Isolation of Leptospira from blood culture bottles

International audienceWith the increasing use of real time PCR techniques, Leptospira isolation has mostly been abandoned for the diagnosis of human leptospirosis. However, there is a great value of collecting Leptospira isolates to better understand the epidemiology of this complex zoonosis and to provide the researchers with different isolates. In this study, we have successfully isolated different Leptospira strains from BacT/Alert aerobic blood culture bottles and suggest that this privileged biological material offers an opportunity to isolate leptospires

Continuous Excretion of Leptospira borgpetersenii Ballum in Mice Assessed by Viability Quantitative Polymerase Chain Reaction.

International audienceRodents are the main reservoir animals of leptospirosis. In this study, we characterized and quantified the urinary excretion dynamics of Leptospira by Mus musculus infected with 2 × 108 virulent Leptospira borgpetersenii serogroup Ballum. Each micturition was collected separately in metabolic cages, at 12 time points from 7 to 117 days post-infection (dpi). We detected Leptospira in all urine samples collected (up to 8 per time point per mouse) proving that Leptospira excretion is continuous with ca. 90% live L. borgpetersenii Ballum, revealed by viability quantitative polymerase chain reaction. Microscopic visualization by Live/Dead fluorescence confirmed this high proportion of live bacteria and demonstrated that L. borgpetersenii Ballum are excreted, at least partly, as bacterial aggregates. We observed two distinct phases in the excretion dynamics, first an increase in Leptospira concentration shed in the urine between 7 and 63 dpi followed by a plateau phase from 63 dpi onward, with up to 3 × 107Leptospira per mL of urine. These two phases seem to correspond to progressive colonization of renal tubules first, then to stable cell survival and maintenance in kidneys. Therefore, chronically infected adult mice are able to contaminate the environment via urine at each micturition event throughout their lifetime. Because Leptospira excretion reached its maximum 2 months after infection, older rodents have a greater risk of contaminating their surrounding environment

Cytokine, iNOS and chemokine gene expression in the kidneys depending on infected animals during <i>Leptospira</i> carrier state.

<p>Mice (open squares) and hamsters (filled squares) were infected with a sublethal dose of <i>L</i>. <i>borgpetersenii</i> serogroup Ballum isolate B3-13S. Total mRNA was extracted from kidneys at 14 and between 21 and 28 days postinfection and RT-qPCR assays were processed as described in Materials and Methods. Results are expressed as expression ratio relative to non-infected animal controls (time 0). Values are means (horizontal line) and individual ratio (dots). Significant difference in the gene expression levels were evaluated between animals and compared to control individual using an unpaired <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.005, ***<i>P</i><0.0005, <i>ns</i>: not significant.</p

Details and sequence of primers used for qPCR assays.

<p>Details and sequence of primers used for qPCR assays.</p

Survival and body weight parameters in hamsters and mice infected with virulent <i>L</i>. <i>borgpetersenii</i> serogroup Ballum isolate B3-13S.

<p>Six- to eight-week-old hamsters or mice were injected with B3-13S isolate at 1 x 10⁸ leptospires and survival (A) and body weight (B) were monitored until 28 days postinfection as described in Materials and Methods. (A) Data represent mean of three independent experiments (<i>N</i> = 6, 9, 17). (B) Values represent mean ± SD for hamsters (<i>N</i> = 15) and mice (<i>N</i> = 10). Significant difference between animals was evaluated using an unpaired <i>t</i>-test. ***<i>P</i><0.0005.</p

Development of fibrosis in hamster kidneys during chronic leptospirosis.

<p>Observation of kidneys from control non-infected hamster (A) and mouse (C) showing normal staining for interstitial structures. (B) Intense staining of the collagen was observed in the interstitium (i) of kidneys from hamster inoculated with B3-13S isolate at D28 postinfection. (C) Intense normal staining of blood vessels (ε) contrasting with no staining of interstitium (i) in mouse kidneys 28 days after infection with B3-13S leptospires. (A-D) Masson’s trichrome stain, bar represents 100 μm. (E) Score of renal fibrosis at 14 and between 21 and 28 days postinfection in hamsters and mice inoculated with virulent B3-13S isolate. (F) Gene expression of cytokine TGF-β in kidneys from infected animals was quantified by RT-qPCR as described in Materials and Methods. (E-F) Data are means (horizontal line) and individual value (dots). Significant difference between animals or time postinfection was evaluated using an unpaired <i>t</i>-test. *<i>P</i><0.05, *<i>P</i><0.005, ***<i>P</i><0.0005, <i>ns</i>: not significant.</p

Inflammatory lesions were observed in the kidneys of animals during chronic carriage of virulent <i>L</i>. <i>borgpetersenii</i> serogroup Ballum isolate B3-13S.

<p>(A) Normal glomerulus (*) with typical renal tubules (t) were observed in sections of kidneys collected from non-infected control hamster (HE stain, Magnification, X200). (B) Focal interstitial infiltration of polymorphonuclear cells (filled arrow) and lymphocytes (open arrow) were observed in kidneys at D28 postinfection. Dilatation of tubules (t) was commonly observed with inflammatory infiltration (i) or hyaline deposit (ε) in the lumen, and congestion (*) of several glomeruli. (C) Dilatation of the Bowman’s space (*) was also noticed at D28 postinfection in hamster kidneys. (D) Typical renal structures with glomerulus (*) and tubules (t) in kidneys from control non-infected mouse. (E) Histological observations of renal tissues from mouse at D28 postinfection showing normal glomerulus (*) and tubules (t). (A-E) HE stain. Scale bar represents 100 μm. (F) Lesion score was calculated for each individual at 14 and between 21–28 days postinfection. Values are means (horizontal line) and individual score (dots). Significant difference between animals or time postinfection was evaluated using an unpaired <i>t</i>-test. *<i>P</i><0.05, ***<i>P</i><0.0005, <i>ns</i>: not significant.</p

Phylogenetic analysis of leptospiral <i>lfb-1</i> gene sequences.

<p>Sequences from the 4 patients are shown in red while clones from environmental soils samples obtained during the retrospective study are shown in blue (first environmental sampling) or green (late environmental sampling). Note the presence of the <i>L</i>. <i>interrogans</i> Pyrogenes NC <i>lfb-1</i> sequence from the patient in all the corresponding investigated soils. <i>lfb-1</i> sequence from Site 2 soil 2 was identify as <i>L</i>. <i>interrogans</i> Australis and indeed cluster with <i>L</i>. <i>interrogans</i> Australis NC. Several sequences for Site 4 either cluster with <i>L</i>. <i>kmetyi</i> (Soil1) or form a new branch within pathogenic <i>Leptospira</i> species (Soil 4 sequence 1, 2 and 3). Phylogenetic tree was built using Phylo-win program with 500 bootstrap replicates applying Neighbour Joining method and Kimura’s 2-parameter distances.</p

Summary of sampling data and results for the six areas investigated.

<p>Summary of sampling data and results for the six areas investigated.</p

qPCR and v-PCR results and <i>lfb-1</i> sequence analysis for the six investigated areas.

<p>qPCR and v-PCR results and <i>lfb-1</i> sequence analysis for the six investigated areas.</p