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			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Prevalence and Molecular Detection of Intimin (<i>eaeA</i>) Virulence Gene in <i>E. coli</i> O157:H7 in Calves</p>
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			<p class="Authors ParaOverride-1">&nbsp;</p>
			<p class="Authors ParaOverride-1"><span class="CharOverride-3">Afaf Abdulrahman Yousif*, Mohammed Ali Hussein</span></p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Affiliations ParaOverride-2">Department of Internal and Preventive veterinary Medicine, College of Veterinary Medicine, University of Baghdad, Iraq.</p>
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		<div>
			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Abstract</span> | This study was carried out to investigate the prevalence of <span class="CharOverride-6">Escherichia coli</span> O157:H7 serotype from diarrheic and non-diarrheic calves. The study was out in Baghdad, a province in Iraq. A total of 350 faecal samples from 35 diarrheic calves and 315 non<span class="CharOverride-5">- </span>diarrheic calves with different ages (up to 1 year) and from both sexes. After initially enrichment, samples were streaked on sorbitol MacConkey agar plus cifixime potassium tellurite (SMA-CT) and Chrom agar™ <span class="CharOverride-6">E .coli </span>O157:H7. Non-sorbitol fermenting (NSF) <span class="CharOverride-6">E</span>. <span class="CharOverride-6">coli </span>isolates were conducted to serotyping using commercial Latex agglutination test for detection of O157 and H7 antigen. <span class="CharOverride-6">E. coli</span> Isolates were additionally tested for virulence factor <span class="CharOverride-6">eae </span>by PCR techniques. Four isolates (11.42%) belonged to <span class="CharOverride-6">E. coli </span>O157:H7 in 35 diarrheic calves and 28 (8.88%) in non- diarrheic calves<span class="CharOverride-6">. </span>All four isolates<span class="CharOverride-6"> </span>from diarrheic calves<span class="CharOverride-6"> </span>were found positive for intimin (<span class="CharOverride-6">eaeA</span>) gene (100%) and only 13 from 28 isolates (46.42%) were possessing (<span class="CharOverride-6">eaeA</span>) gene in none diarrheic calves. In conclusion, this study revealed the importance of calves to act as a reservoir for <span class="CharOverride-6">E. coli </span>O157:H7. Also, the <span class="CharOverride-6">eaeA </span>genes in a high percentage in most calves suggest that they may be virulent for humans. </p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Keywords </span>| <span class="CharOverride-6">E. coli </span>O157:H7<span class="CharOverride-6">,</span><span class="CharOverride-5"> </span>Intimin (<span class="CharOverride-6">eaeA</span>)<span class="CharOverride-5"> </span>gene<span class="CharOverride-6">, </span>PCR<span class="CharOverride-6">, E. coli </span>O157:H7<span class="CharOverride-6"> </span>in calves</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
		</div>
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			<p class="Editor----Citation">&nbsp;</p>
			<p class="Editor----Citation"><span class="CharOverride-7">Editor</span> | Muhammad Abubakar, National Veterinary Laboratories, Islamabad, Pakistan.</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Received</span> | May 20, 2015; <span class="CharOverride-5">Revised</span> | June 04, 2015; <span class="CharOverride-5">Accepted</span> | June 05, 2015; <span class="CharOverride-5">Published</span> | June 12, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-5">*Correspondence</span> | Afaf  Abdulrahman Yousif, University of Baghdad, Iraq; <span class="CharOverride-5">Email:</span> Afaf_a.rahman@yahoo.com</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Citation</span> | Yousif AA, Hussein MA (2015). Prevalence and molecular detection of intimin (<span class="CharOverride-40">eaeA</span>) virulence gene in <span class="CharOverride-40">E. coli</span> O157:H7 in calves. Res. J. Vet. Pract. 3(3): 47-52.</p>
			<p class="Editor----Citation"><span class="CharOverride-7">DOI</span><span class="CharOverride-8"> | </span><a href="http://dx.doi.org/10.14737/journal.rjvp/2015/3.3.47.52"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.rjvp/2015/3.3.47.52</span></a></p>
			<p class="Editor----Citation"><span class="Editor---Citation CharOverride-5" xml:lang="en-US">ISSN</span> | 2308-2798</p>
			<p class="Editor----Citation"><span class="CharOverride-7">Copyright </span>© 2015 Yousif and Hussein. 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----">Introduction  </p>
