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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

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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

BACKGROUNDGiardia 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 weekly6. 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*

GiardiaTPI 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