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<body id="AAVS_MH20150328090321_Gupta-et-al" xml: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">Immunoreactivity to Culture Filtrate Proteins of <i>Mycobacterium avium</i> Subspecies <i>paratuberculosis</i> in Naturally Infected Goat and Sheep Sera</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Authors ParaOverride-1">&nbsp;</p>
			<p class="Authors ParaOverride-1"><span class="CharOverride-2">Saurabh Gupta</span><span class="CharOverride-3">1, 2</span><span class="CharOverride-2">, Kundan Kumar Chaubey</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Shoor Vir Singh</span><span class="CharOverride-3">1*</span><span class="CharOverride-2">, Ashok Kumar Bhatia</span><span class="CharOverride-3">2</span><span class="CharOverride-2">, Naveen Kumar</span><span class="CharOverride-3">1</span><span class="CharOverride-2">,</span><span class="CharOverride-3"> </span><span class="CharOverride-2">Anjana Goel</span><span class="CharOverride-3">2</span><span class="CharOverride-2">,</span><span class="CharOverride-3"> </span><span class="CharOverride-2">Tarun Kumar Sachan</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Krishan Dutta Rawat</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Jagdip Singh Sohal</span><span class="CharOverride-3">3</span><span class="CharOverride-2">, Kuldeep Dhama</span><span class="CharOverride-3">4</span></p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Affiliations ParaOverride-1"><span class="CharOverride-4">1</span>Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO-Farah, Mathura, Uttar Pradesh, India; <span class="CharOverride-4">2</span>Department of Biotechnology, GLA University, Chaumuhan, Mathura, Uttar Pradesh, India; <span class="CharOverride-4">3</span>Amity Institute of Microbial Technology, Amity University Rajasthan, Jaipur, India; <span class="CharOverride-4">4</span>Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 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">Mycobacterium avium</span>&#160;subspecies&#160;<span class="CharOverride-6">paratuberculosis </span>(MAP), the cause of granulomatous chronic enteritis in ruminants (Johne’s disease) is under reported due to difficulties in diagnosing pre-clinical cases. Compromised specificity is a problem due to extensive sharing of antigens /epitopes among MAP and<span class="CharOverride-6"> </span>other mycobacterial strains. Culture filtrate (CF) proteins profile of native ‘Indian Bison Type’ strain of MAP<span class="CharOverride-6"> </span>was studied at different harvesting times (2-8 weeks of growth) in Middlebrook 7H9 medium supplemented with ADC, PANTA antibiotics and mycobactin J. Analysis of harvested CF proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the greater part of CF proteins had molecular masses (&lt;70 kDa) as 14, 19, 26, 34-41, 52-55, and 70 kDa. Immunoblotting showed reactivity of CF proteins commonly recognised (28, 34-36, 38-42, 45, and 56 kDa) with all four MAP infected goat and sheep sera at 2-8 weeks of growth. Collectively, these immunoreactive MAP CF proteins could be the potential targets for developing diagnostics against Johne’s disease with improved sensitivity and high specificity instead of whole cell sonicated crude protoplasmic extracts (PPA).</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Keywords</span> | Johne’s disease, <span class="CharOverride-6">Mycobacterium avium </span>subspecies <span class="CharOverride-6">paratuberculosis, </span>Culture filtrate, SDS-PAGE, Immunoblotting</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Editor----Citation">&nbsp;</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Editor</span> | Muhammad Zubair Shabbir, Assistant Professor, Quality Operations Laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan.</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Received</span> | March 28, 2015; <span class="CharOverride-5">Revised</span> | April 23, 2015; <span class="CharOverride-5">Accepted</span> | April 24, 2015; <span class="CharOverride-5">Published</span> | May 17, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-5">*Correspondence</span> | Shoor Vir Singh, Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh, India; <span class="CharOverride-5">Email: </span>shoorvir_singh@rediffmail.com</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Citation </span>| Gupta S, Chaubey KK, Singh SV, Bhatia AK, Kumar N, Goel A, Sachan TK, Rawat KD, Sohal JS, Dhama K (2015). Immunoreactivity to culture filtrate proteins of <span class="CharOverride-19">Mycobacterium avium</span> subspecies <span class="CharOverride-19">paratuberculosis</span> in naturally infected goat and sheep sera. Adv. Anim. Vet. Sci. 3(6): 347-353.  </p>
