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<body id="Nexus-632" lang="en-GB">
		<div class="Basic-Text-Frame">
			<p class="Type-of-Article">&nbsp;</p>
			<p class="Type-of-Article">Research Article</p>
	</div>
		<div class="Basic-Text-Frame">
			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Characterization of <i>Rhodococcus equi</i> Isolates from Foals with Respiratory Problems using a Multiplex PCR for the <i>vap</i> Genes</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Authors ParaOverride-1">&nbsp;</p>
			<p class="Authors ParaOverride-1"><span class="CharOverride-2">Sourabh Chhabra</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Sandip Kumar Khurana</span><span class="CharOverride-3">2*</span><span class="CharOverride-2">, Pradeep Kumar Kapoor</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Harisankar Singha</span><span class="CharOverride-3">2</span><span class="CharOverride-2">, Yarvendra Singh</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Richa Khirbat</span><span class="CharOverride-3">3</span></p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Affiliations ParaOverride-1"><span class="CharOverride-4">1</span>Department of Veterinary Public Health and Epidemiology, LUVAS, Hisar; <span class="CharOverride-4">2</span>National Research Centre on Equines, Hisar; <span class="CharOverride-4">3</span>Department of Animal Biotechnology, LUVAS, Hisar, India.</p>
		</div>
		<div>
			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Abstract</span> | <span class="CharOverride-6">Rhodococcus equi</span> is widely associated with subacute or chronic suppurative bronchopneumonia, ulcerative lymphangitis, lymphadenitis and enteritis in foals aged 1-4 months. Identification of the virulence plasmids associated with <span class="CharOverride-6">R. equi</span> is of great importance in ascertaining pathogenicity in equines. Virulence factor is associated with presence of 85-90kb plasmid which encodes various virulence associated proteins (VAP -A,-B,-C,-D,-E,-F,-G,-H). VAP A is essential for virulence in foals. This study was aimed at the evaluation of molecular characteristics of <span class="CharOverride-6">R. equi </span>isolates<span class="CharOverride-6"> </span>from foals with respiratory problems by multiplex PCR assay that amplifies the <span class="CharOverride-6">vap</span> gene family (<span class="CharOverride-6">vap-</span>A,-B,-C,-D,-E,-F,-G,-H). Twenty-eight isolates of <span class="CharOverride-6">R. equi</span> from foals with respiratory problems were used in this study. The PCR amplification reaction was performed using specific primers for <span class="CharOverride-6">vap</span> gene family <span class="CharOverride-6">viz.</span> <span class="CharOverride-6">vap-</span>A,-B,-C,-D,-E,-F,-G,-H which yielded the bands of 350, 400, 450, 500, 600, 650 and 700 bp, respectively. All the 28 isolates were positive for <span class="CharOverride-6">vap </span>A,C,D,E,F,G and H and negative for <span class="CharOverride-6">vap </span>B gene which was characteristic of virulent <span class="CharOverride-6">R. equi</span> isolates of equine origin. The multiplex PCR assay for the <span class="CharOverride-6">vap</span> gene family allowed identification and characterization of virulent isolates in a simple one-step reaction. This is a significant advantage in comparison with the conventional diagnostic methodologies of <span class="CharOverride-6">R. equi</span> strains and simultaneously evaluated the presence of the <span class="CharOverride-6">vap </span>A gene which is responsible for virulence in foals.