Studies using luciferase-expressing Leishmania donovani parasites

Visceral leishmaniasis is a life threatening illness that affects 12 million people worldwide and 350 million people across 88 countries are at risk of infection. The disease is caused by Leishmania donovani, a parasite transmitted by the female sand fly. Resistance to the drugs used to treat the disease is becoming more common in some parts of the world and there are very few alternatives available for treatment. In this study two strains of L. donovani (LV82 and 200011), which express different luciferase genes and thus emit light of different wavelengths (red or green) were assessed for their ability to act in drug screening studies. We assessed if the insertion of the luciferase gene into L. donovani parasites altered their susceptibility to commonly used anti-leishmanial drugs and their ability to infect cells and animals. Four different anti-leishmanial drugs were used in these studies i.e. sodium stibogluconate, amphotericin B, miltefosine and paromomycin. Intraperitoneal or bone marrow derived macrophages infected with L. donovani were incubated with different concentrations of each drug (sodium stibogluconate, 4-0.02mg/ml; amphotericin B, 4-0.02μg/ml: miltefosine, 4-0.02μg/ml; paromomycin, 4-0.02μg/ml) and the amount of parasites was assessed by determining the amount of light released using the IVIS® imaging system. The insertion of the luciferase gene into the L. donovani parasites resulted in a change in infectivity compared to the relevant wild-type (p<0.05). Promastigote drug screening was determined using a resazurin based assay incubated with amphotericin B, 1-0.005μg/ml; miltefosine, 20-1.25μg/ml; paromomycin, 300-17.5μg/ml. Furthermore, insertion of either gene into either parasite strain did not result in any change in susceptibility of promastigotes to paromomycin, however there were differences in susceptibility of promastigotes to amphotericin B and miltefosine. When infecting intraperitoneal macrophages the insertion of either luciferase gene into LV82 or 200011 L. donovani parasites did not affect their susceptibility to sodium stibogluconate, amphotericin B, miltefosine or paromomycin. However, when infecting bone marrow-derived macrophages the insertion of either luciferase gene into either parasite strain did not affect their susceptibilities to amphotericin B, miltefosine or paromomycin. In vivo studies were also carried out to determine if non-ionic surfactant vesicles (NIV) were capable of enhancing luciferin delivery and increasing parasite detection sensitivity by measuring bioluminescence using the IVIS® imaging system. Treatment with luciferin-non-ionic surfactant vesicles resulted in higher levels of bioluminescence (parasite detection sensitivity) than luciferin solution treatment (p<0.05).

Name dc:type.qualificationname

mres

Level dc:type.qualificationlevel

masters-pg

Grantor dc:publisher.institution

University of Strathclyde

Year dc:date.issued

2013

Author dc:creator

• Carruthers, John Mathew

DOI

10.48730/9pht-nd33

Identifier

T13788

OAI identifier oai:identifier

oai:strathclyde:mg74qm120