In-vitro culture method of grouper enterospora

Abstract

The invention belongs to the technical field of aquatic disease prevention and control and parasitology research, and particularly relates to an in-vitro culture method of grouper enterosporidia, which comprises the following steps: obtaining purified spores of the grouper enterosporidia; and culturing the purified spores of the enterosporidia of the epinephelus coioides by utilizing a culture of an intestinal cell line ECGI-21 of the epinephelus coioides. The invention establishes an efficient and repeatable enterosporidium grouper in-vitro culture system, is suitable for fundamental research, pathogenic mechanism exploration, drug screening and vaccine development of the pathogen, and has the advantages of easily available experimental materials, simplicity and convenience in operation, high repeatability and the like.

Description

Technical Field

[0001] This invention belongs to the field of aquatic disease control and parasitology research technology, specifically relating to an in vitro culture method for Enterobacter spp. of grouper. Background Technology

[0002] Microsporidia are obligate intracellular parasites. Globally, approximately 220 genera and over 1700 species of microsporidia have been reported, with new species being reported 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, silkworms, and bees, causing serious losses to human health and economic development. However, to date, there are no effective means to control microsporidiasis.

[0003] Grouper is a commercially valuable marine species and has become an important pillar of the economy in the southern coastal areas of China. Its farming methods include pond culture, cage culture, and intensive farming. However, with the expansion of farming scale and the increase in intensification, the frequency of bacterial, viral, and parasitic diseases in grouper farming is constantly rising, causing huge economic losses. In recent years, microsporidiasis has become widespread in the grouper fry stage in Hainan Province, with a mortality rate reaching 100%. Microsporidia have become one of the main pathogenic factors of microsporidiasis. Despite the serious harm caused by this disease, the pathogenic mechanism of microsporidia in grouper remains unclear, and effective control measures are lacking.

[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 has been slower. Numerous studies have attempted to culture fish microsporidia in primary cells and cell lines. However, research on *Enterocera stylosa* remains relatively limited, especially as its continuous proliferation and life cycle observation cannot be achieved under in vitro conditions, significantly hindering the study of its pathogenic mechanisms, drug screening, and vaccine development. Therefore, there is an urgent need to establish an in vitro culture system for *Enterocera stylosa* to provide an experimental foundation for in-depth research. Summary of the Invention

[0005] This invention provides an in vitro culture method for grouper enterospora, which enables the grouper enterospora to be cultured and stably proliferated in exogenous cell lines.

[0006] The technical solution adopted in this invention is: This invention provides a method for in vitro culture of Enterobacteriaceae from grouper, comprising the following steps: To obtain purified spores of *Enterocera prolifera* from grouper; The purified spores of *Enterocera stylosaurus* were cultured using the *C. stylosaurus* intestinal cell line ECGI-21. The *C. stylosaurus* intestinal cell line ECGI-21, with accession number GDMCC No:62408, is deposited at the Guangdong Provincial Microbial Culture Collection Center. The *C. stylosaurus* intestinal cell line ECGI-21 used in this invention was donated by Zhou Sheng of South China Agricultural University.

[0007] Preferably, the process of culturing the purified spores of *Enterocera stylosaurus* using the *C. stylosaurus* intestinal cell line ECGI-21 is as follows: The ECGI-21 intestinal cell line of the oblique grouper was cultured in a culture medium to obtain the culture of the ECGI-21 intestinal cell line of the oblique grouper. The purified spores of *Enterocera stylosa* were diluted with culture medium and then inoculated into the culture of the *ECGI-21* intestinal cell line of *Odontocera stylosa*.

[0008] Preferably, the ECGI-21 intestinal cell line of the oblique grouper is cultured at 28°C in the absence of CO2 for 8-16 hours.

[0009] Preferably, the ratio of the number of purified spores of the grouper intestinal cell line ECGI-21 to that of grouper enterospora is 1:1.

[0010] Preferably, the culture medium is formulated as follows: Each milliliter of Leibovitz L-15 medium was supplemented with v / v 10% fetal bovine serum, 100 IU penicillin, 0.1 mg streptomycin, 0.05 mg 4-hydroxyethylpiperazine ethanesulfonic acid, and 2.66 mg NaCl.