</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Keywords </span>| <span class="CharOverride-6">Rhodococcus equi</span>, Equine, <span class="CharOverride-6">vap </span>gene, Virulence- associated protein</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Editor----Citation"><span class="CharOverride-7">Editor</span> | Minakshi Prasad, Head of the Department of Animal Biotechnology, LUVAS, Hisa Lala Lajpat Rai University of Veterinary &amp; Animal Sciences (LUVAS), Hisar, Haryana, 125004, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-7">Special Issue</span> | 1(2015) “Biotechnological and molecular approaches for diagnosis, prevention and control of infectious diseases of animals”</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Received</span> | March 12, 2015; <span class="CharOverride-5">Revised</span> | April 16, 2015; <span class="CharOverride-5">Accepted</span> | April 17, 2015; <span class="CharOverride-5">Published</span> | April 21, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-5">*Correspondence</span> | Sandip Kumar Khurana, National Research Centre on Equines, Hisar, India; <span class="CharOverride-5">Email:</span> sandipkk2003@yahoo.co.in</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Citation</span> | Chhabra S, Khurana SK, Kapoor PK, Singha H, Singh Y, Khirbat R (2015). Characterization of <span class="CharOverride-21">Rhodococcus equi</span> isolates from foals with respiratory problems using a multiplex PCR for the <span class="CharOverride-21">vap</span> genes. Adv. Anim. Vet. Sci. 3(1s): 28-32.  </p>
			<p class="Editor----Citation"><span class="CharOverride-7">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.1s.28.32"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.1s.28.32</span></a></p>
			<p class="Editor----Citation"><span class="Editor---Citation CharOverride-5" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-5" lang="en-US">(</span><span class="Editor---Citation CharOverride-5" lang="en-US">Online)</span> | 2307-8316; <span class="Editor---Citation CharOverride-5" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-5" lang="en-US">(Print)</span> | 2309-3331</p>
			<p class="Editor----Citation"><span class="CharOverride-7">Copyright </span>© 2015 Chhabra et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Heading-1--Introduction----">&nbsp; </p>
		  <p class="Heading-1--Introduction----">Introduction</p>
			<p class="Caps-on-First-Para">&nbsp;</p>
		  <p class="Caps-on-First-Para"><span class="_idGenDropcap-1">R</span><span class="CharOverride-6">. </span><span class="CharOverride-6">equi</span> is Gram positive and facultative intracellular pathogen belonging to order <span class="CharOverride-6">Actinomycetales</span>. These are coccobacillary, aerobic and non-motile organisms formerly known as <span class="CharOverride-6">Corynebacterium equi</span>. Infection causes subacute or chronic abcessating or suppurative bronchopneumonia, ulcerative lymphangitis and enteritis (<a href="#Meijer-WG--Prescott-JF--2004"><span class="Hyperlink">Meijer and Prescott, 2004</span></a>) in foals aged 1-4 months (<a href="#Tkachuksaad-O--Prescott-J--1991"><span class="Hyperlink">Tkachuksaad and Prescott, 1991</span></a>; <a href="#Yager-JA--Prescott-CA--Kramar-DP--Hannah-H--Balson-GA--Croy-BA--1991"><span class="Hyperlink">Yager et al., 1991</span></a>; <a href="#Khurana-SK--Malik-P--Virmani-N--Singh-BR--2009-"><span class="Hyperlink">Khurana et al., 2009</span></a>; <a href="#Von-Bargen-K--Haas-A--2009"><span class="Hyperlink">Von Bargen and Haas, 2009</span></a>; <a href="#Khurana-SK--2015a"><span class="Hyperlink">Khurana, 2015a</span></a>, <a href="#Khurana-SK--2015b"><span class="Hyperlink">2015b</span></a>; <a href="#Khurana-SK--Dhama-K--Prasad-M--Karthik-K--Tiwari-R--2015"><span class="Hyperlink">Khurana et al., 2015</span></a>) months old. <span class="CharOverride-6">R. equi</span> has been reported to infect number of domestic animals including pig, sheep, camel and cattle and has been also recognized as opportunistic pathogen in both immunocompetent and immunocompromised humans and readily cause infection in HIV positive patients (<a href="#Takai-S--Ikeda-T--Sasaki-Y--Watanabe-Y--Ozawa-T--Tsubaki-S--Sekizaki-T--1995"><span class="Hyperlink">Takai et al., 1995</span></a>; <a href="#Mizuno-FS--Sakamoto-M--Yoshikawa-MK--2005"><span class="Hyperlink">Mizuno et al., 2005</span></a>; <a href="#Napolea-o-F--Damasco-PV--Camello-TCF-et-al.