to detect the proliferation and maturation of purified spores in the infected host cell samples.

[0088] The fluorescent staining method includes the following steps: Host cell samples infected with *Hepatocystis hepatica* were stained with fluorescent dyes. The stained host cells were collected, and the formation of mature spores within the stained cells was observed. The fluorescent dyes included DY96 and DAPI. Details are as follows:

[0089] 1. Materials and Methods 1.1 Cell lines and culture conditions Rabbit kidney cell lines were cultured in modified Leibovitz L-15 medium supplemented with 10% fetal bovine serum, 100 IU / mL penicillin, and 0.1 mg / mL streptomycin at 37°C. Unless otherwise specified, all cell cultures were performed in 24-well plates.

[0090] The rabbit kidney cell line is RK13, derived from a laboratory cryopreservation of aquatic parasites and aquatic parasitic diseases. The applicant holds the cells and guarantees to make them available to the public for 20 years from the date of application.

[0091] Among them, the full name of RK13 cells is Rabbit Kidney-13.

[0092] 1.2 Main Reagents Trypsin, modified Leibovitz L-15 medium, fetal bovine serum, penicillin, and streptomycin were all purchased from Gibco.

[0093] 1.3 Inoculation with shrimp hepatic enterospora. (1) Cell inoculation: One day before the experiment, the required amount of rabbit kidney cell suspension was inoculated into 24-well plates at a volume of 500 μL per well. When the host cells reached 80% confluence, purified spores of shrimp hepatic enterospora were inoculated into the host cells and co-cultured.

[0094] The conditions for co-cultivation were 37℃, 5% CO2, and 95% humidity.

[0095] (2) Microsporidia inoculation: Add 500 μL of diluted microsporidia suspension to each well of the cells cultured overnight in a 24-well plate, ensuring that the number of microsporidia in each well is 10. 6 They were then placed in an incubator at 37°C and cultured for different durations.

[0096] The method for obtaining the microsporidian fluid is the same as the purification method for shrimp hepatospira.

[0097] Overnight culture refers to a culture time of ≥12 hours.

[0098] (3) Time point sampling: Set time gradients: 1 hour, 3 hours, 5 hours, 7 hours, 9 hours and 12 hours, and take 3 wells at each time point. Discard the culture medium and wash twice with PBS solution. Add 200 μL of trypsin solution, lay the 24-well plate flat, ensuring that the liquid covers the cell layer, and let it stand for digestion for 2 minutes. Add 500 μL of DMEM complete culture medium, gently pipette to form a single-cell suspension, collect it into a centrifuge tube, and use it for microsporidia DNA extraction.

[0099] The pancreatic enzyme solution had a mass / volume percentage concentration of 25% and was purchased from Shandong Sikejie Biotechnology Co., Ltd.

[0100] 1.4 Establishment of the absolute quantitative standard curve In the LightCycler 96 real-time PCR detection system, quantitative real-time PCR was performed in 96-well plates using ChamQ Universal SYBR qPCRMaster Mix. The primers were qPCR-18s-F and qPCR-18s-R, and the DNA template was the standard plasmid pET-32a-18s constructed in Example 1. The PCR amplification reaction system and reaction conditions are shown in Tables 4 and 5, respectively.

[0101] The nucleotide sequence of qPCR-18s-F is shown in SEQ ID NO.3: 5'-AAAGTGATTAGACACCGCTGTAGTT-3'.

[0102] The nucleotide sequence of qPCR-18S-R is shown in SEQ ID NO.4: 5'-TACGATAGACTTGACCCTGGTAAGT-3'.

[0103] Table 4 Absolute Quantitative PCR Reaction System .

[0104] Table 5. Absolute Quantitative PCR Reaction Conditions .

[0105] 1.5 Determination of growth curve of shrimp hepatic enterospora. The qPCR reaction system and conditions are as described above. The genome copy number of *Enterobacter hepatisporus* in host cells infected with *Enterobacter hepatisporus* was calculated. Host cells infected with *Enterobacter hepatisporus* are also known as *Enterobacter hepatisporus*-infected rabbit kidney cells.

[0106] 1.6 Staining Experiment (1) Cell fixation and permeabilization: Rabbit kidney cells infected with shrimp enterocystis hepatis were gently washed twice with PBS solution to remove the culture medium. 4% paraformaldehyde was added for fixation at room temperature for 15 minutes. The cells were washed three times with PBS solution for 5 minutes each time. 500 μL of Triton X-100 (0.1% v / v) was added for treatment for 1 hour, followed by three more washes with PBS solution for 5 minutes each time.

[0107] (2) DY96 staining: Under light-protected conditions, add 100 μL of diluted DY96 staining solution and incubate at room temperature for 10 minutes. Wash three times with PBS solution for 5 minutes each time to remove unbound dye. The preparation method of DY96 staining solution is as follows:

[0108] 10 mg of DY96 powder was dissolved in 2 mL of DMSO solution to prepare a 5 mM stock solution for the first staining solution. This stock solution was then wrapped in aluminum foil and stored at -20°C to protect it from light. Before staining, the stock solution was diluted 1000 times in PBS to prepare a final DY96 staining solution with a concentration of 5 μM. The DY96 powder was purchased from Beyotime Biotechnology Co., Ltd.

