1 Â 10
3 to 10
4 bacteria/ml (50–500 times ED 50 ) (see Note
11).
3. On day 35 (1 week after the last immunization), challenge
hamsters intraperitoneally with 1 ml of Leptospira suspension
using an insulin syringe U-100 with a 26 G Â ½
00 needle (see
Note 12).
4. Weigh animals daily and monitor them twice a day after challenge for endpoint criteria, i.e., loss of appetite, gait or breathing difficulty, prostration, ruffled fur, or weight loss of !10% of
the animal’s maximum weight.
5. Animals that meet endpoint criteria are euthanized by isoflurane inhalation followed by immediate thoracotomy.
6. Twenty-eight days post-challenge, euthanize any hamsters that
survived the infection using the same protocol.
7. Draw survival curves for each group (Fig. 1) and analyze statistical differences among groups using the Fisher test.
8. Collect the blood by postmortem cardiac puncture using 3 ml
syringe with a 25 G Â 5/8
00 needle. Transfer the blood into a
5 ml EDTA tube and mix well by inverting the tube five to six
times. Immediately extract the DNA as described in Subheading 3.6, step 2.
9. Collect one kidney from each animal and inoculate in semisolid
EMJH for culture as described in Subheading 3.5. Collect the
other kidney and liver in individual dry tubes for quantification
of tissue colonization by qPCR as described in Subheading 3.6.
3.4 Evaluation
of the IgG Response by
Enzyme-Linked
Immunosorbent Assay
(ELISA)
Allow all ELISA reagents to reach room temperature before use.
1. On the day of collection (i.e., before immunization and at day
4, 11, 18, 25, 32), prepare serum from the blood collected via
retro-orbital bleed. Incubate tube containing the blood at
room temperature for 30–45 min to allow clotting. Spin the
blood at 2000 Â g for 15 min in a refrigerated centrifuge.
Aspirate the supernatant (serum) and transfer it into a clean
microcentrifuge tube. Keep serum at À80
C until use (see
Note 13).
2. Add 100 μl of PBS containing 1 μg purified protein into a
microtiter plate well, in triplicate (3 wells for each sample to
be tested). Cover the plate and incubate overnight at 4
C.
3. Aspirate or decant solution from wells and discard the liquid.
Wash wells three times with 200 μl of PBS using a multichannel
pipettor or an automated 96-well plate washer (see Note 14).
4. Add 200 μl of protein-free blocking buffer (PFB) into the
wells. Incubate for 1 h at RT.
Leptospirosis Vaccine Studies in Hamsters
263
3 to 10
4 bacteria/ml (50–500 times ED 50 ) (see Note
11).
3. On day 35 (1 week after the last immunization), challenge
hamsters intraperitoneally with 1 ml of Leptospira suspension
using an insulin syringe U-100 with a 26 G Â ½
00 needle (see
Note 12).
4. Weigh animals daily and monitor them twice a day after challenge for endpoint criteria, i.e., loss of appetite, gait or breathing difficulty, prostration, ruffled fur, or weight loss of !10% of
the animal’s maximum weight.
5. Animals that meet endpoint criteria are euthanized by isoflurane inhalation followed by immediate thoracotomy.
6. Twenty-eight days post-challenge, euthanize any hamsters that
survived the infection using the same protocol.
7. Draw survival curves for each group (Fig. 1) and analyze statistical differences among groups using the Fisher test.
8. Collect the blood by postmortem cardiac puncture using 3 ml
syringe with a 25 G Â 5/8
00 needle. Transfer the blood into a
5 ml EDTA tube and mix well by inverting the tube five to six
times. Immediately extract the DNA as described in Subheading 3.6, step 2.
9. Collect one kidney from each animal and inoculate in semisolid
EMJH for culture as described in Subheading 3.5. Collect the
other kidney and liver in individual dry tubes for quantification
of tissue colonization by qPCR as described in Subheading 3.6.
3.4 Evaluation
of the IgG Response by
Enzyme-Linked
Immunosorbent Assay
(ELISA)
Allow all ELISA reagents to reach room temperature before use.
1. On the day of collection (i.e., before immunization and at day
4, 11, 18, 25, 32), prepare serum from the blood collected via
retro-orbital bleed. Incubate tube containing the blood at
room temperature for 30–45 min to allow clotting. Spin the
blood at 2000 Â g for 15 min in a refrigerated centrifuge.
Aspirate the supernatant (serum) and transfer it into a clean
microcentrifuge tube. Keep serum at À80
C until use (see
Note 13).
2. Add 100 μl of PBS containing 1 μg purified protein into a
microtiter plate well, in triplicate (3 wells for each sample to
be tested). Cover the plate and incubate overnight at 4
C.
3. Aspirate or decant solution from wells and discard the liquid.
Wash wells three times with 200 μl of PBS using a multichannel
pipettor or an automated 96-well plate washer (see Note 14).
4. Add 200 μl of protein-free blocking buffer (PFB) into the
wells. Incubate for 1 h at RT.
Leptospirosis Vaccine Studies in Hamsters
263