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		<div class="Basic-Text-Frame">
			<p class="Type-of-Article">&nbsp;</p>
			<p class="Type-of-Article"><span class="CharOverride-1">Research Article</span></p>
	</div>
		<div class="Basic-Text-Frame">
			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Characterization and Transcriptional Profile of Toll-Like Receptors of <i>Bubalus bubalis</i> with Endometritis</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Authors ParaOverride-1">&nbsp;</p>
			<p class="Authors ParaOverride-1"><span class="CharOverride-3">Noha Mohamed Osman</span><span class="CharOverride-4">1,2*</span><span class="CharOverride-3">, Ahlam Ahmed  Abou Mossallam</span><span class="CharOverride-4">2</span><span class="CharOverride-3">, Fawzy Reyad El Seedy</span><span class="CharOverride-4">3</span><span class="CharOverride-3">, Eman Roshdy Mahfouz</span><span class="CharOverride-4">2</span><span class="CharOverride-3">, Rui Li</span><span class="CharOverride-4">4</span><span class="CharOverride-3">, Michal Mikula</span><span class="CharOverride-4">5</span></p>
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		<div class="Basic-Text-Frame">
			<p class="Affiliations ParaOverride-1"><span class="CharOverride-6">1</span>Department of Molecular and Computational Biology, University of Southern California, 1050 Childs Way RRI 316 Los Angeles, CA 90089, USA; <span class="CharOverride-6">2</span>Department of Cell Biology, Genetic Engineering Division, National Research Center, Dokki, Giza, Egypt; <span class="CharOverride-6">3</span>Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt; <span class="CharOverride-6">4</span>Department of Animal Sciences, Washington State University, Pullman, WA 99164-6351, USA; <span class="CharOverride-6">5</span>Department of Oncological Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland.</p>
		</div>
		<div>
			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-7">Abstract </span>| Toll-like receptors (TLRs) are a vital component of innate immune system and perform an important role in disease resistance through recognition of pathogen associated molecular patterns (PAMPs) and initiation of a convenient immune response. Endometritis is the most important disease causing infertility in cattle and buffalo and TLRs play a critical role in elimination of that disease. The present study aims to characterize the transcripts of TLRs: TLR1, TLR2, TLR4, TLR6 and TLR10 in <span class="CharOverride-8">Bubalus bubalis</span> with endometritis. Quantitative real-time (q-PCR) assays were performed to detect transcript expression profiles of these TLRs in liver, mammary gland, ovary and uterus of <span class="CharOverride-8">Bubalus bubalis</span> with and without endometritis. The result showed that the transcript profiles of TLRs especially TLR2 and TLR4 were significantly differences in mammary glands, ovary and uterus but on the othe hands, there is no significant difference in the transcript profile of TLRs in liver with or without endometritis. TLRs nucleotide sequences have more than 97% nucleotide homology between <span class="CharOverride-8">Bubalus bubalis</span> with and without endometritis. Simple modular architecture tool (SMART) analysis revealed the presence of TIR domains and different numbers of leucine rich repeat motifs in tested TLRs. <span lang="en-US">In conclusion, for animals infected with endometritis, there was a significant difference of TLR2 and TLR4 in uterine tissue compared to uninfected animals. Therefore, it is suggested to consider the TLRs results as detection markers for endometritis.</span></p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-7">Keywords</span> | Toll-Like receptors (TLRs), <span class="CharOverride-8">Bubalus bubalis</span>, Endometritis, Gene expression</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Editor----Citation"><span class="CharOverride-10">Editor</span> | Tahir Yaqub, University of Veterinary and Animal Sciences, Lahore, Pakistan.</p>
			<p class="Editor----Citation"><span class="CharOverride-7">Received</span> | January 11, 2015; <span class="CharOverride-7">Revised</span> | February 17, 2015; <span class="CharOverride-7">Accepted</span> | February 19, 2015; <span class="CharOverride-7">Published</span> | March 03, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-7">*Correspondence</span> | Noha Mohamed Osman, University of Southern California, Los Angeles, USA; <span class="CharOverride-7">Email: </span>nmo_138@usc.edu, Osman.noha@yahoo.com</p>
			<p class="Editor----Citation"><span class="CharOverride-7">Citation</span> | Osman NM, Mossallam AAA, El Seedy FR, Mahfouz ER, Li R, Mikula R (2015). Characterization and transcriptional profile of toll-like receptors of <span class="CharOverride-28">Bubalus bubalis</span> with endometritis. J. Inf. Mol. Biol. 3(2): 34-46.</p>
			<p class="Editor----Citation"><span class="CharOverride-10">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.jimb/2015/3.2.34.46"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.jimb/2015/3.2.34.46</span></a></p>
			<p class="Editor----Citation"><span class="CharOverride-10">I</span><span class="CharOverride-7">SSN (Online) </span>| 2307-5465; <span class="CharOverride-7">ISSN (Print)</span> | 2307-5716</p>
			<p class="Editor----Citation"><span class="CharOverride-10">Copyright </span>© 2015 Osman et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
		  <p class="Heading-1--Introduction----">Introduction</p>
