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<body id="AAVS-MH20141216021212_Chaitanya-et-al" lang="en-GB">
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			<p class="Type-of-Article">&nbsp;</p>
			<p class="Type-of-Article"><span class="CharOverride-1">Research Article</span></p>
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			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Strain Typing of <i>Mycobacterium avium</i> subsp. <i>paratuberculosis </i>from Tamil Nadu, India based on Polymorphisms in MAP <i>1506</i> Locus and IS <i>1311</i> PCR-REA</p>
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			<p class="Authors">&nbsp;</p>
			<p class="Authors"><span class="CharOverride-3" lang="en-US">Radha Krishna </span><span class="CharOverride-3">Chaitanya</span><span class="CharOverride-4">1</span><span class="CharOverride-3">, </span><span class="CharOverride-3" lang="en-US">Yarabolu Krishna Mohan </span><span class="CharOverride-3">Reddy</span><span class="CharOverride-4">2</span><span class="CharOverride-3">, </span><span class="CharOverride-3" lang="en-US">Arthanari </span><span class="CharOverride-3">Thangavelu</span><span class="CharOverride-4">3</span></p>
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			<p class="Affiliations ParaOverride-1"><span class="CharOverride-5">1</span>Department of Veterinary Microbiology, College of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh - 517502, India; <span class="CharOverride-5">2</span>Vaccine Research Centre-Viral Vaccines, Centre for Animal Health studies, Madhavaram milk colony, Tamil Nadu Veterinary and Animal Sciences University, Tamil Nadu- 600051, India; <span class="CharOverride-5">3</span>Department of Veterinary Microbiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Tamil Nadu- 600007, India.</p>
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			<p class="Abstract">&nbsp;</p>
			<p class="Abstract"><span class="CharOverride-6">Abstract</span> | <span class="CharOverride-7">Mycobacterium avium</span> subsp. <span class="CharOverride-7">paratuberculosis</span> (MAP) causes Johne’s disease, a progressive chronic granulomatous enteritis affecting the domestic ruminants. Dung samples from cattle, sheep and goat were collected from the suspected clinical cases in livestock farms of different regions of  Tamil Nadu state, India. All the samples were subjected to IS <span class="CharOverride-7">900 </span>PCR for the detection of MAP and 45% were found to be positive. All the positive samples were subjected to IS <span class="CharOverride-7">1311</span> PCR-REA that confirmed MAP ‘Bison type’ is most prevalent cause of JD among the domestic ruminants in Tamil Nadu. MAP isolation has been attempted from the tissue samples collected from the animals that were slaughtered being positive for JD. A total of 4 isolates were obtained and were characterized by IS<span class="CharOverride-7">1311 </span>PCR-REA, MAP<span class="CharOverride-7">1506</span> PCR and sequence analysis which identified the single nucleotide polymorphisms  that allow strain typing. One isolate from sheep intestine was identified as “Intermediate type” strain by MAP <span class="CharOverride-7">1506</span> PCR-sequence analysis. To the best of our understanding, this is the first report of ‘Intermediate type’ strain of MAP from sheep from India.</p>
		  <p class="Abstract">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-6">Keywords </span>| <span class="CharOverride-7">Mycobacterium avium</span> subsp. <span class="CharOverride-7">paratuberculosis, </span>strain typing, Tamil Nadu, IS <span class="CharOverride-7">1311</span> PCR-REA, MAP<span class="CharOverride-7">1506</span> PCR and sequence analysis.</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
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			<p class="Editor----Citation"><span class="CharOverride-9">Editor</span> | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-6">Received</span> | December  16, 2014; <span class="CharOverride-6">Revised</span> | January 05, 2015; <span class="CharOverride-6">Accepted</span> | January 06, 2015; <span class="CharOverride-6">Published</span> | April 10, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-6">*Correspondence</span> | Radha Krishna Chaitanya, Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh, India; <span class="CharOverride-6">Email: </span>chaitanyaerk@gmail.com</p>
			<p class="Editor----Citation"><span class="CharOverride-6">Citation</span> | Chaitanya RK, Krishna Mohan Reddy Y, Thangavelu A (2015). Strain typing of <span class="CharOverride-28">Mycobacterium avium</span> subsp. <span class="CharOverride-28">paratuberculosis</span> from Tamil Nadu, India based on polymorphisms in MAP<span class="CharOverride-28"> 1506</span> locus and IS<span class="CharOverride-28"> 1311</span><span class="CharOverride-6"> </span>PCR-REA. Adv. Anim. Vet. Sci. 3(5): 289-294.  </p>
