[Leish-l] Confirm hits to Leishmania spp. in whole blood of mammals in the US?

Fri Apr 3 18:13:41 BRT 2015

Dear colleagues,

I wish to say the following after seeing the original and responding
 messages:

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.

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.

3. There are two ways to isolate Leishmania from PCR-positive samples:

[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;

[2] Inoculation of the samples into a susceptible lab animal for
intracellular parasitism of macrophages with amastigotes.

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.

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.

 KP

Kwang Poo Chang, PhD
Professor of Microbiology/Immunology
Chicago Medical School/RFUMS
N Chicago, IL 60064, USA

tel 847-578-8837
fax 847-578-3349
e-mail kwangpoo.chang at rosalindfranklin.edu

On Fri, Mar 6, 2015 at 2:24 PM, Bogdan, Christian <
Christian.Bogdan at uk-erlangen.de> wrote:

>  Dear Dr. Sayler,
>
>
>
> 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.
>
>
>
> With regards,
>
>
>
> Christian Bogdan
>
>
>
>
>
> Christian Bogdan, M.D.
>
> Professor of Medical Microbiology and Infectious Disease Immunology, Head
> of the Institute
>
> Microbiology Institute – Clinical Microbiology, Immunology and Hygiene
>
> Universitätsklinikum Erlangen
>
> Friedrich-Alexander Universität Erlangen-Nürnberg
>
> Wasserturmstraße 3/5
>
> D-91054 Erlangen, Germany
>
> Tel. (+49)-9131-852-2551 (office), -2281 (secretary)
>
> Fax (+49)-9131-852-2573 or -9131-85-1001
>
> E-mail: christian.bogdan at uk-erlangen.de
>
> http://www.mikrobiologie.uk-erlangen.de/
>
>
>
>
>
> *Von:* leish-l-bounces at lineu.icb.usp.br [mailto:
> leish-l-bounces at lineu.icb.usp.br] *Im Auftrag von *Sayler,Katherine A
> *Gesendet:* Freitag, 6. März 2015 00:30
> *An:* leish-l at lineu.icb.usp.br
> *Betreff:* [Leish-l] Confirm hits to Leishmania spp. in whole blood of
> mammals in the US?
>
>
>
> Dear readers,
>
>
>
> Thank you sincerely for your time. I am a tick-borne disease biologist by
> trade, so this is outside of my realm.
>
>
>
> I am trying to confirm some preliminary data that suggests the
> *Leishmania* 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.
>
>
>
> Thank you again for reading my message,
>
> Katherine
>
>
>
> Katherine Sayler, M.Ed., Ph.D.
>
> Postdoctoral Associate | Department of Wildlife Ecology and Conservation
>
> Molecular Ecology Laboratory | 2322 Mowry Rd. Gainesville, FL 32611
>
> Phone: 352-871-3259
>
>
>
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