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<body id="AAVS_Nexus-621" lang="en-GB">
		<div class="Basic-Text-Frame">
			<p class="Type-of-Article">&nbsp;</p>
			<p class="Type-of-Article"><span class="CharOverride-1">Research Article</span></p>
	</div>
		<div class="Basic-Text-Frame">
			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Detection of Classical Swine Fever Virus Antigen and Nucleic Acid on Blood of Experimentally Infected Piglets</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Authors ParaOverride-1">&nbsp;</p>
			<p class="Authors ParaOverride-1"><span class="CharOverride-2">Sachin Dattatraya Raut</span><span class="CharOverride-3">1*</span><span class="CharOverride-2">, Kaushal Kishore Rajak</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Ravi Kumar</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Amol Ramdas Gurav</span><span class="CharOverride-3">2</span><span class="CharOverride-2">, Manu Mohan</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Dhanesh Valiyavalappil</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Dhanavelu Muthuchelvan</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Pranob Dhar</span><span class="CharOverride-3">3</span><span class="CharOverride-2">, Awadh Bihari Pandey</span><span class="CharOverride-3">1</span></p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Affiliations ParaOverride-1"><span class="CharOverride-4">1</span>Classical Swine Fever Virus Laboratory, Division of Virology, Indian Veterinary Research Institute; <span class="CharOverride-4">2</span>Division of TAH, IVRI, Campus Mukteswar, Nainital 263 138, Uttarakhand, India; <span class="CharOverride-4">3</span>Division of Biological Standardization, IVRI, Campus Izatnagar-243122, UttarPradesh, India.</p>
		</div>
		<div>
			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Abstract</span> | Classical swine fever (CSF) is an economically important contagious viral disease, responsible for high mortality in young swine population. Classical swine fever virus (CSFV), the causative agent of CSF, belongs to genus <span class="CharOverride-6">Pestivirus</span> under family <span class="CharOverride-6">Flaviviridae</span>. Early detection of virus /genome is important for employing the disease containment measures. In present study, kinetics of antigen in experimentally infected piglets was determined by antigen ELISA and RT-PCR. Out of 12 sero-negative piglets, six were infected with virulent CSFV by intramuscular route, while other six were kept as control group. Piglets were regularly observed on the days post infection (dpi) for development of clinical manifestations of infection. Blood samples were collected on 0, 3, 6, 9, 12, 15, 18 and 21 dpi or till animal succumb of disease. Samples were screened by commercially available antigen ELISA and RT-PCR, for detection of viral proteins and nucleic acid to confirm the disease, prior to onset of clinical signs. Typical symptoms of disease were observed in the infected piglets on 10-12 dpi. Presence of CSFV antigen in blood sample from day’s 6<span class="CharOverride-4">th </span>to7<span class="CharOverride-4">th</span> post infection and viral nucleic acid on 6<span class="CharOverride-4">th</span> and 9<span class="CharOverride-4">th</span> dpi<span class="CharOverride-7"> </span>were confirmed by antigen ELISA and RT-PCR respectively. It was concluded by the present study that CSFV antigen can be detected even before the appearance of clinical signs. Such early detection can be used for field samples to test their repeatability and to prove as tools to control the spread of disease and to minimize economic losses.</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Keywords </span>| CSFV, Experimental Infection, RT-PCR, ELISA</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Editor----Citation"><span class="CharOverride-5">Editor </span>| MA Ramakrishnan, M.V.Sc., PhD., Post Doc (USA), Senior Scientist, Division of Virology, Indian Veterinary Research Institute, Mukteswar, Nainital, Uttarakhand, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-9">Special Issue</span> | 5(2015) “Emerging, Re-Emerging and Important Infectious Diseases of Animals”</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Received</span> | January 24, 2015; <span class="CharOverride-5">Revised</span> | February 18, 2015; <span class="CharOverride-5">Accepted</span> | February 20, 2015; <span class="CharOverride-5">Published</span> | March 03, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-5">*Correspondence</span> | Sachin Dattatraya Raut, Indian Veterinary Research Institute, Nainital, Uttarakhand, India; <span class="CharOverride-5">Email: </span>dr.sachindraut@rediffmail.com</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Citation</span> | Raut SD, Rajak KK, Kumar R, Gurav AR, Manu M, Dhanesh VV, Muthuchelvan D, Dhar P, Pandey AB (2015). Detection of classical swine fever virus antigen and nucleic acid on blood of experimentally infected piglets. Vet. Sci. 3(5s): 1-6.  </p>
