Leptospirosis research: fast, easy and reliable enumeration of mobile leptospires

Schreier, Stefan; Triampo, Wannapong; Doungchawee, Galayanee; Triampo, Darapond; Chadsuthi, Sudarat

doi:10.4067/s0716-97602009000100001

Cited by 18 publications

(23 citation statements)

References 13 publications

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“…Briefly, freshly cultured L. interrogans strain Lai was harvested by a 17 200 g centrifugation step at 15°C for 15 min. After washing with autoclaved 0.01 M phosphate-buffered saline (PBS, pH 7.4), the leptospiral precipitate was suspended in antibiotic-free 2.5% FCS RPMI-1640 liquid medium for counting leptospires under a dark-field microscope with a Petroff-Hausser counting chamber (Fisher Scientific, USA) (Schreier et al, 2009). THP-1 or J774A.1 cells (1 ¥ 10 5 per well) were seeded in 12-well culture plates (Corning, USA) containing a 12 ¥ 12 mm coverslip per well for incubation overnight at 37°C.…”

Section: Detection Of Intracellular Rosmentioning

confidence: 99%

p53 signalling controls cell cycle arrest and caspase-independent apoptosis in macrophages infected with pathogenicLeptospiraspecies

Ojcius

Sun

et al. 2013

Cell Microbiol

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SummaryPathogenic Leptospira species, the causative agents of leptospirosis, have been shown to induce macrophage apoptosis through caspaseindependent, mitochondrion-related apoptosis inducing factor (AIF) and endonuclease G (EndoG), but the signalling pathway leading to AIF/EndoGbased macrophage apoptosis remains unknown. Here we show that infection of Leptospira interrogans caused a rapid increase in reactive oxygen species (ROS), DNA damage, and intranuclear foci of 53BP1 and phosphorylation of H2AX (two DNA damage indicators) in wild-type p53-containing mouse macrophages and p53-deficient human macrophages. Most leptospire-infected cells stayed at the G 1 phase, whereas depletion or inhibition of p53 caused a decrease of the G1-phase cells and the early apoptotic ratios. Infection with spirochaetes stimulated a persistent activation of p53 and an early activation of Akt through phosphorylation. The intranuclear translocation of p53, increased expression of p53-dependent p21 Cip1/WAF1

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Section: Detection Of Intracellular Rosmentioning

confidence: 99%

p53 signalling controls cell cycle arrest and caspase-independent apoptosis in macrophages infected with pathogenicLeptospiraspecies

Ojcius

Sun

et al. 2013

Cell Microbiol

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“…Leptospires were cultured in medium containing 10% Leptospira enrichment Elinghausen-McCullough-Johnson-Harris (EMJH) medium and 90% Leptospira medium base EMJM medium (Difco, Sparks, MD) at 28°C for 5 to 7 days (14,38). Prior to addition to the cells, the number of leptospires was determined by direct counting in a counting chamber under dark-field microscopy (18,25).…”

Section: Methodsmentioning

confidence: 99%

Leptospira santorosai Serovar Shermani Detergent Extract Induces an Increase in Fibronectin Production through a Toll-Like Receptor 2-Mediated Pathway

Tian

Hung

et al. 2011

Infect Immun

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Leptospirosis can activate inflammatory responses through Toll-like receptors (TLRs) and may cause renal tubulointerstitial fibrosis characterized by the accumulation of extracellular matrix (ECM). We have previously demonstrated that Leptospira santorosai serovar Shermani detergent extract stimulates ECM accumulation in vitro. The aim of this study was to examine the mechanistic basis of these previous observations and, in particular, to examine the potential involvement of TLRs. The addition of serovar Shermani detergent extract led to an increase in fibronectin gene expression and production. Inhibition of TLR2 but not TLR4 expression abrogated serovar Shermani detergent extract-mediated increases in fibronectin production. This response was also blocked by the knockdown of the gene expression of the TLR2 downstream transducers myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). Serovar Shermani detergent extract also activated nuclear factor-B, and its inhibition by curcumin-attenuated serovar Shermani detergent extract induced increases in fibronectin production. These effects were also mimicked by the specific TLR2 agonist, Pam(3)CsK(4), a response that was also abrogated by the knockdown of MyD88 and TRAF6. Similarly, the administration of live leptospires to cells also induced fibronectin production that was blocked by inhibition of TLR2 and MyD88 expression. In conclusion, serovar Shermani detergent extract can induce fibronectin production through the TLR2-associated cascade, providing evidence of an association between TLRs and leptospirosis-mediated ECM deposition.