			<p class="Editor----Citation"><span class="CharOverride-9">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.5.289.294"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.5.289.294</span></a></p>
			<p class="Editor----Citation"><span class="Editor---Citation CharOverride-6" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-6" lang="en-US">(</span><span class="Editor---Citation CharOverride-6" lang="en-US">Online</span><span class="Editor---Citation CharOverride-6" lang="en-US">)</span> | 2307-8316; <span class="Editor---Citation CharOverride-6" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-6" lang="en-US">(Print) </span> | 2309-3331</p>
			<p class="Editor----Citation"><span class="CharOverride-9">Copyright </span>© 2015 Chaitanya 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>
			<p class="Editor----Citation">&nbsp;</p>
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			<p class="Heading-1--Introduction----">Introduction</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">J</span>ohne’s disease (JD) caused by <span class="CharOverride-7">Mycobacterium avium</span> subsp. <span class="CharOverride-7">paratuberculosis</span> (MAP) affects wide range of hosts and is responsible for significant economic losses to the livestock industry, due to poor productivity, wasting and early culling of animals. MAP strains have been classified into three groups: Type I (Sheep type), Type II (Cattle type) and Type III (Intermediate type) based on IS <span class="CharOverride-7">900</span> PCR- Restriction fragment length polymorphism (RFLP), pulsed field gel electrophoresis (<a href="#Juan-L--Mateos-A--Dominguez-L--Sharp-JM--Stevenson-K--2005"><span class="Hyperlink">Juan et al., 2005</span></a>); analysis of sequences of gyr A, gyr B and inh A genes (<a href="#Castellanos-E--Aranaz-A--Romero-B--Juan-LD--Alvarez-J--Bezos-J--Rodriguez-S--Stevenson-K--Malteos-A-"><span class="Hyperlink">Castellanos et al</span><span class="Hyperlink">., 2007</span></a>) and based on polymorphisms in MAP <span class="CharOverride-7">1506</span> locus (<a href="#Griffiths-TA--Rioux-K--Buck-JD--2008-.-S"><span class="Hyperlink">Griffiths et al., 2008</span></a>). MAP strains isolated from bison in Montana, USA were identified as ‘Bison type” based on polymorphisms in insertion sequence IS <span class="CharOverride-7">1311 </span>by <a href="#Whittington-RJ--Marsh-IB--Whitlock-RH--2001-."><span class="Hyperlink">Whittington et al. (2001)</span></a> who developed a PCR-Restriction Enzyme Analysis (REA) method that differentiate Cattle, Sheep, and Bison type strains of MAP. MAP strains from India have also been identified as belong to ‘Bison type’ but are different from those of USA and represent a new biotype and have wide host range. Recent reports (<a href="#Singh-AV--Singh-SV--Singh-PK--Sohal-JS--2010-."><span class="Hyperlink">Singh et al., 2010</span></a>; <a href="#Kaur--Filia-G--Singh-SV--Patil-PK--Ravi-Kumar-GVPPS--Sandhu-KS--2011-"><span class="Hyperlink">Kaur et al., 2011</span></a>) have pointed out the existence of ‘cattle type/ Type I’ strains in cattle, buffalo and goats in north India. Also there is evidence of strain sharing and interspecies transmission among different host species (<a href="#Pavlik-I--Horvathova-A--Dvorska-L--Bartl-J--Svastova-P--Du-MR--Rychlik-I--1999-"><span class="Hyperlink">Pavlik et al., 1999</span></a>; <a href="#Whittington-RJ--Marsh-IB--Whitlock-RH--2001-."><span class="Hyperlink">Whittington et al., 2001</span></a>). </p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Strain typing can also be achieved by identifying single nucleotide polymorphisms SNPs in MAP <span class="CharOverride-7">1506</span> gene locus. MAP <span class="CharOverride-7">1506</span> (MACPPE23) is a member of PPE protein family and contains MAP type specific SNPs in its sequence. Proline-Proline-Glutamic acid (PPE) proteins constitute a polymorphic protein family that is restricted to mycobacteria. Previously, these SNPs were targeted to type MAP isolates by <a href="#Griffiths-TA--Rioux-K--Buck-JD--2008-.-S"><span class="Hyperlink">Griffiths et al. (2008)</span></a> and <a href="#Castellanos-E--Aranaz-A--Buck-JD--2010"><span class="Hyperlink">Castellanos et al. (2010)</span></a>. The objective of the present study was to identify the strain types of MAP affecting the domestic ruminant population in Tamil Nadu, India. An IS <span class="CharOverride-7">1311 </span>PCR- REA, MAP <span class="CharOverride-7">1506</span> PCR and sequence analysis were used to differentiate between MAP isolates from cattle, sheep and goats.