Detection of Leishmania Parasites via

Flow Cytometry

Analiese Wenger

What are Leishmania?♦ Single-cell parasite♦ Infect humans and other mammalian

hosts

Life Cycle

Leishmaniasis♦ Manifestations:• Cutaneous

• Mucosal

• Visceral

♦ Recognition of lipophosphoglycan (LPG)

♦ Promastigote engulfment♦ Leishmania live in vesicles protected from:• Antibodies• Killer T cells

♦ Inhibition of phagosome-endosome fusion

♦ Recognition of lipophosphoglycan (LPG)

♦ Promastigote engulfment♦ Leishmania live in vesicles protected from:• Antibodies• Killer T cells

♦ Inhibition of phagosome-endosome fusion

Immunological Subversion

Distribution♦ 98 endemic countries• 0.2 to 0.4 million new VL cases; 0.7 to 1.2 million new

CL cases each year worldwide♦ Restricted to tropical and temperate regions

Scientific Question

Can macrophages infected with Leishmania be detected using a flow

cytometer?

Predictions and Hypotheses♦ Predictions – The intracellular staining

will enable detection of Leishmania species inside macrophages

♦ Alternative hypothesis – flow cytometry will detect Leishmania

♦ Null hypothesis – flow cytometry will not detect Leishmania

Flow Cytometry

♦ Counts individual cells using laser technology• Forward-scattered light (FSC)• Side-scattered light (SSC)

♦ Fluorescence dyes (fluorochromes) for sensing target proteins

♦ Counts individual cells using laser technology• Forward-scattered light (FSC)• Side-scattered light (SSC)

♦ Fluorescence dyes (fluorochromes) for sensing target proteins

Flow Cytometry Data

Experimental Design

♦ Macrophage Cells• Mouse cell line

J774A.1

♦ Leishmania species• L. major - cutaneous• L. infantum - visceral

Experimental Design♦ L. major and L. infantum treatments:• Control without fluorescein isothiocyanate (FITC)• Experimental group with FITC

♦ Anti-Leishmania (GP-63) Monoclonal Antibody

♦ Visualized parasites following flow acquisition

Techniques♦ Preliminary experimentation• FSC & SSC of macrophages

♦ Photomicroscopy♦ Intracellular staining• Modified protocol

FSC & SSC Results

Figure 1 Control J774 macrophages

Figure 2 Macrophages infected with L. major

Figure 3 Macrophages infected with L. infantum

Figure 4 Macrophages infected with L. infantum mixed with control

Figure 1 Control J774 macrophages

Figure 2 Macrophages infected with L. major

Figure 3 Macrophages infected with L. infantum

Figure 4 Macrophages infected with L. infantum mixed with control

Techniques♦ Preliminary experimentation• FSC & SSC of macrophages

♦ Photomicroscopy♦ Intracellular staining• Modified protocol

Photomicroscopy Results

L. major

L. infantum

Techniques♦ Preliminary experimentation• FSC & SSC of macrophages

♦ Photomicroscopy♦ Intracellular staining• Modified protocol

Parasite Flow Results

Figure 1 L. major: (left) without FITC and (right) with FITC

Figure 2 L. infantum: (left) without FITC and (right) with FITC

Figure 3 Histogram plot of L. major: (left) without FITC and (right) with FITC

Figure 4 Histogram plot of L. infantum: (left) without FITC and (right) with FITC

FL1 FL1

FL3

FL3

FL1 FL1 FL1 FL1

FL3

FL3

FL1FL1

Intracellular Staining Results

Figure 6 L. major infected macrophages

Figure 7 L. infantum infected macrophagesFigure 8 L. infantum infected macrophages mixed with control

Figure 5 Control macrophagesFigure 5 Control macrophages Figure 6 L. major infected macrophages

Figure 7 L. infantum infected macrophagesFigure 8 L. infantum infected macrophages mixed with control

Conclusions♦ Anti-leishmania antibody does attach

to the major surface protease of L. infantum and L. major

♦ L. infantum binds with higher specificity to antibody than L. major

♦ Incomplete separation of infected versus uninfected macrophages

Acknowledgements• Dr. Gabrielle Stryker –

Mentor• Dr. Blaise Dondji –

Leishmania expert & donor• W. M. Keck Foundation and

Murdock Fund• Mark Young – FlowJo Wizard• Eric Foss –

Photomicroscopy Technician

• Heidi Anderson• Mercedes Cheslock

ReferencesColomer - Gould, V., L. G. Quintao, J. Keithly, N. Nogueira. (1985). A common surface antigen on amastigoes and promastigotes of Leishmania species. J. Exp. Med., 162(902).Desjardins, M., & Descoteaux, A. (1997). Inhibition of phagolysosomal biogenesis by the Leishmania  lipophosphoglycan. The Journal of Experimental Medicine, 185(12), 2061–2068.Russel, D.G., and H. Wilhelm. (1986). The involvement of the major surface glycoprotein (gp63) of Leishmania in attachment to macrophages. J. Immunol., 136(2616).Kram, D., Thale, C., Kolodziej, H., and Kiderlen, A. (2008). Intracellular parasite kill: Flow cytometry and NO detection for rapid discrimination between anti-leishmanial activity and macrophage activation. Journal of Immunological Methods, 333(20).

Questions?