The study of Toll-Like Receptor 9 and its induction after exposure to CpG ligand in cobia (Rachycentron canadum)

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Cobia culture is hindered by bacterial infection (Photobacterium damselae subsp. piscicida) and in order to study the effect of P. damselae subsp. piscicida challenge and CpG oligodeoxynucleotides (CpG-ODN) stimulation on cobia Toll like receptor 9 (RCTLR9), we cloned the 3102 bp cobia Toll-like Receptor 9 (RCTLR9) and found a homology with other known TLR9 of fish. Further, we stimulated cobia in-vivo with different CpG-ODNs and evaluated induction of RCTLR9, myxovirus resistant protein (Mx), interleukin-1β (IL-1β), chemokine CC and immunoglobulin M (IgM) in spleen and liver by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that, in treated fish majority of the tested CpG-ODNs exhibited induction of these genes which are dependent on time and type of CpG-ODNs. In spleen, CpG-ODN 1668 and 2006 stimulated group significantly increased expression of RCTLR9, IL-1β after 6 days of post stimulation when compared to other groups. While CpG-ODN 2395 had induced significantly increased expression of Mx in spleen when compared to other groups. We also modified the CpG-ODN sequence into CpT, CpA, CpC and evaluated their efficacies on RCTLR9 expression. Results demonstrated that phosphorothioate backbone CpT-ODN (PS-CT-ODN), stimulated significant higher RCTLR9 expression compared to CpA and CpC. Furthermore, we determined the adjuvanticity of different CpG-ODNs by formulation with inactivated bacteria. Results showed that, formulation with CpG-ODN 1668 significantly increased the expression of RCTLR9 and IL-1β in immunized fish. The highest lysozyme and peroxidase activities were also observed in fish immunized with CpG-ODN 1668 formulation. In addition, comparing to other treatments, serum bactericidal activity using the bacterial survival percentage was reduced in CpG-ODN 1668 formulation (25 %). The antibody titer was increased from 6 dpi and reached peak at 21 dpi from CpG-ODN 1668 (OD = 0.27 ± 0.02) and CpG-ODN 2006 (OD=0.24 ± 0.018) formulation. The liver cells of stimulated fish showed a higher lipid content at 6 and 14 dpi. CpG-ODN 1668 and 2395 group with an increased white pulp area was found in the spleen at 14 dpi. Immunization with CpG-ODN 1668 and 2395 formulation protected cobia and increased the survival rate to 90 &; 70 %, respectively in response to challenge with live Photobacterium damselae subsp. piscicida until 3 weeks of post-challenge. Altogether, this study demonstrated that different TLR9 agonists CpG-ODNs have different abilities to augment immune response and protection against Photobacterium. CpG-ODN 1668 can be an adjuvant for cobia vaccination.