</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="Body-Text ParaOverride-1">The study includes a total of 213 dung samples collected from cattle (n=93), sheep (n=68) and goat (n=52) with long term history of emaciation from the organized livestock farms of Tamil Nadu. Intestine samples from cattle (n=4) and a sheep (n=1) were also collected from the animals that were slaughtered after being confirmed as positive for JD. Genomic DNA from the dung and tissue samples was extracted using Quiagen stool DNA mini kit and Quiagen tissue DNA extraction kits, respectively following the manufacturer’s instructions. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Detection of MAP by IS<span class="CharOverride-2">900 </span>PCR</p>
			<p class="Body-Text ParaOverride-1">Polymerase chain reaction to amplify a 229 bp product of IS <span class="CharOverride-7">900</span> gene was carried out as described by <a href="#Vary-PH--Andersen-PR--Green-E--Taylor-JH--Mc-Fadden-JJ--1990-."><span class="Hyperlink">Vary et al. (1990)</span></a> with few modifications.<span class="CharOverride-6"> </span>The reaction mixture (25 µl volume) for IS <span class="CharOverride-7">900 </span>PCR for each sample include 4µl of DNA template, 12.5 µl of master mix, 1µl each of Forward and Reverse primers (10 pico moles concentration) and 6.5 µl nuclease free water (NFW). The PCR conditions were initial denaturation at 94ºC for 5 min., and 35 cycles of 94ºC for 30 sec., 55ºC for 15 sec., 72ºC for 30 sec., and final extension at 72ºC for 7 min.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">IS <span class="CharOverride-2">1311 </span>PCR – REA for Strain Identification</p>
			<p class="Body-Text ParaOverride-1">The samples found positive for MAP by IS <span class="CharOverride-7">900</span> PCR were subjected to PCR for amplification of a 608 bp region of IS <span class="CharOverride-7">1311 </span>gene of MAP and restriction enzyme analysis as described by <a href="#Marsh-IB--Whittington-R--Cousins-D--1999-"><span class="Hyperlink">Marsh et al. (1999)</span></a>.The reaction mixture for IS <span class="CharOverride-7">1311</span> PCR includes 3µl of DNA template, 14 µl of master mix, 1µl each of Forward and Reverse primers (10 pico moles concentration) and 6 µl NFW. The PCR conditions were initial denaturation at 94ºC for 5 min and 35 cycles of 94ºC for 30 sec., 62ºC for 15 sec., 72ºC for 60 sec., and final extension at 72ºC for 7 min. The amplicons of IS <span class="CharOverride-7">1311</span> gene were subjected to restriction enzyme analysis using <span class="CharOverride-7">Hinf 1</span> and <span class="CharOverride-7">Mse 1</span>. Reaction mixture for restriction enzyme analysis includes 8 µl of IS <span class="CharOverride-7">1311</span> PCR amplicon, 1 µl each of <span class="CharOverride-7">Hinf 1</span> and <span class="CharOverride-7">Mse 1</span> , 1.6 µl each of RE Buffer and bovine serum albumin (BSA) which was made up to 16 µl with NFW. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Isolation of MAP and Strain Typing</p>
			<p class="Body-Text ParaOverride-1">Isolation was carried out in Middlebrook 7H9 broth and <span lang="en-US">Herrold’s Egg Yolk Medium (HEYM)</span> from all the five tissue samples as per OIE, 2008. The isolates were characterized by IS <span class="CharOverride-7">900</span>, IS <span class="CharOverride-7">1311 </span>PCR- REA, MAP<span class="CharOverride-7">1506 </span>PCR and sequence analysis. The sequences of IS <span class="CharOverride-7">1311</span> amplicons of all the isolates were analysed for the presence of restriction enzyme sites using NEB cutter.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">MAP <span class="CharOverride-2">1506 </span>PCR</p>
			<p class="Body-Text ParaOverride-1">The isolates were characterized by PCR for amplification of 499 bp region of MAP <span class="CharOverride-7">1506 </span>gene as per the method of <a href="#Castellanos-E--Aranaz-A--Buck-JD--2010"><span class="Hyperlink">Castellanos et al. (2010)</span></a> and sequence analysis. The sequences were compared with gene sequences of MAP K-10 reference strain and MAA 104 available with the GenBank. Multiple alignment of nucleotide sequence of partially amplified MAP <span class="CharOverride-7">1506 </span>PCR products was done by CLUSTAL-W method and a phylogenetic tree was drawn by Neighbour-Joining method using MEGA 5 software. The pair wise identity matrix was done in MegAlign software (DNASTAR Inc).</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="Body-Text ParaOverride-1">The concentration of DNA extracted from dung samples assessed by Biophotometer Plus (Eppendorf) ranged from 10-25 μg/ml and DNA from tissue samples ranged from 22-35 μg/ml.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Detection of MAP by IS <span class="CharOverride-2">900 </span>PCR</p>
