2.5 Quantification of
Bacterial Burden
1. Blood (stabilized in EDTA collector tubes) and organs
(prepared in MagNA Lyser Green Beads tubes) collected
from LiVV-infected and control hamsters.
2. MagNA Lyser Instrument.
3. QIAamp Blood Kit (Qiagen).
4. QIAamp DNA Mini Kit (Qiagen).
5. LightCycler FastStart DNA Master SYBR Green I (Roche Life
Sciences).
6. Spectrophotometer for DNA and RNA quantification.
2.6 Quantification of
Cytokine Gene
Expression
1. Blood (stored in PAXgene RNA tubes) and organs (stored in
RNAlater
® reagent) from LiVV-infected and control hamsters.
2. High Pure tissue RNA Isolation Kit (Roche Life Science).
3. Transcriptor First Strand cDNA Synthesis Kit (Roche Life
Science).
4. TURBO™ DNase (Thermo Fisher Scientific).
5. LightCycler FastStart DNA Master SYBR Green I (Roche Life
Science).
6. Spectrophotometer for DNA and RNA quantification.
3 Methods
Carry out experiments at room temperature unless otherwise
specified.
3.1 Experimental
Infections
1. At 24 h prior to animal infections, house hamsters in individual
cages for proper acclimatization (see Note 5). If animals were
purchased from external suppliers, please consider longer acclimation period of time (>48 h) before experimental infection.
All cages must be fitted with an individual ventilation system or
kept in a ventilated storage unit, in a procedure room, separated from the animal maintenance facility. All animals should
be carefully examined and weighted prior to Leptospira
challenge.
2. Evaluate the density of the LiVV culture by dark-field microscopy and using a Petroff-Hausser or similar counting chamber
(see Note 6).
3. Prepare the appropriate dose of leptospires that will be used for
the animal infections. From previous published results, we
know, for instance, that the LD50 is 10
8 leptospires per animal
when using the hamster model and the LiVV strain (see Note
7). EMJH liquid medium is used to dilute the initial culture as
well as control injectate for uninfected animals unless otherwise
Hamsters as a Model to Study Leptospirosis
247
Bacterial Burden
1. Blood (stabilized in EDTA collector tubes) and organs
(prepared in MagNA Lyser Green Beads tubes) collected
from LiVV-infected and control hamsters.
2. MagNA Lyser Instrument.
3. QIAamp Blood Kit (Qiagen).
4. QIAamp DNA Mini Kit (Qiagen).
5. LightCycler FastStart DNA Master SYBR Green I (Roche Life
Sciences).
6. Spectrophotometer for DNA and RNA quantification.
2.6 Quantification of
Cytokine Gene
Expression
1. Blood (stored in PAXgene RNA tubes) and organs (stored in
RNAlater
® reagent) from LiVV-infected and control hamsters.
2. High Pure tissue RNA Isolation Kit (Roche Life Science).
3. Transcriptor First Strand cDNA Synthesis Kit (Roche Life
Science).
4. TURBO™ DNase (Thermo Fisher Scientific).
5. LightCycler FastStart DNA Master SYBR Green I (Roche Life
Science).
6. Spectrophotometer for DNA and RNA quantification.
3 Methods
Carry out experiments at room temperature unless otherwise
specified.
3.1 Experimental
Infections
1. At 24 h prior to animal infections, house hamsters in individual
cages for proper acclimatization (see Note 5). If animals were
purchased from external suppliers, please consider longer acclimation period of time (>48 h) before experimental infection.
All cages must be fitted with an individual ventilation system or
kept in a ventilated storage unit, in a procedure room, separated from the animal maintenance facility. All animals should
be carefully examined and weighted prior to Leptospira
challenge.
2. Evaluate the density of the LiVV culture by dark-field microscopy and using a Petroff-Hausser or similar counting chamber
(see Note 6).
3. Prepare the appropriate dose of leptospires that will be used for
the animal infections. From previous published results, we
know, for instance, that the LD50 is 10
8 leptospires per animal
when using the hamster model and the LiVV strain (see Note
7). EMJH liquid medium is used to dilute the initial culture as
well as control injectate for uninfected animals unless otherwise
Hamsters as a Model to Study Leptospirosis
247