			<p class="Editor----Citation"><span class="CharOverride-5">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.6.347.353"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.6.347.353</span></a></p>
			<p class="Editor----Citation"><span class="Editor---Citation CharOverride-5">ISSN (Online)</span> | 2307-8316; <span class="Editor---Citation CharOverride-5">ISSN (Print)</span> | 2309-3331</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Copyright</span> © 2015 Gupta 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 ParaOverride-1">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">M</span><span class="CharOverride-6">ycobacterium </span><span class="CharOverride-6">avium </span>subspecies <span class="CharOverride-6">paratuberculosis </span>(MAP), a cause of Johne’s disease (Paratuberculosis) is the most wide-spread and highly prevalent due to difficulties in diagnosing pre-clinical cases. MAP has wide host range including domestic and wild ruminants, free grazing animals and also the human beings (<a href="#Singh-AV--Singh-SV--Singh-PK--Sohal-JS--2010-"><span class="Hyperlink">Singh et al., 2010</span></a>; <a href="#Singh-SV--Kumar-N--Sohal-2014a"><span class="Hyperlink">Singh et al., 2014a</span></a>; <a href="#Singh-SV--Singh-PK--Singh-2014b"><span class="Hyperlink">Singh et al., 2014b</span></a>). Paratuberculosis has high economic impact on dairy industry and &gt; USD 250 million economic losses has been reported in US alone (<a href="#Ott-SL--Wells-SJ--Wagner-BA--1999"><span class="Hyperlink">Ott et al., 1999</span></a>). Though disease is endemic in India and economic losses in dairy farm was estimated from reproductive disorders (Rs. 23400.0/cow/year), forced removal (Rs. 41,750.0/cow/year), reduced milk yield (Rs. 5,712.0/cow/ year) and increased mortality (Rs. 11,666.0/cow/year) (<a href="#Rawat-KD--Chaudhary-S--Gupta-2014"><span class="Hyperlink">Rawat et al., 2014</span></a>). Control of paratuberculosis has been hindered due to lack of efficient and accurate diagnostic tests. Lower specificity of tests can also be a problem due to the sharing of antigens or epitopes among MAP and other mycobacteria (<a href="#Collins-MT--Wells-SJ--Petrini-KR--Collins-JE--Schultz-RD--Whitlock-RH--2005"><span class="Hyperlink">Collins et al., 1991</span></a>). Sensitivity of commercially available ELISA kits prepared using protoplasmic antigen has been reported comparatively low, 13.6-33.3% (<a href="#Singh-SV--Singh-AV--Singh-PK--Sohal-JS--Singh-NP--2007"><span class="Hyperlink">Singh et al., 2007</span></a>).</p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text">Recent studies have focused on developing improved serodiagnostics using species-specific multiple protein antigens (<a href="#Singh-SV--Sohal-JS--Kumar-2014c"><span class="Hyperlink">Singh et al., 2014c</span></a>). Microfluidics and Lab-on-Chip are some of the recent technologies that can predicate development of laboratory-free diagnostic devices for mycobacterial infections (<a href="#Wadhwa-A--Hickling-GJ--and-Shigetoshi-Eda-S--2012a"><span class="Hyperlink">Wadhwa et al., 2012a</span></a>; <a href="#Li-LL--Munir-S--Bannantine-JP--Sreevatsan-S--Kanjilal-S--Kapur-V--2007"><span class="Hyperlink">Li et al., 2011</span></a>). Earlier reports have identified several antigens inducing strong antibody responses but most of them have been found unsuitable for serodiagnosis because they are highly conserved within mycobacteria and hence cross-react with other mycobacterial pathogens. Recently, various MAP specific proteins/genes has been characterized, cloned, expressed and the protein was evaluated for their diagnostic potential (<a href="#Cho-D--Shin-SJ--Tallat-AM--Collins-MT--2007"><span class="Hyperlink">Cho et al., 2007</span></a>). However, strain of MAP may also tune the sensitivity and specificity of test. Present study investigated CF protein profile of native Indian bison type (‘S 5’) MAP strain for the first time with reference to determine the antigenicity / reactivity of the CF proteins by immunoblotting with polyclonal antisera of goat and sheep infected with MAP and to develop diagnostic tests  of higher sensitivity and specificity.