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<body id="AAVS_MH20141219041226_Singh-PL" lang="en-GB">
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			<p class="Type-of-Article">&nbsp;</p>
			<p class="Type-of-Article"><span class="CharOverride-1">Short Communication</span></p>
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		<div class="Basic-Text-Frame">
			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Detection of Bordetella bronchiseptica in Serum of Apparently Healthy and Clinically Sick Pet Dogs</p>
		</div>
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			<p class="Authors ParaOverride-1">&nbsp;</p>
			<p class="Authors ParaOverride-1"><span class="CharOverride-3">Polem Lenin Singh</span><span class="CharOverride-4">1</span><span class="CharOverride-3">, Bhoj Raj Singh</span><span class="CharOverride-4">1*</span><span class="CharOverride-3">, Monika Bhardwaj</span><span class="CharOverride-4">2</span><span class="CharOverride-3">, Prassanvadhana</span><span class="CharOverride-4">2</span><span class="CharOverride-3">, </span><span class="CharOverride-3" lang="en-US">Dharmendra Kumar</span><span class="CharOverride-3"> Sinha</span><span class="CharOverride-4">1</span><span class="CharOverride-3">, </span><span class="CharOverride-3" lang="en-US">Nongthombam </span><span class="CharOverride-3">Boby</span><span class="CharOverride-4">3</span><span class="CharOverride-3">, Ravi Kant Agrawal</span><span class="CharOverride-4">4</span><span class="CharOverride-3">, Abhijit M Pawde</span><span class="CharOverride-4">5</span></p>
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			<p class="Affiliations ParaOverride-1"><span class="CharOverride-5">1</span>Division of Epidemiology, <span class="CharOverride-5">2</span>Division of Bacteriology and Mycology, <span class="CharOverride-5">3</span>Division of Animal Biotechnology, <span class="CharOverride-5">4</span>Division of Livestock Products Technology, <span class="CharOverride-5">5</span>Referral Veterinary Polyclinic, Indian Veterinary Research Institute (IVRI), Izatnagar-243122, Bareilly, India.</p>
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			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-6">Abstract</span> | DNA extracted from serum samples collected from 114 apparently healthy and 71 clinically sick pet dogs of different age, sex, breed and place was used as templet for detection of <span class="CharOverride-7">Bordetella</span> infection through genus specific PCR (gPCR) targeting <span class="CharOverride-7">alc</span> gene and species specific PCR (sPCR) targeting <span class="CharOverride-7">fim</span> gene. <span class="CharOverride-7">Bordetella</span> DNA could be detected in serum of 7 apparently healthy and 7 sick dogs both with gPCR and sPCR. Clinical status (p= 0.35), age (p= 0.31) and sex (p= 0.72) had no significant association with detection of <span class="CharOverride-7">Bordetella</span> DNA in serum samples. Of the 14 samples positive 12 (85.7%) were from Kerala and two (14.3%) from Manipur dogs. Sequencing of the gel purified gPCR product targeting <span class="CharOverride-7">alc</span> gene revealed 95% homology with <span class="CharOverride-7">alc</span> gene sequence of several species of <span class="CharOverride-7">Bordetella</span> and sequencing of the gel purified sPCR product targeting <span class="CharOverride-7">fim</span> gene revealed 100% identity with <span class="CharOverride-7">Bordetella bronchiseptica fim gene.</span> The study suggested that <span class="CharOverride-7">B. bronchiseptica</span> might be the most common bordetellae affecting pet dogs irrespective of clinical illness. The study concluded that PCR with serum extracted DNA template can be an option for rapid detection of <span class="CharOverride-7">Bordetella</span> infection in pet dogs.</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-6">Keywords </span>| Bordetellosis, <span class="CharOverride-7">Bordetella bronchiseptica</span>, PCR, Serum-PCR, dogs, Kennel cough</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
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			<p class="Editor----Citation"><span class="CharOverride-9">Editor</span> | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-6">Received</span> | December  19, 2014; <span class="CharOverride-6">Revised</span> | January 13, 2015; <span class="CharOverride-6">Accepted</span> | January 14, 2015; <span class="CharOverride-6">Published</span> | January 23, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-6">*Correspondence </span>| Bhoj Raj Singh, Indian Veterinary Research Institute, Izatnagar, Bareilly, India; <span class="CharOverride-6">Email:</span> brs1762@gmail.com</p>
