<div dir="ltr"><span style="font-size:12.8000001907349px">Dear colleagues,</span><div style="font-size:12.8000001907349px"><br></div><div style="font-size:12.8000001907349px">I wish to say the following after seeing the original and responding messages:</div><div style="font-size:12.8000001907349px"><br></div><div style="font-size:12.8000001907349px">1. Finding Leishmania DNA in tissue samples from vectors, reservoir animals and human subjects by PCR is doable by using specific primer sets. This is suggestive of the presence of Leishmania, especially after confirmation by sequencing. </div><div style="font-size:12.8000001907349px"><br></div><div style="font-size:12.8000001907349px">2. Isolation of Leishmania from PCR-positive samples will be necessary as the ultimate proof for its actual existence as live cells and, if interested, for experimental studies, e. g. animal infectivity, vector transmission, etc. </div><div style="font-size:12.8000001907349px"><br></div><div style="font-size:12.8000001907349px">3. There are two ways to isolate Leishmania from PCR-positive samples:</div><div style="font-size:12.8000001907349px"> </div><div style="font-size:12.8000001907349px">[1] Inoculation of freshly collected materials into suitable media looking for the emergence of promastigotes. "Microculture in capillary glass tubes" is probably the simplest method;</div><div style="font-size:12.8000001907349px"> </div><div style="font-size:12.8000001907349px">[2] Inoculation of the samples into a susceptible lab animal for intracellular parasitism of macrophages with amastigotes. </div><div style="font-size:12.8000001907349px"><br></div><div style="font-size:12.8000001907349px">Replication of Leishmania under either condition is necessary for visualization of the parasite as the proof of its presence as live cells and for isolation of the parasite. Keep in mind that negative finding in either case is inconclusive and also the possibility for the emergence of avirulent genetic variants. Of relevance to the last point is a need of genomic sequence comparison between Leishmania in the original tissue samples and those isolated into pure culture.</div><div style="font-size:12.8000001907349px"><br></div><div style="font-size:12.8000001907349px">4. Leishmani infatum has been isolated from Tarim hare in Taklamahan desert of Xinjiang Province in China. Together with the subsequent similar finding in Spain, the observation suggests that this and related animals may be a potential reservoir of leishmaniasis. Transmission experiments are still needed to prove this point.</div><div style="font-size:12.8000001907349px"><br></div><div style="font-size:12.8000001907349px"> KP</div><div style="font-size:12.8000001907349px"><br></div><div style="font-size:12.8000001907349px">Kwang Poo Chang, PhD</div><div style="font-size:12.8000001907349px">Professor of Microbiology/Immunology</div><div style="font-size:12.8000001907349px">Chicago Medical School/RFUMS</div><div style="font-size:12.8000001907349px">N Chicago, IL 60064, USA</div><div style="font-size:12.8000001907349px"><br></div><div style="font-size:12.8000001907349px">tel <a href="tel:847-578-8837" value="+18475788837" target="_blank">847-578-8837</a></div><div style="font-size:12.8000001907349px">fax <a href="tel:847-578-3349" value="+18475783349" target="_blank">847-578-3349</a></div><div style="font-size:12.8000001907349px">e-mail <a href="mailto:kwangpoo.chang@rosalindfranklin.edu" target="_blank">kwangpoo.chang@rosalindfranklin.edu</a></div></div><div class="gmail_extra"><br><div class="gmail_quote">On Fri, Mar 6, 2015 at 2:24 PM, Bogdan, Christian <span dir="ltr"><<a href="mailto:Christian.Bogdan@uk-erlangen.de" target="_blank">Christian.Bogdan@uk-erlangen.de</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1f497d">Dear Dr. Sayler,<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1f497d"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1f497d">in proven reservoir hosts (e.g., small mammals) it is no problem to detect Leishmania DNA by PCR in the circulating blood if these animals are
infected. However, one has to be very cautious with the selection of primers. For example, PCR methods based on primers targeting the small subunit rRNA gene or the 18S rRNA gene can yield positive results, but sequencing of the PCR products reveal the presence
of apathogenic Trypanosomes rather than Leishmania.<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1f497d"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1f497d">With regards,<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1f497d"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1f497d">Christian Bogdan<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1f497d"><u></u> <u></u></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-GB" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">Christian Bogdan, M.D.