			<p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">T</span>he innate immune system is a global system of host defense against pathogens. Recognition of pathogens relied on a set of germ line-encoded receptors recognized pattern-recognition receptors (PRRs) as a part of innate immune recognition. These receptors have the ability to recognize conserved pathogen-associated molecular patterns (PAMPs) produced by large groups of pathogens (<a href="#Akira-S--2001-."><span class="Hyperlink">Akira, 2001</span></a>; <a href="#Janeway-CA--Medzhitov-R--2002-."><span class="Hyperlink">Janeway and Medzhitov, 2002</span></a>). </p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Ten members of the TLR receptor family are broadly expressed in the mammalian genome (<a href="#Akira-S--Uematsu-S--Takeuchi-O--2006-"><span class="Hyperlink">Akira et al., 2006</span></a>). TLR1, TLR2 and TLR6 recognize bacterial lipids such as lipoteichoic acid (LTA), whereas TLR3, TLR7, and TLR8 as well as TLR9 recognize nucleic acids, often from viruses. TLR9 also identifies bacterial DNA. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria such as <span class="CharOverride-8">E. coli</span>, while TLR5 binds to flagellin. The ligand for  TLR10 is still not known (<a href="#Hirata-T--Osuga-Y--Hamasaki-K--Hirota-Y--Nose-E--Morimoto-C--Harada-M--Takemura-Y--Koga-K--Yoshino-O"><span class="Hyperlink">Hirata et al., 2007</span></a>; <a href="#Regan-T--Nally-K--Carmody-R--Houston-A--Shanahan-F--MacSharry-J--Brint-E--2013--"><span class="Hyperlink">Regan et al., 2013</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">TLRs are type 1 transmembrane proteins with N terminal extracellular leucine-rich repeat (LRR) and C terminal intracellular TIR (Toll/IL 1 receptor) domain. A TIR domain is required for initiating intracellular signaling and inducing downstream effector molecules, while LRRs are responsible for the recognition of PAMPs. (<a href="#Akira-S--Uematsu-S--Takeuchi-O--2006-"><span class="Hyperlink">Akira et al., 2006</span></a>; <a href="#Kumar-H--Kawai-T--Akira-S--2009-"><span class="Hyperlink">Kumar et al., 2009</span></a>; <a href="#Dubey-PK--Goyal-S--Kathiravan-P--Mishra-BP--Gahlawat-SK--Kataria-RS--2013"><span class="Hyperlink">Dubey et al., 2013</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">TLRs are expressed broadly in Antigen-presenting cells, and regulate the activation of both innate and adaptive immunity through recognition of specific patterns of microbial components (<a href="#Medzhitov-R--2001"><span class="Hyperlink">Medzhitov, 2001</span></a>; <a href="#Matsushima-N--Tanaka-T--Enkhbayar-P--Mikami-T--Taga-M--Yamada-K--Kuroki-Y--2007-."><span class="Hyperlink">Matsushima et al., 2007</span></a>).  Their expression and distribution are associated with altered immune responsiveness (<a href="#Helmby-H--Grencis-RK--2003"><span class="Hyperlink">Helmby and Grencis, 2003</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">TLRs play an important role in regulation of ovulation, fertilization, gestation and parturition, as well as in pathological conditions such as endometritis and mastitis (<a href="#Kannaki-TR--Shanmugam-M--Verma-PC--2011-."><span class="Hyperlink">Kannaki et al., 2011</span></a>). The endometrium is the first line of defense against microbial pathogens. The endometrial cells have ability to develop innate immune defense in cattle, humans and rodents (<a href="#Herath-S--Fischer-DP--Werling-D--Williams-EJ--Lilly-ST--Dobson-H--Bryant-CE--Sheldon--IM--2006-."><span class="Hyperlink">Herath et al., 2006</span></a>; <a href="#Davies-D--Meade-KG--Herath-S--Eckersall-PD--Gonzalez-D--White--JO--Conlan--RS--O-Farrelly--C--Sheldo"><span class="Hyperlink">Davies et al., 2008</span></a>). Endometritis is the most common uterine disease observed in buffaloes slaughtered at abattoirs, and is one of the main causes of infertility in the cattle and buffalo (<a href="#Azawi-OI--Omran-SN--Hadad-JJ--2008-."><span class="Hyperlink">Azawi et al., 2008</span></a>; <a href="#Ajevar-G--Muthu-S--Sarkar-M--Kumar-H--Das-GK--Krishnaswamy-N--2014-."><span class="Hyperlink">Ajevar et al., 2014</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text">Microbial disease of the female genital tract is most common, and has greatest economic importance in both humans and animals. Microbial infections of the genital tract cause infertility by disrupting uterine and ovarian function (<a href="#O-Neill-LA--2008"><span class="Hyperlink">O’Neill, 2008</span></a>). Establishment of infection in the endometrium occurs by the interaction between the virulence factors of microbial invaders and the endometrial immune defenses (<a href="#Sheldon-IM--Rycroft-AN--Dogan-2010"><span class="Hyperlink">Sheldon et al., 2010</span></a>). </p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-8">Bubalus bubalis</span> population (195 million heads) includes both river and swamp buffaloes. Egyptian buffaloes are of the river type with a population of 4 million heads; it ranks the third after Indian (115 millions) and Pakistani (31.7 millions) buffalo, which plays an important role in the economy of developing countries (<a href="#FAOstat--2013-"><span class="Hyperlink">FAOstat, 2013</span></a>). The present study was aimed to characterize the TLRs genes and their transcriptional profile in <span class="CharOverride-8">Bubalus bubalis</span> with and without endometritis.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Materials and Methods</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Tissue Samples</p>
			<p class="Body-Text ParaOverride-1">Tissue samples from four different organs; liver, ovary, mammary gland and uterus, were obtained from thirty animals with and without endometritis from a local abattoir immediately after slaughter and kept on ice until processing. The uteri of animals with endometritis had abnormal secretions as profuse purulent exudate, with signs of inflammation as swelling, redness and hardness (<a href="#Williams-EJ--Fischer-DP--Pfeiffer-DU--England-GC--Noakes-DE--Dobson-H--Sheldon-IM--2005"><span class="Hyperlink">Williams et al., 2005</span></a>; <a href="#Sheldon-IM--Lewis-GS--LeBlanc-SJ--Gilbert-RO--2006"><span class="Hyperlink">Sheldon et al., 2006</span></a>). These animals had no evidence of other diseases based on visual inspection and culture of bacteria using standard microbiological techniques. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">RNA Isolation and Reverse Transcription</p>
			<p class="Body-Text">Total RNA was extracted from liver, ovary, mammary gland and uterus using Easy –RED™ kit (iNtRON Biotechnology) according to the manufacturers’ protocols. <span lang="en-US">The RNA quality and purity was determined by NanoDrop 1000 (Thermo Scientific) and cDNA was synthesized as per manufacturer’s procedure (RevertAid First Strand Synthesis Kit, Thermo Scientific).</span></p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Primer Design and Optimization of RT-PCR</p>