			<p class="Editor----Citation"><span class="CharOverride-6">Citation</span> | Singh PL, Singh BR, Bhardwaj M, Prassanvadhana, Sinha DK, Boby N, Agrawa RK, Pawde AM (2015). Detection of Bordetella bronchiseptica in serum of apparently healthy and clinically sick pet dogs. Adv. Anim. Vet. Sci. 3(2): 123-127.  </p>
			<p class="Editor----Citation"><span class="CharOverride-9">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.2.123.127"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.2.123.127</span></a></p>
			<p class="Editor----Citation"><span class="Editor---Citation CharOverride-6" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-6" lang="en-US">(</span><span class="Editor---Citation CharOverride-6" lang="en-US">Online</span><span class="Editor---Citation CharOverride-6" lang="en-US">)</span> | 2307-8316; <span class="Editor---Citation CharOverride-6" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-6" lang="en-US">(Print) </span> | 2309-3331</p>
			<p class="Editor----Citation"><span class="CharOverride-9">Copyright </span>© 2015 Singh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
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			<p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">K</span>ennel cough is a multi-etiological disease and <span class="CharOverride-7">Bordetella bronchiseptica </span>is the main etiology in dogs (<a href="#Keil-DJ--Fenwick-B--1998-."><span class="Hyperlink">Keil and Fenwick, 1998</span></a>). <span class="CharOverride-7">Bordetella</span> affects dogs of all the age groups (<a href="#Appel-M--Binn-LN--1987-"><span class="Hyperlink">Appel et al., 1987</span></a>; <a href="#Ford-RB--2006-."><span class="Hyperlink">Ford, 2006</span></a>) and has a zoonotic aspect too (<a href="#Woolfrey-FB--Moody-JA--1991-."><span class="Hyperlink">Woolfrey et al., 1991</span></a>) affecting immunocompromised (<a href="#Bauwens-JE--Spach-DH--Schacker-TW--Mustafa-MM--Bowden-RA--1992-."><span class="Hyperlink">Bauwens et al., 1992</span></a>) as well as healthy humans (<a href="#Lo-Re-V--Brennan-PJ--Wadlin-J--Weaver-R--Nachamkin-I--2001-."><span class="Hyperlink">Lo et al., 2001</span></a>). Many conventional bacteriological methods, immunological (agglutination, microagglutination and enzyme linked immunosobant assay) and molecular tools (polymerase chain reaction, PCR; real time PCR) have been used to detect <span class="CharOverride-7">Bordetella</span> infection in animals. However, diagnosis of bordetellosis is a problem because of slow growing and fastidious nature of the pathogen and contaminants in the clinical samples (<a href="#Kumar-S--Singh-BR--Bhardwaj-M--Singh-V--2014-."><span class="Hyperlink">Kumar et al., 2014</span></a>; <a href="#Bhardwaj-M--Singh-BR--Kumar-S--Pawde-AM--2013a-."><span class="Hyperlink">Bhardwaj et al., 2013a</span></a>, <a href="#Bhardwaj-M--Singh-BR--Vadhana-P--2013b-."><span class="Hyperlink">b</span></a>; <a href="#Bhardwaj-M--2013-"><span class="Hyperlink">Bhardwaj, 2013</span></a>; <a href="#Rapuntean-Gh--Rapuntean-S--2010-."><span class="Hyperlink">Rapuntean and Rapuntean, 2010</span></a>; <a href="#Sacco-RE--Register-KB--Nordholm-GE--2000-."><span class="Hyperlink">Sacco et al., 2000</span></a>). </p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Attempts have been made to identify circulating DNA in blood and or serum where isolation and identification of the pathogen from clinical samples is difficult even after using the best methods (<a href="#Bougnoux-ME--Dupont-C--Mateo-J--Saulnier-P--Faivre-V--Payen-D--Nicolas-Chanoine-MH--1999-"><span class="Hyperlink">Bougnoux et al., 1999</span></a>; <a href="#Kawamura-S--Maesaki-S--Noda-T--Hirakata-Y--Tomono-K--Tashiro-T--Kohno-S--1999-"><span class="Hyperlink">Kawamura et al., 1999</span></a>; <a href="#Murdoch-DR--Walford-EJ--Jennings-LC--Light-GJ--Schousboe-MI--Chereshsky-AY--Town-GI--1996-."><span class="Hyperlink">Murdoch et al., 1996</span></a>; <a href="#Zerva-L--Bourantas-K--Mitka-S--Kansouzidou-A--Legakis-NJ--2001-."><span class="Hyperlink">Zerva et al., 2001</span></a>) or when etiology is obscure (<a href="#De-Madaria-E--Martinez-J--Lozano-B--Sempere-L--Benlloch-S--Such-J--P-rez-Mateo-M--2005-"><span class="Hyperlink">De-Madaria et al., 2005</span></a>). For diagnosis of brucellosis (<a href="#Zerva-L--Bourantas-K--Mitka-S--Kansouzidou-A--Legakis-NJ--2001-."