Evaluation of the constitutive expression levels of ch-TLR 3, ch-TLR 4, ch-TLR 15 and ch-TLR 21 genes in the Peripheral Blood Mononuclear Cells of native Indian poultry breeds, Aseel and Kadaknath

Aim: To assess the basal constitutive expression levels of ch-TLR3, ch-TLR 4, ch-TLR 15 and ch-TLR 21 in the peripheral blood mononuclear cells (PBMCs) in Aseel and Kadaknath chicks (Indian native poultry breeds) and to evaluate the differences in their general innate immune competence. Materials and Methods: PBMCs were isolated from 21 day old Aseel and Kadaknath chicks (n=4) and were subjected to RNA isolation and cDNA synthesis. The basal expression of ch-TLR 3, 4, 15 and 21 was studied using real time PCR with SYBR green chemistry using 18 S-rRNA as the housekeeping gene. Results: PBMCs isolated from Kadaknath chicks exhibited a significantly higher (p<0.05) constitutive expression of ch-TLR 3, ch-TLR 15 and ch-TLR 21 genes when compared to Aseel chicks. In comparison to Aseel, Kadaknath chicks recorded 14.774, 7.182 and 3.507 fold higher expressions of ch-TLR 3, ch-TLR 15 and ch-TLR 21 genes, respectively. In contrast, the constitutive expression of ch-TLR 4 was found to be higher (by 1.733 fold) in Aseel chicks. Conclusion: Our results indicate that Kadaknath chicks are equipped with a better innate immune competence in comparison to Aseel chicks.


Introduction
of a small repertoire of receptors.The efficiency of this system can be appreciated by the fact that recognition Innate arm of the immune system, once of LPS through TLR4 allows innate arm to recognise considered to play only a limited role in eliciting a almost all of the gram negative bacteria.Interaction of protective immune response, has now acquired a centre these TLR ligands with the respective TLRs leads to stage and is now believed to play a pivotal role in the either upregulation or downregulation of various initiation as well as shaping the subsequent adaptive immune related genes which have an important bearing immune responses [1].Cells of the innate arm are on shaping subsequent adaptive responses into a more capable of recognizing a large number of invading precise response [4].pathogens using only a few germline encoded Extensive breeding and selection programmes to pathogen recognition receptors (PRRs), out of which improve economic attributes in commercial poultry the Toll like receptors (TLRs) are of prime importance flocks have compromised its innate immune compe-[2].TLRs recognise molecular patterns known as tence in comparison to their native counterparts.The "Pathogen associated molecular patterns (PAMPs)" discoveries related to functioning of innate arm of the that are unique to microbes viz., Lipoteichoic acid immune system have widely attracted the attention and (LTA), double stranded RNA, lipopolysaccharide (LPS), infused a lot of interest in poultry scientists whose flagellin, unmethylated Cytosine triphosphate-phostarget is to improve the innate resistance of commercial phodiester link-Guanine triphosphate (CpG) etc.
flocks by looking for a better germplasm [5].Generally, which are recognised in chicken by ch-TLR2, ch-TLR the immune competence of birds is assessed based on 3, ch-TLR 4, ch-TLR 5 and ch-TLR 21 genes [3].This their level of complement activity or antibody response pathogen sensing mechanism enables innate arm to to Sheep RBCs (SRBCs).In the recent past, after the recognise majority of the pathogens only with the help establishment of pivotal role of TLRs in pathogen recognition and generation of both innate and adaptive immune response, the constitutive and inducible expression of TLRs and other related genes has been correlated with general immune competence of the doi:10.5455/vetworld.2013.568-572 fowl [6].A better disease tolerance of Aseel and sigma-Aldrich) and centrifuged at 1800 rpm for 20 Kadaknath, Indian chicken breeds, in comparison to minutes in swing out rotors.White ring at the interface their commercial counterparts is well documented containing the mononuclear cells/PBMCs was [7,8].Nevertheless, majority of the studies that were carefully aspirated and washed twice with HBSS aimed at determining the immune competence, on the followed by centrifugation at 800-1000 rpm for 10 basis of constitutive and inducible expression of chminutes.TLRs and other related genes, in fowls were conducted 6 Relative expression study: PBMCs @ 2 X 10 were in commercial breeds and the knowledge related to the subjected to RNA isolation, c-DNA preparation and expression of these innate immune markers in native relative quantification using real time PCR (ABI, poultry breeds such as Aseel and Kadaknath is largely 7500).The primers used in the study were designed unknown.
using the softwares available online (Table -1).The Thus, we designed this study with an objective to primers were of small amplicon size (100-300 bp), evaluate the constitutive expression of genes that 0 amplified at 58 C and showed primer efficiency in the encode four important chicken TLRs i.e., ch-TLR 3, range of 1.8-2.0.18S rRNA was considered as the ch-TLR 4, ch-TLR 15 and ch-TLR 21 in the peripheral housekeeping gene for this study.The reaction blood mononuclear cells (PBMCs) of Indian native breeds; Aseel and Kadaknath.
conditions were standardized with SYBR green chemistry and all the primers produced a single peak in