			<p class="Body-Text ParaOverride-1">The TLRs coding regions were amplified by designing primers using <span class="CharOverride-8">Bubalus bubalis </span>transcripts sequences as following: TLR1 (HQ327989), TLR2 (HM756162), TLR4 (HQ343416), TLR6 (HQ327992), TLR10 (HQ327991) as presented in <a href="#Table-1"><span class="Hyperlink">Table 1</span></a> using the Primer3 software (<a href="#Rozen-S--Skaletsky-H--2000-"><span class="Hyperlink">Rozen and Skaletsky, 2000</span></a>). These primers were used for PCR analysis and Real time quantitative PCR (qRT-PCR). The primers were synthesized by Amersham Pharmacia Biotech Company. </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-7"><a id="Table-1"></a>Table 1:</span> Primers sequences of TLR1, 2, 4, 6 and 10 used in PCR</p>
			<table width="657" height="968" class="Table-Style-1" id="table-1">
				<colgroup>
					<col class="_idGenTableRowColumn-1" />
					<col class="_idGenTableRowColumn-2" />
					<col class="_idGenTableRowColumn-3" />
					<col class="_idGenTableRowColumn-4" />
					<col class="_idGenTableRowColumn-5" />
					<col class="_idGenTableRowColumn-6" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Gene</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Name</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Forward primer (5’-3’)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Reverseprimer (5’-3’)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Annealing</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Size(bp)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td>
							<p class="Basic-Paragraph ParaOverride-3"><span class="CharOverride-14">TLR1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR1.1</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR1.2</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR1.3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">ATG CCT GAC ATC CTC TCA CT</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TGA CCC AGG AAA TGA AAT CT</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TCA ACC AGG AAC TGG AAT ACT TGG A</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">ACC TCC TTG GAT CGA GGT CC</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">CAG CAG AAA TAC ATT AAC ACG G</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TGG GCA GGG CAT CAA ATG GA</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">63.9°C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">63.9°C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">60°C</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1165 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1195 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">120 bp</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Basic-Paragraph ParaOverride-3"><span class="CharOverride-14">TLR2</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR2.1</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR2.2</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR2.3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">AGA GTG GAG GTC AAA TCA CTG GAC A</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GCC TGT AAG GAT GCC TGG CCC</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GGT TTT AAG GCA GAA TCG TTT G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TGC CTG AAA CTT GTC GGT GGC C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GGT CAG TCT TGG TCT GGT GGT</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">AAG GCA CTG GGT TAA ACT GTG T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">68.6°C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">68.6°C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">60°C</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1329 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1253 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">190 bp</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td>
							<p class="Basic-Paragraph ParaOverride-3"><span class="CharOverride-14">TLR4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR4.1</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR4.2</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR4.3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">CGG CAC AGA CAG AGG GTC ATG C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TTT TGG GAC AAC CAA CCT GAA GCA T</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">CTT GCG TAC AGG TTG TTC CTA A</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GCA AAC CTT GAA AAT GGC AGG CAA</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">CAA GAA GTG ACA ACC TCC ACC TGA</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">CAT AAC TTC TCC AGC TTC CCA G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">62.2°C </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">63.6°C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">60°C</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1500 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1342 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">153 bp</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Basic-Paragraph ParaOverride-3"><span class="CharOverride-14">TLR6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR6.1</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR6.2</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR6.3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">ATC AGC AGC AAC CCT CCG GG</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">ACA CAT GCT TTG TCC TCA GGA ACC A</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">AAG TGA ATG ATG ACA ATT GTC</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TGG GTG TGG TAT CTT TAG CAG CCT</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15"> </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">AAT TTA TCC CCT GCT GCG TTT GGA</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15"> </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">AGT GAC TTC AAT GTC GTT T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">67°C </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">65°C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">60°C</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1532 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1388 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">235 bp</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td>
