C4 and C2 (Classical and Lectin Pathways, [8]), and C5-C9
proteins (Terminal Pathway, [9]). Proteases present in the
supernatants of nonpathogenic L. biflexa serovar Andamana
strain CH11 or L. biflexa serovar Patoc strain Patoc I do not
have a significant proteolytic activity on these substrates.
4. Inhibition of Complement activation by proteases secreted by
pathogenic Leptospira—The proteolytic activity of secreted proteases from pathogenic Leptospira cultures cleaves several Complement proteins either purified or present in NHS, affecting
global activation of the Alternative, the Classical, and the Lectin Pathways. Proteases present in the supernatants of nonpathogenic Leptospira cultures have no significant effect on
the cleavage of Complement proteins [8], and they do not
interfere with Complement activation. The activation of each
Complement Pathway is evaluated independently, according to
Roos et al. [12] and Fraga et al. [8].
2 Materials
2.1 Serum
Susceptibility Assay
1. Leptospira cultures. Cultivate in modified EllinghausenMcCullough-Johnson-Harris (EMJH) medium. Alternatively,
leptospires may also be cultivated in EMJH medium supplemented with Difco™ Leptospira Enrichment EMJH (Becton
Dickinson) (see Note 1). Use Leptospira cultures in mid to late
logarithmic phase.
2. Modified EMJH medium: Difco™ Leptospira Medium Base
EMJH (Becton Dickinson) supplemented with 10% normal
rabbit serum previously inactivated for 30 min at 56
C, containing 0.015% L-asparagine, 0.001% sodium pyruvate, 0.001%
calcium chloride, 0.001% magnesium chloride, 0.03% peptone,
and 0.03% meat extract.
3. Phosphate-buffered saline (PBS): 0.01 M Na 2 HPO 4 ,
0.0018 M KH 2 PO 4 , pH 7.4, 0.137 M NaCl, 0.0027 M KCl.
4. A pool of normal human serum (NHS): Serum samples can be
used either fresh or thawed from aliquots stored at À80
C (see
Note 2). Heat-inactivated NHS (HI-NHS) is obtained by
incubating NHS in a water bath or in a dry block incubator
for 30 min at 56
C.
5. Refrigerated centrifuge with appropriate adaptors for 50 mL
tubes or for microfuge tubes.
6. Microscope with a dark-field condenser.
7. Bacterial counting chamber [cell depth: 0.02 mm (1/50 mm)].
8. Water bath/dry block incubator.
Complement Resistance Assays
189
proteins (Terminal Pathway, [9]). Proteases present in the
supernatants of nonpathogenic L. biflexa serovar Andamana
strain CH11 or L. biflexa serovar Patoc strain Patoc I do not
have a significant proteolytic activity on these substrates.
4. Inhibition of Complement activation by proteases secreted by
pathogenic Leptospira—The proteolytic activity of secreted proteases from pathogenic Leptospira cultures cleaves several Complement proteins either purified or present in NHS, affecting
global activation of the Alternative, the Classical, and the Lectin Pathways. Proteases present in the supernatants of nonpathogenic Leptospira cultures have no significant effect on
the cleavage of Complement proteins [8], and they do not
interfere with Complement activation. The activation of each
Complement Pathway is evaluated independently, according to
Roos et al. [12] and Fraga et al. [8].
2 Materials
2.1 Serum
Susceptibility Assay
1. Leptospira cultures. Cultivate in modified EllinghausenMcCullough-Johnson-Harris (EMJH) medium. Alternatively,
leptospires may also be cultivated in EMJH medium supplemented with Difco™ Leptospira Enrichment EMJH (Becton
Dickinson) (see Note 1). Use Leptospira cultures in mid to late
logarithmic phase.
2. Modified EMJH medium: Difco™ Leptospira Medium Base
EMJH (Becton Dickinson) supplemented with 10% normal
rabbit serum previously inactivated for 30 min at 56
C, containing 0.015% L-asparagine, 0.001% sodium pyruvate, 0.001%
calcium chloride, 0.001% magnesium chloride, 0.03% peptone,
and 0.03% meat extract.
3. Phosphate-buffered saline (PBS): 0.01 M Na 2 HPO 4 ,
0.0018 M KH 2 PO 4 , pH 7.4, 0.137 M NaCl, 0.0027 M KCl.
4. A pool of normal human serum (NHS): Serum samples can be
used either fresh or thawed from aliquots stored at À80
C (see
Note 2). Heat-inactivated NHS (HI-NHS) is obtained by
incubating NHS in a water bath or in a dry block incubator
for 30 min at 56
C.
5. Refrigerated centrifuge with appropriate adaptors for 50 mL
tubes or for microfuge tubes.
6. Microscope with a dark-field condenser.
7. Bacterial counting chamber [cell depth: 0.02 mm (1/50 mm)].
8. Water bath/dry block incubator.
Complement Resistance Assays
189