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			<p class="Type-of-Article" lang="en-GB">&nbsp;</p>
			<p class="Type-of-Article" lang="en-GB"><span class="CharOverride-1">Short Communication</span></p>
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			<p class="title- ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="title- ParaOverride-1" lang="en-GB">Genetic Variation in the rDNA ITS-2 Sequence of Haemonchus placei from Cattle Host </p>
		</div>
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			<p class="Authors ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Authors ParaOverride-1" lang="en-GB"><span class="CharOverride-3" lang="en-US">Qasim Ali</span><span class="CharOverride-4" lang="en-US">1</span><span class="CharOverride-3" lang="en-US">, Muhammad Imran Rashid</span><span class="CharOverride-4" lang="en-US">1</span><span class="CharOverride-3" lang="en-US">, </span><span class="CharOverride-3" lang="en-US">Kamran Ashraf</span><span class="CharOverride-4" lang="en-US">1</span><span class="CharOverride-3" lang="en-US">, </span><span class="CharOverride-3" lang="en-US">Muhammad Nauman Zahid</span><span class="CharOverride-4" lang="en-US">2</span><span class="fwb CharOverride-5" lang="en-US">, </span><span class="CharOverride-3" lang="en-US">Shoaib Ashraf</span><span class="CharOverride-4" lang="en-US"> 4</span><span class="CharOverride-3" lang="en-US">, Umer Chaudhry</span><span class="CharOverride-4" lang="en-US"> 3 </span><span class="CharOverride-3">&#160;</span><span class="CharOverride-3" lang="en-US">  </span><span class="CharOverride-3">   </span></p>
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			<p class="Normal ParaOverride-1"><span class="CharOverride-7" lang="en-GB">1</span><span class="CharOverride-8">Department of Parasitology, University of Veterinary and Animal Science, Lahore, Pakistan; </span><span class="CharOverride-7">2</span><span class="CharOverride-8"> Department of Microbiology, University of Veterinary and Animal Science, Lahore, Pakistan; </span><span class="CharOverride-7">3 </span><span class="CharOverride-8">Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Alberta, Canada; </span><span class="CharOverride-7">4 </span><span class="CharOverride-8">Department of Pharmacology, University of Veterinary and Animal Science, Lahore, Pakistan. </span></p>
		</div>
		<div>
			<p class="Abstract ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Abstract ParaOverride-1" lang="en-GB"><span class="CharOverride-9">Abstract </span>| The<span class="CharOverride-10"> </span>large stomach worm, <span class="CharOverride-10">Haemonchus</span>, commonly known as the barber’s pole worm, is a blood sucking nematode found in the abomasa of small and large ruminants. Allele-specific amplification of the rDNA internal transcribed spacer-2 (ITS-2) sequences was performed from the total of 78 individual adult worms to screen <span class="CharOverride-10">Haemonchus placei</span> at species level, which is the significant diagnostic tool to identify this major economically important species. Further full sequences analysis of the ITS-2 region revealed that there are 4 sites shows intraspecific variations at position<span class="CharOverride-10"> </span>65, 111, 125 and 148. For instance this study is the first documented report of intraspecific genetic variations in the rDNA ITS-2 sequences of <span class="CharOverride-10">H. placei</span> from cattle in Pakistan and the results shows that <span class="CharOverride-10">H. placei</span> is genetically different from the isolates studied previously. However, detailed and large size samples strategy will be required to identify the co-infection and interspecies hybridization between <span class="CharOverride-10">H. placei</span> and <span class="CharOverride-10">Haemonchus contortus</span> in cattle.       </p>
		  <p class="Abstract ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Abstract ParaOverride-1" lang="en-GB"><span class="CharOverride-9">Keywords</span> | <span class="CharOverride-10" lang="en-US">Haemonchus placei</span><span lang="en-US">, rDNA ITS-2, Pakistan</span></p>
		  <p class="Abstract ParaOverride-1" lang="en-GB">&nbsp;</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-12">Editor</span> | Tahir Yaqub, University of Veterinary and Animal Sciences, Lahore, Pakistan.</p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-9">Received</span> | August 20, 2014; <span class="CharOverride-9">Revised</span> | October 13, 2014; <span class="CharOverride-9">Accepted</span> | October 25, 2014; <span class="CharOverride-9">Published</span> | December 02, 2014&#9;&#9;</p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-9">*Correspondence</span> | Umer Chaudhry, University of Calgary, Alberta, Canada; <span class="CharOverride-9">Email:</span> unchaudh@ucalgary.ca</p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-9">Citation</span> | Ali Q, Rashid MI, Ashraf K, Zahid MN, Ashraf S, Chaudhry U (2015). Genetic variation in the rDNA ITS-2 sequence of Haemonchus placei from cattle host. J. Inf. Mol. Biol. 3(1): 13-18.</p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-12">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.jimb/2015/3.1.13.18"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.jimb/2015/3.1.13.18</span></a>  </p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-12">I</span><span class="CharOverride-9">SSN (Online)</span> |  2307-5465; <span class="CharOverride-9">ISSN (Print)</span>  | 2307-5716</p>