							<p class="Basic-Paragraph ParaOverride-3"><span class="CharOverride-14">TLR10</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR10.1</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR10.2</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR10.3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TCT GTG GAG CCG CTG CAA GC </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GCC AGC AAC ACA TCC TTG AGG C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GCT GCA AGC CTG AGG GAT TT</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TGG ACT TTT TCC AGA GCT GCC AGG </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GCC TGT AGA GCC CCA CCC ACT </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">AGG ATT TTA TCA CTG GTT CAC CTC</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">62°C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">54°C</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">60°C</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1654 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">1270 bp</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-15">93 bp</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">RPL0</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">RPL0</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">CAA CCC TGA AGT GCT TGA CAT</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">AGG CAG ATG GAT CAG CCA</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">60°C</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">220 bp</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-11">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GAPDH</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GAPDH</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">CCT GGA GAA ACC TGC CAA GT</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GCC GTA TTC ATT GTC ATA CCA</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">60°C</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">214 bp</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">For qPCR analysis, large ribosomal protein (RPL0) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an internal calibrator due to their threshold cycle (CT) values are close to those of TLRs. Each PCR reaction was performed using 2 µl of 50-60 ng cDNA template, 12.5 μl of RNAse DNAse free water, 2.5μl of 2mM dNTPs, 2.5 μl of 10X taq polymerase buffer, and 2.5 μl forward and reverse primers (10 µM), as well as 0.25µl (1U) of Taq polymerase in 25 µl reaction final volume. The following cycling conditions were used: 3 min. at 94ºC; 35 cycles for 1 min at 94ºC; 2 min at specific annealing of each primer as in table (1); 1 min at 72ºC and a final extension for 10 min at 72ºC. Following optimization, the presence of a single product was confirmed on a 1.5% agarose gel by electrophoresis. The gels were inspected by UV light and photographed by Gel documentation system (In Genius, Syngenebioimaging). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The PCR products were purified using (Thermo Scientific, USA) purification kit, according to the manufacturer’s instructions. The purified product was sequenced using ABI PRISM Dye Terminator Cycle Sequencing Kit (Applied Biosystems) in an automatic sequencer (ABI Prism3100, Genetic analyzer (Applied Biosystems). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Sequence Analysis</p>
			<p class="Body-Text ParaOverride-1">cDNAs infected uterus were used to amplify TLR1, TLR2, TLR4, TLR6 and TLR10 genes. Then, the amplified PCR products were sequenced using reverse and forward primers.<span class="CharOverride-2"> </span></p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Sequence alignment was carried out using NCBI/BLAST (<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi"><span class="Hyperlink">http://blast.ncbi.nlm.nih.gov/Blast.cgi</span></a>) between coding regions (CDs) and amino acids of tested TLRs in infected uterus of <span class="CharOverride-8">Bubalus bubalis</span> with endometritis and <span class="CharOverride-8">Bubalus bubalis</span> sequences in GenBank database. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Characterization of the TLRs Domains </p>
			<p class="Body-Text ParaOverride-1">The different functional domains of TLRs (The transmembrane regions (TM); Leucine-rich repeats (LRR); <span class="Heading-3-Char CharOverride-15" lang="en-US">LRR C-terminal (LRR-CT) and</span> Toll/IL-1 receptor (TIR) was predicted by using simple modular architecture analysis tool (SMART), available at <a href="http://smart.embl-heidelberg.de/"><span class="Hyperlink">http://smart.embl-heidelberg.de/</span></a> (<a href="#SchuItz-J--Milpetz-F--Bork-P--Ponting-CP--1998"><span class="Hyperlink">Schultz et al., 1998</span></a>) and LRRfinder (<a href="http://www.lrrfinder.com"><span class="Hyperlink">www.lrrfinder.com</span></a>) (<a href="#Offord-V--Coffey-TJ--Werling-D--2010-"><span class="Hyperlink">Offord et al., 2010</span></a>). The amino acid sequence of TIR domains of tested TLRs of infected animals with endometritis was subjected to align with TIR amino acid sequence of <span class="CharOverride-8">Bubalus bubalis</span> sequences in GenBank database for detecting the percentage of the identity.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Quantitative Real Time PCR (qRT-PCR)</p>
			<p class="Body-Text ParaOverride-1">qRT-PCR was used to estimate TLR expression levels in the examined samples. For each sample, cDNA was diluted 1:25 with 10Mm Tris pH 7.5. For each sample, 1.9 µl of diluted cDNA template was combined with 2 µl of 2X Master Mix (Bioline) and 0.1 µl of 10 µM primer set for a final reaction volume 4 µl in MicroAmp optical 384 well reaction plate in triplicates along with no template control (NTC) to all wells. Cycling was performed on 7900HT Real-Time PCR system (Applied Biosystems, Life Technologies Corporation) using the following cycling conditions: 95°C for 10 min (hold), 95°C for 15 sec (melt) and 60°C for 1 min (anneal/extend) for 40 cycles. Dissociation curves were generated to ensure that a single amplicon had been produced. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Statistical Analysis</p>
