needed (see Note 8). The volume that will be injected should
not exceed 1 mL in total for each hamster.
4. Fill 1 mL syringes equipped with 25G needles
(0.5 mm  16 mm) with the volume that will be injected to
animals, whether bacterial dose or control injectate. Do not
lock the needles’ caps back to avoid accidental infection.
5. Manually restrain the animals, exposing the abdomen and
directing the head downward (see Note 5 and [18]).
6. Spread the animals’ abdomen with 70% ethanol for
disinfection.
7. Inject the hamsters intraperitoneally with the appropriate dose
of leptospires or the same volume of control injectate (see Note
5). Needle should be gently inserted into the left or right lower
quadrant, avoiding the abdomen midline, and injections
should preferably be done at once (see Note 9).
8. Following injections, hamsters should be weighted daily and
examined twice per day for the onset of clinical signs and
reaching of endpoint criteria (see Note 10). From that stage
on, the entire content of infected animal cages, as well as their
uninfected counterparts for safety, should be considered as
contaminated since infected animals shed virulent bacteria in
their urine.
3.2 Blood and Tissue
Collection
1. LiVV-infected and uninfected control hamsters are subjected
to a deep anesthesia through CO 2 inhalation. This procedure is
considered as a deep and final anesthesia since animals will not
recover from it. However blood collection is easier when the
heart is still beating. Consequently, we consider that anesthesia
is deep enough when breathing rate is lower than 3–5/min and
animals no longer respond to pain stimuli (usually assessed
through toe pinch).
2. Place deeply unconscious hamsters on their back, and pin them
down on a dissecting table. Animals should be entirely disinfected with 70% ethanol and dissected in a laminar flow
biological safety cabinet (see Note 3). Please note that the
following next steps give storage conditions for tissue and
blood samples according to the use they are destined to, especially for Leptospira burden quantification and cytokine gene
expression, and are summed up in Table 1.
3. Collect blood through cardiac puncture [19] using 1 mL syringes with 25G needles (see Note 9), and immediately process
and store:
(a) In EDTA collector tubes that will be stored at 4
C until
DNA extraction.
248
Julie Cagliero et al.
not exceed 1 mL in total for each hamster.
4. Fill 1 mL syringes equipped with 25G needles
(0.5 mm  16 mm) with the volume that will be injected to
animals, whether bacterial dose or control injectate. Do not
lock the needles’ caps back to avoid accidental infection.
5. Manually restrain the animals, exposing the abdomen and
directing the head downward (see Note 5 and [18]).
6. Spread the animals’ abdomen with 70% ethanol for
disinfection.
7. Inject the hamsters intraperitoneally with the appropriate dose
of leptospires or the same volume of control injectate (see Note
5). Needle should be gently inserted into the left or right lower
quadrant, avoiding the abdomen midline, and injections
should preferably be done at once (see Note 9).
8. Following injections, hamsters should be weighted daily and
examined twice per day for the onset of clinical signs and
reaching of endpoint criteria (see Note 10). From that stage
on, the entire content of infected animal cages, as well as their
uninfected counterparts for safety, should be considered as
contaminated since infected animals shed virulent bacteria in
their urine.
3.2 Blood and Tissue
Collection
1. LiVV-infected and uninfected control hamsters are subjected
to a deep anesthesia through CO 2 inhalation. This procedure is
considered as a deep and final anesthesia since animals will not
recover from it. However blood collection is easier when the
heart is still beating. Consequently, we consider that anesthesia
is deep enough when breathing rate is lower than 3–5/min and
animals no longer respond to pain stimuli (usually assessed
through toe pinch).
2. Place deeply unconscious hamsters on their back, and pin them
down on a dissecting table. Animals should be entirely disinfected with 70% ethanol and dissected in a laminar flow
biological safety cabinet (see Note 3). Please note that the
following next steps give storage conditions for tissue and
blood samples according to the use they are destined to, especially for Leptospira burden quantification and cytokine gene
expression, and are summed up in Table 1.
3. Collect blood through cardiac puncture [19] using 1 mL syringes with 25G needles (see Note 9), and immediately process
and store:
(a) In EDTA collector tubes that will be stored at 4
C until
DNA extraction.
248
Julie Cagliero et al.