</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Heading-1--Introduction----">Materials and methods</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Mycobacterial Strain</p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-6">Mycobacterium avium</span>&#160;subspecies&#160;<span class="CharOverride-6">paratuberculosis </span>(‘S 5’) strain was procured from the mycobacterial repository, Central Institute for Research on Goats (CIRG), Makhdoom. The strain was maintained on Modified Herrold’s egg yolk medium with mycobactin J (HEYM) as per <a href="#Singh-AV--Singh-SV--Singh-PK--Sohal-JS--2010-"><span class="Hyperlink">Singh et al. (1996)</span></a> and was sub-cultured<span class="CharOverride-5"> </span>in<span class="CharOverride-5"> </span>Middlebrook 7H9 medium (as per the manufacturer, Becton Dickinson, BD) supplemented with ADC (10% or 100ml/l), PANTA antibiotics and mycobactin J (2mg/l).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Growth Pattern</p>
			<p class="Body-Text ParaOverride-1">Growth of MAP<span class="CharOverride-6"> </span>in middlebrook 7H9 medium was monitored by taking absorbance (OD) of the culture at third day, then every week at 600nm for a period of 8 weeks (<a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>). Growth curves were obtained by plotting the absorbance (OD) values versus incubation time in weeks. Turbidity in the medium was measured by Mcfarland standards (0.5, 1, 2, 3, 4, 5 and 6) to check the growth of the culture. To avoid clump formation the cultures were constantly shaken at 100 rpm during incubation.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Culture Filtrate (CF)</p>
			<p class="Body-Text ParaOverride-1">CF were&#160;obtained by harvesting (4000×<span class="CharOverride-6">g</span>, 20 min, 4°C) bacterial growth at different times points (2, 4, 6 and 8 weeks of incubation) and was filtered (using 0.22-μm pore size syringe filter, Millipore). CF were precipitated with saturated ammonium sulfate (Rankem) due to the high amount of albumin in the broth followed by extensive dialysis overnight against 10 mM phosphate-buffered saline (pH 7.4) until free of ammonium ions at 4°C. Dialysed CF were concentrated 10-fold using vacuum concentrator (SPD Speedvac, Thermo Savant). Concentration of CF proteins was quantified by Bradford protein assay kit (Genei) and was stored at -20°C.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS- PAGE)</p>
			<p class="Body-Text ParaOverride-1">CF protein profile was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and stained with Coomassie brilliant blue as per method given by<span class="CharOverride-5"> </span><a href="#Laemmli-UK--1970-."><span class="Hyperlink">Laemmli (1970)</span></a>.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Immunoblotting </p>
			<p class="Body-Text">The electrophoresed CF proteins were transferred on to PVDF-plus membrane (pore size 0.45 µm, Millipore) using Mini Trans-blot cell (Bio-Rad) for 1.5 hrs at 60 V, 100 mA as per <a href="#Towbin-H--Staehelin-T--Gordon-J--1979"><span class="Hyperlink">Towbin et al. (1979)</span></a><span class="CharOverride-5"> </span>with some modifications<span class="CharOverride-5">.</span> Membrane was blocked for 1 hr at room temperature in 5% skim milk (Himedia) containing TBS with 0.05% Tween-20 (TBST) and were probed with polyclonal primary antibody (goat serum) diluted 1:100 in 1X TBST for 2 hrs at room temperature. After incubation membrane was washed 3 times with 1X TBST. Reactivity was seen by incubating the PVDF-plus membrane with peroxidase-conjugated anti-goat IgG (Sigma, USA) in 1:2000 dilution for 1 hr at room temperature followed by washing 5 times (5 min each) with TBST. Visualization of immuno-reactive protein bands was done by diaminobenzidine (DAB) (Sigma).</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Statistical Analysis</p>
			<p class="Body-Text ParaOverride-1">The correlation (<span class="CharOverride-6">R</span><span class="CharOverride-9">2</span>) and Standard deviation of OD600 with time interval of growth measurements were analyzed using Graph Pad InStat. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Results</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Growth Pattern </p>