			<p class="Body-Text ParaOverride-1">The expression of candidate genes was evaluated using a 2<span class="CharOverride-6">-ΔΔCt</span> method (<a href="#Livak-KJ--Schmittgen-TD--2001-"><span class="Hyperlink">Livak and Schmittgen 2001</span></a>) .The Geometric mean of reference genes (RPLP0 and GAPDH) acted as an internal calibrator within each sample to calculate <span class="CharOverride-6">Δ</span> Ct.The <span class="CharOverride-6">ΔΔ</span> Ct was calculated by <span class="CharOverride-6">Δ</span>Ct (treatment) − <span class="CharOverride-6">Δ</span>Ct (control). The fold change in expression was then used as a relative measure of gene expression. Data analysis was carried out by non-parametric Mann-Whitney test. Significance was set at <span class="CharOverride-8">P</span>&lt;0.05 and the results were reported as mean ± SEM.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Results </p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Sequencing of TLRs genes</p>
			<p class="Body-Text ParaOverride-1">The full-length of TLR1, TLR2, TLR4, TLR6, and TLR10 cDNAs coding regions of <span class="CharOverride-8">Bubalus bubalis</span> with endometritis have been submitted to GenBank and their accession numbers are presented in <a href="#Table-2-"><span class="Hyperlink">Table 2</span></a>. The percentage of nucleotide and aminoacid identity of <span class="CharOverride-8">Bubalus bubalis</span> with and without endometritis is shown in <a href="#Table-2-"><span class="Hyperlink">Table 2</span></a>. There was a highly degree of identity between <span class="CharOverride-8">Bubalus bubalis</span> with and without endometritis.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Analysis of Functional Domains of Tested TLRs</p>
			<p class="Body-Text ParaOverride-1">The results showed that all TLRs of <span class="CharOverride-8">Bubalus bubalis</span> with and without endometritis have LRR and TIR domains and one transmembrane domains (TM) in all tested TLRs except TLR6, which has two transmembrane domains (TM). LRRCT and LRRNT (cysteine flanking regions of LRR at C and N terminals, respectively) were examined in tested TLRs, we found all TLRs contain LRRCT, while LRRNT was absent in all TLRs as shown in <a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Next, we used LRR finder to predict the repeat number of LRR motifs present in TLRs. As shown in <a href="#Table-3-"><span class="Hyperlink">Table 3</span></a>, we found an equal number of LRRs (20) in TLR2, TLR6 and TLR10 <span class="CharOverride-8">Bubalus bubalis</span> with and without endometritis but on the other hand TLR1 of <span class="CharOverride-8">Bubalus bubalis</span> with and without endometritis has (18) and (19) LRR respectively, while TLR4 of <span class="CharOverride-8">Bubalus bubalis </span>with and without endometritis has (21) and (22) LRRs with 2 LRRs predicted overlapped respectively.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-7"><a id="Table-2-"></a>Table 2:</span> Coding DNA, translated amino acids and TIR domain sequence similarity of TLRs between <span class="CharOverride-8">Bubalus bubalis </span>with and without endometritis</p>
			<table width="657" height="140" class="Table-Style-1" id="table-2">
				<colgroup>
					<col class="_idGenTableRowColumn-1" />
					<col class="_idGenTableRowColumn-12" />
					<col class="_idGenTableRowColumn-13" />
					<col class="_idGenTableRowColumn-14" />
					<col class="_idGenTableRowColumn-15" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">    TLR</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">  CDs identity (%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Amino acid identity (%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">TIR domain identity(%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Accession number</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.5</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">100</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">KM058706</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR2</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">KM058707</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.5</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">98.9</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">KM058708</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.8</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.2</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">98.6</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">KM058709</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TLR10</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">99.5</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">98.9</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">97.9</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">KM058710</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-7"><a id="Table-3-"></a>Table 3:</span> <span class="CharOverride-19">Leucine-rich repeats in ami</span><span class="CharOverride-19">no acid sequences of tested TLRs </span></p>
			<table width="657" height="71" class="Table-Style-1" id="table-3">
			  <colgroup>
			    <col class="_idGenTableRowColumn-17" />
			    <col class="_idGenTableRowColumn-18" />
			    <col class="_idGenTableRowColumn-19" />
			    <col class="_idGenTableRowColumn-20" />
			    <col class="_idGenTableRowColumn-21" />
			    <col class="_idGenTableRowColumn-22" />
		      </colgroup>
			  <tbody>
			    <tr class="_idGenTableRowColumn-10">
			      <td><p class="Basic-Paragraph"><span class="CharOverride-14">Species</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-14">TLR1</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-14">TLR2</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-14">TLR4</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-14">TLR6</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-14">TLR10</span></p></td>
		        </tr>
			    <tr class="_idGenTableRowColumn-16">