			<p class="Editor----Citation"><span class="CharOverride-5">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.5s.1.6"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.5s.1.6</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><span class="Editor---Citation CharOverride-5" lang="en-US">)</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-9">Copyright </span>© 2015 Raut 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----">&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">C</span>SF is a highly contagious viral hemorrhagic disease of domestic pigs causing serious economic losses (<a href="#Paton-DJ--Greiser-Wilke-I--2003-."><span class="Hyperlink">Paton and Greiser-Wilke, 2003</span></a><span class="apple-style-span">)</span>. The causative agent of disease, CSFV, belongs to the <span class="CharOverride-6">Pestivirus </span>genus of the <span class="CharOverride-6">Flaviviridae</span> family (<a href="#Wengler-G--1991-"><span class="Hyperlink">Wengler, 1991</span></a>). In India, most of the outbreaks have been reported from North-East states, where a large number of country’s pig population is reared. The mean prevalence of CSFV antibodies in different parts of India has been found to be 63.3%. On the other hand 76.7% of the suspected samples were found to contain CSFV antigen (<a href="#Nandi-S--Muthuchelvan-D--Ahuja-A--Bisht-S--Chander-V--Pandey-AB-Singh-R-K--2011-"><span class="Hyperlink">Nandi et al., 2011</span></a>). Virus is transmitted oronasally by direct or indirect contact with infected pigs or by feeding virus contaminated feed (<a href="#Fritzemeier-J--Teuffert-J--Greiser-Wilke-I--Staubach-C--Schluter-H--Moennig-V--2000"><span class="Hyperlink">Fritzemeier et al., 2000</span></a>; <a href="#Moennig-V--Floegel-Niesmann-G--Greiser-Wilke-I---2003-"><span class="Hyperlink">Moennig et al., 2003</span></a>). CSF diagnosis in a herd in the early phase of infection has got paramount importance for both economical and epidemiological points of view (<a href="#Dewulf-J--Koenen-F--Mintiens-K--Denis-D--Ribbens-S-de-Kruif-A--2004-"><span class="Hyperlink">Dewulf et al., 2004</span></a>; <a href="#Lipowski-A--Pejsak-Z--Thibault-JC--Crevat-D--1996"><span class="Hyperlink">Lipowski et al., 1996</span></a>). The standard procedure for the detection of CSFV infection is based on virus isolation, which is then confirmed by the use of antigen detection ELISA and RT-PCR (<a href="#McGoldrick-A--Bensaude-E--Ibata-G--Sharp-G--Paton-DJ--1999"><span class="Hyperlink">McGoldrick et al., 1999</span></a>; <a href="#De-Smit-AJ--2000-"><span class="Hyperlink">De Smit, 2000</span></a>). RT-PCR has been found to be the most sensitive method for detection of CSFV (<a href="#Le-Dimna-M--Vrancken-R--Koenen-F--Bougeard-S--Mesplede-A--Hutet-E--Kuntz-Simon-G--Le-Potier-MF--2008"><span class="Hyperlink">Le Dimna et al., 2008</span></a>). During the phase of CSF epidemics, when a large number of samples need to be screened, antigen detection ELISA was found promising (<a href="#Shannon-AD--Morrissy-C--Mackintosh-SG-Westbury-HA--1993-"><span class="Hyperlink">Shannon et al., 1993</span></a>). A wide variety of samples are suitable for disease diagnosis, which helps in detection of viral antigen in early phase of infection which reduces the time of diagnosis and spread of disease. The aim of present study was to detect CSF virus from blood samples of experimentally infected piglets by antigen ELISA and RT-PCR. This will help to confirm the infection of CSFV well before onset of the clinical signs and symptoms and to implement control strategy more effectively. </p>
			<p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
		  <p class="Caps-on-First-Para ParaOverride-1">&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-">Experimental Animals</p>
