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			<p class="Type-of-Article">&nbsp;</p>
			<p class="Type-of-Article">&nbsp;</p>
			<p class="Type-of-Article">Research Article</p>
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			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Isolation and Characterization of <i>Listeria</i> spp. from Organized and Migratory Sheep Flocks in India</p>
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			<p class="Authors ParaOverride-1">&nbsp;</p>
			<p class="Authors ParaOverride-1"><span class="CharOverride-2">Shivaramu Keelara</span><span class="CharOverride-3">1</span><span class="CharOverride-2">*, </span><span class="CharOverride-2" xml:lang="en-US">Satya Veer Singh </span><span class="CharOverride-2">Malik</span><span class="CharOverride-3">2</span><span class="CharOverride-2">, Shivasharanappa Nayakvadi</span><span class="CharOverride-3">3</span><span class="CharOverride-2">, Samir Das</span><span class="CharOverride-3">2</span><span class="CharOverride-2">, </span><span class="CharOverride-2" xml:lang="en-US">Sukhadeo Baliram Barbuddhe</span><span class="CharOverride-3">4</span></p>
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			<p class="Affiliations ParaOverride-1"><span class="CharOverride-4">1</span>Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA; <span class="CharOverride-4">2</span>Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, 243122, India; <span class="CharOverride-4">3</span>Animal Sciences Section, Central Coastal Agricultural Research Institute, Goa, 403402, India; <span class="CharOverride-4">4</span>National Institute of Biotic Stresses Management, Raipur, Chhattisgarh, India.</p>
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			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Abstract</span> | In the present investigation, a total of 880 clinical samples comprising blood (n=215), vaginal swabs (n=220), fecal swabs (n=220), placenta (n=10) and sera (n=215) from ewes with abortion or history of abortion as well as from apparently healthy ewes of organized and migratory flocks were collected for screening listeric infection by isolation, serological and molecular diagnostic methods. 23 different isolates were recovered which comprised of 15 <span class="CharOverride-6">L. monocytogenes,</span> 2 <span class="CharOverride-6">L. ivanovii</span> and<span class="CharOverride-6"> </span>6 other listeriae. Among these, four haemolytic isolates<span class="CharOverride-6"> </span>(<span class="CharOverride-6">L</span>. <span class="CharOverride-6">ivanovii</span>-2 and <span class="CharOverride-6">L. monocytogenes</span>-2) were found pathogenic based on hemolysis on sheep blood agar, CAMP test, PI-PLC activity, virulence-associated genes (<span class="CharOverride-6">prf</span>A, <span class="CharOverride-6">plc</span>A, <span class="CharOverride-6">act</span>A, <span class="CharOverride-6">hly</span>A and <span class="CharOverride-6">iap</span>) as well as by <span class="CharOverride-6">in vivo</span> pathogenicity tests namely, chick embryo and mice inoculation tests. Indirect plate ELISA revealed 41.96% seropositivity for antibodies against listeriolysin O in ewes with abortion or history of abortion and 26.21% in apparently healthy ewes, which after adsorption of sera with Streptolysin O (SLO) reduced to 18.75% and 9.70% in respective groups. On over all basis, out of 215 sheep sera, 74 (34.42%) sera showed positivity for ALLO, which was reduced to 31(14.42%) following adsorption with SLO, indicating the need for sera adsorption for removing the cross-reactivity. The study had significant implications in understanding the epidemiology of listeric infection in migratory flocks.</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Keywords</span> | Listeria spp, Migratory sheep, Abortion, LLO, ELISA</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
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			<p class="Editor----Citation"><span class="CharOverride-5">Editor</span> | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Received</span> | March 28, 2015; <span class="CharOverride-5">Revised</span> | April 29, 2015; <span class="CharOverride-5">Accepted</span> | April 30, 2015; <span class="CharOverride-5">Published</span> | May 07, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-5">*Correspondence</span> | Shivaramu Keelara, University of Maryland, College Park, USA; <span class="CharOverride-5">Email:</span> shivakeelara@gmail.com</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Citation</span> | Keelara S, Malik SSV, Nayakvadi S, Das S, Barbuddhe SB (2015). Isolation and characterization of <span class="CharOverride-19">Listeria</span> spp. from organized and migratory sheep flocks in India. Adv. Anim. Vet. Sci. 3(6): 325-331.  </p>