			      <td><p class="Basic-Paragraph"><span class="CharOverride-20">Bubalus bubalis</span><span class="CharOverride-15"> with endometritis</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">18</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">20</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">21 and 2 predicted overlapped</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">20</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">20</span></p></td>
		        </tr>
			    <tr class="_idGenTableRowColumn-10">
			      <td><p class="Basic-Paragraph"><span class="CharOverride-20">Bubalus bubalis</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">19</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">20</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">22 and 2 predicted overlapped</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">20</span></p></td>
			      <td><p class="Basic-Paragraph"><span class="CharOverride-15">20</span></p></td>
		        </tr>
		      </tbody>
		  </table>
			
		  <p>&nbsp;</p>
			<p><br>
		  </p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150303231411.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150303231411.png" width="80" height="80"></a>
            
          <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-7"><a id="Figure-1-"></a>Figure 1:</span><a href="http://nexusacademicpublishers.com/uploads/figures/20150303231411.png"> predicted functional domains in tested TLRs of <span class="CharOverride-8">Bubalus bubalis, </span>with and without endometritis (vertical bar is transmembrane domain (TM)</a></p>
       </div><br>

			
		  
		  
			
            <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150303230114.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150303230114.png" width="80" height="80"></a>
            
         <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-7"><a id="Figure--2A--"></a>Figure (2A): </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150303230114.png"> Expression levels of TLR1,TLR2, TLR4,TLR6 and TLR10 genes in liver tissues collected from animals with and without  endometritis. The expression levels of these genes were normalized to RPL0 and GAPDH and calibrated with the average of control. Expression levels were obtained from three pooled biological replicate samples ± S.E.M.</a></p>
       </div><br>
       
			
		  <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150303234714.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150303234714.png" width="80" height="80"></a>
            
         <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-7"><a id="Figure--2B--"></a>Figure (2B): </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150303234714.png"> Expression levels of TLR1,TLR2, TLR4,TLR6 and TLR10 genes in mammary gland tissues collected from animals with and without  endometritis. The expression levels of these genes were normalized to RPL0 and GAPDH and calibrated with the average of control. Expression levels were obtained from three pooled biological replicate samples ± S.E.M.</a></p>
       </div>
       
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-7"><a id="Figure--2B--"></a>Figure (2B): </span>Expression levels of TLR1,TLR2, TLR4,TLR6 and TLR10 genes in mammary gland tissues collected from animals with and without  endometritis. The expression levels of these genes were normalized to RPL0 and GAPDH and calibrated with the average of control. Expression levels were obtained from three pooled biological replicate samples ± S.E.M.</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150303230816.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150303230816.png" width="80" height="80"></a>
            
        <p class="Figure--and-Table-Heading ParaOverride-1"><a id="Figure--2C--"></a><span class="CharOverride-7">Figure (2C): </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150303230816.png"> Expression levels of TLR1,TLR2, TLR4,TLR6 and TLR10 genes in ovarian tissues collected from animals with and without  endometritis. The expression levels of these genes were normalized to RPL0 and GAPDH and calibrated with the average of control. Expression levels were obtained from three pooled biological replicate samples ± S.E.M.</a></p>
       </div>
			<p class="Figure--and-Table-Heading ParaOverride-1"><a id="Figure--2C--"></a><span class="CharOverride-7">Figure (2C): </span>Expression levels of TLR1,TLR2, TLR4,TLR6 and TLR10 genes in ovarian tissues collected from animals with and without  endometritis. The expression levels of these genes were normalized to RPL0 and GAPDH and calibrated with the average of control. Expression levels were obtained from three pooled biological replicate samples ± S.E.M.</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			
            <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150303234916.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150303234916.png" width="80" height="80"></a>
            
        <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-7"><a id="Figure--2D--"></a>Figure (2D): </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150303234916.png"> Expression levels of TLR1,TLR2, TLR4,TLR6 and TLR10 genes in uterine tissues collected from animals with and without  endometritis. The expression levels of these genes were normalized to RPL0 and GAPDH and calibrated with the average of control. Expression levels were obtained from three pooled biological replicate samples ± S.E.M.</a></p>
       </div>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-7"><a id="Figure--2D--"></a>Figure (2D): </span>Expression levels of TLR1,TLR2, TLR4,TLR6 and TLR10 genes in uterine tissues collected from animals with and without  endometritis. The expression levels of these genes were normalized to RPL0 and GAPDH and calibrated with the average of control. Expression levels were obtained from three pooled biological replicate samples ± S.E.M.</p>
			
            <p class="Heading-2--History-in-MM-">&nbsp;</p>
            <p class="Heading-2--History-in-MM-">&nbsp;</p>
          <p class="Heading-2--History-in-MM-">Results of Expression of TLRs Genes</p>