			<p class="Editor----Citation" lang="en-GB"><span class="CharOverride-12">Copyright </span>© 2015 Ali 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>
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			<p class="Caps-on-First-Para" lang="en-GB">&nbsp;</p>
			<p class="Caps-on-First-Para" lang="en-GB">&nbsp;</p>
			<p class="Caps-on-First-Para" lang="en-GB"><span class="_idGenDropcap-1">G</span>astrointestinal parasitic (GI) nematodes are extremely important agents of both human and animal disease <span lang="en-US">(</span><a href="#Sutherland-IA--Leathwick-DM--2011"><span class="Hyperlink" lang="en-US">Sutherland </span><span class="Hyperlink CharOverride-14" lang="en-US">and Leathwick, 2011</span></a><span class="CharOverride-15" lang="en-US">)</span>. GI nematode infections constitute a significant threat to the health and welfare of grazing livestock throughout the world and impose a significant cost in terms of productivity in grazing animals. GI parasitic infections of livestock are among the most economically important diseases in the livestock of Pakistan <span lang="en-US">(</span><a href="#Annonymous-2012-2013."><span class="Hyperlink" lang="en-US">Annonymous, </span><span class="Hyperlink CharOverride-14" lang="en-US">2012-2013</span></a><span lang="en-US">)</span>.  </p>
		  <p class="Caps-on-First-Para" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">The major GI nematode species in cattle are <span class="CharOverride-10">H. placei</span>, <span class="CharOverride-10">Ostertagia ostertagi</span>, <span class="CharOverride-10">Mecistocirrus digitatus</span> and <span class="CharOverride-10">Trichostrongylus axei</span>, <span class="CharOverride-10">Strongyloides papillosus</span>, <span class="CharOverride-10">Cooperia</span> and <span class="CharOverride-10">Nematodirus</span> and <span class="CharOverride-10">Oesophagostomum radiatum</span> found in the abomasum, small intestine and large intestine <span lang="en-US">(</span><a href="#Oku-Y--Nakazawa-M--Hatakeyama-S--Miyaji-S--Kitaguchi-T--Cabrera-Lopez-CA--Okamoto-M--Kamiya-M--Ohbay"><span class="Hyperlink" lang="en-US">Oku et al., 1987</span></a><span lang="en-US">; </span><a href="#Fukumoto-S--Etani-K--Toi-K--Hanadate-M--Hidaka-M--Yokoya-K--Hiramatsu-T--Iguchi-T--Kudo-S--Miyamoto"><span class="Hyperlink" lang="en-US">Fukumoto et al., 1990</span></a><span lang="en-US">)</span>. Of these species, <span class="CharOverride-10">H. placei</span>, <span class="CharOverride-10">O. ostertagi</span> and <span class="CharOverride-10">Cooperia oncophora</span> are considered the most prevalent in cattle; in particular, the infection rate of <span class="CharOverride-10">H. placei</span> was high. This nematode is commonly described as a large stomach worm, namely, a trichostrongyloid nematode, and is an important blood-sucking nematode present in the abomasum of cattle. This parasite may cause mucosal inflammation, haemorrhage, ulcers and necrosis in the abomasum <span lang="en-US">(</span><a href="#Gennari-SM--Bressan-MC--Rogero-JR--MacLean-JM--Duncan-JL--1991"><span class="Hyperlink" lang="en-US">Gennari et al., 1991</span></a><span lang="en-US">)</span>. It appears to be confined mainly to Asian countries; however, it has also been found in North America, Australia, and Brazil. The movement of its hosts for agricultural purposes has resulted in the global spread of this parasite. </p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Generally, ruminants are concurrently infected with more than one species of GI nematodes, each having a different pathological effect on the host. It has been a difficult task to eradicate GI nematodes from grazing ruminants due to the variation in host susceptibility to the parasite, the wide distribution of nematodes in nature and the presence of wild ruminants. An effective method is required for the control of nematodes by reducing their infection rate and their transmission to the host in order to protect cattle for production losses. Strategies for the control of nematodes by using anthelmintic drugs should be devised based on the quantitation and identification of species <span lang="en-US">(</span><a href="#Mochizuki-R--Endoh-D--Onuma-M--Fukumoto-S--2006-"><span class="Hyperlink" lang="en-US">Mochizuki et al., 2006</span></a><span lang="en-US">)</span>. </p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">The rDNA ITS-2 sequences are the most commonly used markers to discriminate among nematode species <span lang="en-US">(</span><a href="#Gasser-RB--Newton-SE--2000"><span class="Hyperlink" lang="en-US">Gasser and Newton, 2000</span></a><span lang="en-US">). This sequence has been a popular choice because of its variability and is under concerted evolution (</span><a href="#Nadler-SA--Hoberg-EP--Hudspeth-DS--Rickard-LG--2000-."