			<p class="Body-Text ParaOverride-1"><span class="CharOverride-6">Mycobacterium avium</span>&#160;subspecies&#160;<span class="CharOverride-6">paratuberculosis </span>(‘S 5’) strain developed a granular growth with visible clumps suspended in the 7H9 medium up to 6 weeks, afterwards, clusters of floating cells were observed (<a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>). The growth curve  of MAP ‘S 5’ strain in 7H9 medium showed sharp bacterial growth phases delineation; however, after 3 weeks of incubation the culture was in log phase (early exponential) upto 8 weeks (mid to late exponential) in 7H9 medium (<a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>). Total bacterial mass was with a maximum absorbance of 1.43 at 8 weeks. OD600 and time interval of growth measurements were highly correlated (<span class="CharOverride-6">R</span><span class="CharOverride-9">2</span> = 0.997, <a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>). We determined in Indian Bison type ‘S 5’<span class="CharOverride-6"> </span>strain of MAP that 1 McFarland unit was nearly equivalent to 0.26 OD600.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Table-1-"></a>Table 1: </span>History of goats and sheep whose serum were used in the immunoblotting</p>
			<table width="658" height="326" class="Table-Style-1" id="table-1">
				<colgroup>
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-10">Sn</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-10">Animal type/Breed</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-10">Age/ Sex</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-10">Physical condition</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-10">Diarrhea</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-10">Fecal</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-10">Shedding (Microscopy)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-10">IS900 blood PCR</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-10">Serology (‘Indigenous ELISA’)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Sheep </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-11">(Muzzaffarnagri)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Adult female</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Extremely weak and emaciated</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Yes</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Yes</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Positive</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Positive</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">2</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Goat 1 </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-11">(Barbari)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Adult female</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Weak and emaciated</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Yes</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Yes</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Positive</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Positive</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Goat 2 </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-11">(Jamunapari)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Adult female</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Weak and emaciated</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">No</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Yes</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Positive</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Positive</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Goat 3 </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-11">(Jakhrana)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Adult male</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Weak</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Yes</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Yes</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Negative</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-11">Positive</span></p>
						</td>
					</tr>
				</tbody>
			</table><br>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150520005026.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150520005026.png" width="80" height="80"></a>
            
           <p class="Body-Text ParaOverride-1"><span class="CharOverride-13"><a id="Figure-1-"></a>Figure 1: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150520005026.png"><span class="CharOverride-14"> Growth curve of </span><span class="CharOverride-15">Mycobacterium avium</span><span class="CharOverride-14">&#160;subspecies&#160;</span><span class="CharOverride-15">paratuberculosis </span><span class="CharOverride-14">(‘S 5’) strain upto 8 weeks by taking OD at 600nm. Each point correlates to the mean of three determinations ± standard deviation</span></a></p>
