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Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miamiensis avidus
Shogo Motokawa1, Yukie Narasaki1, Jun-Young Song2
1Graduate School of Science and Engineering, Ehime University, Matsuyama 790-8577, Japan; Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama 790-8577, Japan.
Abstract:
The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30kDa; serotype II, 38kDa; serotype III, 34kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores-<51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.
Insights
Miamiensis avidus causes scuticociliatosis. This study identified serotype-specific polypeptides as ciliary membrane antigens and developed PCR methods for accurate serotyping of this fish pathogen.
Area of Science:
- Parasitology
- Marine Biology
- Molecular Biology
Background:
- Miamiensis avidus is a ciliate responsible for scuticociliatosis in Japanese flounder.
- Three distinct serotypes of M. avidus have been previously identified based on specific antigenic polypeptides.
Purpose of the Study:
- To determine the localization of serotype-specific polypeptides in M. avidus.
- To identify the genes encoding these polypeptides.
- To develop molecular tools for serotyping M. avidus.
Main Methods:
- SDS-PAGE and immunoblot analysis of ciliate components (cilia, membrane proteins, cytoskeleton).
- Scanning electron microscopy of ciliates treated with homologous antiserum.
- DNA sequencing and PCR assay development using serotype-specific primers.
Main Results:
- Serotype-specific polypeptides (30kDa, 38kDa, 34kDa) were localized to the ciliary membrane.
- Genes encoding these polypeptides showed low sequence identity (<51.5%) between serotypes.
- PCR-based serotyping results were consistent with traditional methods, classifying 20 of 21 isolates.
Conclusions:
- The identified polypeptides function as ciliary membrane immobilization antigens.
- Serotype-specific PCR assays provide a reliable method for differentiating M. avidus serotypes.
- Combining species-specific and serotype-specific PCR is valuable for M. avidus identification and surveillance.