><span class="Hyperlink" lang="en-US">Nadler et al., 2000</span></a><span lang="en-US">)</span>. Thus the ITS-2 gene has been widely applied in species identification within the genus <span class="CharOverride-10">Haemonchus </span>(<a href="#Gasser-RB--1999-.-PC"><span class="Hyperlink">Gasser, 1999</span></a>; <a href="#Heise-M--Epe-C--Schnieder-T--1999"><span class="Hyperlink">Heise et al., 1999</span></a>). For example, the separate species status of <span class="CharOverride-10">H. placei</span> and <span class="CharOverride-10">H. contortus</span> was supported by ITS-2 data, where three fixed nucleotide differences at position 24, 205 and 219 were reported by <a href="#Stevenson-LA--Chilton-NB--Gasser-RB--1995-."><span class="Hyperlink">Stevenson et al. (1995)</span></a> and <a href="#Chaudhry-UN--E.M.R.--Muthusamy-R--Abbas-M--Gilleard-JS--2014-"><span class="Hyperlink">Chaudhry et al. (2014)</span></a>. There have only been a few documented studies on the genetic variation within the isolates of <span class="CharOverride-10">H. placei</span>, however the variations within <span class="CharOverride-10">H. contortus</span> ranged between 4.0 to 5.2% <span lang="en-US">(</span><a href="#Heise-M--Epe-C--Schnieder-T--1999"><span class="Hyperlink">Heise et al., 1999</span></a><span lang="en-US">; </span><a href="#Stevenson-LA--Chilton-NB--Gasser-RB--1995-."><span class="Hyperlink">Stevenson et al., 1995</span></a><span lang="en-US"> and </span><span class="Hyperlink">Chaudhry et al., 2014</span><span lang="en-US">). Genetic characterization is important for accurate identification and effective control due to the anthelmintic resistance problem in this nematode (</span><a href="#Gasser-RB--2006-."><span class="Hyperlink" lang="en-US">Gasser, 2006</span></a><span lang="en-US">)</span>. The present study contributes to validate the allele<span class="CharOverride-9">-</span>specific molecular base marker and genetic variations in the ITS-2 region of this important nematode species from cattle host. </p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Adult worms were harvested on necropsy from the abomasa of ruminant hosts collected from abattoirs in Punjab province of Pakistan (4 populations) (<a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>). Following ethanol fixation, worms were examined under a dissecting microscope to determine whether they belongs to <span class="CharOverride-10">Haemonchus</span> genera based on size and gross appearance <span lang="en-US">(</span><a href="#Lichtenfels-JR--Pilitt-PA--2000-."><span class="Hyperlink" lang="en-US">Lichtenfels and Pilitt, 2000</span></a><span lang="en-US">)</span>. Species identity was subsequently confirmed by molecular methods as described below. Overall, the dataset was composed of 78 individual specimens of adult worms from the genus <span class="CharOverride-10">Haemonchus</span> distributed among four different populations from individual cattle host.</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Adult worms were fixed in 70% ethanol immediately following removal from the host abomasum. The heads of individual worms were dissected and lysed in single 0.2ul tube containing 50µl of proteinase K lysis buffer and stored at -80°C as previously described <span lang="en-US">(</span><a href="#Redman-E--Packard-E--Grillo-V--Smith-J--Jackson-F--Gilleard-JS--2008-.-Microsatellite-analysis-revea"><span class="Hyperlink" lang="en-US">Redman et al., 2008</span></a><span lang="en-US">)</span>. A neat single worm lysate dilution, 1µl of 1:5, was used as PCR template and identical dilutions of lysate buffer, made in parallel, were used as negative controls. </p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">The rDNA ITS-2 region was amplified from individual <span class="CharOverride-10">Haemonchus</span> adult worm lysates using a allele-specific forward primer complementary to 5’ prim end of rDNA ITS-2 coding sequence (HpITS-2F: 5’-<span class="CharOverride-17">atactacaatgtggctag-</span>3’) and reverse primer complimentary to the 3’ prim end of rDNA coding sequence (HpITS-2R: 5’- TGATAAAAGAACATCGTT-3’). A 231bp fragment spanning the entire ITS-2 rDNA region was PCR amplified using a 50µl master mix containing final concentrations of 1X thermopol