       </div><br>

			
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150520005128.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150520005128.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/20150520005128.png">SDS-PAGE of CF proteins harvested at different incubation periods of MAP</a></p>
       </div>
       
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-16">Lane 1</span><span class="CharOverride-7">: 2 weeks; </span><span class="CharOverride-16">lane 2</span><span class="CharOverride-7">: 4 weeks; </span><span class="CharOverride-16">lane 3</span><span class="CharOverride-7">: 6 weeks; </span><span class="CharOverride-16">lane 4</span><span class="CharOverride-7">: 8 weeks.</span></p><br>
			
            <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150520000630.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150520000630.png" 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/20150520000630.png">Immunoblots with CF proteins of MAP in naturally infected sheep serum</a></p>
       </div>
       
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-16">Lane 1</span><span class="CharOverride-7">: 0 Day; </span><span class="CharOverride-16">lane 2</span><span class="CharOverride-7">: 2 weeks; </span><span class="CharOverride-16">lane 3</span><span class="CharOverride-7">: 4 weeks; </span><span class="CharOverride-16">lane 4</span><span class="CharOverride-7">: 6 weeks; </span><span class="CharOverride-16">lane 5</span><span class="CharOverride-7">: 8 weeks.</span></p><br>
            
		  <p class="Heading-2--History-in-MM- ParaOverride-1">Immunoblot of <span class="CharOverride-1">Mycobacterium avium</span>&#160;Subspecies<span class="CharOverride-1"> paratuberculosis</span> Secreted Proteins</p>
		  <p class="Body-Text ParaOverride-1">The initial antigenic profiles were detected at 2, 4, 6 and 8 weeks of incubation in 7H9 medium. Analysis of harvested CF proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the greater part of CF proteins had molecular masses (&lt;70 kDa) as 14, 19, 26, 34-41, 52-55, 68-70 and 90-92 kDa (<a href="#Figure-2-"><span class="Hyperlink">Figure 2</span></a>). The antibody recognition patterns of secreted proteins using 4 positive sera of goats and sheep (<a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>) showed a high degree of variability among MAP infected animals (heavy shedders). Multiple proteins were bound by all the four positive sera, but the specific proteins bound by each serum sample were variable. However, the antigenic CF proteins of MAP ‘S 5’ strain which showed wide variation in recognition pattern with sera from clinical cases were ranged from 19 to 62 kDa at 2 to 8 weeks of growth (<a href="#Figure-3-"><span class="Hyperlink">Figure 3</span></a>, <a href="#Figure-4-"><span class="Hyperlink">4</span></a> and <a href="#Figure-5-"><span class="Hyperlink">5</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Antibody Recognition Pattern to Secreted proteins using sera from 4 Clinical Cases</p>
			<p class="Body-Text ParaOverride-1">Polyclonal sera from 3 goats and 1 sheep naturally infected with <span class="CharOverride-6">Mycobacterium avium&#160;subspecies paratuberculosis</span> (confirmed by fecal microscopy, IS<span class="CharOverride-6">900</span> blood PCR and ‘Indigenous serum ELISA kit’) were tested for reactivity pattern to CF proteins. The CF proteins commonly recognised with all four MAP infected goats and sheep sera at 2 weeks (early growth) were approximately, 38-42, 45, and 56 kDa and at 8 weeks growth were approximately, 28, 34-36, 38-42, 45, and 56 kDa. Sera from goat 1, 2 and 3 reacted with 28, 38, 45 and 65 kDa secreted proteins at 2-8 weeks of growth (<a href="#Figure-4-"><span class="Hyperlink">Figure 4</span></a> and <a href="#Figure-5-"><span class="Hyperlink">5</span></a>). However, 38-42, 45 and 56 kDa CF proteins reacted with sheep serum (<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/20150520003730.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150520003730.png" width="80" height="80"></a>
            
          <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Figure-4-"></a>Figure 4: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150520003730.png">Immunoblots with CF proteins of MAP in naturally infected goat 1 serum</a></p>
       </div>
       
			