			<p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">E</span><span>nterohemorrhagic&#160;</span><span class="CharOverride-6">Escherichia </span><span class="CharOverride-6">coli</span>&#160;(EHEC) strains, of which&#160;<span class="CharOverride-6">E. coli</span>&#160;O157:H7 is the best-studied serotype, Shiga toxin-producing&#160;<span class="CharOverride-6">Escherichia coli</span>&#160;O157:H7 causes foodborne infections, and cattle are the primary reservoir which harbour the bacteria in their intestinal tracts without showing clinical symptoms (<a href="#Kieckens-E--Rybarczyk-J--De-Zutter-L--Duchateau-D--Vanrompay-D--Cox-E--2015-."><span class="Hyperlink">Kieckens et al., 2015</span></a>; <a href="#Katani-R--Cote-R--Raygoza-Garay-JA--Li-L--Arthur-TM--DebRoy-C--Mwangi-MM--Kapur-V--2015"><span class="Hyperlink">Katani et al., 2015</span></a>).</p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Enterohemorrhagic <span class="CharOverride-6">Escherichia coli </span>(EHEC) O157:H7 responsible for frequent haemorrhagic colitis and haemolytic uremic syndrome in humans. In 1982 <span class="CharOverride-6">E. coli </span>O157:H7 was first recognized as a human pathogen (<a href="#Riley-LW--Remis-RS--Helgerson-SD--McGee-HB--Wells-JG"><span class="Hyperlink">Riley et al., 1982</span></a>). As it was associated with consumption of undercooked ‘hamburgers’. As it has been found that healthy cattle can harbour the bacterium, ruminants are now regarded as its main reservoir, though STEC O157:H7 has been isolated from other animal species such as pigs, sheep, geese, gulls, geese and pet animals (<a href="#Gyles-CL--2007-"><span class="Hyperlink">Gyles, 2007</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The ability of the organism to survive in feed, water, soil and manure has important implications for its persistence in cattle herds and contamination of water supplies and crops. Effective measures to reduce or eliminate <span class="CharOverride-6">E. coli </span>O157:H7 in cattle will reduce not only food borne illness but also the risk of transmission of the organism into the environment (<a href="#Bach-SJ--McAllister-TA--Baah-J--Yanke-LJ--Veira-DM--Gannon-VP--Holley-RA--2002"><span class="Hyperlink">Bach et al., 2002</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">A wide range of prevalence estimates ranging from 0.1% to 62% of <span class="CharOverride-6">E. coli</span> O157 in cattle was reported worldwide (<a href="#Lin-YL--Chou-C--Pan-T--2001"><span class="Hyperlink">Lin et al., 2001</span></a>; <a href="#Reinstein-SJ--Fox-T--Shi-X--Alam-MJ--Renter-DG--2009-"><span class="Hyperlink">Reinstein et al., 2009</span></a>; <a href="#Pennington-H--2010-."><span class="Hyperlink">Pennington, 2010</span></a>). The inconsistent prevalence estimates of <span class="CharOverride-6">E. coli</span> O157 reported in cattle in various geographical locations might be, to some extent, due to variable methodological modus operandi to identify the organism, such as sampling strategy, type of samples, enrichment procedures, immunomagnetic separation and cultural media of choice. Therefore, the factors that contribute to the variability in the detection of the organism and thus in the prevalence estimate need to be identified by analysing the available published reports (<a href="#Islam-Z--Musekiwa-A--Islam-K--Ahmed-S--Chowdhury-S--Abdul-Ahad--Biswas-PK--2014-"><span class="Hyperlink">Islam et al., 2014</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The “top five” EHEC serotypes are defined as <span class="CharOverride-6">E. coli </span>strains harbouring Shiga toxin (<span class="CharOverride-6">stx</span>) and intimin (<span class="CharOverride-6">eae</span>) genes and belonging to one of the following serotypes:O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 (<a href="#French-Agency-for-Food--Environmental-and-Occupational-Health-and-Safety--ANSES---2010-."