In Vitro Cultivation for Glugea plecoglossi (Microsporidia) Isolated from Ayu (Plecoglossus altivelis)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fish Maintenance
2.2. G. plecoglossi Spores Purification
2.3. Culture of Primary MO/MΦ, EPC Cells and RAW264.7 Cells
2.4. In Vitro Infection of Primary Cultures and Cell Lines Using G. plecoglossi Spores
2.5. Cell Staining
2.6. Observation of G. plecoglossi Spores in Primary Cell and Cell Lines Using Transmission Electron Microscopy (TEM)
2.7. Cytokines Detection Using Real-Time Fluorescent Quantitative PCR
2.8. Re-Infection of Both Cell Lines and In Vivo Infection of Ayu Using EPC-Cultured G. plecoglossi Spores
3. Results
3.1. Light Microscopy Observation of Ayu MO/MΦ Infected with G. plecoglossi
3.2. Light Microscopy Observation of RAW264.7 Cells Infected with G. plecoglossi
3.3. Light Microscopy Observation of EPC Cells Infected with G. plecoglossi
3.4. TEM Observation of G. plecoglossi Spores in Primary Cell and Cell Lines
3.5. The Cytokines Expression in MO/MΦ, RAW264.7, and EPC Cells upon G. plecoglossi Infection
3.6. The Infectivity of Spores Cultured in EPC on Cell Lines and Ayu
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Gene | GenBank No. | Primer | Nucleotide Sequence (5′→3′) | Resource |
---|---|---|---|---|
PaTNF-α | JP740414 | PaTNF-αF | ACATGGGAGCTGTGTTCCTC | [25] |
PaTNF-αR | ACATGGGAGCTGTGTTCCTC | |||
PaIL-1β | HF543937 | PaIL-1βF | TACCGGTTGGTACATCAGCA | |
PaIL-1βR | TGACGGTAAAGTTGGTGCAA | |||
PaTGF-β | JP742920 | PaTGF-βF | CTGGAATGCCGAGAACAAAT | |
PaTGF-βR | CTGGAATGCCGAGAACAAAT | |||
PaIL-10 | JP758157 | PaIL-10F | TGCTGGTGGTGCTGTTTATGTGT | |
PaIL-10R | AAGGAGCAGCAGCGGTCAGAA | |||
Pa18S rRNA | FN646593 | Pa18SF | GAATGTCTGCCCTATCAACT | |
Pa18SR | GATGTGGTAGCCGTTTCT | |||
RAWTNF-α | NM013693 | RAWTNF-αF | CCTCACACTCAGATCATCTTCTC | [27] |
RAWTNF-αR | AGATCCATGCCGTTGGCCAG | |||
RAWIL-1β | NM008361 | RAWIL-1βF | CTTTGAAGAAGAGCCCATCC | [28] |
RAWIL-1βR | TTTGTCGTTGCTTGGTTCTC | |||
RAWTGF-β | NM011577 | RAWTGF-βF | CAAGGAGACGGAATACAGGG | [29] |
RAWTGF-βR | CGCACACAGCAGTTCTTCTC | |||
RAWIL-10 | NM010548 | RAWIL-10F | GCTCTTACTGACTGGCATGAG | [30] |
RAWIL-10R | CGCAGCTCTAGGAGCATGTG | |||
RAWβ-actin | NM007393 | RAWβ-actinF | CCTAAGGCCAACCGTGAAAAG | [31] |
RAWβ-actinR | TCTTCATGGTGCTAGGAGCCA | |||
EPCTNF-α | JN412133 | EPCTNF-αF | CAAGCAATTGGCGAGTGTGT | [32] |
EPCTNF-αR | CAGTTCCACTTTCCTGATTACTCTGA | |||
EPCIL-1β | AJ245635 | EPCIL-1βF | AGACCAATCTCTACCTCGCTTGTAC | |
EPCIL-1βR | TTAATGGTGTTTAATGTTTCACTGATCTC | |||
EPCTGF-β | JN412135 | EPCTGF-βF | TGTATAACAGCACTGTCGAGCTAAGC | |
EPCTGF-βR | TCCCTTCTCATTAGGATCTTCTACATC | |||
EPCIL-10 | JN412134 | EPCIL-10F | GATGTCACGTCATGGACGAGAT | |
EPCIL-10R | GGACTGGAAGTGGTTCTTCTGTACA | |||
EPC40S | JN412136 | EPC40SF | TTTTGAGAAGAGGCATAAGAACATGT | |
EPC40SR | GTAACGATGTCACCAACAGTCACA |
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Xu, G.; Zhang, Z.; Zhou, Q.; Song, M.; Yang, G.; Kang, J.; Xu, Z.; Chen, F.; Chen, J. In Vitro Cultivation for Glugea plecoglossi (Microsporidia) Isolated from Ayu (Plecoglossus altivelis). Microorganisms 2024, 12, 522. https://doi.org/10.3390/microorganisms12030522
Xu G, Zhang Z, Zhou Q, Song M, Yang G, Kang J, Xu Z, Chen F, Chen J. In Vitro Cultivation for Glugea plecoglossi (Microsporidia) Isolated from Ayu (Plecoglossus altivelis). Microorganisms. 2024; 12(3):522. https://doi.org/10.3390/microorganisms12030522
Chicago/Turabian StyleXu, Guizong, Zengyi Zhang, Qianjin Zhou, Mingyan Song, Guanjun Yang, Jinwei Kang, Zhongjie Xu, Fangjie Chen, and Jiong Chen. 2024. "In Vitro Cultivation for Glugea plecoglossi (Microsporidia) Isolated from Ayu (Plecoglossus altivelis)" Microorganisms 12, no. 3: 522. https://doi.org/10.3390/microorganisms12030522
APA StyleXu, G., Zhang, Z., Zhou, Q., Song, M., Yang, G., Kang, J., Xu, Z., Chen, F., & Chen, J. (2024). In Vitro Cultivation for Glugea plecoglossi (Microsporidia) Isolated from Ayu (Plecoglossus altivelis). Microorganisms, 12(3), 522. https://doi.org/10.3390/microorganisms12030522