reaction buffer, 2mM MgSO<span class="CharOverride-18">4, </span>100uM of each dNTPs, 0.1uM forward and reverse primers and 1.25U Taq DNA polymerase (New England Biolabs). Thermo-cycling parameters were 95<span class="CharOverride-19">o</span>C for 5 min followed by 35 cycles of 95<span class="CharOverride-19">o</span>C for 1 min, 55<span class="CharOverride-19">o</span>C for 1 min and 72<span class="CharOverride-19">o</span>C for 1 min with a single final extension cycle of 72<span class="CharOverride-19">o</span>C for 5 min. </p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text" lang="en-GB">The entire rDNA ITS-2 region was amplified from <span class="CharOverride-10">Haemonchus</span> (identified as a <span class="CharOverride-10">H. placei</span> section 3.1) adult worm lysates using a “universal” forward primer complementary to 5.8S rDNA coding sequence (NC1F: 5’-<span class="CharOverride-17">acg tct ggt tca ggg ttg tt- </span>3’) and reverse primer complimentary to the 28S rDNA coding sequence (NC2R: 5’-TTA GTT TCT TTT CCT<span class="CharOverride-20"> </span>CCG CT- 3’) <span lang="en-US">(Stevenson et al., 1995)</span>. A 321bp fragment spanning the entire ITS-2 rDNA region was PCR amplified using a 50µl master mix containing final concentrations of 1X thermopol reaction buffer, 2mM MgSO<span class="CharOverride-18">4, </span>100uM of each dNTPs, 0.1uM <span class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-11">Positions 24, 205 and 219 (indicated in larger font) have fixed interspecies polymorphisms whereas the other listed positions show intra-specific variation. rDNA ITS-2 sequence polymorphisms identified from a total of 66 individual derived from four </span><span class="CharOverride-27">H. placei</span><span class="CharOverride-11"> populations: H1C, H2C, H3C, and H4C</span></span> <span class="Body-Text ParaOverride-1">forward and reverse primers and 1.25U Taq DNA polymerase (New England Biolabs). Thermo-cycling parameters were 95<span class="CharOverride-19">o</span>C for 5 min followed by 35 cycles of 95<span class="CharOverride-19">o</span>C for 1 min, 57<span class="CharOverride-19">o</span>C for 1 min and 72<span class="CharOverride-19">o</span>C for 1 min with a single final extension cycle of 72<span class="CharOverride-19">o</span>C for 5 min.  rDNA ITS-2 amplicons from individual worm lysates were further were sequenced using the reverse primer (NC2R: 5-TTA GTT TCT TTT CCT<span class="CharOverride-20"> </span>CCG CT- 3’) on an ABI Prism 377 capillary sequencer. Sequences were edited and aligned with <span class="CharOverride-10">H. placei</span> ITS-2 sequences available in Genebank (Acc No X78812) using Geneious Pro 5.4 software <span class="CharOverride-28">(</span><a href="http://www.geneious.com/"><span class="Hyperlink CharOverride-28">http://www.geneious.com/</span></a><span class="CharOverride-28">)</span>. </span></p>
			<p class="Body-Text" lang="en-GB">&nbsp;</p>
		  <p class="Body-Text" lang="en-GB">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB"><span class="CharOverride-9"><a id="Table-1-"></a>Table 1:</span> Summary of the field populations of <span class="CharOverride-10">Haemonchus</span> collected from cattle host from Punjab (PN) region of Pakistan</p>
			<table width="657" height="117" 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" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">Field populations ID</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-21">Host </span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-21">Worm</span><span class="Title CharOverride-22"> </span><span class="Title CharOverride-21">number         </span><span class="Title CharOverride-23"> </span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-21">Origin</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-1"><span class="Title CharOverride-21">District</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">H1C</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">Cattle</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">33</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">Abattoir</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-22">Sargodha</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">H2C</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">Cattle</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">23</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">Abattoir</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-22">Lahore</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">H3C</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">Cattle</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">7</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">Abattoir</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-22">Okara</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">H4C</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">Cattle</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">16</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-2"><span class="Title CharOverride-22">Abattoir</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-22">Sahiwal </span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Normal">&nbsp;</p>