><span class="Hyperlink">ANSES, 2010</span></a>). Intimin (eaeA<span class="CharOverride-11">&#160;</span>gene) and Tir (tir<span class="CharOverride-11">&#160;</span>gene) are key colonization factors, which paly significant roles in E. coli<span class="CharOverride-11">&#160;</span>O157:H7attachment to host epithelium (<a href="#McNeilly-TN--Mitchell-MC--Rosser-T--McAteer-S-and-Low-JC--2010-"><span class="Hyperlink">McNeilly et al., 2010</span></a>; <a href="#Zhang-X--Yu-Z--Zhang-S--He-K--2014"><span class="Hyperlink">Zhang et al., 2014</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><a href="#Blanco-M--Blanco-2004"><span class="Hyperlink">Blanco et al. (2004)</span></a> recorded that Intimin is required for intimate bacterial adhesion to epithelial cells inducing a characteristic histopathological lesion defined as “attaching and effacing” (A/E). This lesion is governed by a large pathogenicity island named the locus of enterocyte effacement (LEE). The products of LEE are a type III secretion system, intimin and its translocated intimin receptor, and other secreted proteins. The secretion system is a molecular syringe for which secreted proteins are transferred into host cell cytoplasm. Intimin is encoded by <span class="CharOverride-6">eae </span>gene that presents heterogeneity in their 3’ end, involved in binding to the enterocytes.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text">This study was the first in Iraq aimed to isolate and confirmed <span class="CharOverride-6">E. coli </span>O157:H7serotypes in the faecal samples of calves located place around Baghdad province and to determine the <span class="CharOverride-6">eaeA</span> virulence genes in these strains by PCR technique.</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Heading-1--Introduction----">Materials and Methods</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-13">Bacteriological Examination</span></p>
			<p class="Body-Text ParaOverride-1">Three hundred fifty calves aged between (up to one year), from both sexes found in field at different places in Baghdad city, for six months (November 2014 to April 2015). All methods of culturing, Gram stain and biochemical test were done according to (<a href="#Markey-B--Leonard-F--Archambault-M--Cullinane-A--Maguire-D--2014"><span class="Hyperlink">Marky et al., 2014</span></a>). Samples were cultured on MacConkey agar and Eosin Methylene blue agar and incubated aerobically at 37°C for 24- 48 hours<span class="CharOverride-5">; </span>the growing colonies were examined by naked eye concerning their shape, size and color. Then bacterial cells were stained by gram stain. IMViC” tests (Indole,<span class="CharOverride-5"> </span>Methyl Red, Voges-Proskauer and Citrate)<span class="CharOverride-10"> </span>and Triple sugar iron medium. Culturing on two specific media, Cefixime Tellurite - Sorbitol MacConkey agar (CT-SMAC)[ LABM™ (England) ] and CHROM agar O157 [The Pioneer of Chromogenic Media/Paris] according to (<a href="#Chow-VTK--Inglis-TJJ--Peng-Song-K--2006"><span class="Hyperlink">Chow et al., </span><span class="Hyperlink">2006</span></a>) and confirmation by using Latex agglutination Test for<span class="CharOverride-6"> E. coli</span> O157:H7. This test was used for serotyping of <span class="CharOverride-6">E. coli</span> O157:H7 by using commercial kit (Wellcolex <span class="CharOverride-6">E. coli</span> O157:H7, Remel) to detect both the somatic antigen O157 and the flagellar antigen H7 according to the manufacturer company.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp; </p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">PCR Assay for Detection of <span class="CharOverride-15">eaeA</span><span class="CharOverride-2"> </span>gene in Isolated<span class="CharOverride-6"> </span><span class="CharOverride-2">E. coli </span>O157:H7.</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-5">DNA extraction:</span> Genomic DNA of <span class="CharOverride-6">E. coli </span>O157:H7<span class="CharOverride-6"> </span>isolate was extracted by using (Presto™ Mini g DNA Bacteria Kit Geneaid. USA) according to manufacture procedure.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-5">Oligonucleotide primer: </span>The oligonucleotide primers for <span class="CharOverride-6">eaeA</span> gene were:</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-3">F -5’ GAC CCG GCA CAA GCA TAA GC -3’</p>
			<p class="Body-Text ParaOverride-1">and </p>
			<p class="Body-Text ParaOverride-3">R -5’ CCA CCT GCA GCA ACA AGA GG -3’ </p>
		  <p class="Body-Text ParaOverride-3">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The product size was 384 pb were designed by (<a href="#Paton-AW--Paton-JC--1998-"><span class="Hyperlink">Paton and Paton, 1998</span></a>). The purity and concentration of extracted DNA was measured using Nanodrop spectrophotometer (NuDrops)™ [ActGene(USA)] .</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-5">Gel electrophoresis for check extracted DNA: </span>It was very important step to complete PCR assay, which was used to check the extracted DNA by loading the eluted DNA by agarose gel electrophoresis.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-5">Preparation of PCR Master Mix: </span>All required reagents were thawed completely and put them on ice, and reagent was mixed well by inversion and spins them down prior to pipetting. PCR master mix reaction was prepared by using GoTaq® Green Master Mix from Promega, USA.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp; </p>
