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			<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">Detection of <i>Mycoplasma equigenitalium</i> from Equines by Capture-ELISA</p>
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			<p class="Authors ParaOverride-1">&nbsp;</p>
			<p class="Authors ParaOverride-1"><span class="CharOverride-2">Sandip Kumar Khurana</span><span class="CharOverride-3">1*</span><span class="CharOverride-2">, Daya Nand Garg</span><span class="CharOverride-3">2</span><span class="CharOverride-2">, Yarvendra Singh</span><span class="CharOverride-3">2</span></p>
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			<p class="Affiliations ParaOverride-1"><span class="CharOverride-4">1</span>National Research Centre on Equines, Hisar, 125 001, Haryana, India; <span class="CharOverride-4">2</span>Department of Veterinary Public Health, College of Veterinary Sciences, LUVAS, Hisar, 125 004, Haryana, India.</p>
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			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Abstract</span> | Mycoplasmas are smallest self-replicating prokaryotes and responsible for varied genital and respiratory disease conditions in animals as well as human beings. They assume greater importance due to their resistance for common antibiotics and ability to survive the temperature of cryopreservation during artificial insemination. <span class="CharOverride-6">Mycoplasma equigenitalium </span>has been implicated in reproductive and genital problems of mares and stallions. The conventional cultural methods are quite cumbersome and time consuming. In this study detection rate by capture-ELISA (9.6%) was higher than cultural methods (4.8%) for diseased equines. Overall detection rate by capture-ELISA (3.6%) was also higher than cultural methods (1.8%). Therefore capture-ELISA becomes a simple, rapid and sensitive alternative tool for detection of <span class="CharOverride-6">M.</span> <span class="CharOverride-6">equigenitalium.</span></p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Keywords </span>| Mycoplasma equigenitalium, Capture-ELISA</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
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			<p class="Editor----Citation"><span class="CharOverride-7">Editor</span> | Minakshi Prasad, Head of the Department of Animal Biotechnology, LUVAS, Hisa Lala Lajpat Rai University of Veterinary &amp; Animal Sciences (LUVAS), Hisar, Haryana, 125004, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-7">Special Issue</span> | 1(2015) “Biotechnological and molecular approaches for diagnosis, prevention and control of infectious diseases of animals”</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Received</span> | March 15, 2015; <span class="CharOverride-5">Revised</span> | April 15, 2015; <span class="CharOverride-5">Accepted</span> | April 18, 2015; <span class="CharOverride-5">Published</span> | April 21, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-5">*Correspondence</span> | Sandip Kumar Khurana, National Research Centre on Equines, Hisar, India; <span class="CharOverride-5">Email:</span> sandipkk2003@yahoo.co.in</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Citation</span> | Khurana SK, Garg DN, Singh Y (2015). Detection of <span class="CharOverride-17">Mycoplasma equigenitalium</span> from equines by capture-ELISA. Adv. Anim. Vet. Sci. 3(1s): 33-37.  </p>
			<p class="Editor----Citation"><span class="CharOverride-7">DOI</span><span class="CharOverride-8"> | </span><a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.1s.33.37"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.1s.33.37</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-7">Copyright </span>© 2015 Khurana 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----">&nbsp;</p>
		  <p class="Heading-1--Introduction----">INTRODUCTION</p>
			<p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">M</span>ycoplasmas are smallest wall-less prokaryotes capable of self replication and are common inhabitants of genital and respiratory tract of animals and human beings. They have been isolated from equines with reproductive problems as well as apparently healthy equines from vagina, cervix, uterus and clitoral fossa of mares, and from prepuce, urethra and semen of stallions (<a href="#Garg-DN--1999-"><span class="Hyperlink">Garg, 1999</span></a>). <span class="CharOverride-6">Mycoplasma equigenitalium </span>was first isolated from cervix region of mares (<a href="#Kirchhoff-H--1978-."