			<p class="Body-Text ParaOverride-1">Out of 213 samples of dung subjected to IS <span class="CharOverride-7">900</span> PCR, 95 samples (45 from cattle, 32 from sheep and 18 from goat) (45 per cent) revealed specific amplicons of 229 bp size suggestive of MAP infection. All the tissue samples (100 per cent) were found positive for MAP. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">IS <span class="CharOverride-2">1311</span> PCR-Restriction Enzyme Analysis for Strain Identification</p>
			<p class="Body-Text ParaOverride-1">All the samples which were positive by IS <span class="CharOverride-7">900 </span>PCR were also confirmed for the IS <span class="CharOverride-7">1311</span> specific amplicons of 608 bp size. Restriction digestion of amplicons using <span class="CharOverride-7">Hinf 1</span> and <span class="CharOverride-7">Mse 1 </span>restriction enzymes revealed three bands of sizes 67 bp, 218 bp and 323 bp in all the samples confirming them as ‘bison type’. The intestine sample from Ooty sheep revealed two bands of 323 bp and 285bp.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Isolation of MAP and Strain Typing</p>
			<p class="Body-Text ParaOverride-1">Four isolates <span class="CharOverride-7">viz</span>., Ooty sheep, MVC cattle, MVC goat, URF cattle were obtained in MB 7H9 broth after 12-16 weeks. Growth in culture was confirmed as MAP by Ziehl-Neelsen staining followed by IS <span class="CharOverride-7">900 </span>PCR (<a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>). All the four MAP isolates obtained in the present study along with ABT cattle isolate received from the Department of Animal biotechnology, Madras Veterinary College, TANUVAS and BV-I and BV-II sheep isolates received from Bacterial Vaccines division, TANUVAS were characterized. All the seven isolates generated IS <span class="CharOverride-7">1311</span> specific amplicons of 608 bp size (<a href="#Figure-2-"><span class="Hyperlink">Figure 2</span></a>). Restriction enzyme digestion of IS <span class="CharOverride-7">1311</span> amplicons with <span class="CharOverride-7">Hinf 1</span> and <span class="CharOverride-7">Mse 1</span> resulted in two fragments of 323 bp and 285 bp for Ooty sheep isolate, a band pattern specific for MAP ‘Sheep type’. All other isolates revealed three fragments of 323 bp, 218 bp and 67 bp, a band pattern specific for ‘Bison type’ strains of MAP upon electrophoresis of RE digested product in 4 per cent agarose gel (<a href="#Figure-3-"><span class="Hyperlink">Figure 3</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			
          <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150414030724.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150414030724.png" width="80" height="80"></a>
            
           <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-1-"></a>Figure 1: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150414030724.png">IS <span class="CharOverride-7">900</span> PCR for MAP confirmation of the isolates showing 229 bp amplicon</a></p>
       </div>

			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-13">Lane 1 &amp; 5:</span><span class="CharOverride-12">100 bp DNA marker;</span><span class="CharOverride-13"> Lane 2:</span><span class="CharOverride-12"> ABT Cattle isolate; </span><span class="CharOverride-13">Lane 3: </span><span class="CharOverride-12">URF Cattle isolate; </span><span class="CharOverride-13">Lane 4: </span><span class="CharOverride-12">Negative control: </span><span class="CharOverride-13">Lanes 6, 7, 8:</span><span class="CharOverride-12"> MVC Goat, MVC cattle and Ooty sheep isolates, respectively.</span></p>
		  <p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150414031125.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150414031125.png" width="80" height="80"></a>
            
          <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-2-"></a>Figure 2: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150414031125.png">IS <span class="CharOverride-7">1311</span> PCR of the isolates showing 608 bp amplicon</a></p>
       </div>
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-13">Lane 1:</span><span class="CharOverride-12"> 100 bp DNA marker;</span><span class="CharOverride-13"> Lanes 2, 3, 4: </span><span class="CharOverride-12">Ooty sheep, URF cattle, MVC cattle isolates respectively; </span><span class="CharOverride-13">Lane 5:</span><span class="CharOverride-12"> Negative control: </span><span class="CharOverride-13">Lane 6:</span><span class="CharOverride-12"> MVC goat isolate.</span></p>
