2. cDNA from previous steps are analyzed by qPCR assay (see
Subheading 3.3.3 and Note 11). Primer sequences for the
main immune mediators targeted in our experiments are
provided in Table 2.
3. Gene expression results are expressed as relative normalized
target gene expression (e.g., of immune mediators). Values
can be calculated using the ratio of the level of expression in
infected animals to the level of expression in uninfected animals
used as calibrators (see Notes 12 and 13).
4 Notes
1. All animal experiments are prepared and conducted according
to the guidelines of the Animal Care and Use Committees of
the Institut Pasteur, follow ethical and regulatory standards of
the European Union Legislation governing the care and use of
laboratory animals (Directive 2010/63/EU), and are performed by an authorized trained person.
2. Although it was suggested that men are more prone to develop
severe forms of leptospirosis and be hospitalized, studies comparing data from male and female patients with the same level
of exposure show no difference in the severity of the illness
[20–22]. However, the impact of sex on the severity of leptospirosis remains an open question, and, in two recent studies
comparing experimentally infected male and female hamsters,
the authors conclude that males are more susceptible to infection than females [23, 24]. We thus recommend matching of
animals by gender.
3. Pathogenic Leptospira species are Biosafety Level 2 (BSL-2)
pathogens, and L. interrogans is described as pathogenic for
humans. Ensure that all experiments involving viable bacteria
are completed in a containment level 2 laboratory and that
appropriate risk assessments are in place. Cultures and infected
materials must be handled in a laminar flow biological safety
cabinet. Any material that has been in contact with infected
animals is considered as contaminated and should be disinfected with bleach or autoclaved for appropriate waste disposal.
4. Alternately, a commercial enrichment additive, Difco™ Leptospira Enrichment EMJH (Becton Dickenson), can be purchased to supplement the EMJH medium.
5. Personnel involved in the procedure should be trained for
proper hamsters handling and restraint, to avoid injury
and/or contamination. Indeed, hamsters are generally calm
and docile animals, but they may bite or scratch if they are
stressed or surprised. For handling, always approach hamsters
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Subheading 3.3.3 and Note 11). Primer sequences for the
main immune mediators targeted in our experiments are
provided in Table 2.
3. Gene expression results are expressed as relative normalized
target gene expression (e.g., of immune mediators). Values
can be calculated using the ratio of the level of expression in
infected animals to the level of expression in uninfected animals
used as calibrators (see Notes 12 and 13).
4 Notes
1. All animal experiments are prepared and conducted according
to the guidelines of the Animal Care and Use Committees of
the Institut Pasteur, follow ethical and regulatory standards of
the European Union Legislation governing the care and use of
laboratory animals (Directive 2010/63/EU), and are performed by an authorized trained person.
2. Although it was suggested that men are more prone to develop
severe forms of leptospirosis and be hospitalized, studies comparing data from male and female patients with the same level
of exposure show no difference in the severity of the illness
[20–22]. However, the impact of sex on the severity of leptospirosis remains an open question, and, in two recent studies
comparing experimentally infected male and female hamsters,
the authors conclude that males are more susceptible to infection than females [23, 24]. We thus recommend matching of
animals by gender.
3. Pathogenic Leptospira species are Biosafety Level 2 (BSL-2)
pathogens, and L. interrogans is described as pathogenic for
humans. Ensure that all experiments involving viable bacteria
are completed in a containment level 2 laboratory and that
appropriate risk assessments are in place. Cultures and infected
materials must be handled in a laminar flow biological safety
cabinet. Any material that has been in contact with infected
animals is considered as contaminated and should be disinfected with bleach or autoclaved for appropriate waste disposal.
4. Alternately, a commercial enrichment additive, Difco™ Leptospira Enrichment EMJH (Becton Dickenson), can be purchased to supplement the EMJH medium.
5. Personnel involved in the procedure should be trained for
proper hamsters handling and restraint, to avoid injury
and/or contamination. Indeed, hamsters are generally calm
and docile animals, but they may bite or scratch if they are
stressed or surprised. For handling, always approach hamsters
252
Julie Cagliero et al.