><span class="Hyperlink">Kirchhoff, 1978</span></a>), since then this organism has been isolated from various sites from reproductive tract of equines (<a href="#Kirchhoff-H--Naglic-T--Heitmann-J--1979"><span class="Hyperlink">Kirchhoff et al., 1979</span></a>; <a href="#Kirchhoff-H--Heitmann-J--Bisping-W--1980"><span class="Hyperlink">Kirchhoff et al., 1980</span></a>; <a href="#Naglic-T--Topolnik-E--Sukalic-M--Hajsig-D--1980"><span class="Hyperlink">Naglic et al., 1980</span></a>; <a href="#Mani-P--Incastrone-G--Chiavacci-L--1985"><span class="Hyperlink">Mani et al., 1985</span></a>; <a href="#Bermudez-V--Miller-R--Johnson-W--Rosendal-S--Ruhnke-L--1987"><span class="Hyperlink">Burmudez et al., 1987</span></a>; <a href="#Copes-J--Blanco-A--Giaboni-G--Stanchi-N--Cerda-R--Nievas-V--1995"><span class="Hyperlink">Copes et al., 1995</span></a>; <a href="#Khurana-SK--Garg-DN--2001-."><span class="Hyperlink">Khurana and Garg, 2001</span></a>). Seroprevalence of <span class="CharOverride-6">M. equigenitalium</span> has also been reported (<a href="#Khurana-SK--Garg-DN--Kumar-A--2004"><span class="Hyperlink">Khurana et al., 2004</span></a>; <a href="#Khurana-SK--Malik-P--2009"><span class="Hyperlink">Khurana and Malik, 2009</span></a>). Mycoplasmas are important because of their resistance to conventional antibiotics and ability to survive the temperature of cryopreservation, thus may be transmitted during artificial insemination.  </p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Cytadosorption localizes the mycoplasmas on membrane of infected cells, these can be demonstrated in fluid expressed from internal organs of equines including aborted fetus by light microscopy after intensified Giemsa staining. Advanced procedures like UV microscopy following DNA binding flurochrome staining, acridine orange staining, fluorescent antibody, immunoperoxidase, counter-immuno-electrophoresis, ELISA, immune adherence haemagglutination tests are all capable of detecting mycoplasma antigen  in body tissues and fluids but their application and value to equine mycoplasmosis have not been explored.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp; </p>
			<p class="Body-Text ParaOverride-1"><a href="#Boothby-JT--Mueller-R--Jasper-DE--Thomos-CB--1986"><span class="Hyperlink">Boothby et al. (1986)</span></a> performed<span class="CharOverride-6"> </span>capture-ELISA using monoclonal antibodies to detect <span class="CharOverride-6">M. bovis</span> in milk samples from dairy experiencing an epizootic of mastitis. This method was reported to be 100 per cent specific. Broth enrichment increased the sensitivity from 65 % to 86% compared with standard culture methods. <a href="#Nielson-KH--Stewart-RB--Garcia-MM--Eaglesome-MD--1987"><span class="Hyperlink">Nielson et al.</span><span class="Hyperlink"> (1987)</span></a> have also used this technique for detection of <span class="CharOverride-6">M. bovis</span> antigen in semen and perpetual washings of bulls. The technique had excellent specificity and the sensitivity  of assay was approximately 200 colour changing units/ml. <a href="#Ball-HJ--Finlay-D--Reilly-GAC--1994-."><span class="Hyperlink">Ball et al. (1994)</span></a> have used an antigen capture ELISA  for detection of <span class="CharOverride-6">M. bovis</span>  in pneumonic calf lungs and nasal swabs. <a href="#Rodriguez-F--Ball-HJ--Finlay-D--Campbell-D--Mackie-DP--1996"><span class="Hyperlink">Rodriguez et al. (1996)</span></a> used capture- ELISA for detection of <span class="CharOverride-6">M. mycoides </span>subsp<span class="CharOverride-6"> mycoides</span> using monoclonal using monoclonal antibodies. The test was found to be specific and the sensitivity increased by overnight or 48 hr enrichment. <a href="#Spergser-J--Aurich-C--Aurich-JE--Rosengarten-R--2002"><span class="Hyperlink">Spergser et al. (2002)</span></a> have reported high prevalence of mycoplasma in genital tract of stallion by PCR assay and cultural methods. <a href="#Dahiya-SK--Tiwari-SC--Batra-M--Sharma-A--2009"><span class="Hyperlink">Dahiya et al. (2009)</span></a> have found capture-ELISA more sensitive than cultural isolation for <span class="CharOverride-6">M. mycoides </span>subsp<span class="CharOverride-6"> mycoides. </span><a href="#Brooks-C--Finlay-D--Blackburn-P--Ball-H-J--2009"><span class="Hyperlink">Brooks et al. (2009)</span></a> have developed a simple and specific capture/ sandwich ELISA for detection of <span class="CharOverride-6">M. mycoides </span>subsp<span class="CharOverride-6"> mycoides</span> (SC) for screening large number of samples for CBPP. <a href="#Soderland-R--Bolske-G--Holst-Bs--Aspan-A--2011-."><span class="Hyperlink">Soderlund et al. (2011)</span></a> have developed a real time PCR for detection of <span class="CharOverride-6">M. felis</span>. <a href="#Guimaraes-AM--Santos-AP--Timenetsky--Bower-LP--Strait-E--Messik-JB--2014"><span class="Hyperlink">Guimaraes et al. (2014)</span></a> have developed a multiplex microbead immunoassay for detection of <span class="CharOverride-6">M. suis</span> antigens, which was found to be more sensitive than qPCR. <a href="#Meng-K--Sun-W--Zhao-P--Zhang-L--Cai-D--Cheng-Z--Guo-H--Lin-J--Yang-D--Wang-S--Hai-T--2014"><span class="Hyperlink">Meng et al. (2014)</span></a> have developed a colloidal gel-based immunochromatographic assay for highly specific, sensitive and rapid detection of <span class="CharOverride-6">M.suis</span>. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Capture ELISA has not been used for detection of equine mycoplasmas. But, it is useful in detection of mycoplasmal antigens from various species as evident from various refences cited here earlier. It could be a very easy and useful method for dectecting <span class="CharOverride-6">M. equigenitalium</span> anigen in swabs, fluids where it might not be possible by cultural 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="Body-Text ParaOverride-1">A capture ELISA technique (<a href="#Nielson-KH--Stewart-RB--Garcia-MM--Eaglesome-MD--1987"><span class="Hyperlink">Nielson et al., 1987</span></a>) with some modifications using polyclonal antiserum raised in rabbit and donkey foal against <span class="CharOverride-6">M. equigenitalium</span> (NCTC 10176/T-37) was adopted for detection of <span class="CharOverride-6">M. equigenitalium</span>-anigen in vagival swab/ preputial swab samples and specimens (heart blood, stomach content and tissue from lungs, heart, liver, spleen, kidneys) from aborted foals. The brief methodology is given below.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Test-sample Preparation </p>
			<p class="Body-Text ParaOverride-1">The vaginal swabs/preputial swabs from equines and other specimen from aborted fetuses were processed as per <a href="#Ball-HJ--Finlay-D--Reilly-GAC--1994-."><span class="Hyperlink">Ball et al. (1994)</span></a> with some modifications. Specimens were transferred in Standard Liquid Medium (SLM) containing PPLO broth, yeast extract, inactivated horse serum, calf thymus DNA, thallium acetate and benzyl penicillin, then incubated at 37°C for 4-5 days, the enriched samples were centrifuged at 10,000 g at 4°C for 30 min, washed thrice with PBS (7.2), followed by 3 cycles of freezing and thawing process and kept at -20°C until used in capture-ELISA as antigen. The known positive antigen sample was prepared by adding <span class="CharOverride-6">M. equigenitalium</span> (NCTC 10176/T-37) organisms in PBS (10<span class="CharOverride-4">5</span> CFU /ml) which was also processed for three cycles of freezing and thawing. The PBS without any mycoplasma organisms served as negative control.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-"><span class="CharOverride-1">M. equigenitalium</span> Antisera </p>
			<p class="Body-Text ParaOverride-1">The freeze-dried culture of <span class="CharOverride-6">M. equigenitalium</span> (NCTC 10176/T-37) was reconstituted in 0.5 ml of SLM, incubated at 37°C for 4-5 days. The culture was grown in gradually increasing volumes of SLM with final volume of 1 litre. The resultant growth in gradually in one litre SLM was centrifuged at 10000xg at 4°C for 30 min. pellet of organisms was washed thrice with PBS (pH 7.2) and suspended in 10 ml PBS which was stored at 4°C. It was sonicated at 180-200 watts (Braunsonic–1510) for 3 cycles of 4 min each and protein content was estimated (<a href="#Lowry-OH--Rosenbrough-NJ--Farr-AL--Randall-RJ--1951-."><span class="Hyperlink">Lowry et al., 1951</span></a>). It served as stock antigen for immunization of rabbit and donkey foal.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Healthy mycoplasma free rabbits (New Zealand white) and donkey foals (6 months) were immunized as per schedule (<a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>) and bled one week after the last injection to harvest <span class="CharOverride-6">M. equigenitalium</span> hyperimmune serum. Pre-immune serum of rabbit and donkey foal were collected for their usage as known negative serum for conducting capture-and indirect-ELISA.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Enzyme-antiglobulin-conjugate and Substrate </p>
			<p class="Body-Text ParaOverride-1">Horse-radish-peroxidase labelleed rabbit anti-horse IgG (Prod. Cat. No. A9292, Sigma, USA) was used as<span class="CharOverride-5"> </span>enzyme-antiglobulin-conjugate. A solution of orthophenylene diamine dihydro-chloride (OPD, Sigma, USA) prepared fresh was used as substrate.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Optimization of Antisera and Conjugate </p>
			<p class="Body-Text">The optimum dilution of equine anti-<span class="CharOverride-6">M. equigenitalium</span> (NCTC 10176/T-37) serum was determined by chequer-board titration with known positive and negative samples. Rabbit anti-<span class="CharOverride-6">M. equigenitalium</span> serum was diluted 1:40,1:80, 1:160, and 1:320 in 0.1M carbonate-bicarbonate  buffer (pH 9.6) before coating the wells of microplates. The equine anti-<span class="CharOverride-6">M. equigenitalium</span> serum was diluted to 1:40,1:80, 1:160 and 1:320 in 0.1 M PBS with 0.05per cent tween-20 (PBST). The dilution of conjugate 1:1000, 1:2000, 1:3000 and 1:4000 were prepared in PBST. The capture-ELISA was performed using the above prepared dilutions of rabbit and equine <span class="CharOverride-6">M. equigenitalium</span> sera and conjugate. Optimum dilution of conjugate was 1:2000 whereas dilution for both rabbit and equine <span class="CharOverride-6">M. equigenitalium</span> serum  was 1:160, which were the highest dilutions resulting in maximum colour  change and contrast between known positive  and known negative antigen samples. In each plate, positive control consisted of <span class="CharOverride-6">M. equigenitalium</span> (NCTC 10176/T-37) organisms (10⁷ CFU/ml) in PBS (pH 7.2) which were serially two-fold diluted; from 1:10 to 1:1280.</p>