</span><span lang="EN-US" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d"><u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-GB" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">Professor of Medical Microbiology and Infectious Disease Immunology, Head of the Institute<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-GB" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">Microbiology Institute – Clinical Microbiology, Immunology and Hygiene</span><span lang="EN-US" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d"><u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">Universitätsklinikum Erlangen<u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">Friedrich-Alexander Universität Erlangen-Nürnberg<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">Wasserturmstraße 3/5<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-GB" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">D-91054 Erlangen, Germany</span><span lang="EN-US" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d"><u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-GB" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">Tel. <a href="tel:%28%2B49%29-9131-852-2551" value="+4991318522551" target="_blank">(+49)-9131-852-2551</a> (office), -2281 (secretary)</span><span lang="EN-US" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d"><u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-GB" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">Fax <a href="tel:%28%2B49%29-9131-852-2573" value="+4991318522573" target="_blank">(+49)-9131-852-2573</a> or -9131-85-1001</span><span lang="EN-US" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d"><u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d">E-mail:
</span><span lang="EN-GB" style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d"><a href="mailto:christian.bogdan@uk-erlangen.de" target="_blank"><span lang="DE">christian.bogdan@uk-erlangen.de</span></a></span><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d"><u></u><u></u></span></p>
<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#1f497d"><a href="http://www.mikrobiologie.uk-erlangen.de/" target="_blank">http://www.mikrobiologie.uk-erlangen.de/</a><u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:#1f497d"><u></u> <u></u></span></p>
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<p class="MsoNormal"><b><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif"">Von:</span></b><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif""> <a href="mailto:leish-l-bounces@lineu.icb.usp.br" target="_blank">leish-l-bounces@lineu.icb.usp.br</a> [mailto:<a href="mailto:leish-l-bounces@lineu.icb.usp.br" target="_blank">leish-l-bounces@lineu.icb.usp.br</a>]
<b>Im Auftrag von </b>Sayler,Katherine A<br>
<b>Gesendet:</b> Freitag, 6. März 2015 00:30<br>
<b>An:</b> <a href="mailto:leish-l@lineu.icb.usp.br" target="_blank">leish-l@lineu.icb.usp.br</a><br>
<b>Betreff:</b> [Leish-l] Confirm hits to Leishmania spp. in whole blood of mammals in the US?<u></u><u></u></span></p>
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<p class="MsoNormal"><u></u> <u></u></p>
<p><span style="font-family:"Calibri","sans-serif";color:black">Dear readers, <u></u>
<u></u></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black"><u></u> <u></u></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black">Thank you sincerely for your time. I am a tick-borne disease biologist by trade, so this is outside of my realm.
<u></u><u></u></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black"><u></u> <u></u></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black;background:white">I am trying to confirm some preliminary data that suggests the
<em><span style="font-family:"Calibri","sans-serif"">Leishmania</span></em> spp. are circulating in limited geographic locales in southern Florida. Is it at all reasonable to expect to detect the DNA of the trypanosomatids in the blood of infected wildlife?
My suspicion is that this is completely improbable, as it appears that in order to detect DNA in enough quantity or quality for sequencing, you need cultured organisms. From sandflies it appears that it is possible to PCR amplify and sequence directly from
the vector, but never from the reservoir (or potential reservoir) host. Is this true? Any advice or insight would be greatly appreciated and I am tremendously naïve and this is clearly not my area of expertise</span><span style="font-family:"Calibri","sans-serif";color:black">.<u></u><u></u></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black"><u></u> <u></u></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black">Thank you again for reading my message,<u></u><u></u></span></p>
<p><span style="font-family:"Calibri","sans-serif";color:black">Katherine<u></u><u></u></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:black">Katherine Sayler, M.Ed., Ph.D.</span><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black"><u></u><u></u></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:black">Postdoctoral Associate | Department of Wildlife Ecology and Conservation</span><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black"><u></u><u></u></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:black">Molecular Ecology Laboratory | 2322 Mowry Rd. Gainesville, FL 32611</span><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black"><u></u><u></u></span></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:black">Phone: <a href="tel:352-871-3259" value="+13528713259" target="_blank">352-871-3259</a> </span><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black"><u></u><u></u></span></p>
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