			<p class="Editor----Citation"><span class="CharOverride-5">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.6.325.331"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.6.325.331</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 Keelara 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="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">L</span><span>isteriosis </span>caused by pathogenic strains of <span class="CharOverride-6">Listeria monocytogenes </span>and <span class="CharOverride-6">L.ivanovii</span>, is a serious invasive disease characterized by three forms namely; encephalitis, septicaemia and abortion in animals (<a href="#Low-JC--Donachie-W--1997"><span class="Hyperlink">Low and Donachie, 1997</span></a>). Listeriosis leads to septicaemia, abortion, stillbirth, perinatal infections, meningitis, gastroenteritis and meningoencephalitis, especially in immunocompromised individuals (<a href="#Barbuddhe-SB--Malik-2012"><span class="Hyperlink">Barbudde et al., 2012</span></a>). The occurrence of listeric infections in the Indian subcontinent has been extensively reviewed by many workers (<a href="#Malik-SVS--Barbuddhe-S"><span class="Hyperlink">Malik et al., 2002</span></a>; <a href="#Barbuddhe-SB--Malik-2012"><span class="Hyperlink">Barbudde et al., 2012</span></a>). In India, we have recorded outbreaks of listeriosis in animal populations (<a href="#Kumar-H--Singh-BB--Kaur-BK--Singh-R--Sidhu-PK--Sandhu-KS--2007"><span class="Hyperlink">Kumar et al., 2007</span></a>; <a href="#Yadav-MM--Roy-A--2008-"><span class="Hyperlink">Yadav and Roy. 2008</span></a>; <a href="#Kaur-S--Malik-S-2010"><span class="Hyperlink">Kaur et al., 2010</span></a>). Recently, a database of <span class="CharOverride-6">Listeria </span>strains isolated in India from various sources, Indian <span class="CharOverride-6">Listeria </span>Culture Database (ILCD) has been established (<a href="#Jangam-AK--Barbuddhe-SB--Kalekar-S--R"><span class="Hyperlink">Jangam et al., 2010</span></a>, <a href="#Barbuddhe-SB--Malik-2000"><span class="Hyperlink">Barbuddhe et al., 2000</span></a>; <a href="http://www.icargoa.res.in/ilcd"><span class="Hyperlink">http://www.icargoa.res.in/ilcd</span></a>). The database provides visualization of geographical source of the strain, its lineage, serotype, source of isolation (animal/human), year of isolation, phenotypic and genotypic characteristics as well as antibiotic sensitivity patterns. Listeric infection in animals is usually acquired by consumption of spoiled silage, in which these bacteria multiply, resulting in herd outbreaks. Though the reliable diagnosis of listeriosis is made by isolation and identification of the pathogen but it is time consuming (<a href="#Rocourt-J--Schrettenbrunner-A--Seeliger-HPR--1983-.-Differenciation-biochimique-des-groupe"><span class="Hyperlink">Rocourt et al., 1983</span></a>). Therefore, the diagnosis of pathogenic <span class="CharOverride-6">Listeria</span> spp. and listeric infection can be made on the basis of virulence genes (<a href="#Notermans-SHW--Dufre-1991b"><span class="Hyperlink">Notermans et al., 1991b</span></a>). Listeriolysin-O (LLO), an extracellular 58 kDa haemolysin, is a major virulence factor of <span class="CharOverride-6">L. monocytogenes</span> (<a href="#Gaillard-JL--Berche-P--Sansonetti-P--1986-"><span class="Hyperlink">Gaillard et al., </span><span class="Hyperlink">1986</span></a>) and is produced by all the pathogenic strains (<a href="#Geoffroy-C--Gaillard-JL--Alouf-JE--Berche-P--1989"><span class="Hyperlink">Geoffrey et al., 1989</span></a>). Listeriolysin-O (LLO) expressed by the <span class="CharOverride-6">hly</span> gene serves as an ideal virulence marker as it is produced by only virulent strains of <span class="CharOverride-6">L. monocytogenes</span>. </p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Detection of virulence factors (LLO and PI-PLC) of pathogenic <span class="CharOverride-6">Listeria</span> spp. (<a href="#Notermans-SHW--Dufre-1991b"><span class="Hyperlink">Notermans et al., 1991b</span></a>) and/or of genes responsible for their expression by PCR assay (<a href="#Kotlowski-R--Kaczmalek-M--Kur-J-"><span class="Hyperlink">Kotlowski et al., 1996</span></a>) and their correlation with seropositivity in monoclonal or polyclonal antibodies-based ELISA would be an ideal approach for ascertaining the virulence of <span class="CharOverride-6">Listeria</span> isolates from clinical sample. In India, sheep are mainly reared under intensive and semi-intensive rearing systems. Studies on assessment of the extent of listeric infections among sheep population in organized farms/ migratory flocks are lacking. The objective of the study was to investigate the listeric infection in sheep having reproductive disorders and apparently healthy sheep employing various methods.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Materials and methods</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Study Population and Collection of Samples</p>
			<p class="Body-Text ParaOverride-1">A total of 880 samples comprising blood (n=215), faecal swabs (n=220), vaginal swabs (n=220), placenta (n=10) and serum (n=215) were aseptically collected from ewes (n=220). The animals were apparently healthy (n=103) and clinical cases in ewes with abortion or with a history of abortion (n=117). The samples were collected from one organized sheep farm from Himachal Pradesh (Northern India) as well as three migratory flocks from Karnataka (Southern India) and Himachal Pradesh. All the samples were collected aseptically and transported to the laboratory under chilled conditions and stored at 4<span class="CharOverride-8">o</span>C till processed.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Bacteria</p>