			<p class="Body-Text ParaOverride-1">Twelve apparently healthy seronegative for CSFV white large Yorkshire (10-12<span class="CharOverride-4">th</span> week’s age and 20-30 kg in weight) piglets of either sex were used in present study. Seronegativity was tested using commercially available ELISA kit (IDEXX CSFV antibody test kit). All the animals were housed comfortably and provided feed and water <span class="CharOverride-6">ad libidum. </span></p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Virulent Virus </p>
			<p class="Body-Text ParaOverride-1">Virulent CSFV (Mhow/2005) being maintained by needle passage in pigs, at Division of Biological Standardization, IVRI, Izatnagar was used for experimental infection. Identity of the virus was re-confirmed by RT-PCR before inoculating to the animals. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Experimental Infection and Sample Collection</p>
			<p class="Body-Text ParaOverride-1">Twelve ear tagged seronegative piglets were divided into two groups (A and B), of six piglets each. Both groups were housed and handled separately during entire experimental period. Six piglets from group A were inoculated intramuscularly following OIE guidelines, with an amount of virus corresponding to 10<span class="CharOverride-4">5</span> PID<span class="CharOverride-15">50</span> (median pig infectious dose), with 5ml of 10% spleenic suspension of virulent CSF virus. Onset of disease and clinical symptoms were observed daily post infection. Piglets of group B were kept as healthy control. All the animals were observed to evaluate clinical score based on body temperature (Peak level: +++) and typical symptoms of CSF (<span class="CharOverride-5">++</span>: huddling, inco-ordination, <span class="CharOverride-5">+++</span>: reddening skin at the base of ear, inner side of abdomen, legs etc.) till survival/ completion of experiment. Blood samples were collected from all the animals in vacutainers containing EDTA on 0,3,6,9,12,15,18 and 21 dpi. All the samples were placed into a freezer (-80°C) until time for examination. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Detection of CSFV Antigen and Nucleic Acid </p>
			<p class="Body-Text ParaOverride-1">CSFV antigen from blood samples was tested by commercially available priocheck CSFV Ag ELISA following manufacture’s protocol; whereas viral nucleic acid was detected by RT-PCR. RNA Extraction was done by using commercial kit (Macherey-Nagel Nucleospin RNA Blood Midi) and quantity and purity of total RNA was checked by nanovue spectrophotometer. Approximately 1000ng of RNA was reverse transcribed using RevertAid<span class="CharOverride-4">TM</span> First Strand cDNA Synthesis Kit (M/s MBI Fermentas Life Sciences, Maryland, USA). Primers for amplification of CSFV 5’UTR gene were used as per published reference (<a href="#Paton-D-J--McGoldrick-A--Greiser-Wilke-2000"><span class="Hyperlink">Paton et al., 2000</span></a>). The stepwise cyclic conditions were: step I, 1 cycle, 95°C for 3 min; step II, 35 cycles, 95°C for 30 sec, 50<span class="CharOverride-4">o</span>C for 45 sec., 72<span class="CharOverride-4">o</span>C for 1 min; step III, 72<span class="CharOverride-4">o</span>C for 10 min. The PCR products were analysed by electrophoresis using 1.5 % agarose gel along with molecular weight marker.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</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-">Screening of Piglets for Seronegativity   </p>
			<p class="Body-Text ParaOverride-1">Maximal colour development at the end of reaction in blocking ELISA using pre-infected sera confirmed the absence of CSFV specific antibodies. These observations were interpreted by calculating the blocking percentage value, which was found to be less than 30 percent (&lt;30%).  This indicated that piglets were seronegative against CSFV.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150304024611.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150304024611.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/20150304024611.png"> Rectal temperature of experimentally infected piglets showing rise in temperature from 3-4th dpi with peak on 14-15th dpi</a></p>
       </div><br>

			