[0011] Preferably, the method for obtaining purified spores of grouper enterospora is as follows: Intestinal tissue was collected from fish infected with grouper enterospora disease, ground, filtered and washed, and purified spores of grouper enterospora were obtained by ultracentrifugation using a sucrose density gradient.

[0012] Preferably, when using sucrose density gradient ultracentrifugation, the sucrose solution used has a mass fraction of 90%, 60%, and 30%.

[0013] Preferably, the conditions for sucrose density gradient ultracentrifugation are 40,000g, 4℃ for 40 min.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for in vitro culture of Enterobacteriaceae from grouper, comprising the following steps: Purified spores of *Enterocera dorsalis* were obtained. These purified spores were cultured using the *ECGI-21* intestinal cell line of *O. dorsalis* (GDMCC No. 62408), deposited at the Guangdong Provincial Microbial Culture Collection Center. This invention obtained a homogenate of *Enterocera dorsalis*-infected intestinal tissue through tissue processing, purified it, and then inoculated it into the *ECGI-21* intestinal cell line for co-culture. The mature spores of *Enterocera dorsalis* were then stained with staining solution, and their development and maturation within the cells were observed. Results showed that mature spores were produced in the host cells within 2 hours of infection. The genome copy number of *Enterocera dorsalis* was determined using qPCR, showing rapid genome replication within 4 hours of infection, indicating that *Enterocera dorsalis* can be cultured and stably proliferated in the *ECGI-21* intestinal cell line. The method of this invention provides a key technology platform for basic research, pathogenic mechanism analysis, drug screening and vaccine development of Enterobacter spp. in grouper. The method of this invention has the advantages of convenient acquisition of experimental materials, simple operation and strong reproducibility, and is suitable for in vitro culture and proliferation of this pathogen in the laboratory. Attached Figure Description

[0015] Figure 1 The proliferation of *Enterocera stylosa* in the intestinal cell line ECGI-21 of *Enterocera stylosa* is shown in Figure A, which is the standard curve result; Figure B is the proliferation curve of mature spores of *Enterocera stylosa* in the intestinal epithelial cells of *Enterocera stylosa*.

[0016] Figure 2 Images of *Enteromorpha spp.* in the intestinal cell line ECGI-21 of *Odontomorpha spp.* at different time points after infection: A: 2 h post-infection; B: 3 h post-infection; C: 4 h post-infection. Detailed Implementation

[0017] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0018] The inventive concept of this invention is as follows: This invention successfully reconstructed a microenvironment suitable for the infection, colonization, and proliferation of grouper enterospora in the cell nucleus using the ECGI-21 intestinal cell line derived from the grouper. This breakthrough overcomes the limitation that heterologous cell lines cannot support the completion of the grouper's life cycle and solves the problem that grouper enterospora cannot proliferate continuously in vitro. This provides technical support for establishing a stable in vitro culture system for grouper enterospora.

[0019] The ability of *Enterocera dorsalis* to infect, colonize, and proliferate in the ECGI-21 intestinal epithelial cell line derived from *Odontocera obliquee* is fundamentally due to the species-specific microenvironment highly congruent with that of the natural host. ECGI-21 cells retain intestinal epithelial-specific receptors, nuclear factors, and metabolic conditions, providing essential physiological support for the invasion, colonization, and sustained in vitro proliferation of *Enterocera dorsalis*. Compared to heterologous cell lines that lack key factors and thus cannot complete their life cycle, the species specificity and suitable cellular physiological state of ECGI-21 form the technological basis for a stable in vitro culture system.

[0020] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0021] Example 1 An in vitro culture method for Enterobacteriaceae of grouper is as follows: 1. Obtain purified spores of *Enterocera stylosa* from grouper.

[0022] 1) Preparations.

[0023] Solution preparation: Prepare sucrose solutions with concentrations of 90%, 60%, and 30% wt in 15 mL centrifuge tubes and autoclave them.