		  <p class="Normal">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB"><span class="CharOverride-9"><a id="Table-2"></a>Table 2:</span> Summary of inter- and intra-species variation in the <span class="CharOverride-10">H. placei</span> rDNA ITS-2 sequence. </p>
			<table width="657" height="186" class="Table-Style-1" id="table-2">
				<colgroup>
					<col class="_idGenTableRowColumn-1" />
					<col class="_idGenTableRowColumn-10" />
					<col class="_idGenTableRowColumn-3" />
					<col class="_idGenTableRowColumn-11" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-12">
						<td>
							<p class="Normal"><span class="Title CharOverride-26">Species</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-26">Alignment position</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-26">Nucleotide</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-26">Type of base exchange</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-13">
						<td rowspan="7">
							<p class="Normal"><span class="Title CharOverride-8">H. placei</span><span class="Title CharOverride-24"> populations</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-26">24</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-26">G</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-24" lang="fr-FR">Fix SNP</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-14">
						<td>
							<p class="Normal"><span class="Title CharOverride-24">65</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-24">A/T</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-24">Transversion</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td>
							<p class="Normal"><span class="Title CharOverride-24">111</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-24">A/G</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-24">Transition</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-15">
						<td>
							<p class="Normal"><span class="Title CharOverride-24">125</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-24">C/T</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-24">Transition</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Normal"><span class="Title CharOverride-24">148</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-24">A/T</span></p>
						</td>
						<td>
							<p class="Normal ParaOverride-3"><span class="Title CharOverride-24">Transversion</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-15">
						<td>
							<p class="Normal"><span class="Title CharOverride-24">205</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-24">A</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-24" lang="fr-FR">Fix SNP</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td>
							<p class="Normal"><span class="Title CharOverride-24">219</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-24">G</span></p>
						</td>
						<td>
							<p class="Normal"><span class="Title CharOverride-24" lang="fr-FR"> Fix SNP</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150117040341.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150117040341.png" width="80" height="80"></a>
            
            <p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB"><span class="CharOverride-9"><a id="Figure-1-"></a>Figure 1:</span><span class="CharOverride-14"> </span><span class="CharOverride-15"><a href="http://nexusacademicpublishers.com/uploads/figures/20150117040341.png">(A, B)</a></p>
       </div>

		  <p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB"> (A) <span class="CharOverride-10">H. placei</span> has fixed nucleotide sequence differences at position 24 and 205 of the ITS-2 sequence. This allowed the design of forward and reverse primers for which the final 3’ base was complementary to these positions and so specific respective species. (B) ITS-2 products amplified with <span class="CharOverride-10">H. placei</span> allele<span class="CharOverride-9">-</span>specific primers. M, 100bp ladder; Tracks 1-4; amplicons from <span class="CharOverride-10">H. placei </span>single worm DNA templates, Tracks 5-8; (NC) no template  negative control; HP PC, <span class="CharOverride-10">H. placei</span> positive control.		  </p>
		  <p class="Body-Text" lang="en-GB">
	      <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150117045041.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150117045041.png" width="80" height="80"></a>
            