><span class="Hyperlink">Zerva et al., 2001</span></a>) and candidiasis (<a href="#Bougnoux-ME--Dupont-C--Mateo-J--Saulnier-P--Faivre-V--Payen-D--Nicolas-Chanoine-MH--1999-"><span class="Hyperlink">Bougnoux et al., 1999</span></a>) in human DNA template extracted from serum has proved<span class="CharOverride-7"> </span>more sensitive and specific template than extracted from whole blood samples, probably due to co-purification of PCR inhibitory substances with pathogen DNA (<a href="#Akane-A--Matsubara-K--Nakamura-H--Takahashi-S--Kimura-K--1994-"><span class="Hyperlink">Akane et al., 1994</span></a>). The well proven diagnostic method for infections in human has little known utility of the serum extracted pathogen DNA in diagnosis of infections in animals including for diagnosis of <span class="CharOverride-7">Bordetella</span> infection. As collection of deep nasal swabs required for diagnosis of bordetellosis is difficult to collect even from well-trained dog and thus the diagnosis. Antibody detection methods though simple but lacks specificity thus have no diagnostic utility (<a href="#Kumar-S--Singh-BR--Bhardwaj-M--Singh-V--2014-."><span class="Hyperlink">Kumar et al., 2014</span></a>; <a href="#Bhardwaj-M--Singh-BR--Kumar-S--Pawde-AM--2013a-."><span class="Hyperlink">Bhardwaj et al., 2013a</span></a>, <a href="#Bhardwaj-M--Singh-BR--Vadhana-P--2013b-."><span class="Hyperlink">b</span></a>).  This study was undertaken to explore the utility of serum samples for detection of antigen rather than antibodies for specific diagnosis of bordetellosis through identifying circulating <span class="CharOverride-7">Bordetella bronchiseptica</span> DNA in the serum samples of apparently healthy and clinically sick pet dogs.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Out of 185 serum samples, 14 (7.6%) samples were positive for both genus specific amplicon (gPCR) and species specific PCR targeting <span class="CharOverride-7">alc </span>(<a href="#Figure-1"><span class="Hyperlink">Figure 1</span></a>) and<span class="CharOverride-7"> fim</span> (<a href="#Figure-2"><span class="Hyperlink">Figure 2</span></a>) gene, respectively (<a href="#Table-2"><span class="Hyperlink">Table 2</span></a>). In earlier studies on the same lot of dogs using DNA template extracted from deep nasal swabs only four dogs were detected positive for <span class="CharOverride-7">B. bronchiseptica</span> infection and <span class="CharOverride-7">B. bronchiseptica</span> could be isolated from only one sample without showing any correlation between detection of <span class="CharOverride-7">B. bronchiseptica</span> and kennel cough in dogs (<a href="#Bhardwaj-M--2013-"><span class="Hyperlink">Bhardwaj, 2013</span></a>). All the five dogs positive for Bordetella in nasal swabs were also positive for PCR using DNA templet extracted from respective serum samples indicating 100% sensitivity of the serum PCR method. Probability of detection of <span class="CharOverride-7">B. bronchiseptica </span>was significantly (p= 0.016) higher in serum extracted DNA than detection of <span class="CharOverride-7">Bordetella </span>DNA in nasal swabs of dogs (<a href="#Bhardwaj-M--2013-"><span class="Hyperlink">Bhardwaj, 2013</span></a>). Findings indicated the superiority of serum samples for PCR based detection of <span class="CharOverride-7">Bordetella bronchiseptica</span> infection over nasal swabs. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text">Detection of <span class="CharOverride-7">B. bronchiseptica</span> by PCR in serum samples had no significant (p= 0.35) association with health status either in male (p= 0.75) or female (p= 0.34) dogs. Further there appeared to be no significant (p= 0.72) association of sex of dogs with probability of detection of <span class="CharOverride-7">Bordetella </span>DNA in serum samples. In earlier studies too detection of <span class="CharOverride-7">Bordetella</span> in nasal swabs of dogs could not be associated with kennel cough however, detection of <span class="CharOverride-7">B. bronchiseptica</span> in bitches was significantly higher (p= 0.03) than in male dogs and sex has been suggested an important predisposing determinant associated with the infection (<a href="#Bhardwaj-M--Singh-BR--Kumar-S--Pawde-AM--2013a-."><span class="Hyperlink">Bhardwaj et al., 2013a</span></a>). The observations indicated that we may reach on different conclusions depending on sensitivity of methods used in the study. </p>