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-16">Lane 1</span><span class="CharOverride-7">: 2 weeks; </span><span class="CharOverride-16">lane 2</span><span class="CharOverride-7">: 4 weeks; </span><span class="CharOverride-16">lane 3</span><span class="CharOverride-7">: 6 weeks; </span><span class="CharOverride-16">lane 4</span><span class="CharOverride-7">: 8 weeks.</span></p>
		  <p>&nbsp;</p>
			<p><br>
		  </p>
			<p class="Heading-1--Introduction----">Discussion</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Current immuno-diagnostics for Johne’s disease, a chronic enteritis in ruminants, that is responsible for extensive economic losses to farmers and dairy industry worldwide caused by <span class="CharOverride-6">Mycobacterium avium</span> subspecies <span class="CharOverride-6">paratuberculosis</span> (MAP) are hampered by the lack of specific antigens. The capability of rapidly diagnosing the disease and identifying its causative agent is critical to combat diseases and halt epidemics (<a href="#Li-LL--Munir-S--Bannantine-JP--Sreevatsan-S--Kanjilal-S--Kapur-V--2007"><span class="Hyperlink">Li et al., 2014</span></a>). Test results from the sub-clinically MAP infected animals are challenge to interpret, because clinical signs are not present to assist the interpretation. Transmission of infection usually takes place prior the tests becoming positive and before clinical signs developed. Most frequently used test for diagnosis of MAP infection include bacterial culture, IS<span class="CharOverride-6">900</span> PCR amplification, interferon-<span class="CharOverride-17">γ</span><span class="CharOverride-6"> </span>assay and serum antibody detection using an ELISA platform. <a href="#Wadhwa-A--Bannantine-JP--Byrem-2012c"><span class="Hyperlink">Wadhwa et al. (2012)</span></a> used complement fixation, agar gel immunodiffusion (AGID), and ELISA to determine the antibody response in JD. The complement fixation and agar gel immunodiffusion tests both suffer poor sensitivity (<a href="#Sherman-DM--Gay-JM--Bouley-DS--and-Nelson-GH--1990-."><span class="Hyperlink">Sherman et al., 1990</span></a>; <a href="#Pahangchopi-D--Singh-RV--Singh-SV--Das-2014"><span class="Hyperlink">Pahangchopi et al., 2014</span></a>), series of studies reported that immunological tests like ELISA is the best method as compared to complement fixation and AGID for controlling JD in dairy and beef herds (<a href="#Wynne-JW--Bull-TJ--Seemann-2011"><span class="Hyperlink">Wynne et al., 2011</span></a>). Recent studies have focused to develop improved serodiagnostics using species-specific multiple protein antigens. Detection of MAP using ELISA have been reported in many previous studies using different antigens as protoplasmic antigen (PPA) (<a href="#Collins-MT--Wells-SJ--Petrini-KR--Collins-JE--Schultz-RD--Whitlock-RH--2005"><span class="Hyperlink">Collins et al., 2005</span></a>; <a href="#Singh-AV--Singh-SV--Singh-PK--Sohal-JS--2010-"><span class="Hyperlink">Singh et al., 2007</span></a>; <a href="#Sharma-G--Singh-SV--Sevilla-2008"><span class="Hyperlink">Sharma et al., 2008</span></a>), lipoarabinomannan (LAM) (<a href="#Sweeney-RW--Whitlock-RH--Buckley1994"><span class="Hyperlink">Sweeney et al., 1994</span></a>), culture filtrate of MAP (<a href="#Shin-SJ--Cho-D--Collins-MT--2008"><span class="Hyperlink">Shin et al., 2008</span></a>), and MAP proteins (Map1152, Map1156, Map2609, Map2942c and Map0210c) (<a href="#Bannantine-JP--Paulson-AL--Chacon-O--Fenton-RJ--Zinniel-DK--McVey-DS--Smith-DR--Czuprynski-CJ--Barle"><span class="Hyperlink">Bannatine et al., 2011</span></a>; <a href="#Willemsen-PTJ--Westerveen-J--Dinkla-2006"><span class="Hyperlink">Willemsen et al., 2006</span></a>) for testing antibodies against MAP. Although PPA contains proteins very similar to proteins commonly found in closely related mycobacteria species. LAM is one of the constituent of the cell wall of mycobacteria species and its core structure is shared among mycobacterial species (<a href="#Mishra-AK--Driessen-NN--Appelmelk-BJ--Besra-GS--2011"><span class="Hyperlink">Mishra et al., </span><span class="Hyperlink">2011</span></a>). Some studies also suggested that secreted proteins may be better as solid-phase ELISA antigens resulting in a more sensitive assay (<a href="#Cho-D--Collins-MT--2006-"><span class="Hyperlink">Cho and Collins, 2006</span></a>; <a href="#Willemsen-PTJ--Westerveen-J--Dinkla-2006"><span class="Hyperlink">Willemsen et al., 2006</span></a>). On the other hand, <a href="#Sung-N--Collins-MT--2003"><span class="Hyperlink">Sung and Collins (2003)</span></a> have reported that expression of MAP CF protein depends on the type and culture conditions. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			