			<p class="Body-Text">The strains of <span class="CharOverride-6">L. monocytogenes</span> 4b (MTCC 1143), <span class="CharOverride-6">Staphylococcus aureus</span> (MTCC 1144), <span class="CharOverride-6">Rhodococcus equi</span> (MTCC 1135), <span class="CharOverride-6">Streptococcus faecalis</span> (MTCC 439), <span class="CharOverride-6">Bacillus cereus</span> (MTCC 1272), <span class="CharOverride-6">Escherichia coli</span> (MTCC 443), <span class="CharOverride-6">Aeromonas hydrophila</span> (MTCC 646) used in the study were obtained from the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India.</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The reference strains of <span class="CharOverride-6">Listeria</span> namely, <span class="CharOverride-6">L. monocytogenes</span> 4b (NCTC 11994), <span class="CharOverride-6">L</span>. <span class="CharOverride-6">monocytogenes</span> 1/2a (NCTC 7973), <span class="CharOverride-6">L. monocytogenes</span> 1/2b (NCTC 10887), <span class="CharOverride-6">L. ivanovii</span> (NCTC 11846), <span class="CharOverride-6">Listeria innocua</span> (NCTC 11288), <span class="CharOverride-6">Listeria seeligeri</span> (NCTC 11856), <span class="CharOverride-6">Listeria grayi</span> (NCTC 10812), <span class="CharOverride-6">Listeria welshimeri</span> (NCTC 11857) were kindly provided by Prof. K. L. Morgan, University of Liverpool, U.K. The strains of <span class="CharOverride-6">Salmonella</span> (1117) and <span class="CharOverride-6">Vibrio cholerae</span> (0139) were procured from Division of Veterinary Public Health, Indian Veterinary Research Institute, India. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Isolation of Listeria</p>
			<p class="Body-Text ParaOverride-1">Isolation of listeriae from the blood, faecal, vaginal, and placental bits was carried out as per the US Department of Agriculture (USDA) method described by <a href="#McClain-D--Lee-WH--1988-"><span class="Hyperlink">McClain and Lee (1988)</span></a> after making necessary modifications. Briefly, approx. 5 ml of each of the blood and 25 gm of the placental tissue were inoculated into 50 ml and 225 ml of Pre-enrichment Broth (PEB; Tryptic Soy broth with 0.6% yeast extract; Himedia Laboratories, Mumbai, India). After incubating for 24 h at 30<span class="CharOverride-8">o</span>C, 10 ml of PEB was transferred to 90 ml of University of Vermont-1 (UVM-1) broth (Himedia Laboratories) and incubated overnight at 30<span class="CharOverride-8">o</span>C. Each of the collected swabs (fecal and vaginal) was aseptically inoculated into 10ml of PEB. After incubation, the inoculum was transferred to UVM-1 and incubated overnight at 30<span class="CharOverride-8">o</span>C. The enriched UVM-1 inoculum (0.1ml) was then transferred to UVM-2 broth and again incubated overnight at 30<span class="CharOverride-8">o</span>C. The inoculum enriched in UVM-2 was streaked directly on Dominguez-Rodriguez isolation agar (<a href="#Dominguez-Rodriguez-L--Suarez-Fernandez-G--Fernandez-Garayzobal-J--Rodriguez-Ferri-E--1984"><span class="Hyperlink">Dominguez-Rodriguez et al., 1984</span></a>) and the plates were incubated at 30<span class="CharOverride-8">o</span>C for 48 h. The greenish-yellow glistening, iridescent and pointed colonies of about 0.5mm diameter surrounded by a diffuse black zone of aesculin hydrolysis were suspected to be listeriae. The presumed colonies of <span class="CharOverride-6">Listeria</span> (at least three per plate) were further confirmed.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Confirmation of the Isolates</p>
			<p class="Body-Text ParaOverride-1">Morphologically typical colonies were verified by Gram’s staining, catalase reaction, tumbling motility at 20-25<span class="CharOverride-8">o</span>C, methyl red-Voges Proskauer (MR-VP) reactions, CAMP test with <span class="CharOverride-6">S. aureus</span> and <span class="CharOverride-6">R. equi,</span> nitrate reduction, fermentation of sugars (rhamnose, xylose, mannitol and <span class="CharOverride-9">α</span>-methyl-D-mannopyranoside) and hemolysis on 5% sheep blood agar (SBA). The DL-alanine <span class="CharOverride-9">β</span>-napthylamide (DLABN) test was performed as described by <a href="#McLauchlin-J--1997-."><span class="Hyperlink">McLauchlin (1997)</span></a> in order to differentiate the listerial isolates.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The biochemically characterized <span class="CharOverride-6">Listeria </span>isolates were plated on ALOA (Himedia Laboratories, Mumbai, India), a chromogenic selective medium for identification of <span class="CharOverride-6">Listeria </span>spp. and their pathogenic potential as described by <a href="#Ottaviani-F--Ottaviani-M--Agosti-M--1997"><span class="Hyperlink">Ottaviani et al. (1997)</span></a>. Each <span class="CharOverride-6">Listeria </span>isolate was spot inoculated separately on ALOA plates and incubated at 37oC and then observed for typical blue-green colonies with clearly defined opaque halo up to 5 days.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The pathogenicity testing of the <span class="CharOverride-6">Listeria</span> isolates was done by mice inoculation test (<a href="#Menudier-A--Bosiraud-C--Nicolas-JA--1991"><span class="Hyperlink">Menudier et al., 1991</span></a>) and chick embryo inoculation (<a href="#Notermans-SHW--Dufre-1991b"><span class="Hyperlink">Notermans et al., 1991b</span></a>) and permission from institute animal ethical committee (IAEC) was obtained before testing.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Phosphatidylinositol-specific Phospholipase C (PI-PLC) Assay</p>