		  <p class="Body-Text">&nbsp;</p>
			
            <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150125023720.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150125023720.png" width="80" height="80"></a>
            
            <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-1"></a>Figure 1:</span><a href="http://nexusacademicpublishers.com/uploads/figures/20150125023720.png"> Genus Specific PCR (<span class="CharOverride-7">alc</span><span class="CharOverride-6"> </span>gene)</a></p>
       </div>

			
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-12">Lanes 1-3:Study samples; Lane 4: Positive control; Lane 5: Negative control; Lane M: 100bp DNA ladder</span></p>
		  <p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			
          <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150125021122.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150125021122.png" width="80" height="80"></a>
            
           <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-6"><a id="Figure-2"></a>Figure 2:</span><a href="http://nexusacademicpublishers.com/uploads/figures/20150125021122.png"> Genus Specific PCR (<span class="CharOverride-7">fim</span> gene)</a></p>
       </div>
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-12">Lane M: 100bp DNA ladder; Lane 1: Positive control; Lane 2: Negative control; Lanes 3-4: Study samples</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-6"><a id="Table-1-"></a>Table 1: </span>Primers for <span class="CharOverride-7">Bordetella bronchiseptica </span>detection</p>
			<table width="657" 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"><span class="CharOverride-13">Name of primers</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">   Sequence 5&apos; </span><span class="CharOverride-14">→</span><span class="CharOverride-13"> 3&apos;&#9;</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Target gene and product length (bp)  </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">References</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">B688Bbalc-F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">ACCAACCGCATTTATTCCTACTA</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-15">alc, 324</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-15">Bharadwaj, 2013</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">B1012Bbalc-R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">GGCCCTGGAGTTCGTATTTATG</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">425BBfim-1 F</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TGAACAATGGCGTGAAAGC</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-15">fim, 425</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-15">Xin et al., 2008</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-9">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">425BBfim-2 R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">TCGATAGTAGGACGGGAGGAT</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading">&nbsp;</p>
		  <p class="Figure--and-Table-Heading">Note: (F), Forward primer; (R), Reverse primer&#160;<span class="CharOverride-6">&#9;</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-6"><a id="Table-2"></a>Table 2: </span>Detection of <span class="CharOverride-7">Boretella</span> DNA in serum of pet dogs in relation to their sex and health status</p>
			<table width="657" height="140" class="Table-Style-1" id="table-2">
				<colgroup>
					<col class="_idGenTableRowColumn-10" />
					<col class="_idGenTableRowColumn-3" />
					<col class="_idGenTableRowColumn-11" />
					<col class="_idGenTableRowColumn-12" />
					<col class="_idGenTableRowColumn-13" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-7">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Sex</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Health status</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Total samples tested</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Positive for gPCR</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Positive for sPCR</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-15">Male</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">Apparently healthy</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">77</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">5</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">5</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-14">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">Sick </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">37</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">3</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-8">