			<p class="Body-Text">&nbsp;</p>
			<p class="Body-Text">&nbsp;</p>
			
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Table-1-"></a>Table 1: </span>Immunization schedule for raising hyperimmune serum against <span class="CharOverride-6">M. equigenitalium</span>   in rabbit and donkey foal<span class="CharOverride-5"> </span></p>
			<p class="Figure--and-Table-Heading ParaOverride-1">                                            </p>
			<p class="Figure--and-Table-Heading ParaOverride-2">Schedule for rabbit </p>
			<table width="657" height="178" class="Table-Style-1" id="table-1">
				<colgroup>
					<col class="_idGenTableRowColumn-1" />
					<col class="_idGenTableRowColumn-2" />
					<col class="_idGenTableRowColumn-3" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Week </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Inoculum*</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Distribution and site</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1/7</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1.8 ml killed antigen+1.8 ml CFA **</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Subcutaneously 0.4 ml in each rear foot-pad, 0.8 ml distributed at 4 site on the back  and 2.0 ml intramuscularly in the thigh region</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1.25 ml killed antigen</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1.0 ml intramuscularly and 0.25 ml  intravenously</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0.25 ml killed antigen</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0.25 ml  intravenously</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">5</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0.25 ml killed antigen</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0.25 ml  intravenously</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">8</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0.25 ml killed antigen</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0.25 ml  intravenously</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Body-Text ParaOverride-1">                                            <span class="CharOverride-5">  </span></p>
			<p class="Figure--and-Table-Heading ParaOverride-2">Schedule for donkey foal</p>
			<table width="657" class="Table-Style-1" id="table-2">
				<colgroup>
					<col class="_idGenTableRowColumn-7" />
					<col class="_idGenTableRowColumn-8" />
					<col class="_idGenTableRowColumn-9" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Week </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">                      Inoculum*</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">                        Distribution and site</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1/7</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7.5 ml killed antigen + 7.5 ml CFA </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">15 ml distributed subcutaneously at prescapular region</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4/7</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7.5 ml killed antigen</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7.5 ml distributed subcutaneously at prescapular region</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">2</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7.5 ml killed antigen + 7.5 ml CFA</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">15 ml distributed subcutaneously  at prescapular region</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4.5 ml killed antigen </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4.5 ml  intravenously</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">8</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7.5 ml live antigen (CFU 2X 10⁹ml)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7.5 ml distributed subcutaneously at prescapular region</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">9</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7.5 ml live antigen (CFU 2X 10⁹ml)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7.5 ml distributed subcutaneously at prescapular region</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-11">*Stock antigen diluted to Brown’s opacity tube no. 10 with PBS (pH 7.2); ** CFA = Complete</span><span class="CharOverride-11">Freunds’s adjuvant (Difco, USA).</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>Appearance of precipitin, growth inhibiting and ELISA-antibodies in experimentally antigenized donkey foal with <span class="CharOverride-6">M. equigenitalium</span> (NCTC 10176/T-37)</p>