--2005-"><span class="Hyperlink">Napoleao et al., 2005</span></a>; <a href="#Khurana-SK--2014-"><span class="Hyperlink">Khurana, 2014</span></a>; <a href="#Khurana-SK--Kanu-Priya--Singh-N--Singha-H--Punia-S--2014"><span class="Hyperlink">Khurana et al., 2014</span></a>). It is commonly isolated from soil, feces and gut from healthy and sick animals. Identification of the virulence plasmids associated with <span class="CharOverride-6">R. equi</span> is of great importance in characterizing pathogenicity of disease in equines (<a href="#Monego-F--Maboni-F--Krewer-C--Vargas-A--Costa-M--Loreto-E--2009"><span class="Hyperlink">Monego et al., 2009</span></a>). The virulence is associated with the ability of the bacteria to prevent phagosome-lysosome fusion and replication within macrophages resisting clearance by organism defence (<a href="#Kanaly-ST--Hines-SA--Palmer-SA--1993"><span class="Hyperlink">Kanaly et al., 1993</span></a>; <a href="#Krewer-CC--Spricigo-DA--Botton-SA-et-al.--2008"><span class="Hyperlink">Krewer et al., 2008</span></a>). Virulence factor is associated with presence of 85-90kb plasmid which encodes various virulence associated antigen (<a href="#Byrne--BA--Prescott-JF--Palmer-GH--Takai-S--Nicholson-VM--Alperin-DC--Hines--SA--2001"><span class="Hyperlink">Byrne et al., 2001</span></a>). According to many researchers virulent and intermittently virulent <span class="CharOverride-6">R. equi</span> is presented with virulence associated antigen Vap A (15-17 kDa) and Vap B (20 kDa) respectively. Gene for the <span class="CharOverride-6">vap</span>A antigen is virtually found in all clinical <span class="CharOverride-6">R. equi</span> isolated from foals (<a href="#Costa-MM--Machado-SA--Krewer-CC--Ilha-MRS--Gra-a-DL--Mattos-Guaraldi-AL--Vargas-ACV--2006"><span class="Hyperlink">Costa et al., 2006</span></a>; <a href="#Takai-S--Fukunaga-N--Ochiai-S--Sakai-T--Sasaki-Y--Tsubaki-S--1996"><span class="Hyperlink">Takai et al., 1996</span></a>) and <span class="CharOverride-6">vap </span>B gene on isolates from pigs and humans. Earlier studies identified pathogenicity &#173;&#173;&#173;&#173;&#173;&#173;&#173;&#173;&#173;&#173;&#173;&#173;island within the plasmid which contains seven  <span class="CharOverride-6">vap</span> genes and discovery of more <span class="CharOverride-6">vap</span> proteins has taken this number to nine <span class="CharOverride-6">vap</span> genes <span class="CharOverride-6">vap-</span>A, -B, -C, -D, -E, -F, -G, H  and I. Thus the presence or absence of the <span class="CharOverride-6">vap</span>A gene can be used as a marker to show presence or absence of the <span class="CharOverride-6">vapA</span> encoding virulence plasmid. Virulence plasmid negative strains of <span class="CharOverride-6">R. equi </span>do not express <span class="CharOverride-6">vap</span>A gene and are therefore incapable of causing disease (<a href="#Jain-S--Bloom-BR--Hondalus-MK--2003"><span class="Hyperlink">Jain et al., 2003</span></a>). The multiplex PCR assay for the <span class="CharOverride-6">vap</span> gene family allowed identification and characterization of virulent isolates in a simple one-step reaction. This is a significant advantage in comparison with the diagnostic methodologies presented by <a href="#Halbert-ND--Reitzel-RA--Martens-RJ--Cohen-ND--2005-."><span class="Hyperlink">Halbert et al. 2005</span></a> which allowed identification of <span class="CharOverride-6">R. equi</span> strains and simultaneously evaluated the presence of the <span class="CharOverride-6">vapA </span>gene. The PCR technique has the potential to identify virulent <span class="CharOverride-6">R. equi</span> rapidly by amplification of gene sequences that are unique to the virulence plasmids. This approach is useful not only for epidemiological investigations but also for early diagnosis programs. This study was aimed at the evaluation of molecular characteristics of <span class="CharOverride-6">R. equi </span>isolates<span class="CharOverride-6"> </span>obtained from foals and adult horses from different geographical locations in India by Multiplex PCR assay that amplifies the vap gene family (<span class="CharOverride-6">vap-</span>A,-B,-C,-D,-E,-F,-G,-H).</p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
		  <p class="Heading-1--Introduction----">Materials and methods </p>
			<p class="Heading-2--History-in-MM-">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-">Bacterial Isolates</p>