		  <p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Sequence analysis of IS <span class="CharOverride-7">1311</span> gene of the isolates in comparison with the MAP K-10 reference strain and MAA 104 complete genome revealed point mutations at base positions 68, 236, 422 and 527 that differentiate these from <span class="CharOverride-7">Mycobacterium avium </span>subsp. <span class="CharOverride-7">avium </span>(MAA) and confirming them as MAP. ABT/MVC/URF cattle, MVC goat, BV-I and II sheep isolates showed an additional point mutation/ nucleotide polymorphism (C/T) at base position 223 that causes a second <span class="CharOverride-7">Hinf 1</span> restriction site. Whereas the Ooty sheep isolate has ‘C’ at 223 bp position. The sequences were also analysed for the presence of MAP specific restriction enzyme (<span class="CharOverride-7">Hinf 1</span>) sites using NEB cutter (New England Bio Labs online software). Ooty sheep isolate revealed a single <span class="CharOverride-7">Hinf 1</span> restriction site and ABT isolate had two <span class="CharOverride-7">Hinf 1</span> restriction sites. None of the isolates shown <span class="CharOverride-7">Mse 1</span> restriction site which is specific for MAA.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">MAP <span class="CharOverride-2">1506</span> PCR and Sequence analysis</p>
			<p class="Body-Text ParaOverride-1">All the seven MAP isolates showed specific bands of 499 bp of MAP <span class="CharOverride-7">1506</span> gene (<a href="#Figure-4-"><span class="Hyperlink">Figure 4</span></a>). Sequence analysis of MAP <span class="CharOverride-7">1506</span> gene loci from all the six isolates (ABT/MVC/URF cattle, MVC goat, BV-I and II sheep isolates that were identified as ‘Bison type’ in IS <span class="CharOverride-7">1311</span> PCR-REA and sequence analysis) have nucleotide substitutions (nucleotide 293, 328, 344, 411 and 542) that that categorized them into Cattle/Bison type (Type II) and clearly distinguished them from Type I strains. The Ooty sheep isolate (classified as “Sheep type” by IS <span class="CharOverride-7">1311</span> PCR-REA) has SNPs that classified it into ‘Intermediate type’ and these SNPs are also present in MAP <span class="CharOverride-7">1506</span> homologues in MAA (<a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Phylogenetic analysis revealed that Ooty sheep isolate was closely related to MAA with percent identity of 99.1%. Remaining isolates are closely related to MAP K10 with the percent identity of 99.8 to 100% (<a href="#Figure-5-"><span class="Hyperlink">Figure 5</span></a>). The partial nucleotide sequences of MAP <span class="CharOverride-7">1506 </span>gene of the isolates were submitted to the GenBank and allotted with the accession number JF 794776 for Ooty sheep isolate and KP 326413 to 326418 for ABT cattle, BV-I, BV-II sheep, MVC cattle, MVC goat and URF cattle isolates respectively.</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 clinical samples (dung and tissues) were screened by IS <span class="CharOverride-7">900</span> PCR as this insertion sequence is found to be specific for this subspecies and being present in 14-18 copies in the MAP genome offers high sensitivity of detection by PCR (<a href="#Green-EP--Tizard-MLV--Moss-MT--Thompson-J--Winterbourne-DJ--McFadden-JJ--Taylor-JH--1989"><span class="Hyperlink">Green et al.,1989</span></a>; <a href="#Bull-TJ--Hermon-Taylor-J--Pavlik-I--El-Zaatari-F--Tizard-M--2000-."><span class="Hyperlink">Bull et al., 2000</span></a>; <a href="#Vansnick-EL--de-Rijk-OP--Vercammen-F--Geysen-D--Rigouts-L--Portaels-F--2004"><span class="Hyperlink">Vansnick et al., 2004</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			
		  <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150414035925.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150414035925.png" width="80" height="80"></a>
            
       <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-3-"></a>Figure 3: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150414035925.png">IS <span class="CharOverride-7">1311</span> PCR-Restriction enzyme analysis using <span class="CharOverride-7">Hinf 1, Mse 1 </span>of the isolates</a></p>
       </div>