			<p class="Body-Text">As shown in <a href="#Figure--2D--"><span class="Hyperlink">Figure 2D</span></a>, our results revealed that TLR1<span class="CharOverride-8"> </span>expression levels increased six-fold in the uterine tissues of <span class="CharOverride-8">Bubalus bubalis</span> with endometritis compared to those in the uterine tissues of animals without endometritis (P=0.0173). Similarly, the expression levels of both TLR6 and TLR10 were higher with nine-fold increase for TLR6 (P=0.0303), and an almost ten-fold increase for TLR10 (P=0.0303) in the uterine tissues of animals with endometritis compared to those in the uterine tissues of animals without endometritis. In addition, TLR2 showed twenty-six-fold increase in the uterine tissues of animals with endometritis compared to those in the uterine tissues of animals without endometritis (P=0.0043). Moreover, TLR4 showed a significant increase in the uterine tissues of animals with endometritis compared to those in the uterine tissues of animals without endometritis, with a twelve-fold increase (P= 0.0087).</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">In the ovarian tissues, the expression profile of candidate genes showed that the expression of TLR1, TLR6 and TLR10 had the same level of expression between animals with and without endometritis (P =0.0173, <a href="#Figure--2C--"><span class="Hyperlink">Figure 2C</span></a>). In addition, there was a significant increase in the expression levels of TLR2 and TLR4<span class="CharOverride-8"> </span>genes in the ovary tissues of animals with endometritis compared with those without endometritis (P=0.0043; <a href="#Figure--2C--"><span class="Hyperlink">Figure 2C</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">A comparison of the expression levels of  TLR1<span class="CharOverride-8">, </span>TLR2<span class="CharOverride-8">, </span>TLR4<span class="CharOverride-8">, </span>TLR6 and TLR10 genes in mammary gland tissues from animals with and without endometritis were performed. As shown in <a href="#Figure--2B--"><span class="Hyperlink">Figure 2B</span></a>, the expression levels of TLR1 (P=0.7489), TLR6<span class="CharOverride-8"> </span>(P=0.7706) and TLR10<span class="CharOverride-8"> </span>(P=0.7005) genes in the mammary gland tissues had no significant difference between animals with and without endometritis. Conversely, TLR2 showed a highly significant increase in animals with endometritis compared to animals without endometritis, with a 6.6 fold increase (P=0.0303). Similarly,<span class="CharOverride-8"> </span>TLR4 showed a highly significant increase in the mammary glands of animals with endometritis compared to those from animals without endometritis, with a 4.0 fold increase (P=0.0173).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Finally, we carried out a comparison for the expression levels of TLR1<span class="CharOverride-8">, </span>TLR2<span class="CharOverride-8">, </span>TLR4<span class="CharOverride-8">, </span>TLR6 and TLR10 genes in liver tissues from animals with and without endometritis. As shown in <a href="#Figure--2A--"><span class="Hyperlink">Figure 2A</span></a>, these tissues had similar expression levels for TLR1<span class="CharOverride-8">, </span>TLR2<span class="CharOverride-8">, </span>TLR4<span class="CharOverride-8">, </span>TLR6 and TLR10. </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Discussion</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">In the present study, we reported the first sequencing data of TLRs genes and their expression in <span class="CharOverride-8">Bubalus bubalis </span>with endometritis. Toll-like receptors consider the initial defence against microbes in endometrium. Their characterizations are important to evaluate the innate immune system.<span class="CharOverride-8"> </span>TLR1, TLR2, TLR4, TLR6 and TLR10 cDNA, translated amino acids and TIR domain sequences of <span class="CharOverride-8">Bubalus bubalis </span>with endometritis were compared with <span class="CharOverride-8">Bubalus bubalis </span>without endometritis. TLRs cDNA ncleotide sequences of <span class="CharOverride-8">Bubalus bubalis </span>with and without endometritis<span class="CharOverride-8"> </span>were 98 to 99% identical. Furthermore,<span class="CharOverride-8"> </span>TLR6 of <span class="CharOverride-8">Bubalus bubalis </span>with and without endometritis had an insertion of nucleotide triple ‘TTA’ resulting in the addition of tyrosine amino acid (Y) at position 181 compared with <span class="CharOverride-8">Bos Taurus</span> (<a href="#Carl-GF--Charles-AS--Carmen-MC--Allen-WC--Sara-H--Patricia-C--Alan-S--2003"><span class="Hyperlink">Carl et al., 2003</span></a>; <a href="#Dubey-PK--Goyal-S--Kathiravan-P--Mishra-BP--Gahlawat-SK--Kataria-RS--2013"><span class="Hyperlink">Dubey et al., 2013</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">TIR domain has an important role in activation of inflammatory response, in cooperation with the adaptor protein MyD88 (<a href="#Kawai-T--Akira-S--2007-."><span class="Hyperlink">Kawai and Akira, 2007</span></a>). We found that the identity of TIR sequences between with and without endometritis ranged from 97 (TLR10) to 99.3 (TLR2 and TLR4) and 100 %( TLR1). TLR2 recognizes peptidoglycan cell wall components of Gram-positive bacteria, while TLR4 ligands include Gram-negative bacteria. Both of TLR2 and TLR4 have an important role in regulation of innate immune response (<a href="#Herath-S--Fischer-DP--Werling-D--Williams-EJ--Lilly-ST--Dobson-H--Bryant-CE--Sheldon--IM--2006-."><span class="Hyperlink">Herath et al., 2006</span></a>; <a href="#Davies-D--Meade-KG--Herath-S--Eckersall-PD--Gonzalez-D--White--JO--Conlan--RS--O-Farrelly--C--Sheldo"><span class="Hyperlink">Davies et al., 2008</span></a>)<span class="CharOverride-8">.