Genetic diversity of Leptospira interrogans circulating isolates and vaccine strains in China from 1954–2014

Leptospirosis is one of the most important but neglected, infectious tropical diseases worldwide. Leptospira interrogans is now recognized as a leading cause of the disease. Little is known of the genetic diversity and phylogenetic characteristics of L. interrogans within China. To better understand the transmission and genetic diversity of L. interrogans populations, we characterized 271 isolates and seven vaccine strains from China during 1954–2014 using multilocus variable-number tandem repeat analysis (MLVA). 110 different L. interrogans MLVA profiles (MTs) were identified, of which five were predominant, reflecting a high level of genetic diversity in L. interrogans population in China. Different from that of circulating isolates, seven vaccine strains have different MT, of which some are phylogenetically away from the circulating isolates. The results showed that Icterohaemorrhagiae, Hebdomadis, and Canicola ranked as the top three serogroups among L. interrogans strains tested. The cluster analysis demonstrate the clonal links between rodent and human isolates, suggesting the rodent species played a key role in the transmission of leptospirosis to humans, and contributed to the circulation of the pathogen in humans. Taken together, these findings should provide insight into a better knowledge of the epidemiology and molecular evolution of L. interrogans in China. Furthermore, the results should facilitate the selection of candidate vaccine strains in the future

Specific Immunoglobulin G Antibody Detected in Umbilical Blood and Amniotic Fluid from a Pregnant Woman Infected by the Coronavirus Associated with Severe Acute Respiratory Syndrome

Specific immunoglobulin G antibody for severe acute respiratory syndrome (SARS) coronavirus was detected in maternal blood, umbilical blood, and amniotic fluid from a pregnant SARS patient. Potential protection of fetus from infection was suggested

Source tracking of human leptospirosis: serotyping and genotyping of Leptospira isolated from rodents in the epidemic area of Guizhou province, China

BACKGROUND: Sustained human leptospirosis as well as death cases has been reported in Qiandongnan Prefecture, Southeast of Guizhou, China, recently, but these human patients were only clinically diagnosed, and leptospires have never been isolated from patients in these epidemic regions, In order to track the source of infection and understand the etiologic characteristic of leptospirosis, we performed rodent carrier surveillance for leptospirosis in the epidemic area in 2011. The population distribution of rodents in the epidemic regions was revealed. RESULTS: Four strains of leptospire were isolated from Apodemus agrarius. Microscopic agglutination test (MAT) confirmed the four isolates belonged to leptospiral serogroup Icterohaemorrhagiae. Multilocus sequence typing (MLST) indicated that all the four strains were defined as sequence type 1(ST1), which is identical to the three strains isolated from Rattus tanezumi in Rongjiang County in 2007. Clustering analysis of the MLST data indicated that the local isolates exactly matched with reference strain of leptospiral serovar Lai strain 56601, which is consistent with anti-Leptospira antibody detection of patients using MAT. CONCLUSIONS: Apodemus agrarius may be the potentially important carrier of leptospirosis and the potential source of leptospiral infection in human, and serovar Lai maybe the epidemic serovar of Leptospira in the localities

Molecular Typing of Pathogenic <i>Leptospira</i> Serogroup Icterohaemorrhagiae Strains Circulating in China during the Past 50 Years

<div><p>Background</p><p>Leptospirosis is one of the most important neglected tropical infectious diseases worldwide. Icterohaemorrhagiae has been throughout recent history, and still is, the predominant serogroup of this pathogen in China. However, very little in detail is known about the serovars or genotypes of this serogroup.</p><p>Methodology/Principal Findings</p><p>In this study, 120 epidemic strains from five geographically diverse regions in China collected over a 50 year period (1958~2008), and 8 international reference strains characterized by 16S rRNA sequencing and MLST analysis. 115, 11 and 2 strains were identified as <i>L</i>. <i>interrogans</i>, <i>L</i>. <i>borgpetersenii</i>, and <i>L</i>. <i>kirschneri</i>, respectively. 17 different STs were identified including 69 ST1 strains, 18 ST17, 18 ST128, 9 ST143 and 2 ST209. The remaining 12 strains belonged to 12 different STs. eBURST analysis demonstrated that, among the clonal complexes isolated (CCs), CC1 accounted for 73.3% (88/120) strains representing three STs: ST1, ST128 and ST98. ST1 was the most likely ancestral strain of this CC, followed by singleton CC17 (17/120) and CC143 (11/120). Further analysis of adding 116 serogroup Icterohaemorrhagiae strains in the MLST database and studies previously described using global eBURST analysis and MST dendrogram revealed relatively similar ST clustering patterns with five main CCs and 8 singletons among these 244 strains. CC17 was found to be the most prevalent clone of pathogenic <i>Leptospira</i> circulating worldwide. This is the first time, to our knowledge, that ST1 and ST17 strains were distributed among 4 distinct serovars, indicating a highly complicated relationship between serovars and STs.</p><p>Conclusions/Significance</p><p>Our studies demonstrated a high level of genetic diversity in the serogroup Icterohaemorrhagiae strains. Distinct from ST17 or ST37 circulating elsewhere, ST1 included in CC1, has over the past 50 years or so, proven to be the most prevalent ST of pathogenic leptospires isolated in China. Moreover, the complicated relationship between STs and serovars indicates an urgent need to develop an improved scheme for <i>Leptospira</i> serotyping.</p></div