            <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150520000431.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150520000431.png" width="80" height="80"></a>
            
          <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Figure-5-"></a>Figure 5: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150520000431.png">Immunoblots with CF proteins of MAP in naturally infected goat 2 serum</a></p>
       </div>
       
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-16">Lane 1</span><span class="CharOverride-7">: 2 weeks; </span><span class="CharOverride-16">lane 2</span><span class="CharOverride-7">: 4 weeks; </span><span class="CharOverride-16">lane 3</span><span class="CharOverride-7">: 6 weeks; </span><span class="CharOverride-16">lane 4</span><span class="CharOverride-7">: 8 weeks.</span></p><br>
			<p class="Body-Text ParaOverride-1">Recent technological developments have led to the proliferation of new, rapid diagnostic tests that hold promise for the improved management and control of infectious diseases. Mycobacterial infections such as tuberculosis (TB), bovine tuberculosis (bTB), and Johne’s disease (JD) are major infectious diseases of both human and animals (<a href="#Wadhwa-A--Bannantine-JP--Byrem-2012c"><span class="Hyperlink">Wadhwa et al., 2012b</span></a>). For tuberculosis and bovine TB, there has been recent progress in developing laboratory-free diagnostic methods. New technologies such as ‘microfluidics’ (<a href="#Wadhwa-A--Bannantine-JP--Byrem-2012c"><span class="Hyperlink">Wadhwa et al., 2012a</span></a>) and ‘Lab-on- Chip’ (<a href="#Li-LL--Munir-S--Bannantine-JP--Sreevatsan-S--Kanjilal-S--Kapur-V--2007"><span class="Hyperlink">Li et al., 2011</span></a>) are examples of promising new technologies that can underpin development of laboratory-free diagnostic devices for these mycobacterial infections. Although there have been developed a sensitive serum ELISA test, ethanol vortex enzyme-linked immunosorbent assay (EVELISA), using ethanol extract of MAP especially for Johne’s disease (<a href="#Wadhwa-A--Bannantine-JP--Byrem-2012c"><span class="Hyperlink">Wadhwa et al., 2012c</span></a>). Another study was conducted to assess the performance of EVELISA optimized to diagnose bovine TB using serum samples from various groups of red deer and cattle including animals experimentally infected with MAP and suggests that EVELISA can form a basis for development of a sensitive and specific test for bovine TB (<a href="#Wadhwa-A--Bannantine-JP--Byrem-2012c"><span class="Hyperlink">Wadhwa et al., 2013</span></a>; <a href="#Wadhwa-A--Bannantine-JP--Byrem-2012c"><span class="Hyperlink">Wadhwa et al., 2014</span></a>). Furthermore, EVELISA-based control measures increase the annual per capita revenue of US dairy farms when compared to no JD control and ELISA based JD control, respectively (<a href="#Massaro-T--Lenhart-S--Spence-M--drakes-2013"><span class="Hyperlink">Massaro et al., 2013</span></a>). An AC electrokinetic impedance sensing can be used for rapid and sensitive detection of specific antibodies in serum samples and this could be a basis for development of a point of care diagnostic device for human and bovine tuberculosis (<a href="#Cui-H--Li-S--Yuan-Q--Wadhwa-A--Eda-S--Chambers-M--Ashford-R--Jiang-H--Wu-J--2013"><span class="Hyperlink">Cui et al., 2013</span></a>). <a href="#Garg-R--Patil-PK--Singh-SV--Sharma-2015"><span class="Hyperlink">Garg et al. (2015)</span></a> recently reported that the combination of milk ELISA and milk PCR may be adopted as a model strategy for the screening and diagnosis of JD in lactating cattle herds in Indian conditions.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text">Our results demonstrate the correlation between turbidity and OD600 measurements with time period of growth. We determined in Indian Bison type ‘S 5’<span class="CharOverride-6"> </span>strain of MAP that 1 McFarland unit was nearly equivalent to 0.26 OD600. These measurements were not quite as precise, since <span class="CharOverride-6">Mycobacterium avium </span>subsp. <span class="CharOverride-6">paratuberculosis</span> tends to form clumps. Strain to strain variations were reported in growth curves and growth rate of MAP (<a href="#Elguezabal-N--Felix-Bastida-F--Sevilla-IA--Gonzalez-N--Molina-E--Garrido-JM--Juste-RA--2011"><span class="Hyperlink">Elguezabal et al., 2011</span></a>). The present study also showed that 7H9 medium allowed to obtaining more and earlier CF proteins and