			<p class="Body-Text ParaOverride-1">All the biochemically characterized<span class="CharOverride-6"> Listeria</span> isolates were assayed for PI-PLC activity as per the method of <a href="#Notermans-S--Dufrenne-1991a"><span class="Hyperlink">Notermans et al. (1991a)</span></a> with certain modifications. In brief, the<span class="CharOverride-6"> Listeria</span> isolates were grown overnight onto sheep blood agar (SBA) plates at 37<span class="CharOverride-8">o</span>C. The growth of each <span class="CharOverride-6">Listeria</span> isolate harvested from the SBA plate was spot inoculated separately on tryptone soya yeast extract (TSYE, Himedia Labs, Mumbai, India) agar plates supplemented with 2.5 mM CaCl2 and 40 mM MgSO4 in a manner to get a clear visible bacterial growth of approximately 2 mm diameter following an incubation at 37<span class="CharOverride-8">o</span>C for 24 to 48 h. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The L-<span class="CharOverride-9">α</span>-phosphatidylinositol (PI) solution was prepared by dissolving PI substrate (Sigma, USA) in 20 mM Tris–HCl (Sigma) buffer pH 7.0 at 20 mg/ml and subjecting the resultant turbid solution to ultrasonication (5 cycles of 60 s each with 30 s cooling intervals) in an ultrasonicator (Sanyo, U.K.). Subsequently, the agarose solution (1.4%) prepared in 20 mM Tris– HCl buffer, pH 7.0 was cooled to 55<span class="CharOverride-8">o</span>C and added with 100 μg/ ml of chloramphenicol to prevent the growth of contaminants. Finally, an overlay suspension was prepared by mixing the PI solution pre-warmed to 55<span class="CharOverride-8">o</span>C in a water bath with an equal volume of agarose solution held at 55<span class="CharOverride-8">o</span>C just before the use and overlaid at a rate of 4 ml per petri dish (9cm diameter) onto the previously seeded colonies of <span class="CharOverride-6">Listeria </span>isolates on TSYE. The plates were then incubated at 37<span class="CharOverride-8">o</span>C and observed daily for turbid halos around colonies up to 5 days.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Multiplex Polymerase Chain Reaction (mPCR) Assay for the Detection of Virulence Genes of Listeria spp.</p>
			<p class="Body-Text ParaOverride-1">All the <span class="CharOverride-6">Listeria</span> isolates were screened by multiplex polymerase chain reaction (mPCR) as per the protocol and primer sequences used by <a href="#Rawool-DB--Malik-SVS--Shakuntala-I--Sahare-AM--Barbuddhe-SB--2007-."><span class="Hyperlink">Rawool et al. (2007)</span></a> for the detection of virulence associated genes <span class="CharOverride-6">viz.,</span> haemolysin (<span class="CharOverride-6">hly</span>A), Phosphatidylinositol-specific phospholipase<span class="CharOverride-6"> </span>C<span class="CharOverride-6"> </span>(<span class="CharOverride-6">plc</span>A), <span class="CharOverride-6">Act</span>A protein (<span class="CharOverride-6">act</span>A), a surface protein, p60 (<span class="CharOverride-6">iap</span>)<span class="CharOverride-6"> </span>and positive regulatory factor A (<span class="CharOverride-6">prf</span>A). Briefly, the mPCR protocol was standardized employing the standard pathogenic strains of <span class="CharOverride-6">L. monocytogenes</span> 4b (MTCC 1143, NCTC 11994), <span class="CharOverride-6">L. monocytogenes</span> ½a (NCTC 7973), <span class="CharOverride-6">L. monocytogenes</span> ½b (NCTC 10887) and <span class="CharOverride-6">L. ivanovii </span>(NCTC 11846). Subsequently, the test isolates were screened by the standardized mPCR for the detection of aforesaid virulence associated genes of <span class="CharOverride-6">Listeria </span>spp. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">50 μl PCR reaction mix included 5.0 μl of 10× PCR buffer (100 mM Tris–HCl buffer, pH 8.3 containing 500 mM KCl, 15 mM MgCl2 and 0.01% gelatin), 0.2 mM dNTP mix (Sigma, USA), 2 mM MgCl2 and 10 μM of a primer set containing forward and reverse primers (a final concentration of 0.1 μM of each primer), 1 U of Taq DNA polymerase (Sigma, USA), 5 μl of cell lysate and sterilized milliQ water to make up the reaction volume. The DNA amplification reaction was performed in a Master Cycler Gradient Thermocycler (Eppendorf, Germany) with a preheated lid. The cycling conditions for PCR included an initial denaturation of DNA at 95°C for 2 min followed by 35 cycles each of 15 s denaturation at 95°C, 30 s annealing at 60°C and 1 min and 30 s extension at 72°C, followed by a final extension of 10 min at 72°C and held at 4°C. All the five sets of primers for virulence-associated genes were amplified under similar PCR conditions and amplification cycles. The resultant PCR products were further analyzed by agarose gel electrophoresis (1.5%; low melting temperature agarose L), stained with ethidium bromide (0.5μg/ml) and visualized by a UV transilluminator (UVP Gel Seq Software, England).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">ELISA</p>
