3 Methods
All steps involving Leptospira must be performed in a biosafety
cabinet under BSL-2 containment. All animal experiments must
be approved by the local Institutional Animal Care and Use Committee and performed under Animal Biosafety Level 2 (ABSL-2)
conditions.
3.1 Vaccine
Preparation
1. Draw up 2 ml of CFA (or IFA) into a 5 ml glass syringe with a
19 G needle (see Note 5).
2. In another 5 ml glass syringe, draw up 2 ml of purified protein
(0.1–1 mg/ml). For the negative control group, use 2 ml
of PBS.
3. Remove the 19 G needles and connect the two syringes to a
plastic three-way Luer lock.
4. Mix the CFA (or IFA) and the purified protein by plunging the
solutions into opposite syringe 15–20 times to create a white
emulsion (see Note 6). For the control group, follow the same
protocol with PBS.
5. Transfer all of the protein-adjuvant or PBS-adjuvant emulsion
into one syringe and remove the stopcock. Remove the air
bubbles and transfer the emulsion to a clean glass tube.
3.2 Hamster
Immunization
1. Three days prior to the first immunization, collect the blood
from hamsters by retro-orbital bleed using a capillary tube (see
Note 7). Immediately transfer the blood to a microcentrifuge
tube. Process the blood as described in Subheading 3.4.
2. On day 0, inject hamsters subcutaneously with 200 μl of
antigen-CFA emulsion (10–100 μg protein) using a 1 ml
syringe with a 25 G needle (see Note 8). For the negative
control group, use 200 μl of PBS or PBS-CFA emulsion.
3. On day 14 and 28, inject hamsters subcutaneously with 200 μl
of antigen-IFA emulsion (10–100 μg protein) using 1 ml
syringe with a 25 G needle. For the negative control groups,
inject 200 μl of PBS or PBS-IFA emulsion.
4. Starting on day 4 until day 32, collect the blood weekly from
hamsters by retro-orbital bleed. Transfer the blood to a microcentrifuge tube. Process the blood as described in Subheading 3.4.
3.3 Challenge
and Evaluation
of the Protection
1. Determine the culture density of the Leptospira challenge strain
(see Note 9) by dark-field microscopy using a Petroff-Hausser
chamber (see Note 10).
2. Prepare appropriate dilution of the bacteria in liquid EMJH
based on the ED 50 . For L. interrogans serovar Copenhageni
strain Fiocruz L1-130, prepare a suspension with a density of
262
Karen V. Evangelista and Kristel Lourdault
All steps involving Leptospira must be performed in a biosafety
cabinet under BSL-2 containment. All animal experiments must
be approved by the local Institutional Animal Care and Use Committee and performed under Animal Biosafety Level 2 (ABSL-2)
conditions.
3.1 Vaccine
Preparation
1. Draw up 2 ml of CFA (or IFA) into a 5 ml glass syringe with a
19 G needle (see Note 5).
2. In another 5 ml glass syringe, draw up 2 ml of purified protein
(0.1–1 mg/ml). For the negative control group, use 2 ml
of PBS.
3. Remove the 19 G needles and connect the two syringes to a
plastic three-way Luer lock.
4. Mix the CFA (or IFA) and the purified protein by plunging the
solutions into opposite syringe 15–20 times to create a white
emulsion (see Note 6). For the control group, follow the same
protocol with PBS.
5. Transfer all of the protein-adjuvant or PBS-adjuvant emulsion
into one syringe and remove the stopcock. Remove the air
bubbles and transfer the emulsion to a clean glass tube.
3.2 Hamster
Immunization
1. Three days prior to the first immunization, collect the blood
from hamsters by retro-orbital bleed using a capillary tube (see
Note 7). Immediately transfer the blood to a microcentrifuge
tube. Process the blood as described in Subheading 3.4.
2. On day 0, inject hamsters subcutaneously with 200 μl of
antigen-CFA emulsion (10–100 μg protein) using a 1 ml
syringe with a 25 G needle (see Note 8). For the negative
control group, use 200 μl of PBS or PBS-CFA emulsion.
3. On day 14 and 28, inject hamsters subcutaneously with 200 μl
of antigen-IFA emulsion (10–100 μg protein) using 1 ml
syringe with a 25 G needle. For the negative control groups,
inject 200 μl of PBS or PBS-IFA emulsion.
4. Starting on day 4 until day 32, collect the blood weekly from
hamsters by retro-orbital bleed. Transfer the blood to a microcentrifuge tube. Process the blood as described in Subheading 3.4.
3.3 Challenge
and Evaluation
of the Protection
1. Determine the culture density of the Leptospira challenge strain
(see Note 9) by dark-field microscopy using a Petroff-Hausser
chamber (see Note 10).
2. Prepare appropriate dilution of the bacteria in liquid EMJH
based on the ED 50 . For L. interrogans serovar Copenhageni
strain Fiocruz L1-130, prepare a suspension with a density of
262
Karen V. Evangelista and Kristel Lourdault