			<p class="Body-Text ParaOverride-1">The study was conducted on 28 bacterial isolates from foals with respiratory problems from Haryana and Rajasthan previously identified as <span class="CharOverride-6">R. equi</span> by biochemical tests. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">DNA Extraction</p>
			<p class="Body-Text">All twenty eight isolates were cultured 5% sheep blood agar and then transferred to nutrient agar and incubated for 24 hours at 37°C.Then the colonies of all 28 isolates were inoculated in BHI broth and incubated at 37°C for 24 hours. The genomic DNA of all isolates from overnight BHI broth culture was isolated by snap chilling. One ml of overnight BHI broth culture of all the isolates was taken in eppendorf tube and centrifuged at 7000 rpm for 5 min. at 4°C and supernatant was discarded. Again one ml of BHI is added to same tube and above step of centrifugation was repeated and supernatant was discarded. 100-200 µl of nuclease free water (NFW) was added to the precipitate in the eppendorf tube and kept in water bath for 5 min. at 95°C and immediately kept in ice for 5 min. The suspension obtained was centrifuged at 7000 rpm for 5 min. at 4°C. The supernatant was stored in fresh eppendorf tube and used as DNA template for Multiplex PCR. The purity of DNA was evaluated by taking the ratio of optical densities (OD) at 260 nm to that of 280 nm, by spectrophotometer (Biorad Smart SpecTM Plus). The samples having OD ratio between 1.7-1.9 were considered having acceptable purity and used in future experiments. For computing the concentration of plasmid DNA, it is known that one OD value at 260 nm corresponds to 50 µg/ml of double stranded DNA. Hence, the concentration of DNA was calculated using the following formula:</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-6">DNA concentration (µg DNA/ml) = OD 260 </span><span class="CharOverride-12">x</span><span class="CharOverride-6">50 </span><span class="CharOverride-12">x </span><span class="CharOverride-6">dilution factor</span></p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150422181405.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150422181405.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/20150422181405.png">Multiplex PCR amplification of<span class="CharOverride-6"> vap</span> gene family.</a></p>
       </div>

			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-13">Lane 1:</span><span class="CharOverride-11">  Molecular Ladder; </span><span class="CharOverride-13">Lane 2:</span><span class="CharOverride-11"> </span><span class="CharOverride-14">R. equi</span><span class="CharOverride-11"> Standard; </span><span class="CharOverride-13">Lane 3:</span><span class="CharOverride-11"> Negative control; </span><span class="CharOverride-13">Other Lane:</span><span class="CharOverride-11"> Respective </span><span class="CharOverride-14">R. equi isolate</span><span class="CharOverride-11"> number</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 ParaOverride-1"><span class="CharOverride-5"><a id="Table-1-"></a>Table 1:</span> Primers utilized with multiplex PCR for <span class="CharOverride-6">vap</span> gene family amplification</p>
			<table width="657" height="393" class="Table-Style-1" id="table-1">
				<colgroup>
					<col class="_idGenTableRowColumn-1" />
					<col class="_idGenTableRowColumn-2" />
					<col class="_idGenTableRowColumn-3" />
					<col class="_idGenTableRowColumn-4" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-15">Identification</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GeneBank access</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-15">Sequence</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-15">Size (bp)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPA-F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">ACAAGACGGTTTCTAAGGCG</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">550</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPA-R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">TTGTGCCAGCTACCAGAGCC</span></p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPB-F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">RER20KDAVA</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">GAATTCGAAAGCGCAAAGGT</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">650</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPB-R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">RER20KDAVA</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">TTCCGTGAACATCGTACTGC</span></p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPC-F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">GGGTCGTCCATCCAAATCGA</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">700</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPC-R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">GGTCAGGCCTATCACCCTTG</span></p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPD-F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF118814</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">GGTGGTGCGATGTCAGAATG</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">400</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPD-R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF118814</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">TGGAACGTCTTGCCCTTCTT</span></p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPE-F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">ATATGACGACCGTTCACAAG</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">600</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPE-R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">CTCCGATGCCCACCAAACTA</span></p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPF-F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AGAATATGCCTGGTATGGGC</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">350</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPF-R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">TCGTCGTATAGCTGCTGCAG</span></p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPG-F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">TCATTGCCACCCTCCGGTTC</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">450</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPG- R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">GCGAACGCGGAAACTTCAAT</span></p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPH-F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AATTCCTATCAAGGACAGC</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">500</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">VAPH-R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">AF116907</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph ParaOverride-1"><span class="CharOverride-16">ATACCGATTACGGAGCTCAC</span></p>
						</td>
						<td />
					</tr>
				</tbody>
			</table><br>
		  <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150422180212.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150422180212.png" width="80" height="80"></a>
            
        <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Figure-2-"></a>Figure 2: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150422180212.png">Multiplex PCR amplification of <span class="CharOverride-6">vap</span> gene family</a></p>
       </div>
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-13">Lane 1:</span><span class="CharOverride-11">  Molecular Ladder; </span><span class="CharOverride-13">Lane 2:</span><span class="CharOverride-11"> </span><span class="CharOverride-14">R. equi</span><span class="CharOverride-11"> Standard; </span><span class="CharOverride-13">Lane 3:</span><span class="CharOverride-11"> Negative control; </span><span class="CharOverride-13">Other Lane:</span><span class="CharOverride-11"> Respective </span><span class="CharOverride-14">R. equi isolate</span><span class="CharOverride-11"> number</span></p>
		  <p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Multiplex PCR Assay for vap Gene Family</p>