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150304024314.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150304024314.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/20150304024314.png"> Clinical signs in experimentally infected piglets: A. Cyanosis of ear base; B. Dead carcass with cyanotic discoloration at inner side of abdomen and legs</a></p>
       </div><br>
       <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150304025516.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150304025516.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/20150304025516.png"> Detection of CSFV antigen in blood of experimentally infected piglets using priocheck CSFV Ag. Kit. All the animals were found to be positive from 6<span class="CharOverride-4">th</span> dpi with gradual increase in titre till death</a></p>
       </div><br>
			
		  <p class="Heading-2--History-in-MM-">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-">Experimental Infection </p>
			<p class="Body-Text ParaOverride-1">All the piglets of infected group showed increase in rectal temperature; which reached to peak level (106<span class="CharOverride-4">o</span>F-108<span class="CharOverride-4">o</span>F)<span class="CharOverride-5"> </span>after 5-6 dpi of virus (<a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>). Typical symptoms of CSF (huddling, inco-ordination, reddening skin at the base of ear, inner side of abdomen, legs etc.) were observed10-12 dpi onwards (<a href="#Figure-2-"><span class="Hyperlink">Figure 2</span></a>) and all the piglets died in between 14-21 dpi. During postmortem, typical lesions (hemorrhages in the visceral organs like trachea, bladder, intestinal mucosa and caecum) of CSF was observed. Piglets of control group were found normal till the end of the experiment. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Detection of CSFV Antigen and Viral RNA in Blood  </p>
			<p class="Body-Text ParaOverride-1">CSFV antigen was initially detected on day’s 6<span class="CharOverride-4">th</span>-7<span class="CharOverride-4">th</span> post infection and gradually increased till last day/ the day of death (<a href="#Figure-3-"><span class="Hyperlink">Figure 3</span></a>).<span class="CharOverride-5"> </span>Blood samples from control group were found to be negative for CSFV antigen. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Majority of infected piglets exhibited viral nucleic acid in the blood on 9<span class="CharOverride-4">th</span> dpi by amplification of 5’UTR; whereas on 6<span class="CharOverride-4">th</span> dpi, amplification noticed in blood sample of some infected piglet (<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">&nbsp;</p>
			
			
			
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Table-1-"></a>Table 1:</span> Evaluation of CSF disease <span class="CharOverride-6">vis-à-vis </span>antigen kinetics in blood of experimentally infected piglets</p>
			<table width="657" height="462" 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-3" />
					<col class="_idGenTableRowColumn-5" />
					<col class="_idGenTableRowColumn-6" />
					<col class="_idGenTableRowColumn-7" />
					<col class="_idGenTableRowColumn-7" />
					<col class="_idGenTableRowColumn-8" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-9">
						<td rowspan="2">
							<p class="Body-Text ParaOverride-2">Piglet no.</p>
						</td>
						<td rowspan="2">
							<p class="Body-Text ParaOverride-2">Parameters</p>
						</td>
						<td colspan="8">
							<p class="Body-Text ParaOverride-2">Days post infection</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Body-Text ParaOverride-2">0</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">3</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">6</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">9</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">12</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">15</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">18</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">21</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td rowspan="3">
							<p class="Body-Text ParaOverride-2">1</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">Clinical Score</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++/*</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Body-Text ParaOverride-2">ELISA</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Body-Text ParaOverride-2">RT-PCR</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td rowspan="3">