						<td rowspan="3">
							<p class="Basic-Paragraph"><span class="CharOverride-15">Female </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">Apparently healthy</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">37</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">2</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">2</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-15">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">Sick </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">34</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">4</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-16">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">Total </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">185</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">14</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-15">14</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">The association of <span class="CharOverride-7">B. bronchiseptica</span> infection with kennel cough is well documented and reported frequently at several places (<a href="#Appel-M--Binn-LN--1987-"><span class="Hyperlink">Appel et al., 1987</span></a>; <a href="#Keil-DJ--Fenwick-B--1998-."><span class="Hyperlink">Keil and Fenwick, 1998</span></a>; <a href="#Ford-RB--2006-."><span class="Hyperlink">Ford, 2006</span></a>). However, in the present study probability of detection of <span class="CharOverride-7">B. bronchiseptica</span> DNA in serum of sick dogs (9.86%) was only insignificantly higher (p= 0.35) than detection of the pathogen from apparently healthy dogs (6.14%). The findings are in concurrence to earlier studies in India on bordetellosis and kennel cough in dogs (<a href="#Bhardwaj-M--Singh-BR--Kumar-S--Pawde-AM--2013a-."><span class="Hyperlink">Bhardwaj et al., 2013a</span></a>, <a href="#Bhardwaj-M--Singh-BR--Vadhana-P--2013b-."><span class="Hyperlink">b</span></a>). The study also indicated that just colonization of <span class="CharOverride-7">B. bronchiseptica</span> in dog nares or detection of pathogen’s DNA in serum may not be sufficient for diagnosis of bordetellosis and other factors might be important for precipitation of clinical disease in dogs.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">All 14 positive samples with genus specific (<span class="CharOverride-7">alc</span> gene) PCR were also positive for species specific PCR targeting <span class="CharOverride-7">fim</span> gene in the study indicating that only <span class="CharOverride-7">B. bronchiseptica</span> was able to invade the dog tissues. However, earlier study on nasal swabs indicated that although <span class="CharOverride-7">B. bronchiseptica</span> was the major bordetellae colonizing dog nares other bodetellae might be present in nasal swabs of dogs (<a href="#Bhardwaj-M--Singh-BR--Kumar-S--Pawde-AM--2013a-."><span class="Hyperlink">Bhardwaj et al., 2013a</span></a>). In earlier studies  on dogs (<a href="#Bhardwaj-M--2013-"><span class="Hyperlink">Bhardwaj, 2013</span></a>, <a href="#Bhardwaj-M--Singh-BR--Kumar-S--Pawde-AM--2013a-."><span class="Hyperlink">Bhardwaj et al., 2013a</span></a>) out of total 7 nasal swabs positive to gPCR only four samples shown <span class="CharOverride-7">fim</span> and <span class="CharOverride-7">fla</span> genes amplification indicating that other species of <span class="CharOverride-7">Bordetella</span> might be colonizing nasal mucosa of dogs. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Sequencing result of <span class="CharOverride-7">alc</span> and <span class="CharOverride-7">fim </span>gene PCR products revealed that the PCR performed in the study was true and specific in detecting only <span class="CharOverride-7">Bordetella</span>.Sequences alignment of genus specific <span class="CharOverride-7">alc</span> gene sequence amplified in the study had 95% identity with sequences of several species of <span class="CharOverride-7">Bordetella</span> available in database and species specific <span class="CharOverride-7">fim</span> gene sequence matched 100% with <span class="CharOverride-7">B. bronchiseptica </span>sequences in database. The study concluded that PCR with serum extracted DNA might be an option for diagnosis of <span class="CharOverride-7">Bordetella </span>infection in dog however there seems to be no association between kennel cough and <span class="CharOverride-7">Bordetella </span>detection in dogs in India.