Individuals from the Early Medieval Cemetery Aschheim-Bajuwarenring (Germany) that were analyzed in this study and corresponding results of screening for a portion of the <i>Y. pestis</i> specific <i>plasminogen activator gene</i> (<i>pla</i>).

1<p>estimated age by archaeological evidence <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-GutsmiedlSchmann1" target="_blank">[20]</a> for the multiple burial,</p>2<p>estimated age by radiocarbon dating determined for the particular individual (cal 2 sigma),</p>3<p>neg = no amplicon, pos = amplification results, - = not tested.</p

Results of molecular assays carried out on samples from individual A120 in two independent aDNA laboratories.

<p>Abbreviations: anc: ancestral, der: derived, pos: positive, <i>pla</i>: plasminogen activator gene, -: not tested, neg: no amplicon.</p

Global phylogeny for <i>Y.</i><i>pestis</i>.

<p>This global phylogeny for <i>Y. pestis</i> is based upon <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat-1003349-g001" target="_blank">figures 1A</a> and S3B in Cui et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Cui1" target="_blank">[11]</a>. It includes four major branches (0–4) and is rooted with <i>Y. pseudotuberculosis</i>, the ancestor of <i>Y. pestis </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Achtman3" target="_blank">[28]</a>. The identities of many of the major nodes defined by Cui et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Cui1" target="_blank">[11]</a> are presented in blue text. Circles represent specific populations; populations highlighted in gray have, to date, only been found in Asia <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Morelli1" target="_blank">[10]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Cui1" target="_blank">[11]</a>. Note that the location where strain Angola was isolated, which is the sole representative of population 0.PE3, is unknown. The phylogenetic position of Mongolian strain MNG 2972 is indicated with the blue box (see text). Five previously identified <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Morelli1" target="_blank">[10]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Cui1" target="_blank">[11]</a> key SNPs were utilized in the current study: s545, which occurs along the branch between nodes N06 and N07 (not shown); s87 and s89; which occur along the branch between N04 and N05; s82, which occurs along the branch between the branching point of strain MNG 2972 and N04; and s463, which occurs along the branch between the branching point of strain MNG 2972 and N03. It is known that these SNPs occur along these specific branches but the exact position and order of these SNPs along each branch is unknown. Sample A120 possesses ancestral states for SNPs s87, s89, and s545; and derived states for SNPs s82 and s463. Thus, the position of sample A120 in this phylogeny is along the branch between the branching point of strain MNG 2972 and N04, along branch N04-N05, along the branch from N04 to 0.ANT1 (red branches), or along one of the sub-branches within 0.ANT.1 (not shown). The phylogenetic positions of strains from the second pandemic <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Bos1" target="_blank">[3]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Haensch1" target="_blank">[12]</a> are indicated with the yellow boxes according to Cui et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Cui1" target="_blank">[11]</a>. The basal node for the 1.ORI group, which caused the third pandemic, is N14 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Cui1" target="_blank">[11]</a>.</p

High <i>Leptospira</i> Diversity in Animals and Humans Complicates the Search for Common Reservoirs of Human Disease in Rural Ecuador

<div><p>Background</p><p>Leptospirosis is a zoonotic disease responsible for high morbidity around the world, especially in tropical and low income countries. Rats are thought to be the main vector of human leptospirosis in urban settings. However, differences between urban and low-income rural communities provide additional insights into the epidemiology of the disease.</p><p>Methodology/Principal findings</p><p>Our study was conducted in two low-income rural communities near the coast of Ecuador. We detected and characterized infectious leptospira DNA in a wide variety of samples using new real time quantitative PCR assays and amplicon sequencing. We detected infectious leptospira in a high percentage of febrile patients (14.7%). In contrast to previous studies on leptospirosis risk factors, higher positivity was not found in rats (3.0%) but rather in cows (35.8%) and pigs (21.1%). Six leptospira species were identified (<i>L</i>. <i>borgpetersenii</i>, <i>L kirschnerii</i>, <i>L santarosai</i>, <i>L</i>. <i>interrogans</i>, <i>L noguchii</i>, and an intermediate species within the <i>L</i>. <i>licerasiae</i> and <i>L</i>. <i>wolffii</i> clade) and no significant differences in the species of leptospira present in each animal species was detected (χ² = 9.89, adj.p-value = 0.27).</p><p>Conclusions/Significance</p><p>A large portion of the world’s human population lives in low-income, rural communities, however, there is limited information about leptospirosis transmission dynamics in these settings. In these areas, exposure to peridomestic livestock is particularly common and high prevalence of infectious leptospira in cows and pigs suggest that they may be the most important reservoir for human transmission. Genotyping clinical samples show that multiple species of leptospira are involved in human disease. As these genotypes were also detected in samples from a variety of animals, genotype data must be used in conjunction with epidemiological data to provide evidence of transmission and the importance of different potential leptospirosis reservoirs.</p></div

Partial (Munich) and total (Mainz) alignment of amplified consensus sequences regarding several SNPs.

<p>DNA sequences longer than 50 nt are deposited in GenBank (accession numbers are given), SNP positions are indicated by bold letters and a dot in the bottom line. The Y in s82 indicates that either a C or a T was observed at this position, a common artifact attributable to DNA degradation <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003349#ppat.1003349-Paabo1" target="_blank">[23]</a>, whereas lower case letters indicate poor quality nucleotides.</p

Details of assays for detecting pathogenic and intermediate leptospira species.

<p>These two assays amplify the same region but the probes anneal to different targets within the amplified region. See also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004990#pntd.0004990.s004" target="_blank">S3 Fig</a>.</p

Association between rainfall and presence of <i>Leptospira</i> DNA in sera and urine febrile patients during the period of this study.

<p>Data were stratified into low precipitation (< 50 mm) and high precipitation months (>50 mm).</p

<i>Leptospira</i> DNA positivity in samples collected in 2014 and the first 6 months of 2015 from two rural communities in Manabi-Ecuador.

<p><i>Leptospira</i> DNA positivity in samples collected in 2014 and the first 6 months of 2015 from two rural communities in Manabi-Ecuador.</p

Leptospira genotyping results in febrile patients, cattle, pigs and rats sampled at our study sites.

<p>Concentric circles represent the sample size in Site 1 (colored with light gray) and Site 2 (dark gray). The order of these concentric circles is dependent on the sample size at each site. The proportion of each <i>Leptospira</i> genotype is marked with a different color, however white portions represent the percentage of samples for which genotyping was unsuccessful. Sample sizes for each group is detailed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004990#pntd.0004990.t002" target="_blank">Table 2</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004990#pntd.0004990.s010" target="_blank">S6 Table</a>.</p