Materials and Methods
the dissociation curve analysis confirming the high Ethical approval: This study was conducted as per the specificity of the products.To determine the relative provisions of the Institute Animal Ethics Committee.
expression levels of genes investigated between Aseel and Kadaknath breeds, the constitutive expression in Chicks: Day old chicks belonging to Aseel and Kadaknath PBMCs from Aseel breed was considered as a unit breeds were purchased from the Central Poultry (control) for respective genes under comparison.Research Station, Anand.Chicks were maintained in the Poultry Demonstration Unit, Veterinary College, Statistical analysis: Data were analysed using WASP 1 SDAU-Sardarkrushinagar under standard managemental software available online in the statistical package at procedures and during the entire experimental period ICAR-Goa complex site (http://www.icargoa.res.in/they were provided with ad-libitum feed and water. wasp/rbd1.php).In the present study, PBMCs isolated from 21 day expressions of ch-TLR 3, ch-TLR 15 and ch-TLR 21 old Kadaknath chicks had significantly higher ch-TLR 3, ch-TLR 15 and ch-TLR 21 m-RNA transcript genes, respectively.However, an exception was that expression, whereas ch-TLR 4 had apparently higher the constitutive expression level of ch-TLR 4 was expression in Aseel.Differential expression of various found to be apparently higher in PBMCs isolated from important immune-related genes has been correlated Aseel chicks than that of Kadaknath chicks.Though with the disease susceptibility or resistance pattern of non-significant, the expression of TLR 4 was found to the host [24].The difference in ch-TLR 15 gene be 1.733 times higher in Aseel than Kadaknath chicks expression by heterophils of lines A (Salmonella PBMCs (Table -2).resistant) and B (Salmonella susceptible) is speculated

Discussion
to account for some of the observed differences The discovery of toll-like receptors (TLRs), between the lines in their susceptibility to infection [25].The bacterial load in susceptible chickens was particularly on the cells of innate arm of immune significantly higher than that in the resistant chickens system [9,10] and their critical role in the providing and ch-TLR4, ch-TLR2-1 and ch-TLR21 expression immunity has aroused intense curiosity in the scientific was highly diminished in the leukocytes of susceptible arena and the studies conducted in the recent years on chickens when compared with those of resistant this subject has greatly improved our understanding of chickens [26].Higher expression of ch-TLR 4, ch-TLR the functioning of immune system [11].A better 15, ch-TLR 21 and a few other genes involved with understanding of innate arm could also assist poultry TLR activation pathways was reported with scientists to meticulously look for desired immune-Salmonella Enteritidis (SE) infection in chicken line A related genes with a potential to be incorporated in (resistant to Salmonella Enteritidis infection) [27].The marker assisted selective breeding programmes.
significantly higher expression of ch-TLR3, ch-TLR 15 Keeping these facts in mind, this study was conducted and ch-TLR 21 in Kadaknath chicks, observed in the on two Indian native breeds of fowl i.e., Aseel and present study, is suggestive of a better immune compe-Kadaknath which are reported to have enhanced tence of Kadaknath in comparison to Aseel chicks.In abilities to fight infectious diseases when compared to present study, constitutive expression of ch-TLR 4 in abilities of the commercial breeds [12,13].
PBMCs from Aseel chicks was found to be apparently TLRs are a group of highly conserved and germline higher than that of Kadaknath chicks.Contrary to our encoded pattern recognition receptors.Chicken reports, significantly higher expression of ch-TLR 4 genome has orthologs of mammalian TLR 1, TLR 2, and ch-TLR 5 in heterophils from Kadaknath than in TLR 3, TLR 4, TLR 5 and TLR 7 whereas orthologs of heterophils of Aseel, Necked neck, Dwarf and White TLR 8, TLR9 and TLR 10 appears to be either defective Leghorn lines was reported earlier [28].Contrasting or missing [14].TLR  .The present study indicates a better TLR 3, TLR 4, TLR 15 and TLR 21 in host defense immune competence of Kadaknath breed to deal with a against viral and bacterial pathogens is well documented variety of the invading pathogens.In this context, it is [20][21][22][23].These earlier findings formed the premise for important to note that we have not used any infectious selecting ch-TLR 3, ch-TLR 4, ch-TLR 15 and ch-TLR model to ascertain the immune competence of these 21 genes and to study their constitutive expression in two breeds by exposing them to pathogenic challenge.PBMCs of Indian native Aseel and Kadaknath breeds Thus, ingenious experimental designs combined with which enables us to better under-stand the general the efficiency of latest tools and techniques are immune competence of the native Indian poultry required to test this notion further.breeds.The PBMCs were collected from 21 day old Conclusion chicks which coincide with the maturation of bursa at Higher expression of TLR genes has been three weeks of their age and thereby lead to an