			<table width="657" height="324" class="Table-Style-1" id="table-3">
				<colgroup>
					<col class="_idGenTableRowColumn-10" />
					<col class="_idGenTableRowColumn-11" />
					<col class="_idGenTableRowColumn-12" />
					<col class="_idGenTableRowColumn-13" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-14">
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-12">Day/week post immunization</span></p>
						</td>
						<td colspan="3">
							<p class="Basic-Paragraph"><span class="CharOverride-12">Anti M. equigenitalium antibodies detected with </span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">AGIPT </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">GI </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">ELISA titre</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1 day</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4 day</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7 day</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:40</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">2 week*</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:80</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">3 week</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:640</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4 week*</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:640</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">5 week</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:1280</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">6 week</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:1280</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7 week</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:1280</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">8 week*</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">-</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:1280</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">9 week*</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:2560</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">10 week</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">+</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1:5120</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-11">*= booster dose; AGIPT=agar gel immune precipitation test; GI= growth inhibition; + = positive; - = negative</span></p>
			<p class="Heading-2--History-in-MM-">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-">&nbsp;</p>
		  <p class="Heading-2--History-in-MM-">Procedure </p>
			<p class="Body-Text">Distilled water rinsed well of microtitre plate (Linbro, type IS-FB-96, Flow Lab, UK) were coated for 18 hrs. at 37°C with 0.05 ml of rabbit anti- <span class="CharOverride-6">M. equigenitalium</span> serum diluted to 1:160 in 0.1 M carbonate bicarbonate buffer (pH 9.6). The plate wells were washed with 0.1 M PBS (pH 7.4) having 0.05 per cent (v/v) tween-20 (PBST) and air-dried.The coated plates were stored at 4°C till use. Blocking of plates was done with addition of 0.05 ml bovine serum albumin (20%) in each well and incubated at 37°C. After 4 washings and air drying of plate wells, equine <span class="CharOverride-6">M. equigenitalium</span> serum diluted to 1:160 was added to each well (0.05ml) and incubated at 37°C for 1 hr and followed by 4 washings with PBST. The prepared test antigen in PBS (0.05 ml) was added in duplicate to plate wells and incubated for 1 hrs. at 37°C for another hour. After another washing step, optimally diluted anti-equine immunoglobulin horse radish-peroxidase conjugate (1:2000) was added (0.05 ml) and again incubated at 37°Cfor 1 hr. followed by four washings. After air drying the plates, 0.1 ml of enzyme substrate (OPD) was added to each well and incubated at room temperature (25-30°C) for 5 min. Stopping reagent (1 M H<span class="CharOverride-14">2</span>SO<span class="CharOverride-14">4</span><span class="apple-converted-space CharOverride-15">&#160;</span>solution) was added (0.1 ml) to each well and absorbance was measured at 492 nm in a Organon-teknika Reader 530 (Organon, Germany). Known positive and negative antigen samples were included for comparison with each test plate.</p>
		  <p class="Body-Text">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Interpretation </p>
			<p class="Body-Text ParaOverride-1">The mean absorbance of known negative antigen (5 tests) and known positive antigen (5 tests) was calculated. ELISA- value (mean absorbance of test sample mean absorbance of known negative sample) for each sample was calculated. An ELISA-value below 1.5 was considered negative.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">Results and discussion </p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Appearance of Precipitin, Growth Inhibiting and ELISA-antibodies in Experimentally Antigenized Donkey Foal </p>
