from behind and place a hand palm down over the animal,
preferably thumb over the neck. During leptospires injection
procedures, make sure hamsters are secure while their abdomen
exposed by grasping enough loose skin over the neck and back.
Please see [25] for additional information on hamsters handling
and restraint.
6. The chamber is used according to manufacturer’s instructions.
After completing the count, remove the reusable cover glass,
and clean the counting chamber and the cover glass with
ethanol. Dry the counting chamber with a soft cloth or wipe.
7. Previous results established that a dose of 10
8 leptospires from
the LiVV strain per intraperitoneally infected hamster was a
relevant and reproducible model of median lethal dose (LD50)
[16]. However, infectious dose may vary a lot depending on
the bacterial strain and the infection route that are being used
for experimental procedures.
8. Alternatively, leptospires can be centrifuged at 3400 Â g for
10 min and resuspended in PBS. In that case, PBS alone will be
injected in control non-infected animals.
9. Following use, syringes should be promptly disposed in the
sharps container. Needles should not be bent, sheared, replaced
in the needle sheath or guard, or removed from the syringe
following use. Sharps containers must be disposed as hazardous
waste and properly treated prior to final disposal.
10. Examination of challenged hamsters relies on the scoring of
clinical signs of illness, mostly weight loss, low alertness, ruffled
head and fur, hunched posture, prostration, and small eyes. In
case of lethal challenges, death can closely follow the onset of
clinical signs; thus endpoint criteria should be carefully defined
and should include anorexia, dehydration, and weight loss of
>10% of initial weight (see Note 1 and [26]). However these
endpoint criteria largely depend on the infecting Leptospira
strain. In the case of an infection with the LiVV strain, lethally
infected animals usually die at day 4 post-infection, without any
clear clinical signs except for prostration that can be observed a
few hours before death. In any case, moribund animals or
survivors checked for sub-lethal infections are euthanized
through CO 2 asphyxia.
11. qPCR assays are performed on a LightCycler 480 real-time
PCR system using the LightCycler FastStart DNA Master
SYBR Green I and the LightCycler 480 software (version
1.5.0) according to manufacturer’s instructions and as previously described [14]. Acquisition of fluorescence for threshold
cycle (C T or Crossing Point C p ) calculation is processed during
elongation step of the qPCR, each qPCR reaction being performed twice (for experimental replicates). The specificity of
Hamsters as a Model to Study Leptospirosis
253
preferably thumb over the neck. During leptospires injection
procedures, make sure hamsters are secure while their abdomen
exposed by grasping enough loose skin over the neck and back.
Please see [25] for additional information on hamsters handling
and restraint.
6. The chamber is used according to manufacturer’s instructions.
After completing the count, remove the reusable cover glass,
and clean the counting chamber and the cover glass with
ethanol. Dry the counting chamber with a soft cloth or wipe.
7. Previous results established that a dose of 10
8 leptospires from
the LiVV strain per intraperitoneally infected hamster was a
relevant and reproducible model of median lethal dose (LD50)
[16]. However, infectious dose may vary a lot depending on
the bacterial strain and the infection route that are being used
for experimental procedures.
8. Alternatively, leptospires can be centrifuged at 3400 Â g for
10 min and resuspended in PBS. In that case, PBS alone will be
injected in control non-infected animals.
9. Following use, syringes should be promptly disposed in the
sharps container. Needles should not be bent, sheared, replaced
in the needle sheath or guard, or removed from the syringe
following use. Sharps containers must be disposed as hazardous
waste and properly treated prior to final disposal.
10. Examination of challenged hamsters relies on the scoring of
clinical signs of illness, mostly weight loss, low alertness, ruffled
head and fur, hunched posture, prostration, and small eyes. In
case of lethal challenges, death can closely follow the onset of
clinical signs; thus endpoint criteria should be carefully defined
and should include anorexia, dehydration, and weight loss of
>10% of initial weight (see Note 1 and [26]). However these
endpoint criteria largely depend on the infecting Leptospira
strain. In the case of an infection with the LiVV strain, lethally
infected animals usually die at day 4 post-infection, without any
clear clinical signs except for prostration that can be observed a
few hours before death. In any case, moribund animals or
survivors checked for sub-lethal infections are euthanized
through CO 2 asphyxia.
11. qPCR assays are performed on a LightCycler 480 real-time
PCR system using the LightCycler FastStart DNA Master
SYBR Green I and the LightCycler 480 software (version
1.5.0) according to manufacturer’s instructions and as previously described [14]. Acquisition of fluorescence for threshold
cycle (C T or Crossing Point C p ) calculation is processed during
elongation step of the qPCR, each qPCR reaction being performed twice (for experimental replicates). The specificity of
Hamsters as a Model to Study Leptospirosis
253