[0024] Equipment preparation: Prepare a grinding pestle, grinding bowl, 15mL centrifuge tubes, 50mL centrifuge tubes, PBS solution, scissors, tweezers, and nylon cloth. Perform high-temperature and high-pressure sterilization.

[0025] 2) Dissection of diseased fish infected with grouper enterospora: Take out the intestinal tissue of the diseased fish and place it in a mortar and pestle. Add PBS solution and grind it. After filtering through nylon cloth, place it in a 50mL centrifuge tube for later use.

[0026] The grouper infected with Enterobacter spp. was collected from a grouper farm in Wanning City, Hainan Province.

[0027] 3) Centrifuge the solution at 8000g and 4℃ for 5min, and discard the supernatant solution after centrifugation.

[0028] 4) Add 30 mL of PBS solution to the precipitate and vortex. Centrifuge at 1000 g and 4 °C for 5 min.

[0029] 5) Remove the supernatant solution and place it in a new, clean 50mL centrifuge tube. Centrifuge at 8000g and 4℃ for 5 minutes, and discard the supernatant solution.

[0030] 6) Add 30 mL of PBS solution to the precipitate and vortex. Centrifuge the solution at 1000 g and 4 °C for 5 min.

[0031] 7) Take the supernatant solution and place it in a new clean 50mL centrifuge tube. Centrifuge at 8000g and 4℃ for 5min. Discard the supernatant solution and rinse the precipitate with 2mL of PBS solution to obtain the grouper enterospora stock solution. Store at 4℃ for later use.

[0032] 8) Take a new centrifuge tube and slowly add 2 mL of 90% sucrose solution, 2 mL of 60% sucrose solution, 2 mL of 30% sucrose solution, and 2 mL of grouper enterospora stock solution to the tube in sequence, close to the wall. Centrifuge at 40000g and 4℃ for 40 min using an ultra-high-speed refrigerated centrifuge.

[0033] This step utilizes sucrose density gradient ultracentrifugation to achieve layered purification of *Enterocera grouperica* by leveraging the density difference between *Enterocera grouperica* and impurities such as host cell debris and uninfected cells. Removing these impurities yields high-purity *Enterocera grouperica*, ensuring the activity, accuracy, and reliability of subsequent in vitro culture and molecular experiments.

[0034] 9) Take out the middle layer of the 90% sucrose solution and centrifuge at 8000g and 4℃ for 1 min. Discard the upper layer, add 1 mL of PBS solution to resuspend, and then centrifuge at 8000g and 4℃ for 2 min.

[0035] This step selects the 90% sucrose solution layer because the grouper enterospora deposit in this layer, maximizing the enrichment of the grouper enterospora and reducing impurities. Steps 8) and 9) are crucial for purification, removing impurities through density stratification and washing to ensure high purity of the grouper enterospora and the activity required for subsequent experiments.

[0036] 10) Repeat step 9) four times. Dilute in a clean bench and count to obtain purified spores of *Enterocera stylosa*. Store at 4°C for later use.

[0037] 2. The purified spores of the grouper enterospora were cultured using the ECGI-21 intestinal cell line of the oblique grouper.

[0038] 1) Cells and culture conditions.

[0039] The cells used for in vitro culture of grouper enterospora in this invention were obtained from the ECGI-21 intestinal cell line of oblique-banded grouper provided by Zhou Sheng of South China Agricultural University; the culture medium was a modified Leibovitz L-15 medium.

[0040] The ECGI-21 intestinal cell line of the grouper is disclosed in the patent "An intestinal cell line of grouper ECGI-21 and its uses", publication number CN114874974A. The ECGI-21 intestinal cell line of grouper is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 62408. The ECGI-21 intestinal cell line of grouper used in this invention was donated by Zhou Sheng of South China Agricultural University.

[0041] The modified Leibovitz L-15 medium formula is as follows: Leibovitz L-15 medium supplemented with v / v 10% fetal bovine serum, 100 IU / mL penicillin, 0.1 mg / mL streptomycin, 0.05 mg / mL Hepes, and 2.66 mg / mL NaCl. Hepes refers to 4-hydroxyethylpiperazine ethanesulfonic acid.