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-13">Lane 3 &amp; 10:</span><span class="CharOverride-12"> 100 bp DNA marker; </span><span class="CharOverride-13">Lane 1: </span><span class="CharOverride-12">Ooty sheep showing ‘Sheep type’ IS </span><span class="CharOverride-17">1311 </span><span class="CharOverride-12">band profile with 323 bp and 285 bp band;</span><span class="CharOverride-17"> </span><span class="CharOverride-13">Lane 4-9:</span><span class="CharOverride-12"> BV-l &amp; ll sheep, ABT/MVC/URF cattle, MVC goat isolates, repectively, showing ‘bison type’ pattern with 323 bp, 218 bp and 67 bp fragments; </span><span class="CharOverride-13">Lane 11: </span><span class="CharOverride-12">BV-ll isolate.</span></p>
			<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"><span class="CharOverride-6"><a id="Table-1-"></a>Table 1: </span>SNPs identified within the MAP <span class="CharOverride-7">1506</span> gene of isolates with different IS <span class="CharOverride-7">1311</span> profiles</p>
			<table width="657" height="255" 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-4" />
					<col class="_idGenTableRowColumn-4" />
					<col class="_idGenTableRowColumn-4" />
					<col class="_idGenTableRowColumn-5" />
					<col class="_idGenTableRowColumn-6" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-7">
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-19">Isolate</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-19">Host animal</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-19">IS</span><span class="CharOverride-20">1311</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-19">PCR-REA Profile</span></p>
						</td>
						<td colspan="5">
							<p class="Basic-Paragraph"><span class="CharOverride-19">Position on MAP 1506</span><span class="CharOverride-19"> gene</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-19">Strain type</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">bp 293</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">bp 328</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">bp 344</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">bp 411</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">bp 542</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">ABT-Cattle</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Cattle</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Bison type</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Type II</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">MVC-Cattle</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Cattle</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Bison type</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Type II</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">URF- Cattle</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Cattle</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Bison type</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Type II</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">MVC-Goat</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Goat</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Bison type</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Type II</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">BV-I Sheep</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Sheep</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Bison type</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Type II</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">BV-II Sheep</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Sheep</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Bison type</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Type II</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Ooty-Sheep</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Sheep</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Sheep type</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">A</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">A</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">C</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Type III</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td colspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-21">MAA 104 CP000479.1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">avium type</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">A</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">C</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">MAA</span><span class="CharOverride-22">*</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td colspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-21">MAP K-10 AE016958.1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Cattle type</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">G</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">T</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-21">Type II</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1">* <span class="CharOverride-24">Mycobacterium avium</span><span class="CharOverride-14"> subsp.</span><span class="CharOverride-24"> avium</span></p>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150414031127.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150414031127.png" width="80" height="80"></a>
            