			<p class="Body-Text ParaOverride-1">Indirect plate ELISA was standardized to detect antibodies in the sera of spontaneous cases of listeric infection in aborted ewes and also in apparently healthy ewes. Listeriolysin-O (LLO) was prepared and purified from the cell free supernatant (CFA) by ion exchange chromatography in accordance with the method of <a href="#Lhopital-S--Marly-J--Pardon-P--Berche-P--1993"><span class="Hyperlink">Lhopital </span><span class="Hyperlink">et al. (1993)</span></a>. The purity of the LLO was checked by SDS-PAGE, which showed a homogeneous 58.0 kDa protein. The fractions having LLO were pooled and the protein content was estimated, and finally stored at -20<span class="CharOverride-8">o</span>C until used.<span class="CharOverride-5"> </span></p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The indirect plate ELISA was performed as per the methods of <a href="#Low-JC--Donachie-W--1991"><span class="Hyperlink">Low and Donachie (1991)</span></a>. The ELISA was standardized by checker board analysis. Briefly, purified LLO was used in at the optimal concentration 40 ng/well (100µl/well) for coating the microtitre plates (Nunc, Denmark). The plates were covered and incubated at 37<span class="CharOverride-8">o</span>C for 2 h before washing five times with phosphate buffer saline (PBS), pH 7.2 plus 0.05% Tween 80 (PBS-T). Each of the test serum collected from aborted, with history of abortion and apparently healthy ewes was diluted 1:200 in PBS and added (100µl/well) in duplicate to the plates. The sealed plates were incubated at 37<span class="CharOverride-8">o</span>C for 90 min and again washed as before. Subsequently, rabbit anti-sheep IgG HRPO conjugate (Bethyl, India) was diluted to 1:10000 in PBS were added (100 µl/well) to the plates. The plates were again incubated at 37<span class="CharOverride-8">o</span>C for 90 min and washed as described earlier. Finally, ortho-phenylenediamine dihydrochloride (OPD) solution (1 mg/ml) in citrate buffer with 12 µl/100 ml of hydrogen peroxide was added (100µl/well) as a substrate and after 15 min, the reaction was read by ELISA plate reader (Anthos Labtek) at 492 nm. A serum sample at a dilution of 1:200 with a positive to negative (P/N) ratio of more than 2 was considered positive for listeriosis in standardized ELISA.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The detection of specific ALLO which is often present at low titer in ewe sera requires prior adsorption of anti-SLO, as Streptolysin-O (SLO) and Listeriolysin-O (LLO) are antigenically related. The adsorption of anti-SLO in the sera was done as per the method described by <a href="#Berche-P--Reich-KA--Bomichan-M--Beretti-JL"><span class="Hyperlink">Berche et </span><span class="Hyperlink">al. (1990)</span></a> with certain modifications. The purified SLO (Sigma-Aldrich, USA) was coated onto 96-well flat bottom polysorp plates (Nunc, Denmark) at a concentration 60 µg/ml, which was added to wells at the rate of 200µl/well and then incubated for 1 h at 25<span class="CharOverride-8">o</span>C. Each ewe serum (0.5ml), diluted 1/100 and 1/200 in PBS-T was then added to the wells and then incubated for 1 h at 25<span class="CharOverride-8">o</span>C. The SLO-adsorbed ewe sera were then used in the indirect plate ELISA to screen the ewe sera for ALLO as per the method described earlier.</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-">Isolation and Identification of Pathogens</p>
			<p class="Body-Text ParaOverride-1">Different <span class="CharOverride-6">Listeria</span> spp. were isolated from 23 of 665 (225 each of fecal and vaginal swab, 10 placenta and 215 of blood) samples collected from 220 ewes. Out of these 15 isolates were confirmed as <span class="CharOverride-6">L. monocytogenes</span>, two isolates as <span class="CharOverride-6">L. ivanovii</span> and the remaining were <span class="CharOverride-6">Listeria</span> spp. No <span class="CharOverride-6">Listeria</span> spp. could be isolated from blood samples. The overall occurrence of listeric infection was 3.4%. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150507145820.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150507145820.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/20150507145820.png">Multiplex PCR profile of virulence associated genes of <span class="CharOverride-6">L. monocytogenes</span> from sheep</a></p>
       </div>

			
		  <p class="Heading-2--History-in-MM-">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-">Pathogenicity Testing</p>
			<p class="Body-Text ParaOverride-1">Out of these 23 isolates, four haemolytic isolates (<span class="CharOverride-6">L. ivanovii</span>-2 and <span class="CharOverride-6">L. monocytogenes-</span>2) were found to be pathogenic by in vitro pathogenicity tests namely, haemolysis on sheep blood agar (SBA); Christie, Atkins, Munch-Petersen (CAMP) test, phosphatidylinositol-specific phospholipase C (PI-PLC) activity on ALOA medium and in PI-PLC assay as well as by <span class="CharOverride-6">in vivo</span> pathogenicity tests namely, chick embryo and mice inoculation tests<span class="CharOverride-5"> </span>(<a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>). All other <span class="CharOverride-6">Listeria </span>spp. isolates were non-pathogenic.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Multiplex Polymerase Chain Reaction Assay</p>