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“…10,11 Repeated measures analysis of variance and Wilks test were performed to compare the mean OD over time across sequence types under differing conditions 12 ; controlling for temperature, pH, and media, there was no difference over time for early year ST34 versus later year ST34 and/or non-ST34 isolates (P = 0.3). The OD measurements did not correlate with viability.…”

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confidence: 99%

Viability of Leptospira Isolates from a Human Outbreak in Thailand in Various Water Types, pH, and Temperature Conditions

Stoddard¹,

Bui²,

Haberling³

et al. 2014

The American Society of Tropical Medicine and Hygiene

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Abstract. Leptospira spp. isolated from patients during a multiyear outbreak in Thailand were genotyped using multilocus sequence typing and a majority were identified as ST34, especially in earlier years. We tested whether ST34 isolates were better adapted to survive in various pH levels, temperatures, and water sources. Motility and growth were monitored over a 12-week period. Early year ST34 isolates did not appear to have a significant fitness advantage over non-ST34, however, this may have been because a majority of the isolates survived to the termination of the study, with the exception being at high temperature (37 C) and/or basic pH (8.65). Failure to detect a significant fitness advantage of ST34 may be a result of the length of the study or the small sample size. Lengthening the study and looking at virulence and maintenance in the host could yield additional information about this outbreak.Leptospirosis has a broad range of symptoms, from mild flu-like illness to acute renal failure and pulmonary hemorrhage.1,2 The disease is found worldwide, but is more common in countries with a rainy season and warmer climate that promotes the survival of leptospires in the environment. 3 The outbreak could not be explained by climate or regional flooding; the authors hypothesized that the increased incidence may have been caused by the presence of a biologically successful strain of pathogenic Leptospira.3 In this study, we tested whether early year ST34 isolates (2001 and 2003) were better adapted than later year ST34 and/or non-ST34 isolates (2005 and 2006) from this outbreak for survival in water from different sources and at variable pH and temperature.Isolates representing four of the 11 STs from various years of the outbreak were selected (Table 1). Isolates had been maintained at 25-30 C and had gone through 18-23 passages in Ellinghausen, McCullough, Johnson, and Harris (EMJH) semisoild media. Isolates were inoculated into liquid EMJH, grown at 30 C to the logarithmic phase, and adjusted to a McFarland standard of 0.5. [5][6][7][8][9] Spiking was performed by centrifuging 12 mL of the adjusted culture, discarding the supernatant, and then resuspending with 12 mL of suspending fluid. Rice field water and pond water from the outbreak location in Thailand were autoclaved before use. For each suspending fluid, conditions were tested in duplicate with various temperatures (25 C, 30 C, or 37 C) and pH (unadjusted, 5.65 or 8.65) combinations. The unadjusted pH was found to be pH 6.95 in rice field water and pH 7.79 in pond water. Once the Leptospira were resuspended, tubes were placed in a dark incubator at the appropriate temperature for 12 weeks. For each test combination, growth and viability of the Leptospira isolates were determined by measuring optical density (OD), leptospiral motility, and growth in semisolid EMJH (Figure 1).The OD at 400 nm was measured twice each week as a method of monitoring viable Leptospira ( Figure 1A). 10,11 Repeated measures analysis of variance and Wilks test were performed to...

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Leptospirosis research: fast, easy and reliable enumeration of mobile leptospires

Cited by 18 publications

References 13 publications

p53 signalling controls cell cycle arrest and caspase-independent apoptosis in macrophages infected with pathogenicLeptospiraspecies

p53 signalling controls cell cycle arrest and caspase-independent apoptosis in macrophages infected with pathogenicLeptospiraspecies

Leptospira santorosai Serovar Shermani Detergent Extract Induces an Increase in Fibronectin Production through a Toll-Like Receptor 2-Mediated Pathway

Viability of Leptospira Isolates from a Human Outbreak in Thailand in Various Water Types, pH, and Temperature Conditions

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