			<p class="Body-Text ParaOverride-1">The PCR tubes containing an amplification mixture were transferred to thermal-cycler and started the program for amplification as shown in the <a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Table-1-"></a>Table 1: </span>PCR program for detection (<span class="CharOverride-6">eaeA</span>) gene</p>
			<table width="658" height="165" class="Table-Style-1" id="table-1">
				<colgroup>
					<col />
					<col />
					<col />
					<col />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">No. of cycles</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">Time</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">Temperature (</span><span class="Title CharOverride-17" xml:lang="ar-SA">°C</span><span class="Title CharOverride-16">)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">Step</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">5 min.</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">94</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">Initial denaturation</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td rowspan="3">
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">35</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">1min.</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">94</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">Denaturation</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">30 sec.</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">57</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">Annealing</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">1 min.</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">72</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">Extension</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">10min</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">72</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">Final extension</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td height="21" />
						<td />
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">Hold</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-5">PCR Product Analysis (Agarose Gel Electrophoresis): </span>It is a very important step to complete PCR assay, which was used to analyse the PCR product by agarose gel electrophoresis, Finally PCR products (bands) were visualized using a UV transilluminator[ Cleaver Scientific (U.K.)] and photographed by using digital camera. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Ethically Approved </p>
			<p class="Body-Text">This study was approved by the ethical and research committee of Veterinary Medicine of College, University of Baghdad, Ministry of High Education and Scientific Research.</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Heading-1--Introduction----">Results</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM-"><span class="CharOverride-2">E. coli </span>O157:H7<span class="CharOverride-2"> </span>Isolation</p>
			<p class="Body-Text ParaOverride-1">Different morphological shape and colour of <span class="CharOverride-6">E. coli </span>colonies were appeared on different media. The colonies revealed red /pink colour On MacConkey agar and metallic sheen on Eosin Methylene Blue. The Gram stain of suspected <span class="CharOverride-6">E. coli </span>colonies revealed, negative non spore forming rod. The isolated bacteria gave different reaction in biochemical tests. It gave negative for Voges–proskuar, simmon citrate and positive for indole and motility tests. The Triple sugar Iron test (TSI) showed Yellow with/without gas production. The isolated colonies of <span class="CharOverride-6">E. coli</span> appeared small, circular and colourless with smoky centre (1-2) mm in diameter on SMA-CT. On Chrome agar the colonies of <span class="CharOverride-6">E. coli </span>O157<span class="CharOverride-6"> </span>showed mauve colour. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Serotyping Test (Wellcolex <span class="CharOverride-2">E. coli </span>O157:H7, Remel) </p>
