Investigation of genetic polymorphism of TLR3, TLR4, TLR5 and TLR15 receptors in Denizli and Gerze chicken breeds in vivo and in vitro conditions

Microorganisms affecting the immune system in chickens can originate from bacteria, viruses, fungi, and parasites. The avian immune system is divided into two categories: innate immunity and acquired immunity. While acquired immunity provides a basic level of protection against invading pathogens, innate immunity detects the presence of microbial agents through pattern recognition receptors and activates defense mechanisms. Among these receptors, Toll-like receptors play a critical role in regulating immune responses, defending against infections, and maintaining immune homeostasis. Information about these receptors, which are part of the innate immune system, provides fundamental insights into the function of the immune system and disease resistance. In this context, this study was conducted to determine the immune potential of denizli and gerze chicken breeds, to support protection strategies and establish a foundation for future research. The study investigated the polymorphisms of ChTLR3, ChTLR4, ChTLR5 and ChTLR15 receptors associated with diseases and gene expression levels of synthetic class B CpG ODN 2007 and Poly I:C ligand samples after cell culture at different times and concentrations in erythrocyte cells. In the polymorphism analysis of the study, blood samples collected from six individuals from both breeds were used for ChTLR3, ChTLR4, ChTLR5 and ChTLR15 receptor genes by next generation sequencing. The analyses were conducted through Next-Generation Sequencing, the single nucleotide polymorphisms, number of haplotypes, and haplotype diversity, of these genes were determined. For the ChTLR3 gene, 17 SNPs and 7 haplotypes and 0.87 haplotype diversity were identified; for ChTLR4, 26 SNPs and 3 haplotypes and 0.61 haplotype diversity; for ChTLR5, 39 SNPs and 7 haplotypes and 0.88 haplotype diversity; and for ChTLR15, 24 SNPs and 4 haplotypes and 0.67 haplotype diversity were detected. In addition, the sequence data of Denizli and Gerze chicken breeds indicate that ChTLR3, ChTLR4, ChTLR5 and ChTLR15 receptors 9, 9, 10 and 11 polymorphic regions at protein level, respectively. In the cell culture phase, blood samples from four individuals of each breed and erythrocytes were separated from blood samples using histopaque and incubated were incubated with synthetic class B CpG ODN and Poly I:C ligands at different durations and concentrations. Incubations were conduct at 41 ˚C, and then the gene expression levels of the endogenous reference gene GAPDH and four receptors were analyzed by RT-qPCR method. The results showed that the expression levels of ChTLR3, which is associated with viral disease resistance, and ChTLR4, ChTLR5 and ChTLR15 receptor genes, which are associated with bacterial disease resistance, were statistically significantly increased (P<0.05 and P<0.001). This study represents the first investigation into the polymorphisms and gene expression levels of immune-related genes in the local Denizli and Gerze chicken breeds. The findings provide a foundational resource for future molecular breeding studies in these breeds. KEYWORDS: Erythrocyte, Cell Culture, Immune Response, mRNA Expression Polymorphism, Toll-Like Receptors