			<p class="Body-Text ParaOverride-1">Appearance of precipitin, growth inhibiting and ELISA-antibodies in experimentally antigenized donkey foal with <span class="CharOverride-6">M. equigenitalium</span> (NCTC 10176/T-37) is given in <a href="#Table-2-"><span class="Hyperlink">Table 2</span></a>. The results indicated that ELISA antibodies gradually increased from nil (pre-immune titre) to 5-week time and it remained at 1:1280 till 8th week. Subsequently, it increased to 1:2560 and 1:5120 by 9th and 10th week, respectively. The precipitin antibodies also appeared at 5-week post immunization with <span class="CharOverride-6">M. equigenitalium</span> antigens and were observed till 10th week. The growth inhibiting antibodies appeared at 5th week but disappeared at 7th to 8th week. However, <span class="CharOverride-6">M. equigenitalium</span> precipitins were again recorded at 9th and 10th week post- immunization.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1">Detection of <span class="CharOverride-1">M. equigenitalium</span> Antigen in Equines</p>
			<p class="Body-Text ParaOverride-1">All the 219 equine samples, which included frozen semen, neat semen, prepucial swab, vaginal swab, cervical swab, aborted foetus samples, were subjected to detection of <span class="CharOverride-6">M. equigenitalium</span> antigen by capture-ELISA. <span class="CharOverride-6">M. equigenitalium</span> antigen was detected in samples from 4 of 63 (6.4%) repeat breed and 4 of 17 (23.6%) metritis mares. These detection rates were higher in comparison to detection by culture method, which were 3.2 and 11.8 per cent correspondingly. However, from 135 healthy equines <span class="CharOverride-6">M. equigenitalium</span> antigen was not detected in any of samples. The results obtained are given in <a href="#Table-3-"><span class="Hyperlink">Table 3</span></a>.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Table-3-"></a>Table 3: </span>Detection of <span class="CharOverride-6">M. equigenitalium</span> by culture and ELISA (capture)</p>
			<table width="657" class="Table-Style-1" id="table-4">
				<colgroup>
					<col class="_idGenTableRowColumn-15" />
					<col class="_idGenTableRowColumn-7" />
					<col class="_idGenTableRowColumn-16" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-17">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Reproductive health status of equines with total numbers in parentheses</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Culture</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Capture-ELISA</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">(A)</span><span class="CharOverride-13">Diseased</span><span class="CharOverride-12"> </span><span class="CharOverride-13">(84)</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-13">2 (3.2)*</span></p>
						</td>
						<td rowspan="2">
							<p class="Basic-Paragraph"><span class="CharOverride-13">4(6.4)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-18">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Repeat breed (63)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Metritis (17)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">2 (11.8)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4 (23.6)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-18">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Aborted fetus (2)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0(0)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0 (0)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-19">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Balanoposthitis (2)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0(0)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0(0)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-20">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Total</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4(4.8)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">8(9.6)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-19">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">(B)</span><span class="CharOverride-13">Apparently Healthy</span><span class="CharOverride-12"> </span><span class="CharOverride-13">(135)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0(0)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">0(0)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Grand total (219)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4(1.8)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">8 (3.6)</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-11">*Figures in parentheses indicate percentage.</span></p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The overall prevalence of<span class="CharOverride-16"> </span><span class="CharOverride-6">M. equigenitalium</span> was also higher (3.6%) by detection of its antigen by capture ELISA than by cultural methods (1.8%) in equines with reproductive disorders (<a href="#Table-3-"><span class="Hyperlink">Table 3</span></a>).