Screening of immunogenic antigens from scuticociliate in Takifugu rubripes, and the development and evaluation of subunit vaccines

In recent years, scuticociliatosis has emerged as a major parasitic disease in the aquaculture of Takifugu rubripes, inflicting significant economic losses on the pufferfish farming industry. The conventional approach of using chemical drugs to prevent and control parasitic diseases poses substantial risks to the environment and food safety. Consequently, the utilization of vaccines for the prevention and treatment of aquatic animal diseases is currently regarded as the mainstream direction for future aquatic animal disease prevention and control. In this study, utilizing the laboratory-established pathogenic strain of scuticociliates, Pseudocohnilembus persalinus, as the immunogen, we aimed to screen for immunogenic proteins capable of eliciting immune responses in hosts. Through enrichment culture of P. persalinus in vitro, impurities were filtered out using membranes, and the organisms were subsequently collected by centrifugation, frozen-thawed and disrupted to obtain whole-cell proteins. BALB/c mice and T. rubripes served as the immunization subjects, receiving intraperitoneal injections of the proteins to generate polyclonal antisera against scuticociliates. Western blot analysis was then performed, incubating the whole-cell proteins with mouse and T. rubripes antisera. The most intensely stained protein bands were excised, subjected to gel excision and mass spectrometry for identification and comparison. The resulting identified gene, SDP35, belongs to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) family and exhibits the following characteristics: The SDP35-encoded polypeptide chain comprises two protein domains: the N-terminal coenzyme NAD-binding domain and the C-terminal catalytic domain, which are distinctive features of this family of proteins. The SDP35 protein is primarily localized in the cytoplasm, consistent with GAPDH's function in catalyzing glycolysis within the cytoplasm. In addition, the SDP35 gene bears some ciliate-specific traits, notably the presence within its open reading frame of ten non-canonical glutamine-encoding codons (TAA), which serve as stop codons in most other organisms. Toxicity challenge experiments were conducted on T. rubripes before and after vaccination, revealing that the subunit vaccine against scuticociliates significantly reduced the scuticociliate density at the wound site of T. rubripes. The parasite reduction rate reached 80.39 %. Concurrently, the vaccine enhanced the activity of acetylcholinesterase (ACH), peroxidase (POD), and total superoxide dismutase (T-SOD) in T. rubripes, thereby strengthening its anti-infective capability and exerting a preventive effect against scuticociliatosis.