			<p class="Body-Text"><span class="CharOverride-6">Escherichia coli </span>colonies from (SMA-CT) were tested for identification of both O157:H7<span class="CharOverride-6"> </span>antigens by Wellcolex <span class="CharOverride-6">E. coli </span>O157:H7, Remel. The isolates that gave a positive reaction for the O157<span class="CharOverride-6"> </span>antigen were sub-cultured overnight on blood agar for the detection of flagellar antigen (H7). Red colour agglutination indicated a positive result for (O antigen) in comparison to clear red colour of the control and the blue colour agglutination indicated positive result for (H antigen) in comparison to clear blue colour of the control. </p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Prevalence of <span class="CharOverride-2">E. coli</span> O157:H7 in Calves</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-6">E. coli</span> was isolated at a high percentage from samples of Diarrheic and non-diarrheic calves, <span class="CharOverride-6">E. coli</span> O157:H7 appeared in 4 isolates (11.42%) from diarrheic calves, and 28 isolates of <span class="CharOverride-6">E. coli</span> O157:H7 were isolated from non-diarrheic calves (<a href="#Table-2"><span class="Hyperlink">Table 2</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Normal" xml:lang="en-US"><span class="CharOverride-21" xml:lang="en-GB"><a id="Table-2"></a>Table 2:</span><span class="CharOverride-22" xml:lang="en-GB"> Number and rate of infection of </span><span class="CharOverride-23" xml:lang="en-GB">E. coli </span><span class="CharOverride-22" xml:lang="en-GB">O157:H7</span><span class="CharOverride-23" xml:lang="en-GB"> </span><span class="CharOverride-22" xml:lang="en-GB">in diarrheic and healthy calves</span></p>
			<table width="657" height="94" class="Table-Style-1" id="table-2">
				<colgroup>
					<col />
					<col />
					<col />
					<col />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">No. of  </span><span class="Title CharOverride-24">E. coli</span><span class="Title CharOverride-16"> O157:H7</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">No. of  </span><span class="Title CharOverride-24">E .coli</span><span class="Title CharOverride-16"> isolates</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">No. of samples</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">Animals</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">4(11.42%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">32(91.42%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">35</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">Diarrheic calves</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">28(8.88%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">306(97.14%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">315</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">Non-diarrheic calves</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">32(9.14%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">338(96.57)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">350</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">Total</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Heading-2--History-in-MM-">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-"><span class="CharOverride-2">E. coli </span>O157:H7 Confirmation by PCR</p>
			<p class="Body-Text ParaOverride-1">The confirmation process of the <span class="CharOverride-6">E. coli</span> O157:H7 isolates recovered from fecal samples of calves to detect the presence of specific virulence trait <span class="CharOverride-6">eaeA</span> gene by PCR assay, all four isolates from diarrheic calves were possess <span class="CharOverride-6">eaeA</span> gene (100%) and 13 (46.42%) of isolates from non-diarrheic calves were positive for <span class="CharOverride-6">eaeA</span> gene. The study revealed that 17(53.12%) from total isolates gave positive results with <span class="CharOverride-6">eaeA </span>primers equal to target product size(384bp) (<a href="#Table-3-"><span class="Hyperlink">Table 3 </span></a>and <a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text"><span class="CharOverride-25"><a id="Table-3-"></a>Table 3: </span><span class="CharOverride-26">E. coli</span><span class="CharOverride-27"> O157:H7 and % of virulence factor </span><span class="CharOverride-26">eaeA</span></p>
			<table width="657" height="94" class="Table-Style-1" id="table-3">
				<colgroup>
					<col />
