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In vivo bioluminescence imaging can be used to monitor variation in Giardia infection burden
Katherine S. McGowan and Steven Singer
Biology Department, Georgetown University
ABSTRACT
Materials and MethodsMATERIALS AND METHODS
RESULTS
RESULTS
------------------------------------------------------------------------------------------------------------------------------------------------------
Giardia lamblia is a protozoan parasite that replicates in the small intestines of infected individuals and is one of the
leading causes of diarrheal disease around the world. Although most individuals will resolve acute Giardia infections within
a few weeks, approximately 15% of those infected will develop chronic giardiasis. Recent studies suggest that Giardia
strain and infective stage (cyst vs. trophozoite) may mediate infection chronicity, but the influence of these factors on
infection outcome is not yet well understood. To study the role of Giardia strain and infective stage on infection chronicity,
we infected mice with both the cyst and trophozoite form of Giardia that express firefly luciferase, a reporter gene that
enables localization and quantification of parasite burden using bioluminescence imaging. Although in vitro-derived cysts
were non-infectious, long-term bioluminescence monitoring of trophozoite-infected mice over the course of 26-28 days
indicates that bioluminescence imaging can be used to accurately and sensitively measure disease burden in vivo. In the
future, bioluminescence imaging using luciferase-expressing parasites can be used to study long-term infection dynamics
as mediated by various parasite and host characteristics.
CONCLUSIONS
BACKGROUND
Giardia infective stage and assemblage Firefly luciferase imaging
Light
D-luciferin
Oxyluciferin
+
GS-
LUC
Vector
CWP1
Promoter Luciferase
GS, H3
WB
1. Clone luciferase
gene driven by CWP1
promoter into the GS-
AO vector
2. Transfect Giardia GS
and H3 with GS-LUC
Clone luciferase gene Engineer Giardia-LUC Infect and image mice
3. Encyst
Giardia-LUC
trophozoites
4. Infect mice 5. Image mice weekly
6. Count intestinal parasites
and perform real-time PCR
on stool samples
Analyze samples
Figure 5. Luminescence data shows that SCID mice remained
chronically infected throughout the duration of the study while
wild-type mice were able to clear a trophozoite-generated
infection within 26 days. Mice were infected with either 106 WB/C2
trophozoites or 106 WB/C2 cysts and were imaged once a week for
26 days. Wild-type cyst-infected mice showed no signs of
luminescence, indicating that the in vitro cysts were not infectious.
Figure 4. Wild-type mice infected with cysts did not establish infection.
BALB/c mice infected with WB/C2 trophozoites and BALB/c SCID mice infected
with WB/C2 trophozoites showed parasite burden seven days after infection, as
indicated by luminescence. BALB/c mice infected with WB/C2 cysts derived via
encystation using high-bile encystation media (10 mg/mL bovine bile; pH 7.8)4 were
not infected and showed no luminescence.
Wild-Type Troph-Infected SCID Troph-Infected Wild-Type Cyst-Infected
Figure 1. Luciferase-induced bioluminescence of three living Tcrbtm1Mom
(immunocompromised) mice 28 days following infection with WB/C2
luciferase-expressing G. lamblia parasites. Each mouse was given 125 µL of
luciferin (30 mg/mL) via IP injection prior to being sedated and imaged. Presence
of luminescent parasites at day 28 indicates that Tcrbtm1Mom mice were not able to
clear a G. lamblia infection over the course of 4 weeks.
Figure 2. Peak whole-mouse
radiance flux occurs 17 minutes
after injection. Six Tcrbtm1Mom mice
(day 14 of infection) were injected with
125 µL luciferin (30 mg/mL) and
imaged in a series of three-minute
intervals. Average total radiance flux
across the six mice (with standard
deviation) indicates that luminescence
peaks approximately 17 minutes
following injection and declines slowly
afterwards.
A
B C
Figure 3. Bioluminescence imaging can detect regional
differences in Giardia parasite load. The intestines of mice were
dissected from stomach to cecum and cut into three 2-cm. fragments
and three 1-cm. fragments. Intestinal fragments were imaged and
parasites counted. (B) The most parasites were found in the
fragment closest to the stomach. (C) Total parasite count (all
fragments combined) is weakly correlated with whole-mouse
radiance flux (R2 = 0.285).
• Giardia exists in an active (trophozoite) stage
and a dormant (cyst) stage
• Trophozoites generate acute infections in
mice
• Giardia has eight genotypes, but only two (A
and B) are capable of infecting humans1
• Not much is known about how the parasite
influences disease outcome, but mice
infected with H3 (assem. B) cysts showed
disease burden longer than mice infected
with WB (assem. A) trophs in a study at UVA2
Does infection with different strains and/or stages of Giardia
influence disease outcome?
• Luciferase is an enzyme
that catalyzes the oxygen-
ation of luciferin and ATP to
yield oxyluciferin and light3
• Can use parasites that
express luciferase to detect
disease burden in vivo
• Could allow for longitudinal,
real-time disease burden
monitoring
Can imaging with luciferase-expressing parasites be
used to detect Giardia disease burden in vivo?
• Longitudinal Giardia disease burden can be monitored in vivo using parasites that express firefly luciferase.
Imaging data confirms that scid mice remained chronically infected while wild-type mice cleared infection within 26 days.
• Luminescence of infected mice is correlated with actual parasite load. Intestinal parasite counts indicate that
bioluminescence imaging sensitively and accurately detects infection burden in a living mouse.
• Future directions include real-time PCR of stool samples, infection of wild-type mice with infectious cysts, and
successful creation of GS and H3 strains that express luciferase.
PAC*
Giardia
TPI locus
E. coli origin
AmpR
1Thompson, R.C.A. (2009). Echinococcus, Giardia and Crytosporidium: observational studies challenging accepted dogma. Parasitology, 136(12): 1529-1535.
2 Bartlet, L.A., Roche, J., Kolling, G., Bolick, D., Noronha, F., Naylor, C., Guerrant, R. (2013). Persistent G. lamblia impairs growth in a murine malnutrition model. J Clin Invest, 123(6), 2672-2684.
3Marques, Simone, M. & Esteves da Silva, J.C (2009). Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions. IUMBMB life, 61(1), 6-17.
4Kane, A.V., Ward, H.D., Keusch, G.T., & Pereira, M.E. (1991). In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency. J Parasitology, 974-981.
Special thanks to Olga Rodriguez and Chip Albanese for assistance with in vivo imaging, Scott Dawson for provision of WB/C2 parasites,
and Jenny Maloney and Erqiu Li for assistance with infections. Funding: GUROP, Summer 2014
*Puromycin acetyl transferase (PAC) confers drug-
resistance for selection after transfection of parasite