			<p class="Body-Text ParaOverride-1">The PCR reaction was carried out as described by <a href="#Monego-F--Maboni-F--Krewer-C--Vargas-A--Costa-M--Loreto-E--2009"><span class="Hyperlink">Monego et al. (2009)</span></a> with some modifications. The virulence associated protein (vap) family genes viz. vap-A, -C, -D, -E, -F, -G and –H were amplified using gene specific primers. The details of the primers were mentioned in<span class="CharOverride-5"> </span><a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>. The multiplex PCR cycling conditions consisted of initial denaturation for 5min. at 95°C, denaturation for 45sec. at 95°C, primer annealing for 1 min. at 54°C and extension for 1.5 min. at 72°C following final extension which lasted 10 minutes at 72°C. The samples were subjected to 34 cycles of amplification in thermal cycler (XP gradient cycler Bioer Technology PR, China). For each reaction we used 3.0 μl of DNA preparation in 25 μl reaction mixture containing 10.2 μl NFW, 2.5μl of 10X PCR buffer with 15mM MgCl2 (Fermentas), 1.0μl 10mM dNTP mixture (Fermentas), 0.5μl of 10μM of each forward primers, 0.5μl of 10μM of each reverse primers, Taq DNA Polymerase 0.3 μl (5U/ μl stock)(Fermentas). The amplification products (10μl) were submitted to 1.5% agarose gel electrophoresis at 75 volts for 1 hour 25 minutes.   </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">The PCR methodology was applied to analyse 28 isolates. The PCR amplification reaction was performed using the specific primers for vap gene family viz. <span class="CharOverride-6">vap </span>A,-B,-C,-D,-E,-F,-G,-H which yielded the bands of 350, 400, 450, 500, 600, 650 and 700 bp (<a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>, <a href="#Figure-2-"><span class="Hyperlink">2</span></a> and <a href="#Figure-3-"><span class="Hyperlink">3</span></a>). The length of DNA fragments amplified by multiplex PCR was in accordance with previous results of <a href="#Monego-F--Maboni-F--Krewer-C--Vargas-A--Costa-M--Loreto-E--2009"><span class="Hyperlink">Monego et al. (2009)</span></a>. The accuracy of PCR amplification of the <span class="CharOverride-6">vap</span> gene family in our control strains was verified by matching the results with that of standard and negative control used in PCR reaction. All the 28 isolates isolated from foals and adult horses with respiratory problems were positive for <span class="CharOverride-6">vap </span>-A,-C,-D,-E,-F,-G and-H. The amplicon specific for <span class="CharOverride-6">vap</span>-B was absent in all isolates of <span class="CharOverride-6">R. equi.</span></p>
		  <p class="Body-Text">&nbsp;</p>
			
          <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150422181915.jpg" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150422181915.jpg" width="80" height="80"></a>
            
      <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Figure-3-"></a>Figure 3: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150422181915.jpg"> Multiplex PCR amplification of <span class="CharOverride-6">vap</span> gene family</a></p>
       </div>
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-13">Lane 1:</span><span class="CharOverride-11">  Molecular Ladder; </span><span class="CharOverride-13">Lane 2:</span><span class="CharOverride-11"> </span><span class="CharOverride-14">R. equi</span><span class="CharOverride-11"> Standard; </span><span class="CharOverride-13">Lane 3:</span><span class="CharOverride-11"> Negative control; </span><span class="CharOverride-13">Other Lane:</span><span class="CharOverride-11"> Respective </span><span class="CharOverride-14">R. equi isolate</span><span class="CharOverride-11"> number</span></p>
		  <p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Discussion</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The virulence associated antigens (Vap-A) and plasmids are used as epidemiological markers for <span class="CharOverride-6">R. equi</span> virulence in foals (<a href="#Cohen-ND--O-Connor-MS--Chaffin-MK--Martens-RJ--2005-."><span class="Hyperlink">Cohen et al., 2005</span></a>; <a href="#Takai-S--1999-."><span class="Hyperlink">Takai et al., 1999</span></a>). Two categories of plasmids: VapA and VapB family are mainly responsible for pathogenesis and host tropism of <span class="CharOverride-6">R. equi </span>(<a href="#Takai-S--1991a-."