            <p class="Figure--and-Table-Heading ParaOverride-1" lang="en-GB"><span class="CharOverride-9"><a id="Figure-2-"></a>Figure 2: </span><span class="CharOverride-14"> </span><span class="CharOverride-15"><a href="http://nexusacademicpublishers.com/uploads/figures/20150117045041.png">Sequence chromatogram of 1 out of 66 sequences (named<span class="CharOverride-10"> H. placei </span>sequences) obtained from 4 populations showing three fixed species-specific positions (P24, P205, P219) of the rDNA ITS-2 sequence of <span class="CharOverride-10">H. placei</span></a></p>
       </div>
			
		  <p class="Body-Text" lang="en-GB">&nbsp;</p>
		  <p class="Body-Text" lang="en-GB">Previous work has identified three single nucleotide polymorphisms (SNPs) in the rDNA ITS-2 (positions 24, 205 and 219) that show fixed sequence specific differences between <span class="CharOverride-10">H. placei</span> and other closely related species <span class="CharOverride-10">H. contortus</span> <span lang="en-US">(Stevenson et al., 1995)</span>. However this study was based on just two <span class="CharOverride-10">H. placei</span> and eight <span class="CharOverride-10">H. contortus</span> worms obtained from cattle and sheep respectively. Consequently, <a href="#Chaudhry-UN--E.M.R.--Muthusamy-R--Abbas-M--Gilleard-JS--2014-"><span class="Hyperlink">Chaudhry et al. (2014)</span></a> further provided that these inter-species differences were fixed when larger numbers of individuals from geographically diverse populations were examined.  In the present study,  allele-specific PCR primers were developed based on P24 and P219 SNPs, which will then be used to identify <span class="CharOverride-10">Haemonchus</span> populations for the presence of <span class="CharOverride-10">H. placei</span>. For this purpose, allele-specific PCR was then applied to screen all 78 individual worms (from 4 populations) collected from cattle and a total of 66 worms were identified <span class="CharOverride-10">H. placei</span>, rest of the 12 worms did not amplified with this assay (<a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>). </p>
		  <p class="Body-Text" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">The rDNA ITS-2 sequence was PCR amplified and sequenced from DNA derived from 66 worms in four <span class="CharOverride-10">Haemonchus </span>populations (identified <span class="CharOverride-10">H. placei</span> in section 3.1). In all cases, <span class="CharOverride-10">H. placei</span> sequences contained P24 (<span class="CharOverride-29">G</span>), P205 (<span class="CharOverride-29">A</span>), P219 (<span class="CharOverride-29">G</span>) confirming SNPs (<a href="#Table-2"><span class="Hyperlink">Table 2</span></a> and <a href="#Figure-2-"><span class="Hyperlink">Figure 2</span></a>) previously identified by <a href="#Stevenson-LA--Chilton-NB--Gasser-RB--1995-."><span class="Hyperlink" lang="en-US">Stevenson et al. (1995</span></a><span class="Hyperlink" lang="en-US">)</span> and <span class="Hyperlink">Chaudhry et al. (2014)</span>. In addition to confirming the three species-specific fixed SNPs, there were 4 sites that showed intraspecific variation in <span class="CharOverride-10">H. placei</span> at position<span class="CharOverride-10"> </span>65,111, 125 and 148 (<a href="#Table-2"><span class="Hyperlink">Table 2</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">Accurate identification of parasite species is crucial not only for diagnosis, treatment and control but epidemiological studies and clinical trials. Although, sufficient morphological differences exist among adult and larval GI parasitic nematodes to allow their accurate identification, the availability of similar techniques for nematode eggs remains an obstacle to reliable diagnosis. In some cases the structure and size of the egg can be diagnostic; however, in many instances, similarities among eggs from different species and even distinct genera require alternative methods for their differentiation. Presently, the method commonly utilized for GI parasitic nematodes involves in-vitro cultivation of egg to infective third stage larval (L3) recovery and direct collection of adult parasite from slaughtered animals followed by morphological identification. These procedures are labour intensive, time consuming and prone to errors due to variation in egg viability and parasite development in culture.<span class="CharOverride-9"> </span>The advent of DNA technology has provided alternative approaches for the identification of parasites and molecular techniques like allele-specific PCR have proven to be useful in species identification. Studies have shown that the rDNA ITS-2 contain reliable genetic markers to distinguish closely-related species of trichostrongylid nematodes <span lang="en-US">(</span><a href="#Gasser-RB--Chilton-NB--Hoste-H--Beveridge-I--1993"><span class="Hyperlink" lang="en-US">Gasser et al., 1993</span></a><span lang="en-US">; </span><a href="#Campbell-AJ--Gasser-RB--Chilton-NB--1995-"><span class="Hyperlink" lang="en-US">Campbell et al., 1995</span></a><span lang="en-US">; </span><a href="#Chilton-NB--Gasser-RB--Beveridge-I--1995-."