[0042] In this invention, unless otherwise stated, all cell cultures are performed in 24-well plates.

[0043] 2) Main reagents.

[0044] Trypsin, L-15 medium, fetal bovine serum, penicillin, streptomycin, and Hepes were all purchased from GIBCO.

[0045] 3) Inoculate with purified spores of grouper enterospora.

[0046] ECGI-21 intestinal cell line of *Sinocyclocheilus scoparia*: The ECGI-21 required for the experiment was diluted with modified Leibovitz L-15 medium one day before the experiment and inoculated at 10... 6 Cells were seeded at a rate of 500 μL per well in a 24-well plate and cultured overnight at 28°C without CO2 for use in experiments the following day. The culture time for this invention is 12 h; however, it has been verified that culture times of 8 h to 16 h are also acceptable, and will not be specified separately here.

[0047] Inoculation of purified spores of *Enterocera stylosa*: Add 500 μL of purified spores of *Enterocera stylosa* diluted with modified Leibovitz L-15 medium to each well of an overnight cultured 24-well plate, ensuring a final concentration of 10⁻⁶ spores. 6 Continue to incubate in a 28°C incubator.

[0048] Timing and sampling began after inoculation with purified spores of grouper enterospora. Time gradients were set at 0 min, 10 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, and 4 h, with 3 wells taken at each time point.

[0049] Aspirate the culture medium from the 24-well plate and wash twice with PBS. Add 200 μL of trypsin solution, place the 24-well plate flat, ensuring the liquid covers the cell layer, and incubate at 28°C for 2 min for digestion. Add 500 μL of culture medium, gently pipette the cells to form a single-cell suspension, and collect it in a centrifuge tube for DNA extraction from *Enterocera stylosa*.

[0050] The identification of Enterobacteriaceae cultured from grouper is as follows: 1. Establishment of an absolute quantitative standard curve.

[0051] In the LightCycler 96 real-time PCR detection system, quantitative real-time PCR was performed on a 96-well plate using ChamQ Universal SYBR qPCRMaster Mix. The forward primer sequence for quantitative real-time PCR is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2. The amplification reaction system and reaction conditions are shown in Tables 1 and 2, respectively.

[0052] SEQ ID NO. 1: TAACAGCGATGACGAACCCA.

[0053] SEQ ID NO. 2: AGCTTTTTCATGTTCATTCGCC.

[0054] Table 1 Absolute Quantitative PCR Reaction System Table 2 Absolute Quantitative PCR Reaction Conditions 2. Determination of the growth curve of Enterobacter filamentosa in grouper.

[0055] The quantitative real-time PCR reaction system and reaction conditions are as described above. The genome copy number of Enterobacter spp. in infected host cells was calculated.

[0056] 3. Staining experiment.

[0057] 1) Cell fixation and permeabilization: Gently wash cells three times with PBS to remove the culture medium. Add v / v 4% paraformaldehyde for fixation at room temperature for 20 min. Wash three times with PBS, 5 min each time. Add 500 μL of Triton X-100 at a concentration of 0.1% v / v for 1 h. Wash three times with PBS, 5 min each time.

[0058] 2) DY96 staining: Add DY96 under light-protected conditions and incubate at room temperature for 10 min. Wash three times with PBS for 5 min each time to remove unbound dye.

[0059] 3) DAPI counterstaining: Add 100 μL of DAPI working solution and incubate at room temperature in the dark for 10 min. Wash with PBS 3 times, 5 min each time.

[0060] 4) Mounting and observation: Add 10 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.

[0061] The preparation method of the DY96 staining solution used in this invention is as follows: Dissolve 10 mg of DY96 powder in 2 mL of DMSO solution to prepare a 5 mM stock solution for the first staining solution. Wrap the stock solution in aluminum foil and store it at -20°C to protect it from light. Before staining, dilute the stock solution 1000 times in PBS to prepare a DY96 staining solution with a final concentration of 5 μM.

