Non-invasive visualisation and identification of fluorescent

Diaz-Albiter, H. M., Regnault, C., Alpizar-Sosa, E. A., McGuinness, D., Barrett, M. P. and Dillon, R. J. (2018) Non-invasive visualisation and identification of fluorescent. Wellcome Open Research, 3, 160. (doi: 10.12688/wellcomeopenres.14910.1) (PMID:30756095) (PMCID:PMC6367660)

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Background: The leishmaniases are neglected diseases that affect some of the most vulnerable populations in the tropical and sub-tropical world. The parasites are transmitted by sand flies and novel strategies to control this neglected vector-borne disease are needed. Blocking transmission by targeting the parasite inside the phlebotomine vector offers potential in this regard. Some experimental approaches can be best performed by longitudinal study of parasites within flies, for which non-destructive methods to identify infected flies and to follow parasite population changes are required. Methods: Lutzomyia longipalpis were reared under standard insectary conditions at the Wellcome Centre for Molecular Parasitology. Flies were artificially infected with L. tarentolae expressing green fluorescent protein (GFP. Parasite counts were carried out 5 days post-infection and the percentage of infected flies and survival of infected females was established up to days 5 post-infection. Whole living females were visualised using an epifluorescence inverted microscope to detect the presence parasites inferred by a localised green fluorescent region in the upper thorax. Confirmation of infection was performed by localised-fluorescence of dissected flies and estimates of the parasite population. Results: Leishmania tarentolae was successfully transfected and expressed GFP in vitro. L. tarentolae-GFP Infected flies showed similar parasite populations when compared to non-transfected parasites (L. tarentolae-WT). Survival of non-infected females was higher than L. tarentolae-infected groups, (Log-rank (Mantel-Cox) test, p<0.05). L. tarentolae-GFP infected females displayed an intense localised fluorescence in the thorax while other specimens from the same infected group did not. Localised fluorescent flies were dissected and showed higher parasite populations compared to those that did not demonstrate high concentrations in this region (t-test, p<0.005). Conclusion: These results demonstrate the feasibility of establishing a safe non-human infectious fluorescent Leishmania-sand fly infection model by allowing non-destructive imaging to signal the establishment of Leishmania infections in living sand flies.

Item Type:Articles
Additional Information:Version 1; peer review: 1 approved, 2 approved with reservations.
Keywords:GFP, leishmania, lutzomyia, fluorescence, parasite-vector interactions, sand fly
Glasgow Author(s) Enlighten ID:Regnault, Mr Clement and Alpizar Sosa, Edubiel Arturo and Diaz Albiter, Dr Hector and McGuinness, Dr Dagmara and Barrett, Professor Michael
Creator Roles:
Diaz-Albiter, H. M.Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft
Regnault, C.Methodology, Visualization, Writing – original draft
Alpizar-Sosa, E. A.Investigation
McGuinness, D.Data curation, Formal analysis, Investigation, Methodology, Software, Writing – original draft, Writing – review and editing
Barrett, M. P.Funding acquisition, Resources, Writing – review and editing
Authors: Diaz-Albiter, H. M., Regnault, C., Alpizar-Sosa, E. A., McGuinness, D., Barrett, M. P., and Dillon, R. J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Wellcome Open Research
ISSN (Online):2398-502X
Copyright Holders:Copyright © 2018 Diaz-Albiter HM et al.
First Published:First published in Wellcome Open Research 3: 160
Publisher Policy:Reproduced under a Creative Commons License
Data DOI:10.6084/m9.figshare.7283546.v1

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
170547The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOTR)104111/Z/14/ZIII - Parasitology
172823Parasite induced changes to the metabolome of insect vectors of diseaseMichael BarrettThe Royal Society (ROYSOC)NF151329III - Parasitology