							<p class="Body-Text ParaOverride-2">2</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">Clinical Score</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++/*</p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Body-Text ParaOverride-2">ELISA</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Body-Text ParaOverride-2">RT-PCR</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td rowspan="3">
							<p class="Body-Text ParaOverride-2">3</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">Clinical Score</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++/*</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Body-Text ParaOverride-2">ELISA</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Body-Text ParaOverride-2">RT-PCR</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td rowspan="3">
							<p class="Body-Text ParaOverride-2">4</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">Clinical Score</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++/*</p>
						</td>
						<td />
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Body-Text ParaOverride-2">ELISA</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td />
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Body-Text ParaOverride-2">RT-PCR</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td />
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td rowspan="3">
							<p class="Body-Text ParaOverride-2">5</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">Clinical Score</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++/*</p>
						</td>
						<td />
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Body-Text ParaOverride-2">ELISA</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td />
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Body-Text ParaOverride-2">RT-PCR</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td />
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td rowspan="3">
							<p class="Body-Text ParaOverride-2">6</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">Clinical Score</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">-</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">++</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">+++/*</p>
						</td>
						<td />
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Body-Text ParaOverride-2">ELISA</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td />
						<td />
					</tr>
					<tr class="_idGenTableRowColumn-10">
						<td>
							<p class="Body-Text ParaOverride-2">RT-PCR</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">P</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-2">N</p>
						</td>
						<td />
						<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-5">- : </span>No disease<span class="CharOverride-5"> +:</span> Mild; <span class="CharOverride-5">++:</span> Moderate; <span class="CharOverride-5">+++:</span> Severe; *: Death; <span class="CharOverride-5">N:</span> Negative; <span class="CharOverride-5">P:</span> Positive</p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-1--Introduction----">Discussion </p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text">The infected pigs shed large amount of virus in saliva and smaller quantities in urine, nasal and lachrymal fluids that leads to CSF epidemics (<a href="#Edwards-S--2000"><span class="Hyperlink">Edwards, 2000</span></a>). Thus, early tracing of possibly infected herds is focused on the detection of CSFV antigen (<a href="#Shannon-AD--Morrissy-C--Mackintosh-SG-Westbury-HA--1993-"><span class="Hyperlink">Shannon et al., 1993</span></a>) rather than CSFV antibodies and further confirmed by RT-PCR technique, which is specific diagnostic assay that can complement classical serological procedures (<a href="#Depner-K--Hoffmann-B--Beer-M--2007a-"><span class="Hyperlink">Depner et al., 2007</span></a>). Virus isolation is standard procedure for CSFV detection (<a href="#Terpstra-C--1996-"><span class="Hyperlink">Terpstra, 1996</span></a>), however not suitable during outbreak conditions since, it is labor intensive and time consuming for results (<a href="#Terpstra-C--De-Smith-AJ--2000"><span class="Hyperlink">Terpstra and de Smit, 2000</span></a>; <a href="#Dewulf-J--Koenen-F--Mintiens-K--Denis-D--Ribbens-S-de-Kruif-A--2004-"><span class="Hyperlink">Dewulf et al., 2004</span></a>). Early detection of CSFV during outbreak condition helps