					<col />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">No. of </span><span class="Title CharOverride-24">eaeA</span><span class="Title CharOverride-16"> positive</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-16">No. of  </span><span class="Title CharOverride-24">E. coli</span><span class="Title CharOverride-16"> O157:H7</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">4(100%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">4(12.5%)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">13(46.42%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">28(8.80%)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">17(53.12%)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="Title CharOverride-18">32(9.14%)</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p>&nbsp;</p>
			
            <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150614210808.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150614210808.png" width="80" height="80"></a>
            
       <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Figure-1-"></a>Figure 1: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150614210808.png">Agarose gel electrophoresis showed amplification of 384 bp fragments of <span class="CharOverride-6">eaeA</span> genes of <span class="CharOverride-6">E. coli </span>O157:H7</a></p>
       </div>

			
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-19">Lane M</span><span class="CharOverride-29"> shows PCR marker</span></p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">Discussion </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-30">This is the first study which describes the detection and frequency of major virulence genes of STEC isolated from cattle in Baghdad, Iraq. Study revealed 4 isolates of </span><span class="CharOverride-31">E. coli</span><span class="CharOverride-30"> O157:H7 from 35 fecal samples at a percent (</span>11.42%) in diarrheic calves and all these isolates possessed <span class="CharOverride-6">eaeA</span> gene. Non diarrheic calves showed 28(8.88%) positive samples and 13(46.42%) possessed <span class="CharOverride-6">eaeA</span> gene.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-6">Escherichia coli </span>O157:H7 are generally recognized by culturing on different media, on sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC), these results were compatible with <a href="#Garcia-A--Fox-J--Besser-T--2010-"><span class="Hyperlink">Garcia et al. (2010)</span></a> were they found that typical <span class="CharOverride-6">E. coli</span> O157:H7 appeared as colourless colonies and do not fermented sorbitol on SMAC agar while most non-O157 strains ferment sorbitol and appear as pink colour colonies on SMAC agar. Another group of researcher, <a href="#Tahamtan-Y--Pourbakhsh--Hayati-SA--Namdar-M--Shams-N-and-Namvari-ZMM--2011"><span class="Hyperlink">Tahamtan et al. (2011)</span></a> use sorbitol-MacConkey agar plate supplemented with potassium tellurite (2.5 mg/L) and variant cifixime (0.05 mg/L). The <span class="CharOverride-6">E. coli</span> O157:H7 on SMAC agar O157 colonies appear clear due to their inability to ferment sorbitol unlike other <span class="CharOverride-6">E. coli </span>serotypes. <a href="#Laegreid-WW--Elder-RO--Keen-JE--1999"><span class="Hyperlink">Laegreid et al. (1999)</span></a> also used sorbitol-MacConkey agar SMAC plates containing cifixime (0.5 mg), and potassium tellurite (2.5 mg) for isolation of<span class="CharOverride-6"> E. coli</span> O157:H7 from calves, After 18 hour incubation at 37°C the sorbitol negative colonies appear colourless.<span class="CharOverride-6"> </span></p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Our results showed that Chrome agar aids in diagnosis of <span class="CharOverride-6">E. coli</span> O157:H7, it utilizes one of chromogenic substrates which produce mauve colour colonies, while non- <span class="CharOverride-6">E. coli</span> O157:H7 organism may utilize chromogenic substrates resulting in blue to blue green colour colonies, our results are in agreement with <a href="#Tavakoli-H--Bayat-M--Kousha-A--Panahi-P--2008-."><span class="Hyperlink">Tavakoli et al. (2008)</span></a> who recorded that the use of chrome agar allowed presumptive identification of <span class="CharOverride-6">E. coli</span> O157:H7 from the primary isolation plate and differentiation from other organisms. A similar study by <a href="#Yousif-AA--Al-Taii-DH--2014"><span class="Hyperlink">Yousif and Al-Taii (2014</span><span class="Hyperlink">)</span></a> reported that the Chrom agar is useful for diagnosis of <span class="CharOverride-6">Escherichia coli </span>0157:H7.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Latex agglutination test appeared a highly sensitive and specific for the<span class="CharOverride-11">&#160;diagnosis&#160;of</span> <span class="CharOverride-6">E.