amplification is confirmed by analysis of the melting curves of
the PCR products. Results are validated only when C T values
are under the limit value of 40 cycles and with an acceptable
reproducibility between qPCR replicates (<5% of variation).
12. Statistical analysis is performed using appropriate software
(e.g., GraphPad Prism).
13. The level of expression of each target gene is normalized to the
levels
of
glyceraldehyde-3-phosphate
dehydrogenase
(GAPDH) and β-actin as housekeeping (HKG) genes, using
the qbase+ software and considering gene specific amplification
efficiencies (AEs). AEs are determined by generating standard
curves from serial dilutions of a known concentration of purified DNA specific for the target of each reference gene.
References
1. Costa F et al (2015) Global morbidity and
mortality of leptospirosis: a systematic review.
PLoS Negl Trop Dis 9(9):e0003898
2. Adler B, de la Pena Moctezuma A (2010) Leptospira and leptospirosis. Vet Microbiol 140
(3-4):287–296
3. Haake DA, Levett PN (2015) Leptospirosis in
humans. Curr Top Microbiol Immunol
387:65–97
4. Gomes-Solecki M, Santecchia I, Werts C
(2017) Animal models of leptospirosis: of
mice and hamsters. Front Immunol 8:58
5. Levett PN (2001) Leptospirosis. Clin Microbiol Rev 14(2):296–326
6. Naing C, Reid SA, Aung K (2017) Comparing
antibiotic treatment for leptospirosis using network meta-analysis: a tutorial. BMC Infect Dis
17(1):29
7. Vernel-Pauillac F, Werts C (2018) Recent findings related to immune responses against leptospirosis and novel strategies to prevent
infection. Microbes Infect 20(9–10):578–588
8. Picardeau M (2017) Virulence of the zoonotic
agent of leptospirosis: still terra incognita? Nat
Rev Microbiol 15(5):297–307
9. Werts C (2018) Interaction of Leptospira with
the innate immune system. Curr Top Microbiol Immunol 415:163–187
10. Kobayashi Y (2001) Discovery of the causative
organism of Weil’s disease: historical view. J
Infect Chemother 7(1):10–15
11. Inada R et al (1916) The etiology, mode of
infection, and specific therapy of weil’s disease
(spirochaetosis icterohaemorrhagica). J Exp
Med 23(3):377–402
12. Haake DA (2006) Hamster model of leptospirosis. Curr Protoc Microbiol. Chapter 12:
p. Unit 12E 2
13. Cagliero J, Villanueva SYAM, Matsui M (2018)
Leptospirosis pathophysiology: into the storm
of cytokines. Front Cell Infect Microbiol 8:204
14. Matsui M et al (2011) Gene expression profiles
of immune mediators and histopathological
findings in animal models of leptospirosis:
comparison between susceptible hamsters and
resistant
mice.
Infect
Immun
79
(11):4480–4492
15. Matsui M et al (2017) High level of IL-10
expression in the blood of animal models possibly relates to resistance against leptospirosis.
Cytokine 96:144–151
16. Vernel-Pauillac F, Goarant C (2010) Differential cytokine gene expression according to outcome in a hamster model of leptospirosis. PLoS
Negl Trop Dis 4(1):e582
17. Gattermann R et al (2002) Comparative studies of body mass, body measurements and
organ weights of wild-derived and laboratory
golden hamsters (Mesocricetus auratus). Lab
Anim 36(4):445–454
18. Donovan J, Brown P (2006) Handling and
restraint. Curr Protoc Immunol. Chapter 1:
p. Unit 1 3
19. Donovan J, Brown P (2006) Blood collection.
Curr Protoc Immunol. Chapter 1: p. Unit 1 7
20. Morgan J et al (2002) Outbreak of leptospirosis among triathlon participants and community residents in Springfield, Illinois, 1998.
Clin Infect Dis 34(12):1593–1599
21. Van CT et al (1998) Human leptospirosis in the
Mekong delta, Viet Nam. Trans R Soc Trop
Med Hyg 92(6):625–628
254
Julie Cagliero et al.
the PCR products. Results are validated only when C T values
are under the limit value of 40 cycles and with an acceptable
reproducibility between qPCR replicates (<5% of variation).