field veterinarian to eliminate infection from the pig population. Present study initiated with illustration of absence of CSFV specific antibodies in 10-12<span class="CharOverride-4">th</span> week’s age old piglets and infected with virulent CSF virus. Pyrexia is one of the first signs of CSF and is accompanied by viremia. During experimental and horizontal infection of CSF virus, there is variable latency period from inoculation to viraemia and it was reported that, first viraemia is detected at 4 dpi in experimental infection whereas on 12 &amp; 14 dpi in horizontal infection (<a href="#Laevens-H--Koenen--F--Deluyker-H--Berkvens-D--de-Kruif-A--1998-."><span class="Hyperlink">Laevens et. al., 1998</span></a>). In this study, infected piglets, showed initiation of increase in rectal temperature on 3-4 dpi followed by peak level (106<span class="CharOverride-4">0</span>F-108<span class="CharOverride-4">0</span>F)<span class="CharOverride-5"> </span>on 14-15 dpi. Piglets remain vireamic during 6-9 dpi.  Similar findings have been reported that pigs get infection by oro-nasal route, develop high fever on 4-6 dpi (<a href="#Dahle-J--Moennig-V--Coulibaly-CO--Liess-B--1991-."><span class="Hyperlink">Dahle et al., 1991</span></a>, <a href="#Dewulf-J--Koenen-F--Mintiens-K--Denis-D--Ribbens-S-de-Kruif-A--2004-"><span class="Hyperlink">Dewulf et al., 2004</span></a>) and viremia was detected between the first and third day from the beginning of the febrile conditions in infected animals (<a href="#Milanov-D--Asanin-R--Durisic-Suvajdzic-L--Lazic-S--2002"><span class="Hyperlink">Milanov et al., 2002</span></a>). In infected piglets, course of the disease was influenced by the virulence of the virus and symptoms may vary hence, the antigen ELISA, based on the detection of viral antigen (<a href="#Depner-KR--Muller-A--Gruber-A--Rodriguez-A--Bickhardt-K--Liess-B--1995a-"><span class="Hyperlink">Depner et al., 1995</span></a>) and RT-PCR become helpful for rapid detection of CSFV (<a href="#Le-Dimna-M--Vrancken-R--Koenen-F--Bougeard-S--Mesplede-A--Hutet-E--Kuntz-Simon-G--Le-Potier-MF--2008"><span class="Hyperlink">Le Dimna et al., 2008</span></a>). In infected piglets, course of the infection was evaluated by using antigen ELISA and confirmed by RT-PCR technique. CSFV antigen was found in 33 blood samples of infected piglets, collected on 6-7<span class="CharOverride-4">th</span> dpi onwards. It was found that 5’UTR region of CSFV was amplified in 10 samples, collected from infected animals on day 6<span class="CharOverride-4">th</span> and 9<span class="CharOverride-4">th</span> post infection only. This is in agreement with <a href="#Jasna-P--Dosen-R--Pusic-I--Bugarski-D--Valcic-M--2007"><span class="Hyperlink">Jasna et al. (2007)</span></a> who reported that CSFV antigen (viraemia) is detected on 7<span class="CharOverride-4">th</span> and 9<span class="CharOverride-4">th</span> dpi by ELISA in piglets of different age group (28, 35, 44 and 54 days old) exposed to contact infection of CSFV and these findings were confirmed by applying RT-PCR technique, the presence of viral RNA was detected only in 3 piglets from blood samples. Hence detectable level of CSFV antigen was observed on day 6-7<span class="CharOverride-4">th</span> post infection and gradually increased till death. The typical symptoms of CSF (Anorexia, conjunctivitis, in-coordination, diarrhea, reddening of the base of ear etc) observed 10-12 dpi onwards and was found to be similar to those reported by <a href="#Cariolet-R--Bougeard-S--Rault-JC--Jan-B--Keranflech-A--Le-Diguerher-G--Pol-F--Kuntz-Simon-G--Le-Poti"><span class="Hyperlink">Cariolet et al. (2008)</span></a>. All the piglets showed almost same pathogenesis and no individual variation could be noticed. This may be due to similar origin of piglets. Postmortem examination revealed typical lesions (hemorrhages in the visceral organs like kidney, trachea, bladder, intestinal mucosa and caecum) of CSF. Hence it can be concluded that choice of laboratory test depends on the goal, in this study the antigen ELISA and RT-PCR technique found to be promising to detect early infection of CSF virus in experimentally infected piglets well before clinical manifestations. Hence the present study will be helpful to reduce the time of diagnosis and spread of disease which ultimately leads to effective control of the disease.</p>
