El Niño Southern Oscillation and Leptospirosis Outbreaks in New Caledonia

<div><p>Leptospirosis is an important cause of seasonal outbreaks in New Caledonia and the tropics. Using time series derived from high-quality laboratory-based surveillance from 2000–2012, we evaluated whether climatic factors, including El Niño Southern Oscillation (ENSO) and meteorological conditions allow for the prediction of leptospirosis outbreaks in New Caledonia. We found that La Niña periods are associated with high rainfall, and both of these factors were in turn, temporally associated with outbreaks of leptospirosis. The sea surface temperature in El Niño Box 4 allowed forecasting of leptospirosis outbreaks four months into the future, a time lag allowing public health authorities to increase preparedness. To our knowledge, our observations in New Caledonia are the first demonstration that ENSO has a strong association with leptospirosis. This association should be tested in other regions in the South Pacific, Asia or Latin America where ENSO may drive climate variability and the risk for leptospirosis outbreaks.</p></div

Observed (grey) and predicted (red) cases of leptospirosis occurring in each month in New Caledonia, 2000–2012.

<p>Observed (grey) and predicted (red) cases of leptospirosis occurring in each month in New Caledonia, 2000–2012.</p

Seasonal baseline (solid line) and upper limit of the 95% confidence interval (dashed line).

<p>The observed number of leptospirosis cases in each month is shown for 2007–2012. Blue circles indicate months that had been forecasted to be below the epidemic threshold and red crosses indicate months where the forecast predicted an epidemic. When the red crosses are above the 95% confidence interval, this indicates that the forecast had correctly predicted an epidemic intensity in that month.</p

Identification of Cell-Binding Adhesins of <i>Leptospira interrogans</i>

<div><p>Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus <i>Leptospira</i>. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic <i>Leptospira</i> species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic <i>Leptospira interrogans</i> binds to host cells more efficiently than to ECM components. Using <i>in vitro</i> phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate <i>L. interrogans</i> sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its <i>L. biflexa</i> homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for <i>L. interrogans</i>. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in <i>L. interrogans</i> infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and vaccines.</p></div

Intervention timeline.

<p>Events in 2010 are listed on the left alongside the timeframe for the simulated interventions: (I) RDT screening, (IIa) time-of-departure prophylaxis with antimicrobial drugs, (IIb) early-initiated prophylaxis with antimicrobial drugs beginning 7 d prior to deployment, (III) two-dose OCV immunization at 36 and 22 d prior to deployment, (IVa) two-dose OCV immunization combined with time-of-departure chemoprophylaxis, and (IVb) two-dose OCV immunization combined with early-initiated chemoprophylaxis.</p

<i>L. interrogans</i> genes encoding known or putative surface proteins selected for binding to endothelial cells.

<p>Phage clones pre-adsorbed on ECM and subjected to three rounds of selection in EA.hy926 cells were analyzed to identify the inserted <i>L. interrogans</i> sv. Copenhageni st. Fiocruz L1–130 gene. Out of the 931 phage clones selected, 779 have <i>L. interrogans</i> DNA inserts that represent 185 unique genes. Thirty-seven of these genes contain signal sequence. Four genes shown below were prioritized for further assessment.</p><p>*During translocation, the signal sequence of lipoproteins and transmembrane OMPs are cleaved by lipoprotein signal transpeptidase (Lsp) and leader peptidase (Lep), respectively.</p><p><i>L. interrogans</i> genes encoding known or putative surface proteins selected for binding to endothelial cells.</p

Localization of proteins after Triton X-114 fractionation of <i>L. interrogans</i> sv. Copenhageni.

<p><i>L. interrogans</i> was phase partitioned using Triton X-114 as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003215#s2" target="_blank">Materials and Methods</a>. Equivalents of 5×10⁸ or 2.5×10⁹ (*) spirochetes/lane of the whole cell lysate (WCL), protoplasmic cylinder (PC), aqueous (AQ) and detergent (DET) fractions were separated on 12.5 or 15% SDS-PAGE electrophoresis, transferred to PVDF membrane and probed with rabbit immune sera against periplasmic protein flagellin A1 (FlaA1), inner membrane protein LipL31, outer membrane proteins OmpL47 and OmpL1, and mouse immune sera against candidate adhesins LIC10508, LIC11574, LIC12341, and LIC13411. A duplicate gel was silver stained.</p

<i>L. interrogans</i> candidate adhesin LIC11574, but not its <i>L. biflexa</i> ortholog, LBF1629, binds to cells.

<p>MBP fusions of LIC11574 and LBF1629, along with control β-galactosidase (β-gal) were diluted to 0.3 µM and incubated with confluent monolayers of HMEC-1 endothelial (Panel A) or HEp-2 epithelial (Panel B) cells for 1 hr at 37°C, 5% CO₂. The unbound proteins were removed by washing and the bound adhesins were detected by ELISA using anti-MBP antibody. Results are expressed as the difference in the absorbance reading between wells with or without cells. The data shown are means ± standard errors from 8 independent assays with n = 16–28. Asterisks (*) indicate significant difference in binding when compared to the MBP fusion to β-gal, the negative control (two-tailed <i>t-</i>test, *<i>P</i><0.05, ***<i>P</i><0.001). Results show that LBF1629 (the <i>L. biflexa</i> ortholog of LIC11574) does not have cell-binding activity.</p

Epidemiological dynamics.

<p>We illustrate model-data concordance by plotting case observations (points) along with sample paths from stochastic realizations of the model (<i>n =</i> 100) under the status-quo scenario used for fitting. We plot instances (<i>n</i> = 79) in which transmission ensued and omit those where no epidemic occurred (<i>n</i> = 21). (<b>A</b>): Cases in the Artibonite-adjacent communes; (<b>B</b>): cases in all other communes.</p

Selected phage clones bind to endothelial and epithelial cells.

<p>Individual phage clones (∼10¹⁰) were added with confluent endothelial EA.hy926 or epithelial HEp-2 monolayers, or to wells containing cell medium only for 2 hr at 37°C under 5% CO₂. Phage attachment was quantified by ELISA using an anti-M13 (fd phage coat) antibody. Vector phage clone fdDOG <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003215#pntd.0003215-Coburn1" target="_blank">[37]</a> serves as a negative control. Binding is expressed as fold difference in the absorbance between monolayers vs. wells containing medium alone. Results are expressed as means ± standard errors, representative of at least two independent experiments repeated in quadruplicate. Error bars represent standard error while asterisks (*) indicate significant difference in attachment of candidate adhesins to cells compared to fdDOG (two-tailed <i>t-</i>test, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p