activity can be measured and quantified in real time providing some
spatiotemporal features about the disease progression in an intact
living host [9, 10].
In the lab, bioluminescent pathogenic and saprophytic strains
of Leptospira have been constructed by transposon insertion of
a cassette expressing the firefly luciferase gene under the control
of a leptospiral promoter, into the genome [9]. The firefly luciferase
is a useful reporter system that can generate visible light through
the oxidation of D-luciferin, its substrate, in the presence of oxygen
and ATP as a source of energy. Of note, the reaction is catalyzed
only in metabolically active bacteria producing both ATP and the
luciferase. Therefore, we can evaluate the spread of live bacteria by
tracking bioluminescence. The light generated is measured in a
light-tight box using new generation optical cameras that enable a
highly sensitive detection of the photons transmitted. Moreover,
bioluminescence imaging based on luciferase doesn’t require excitation, avoiding high background signal. The absolute luciferase
activity can be analyzed and quantified using the In Vivo Imaging
Software (IVIS) and compared with a baseline or control
conditions [11].
With this method, we were able to observe, in live mice, the
spatiotemporal evolution and intensity of an experimental infection
with leptospires [9]. Moreover, this method proves to be reliable
for the determination of critical experimental end points.
2 Materials
All solutions are reconstituted, prepared, or diluted in sterile Dulbecco’s phosphate buffered saline (DPBS) or guaranteed
endotoxin-free reagents. Prepare and store aliquots of working
solutions at À20
C (unless indicated otherwise). Thaw and warm
all solutions at room temperature before administration to animals.
Diligently follow all waste disposal regulations when disposing
soiled sharp objects or other waste materials.
1. Leptospira biflexa sevorar Patoc strain PFLum7 (saprophytic
bioluminescent leptospires).
2. Leptospira interrogans serovar Manilae strain MFLum1 (virulent L. interrogans bioluminescent strain).
3. Bacterial culture medium Ellinghausen-McCullough-JohnsonHarris (EMJH): 1.0 g/L sodium phosphate dibasic, 0.3 g/L
monopotassium phosphate, 1.0 g/L sodium chloride, 0.25 g/
L ammonium chloride, 5 mg/L thiamine. EMJH medium is
supplemented with 10 g/L bovine serum albumin, 4 mg/L
zinc sulfate heptahydrate, 50 mg/L iron sulfate heptahydrate,
15 mg/L hexahydrate magnesium chloride, 15 mg/L sodium
chloride dihydrate, 0.1 g/L sodium pyruvate, 0.12 mg/L
vitamin B12, 0.4 g/L glycerol, and 1.25 g/L Tween 80.
150
Fre ´ de ´ rique Vernel-Pauillac and Catherine Werts
spatiotemporal features about the disease progression in an intact
living host [9, 10].
In the lab, bioluminescent pathogenic and saprophytic strains
of Leptospira have been constructed by transposon insertion of
a cassette expressing the firefly luciferase gene under the control
of a leptospiral promoter, into the genome [9]. The firefly luciferase
is a useful reporter system that can generate visible light through
the oxidation of D-luciferin, its substrate, in the presence of oxygen
and ATP as a source of energy. Of note, the reaction is catalyzed
only in metabolically active bacteria producing both ATP and the
luciferase. Therefore, we can evaluate the spread of live bacteria by
tracking bioluminescence. The light generated is measured in a
light-tight box using new generation optical cameras that enable a
highly sensitive detection of the photons transmitted. Moreover,
bioluminescence imaging based on luciferase doesn’t require excitation, avoiding high background signal. The absolute luciferase
activity can be analyzed and quantified using the In Vivo Imaging
Software (IVIS) and compared with a baseline or control
conditions [11].
With this method, we were able to observe, in live mice, the
spatiotemporal evolution and intensity of an experimental infection
with leptospires [9]. Moreover, this method proves to be reliable
for the determination of critical experimental end points.
2 Materials
All solutions are reconstituted, prepared, or diluted in sterile Dulbecco’s phosphate buffered saline (DPBS) or guaranteed
endotoxin-free reagents. Prepare and store aliquots of working
solutions at À20
C (unless indicated otherwise). Thaw and warm
all solutions at room temperature before administration to animals.
Diligently follow all waste disposal regulations when disposing
soiled sharp objects or other waste materials.
1. Leptospira biflexa sevorar Patoc strain PFLum7 (saprophytic
bioluminescent leptospires).
2. Leptospira interrogans serovar Manilae strain MFLum1 (virulent L. interrogans bioluminescent strain).
3. Bacterial culture medium Ellinghausen-McCullough-JohnsonHarris (EMJH): 1.0 g/L sodium phosphate dibasic, 0.3 g/L
monopotassium phosphate, 1.0 g/L sodium chloride, 0.25 g/
L ammonium chloride, 5 mg/L thiamine. EMJH medium is
supplemented with 10 g/L bovine serum albumin, 4 mg/L
zinc sulfate heptahydrate, 50 mg/L iron sulfate heptahydrate,
15 mg/L hexahydrate magnesium chloride, 15 mg/L sodium
chloride dihydrate, 0.1 g/L sodium pyruvate, 0.12 mg/L
vitamin B12, 0.4 g/L glycerol, and 1.25 g/L Tween 80.
150
Fre ´ de ´ rique Vernel-Pauillac and Catherine Werts