			<p class="Body-Text">&nbsp;</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Heading-1--Introduction----">Acknowlegement</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The authors are grateful to Director of the Institute for providing the necessary facilities to carry out this work. Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, New Delhi is highly acknowledged for providing necessary funds under DBT-CSF network project to carry out this study.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</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.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">References </p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			
			  <li class="References ParaOverride-4" lang="en-US"><a id="Cariolet-R--Bougeard-S--Rault-JC--Jan-B--Keranflech-A--Le-Diguerher-G--Pol-F--Kuntz-Simon-G--Le-Poti"></a>Cariolet R, Bougeard S, Rault JC, Jan B, Keranflech A, Le Diguerher G, Pol F, Kuntz-Simon G, Le Potier MF (2008). Importance des observations cliniques et necroscopiques dans la detection precoce dun cas de peste porcine classique. Journees de la Recherche Porcine. 40: 45 - 48.</li>
				<li class="References ParaOverride-4" lang="en-US"><a id="Dahle-J--Moennig-V--Coulibaly-CO--Liess-B--1991-."></a>Dahle J, Moennig V, Coulibaly CO, Liess B (1991). Clinical post mortem and virological findings after simultaneous inoculation of pigs with hog cholera and bovine viral diarrhoea virus. Zentralbl. Veterinarmed. B. 38(10): 764 - 772.</li>
				<li class="References ParaOverride-4" lang="en-US"><a id="De-Smit-AJ--2000-"></a>De Smit AJ (2000). Classical Swine Fever, Efficacy of marker vaccines and laboratory diagnosis, PhD Thesis, Universiteit Utrecht. 9-22.</li>
				<li class="References ParaOverride-4" lang="en-US"><a id="Depner-K--Hoffmann-B--Beer-M--2007a-"></a>Depner K, Hoffmann B, Beer M (2007). Evaluation of real-time RT-PCR assay for the routine intra vitam diagnosis of classical swine fever. Vet. Microbiol. 121(3-4): 338- 343. <a href="http://dx.doi.org/10.1016/j.vetmic.2006.12.027"><span class="Hyperlink">http://dx.doi.org/10.1016/j.vetmic.2006.12.027</span></a></li>
				<li class="References ParaOverride-4" lang="en-US"><a id="Depner-KR--Muller-A--Gruber-A--Rodriguez-A--Bickhardt-K--Liess-B--1995a-"></a>Depner KR, Muller A, Gruber A, Rodriguez A, Bickhardt K, Liess B (1995). Classical swine fever in wild boar (Sus scrofa) experimental infections and viral persistence. Dtsch. Tierarztl. Wochenschr. 102(10): 381-4.</li>
				<li class="References ParaOverride-4" lang="en-US"><a id="Dewulf-J--Koenen-F--Mintiens-K--Denis-D--Ribbens-S-de-Kruif-A--2004-"></a>Dewulf J, Koenen F, Mintiens K, Denis D, Ribbens S de Kruif A (2004). Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection. J. Virol. Methods. 119(2): 137-143. <a href="http://dx.doi.org/10.1016/j.jviromet.2004.03.010"><span class="Hyperlink">http://dx.doi.org/10.1016/j.jviromet.2004.03.010</span></a></li>
				<li class="References ParaOverride-4" lang="en-US"><a id="Edwards-S--2000"></a>Edwards S (2000). Survival and inactivation of classical swine fever virus. Vet. Microbiol. 73(2-3): 175-181. <a href="http://dx.doi.org/10.1016/S0378-1135(00)00143-7"><span class="Hyperlink">http://dx.doi.org/10.1016/S0378-1135(00)00143-7</span></a> <a href="http://dx.doi.org/10.1016/S0378-1135(00)00138-3"><span class="Hyperlink">http://dx.doi.org/10.1016/S0378-1135(00)00138-3</span></a></li>
				<li class="References ParaOverride-4" lang="en-US"><a id="Fritzemeier-J--Teuffert-J--Greiser-Wilke-I--Staubach-C--Schluter-H--Moennig-V--2000"></a>Fritzemeier J, Teuffert J, Greiser-Wilke I, Staubach C, Schluter H, Moennig V( 2000). Epidemiology &#9;of classical swine fever in Germany in the 1990s. Vet. Microbiol. 77(1-2): 29-41. <a href="http://dx.doi.org/10.1016/S0378-1135(00)00254-6"><span class="Hyperlink">http://dx.doi.org/10.1016/S0378-1135(00)00254-6</span></a></li>
				<li class="References ParaOverride-4" lang="en-US"><a id="Jasna-P--Dosen-R--Pusic-I--Bugarski-D--Valcic-M--2007"></a>Jasna P, Dosen R, Pusic I, Bugarski D, Valcic M (2007). Passive immunity evaluation in piglets originating from sows vaccinated with china strain of classical swine fever virus. Acta Veterinaria (Beograd). 57 (5-6): 413-427. <a href="http://dx.doi.org/10.2298/AVB0706413P"><span class="Hyperlink">http://dx.doi.org/10.2298/AVB0706413P</span></a></li>
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