</span><span class="CharOverride-11">&#160;</span><span class="CharOverride-34">coli</span><span class="CharOverride-11">&#160;O157:H7</span>, this results in agreement with <a href="#Yousif-AA--Al-Taii-DH--2014"><span class="Hyperlink">Yousif and Al-Taii (2014)</span></a>, <a href="#Al-Dawmy-FAA--Yousif-AA--2013"><span class="Hyperlink">Al-Dawmy and Yousif (2013)</span></a> and <a href="#Karmali-MA--Petric-M--Bielaszewska-M--1999-."><span class="Hyperlink">Karmali </span><span class="Hyperlink">et al. (1999)</span></a> who used latex agglutination test for serotyping of <span class="CharOverride-6">E. coli</span> O157:H7. And describe it as a rapid, reliable, easy to perform and interpret, and it should allow testing for VT to become more widely performed.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The percentage of <span class="CharOverride-6">E. coli</span> O157 isolation from calves were compatible with <a href="#Omisakin-F--MacRae-M--Ogden-ID--Strachan-NJ--2003-"><span class="Hyperlink">Omisakin et al. (2003)</span></a><span class="CharOverride-5"> </span>they reported the prevalence of carriage of <span class="CharOverride-6">E. coli</span> O157 in faeces of cattle was 7.5% and with study of <a href="#Alam-MJ--Zurek-L--2006"><span class="Hyperlink">Alam and Zurek (2006)</span></a><span class="CharOverride-35"> </span>who found the prevalence of <span class="CharOverride-6">Escherichia coli</span> O157:H7 in beef cattle faeces was (9.2%) Another study conducted by <a href="#Kang-SJ--Ryu-SJ--Chae-JS--Eo-SK--Woo-GJ--Lee-JH--2004"><span class="Hyperlink">Kang et al. (2004)</span></a> was compatible with our study as they found the prevalence of E. coli O157 in diarrheic calves at percentage 9.8% and with<span class="CharOverride-5"> </span><a href="#Kuyucuoglu-Y---eker-E--Uguz-C--Saryyupoglu-B--Konak-S--2011"><span class="Hyperlink">Kuyucuoglu et al. (2011)</span></a> as they estimate the prevalence of <span class="CharOverride-6">E. coli</span> O157:H7 in diarrheic calves at percentage (10.6). Whereas <a href="#Blanco-J--Blanco-M--Blanco-JE--Alonso-MP--1993-"><span class="Hyperlink">Blanco et al. (1993)</span></a> found that the prevalence of<span class="CharOverride-6"> Eschrechia coli </span>O157:H7 in the faeces of dairy calves and feedlot cattle is low (0.3 to 2.2%) in the United States, the United Kingdom, Germany, and Spain. While <a href="#Mechie-SC--Chapman-PA--Siddons-CA--1997-"><span class="Hyperlink">Mechie et al. (1997)</span></a> recorded the prevalence of <span class="CharOverride-6">Escherichia coli </span>O157:H7 in calves a high percentage (56%) in England.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The prevalence of <span class="CharOverride-6">E. coli</span> O157:H7 in the current study was higher than that reported by <a href="#El-Shehedi-MA--Mostafa-ME--Aisha-RA--2013-."><span class="Hyperlink">El-Shehedi et al. (2013)</span></a> in AL-Qalyoubia Governorate in Egypt in diarrhoeic calves at level 6.97%.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">In non-diarrheic calves, the results showed that the prevalence of <span class="CharOverride-6">E. coli</span> O157:H7(8.88%) was higher than the percentage recorded by <a href="#Kuyucuoglu-Y---eker-E--Uguz-C--Saryyupoglu-B--Konak-S--2011"><span class="Hyperlink">Kuyucuoglu et al. (2011)</span></a>, as they found 2.6% of healthy calves infected with <span class="CharOverride-6">E. coli</span> O157:H7.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The occurrence of <span class="CharOverride-6">E .coli</span> O157:H7 were also detected in different regions of Turkey. For instances, <span class="CharOverride-6">E. coli</span> O157 was found in 14 individuals among 330 cattle slaughtered in five different abattoir in Istanbul (<a href="#Yilmaz-A--Gun-H--Yilmaz-H--2002"><span class="Hyperlink">Yilmaz et al., 2002</span></a>) and <span class="CharOverride-6">E. coli</span> O157:H7 were isolated in 4 individuals among 312 cattle in the eastern region of Turkey (<a href="#Aslantas----Erdogan-S--Cantekin-Z--G-lacti-I--Evrendilek-GA--2006"><span class="Hyperlink">Aslantas et al., 2006</span></a>). In another study, the rate of <span class="CharOverride-6">E. coli</span> O157:H7 infection was found to be 13.6% (<a href="#Cabalar-M--Boynukara-B--G-lhan-T--Ekin-IH--2001"><span class="Hyperlink">Cabalar et al., 2001</span></a>), this point was very important because turkey was a neighbouring country to Iraq. