EXPRESSION OF TOLL LIKE RECEPTOR3 (TLR3) IN Pangasius pangasius EXPERIMENTALLY INDUCED WITH Edwardsiella tarda

Toll-Like Receptors (TLRs) are germ-line encoded Pattern Recognition Receptors (PRRs) that serve as an important component of innate immune system. TLRs recognize the invading pathogens by sensing the components present on the microbes which are referred as Pathogen Associated Molecular Patterns (PAMPs). Among the 17 predicted TLR genes in fishes, TLR3-mediated activation of immune response depends on viral double-stranded RNA intermediates. As TLR3 gene has not been reported in P.pangassius and its ligand has not been identified clearly, this study was carried out with an objective to identify TLR3 in P.pangassius and to study its expression by inducing with Edwardsiella tarda, a gram-negative bacterial pathogen that infects fish.

Healthy P.pangasius of 40-50g size were procured and acclimatized to the lab conditions.  Control and treatment groups of fishes (6 nos each) were maintained in triplicates. A virulent E.tarda isolate (SRLAAH-ET) was used for experimental induction of TLR3 in P.pangasius in the treatment group. Healthy, uninfected fish group was maintained as the control. Tissue samples (skin, muscle, gill, brain, liver, intestine, kidney and spleen) were collected from the control and treatment groups at 2 h, 4 h, 6 h, 8 h, 12 h and 24 h post-induction.  RNA was extracted from the tissue and cDNA was synthesized using a commercial kit. PCR amplification of TLR3 and β-actin internal control were carried out using self-designed primers. TLR3 expression was assessed by Quantitative Real time PCR (qPCR) using Ct values of the samples. ΔCt values for the tissue at various time of the study were arrived based on the differences between expression of the internal control (β-actin) and the Ct values of TLR3 gene. Three replicate values of 40-ΔCt were performed using one-way ANOVA and mean comparisons were performed by Tukey's multiple range test using two way ANOVA with mean + SD.

The expression profiles of TLR3 in various tissue of P.pangasius is presented in Figure 1. Significant up and down regulations(P<0.001) were induced by the gram negative ligand of E.tarda in various tissue of P.pangasius at various time intervals studied. The findings of this study suggest the possible role of gram negative bacterial ligand for TLR3. This is the first report on the study of expression of TLR3 in P.pangasius.