7. 19 G needles.
8. Pipettors and sterile barrier pipette tips.
2.2 Animals
1. Female golden Syrian hamsters (Mesocricetus auratus), 4 weeks
old (see Note 4).
2. Precision balance.
2.3 Bacterial Strain
1. Leptospira culture for challenge: low in vitro passage pathogenic strain. For example, L. interrogans serovar Copenhageni
strain Fiocruz L1-130 [12].
2. Ellinghausen-McCullough-Johnson-Harris (EMJH) basal
medium [13, 14]: Use sterile double-deionized water in all
media and media component preparation. Dissolve 1 g of
Na 2 PO 4 , 0.3 g of KH 2 PO 4 , and 1 g of NaCl in 900 ml sterile
water. Add the following stock solutions prepared in sterile
water: 1 ml of glycerol (stock, 10 ml/100 ml), 1 ml of
NH 4 Cl (stock, 25 g/100 ml), and 1 ml of thiamine (stock,
0.5 g/100 ml). Adjust pH to 7.4 using NaOH then bring
volume to 1 l with sterile water. Sterilize by autoclaving.
3. EMJH supplement: Dissolve 10 g of bovine serum albumin
(BSA) in 50-ml sterile water with slow and constant stirring.
Once the BSA is dissolved, add the following stock solutions
prepared in sterile water: 1 ml of calcium chloride (stock, 1 g
CaCl 2 ·2H 2 O/100 ml), 1 ml of magnesium chloride (stock, 1 g
MgCl 2 ·2H 2 O/100 ml), 1 ml of zinc sulfate (stock, 0.4 g of
ZnSO 4 ·7H 2 O/100 ml), 0.1 ml of copper sulfate (stock, 0.3 g
CuSO 4 ·5H 2 O/100 ml), 10 ml of ferrous sulfate (stock, 0.5 g
FeSO 4 ·7H 2 O/100 ml), 1 ml of vitamin B 12 (stock, 0.02 g/
100 ml), and 12.5 ml of Tween 80 (stock, 10 g/100 ml).
Adjust pH to 7.4 using NaOH and then bring volume to
100 ml with sterile water. Sterilize by filtration through a
0.22 μm filter.
4. EMJH liquid medium: Add 100 ml of EMJH supplement to
890 ml of EMJH basal medium. Add 1 g of lactalbumin
hydrolysate, 1 mg of superoxide dismutase (SOD), 40 mg of
sodium pyruvate, and 10 ml of heat-inactivated (56
C for
30 min) rabbit serum. Adjust pH to 7.4 if necessary and filter
sterilize (0.22 μm filter). Aliquot into smaller volumes and
store at 4
C.
2.4 Immunization
and Challenge
1. 1 ml syringes with 25 G needles.
2. 1 ml insulin syringes U-100 with 26 G x 1/2
00 needles.
3. 1.5 ml microcentrifuge tubes.
4. Refrigerated centrifuge.
260
Karen V. Evangelista and Kristel Lourdault
8. Pipettors and sterile barrier pipette tips.
2.2 Animals
1. Female golden Syrian hamsters (Mesocricetus auratus), 4 weeks
old (see Note 4).
2. Precision balance.
2.3 Bacterial Strain
1. Leptospira culture for challenge: low in vitro passage pathogenic strain. For example, L. interrogans serovar Copenhageni
strain Fiocruz L1-130 [12].
2. Ellinghausen-McCullough-Johnson-Harris (EMJH) basal
medium [13, 14]: Use sterile double-deionized water in all
media and media component preparation. Dissolve 1 g of
Na 2 PO 4 , 0.3 g of KH 2 PO 4 , and 1 g of NaCl in 900 ml sterile
water. Add the following stock solutions prepared in sterile
water: 1 ml of glycerol (stock, 10 ml/100 ml), 1 ml of
NH 4 Cl (stock, 25 g/100 ml), and 1 ml of thiamine (stock,
0.5 g/100 ml). Adjust pH to 7.4 using NaOH then bring
volume to 1 l with sterile water. Sterilize by autoclaving.
3. EMJH supplement: Dissolve 10 g of bovine serum albumin
(BSA) in 50-ml sterile water with slow and constant stirring.
Once the BSA is dissolved, add the following stock solutions
prepared in sterile water: 1 ml of calcium chloride (stock, 1 g
CaCl 2 ·2H 2 O/100 ml), 1 ml of magnesium chloride (stock, 1 g
MgCl 2 ·2H 2 O/100 ml), 1 ml of zinc sulfate (stock, 0.4 g of
ZnSO 4 ·7H 2 O/100 ml), 0.1 ml of copper sulfate (stock, 0.3 g
CuSO 4 ·5H 2 O/100 ml), 10 ml of ferrous sulfate (stock, 0.5 g
FeSO 4 ·7H 2 O/100 ml), 1 ml of vitamin B 12 (stock, 0.02 g/
100 ml), and 12.5 ml of Tween 80 (stock, 10 g/100 ml).
Adjust pH to 7.4 using NaOH and then bring volume to
100 ml with sterile water. Sterilize by filtration through a
0.22 μm filter.
4. EMJH liquid medium: Add 100 ml of EMJH supplement to
890 ml of EMJH basal medium. Add 1 g of lactalbumin
hydrolysate, 1 mg of superoxide dismutase (SOD), 40 mg of
sodium pyruvate, and 10 ml of heat-inactivated (56
C for
30 min) rabbit serum. Adjust pH to 7.4 if necessary and filter
sterilize (0.22 μm filter). Aliquot into smaller volumes and
store at 4
C.
2.4 Immunization
and Challenge
1. 1 ml syringes with 25 G needles.
2. 1 ml insulin syringes U-100 with 26 G x 1/2
00 needles.
3. 1.5 ml microcentrifuge tubes.
4. Refrigerated centrifuge.
260
Karen V. Evangelista and Kristel Lourdault