RESUMEN
Generally, the injection method is recommended as the best efficient method for vaccine applications in fish. However, labor-intensive and difficult injection for certain fish sizes is always considered as a limitation to aquatic animals. To demonstrate the effectiveness of a novel oral delivery system for the piscine vaccine with nano-delivery made from nano clay, halloysite nanotubes (HNTs) and their modified forms were loaded with killed vaccines, and we determined the ability of the system in releasing vaccines in a mimic digestive system. The efficaciousness of the oral piscine vaccine nano-delivery system was evaluated for its level of antibody production and for the level of disease prevention in tilapia. Herein, unmodified HNTs (H) and modified HNTs [HNT-Chitosan (HC), HNT-APTES (HA) and HNT-APTES-Chitosan (HAC)] successfully harbored streptococcal bivalent vaccine with inactivated S. agalactiae, designated as HF, HAF, HCF and HACF. The releasing of the loading antigens in the mimic digestive tract demonstrated a diverse pattern of protein releasing depending on the types of HNTs. Remarkably, HCF could properly release loading antigens with relevance to the increasing pH buffer. The oral vaccines revealed the greatest elevation of specific antibodies to S. agalactiae serotype Ia in HCF orally administered fish and to some extent in serotype III. The efficacy of streptococcal disease protection was determined by continually feeding with HF-, HAF-, HCF- and HACF-coated feed pellets for 7 days in the 1st and 3rd week. HCF showed significant RPS (75.00 ± 10.83%) among the other tested groups. Interestingly, the HCF-treated group exhibited noticeable efficacy similar to the bivalent-vaccine-injected group (RPS 81.25 ± 0.00%). This novel nano-delivery system for the fish vaccine was successfully developed and exhibited appropriated immune stimulation and promised disease prevention through oral administration. This delivery system can greatly support animals' immune stimulation, which conquers the limitation in vaccine applications in aquaculture systems. Moreover, this delivery system can be applied to carrying diverse types of biologics, including DNA, RNA and subunit protein vaccines.
RESUMEN
Tilapia lake virus (TiLV) now affects Nile tilapia culture worldwide, with no available commercial vaccine for disease prevention. DNA and recombinant protein-based vaccines were developed and tested following viral isolation and characterization. The viral strain isolated from diseased hybrid red tilapia (Oreochromis sp.) shared high levels of morphological and genomic similarity (95.49-99.52%) with other TiLV isolates in the GenBank database. TiLV segment 9 (Tis9) and segment 10 (Tis10) DNA vaccines (pcDNA-Tis9 and pcDNA-Tis10) and recombinant protein vaccines (Tis9 and Tis10) were prepared and tested for their efficacy in juvenile hybrid red tilapia. Fish were immunized with either single vaccines (pcDNA-Tis9, pcDNA-Tis10, Tis9 and Tis10) or combined vaccines (pcDNA-Tis9 + pcDNA-Tis10 and Tis9 + Tis10) by intramuscular injection and intraperitoneal injection for DNA and protein vaccines, respectively. Negative controls were injected with PBS or a naked pcDNA3.1 vector in the same manner. An experimental challenge with TiLV was carried out at 4 weeks post-vaccination (wpv) by intraperitoneal injection with a dose of 1 × 105 TCID50 per fish. Relative percent survival (RPS) ranged from 16.67 ± 00.00 to 61.11 ± 9.62%. The Tis10 and pcDNA-Tis10 vaccines conferred better protection compared to Tis9 and pcDNA-Tis9. Highest levels of protection were observed in pcDNA-Tis9 + pcDNA-Tis10 (61.11 ± 9.62%) and Tis9 + Tis10 (55.56 ± 9.62%) groups. Specific antibody was detected in all vaccinated groups at 1-4 wpv by Dot Blot method, with the highest integrated density at 2 and 3 wpv. In silico analysis of Tis9 and Tis10 revealed a number of B-cell epitopes in their coil structure, possibly reflecting their immunogenicity. Findings suggested that the combination of Tis9 and Tis10 in DNA and recombinant protein vaccine showed high efficacy for the prevention of TiLV disease in hybrid red tilapia.