><span class="Hyperlink">Takai, 1991a</span></a>, <a href="#Takai-S--1991b"><span class="Hyperlink">1991b</span></a>; <a href="#Oldfield-C--Bonella--H--Renwic-L--Dodson-HI--Alderson-G--Goodfellow--M---2004-"><span class="Hyperlink">Oldfield et al., 2004</span></a>; <a href="#Ocampo-Sosa-AA--Lewis-DA--Navas-J--Quigley-F--Callejo-2007"><span class="Hyperlink">Ocampo-Sosa et al., 2007</span></a>). <span class="CharOverride-6">R. equi </span>isolated from clinical cases express only one of these two plasmids and carries the gene(s) encoded either in <span class="CharOverride-6">vap</span>A or <span class="CharOverride-6">vap</span>B families (<a href="#Takai-S--Ikeda-T--Sasaki-Y--Watanabe-Y--Ozawa-T--Tsubaki-S--Sekizaki-T--1995-.-Identification"><span class="Hyperlink">Takai et al., 1995</span></a>). The VapA+ B- type plasmid is associated with the infection in horse (<a href="#Takai-S--Ikeda-T--Sasaki-Y--Watanabe-Y--Ozawa-T--Tsubaki-S--Sekizaki-T--1995-.-Identification"><span class="Hyperlink">Takai et al., 1995</span></a>; <a href="#Ocampo-Sosa-AA--Lewis-DA--Navas-J--Quigley-F--Callejo-2007"><span class="Hyperlink">Ocampo-Sosa et al., 2007</span></a>). This unique plasmid-determined host-specificity has been found only in Rhodococci, however the precise role and mechanism of the <span class="CharOverride-6">vap</span> antigens remain enigmatic. <a href="#Monego-F--Maboni-F--Krewer-C--Vargas-A--Costa-M--Loreto-E--2009"><span class="Hyperlink">Monego et al. (2009)</span></a><span class="CharOverride-6"> </span>identified that none of the 32 <span class="CharOverride-6">vap</span>A-positive isolates showed the presence of vap B gene. The presence of <span class="CharOverride-6">vap</span>-A gene in <span class="CharOverride-6">R. equi</span> clinical isolates and its relationship with lethality of <span class="CharOverride-6">R. equi</span> to susceptible foal was reported by many researchers (<a href="#Takai-S--Ikeda-T--Sasaki-Y--Watanabe-Y--Ozawa-T--Tsubaki-S--Sekizaki-T--1995-.-Identification"><span class="Hyperlink">Takai et al., 1995</span></a>; <a href="#Takai--S--1997"><span class="Hyperlink">Takai, 1997</span></a>; <a href="#Wada-R--Kamada-M--Anzai-T--Nakanishi-A--Kanemaru-T--Takai--S--Tsubaki-S--1997"><span class="Hyperlink">Wada et al., 1997</span></a>). In present study we analyzed 28 <span class="CharOverride-6">R. equi</span> isolates from different geographical regions of India for the presence of <span class="CharOverride-6">vap </span>genes (<span class="CharOverride-6">vap</span>-A,-B,-C,-D,-E,-F,-G,-H) using Multiplex PCR. The molecular profile of the <span class="CharOverride-6">vap</span> gene family found in our study presented <span class="CharOverride-6">vap</span>A, and <span class="CharOverride-6">vap</span>C to <span class="CharOverride-6">vap</span>H genes. The profile all the isolates showed <span class="CharOverride-6">vap</span>A, <span class="CharOverride-6">vap</span>D, and <span class="CharOverride-6">vap</span>G, which in agreement with result data of <a href="#Jacks-S--Gigue-rre-S--Prescott-JF--2007"><span class="Hyperlink">Jacks et al. (2007)</span></a>, which indicate that these <span class="CharOverride-6">vap</span> genes are the most biologically relevant as they are preferentially induced during infection in the natural host. None of the 28 vap-A positive isolates showed the presence of <span class="CharOverride-6">vap</span>-B gene. The results obtained agreed with that of <a href="#Takai-S--Ikeda-T--Sasaki-Y--Watanabe-Y--Ozawa-T--Tsubaki-S--Sekizaki-T--1995-.-Identification"><span class="Hyperlink">Takai et al., (1995</span></a><span class="Hyperlink">)</span> reported that <span class="CharOverride-6">R. equi </span>isolates can express either <span class="CharOverride-6">vap</span>-A or <span class="CharOverride-6">vap</span>-B but not both. In our study all isolates were found positive for <span class="CharOverride-6">vap</span>-A gene and negative for <span class="CharOverride-6">vap</span>-B gene, thus providing a <span class="CharOverride-6">vap</span> A+B- profile, which is specific for virulent <span class="CharOverride-6">R. equi </span>isolates of equine origin.</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 National Research Centre on Equines, Hisar and Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar for providing necessary facilities to carry out the research work.</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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