12. Statistical analysis is performed using appropriate software
(e.g., GraphPad Prism).
13. The level of expression of each target gene is normalized to the
levels
of
glyceraldehyde-3-phosphate
dehydrogenase
(GAPDH) and β-actin as housekeeping (HKG) genes, using
the qbase+ software and considering gene specific amplification
efficiencies (AEs). AEs are determined by generating standard
curves from serial dilutions of a known concentration of purified DNA specific for the target of each reference gene.
References
1. Costa F et al (2015) Global morbidity and
mortality of leptospirosis: a systematic review.
PLoS Negl Trop Dis 9(9):e0003898
2. Adler B, de la Pena Moctezuma A (2010) Leptospira and leptospirosis. Vet Microbiol 140
(3-4):287–296
3. Haake DA, Levett PN (2015) Leptospirosis in
humans. Curr Top Microbiol Immunol
387:65–97
4. Gomes-Solecki M, Santecchia I, Werts C
(2017) Animal models of leptospirosis: of
mice and hamsters. Front Immunol 8:58
5. Levett PN (2001) Leptospirosis. Clin Microbiol Rev 14(2):296–326
6. Naing C, Reid SA, Aung K (2017) Comparing
antibiotic treatment for leptospirosis using network meta-analysis: a tutorial. BMC Infect Dis
17(1):29
7. Vernel-Pauillac F, Werts C (2018) Recent findings related to immune responses against leptospirosis and novel strategies to prevent
infection. Microbes Infect 20(9–10):578–588
8. Picardeau M (2017) Virulence of the zoonotic
agent of leptospirosis: still terra incognita? Nat
Rev Microbiol 15(5):297–307
9. Werts C (2018) Interaction of Leptospira with
the innate immune system. Curr Top Microbiol Immunol 415:163–187
10. Kobayashi Y (2001) Discovery of the causative
organism of Weil’s disease: historical view. J
Infect Chemother 7(1):10–15
11. Inada R et al (1916) The etiology, mode of
infection, and specific therapy of weil’s disease
(spirochaetosis icterohaemorrhagica). J Exp
Med 23(3):377–402
12. Haake DA (2006) Hamster model of leptospirosis. Curr Protoc Microbiol. Chapter 12:
p. Unit 12E 2
13. Cagliero J, Villanueva SYAM, Matsui M (2018)
Leptospirosis pathophysiology: into the storm
of cytokines. Front Cell Infect Microbiol 8:204
14. Matsui M et al (2011) Gene expression profiles
of immune mediators and histopathological
findings in animal models of leptospirosis:
comparison between susceptible hamsters and
resistant
mice.
Infect
Immun
79
(11):4480–4492
15. Matsui M et al (2017) High level of IL-10
expression in the blood of animal models possibly relates to resistance against leptospirosis.
Cytokine 96:144–151
16. Vernel-Pauillac F, Goarant C (2010) Differential cytokine gene expression according to outcome in a hamster model of leptospirosis. PLoS
Negl Trop Dis 4(1):e582
17. Gattermann R et al (2002) Comparative studies of body mass, body measurements and
organ weights of wild-derived and laboratory
golden hamsters (Mesocricetus auratus). Lab
Anim 36(4):445–454
18. Donovan J, Brown P (2006) Handling and
restraint. Curr Protoc Immunol. Chapter 1:
p. Unit 1 3
19. Donovan J, Brown P (2006) Blood collection.
Curr Protoc Immunol. Chapter 1: p. Unit 1 7
20. Morgan J et al (2002) Outbreak of leptospirosis among triathlon participants and community residents in Springfield, Illinois, 1998.
Clin Infect Dis 34(12):1593–1599
21. Van CT et al (1998) Human leptospirosis in the
Mekong delta, Viet Nam. Trans R Soc Trop
Med Hyg 92(6):625–628
254
Julie Cagliero et al.