			<p class="Body-Text ParaOverride-1">In multiplex PCR, both the primer sets employed i.e., one comprising primers for <span class="CharOverride-6">plcA, actA, hlyA</span> and <span class="CharOverride-6">iap,</span> and the other for <span class="CharOverride-6">prfA, actA, hlyA</span> and <span class="CharOverride-6">iap</span>, amplified the DNA fragments of the expected sizes. Out of four haemolytic <span class="CharOverride-6">Listeria</span> isolates ( <span class="CharOverride-6">L.monocytogenes</span> -2 and <span class="CharOverride-6">L.ivanovii</span>-2) The DNA of <span class="CharOverride-6">L. ivanovii</span> isolates recovered from ewes with history of abortion revealed amplified products corresponding to all the five virulence-associated genes namely, <span class="CharOverride-6">plc</span>A, <span class="CharOverride-6">prf</span>A, <span class="CharOverride-6">act</span>A, <span class="CharOverride-6">hly</span>A and <span class="CharOverride-6">iap</span>  in mPCR employing the above indicated sets of primers in 2 separate reactions (<a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">On DNA analysis by the mPCR, out of the two hemolytic, ALOA-positive and PI-PLC-positive <span class="CharOverride-6">L. monocytogenes</span> isolates recovered from abortion case and apparently healthy ewe, respectively, the former revealed the amplified products of all the five virulence-associated genes while the later did not show <span class="CharOverride-6">plc</span>A gene (<a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">ELISA</p>
			<p class="Body-Text ParaOverride-1">The antibodies against LLO (ALLO) were detected in 47 out of 112 (41.96%) serum samples from ewes with a history of abortion and 27 out of 103 (26.21%) apparently healthy cases. However, after adsorption with SLO and retesting by plate ELISA, the positivity for ALLO reduced to 21 (18.75%) of clinical cases and 10 (9.7%) of apparently healthy cases (<a href="#Table-2-"><span class="Hyperlink">Table 2</span></a>). <span class="CharOverride-5"> </span></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>Pathogenicity and PCR profiles of <span class="CharOverride-6">L. monocytogenes </span>and <span class="CharOverride-6">L.ivanovii</span> isolates from healthy and clinical cases in ewes</p>
			<table width="657" height="447" class="Table-Style-1" id="table-1">
				<colgroup>
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-1">
						<td rowspan="4">
							<p class="Basic-Paragraph"><span class="CharOverride-14">Isolate No.</span></p>
						</td>
						<td rowspan="4">
							<p class="Basic-Paragraph"><span class="CharOverride-14">Source of the isolate</span></p>
						</td>
						<td colspan="7">
							<p class="Basic-Paragraph"><span class="CharOverride-14">Pathogenicity profile</span></p>
						</td>
						<td rowspan="2" colspan="5">
							<p class="Basic-Paragraph"><span class="CharOverride-14">PCR profile of virulence-associated genes </span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td colspan="5">
							<p class="Basic-Paragraph"><span class="CharOverride-15">In vitro</span><span class="CharOverride-16">  tests</span></p>
						</td>
						<td colspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-15">In vivo</span><span class="CharOverride-16"> tests</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">Hemolysis on SBA</span></p>
						</td>
						<td colspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">ALOA</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">CAMP with S/R</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">PI-PLC assay</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">Mice lethality</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">Chick embryo lethality</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">plcA</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">prfA</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">actA</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">hlyA</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-16">iap</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Col</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Halo</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Isolate 1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Abortion (Place-</span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-16">nta)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">BG</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+S</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Isolate 2</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">History of abortion (fecal swab)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">BG</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Isolate </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">History of abortion (Vaginal  swab)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">BG</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+R</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Isolate 4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Healthy </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-16">(fecal </span></p>