[0062] DY96, or Direct Yellow 96. The DY96 powder was purchased from Beyotime Biotechnology Co., Ltd.; the PBS was purchased from Solarbio Science & Technology Co., Ltd., with a concentration of 0.01M and a pH of 7.3.

[0063] 4. Results and Analysis.

[0064] 1) Proliferation of grouper enterospora in the ECGI-21 intestinal cell line of grouper.

[0065] Quantitative analysis of the genome copy number of *Enterocera stylosa* in infected cells was performed over a 4-hour time span. A standard curve constructed using a reference sample showed a strong linear correlation between the Ct value and the logarithm of the template number. The results indicated that *Enterocera stylosa* proliferated rapidly in ECGI-21 cells after infection, with its genome increasing approximately three-fold after 4 hours. (See attached results). Figure 1 .

[0066] 2) Maturation of grouper enterospora in the ECGI-21 intestinal cell line of grouper.

[0067] DY96 dye can bind to chitin in the spore wall, thereby marking mature spores. The results of infecting ECGI-21 cells with stained mature spores of *Enterocera stylosa* are shown below. Figure 2 As shown, Figure 2 This includes images of stained mature spores during the period from 2 to 4 hours after infection of ECGI-21 cells. Figure 2 The results showed that mature spores labeled with DY96 were present in the cells 2 hours after infection, and the number of mature spores increased with the extension of infection time, with a large number of mature spores observed in the cells at 4 hours. This indicates that *Enterocera stenosus* can not only infect ECGI-21 cells, but also proliferate within the cells and produce mature spores.

[0068] The above results indicate that *Enterocera stenosus* can rapidly infect ECGI-21 cells and proliferate stably within the cells. This invention represents the first successful in vitro culture of *Enterocera stenosus*.

[0069] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0070] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for in vitro culture of *Enterocera stylosa* from grouper, characterized in that, Includes the following steps: To obtain purified spores of *Enterocera prolifera* from grouper; The purified spores of *Enterocera stylosaurus* were cultured using the ECGI-21 intestinal cell line of *Odontocera stylosaurus*.

2. The in vitro culture method as described in claim 1, characterized in that, The process of culturing purified spores of *Enterocera stylosaurus* using the ECGI-21 intestinal cell line of *Odontocerca fasciatus* is as follows: The ECGI-21 intestinal cell line of the oblique grouper was cultured in a culture medium to obtain the culture of the ECGI-21 intestinal cell line of the oblique grouper. The purified spores of *Enterocera stylosa* were diluted with culture medium and then inoculated into the culture of the *ECGI-21* intestinal cell line of *Odontocera stylosa*.

3. The in vitro culture method as described in claim 2, characterized in that, The ECGI-21 intestinal cell line of the oblique grouper was cultured at 28°C in the absence of CO2 for 8-16 hours.

4. The in vitro culture method as described in claim 2, characterized in that, The ratio of purified spores of the grouper intestinal cell line ECGI-21 to grouper enterospora was 1:

1.

5. The in vitro culture method as described in claim 2, characterized in that, The culture medium has the following formula: Each milliliter of Leibovitz L-15 medium was supplemented with v / v 10% fetal bovine serum, 100 IU penicillin, 0.1 mg streptomycin, 0.05 mg 4-hydroxyethylpiperazine ethanesulfonic acid, and 2.66 mg NaCl.

6. The in vitro culture method as described in claim 1, characterized in that, The method for obtaining purified spores of *Enterocera dorsalis* from grouper is as follows: Intestinal tissue was collected from fish infected with grouper enterospora disease, ground, filtered and washed, and purified spores of grouper enterospora were obtained by ultracentrifugation using a sucrose density gradient.

7. The in vitro culture method as described in claim 6, characterized in that, When performing sucrose density gradient ultracentrifugation, the sucrose solutions used had mass fractions of 90%, 60%, and 30%.

8. The in vitro culture method as described in claim 6, characterized in that, The conditions for sucrose density gradient ultracentrifugation were 40,000g, 4℃, and 40 min.

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