Differential expression of duck Toll-like receptor 7 ( dTLR 7 ) in various organs of indigenous ducks

Aim: The present molecular study was taken up with an aim of investigating the expression profile of duck TLR7 mRNA in various tissues of indigenous ducks of Tamil Nadu. Materials and Methods: A total of 36 ducks which are reared in extensive system have been chosen as research material for the present experiment. Ducks were sacrificed and tissue samples namely lungs, spleen and gastrointestinal tract (duodenum, jejunum, ileum and caecum) were collected in RNA later solution. Total RNA was extracted and converted to cDNA. Gene specific primers were designed and quantitative SYBR Green based Real-time Reverse Transcriptase PCR (qRT-PCR) was performed to study the gene expression levels. The qRT-PCR data was normalized to β-actin, house keeping gene as endogenous control. Results: Real-time quantitative RT-PCR analysis revealed higher expression in lungs and spleen, while expression being lower in digestive organs. Among gut associated tissues, ileum showed highest expression followed by caecum. Statistically no significant difference (P<0.05) in TLR7 expression was found between duodenum and jejunum. Conclusion: These findings have indicated that considerable level of dTLR7 is expressed in different tissues of ducks. The results suggest that TLR7 mediated innate immune response mechanism exists in native ducks, to fight against single stranded RNA viruses.


Introduction
family and plays a fundamental role in pathogen recognition of single stranded virus RNA and Ducks are considered important next only to activation of innate immunity.Acts via TIR domain chicken.India has a population of 26 million ducks [1] containing adapter, myeloid differentiation factor 88 of which 90 to 95 per cent constitutes indigenous or (MYD88).Triggering of TLR7 results in rapid up nondescript type.Ducks, especially the indigenous regulation of proinflammatory cytokines and type are resistant to many diseases despite their interferons (IFNs) which are critical mediators of viral frequent exposure to marshy and grazing areas where defense.TLR7 and TLR8 respond to U-rich singlethe incidence of potential pathogens is relatively high.
stranded RNA (ssRNA), and are pathogen recognition This might be due to their inbuilt strong innate immune receptors (PRRs) for the influenza [6].In duck, only response.Ducks and chicken are considered as TLR7 gene has been characterized molecularly [7] and important hosts for avian influenza virus, however, its gene expression has been investigated by several avian influenza H5N1 strains, which are highly authors [8,9].An understanding of TLR expression pathogenic for chicken, rarely harm ducks [2].Toll-like pattern in different tissue samples will allow more receptor (TLR) signalling pathway is one of the most refined interpretation of immune induction and host critical and by far the best characterized mechanism of pathology relationships [10], and also highlight the innate immune system [3].TLRs are germ line encoded role of TLRs in enhancement of immune status in receptors belonging to a family of evolutionary indigenous ducks reared under free range system, conserved type-I transmembrane glycoproteins [4].
where they are exposed to marshy areas with high The activation of TLRs follows recognition and microbial load and their feeding habit is scavenging.interaction with a wide range of markers of invading However, there is a paucity of information regarding pathogens called Pathogen Associated Molecular expression of TLR7 in ducks especially native type.Patterns (PAMPs) [5].
Therefore this study was taken up with an objective of TLR7 is a member of the Toll-like receptor (TLR) assessing the differential expression of TLR7 in various organs in native ducks of Tamil Nadu.
out on native ducks of Tamil Nadu (n=36) as per the Subsequent melting curve standards was performed to Institute Animal Ethics Committee recommendations.
melt primer dimers by heating to 94ºC for 30 s followed by 55ºC for 1 min, 94ºC for 30 s and 25ºC for 10 s. averaged and expressed as 40-∆Ct mean values.The RNA isolation and cDNA synthesis: Total RNA was higher 40-∆Ct value can be interpreted as a numerical isolated from the collected tissues using TRI Reagent value indicating greater gene expression [11,12].(Sigma-Aldrich, USA).The preliminary concentration Statistical analysis: All the statistical analysis were and purity of the extracted RNA was measured at 260 performed using SPSS software (version 16.0 and 280 nm using the BioPhotometer plus upgraded, Inc, Chicago, IL.).One way Analysis of (Eppendorf).Subsequently, an in-solution DNase Variance (ANOVA) with Duncan's post-hoc test was digestion step (RNase free DNase I, MBI Fermetas, performed.Results are expressed as means ± SE, and USA) was performed to avoid genomic DNA contamithe differences are considered statistically significant nation.The quality and integrity of total RNA was checked at P<0.05, and highly significant at P<0.01.using denaturating 1% agarose gel electrophoresis and