[0109] (3) DAPI counterstaining: Add 100 μL of DAPI working solution with a concentration of 10 μg / mL and incubate at room temperature in the dark for 10 minutes. After treatment, wash three times with PBS solution for 5 minutes each time. The DAPI working solution was purchased from Solarbio.

[0110] (4) Mounting and observation: Add 5 μL of anti-quenching mounting medium to cover the coverslip, avoiding air bubbles. Fix the coverslip by applying nail polish to the edge and observe under a fluorescence microscope.

[0111] 2. Results and Analysis 2.1 Proliferation of Shrimp Hepatic Enterobacteriaceae in Rabbit Kidney Cells Quantitative analysis was performed on rabbit kidney cells infected with *Enterocera hepatica* to detect the genome copy number of *Enterocera hepatica* over a 12-hour time span. A standard curve constructed using reference samples showed a strong linear correlation between the Ct value and the logarithm of the template number. Figure 1 As shown in Figure A. The results showed that *Enterocystis hepatica* proliferated rapidly after infecting rabbit kidney cells, and its genome copy number increased approximately fourfold within 12 hours. See Figure A for details. Figure 1 Figure B in the diagram.

[0112] 2.2 Maturation of Shrimp Hepatocystis in Rabbit Kidney Cells DY96 staining solution can bind to chitin in the spore wall, thereby marking mature spores. The results of infecting rabbit kidney cells with mature spores of *Enterocera hepatica* are shown below. Figure 2 As shown. By Figure 2 The results showed that mature spores labeled with DY96 dye were visible inside the cells 4 hours after infection. This indicates that *Enterocera hepatispora* can not only infect rabbit kidney cells, but also proliferate within the cells and produce mature spores.

[0113] The above results indicate that *Hepatocystis suis* can rapidly infect rabbit kidney cells and proliferate stably within the cells, thus achieving the first in vitro culture of *Hepatocystis suis*.

[0114] In summary, shrimp hepatic enterospora can achieve rapid infection, stable proliferation, and formation of mature spores in rabbit kidney cells. This establishes an in vitro culture method for the first time and verifies the feasibility and effectiveness of the method.

[0115] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0116] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A method for in vitro culture of shrimp hepatic enterospora, characterized in that, Includes the following steps: Purified spores of shrimp hepatospira were obtained; The purified spores of the shrimp hepatic enterospora were inoculated into cultured host cells to obtain host cell samples infected with shrimp hepatic enterospora. The proliferation and maturation of purified spores of *Hepatocystis suis* in host cell samples infected with *Hepatocystis suis* were detected using a fluorescence staining method. The fluorescent staining method includes the following steps: The host cell samples infected with *Hepatopanthera philoxeroides* were stained with fluorescent dyes, and the stained host cells were collected to observe whether mature spores were formed in the stained host cells. The fluorescent dyes included DY96 and DAPI.

2. The in vitro culture method for shrimp hepatic enterospora according to claim 1, characterized in that, The purified spores of *Hepatocystis* were isolated from the hepatopancreatic tissue of shrimp infected with *Hepatocystis* disease.

3. The in vitro culture method for shrimp hepatic enterospora according to claim 1, characterized in that, The staining time is 10 minutes.

4. The in vitro culture method for shrimp hepatic enterospora according to claim 1, characterized in that, The method for inoculating purified spores of the shrimp hepatocystis into cultured host cells for infection includes the following steps: When the host cells being cultured reach a confluence of 80%, the purified spores of the shrimp hepatic enterospora are inoculated into the host cells being cultured and co-cultured. The conditions for co-cultivation were 37℃, 5% CO2, and 95% humidity.

5. The in vitro culture method for shrimp hepatic enterospora according to claim 4, characterized in that, The inoculation amount of purified spores of *Hepatocystis suis* was 10 per well. 6 indivual.

6. The in vitro culture method for shrimp hepatic enterospora according to claim 4, characterized in that, The host cell is a rabbit kidney cell line.

7. The in vitro culture method for shrimp hepatic enterospora according to claim 1, characterized in that, The host cell samples infected with Enterobacter hepatisporum were cell samples harvested 4 hours after infection.

8. The in vitro culture method for shrimp hepatic enterospora according to claim 1, characterized in that, Before staining the host cell samples infected with Enterobius hepatis in shrimp with fluorescent dye, the host cell samples were first washed with PBS solution, and fixed with 4% paraformaldehyde for 15 minutes to obtain fixed host cells. The fixed host cells were then washed with PBS solution before staining.

9. The in vitro culture method for shrimp hepatic enterospora according to claim 8, characterized in that, The PBS solution had a concentration of 0.01 M and a pH of 7.3.

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