</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1"><a href="#Spergser-J--Aurich-C--Aurich-JE--Rosengarten-R--2002"><span class="Hyperlink">Spergser et al. (2002)</span></a> have reported high prevalence of mycoplasmas in the genital tract of asymptomatic stallions in Austria, where <span class="CharOverride-6">M. equigenitalium</span> was isolated from 55 out of 116 stallions. The dominance of <span class="CharOverride-6">M. equigenitalium </span>in the<span class="CharOverride-6"> </span>genital tract of equines has<span class="CharOverride-6"> </span>been confirmed (<a href="#Kirchhoff-H--Naglic-T--Heitmann-J--1979"><span class="Hyperlink">Kirchhoff et al., 1979</span></a>; <a href="#Kirchhoff-H--Heitmann-J--Bisping-W--1980"><span class="Hyperlink">Kirchhoff et al., 1980</span></a>; <a href="#Naglic-T--Topolnik-E--Sukalic-M--Hajsig-D--1980"><span class="Hyperlink">Naglic </span><span class="Hyperlink">et al., 1980</span></a>; <a href="#Zgorniak-Novesielka-IN--Bredlanski-W--Kosiniak--1984"><span class="Hyperlink">Zgorniak-Nowesielka et al., 1984</span></a>; <a href="#Mani-P--Incastrone-G--Chiavacci-L--1985"><span class="Hyperlink">Mani et al., 1985</span></a>; <a href="#Bermudez-V--Miller-R--Johnson-W--Rosendal-S--Ruhnke-L--1987"><span class="Hyperlink">Bermudez et al., 1987</span></a>; <a href="#Copes-J--Blanco-A--Giaboni-G--Stanchi-N--Cerda-R--Nievas-V--1995"><span class="Hyperlink">Copes et al., 1995</span></a>; <a href="#Khurana-SK--Garg-DN--2001-."><span class="Hyperlink">Khurana and Garg, 2001</span></a>). In this study, <span class="CharOverride-6">M. equigenitalium</span> antigen was detected in samples from 4 of 63 (6.4%) repeat breed and 4 of 17 (23.6%) metritis mares, which is significant prevalence and the detection was two-times higher than detection by cultural method. The overall prevalence of<span class="CharOverride-16"> </span><span class="CharOverride-6">M. equigenitalium</span> was higher also (3.6%) by detection of its antigen by capture-ELISA than by cultural methods (1.8%) in equines with reproductive disorders. Therefore, ELISA has been found a very easy, sensitive and rapid method for mass screening of <span class="CharOverride-6">M. equigenitalium</span> anigen in equines especially when compared with cultural methods.</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-3" lang="en-US"><a id="Ball-HJ--Finlay-D--Reilly-GAC--1994-."></a>Ball HJ, Finlay D, Reilly GAC (1994). Sandwich ELISA detection of <span class="CharOverride-6">Mycoplasma bovis</span> in pneumonic calf lungs and nasal swabs. Vet. Rec.<span class="CharOverride-5"> </span>135(22):<span class="CharOverride-5"> </span>531-532. <a href="http://dx.doi.org/10.1136/vr.135.22.531"><span class="Hyperlink">http://dx.doi.org/10.1136/vr.135.22.531</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Bermudez-V--Miller-R--Johnson-W--Rosendal-S--Ruhnke-L--1987"></a>Bermudez V, Miller R, Johnson W, Rosendal S, Ruhnke L (1987). Recovery of <span class="CharOverride-6">Mycoplasma</span> spp. from the reproductive tract of mare during the estrous cycle. Can. Vet. J. 28(8): 519-522.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Boothby-JT--Mueller-R--Jasper-DE--Thomos-CB--1986"></a>Boothby JT, Mueller R, Jasper DE, Thomos CB (1986). Detection of <span class="CharOverride-6">Mycoplasma bovis </span>in milk by<span class="CharOverride-6"> </span>enzyme-linked-immunosorbent-assay, using monoclonal antibodies. Am. J. Vet. Res. 47 (5): 1082-1084.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Brooks-C--Finlay-D--Blackburn-P--Ball-H-J--2009"></a>Brooks C, Finlay D, Blackburn P, Ball H J (2009). Detection of <span class="CharOverride-6">Mycoplasma mycoides</span> subspecies <span class="CharOverride-6">mycoides</span> small colony by a specific capture/enrichment monoclonal anti body-based sandwich ELISA. Res. Vet. Sci. 87(2): 200-203. <a href="http://dx.doi.org/10.1016/j.rvsc.2009.03.007"><span class="Hyperlink">http://dx.doi.org/10.1016/j.rvsc.2009.03.007</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Copes-J--Blanco-A--Giaboni-G--Stanchi-N--Cerda-R--Nievas-V--1995"></a>Copes J, Blanco A, Giaboni G, Stanchi N, Cerda R, Nievas V (1995). Isolation of<span class="CharOverride-6"> Mycoplasma equigenitalium</span> from healthy and infertile mares. Avances en Ciencias-Veterinarias. 10(2): 103-106.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Dahiya-SK--Tiwari-SC--Batra-M--Sharma-A--2009"></a>Dahiya SK, Tiwari SC, Batra M, Sharma A (2009). Comparison of capture-ELISA with cultural isolation for detection of <span class="CharOverride-6">Mycoplasma mycoides </span>subspecies <span class="CharOverride-6">mycoides </span>(LC type, M-30) in experimentally infected lambs. Indian J. Vet. Pathol. 33(1): 69-72.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Garg-DN--1999-"></a>Garg DN (1999). Equine mycoplasmosis: a review. J. Remount Vet. Corps. 38: 39-51.</li>
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