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The results showed that <span class="CharOverride-6">eaeA</span> gene found in a percentage (53.12%) in isolates of <span class="CharOverride-6">E. coli</span> O157:H7 from diarrheic and non-diarrheic calves. This results agreed with <a href="#Galland-JC--Hyatt-DR--Crupper-SS-and-Acheson-DW--2001-"><span class="Hyperlink">Galland et al. (2001)</span></a> who found that 26 from 57 <span class="CharOverride-6">Escherichia coli </span>O157:H7 beef cattle feedlots in southwest Kansas were <span class="CharOverride-6">eaeA </span>gene positive. But other researcher record a high percentage (100%), <a href="#Schouten-JM--Bouwknegt-M--van-de-Giessen-AW--Frankena-K--De-Jong-MC--Graat-EA--2004"><span class="Hyperlink">Schouten et al. (2004)</span></a> found all <span class="CharOverride-6">Escherichia coli </span>O157 isolates on Dutch dairy farms show positive for <span class="CharOverride-6">eaeA</span> gene, <a href="#Synge-BA--Chase-Topping-ME--Hopkins-GF--McKendrick-IJ--Thomson-Carter-F--Gray-D--Rusbridge-SM--Munro"><span class="Hyperlink">Synge et al. (2003)</span></a> recorded that all of the VTEC O157 tested were <span class="CharOverride-6">eaeA</span> positive from beef suckler cows in Scotland and <a href="#Alam-MJ--Zurek-L--2006"><span class="Hyperlink">Alam and Zurek (2006)</span></a> showed that all tested isolates of <span class="CharOverride-6">Escherichia coli</span> O157:H7 in beef cattle were positive for <span class="CharOverride-6">eaeA</span> (Intimin) gene.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Gene <span class="CharOverride-6">eaeA</span> (Intimin) which was a necessary gene for attaching and effacing activity (<a href="#Kaper-JB--Elliott-S--Sperandio-V--Perna-NT--Mayhew-GF--Blattner-FR--1998-."><span class="Hyperlink">Kaper et al., 1998</span></a>). Many investigators have underlined the strong association between carrying <span class="CharOverride-6">eaeA</span> and the capacity of STEC to cause severe human disease, especially HUS (<a href="#Oswald-E--Schmidt-H--Morabito-S--Karch-H--March-s-O--Caprioli-A--2000"><span class="Hyperlink">Oswald et al., 2000</span></a>). In the present study, this important virulence gene was detected in 100% of STEC O157:H7 in diarrheic calves and (46.42%) of non-diarrheic calves isolates. A similar prevalence of the intimin (<span class="CharOverride-6">eaeA</span>) gene has also been found in other studies (<a href="#Blanco-J--Blanco-2001"><span class="Hyperlink">Blanco et al., 2001</span></a>; <a href="#Blanco-M--Blanco-2004"><span class="Hyperlink">Blanco et al., 2004</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Epidemiological studies on EHEC in cattle are very necessary to develop control measures in order to reduce the risk of transmission from cattle to humans. Since isolation procedures are laborious and time-consuming and because of the lack of biochemical features distinguishing most EHEC strains from nonpathogenic <span class="CharOverride-6">E. coli</span>, PCR approaches based on the detection of EHEC-associated genetic markers have been developed<span class="CharOverride-5"> </span>(<a href="#Bibbal-D--Loukiadis-E--K-rour-dan-M--De-Garam-CP--Ferr--F--Cartier-P--Gay-E--Oswald-E--Auvray-F--Bru"><span class="Hyperlink">Bibbal et al., 2014</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">In conclusion, this study revealed the importance of <span class="CharOverride-6">E. coli </span>O157:H7 serotype calves, which represent as a reservoir for strains that transmitted the disease to human. <span class="CharOverride-6">E. coli </span>O157:H7 virulence gene <span class="CharOverride-6">eaeA</span> (intimine) was detected in faeces samples collected from calves using PCR. Therefore, we believe that the isolation of <span class="CharOverride-6">E. coli </span>O157:H7 serotypes in Iraq will be beneficial to the many researchers in this field and for investigation of future epidemiological study.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Acknowledgments</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">This work was supported by College of Veterinary Medicine, Department of Internal and Preventive Veterinary Medicine, University of Baghdad, Iraq. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Author’s cONTRIBUTION</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Both authors contributed equally in all details of this manuscript.</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">Authors declares no conflict of interest.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">References</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
		
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