nearly all these CF proteins were of low molecular weight ranging between 14 and 70 kDa; similar findings regarding CF proteins of MAP have been reported with other field strains elsewhere (<a href="#Olsen-I--Reitan-LJ--Holstad-G--Wiker-HG--2000"><span class="Hyperlink">Olsen et al., 2000</span></a>; <a href="#Cho-D--Collins-MT--2006-"><span class="Hyperlink">Cho and Collins, 2006</span></a>). When immunoblotting test using CF proteins of MAP were performed, a high variability among naturally infected animals in protein binding was observed, similar results were reported by <a href="#Cho-D--Collins-MT--2006-"><span class="Hyperlink">Cho and Collins (2006</span></a><span class="Hyperlink">)</span>. <a href="#Waters-WR--Palmer-MV--Bannantine-2004"><span class="Hyperlink">Waters et al. (2004)</span></a> have shown variable antibody binding patterns in assays with serum from white-tailed deer experimentally infected with <span class="CharOverride-6">M. bovis</span>. High correlation has been shown between the level of shedding of MAP in feces and concentration of serum antibodies (<a href="#Nielsen-SS--Toft-N--2006"><span class="Hyperlink">Nielsen and Toft, 2006</span></a>). However, in agreement, our results showed high intensity and more number of bands were recognized with naturally infected goats and sheep sera. Several other factors may also affect serum antibody levels (<a href="#Hendrick-SH--Kelton-DF--Leslie-KE--Lissemore-2006"><span class="Hyperlink">Hendrick et al., 2006</span></a>), including individual genetic variation (<a href="#Sudgen-EA--Stilwell-K--Michaelides-A--1997"><span class="Hyperlink">Sudgen et al., 1997</span></a>). <a href="#Valentin-Weigand-P--Moriarty-KM--1992"><span class="Hyperlink">ValentinWeigand and Moriarty (1992)</span></a> also reported that during short incubation period MAP may secrete immunoreactive proteins which are not dominant in long term cultures.</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Body-Text">There are studies evaluating sensitivity and specificity of commercial ELISA kits in domestic and wild ruminants derived from bovine strains of MAP (<a href="#Whittington-RJ--Eamens-GJ--Coussens-DV--2003"><span class="Hyperlink">Whittington et al., 2003</span></a>) however, ‘Indigenous ELISA’ developed from ‘Indian Bison type’ strain showed improved sensitivity and specificity as compared to commercial ELISA kits (<a href="#Singh-AV--Singh-SV--Singh-PK--Sohal-JS--2010-"><span class="Hyperlink">Singh et al., 2007</span></a>). Conceptually, the use of specific purified proteins as the solid phase antigen in ELISA assay have suffered from low diagnostic sensitivity (<a href="#Huntley-JF--Stabel-JR--Bannantine-JP--2005"><span class="Hyperlink">Huntley et al., 2005</span></a>). Recombinant antigens can also alter antigenicity due to conformational changes or lack of post-translational modifications (<a href="#Cho-D--Shin-SJ--Tallat-AM--Collins-MT--2007"><span class="Hyperlink">Cho et al., 2007</span></a>; <a href="#Li-LL--Munir-S--Bannantine-JP--Sreevatsan-S--Kanjilal-S--Kapur-V--2007"><span class="Hyperlink">Li et al., 2007</span></a>). The use of MAP<span class="CharOverride-6"> </span>CF proteins as antigen in ELISA assay increased sensitivity by 25% over commercial ELISA kits for low shedding animals (<a href="#Shin-SJ--Cho-D--Collins-MT--2008"><span class="Hyperlink">Shin et al., 2008</span></a>). Also the variability in responses among different animals to recognize single MAP-specific antigen in early and subclinical stages, a cocktail of immuno-reactive native proteins of ‘Indian Bison type’ provide significant advantages for improved serodiagnosis of MAP infection. Moreover, the immunoblotting findings concluded that the CF proteins were quite specific and the immunogenecity of these reactive CF proteins would be further evaluated as biomarkers to develop more sensitive and specific assays.</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Heading-1--Introduction----">Acknowledgement</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Authors are thankful to Directors (Central Institute for Research on Goats, Makhdoom, Farah and GLA University, Mathura) for providing laboratory and aanimal house facilities.</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">No potential conflict of interest to declare.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
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		  <p class="Heading-1--Introduction----">&nbsp;</p>
			
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