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The study concluded that DNA extracted from serum samples can be used in molecular detection of <span class="CharOverride-7">Bordetella</span> DNA for diagnosis of <span class="CharOverride-7">Bordetella</span> infection in pet dogs. Clinical status and sex had no significant association with detection of <span class="CharOverride-7">Bordetella </span>DNA in dog’s serum samples. Genus specific (<span class="CharOverride-7">alc</span> gene) and species specific (<span class="CharOverride-7">fim</span> genes) PCR results revealed that <span class="CharOverride-7">B. bronchiseptica</span> is probably the most common invading <span class="CharOverride-7">Bordetella</span> species affecting dogs.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Samples (deep nasal swabs and serum) were collected from 114 apparently (77 male and 37 female) healthy and 71 clinically sick (37 male and 34 female) pet dogs. Template DNA was extracted from serum samples using QIAmp DNA Mini kit (Qiagen, Germany) following the protocol recommended. From nasal swabs DNa was extracted after pre-enrichment step (<a href="#Bhardwaj-M--Singh-BR--Kumar-S--Pawde-AM--2013a-."><span class="Hyperlink">Bhardwaj et al., 2013a</span></a>) using the same kit. The concentration and purity of the DNA extracted from the serum as well as nasal swab samples were measured by Nano drop (Thermo Scientific, USA) and DNA samples with sufficient amount of concentration and purity indicated by 260/280 showing reading 1.6 to 1.8 were used as template for PCR reaction. The DNA extracted from the serum and nasal swabs was stored at -20°C till used in PCR. The amplification of genus specific and species specific primers were carried out using custom synthesized (Eurofin Pvt. Ltd., India) primers (<a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>). PCR reaction was optimized in 25µl reaction volume using 5 µl of DNA template, 1 µl each of 10 pMol forward and reverse primers, 12.5 µl of master mix and 5.5 µl of nuclease free water.  The amplification of PCR was carried out  in a thermal cycler (Eppendorf, Germany) with an initial denaturation at 95<span class="CharOverride-5">o</span>C for 10 min, 35 cycles of denaturation at 94<span class="CharOverride-5">o</span>C for 30 s, annealing at 53<span class="CharOverride-5">o</span>C for 30 s, extension at 72<span class="CharOverride-5">o</span>C for 45 s, followed by final extension step at 72<span class="CharOverride-5">o</span>C for 7 min. Amplicons of 324 bp for genus specific PCR targeting <span class="CharOverride-7">alc</span> gene and amplicon of 425 bp for species specific PCR targeting <span class="CharOverride-7">fim</span> gene were analysed under UV-gel documentation system (Alpha Innotech Co., USA) after electrophoresis on 1% agarose gel (IBI Scientific, Peosta Lowa)  containing 0.2 mg/ ml ethidium bromide at 80 volts using 1× TBE electrophoresis buffer (Bio Basic Inc. USA). For confirmation of identity of PCR amplicons gel purified (Mini Elute Gel Extraction kit, Qiagen, Germany) amplicons were custom sequenced (Eurofins Genomics Laboratory, India). DNA extracted from reference strain of <span class="CharOverride-7">B. bronchiseptica</span> (MTCC 6838) available in the laboratory was used as positive control.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The study concludes that serum samples may be better sample for diagnosis of bordetellosis for identification of pathogens DNA instead of deep nasal swabs in dogs.</p>
		  <p class="Body-Text ParaOverride-1">&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 Director IVRI, Izatnagar, Joint Director (Academics), and Joint Director (R) IVRI, Izatnagar for providing fellowship to Lenin, Monika and Prasannavadhna, and funds for undertaking the study. We are also thankful to the dog owners, veterinary institutions, and clinicians. Technical assistance by HC Joshi and Laik of Epidemiology Laboratory was instrumental in carrying out this study.</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">There is no conflict of interest among all or any of the authors and also with the funding agencies. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
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