><span class="Hyperlink" lang="en-US">Chilton et al., 1995</span></a><span lang="en-US">; </span><a href="#Stevenson-LA--Chilton-NB--Gasser-RB--1995-."><span class="Hyperlink" lang="en-US">Stevenson et al., 1995</span></a><span lang="en-US">; </span><a href="#Wimmer-B--Craig-BH--Pilkington-JG--Pemberton-JM--2004"><span class="Hyperlink" lang="en-US">Wimmer et al., 2004</span></a><span lang="en-US">)</span>. </p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB"><span class="CharOverride-10">Haemonchus</span> spp. are important, highly pathogenic blood feeding parasites of cattle in many parts of the world, particular in warmer regions with high humidity, such as Asia, south America and the southern USA. <span class="CharOverride-10">H. placei</span> is the most common species traditionally reported from cattle <span lang="en-US">(</span><a href="#Gasbarre-LC--Smith-LL--Lichtenfels-JR--Pilitt-PA--2009a-."><span class="Hyperlink" lang="en-US">Gasbarre et al., 2009a</span></a><span lang="en-US">; </span><a href="#Gasbarre-LC--Smith-LL--Hoberg-E--Pilitt-PA---2009b"><span class="Hyperlink" lang="en-US">Gasbarre et al., 2009b</span></a><span lang="en-US">)</span>. Of the four populations examined (one from Sargodha, one from Lahore, one from Okara and one from Sahiwal), the prevalence of 85% <span class="CharOverride-10">H. placei</span> suggesting this is still the predominant <span class="CharOverride-10">Haemonchus</span> species infecting cattle in Pakistan. </p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Body-Text" lang="en-GB">Indeed, inter<span class="CharOverride-9">-</span>species variation in the ITS-2 sequences between <span class="CharOverride-10">H. placei</span> and <span class="CharOverride-10">H. contortus</span> in cattle, sheep and goats host revealed minor differences such as substitutions of three residues at position P24, P205 and P219, many investigators also indicate the intra-specific variation of ITS-2 sequences among <span class="CharOverride-10">H. contortus</span> from domestic ruminant in the world, however very limited information about the genetic variability in <span class="CharOverride-10">H. placei</span> in ruminants currently available worldwide and particularly on the south Asian region. This work investigates further insight into genetic relationships of <span class="CharOverride-10">H. placei</span> from Pakistani region. Therefore we se quenced ITS-2 region of 66 <span class="CharOverride-10">H. placei</span> individuals and our analysis was conducted from four different cattle host to avoid strain specific nucleotide variations. The results demonstrate three species specific SNPs [P24 (<span class="CharOverride-29">G</span>), P205 (<span class="CharOverride-29">A</span>), P219 (<span class="CharOverride-29">G</span>)] and four intra-specific variations sites at position 65,111, 125<span class="CharOverride-10"> </span>and<span class="CharOverride-10"> </span>148 in <span class="CharOverride-10">H. placei</span> (<a href="#Table-2"><span class="Hyperlink">Table 2</span></a>). Of those, position 65, 123 and 148 have been previously shown in <span class="CharOverride-10">H. placei</span> <span lang="en-US">(</span><a href="#Stevenson-LA--Chilton-NB--Gasser-RB--1995-."><span class="Hyperlink" lang="en-US">Stevenson et al., 1995</span></a><span lang="en-US">; </span><span class="CharOverride-20" lang="en-US"> </span><span class="Hyperlink">Chaudhry et al., 2014</span><span lang="en-US">)</span>. However position 111 polymorphic site had not been identified previously, so we hypothesized that P111 is a new intra-specific variation present in <span class="CharOverride-10">H. placei</span> from Pakistani region. The newly identified genetic variation in the ITS-2 sequence represents a significant addition to databases.  </p>
		  <p class="Body-Text" lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">To our knowledge, this is the first study in Pakistan exploring the genetic variation in <span class="CharOverride-10">H. placei</span>. These results can further use to monitor <span class="CharOverride-10">Haemonchus</span> infection in a context of gastro-intestinal control approaches further integrated to a sustainable agriculture. In or der to confirm the development of <span class="CharOverride-10">H. placei</span> resistant to anthelmintic in cattle further study must be carried out.</p>
			<p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1" lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" lang="en-GB">References</p>
		  <p class="Heading-1--Introduction----" lang="en-GB">&nbsp;</p>
			
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