      <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-4-"></a>Figure 4: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150414031127.png">MAP <span class="CharOverride-7">1506</span> PCR of the isolates showing 499 bp amplified product</a></p>
       </div>
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-13">Lane 4: </span><span class="CharOverride-12">100 bp DNA marker; </span><span class="CharOverride-13">Lane 1: </span><span class="CharOverride-12">ABT cattle isolate; </span><span class="CharOverride-13">Lane 2:</span><span class="CharOverride-12"> Negative Control; </span><span class="CharOverride-13">Lane 3:</span><span class="CharOverride-12"> Ooty sheep isolate; </span><span class="CharOverride-13">Lane 5, 6, 7:</span><span class="CharOverride-12"> MVC goat, MVC cattle and URF cattle isolates, repectively.</span></p>
		  <p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Identification of MAP strains prevailing in a geographical region helps in tracking the origin of infection, the risk factors contributing its transmission, pathogenecity and helps in designing the control programmes (<a href="#Motiwala-AS--Li-L--Kapur-V--Sreevatsan-S--2006-."><span class="Hyperlink">Motiwala et al., 2006</span></a>). Several molecular techniques have been developed by scientists to characterize MAP strains. IS <span class="CharOverride-7">900</span>-RFLP (<a href="#Pavlik-I--Horvathova-A--Dvorska-L--Bartl-J--Svastova-P--Du-MR--Rychlik-I--1999-"><span class="Hyperlink">Pavlik et al., 1999</span></a>), Pulsed field gel electrophoresis analysis (<a href="#Stevenson-K--Hughes-VM--Juan-LD--Inglis-NF--Wright-F--Sharp-JM--2002"><span class="Hyperlink">Stevenson et al., 2002</span></a>; <a href="#Juan-L--Mateos-A--Dominguez-L--Sharp-JM--Stevenson-K--2005"><span class="Hyperlink">Juan et al., 2005</span></a>) of the genome enable characterization of MAP strains into ‘Sheep/Type I’, ‘Cattle/Type II’ and ‘Intermediate/ Type III’. But these techniques require high quantity of pure genomic DNA and hence difficult to employ for field samples directly and extremely slow growing ‘Sheep type’ strains. Rapid tools like DMC-PCR (<a href="#Collins-DM--Zoete-MD--Cavaignac-SM--2002"><span class="Hyperlink">Collins et al., 2002</span></a>) and RDA-PCR (<a href="#Dohmann-K---Strommenger-B--Stevenson-K--Juan-LD--Stratmann-J--Kapur-V--Bull-TJ--Gerlach-GF--2003-."><span class="Hyperlink">Dohmann et al., 2003</span></a>) can distinguish MAP Type II (Cattle) strains from Type I (Sheep) strains. However, these techniques cannot further differentiate type III (Intermediate) strains from type I. Moreover, these techniques are unable to detect “Bison Type” strains which have been reported to be the predominant MAP strains causing JD among livestock in North India (<a href="#Sevilla-I--Singh-SV--Garrido-JM--Aduriz-G--Rodr-guez-S--Geijo-MV--Whittington-RJ--Saunders-V--Whitlo"><span class="Hyperlink">Sevilla et al., 2005</span></a>; <a href="#Sohal-JS---Sheoran-N---Narayanasamy-K--Brahmachari-V--Singh-S--Subodh-S--2009-."><span class="Hyperlink">Sohal et al., 2009</span></a>).Keeping in view these facts, in the present study IS <span class="CharOverride-7">1311</span> PCR and restriction enzyme analysis using <span class="CharOverride-7">Hinf 1 </span>and<span class="CharOverride-7"> Mse 1</span> which can distinguish MAP strains into Cattle type, Sheep type and Bison type were employed to identify the prevalent MAP strains directly from the clinical samples in Tamil Nadu, India. In the current study, it was found that, in all the cases of JD the strain of MAP involved is of ‘Bison type’ except in one case which was identified as ‘Sheep type’ indicating the heterogeneity of MAP in cattle and sheep. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The results of this study are in accordance with the observations of <a href="#Sevilla-I--Singh-SV--Garrido-JM--Aduriz-G--Rodr-guez-S--Geijo-MV--Whittington-RJ--Saunders-V--Whitlo"><span class="Hyperlink">Sevilla et al. (2005</span></a><span class="Hyperlink">)</span>, <a href="#Singh-AV--Singh-SV--Singh-PK--Sohal-JS--2010-."><span class="Hyperlink">Singh et al. (2010)</span></a> and <a href="#Kaur--Filia-G--Singh-SV--Patil-PK--Ravi-Kumar-GVPPS--Sandhu-KS--2011-"><span class="Hyperlink">Kaur et al. (2011)</span></a> who reported that MAP ‘Bison type’ is the predominant strain type affecting livestock population in India. Diversity in MAP strains isolated from cattle and sheep have been reported earlier and were attributed to host-pathogen interactions, specificity or host preference of MAP strains and adaptation to the environmental factors by <a href="#Whittington-RJ--Hope-AF--Marshall-DJ--Taragel-CA--Marsh-I--2000-"><span class="Hyperlink">Whittington et al. (2000)</span></a> and <a href="#Bhide-M--Chakurkar-E--Tkacikova-L--Barbuddhe-S--Novak-M--Mikula-I--2006-"><span class="Hyperlink">Bhide et al. (2006)</span></a>.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150414030328.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150414030328.png" width="80" height="80"></a>
            