visualization under UV light (Figure-1). Two intact bands of 28S and18S with smearing indicated good
In the present study, we provide a comparative quality RNA.cDNA was synthesised using oligo (dT) quantification of constitutive expression of TLR7 in 15 primers and High-capacity cDNA reverse transcriptase various organs of native ducks.TLR7 under investigation enzyme (Applied Biosystems, USA).could be amplified from all the tissues studied.Realtime quantitative RT-PCR analysis revealed significant Primers: Gene specific primers for both dTLR7 and β-(P<0.05)difference in various organs studied with actin (Table -1), used in this study were designed and regard to TLR7 expression (Table -2).Regression custom synthesized from Integrated DNA Technologies analysis of the Ct values generated by the log dilution (USA) and ILS (India).10 series gave slope values for all reactions in the range of Real-time quantitative RT-PCR expression study: All -3.53 for TLR7 and -3.15 for β-actin with the amplification the protocols for Real-time qRT-PCR were carried as efficiencies of 92% and108% respectively.Among all per the guidelines of MIQE (Minimum Information for the organs studied, lungs and spleen showed significantly Publication of Quantitative Real-Time PCR Experiments).
higher (P<0.05)expression for TLR7 than digestive Quantitative expression analysis was performed on organs.Within the gastrointestinal tract, expression in diluted cDNA using Mx-3000P spectrofluorometric various parts was studied and a considerable difference thermocycler (Stratagene, USA) in real-time PCR in TLR7 expression was noticed.Significantly, highest strips (Axygen).A total volume of reaction mixture expression was recorded in ileum (36.47±0.36),containing forward and reverse primer (each 20 followed by caecum (Figure -2).No significant (P<0.05)picomolar concentration) and nuclease free water difference was found statistically, between duodenum (sufficient quantity)was prepared.Quantitative RTand jejunum.Gut associated tissues also exhibited PCR thermal cycler conditions were set at initial lower TLR7 expression than spleen and lungs, but with denaturation at 95°C for 10 min followed by 40 cycles a variation in expression pattern among themselves. of denaturation at 94°C for 15 s, gene specific annealing at 55°C for 10 s, extension at 72°C for 30 s. previous finding [7].The relatively higher TLR7 TLR7 transcripts in lung of chicken.This might be due expression in ileum and caecum is an indirect indication to species variation (genomic organization) as galliformes of presence of more immune cellular receptors and non galliformes.The later has presence of more (enterocytes and peyer's patches) [23].This dominant resident cells expressing the TLR7 receptor, whilst the expression could be due to more mean retention time of same are absent in the chicken [7].Higher TLR7 feed than duodenum and jejunum [24] may perpetuates expression in lungs of native ducks in this investigation TLR7 due to high antigenic exposure to intestinal could be comparable to that of TLR7's higher expression epithelium.Vandervena, et al. [25] have confirmed that found in the lungs of human beings [14].Assuming the immune response differs in ducks depending upon role of TLR7 against single stranded RNA viruses like strain of virus that alters the TLR7 expression.These avian influenza in ducks, this high pulmonary results correlate with an earlier studyin chicken [11].expression in lungs could be significance in the context The distribution pattern of enterocytes of intestinal of highly pathogenic H5N1 avian influenza, which is tract, those interface with the contents of the gut lumen primarily a lung infection [15] and replicates in the by virtue of their expression of Toll-like receptors in respiratory tract of ducks [16].
mammalian species [26] including poultry [27] can While in spleen it is important because of the also influence the induction of enteric immune poorly developed avian lymphatic vessels and nodes responses that probably reflects their role in early [18].Higher expression in spleen might be due to high detection of invading microorganisms.The tissue and immune response activity and this is necessary to cell distribution is an important characteristic of TLR ensure optimum disease resistance.It is likely that the function, since it influences the capacity to detect lungs and spleen of indigenous ducks in free range microorganisms during their entry and growth in might be sensitive to TLR7 ligand by virtue of its different target tissues [28].expression as this tissue interfaces with pathogens that enter through it.

Conclusion Expression of TLR7 in gastro intestinal tract:
Expression profiling for the presence of TLR7 Intestinal epithelium, which is continually exposed to a transcript in different cells and tissues is an important large variety of commensal bacteria and PAMP, has feature that would characterize the ability of these cells been shown to constitutively express several members and tissues in detecting pathogens.The variation in of the TLR in vitro and in vivo [20,21,22].The current TLR7 expression in different tissues under study might investigation showed a comparatively lower TLR7 be due to differences in the organization of lymphoid expression in gut associated tissues than spleen and tissue and also the presence of resident cells expressing lungs, and are similar with reports of Philbin, et al. [13] the TLR7 receptor.The results of the present basic and MacDonald, et al. [8].However, a variation exists study provide new knowledge about the innate immune among different parts of intestine.Several investigations mechanisms of the duck.As a whole, we conclude that on TLR7 expression in chicken were reported, but Toll-like receptor 7 expression which plays a role in the

Table - 1
. Characteristics of primers used for real-time PCR Discussionstudies on duck TLR7 are lacking.Previous reports [10,13,18] on chicken indicated more or less similar

Expression profile of TLR7 in lungs and spleen:
Our expression pattern of TLR7 in various gut associated findings of elevated TLR7 expression in lungs and tissues while this study indicates variation among spleen are in tune with the reports of [7,10,13].Iqbal, et different parts of intestine.We observed a similar al. [10] and Yilmaz, et al. [17] reported constant and expression in duodenum and jejunum.The lowest even level of expression in chicken.On contrary, TLR7 expression in duodenum is in agreement with the Philbin, et al. [13] reported very weak expression of Luoa, J., Zhoua, K., Dongc, J. and Hea, H. (2011) Immune-related gene expression in response to H5N1 avian studies may focus on other relevant innate immune influenza virus infection in chicken and duck embryonic genes in ducks with a viral challenge to arrive at fibroblasts.Molecular Immunology, 48: 924-930.