							<p class="Basic-Paragraph"><span class="CharOverride-16">swab)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">BG</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+S</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">++</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">+</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-10">PI-PLC</span><span class="CharOverride-7"> = Phosphatidylinositol-specific phospholipase C; </span><span class="CharOverride-10">PCR</span><span class="CharOverride-7"> = Polymerase chain reaction; </span><span class="CharOverride-10">SBA</span><span class="CharOverride-7"> = Sheep blood agar; </span><span class="CharOverride-10">S/R</span><span class="CharOverride-7"> = Staphylococcus aureus / Rhodococcus equi; </span><span class="CharOverride-10">ALOA</span><span class="CharOverride-7"> = Agar </span><span class="CharOverride-17">Listeria </span><span class="CharOverride-7">according to Ottaviani and Agosti; </span><span class="CharOverride-10">ALLO</span><span class="CharOverride-7"> = Antibodies against Listeriolysin-O; </span><span class="CharOverride-10">CAMP</span><span class="CharOverride-7"> = Christie, Atkins, Munch-Petersen; </span><span class="CharOverride-10">BG</span><span class="CharOverride-7"> = Blue green; </span><span class="CharOverride-10">L.m.</span><span class="CharOverride-7"> = L. monocytogenes; </span><span class="CharOverride-10">L.i</span><span class="CharOverride-7"> = L. ivanovii</span><span class="CharOverride-5">                         </span></p>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Table-2-"></a>Table 2: </span>Seropositivity of ewes against listeriolysin-O (LLO) with clinical cases and apparently healthy ewes</p>
			<table width="657" height="114" class="Table-Style-1" id="table-2">
				<colgroup>
					<col />
					<col />
					<col />
					<col />
					<col />
					<col />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Sl No</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Clinical status</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">No of samples collected</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">No of samples Positive</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">% seropositivity </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-14">Adsorption with SLO</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Abortion</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">112</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">47</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">41.96</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">21 (18.75%)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-1">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">2</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Healthy</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">103</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">27</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">26.21</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">10 (9.7%)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-2">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">Total</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">215</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">74</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">34.42</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-16">31 (14.42%)</span></p>
						</td>
					</tr>
				</tbody>
			</table>
				  <p class="Body-Text ParaOverride-1">&nbsp;</p>
				  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Discussion</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The genus-<span class="CharOverride-6">Listeria</span> has two pathogenic species namely, <span class="CharOverride-6">Listeria monocytogenes</span> and <span class="CharOverride-6">L. ivanovii.</span> Of these, <span class="CharOverride-6">L. monocytogenes</span> is a well-known cause of abortion, encephalitis and septicaemia in animals and human beings (<a href="#Rocourt-J--Schrettenbrunner-A--Seeliger-HPR--1983-.-Differenciation-biochimique-des-groupe"><span class="Hyperlink">Rocourt and Seeliger, 1985</span></a>; <a href="#McLauchlin-J--1987"><span class="Hyperlink">McLauchlin, 1987</span></a>; <a href="#Kumar-H--Singh-BB--Kaur-BK--Singh-R--Sidhu-PK--Sandhu-KS--2007"><span class="Hyperlink">Kumar et al., 2007</span></a>). The other pathogenic spp, <span class="CharOverride-6">L. ivanovii</span> was isolated from cases of abortion, still birth and neonatal septicemia in sheep and cattle (<a href="#Jose-A-V-zquez-Boland--Kuhn-M--Be"><span class="Hyperlink">Jose et al., 2001</span></a>). There are many pathogenic spp of <span class="CharOverride-6">Listeria </span>known to be excreted in faeces and vaginal secretions which can act as a source of infection in the environment. The listeric infection in animals is mainly transmitted by consumption of spoiled silage, aborted materials and vaginal secretions in which these bacteria multiply readily, resulting in outbreaks, especially in the sheep (<a href="#Low-JC--Donachie-W--1997"><span class="Hyperlink">Low and Donachie, 1997</span></a>; <a href="#Kumar-H--Singh-BB--Kaur-BK--Singh-R--Sidhu-PK--Sandhu-KS--2007"><span class="Hyperlink">Kumar et al., 2007</span></a>). In our study, the pathogenic and non-pathogenic spp. of <span class="CharOverride-6">Listeria </span>were recovered from both organized and migratory flocks from different regions. Isolation of pathogenic strains such as <span class="CharOverride-6">L. ivanovii</span> and <span class="CharOverride-6">L. monocytogenes</span> from aborted ewes in this study showed that, these two isolates are potent pathogens of reproductive disorders in sheep. The migratory flocks had abortion and a history abortion. It is difficult to comment on the route of infection among these sheep but infection might have transmitted from ingestion of infected material or soil. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text">Pathogenic isolates of <span class="CharOverride-6">L. ivanovii</span> were recovered from 2 cases with a history of abortion while pathogenic isolate of <span class="CharOverride-6">L. monocytogenes </span>was recovered from one case of spontaneous abortion. These results are in agreement of other published work wherein pathogenic <span class="CharOverride-6">L. ivanovii </span>has been found to be associated with 4.6% cases of abortion in ewes (<a href="#Nigam-P--Katoch-RC--Sharma-M--Verma-S--1999"><span class="Hyperlink">Nigam et al., 1999</span></a>) whereas <span class="CharOverride-6">L. monocytogenes</span> has been recovered from 9.1% (<a href="#Nigam-P--Katoch-RC--Sharma-M--Verma-S--1999"><span class="Hyperlink">Nigam et al., 1999</span></a>) and 15.7% (<a href="#Sharma-M--Batta-MK--Katoch-RC--1996-."><span class="Hyperlink">Sharma et al.,1996</span></a>)<span class="CharOverride-6"> </span>cases of abortion in ewes from migratory flocks from Himachal Pradesh.<span class="CharOverride-6"> </span>The results of present investigation are also in line with earlier reports.<span class="CharOverride-6"> L. ivanovii</span> has been implicated as the most frequent cause of abortions in case of sheep (<a href="#McLauchlin-J--1987"><span class="Hyperlink">McLauchlin, 1987</span></a>), with abortion rates ranging from 1.65% (<a href="#Alexander-AV--Walker-RL--Johnson-BJ--Charlton-BR--Woods-LW--1992"><span class="Hyperlink">Alexander et al.,1992</span></a>) to 12% (<a href="#Sergeant-ESG--Love-SCJ--McInnes-A--1991"><span class="Hyperlink">Sergeant et al.,1991</span></a>). One pathogenic <span class="CharOverride-6">L. monocytogenes</span> isolate was recovered from apparently healthy ewe. <span class="CharOverride-6">L. monocytogenes</span> has been found to translocate throughout the digestive tract in asymptomatic sheep (<a href="#Zundel-E--Bernard-S--2006-."><span class="Hyperlink">Zundel et al., 2006</span></a>).</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">A number of factors are involved in the manifestation of virulence of <span class="CharOverride-6">L. monocytogenes</span> (<a href="#Portnoy-DA--Chakraborty-T--Goebel-W--Cossart-P--1992-"><span class="Hyperlink">Portnoy et al.</span><span class="Hyperlink"> 1992</span></a>; <a href="#Vazquez_Bolland-JA--Dominguez-Bernal-G--Gonz"><span class="Hyperlink">Vazquez-Boland et al., 2001</span></a>). It has been clearly demonstrated that <span class="CharOverride-6">L. monocytogenes</span> phospholipases are essential determinants of pathogenicity (<a href="#Smith-GA--Marques-H--Jones-H--1995-.-T"><span class="Hyperlink">Smith et al., 1995</span></a>). In the present investigation, even though expression of haemolytic activity by all 23 isolates only four isolates were found to be pathogenic in all the assays and possessed all the five virulence genes except one <span class="CharOverride-6">L. monocytogenes</span> isolate lacking of the <span class="CharOverride-6">plc</span>A gene. Truncated form or mutation in the <span class="CharOverride-6">plc</span>A gene might have contributed to this observation. Those isolates which are PI-PLC negative turned out negative in mice and chick embryo assays. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">In the present study, a relatively high (41.96%) seropositivity was observed against LLO in ewes with abortion or a history of abortion. In case of apparently healthy ewes, relatively less seropositivity (26.21%) was observed. The observation is comparable to earlier reports as 33% documented by <a href="#Barbuddhe-SB--Malik-2000"><span class="Hyperlink">Barbuddhe et al. (2000)</span></a>. However, on testing of the sera adsorbed by SLO, the per cent seropositivity decreased. Out of 47 sera from ewes with abortion or history of abortion showing seropositivity, 21 were seropositive after adsorption with SLO. Ten sera from apparently healthy ewes were seropositive after adsorption with SLO. The reduction of seropositivity is in agreement with the earlier published report on the reduction of the titer from 100 before adsorption to less than 12.5 after adsorption of the cross-reacting whole human sera with <span class="CharOverride-6">S. aureus</span> (<a href="#Larsen-S--Jones-W--1972-.-E"><span class="Hyperlink">Larsen and Jones, 1972</span></a>). <a href="#Kaur-S--Malik-SVS--Vaidya-VM--Kaur-G--2006"><span class="Hyperlink">Kaur et al. (2006)</span></a> reported reduction in seropositivity from 48% to 16% after adsorption with SLO. Thus, from the present study it is evident that adsorption of the sera eliminates cross-reactions in serological tests. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The recovery of pathogenic isolates of <span class="CharOverride-6">L. monocytogenes</span> and <span class="CharOverride-6">L. ivanovii</span> from clinical cases and apparently healthy ewes indicated association of this pathogen with such cases. The study highlights the role of listeric infection in causing reproductive disorders in organized was well as migratory sheep. This study provides significant implications for understanding the epidemiology of listeric infection and is required for control of infection.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">References</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			
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