     <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-5-"></a>Figure 5:</span><a href="http://nexusacademicpublishers.com/uploads/figures/20150414030328.png">Phylogenetic tree of MAP isolates based on MAP <span class="CharOverride-7">1506</span> locus</a></p>
       </div>
			
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">IS <span class="CharOverride-7">1311</span> PCR-REA cannot differentiate type III (intermediate) strains from type I (Sheep type) strains. Hence sequence analysis of MAP <span class="CharOverride-7">1506</span> locus that can discriminate between MAP type I and III strains is employed to further characterize the isolates. <a href="#Griffiths-TA--Rioux-K--Buck-JD--2008-.-S"><span class="Hyperlink">Griffiths et al. (2008)</span></a> reported sequence differences in MAP 1506 locus between MAP type I, II and intermediate (type III) strains and developed a PCR- degenerating gradient gel electrophoresis that distinguish these strains. Based on the polymorphisms, <a href="#Castellanos-E--Aranaz-A--Buck-JD--2010"><span class="Hyperlink">Castellanos et al.</span><span class="Hyperlink"> (2010)</span></a> developed a real time PCR-High resolution melt analysis targeting 499 bp region of MAP <span class="CharOverride-7">1506</span> locus for strain typing.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The present results are in agreement with <a href="#Castellanos-E--Aranaz-A--Buck-JD--2010"><span class="Hyperlink">Castellanos et al. (2010)</span></a> and <a href="#Griffiths-TA--Rioux-K--Buck-JD--2008-.-S"><span class="Hyperlink">Griffiths et al. (2008)</span></a>, and also in concurrence with the results of IS <span class="CharOverride-7">1311</span> PCR-REA results. It is found that the isolate from Ooty sheep is of ‘Intermediate type’ and is more closely related to MAA based on SNPs in MAP <span class="CharOverride-7">1506 </span>locus. This finding is in agreement with the finding of <a href="#Whittington-RJ--Marsh-I--Choy-E--Cousins-D--1998-."><span class="Hyperlink">Whittington et al. (1998)</span></a> who reported that ‘S’ strains are evolutionary intermediates between MAA and MAP cattle strains based on SNPs in IS <span class="CharOverride-7">1311</span> gene. The same was proved in this study through the phylogenetic analysis of IS <span class="CharOverride-7">1311</span> genes of the isolates. SNPs have been frequently targeted in epidemiological and evolutionary studies to differentiate between closely related species and strains of bacteria. However, strain typing based on SNPs can be more meaningful when they target SNPs in the functional coding regions of the gene, especially those linked with host specificity.&#9;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">In the present study, when comparing the results of IS <span class="CharOverride-7">1311</span> PCR-REA with published data it is evident that “bison type” MAP stains are prevalent and are the major cause of JD among livestock in Tamil Nadu. However, the possibility of existence of other types cannot be overlooked which is evident from the findings of this study. Sheep/ Intermediate strains may be under reported owing to their extremely slow growth rate which precludes their isolation in culture. Further investigations with large number of isolates are required to understand the epidemiological distribution and significance of the MAP types. </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 declare that they have no conflict of interest related to the work.</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 are thankful to the Vice-Chancellor, Tamil Nadu Veterinary and Animal Sciences University, Chennai for providing the necessary facilities for this work. We are also thankful to the Department of Biotechnology, Madras Veterinary College and <span lang="en-US">Vaccine Research Centre-</span>Bacterial Vaccines<span lang="en-US">, TANUVAS, Chennai</span> Division for providing MAP isolates.</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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