</span></p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The number and structure of LRRs of TLRs have a significant effect on the function of pathogen recognition receptor system among species (<a href="#Kumar-H--Kawai-T--Akira-S--2009-"><span class="Hyperlink">Kumar et al., 2009</span></a>; <a href="#Dubey-PK--Goyal-S--Kathiravan-P--Mishra-BP--Gahlawat-SK--Kataria-RS--2013"><span class="Hyperlink">Dubey et al., 2013</span></a>). <a href="#Peter-ME--Kubarenko-AV--Weber-AN--Dalpke-AH--2009-"><span class="Hyperlink">Peter et al. (2009)</span></a> reported that deletion of LRR motif leads to loss of functional integrity of the ligand–binding domain of murine TLRs. In addition, (<a href="#Dubey-PK--Goyal-S--Kathiravan-P--Mishra-BP--Gahlawat-SK--Kataria-RS--2013"><span class="Hyperlink">Dubey et al., 2013</span></a>) demonstrated that the differences in the number of LRRs among buffalo and cattle may lead to possible differences in the specific recognition of TLR ligands between these species. In our study we found that the number of LRR domains of <span class="CharOverride-8">Bubalus bubalis </span>with endometritis and their distribution is similar to <span class="CharOverride-8">Bubalus bubalis </span>without endometritis except TLR1 and TLR4.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Bacterial infection of the female genital tract is common in animals especially after parturition causing infertility and sometimes mortality (<a href="#Davies-D--Meade-KG--Herath-S--Eckersall-PD--Gonzalez-D--White--JO--Conlan--RS--O-Farrelly--C--Sheldo"><span class="Hyperlink">Davies et al., 2008</span></a>). The endometrium is the first defence against pathogens. PAMPs were recognized by TLRs which consider as key components of innate immune system which lead to increase expression of TLRs (<a href="#Akira-S--Uematsu-S--Takeuchi-O--2006-"><span class="Hyperlink">Akira, 2006</span></a>). TLRs expression were evaluated in liver, mammary gland, ovary and uterus of <span class="CharOverride-8">Bubalus bubalis </span>with and without endometritis. The results showed no significant difference in TLRs expression in liver tissues between buffaloes with and without endometritis but on the other hand the expression of  TLRs in mammary gland showed no significant difference in the expression levels of TLR1<span class="CharOverride-8">, </span>TLR6 and TLR10 between animals with and without endometritis. Conversely, TLR2<span class="CharOverride-8"> </span>and TLR4 genes expression significantly increased in mammary glands of animals with endometritis compared to animals without endometritis. In Ovarian tissue,<span class="CharOverride-8"> </span>TLR1<span class="CharOverride-8">, </span>TLR6 and TLR10 had the same level of significant difference (P=0.0173). Similarly, TLR2 and TLR4<span class="CharOverride-8"> </span>had the same level of significant difference (P=0.0043). This indicates that the expression levels of several TLRs vary signiﬁcantly in association with the degree of infection in animal (<a href="#Sharma-BS--Leyva-I--Schenkel-F--Karrow-NA--2006"><span class="Hyperlink">Sharma et al., 2006</span></a>; <a href="#Deborah-L--Ling-Z--Kumudika-JB--Richard-JW--2008-."><span class="Hyperlink">Deborah et al., 2008</span></a>; <a href="#Chapwanya-A--Meade-KG--Doherty-ML--Callanan-JJ--Mee-JF--O-Farrelly-C--2009"><span class="Hyperlink">Chapwanya et al., 2009</span></a>; <a href="#Priyanka-B--Suresh-KG--Jyoti-J--Upasna-S--Madhu-ST--Ramesh-KV--2012-"><span class="Hyperlink">Priyanka et al., 2012</span></a>). The presence of uterine infection due to pathogenic infection had a localized effect on ovarian function (<a href="#Sheldon-IM--Noakes-DE--Rycroft-AN--Pfeiffer-DU--Dobson-H--2002"><span class="Hyperlink">Sheldon et al., 2002</span></a>). This is hypothesis is supported by (<a href="#Williams-EJ--Fischer-DP--Pfeiffer-DU--England-GC--Noakes-DE--Dobson-H--Sheldon-IM--2005"><span class="Hyperlink">Williams et al., 2008</span></a>) who stated that pathogenic agents such as <span class="CharOverride-8">E.coli </span>has the ability to produce endotoxin lipopolysaccharide (LPS), which is released from infected uterine tissue to ovarian tissue and peripheral circulation leading to disruption of the hypothalamus,pituitary and ovary functions in animals.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Previous studies demonstrated that the expression levels of TLRs family members increase upon endometritis. Specifically, the mRNA levels of TLR2<span class="CharOverride-8"> </span>and TLR4<span class="CharOverride-8"> </span>increase in endometritis infection of bacterial origin (<a href="#Ozinsky-A--Underhill-DM--Fontenot-JD--Hajjar-AM--Smith-KD--Wilson-CB--Schroeder-L--Aderem-A--2000-"><span class="Hyperlink">Ozinsky et al., 2000</span></a><span class="CharOverride-23">; </span><a href="#Goldammer-T--Zerbe-H--Molenaar-A--Schuberth-HJ--Brunner-RM--Kata-SR--Seyfert-HM---2004"><span class="Hyperlink">Goldammer et al., 2004</span></a><span class="CharOverride-23">;</span> <a href="#Azawi-OI--Omran-SN--Hadad-JJ--2008-."><span class="Hyperlink">Azawi et al., 2008</span></a>; <a href="#Davies-D--Meade-KG--Herath-S--Eckersall-PD--Gonzalez-D--White--JO--Conlan--RS--O-Farrelly--C--Sheldo"><span class="Hyperlink">Davies et al., 2008</span></a>). With the agreement of these data, we detected overexpression of all five examined TLR genes, particularly TLR2<span class="CharOverride-8"> </span>(26.4 fold) and TLR4 (12.4 fold), in infected uterine tissues with endometritis compared to the uninfected ones. </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Conclusion</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Our data demonstrated that the variations in nucleotides and amino acids sequence between <span class="CharOverride-8">Bubalus bubalis </span>with and without endometritis are not associated with endometritis. In addition, there is an overexpression of examined <span class="CharOverride-8">TLRs </span>genes especially TLR2 and TLR4 in animals with endometritis compared to animals without endometritis specifically in uterine tissue.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Acknowledgements</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The authors would like to thank prof. Ostrowski (Department of Oncological Genetics, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland) for providing all facilities to carry out his work.      </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Conflict of Interest</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The authors declared no conflict of interest. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
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