The Construction and Evaluation of Reference Spectra for the Identification of Human Pathogenic Microorganisms by MALDI-TOF MS

<div><p>Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique for the rapid and high-throughput identification of microorganisms. There remains a dearth of studies in which a large number of pathogenic microorganisms from a particular country or region are utilized for systematic analyses. In this study, peptide mass reference spectra (PMRS) were constructed and evaluated from numerous human pathogens (a total of 1019 strains from 94 species), including enteric (46 species), respiratory (21 species), zoonotic (17 species), and nosocomial pathogens (10 species), using a MALDI-TOF MS Biotyper system (MBS). The PMRS for 380 strains of 52 species were new contributions to the original reference database (ORD). Compared with the ORD, the new reference database (NRD) allowed for 28.2% (from 71.5% to 99.7%) and 42.3% (from 51.3% to 93.6%) improvements in identification at the genus and species levels, respectively. Misidentification rates were 91.7% and 57.1% lower with the NRD than with the ORD for genus and species identification, respectively. Eight genera and 25 species were misidentified. For genera and species that are challenging to accurately identify, identification results must be manually determined and adjusted in accordance with the database parameters. Through augmentation, the MBS demonstrated a high identification accuracy and specificity for human pathogenic microorganisms. This study sought to provide theoretical guidance for using PMRS databases in various fields, such as clinical diagnosis and treatment, disease control, quality assurance, and food safety inspection.</p></div

Molecular phylogenetic analysis between 17 sequence types (STs) of 128 pathogenic <i>Leptospira</i> strains based on neighbor-joining (N-J) tree method.

<p>Phylogenetic relationships based on concatenated sequences of 7-locus MLST scheme (3,102 bp) for the 128 pathogenic strains were inferred using N-J method and 1000 bootstrap replications as implemented in MEGA5. Each bacterial strain is labeled as follows: abbreviation of species name (L.int: <i>L</i>. <i>interrogans</i>, L.kir: <i>L</i>. <i>kirschneri</i> and L. bor: <i>L</i>. <i>borgpetersenii</i>), strain name, isolated time, isolated region and (for the 7-locus MLST scheme) sequence type (ST). The dendrogram displays three major clusters corresponding to 3 <i>Leptospira</i> species analyzed in different colors: Red: <i>L</i>. <i>int</i>; Yellow: <i>L</i>. <i>kri</i>; Blue: <i>L</i>. <i>bor</i>.</p

Phylogenetic analysis based on the nearly full-length <i>rrs</i> gene for the 128 pathogenic <i>Leptospira</i> strains.

<p>The evolutionary analysis for the 128 pathogenic isolates was conducted in MEGA5 and evolutionary history was inferred using the Neighbor-Joining method (Tamura, Peterson, Peterson <i>et al</i>., 2011) and a bootstrap value of 1000. Twenty reference sequences representing 20 <i>Leptospira</i> species originated from GenBank are shown by denoting their species group: pathogenic, intermediate pathogenic, nonpathogenic. <i>Turneriella parva</i> NCTC 11395T (AY293856) and <i>Leptonema illini</i> NCTC 11301T (AY714984) were considered as outgroup. The scale bar represents the number of base pairs differences. Each species is labeled as follows: abbreviation of species name (L.int: <i>L</i>. <i>interrogans</i>, L.kir: <i>L</i>. <i>kirschneri</i>, L.bor: <i>L</i>. <i>borgpetersenii</i>, L.als: <i>L</i>. <i>alstonii</i>, L.san: <i>L</i>. <i>santarosai</i>, L.nog: <i>L</i>. <i>noguchii</i>, L.wei: <i>L</i>. <i>weilii</i>, L.ale: <i>L</i>. <i>alexanderi</i>, L.kme: <i>L</i>. <i>kmetyi</i>, L.ina: <i>L</i>. <i>inadai</i>, L.bro: <i>L</i>. <i>broomii</i>, L.wol: <i>L</i>. <i>wolffii</i>, L.lic: <i>L</i>. <i>licerasiae</i>, L.fai: <i>L</i>. <i>fainei</i>, L.bif: <i>L</i>. <i>biflexa</i>, L.mey: <i>L</i>. <i>meyeri</i>, L.wol: <i>L</i>. <i>wolbachii</i>, L.yan: <i>L</i>. <i>yanagawae</i>, L.ter: <i>L</i>. <i>terpstrae</i>, L.van: <i>L</i>. <i>vanthielii</i>, <i>T</i>.<i>par</i>: <i>T</i>. <i>parva</i> and L ill: <i>L</i>. <i>illini</i>). The dendrogram displays that the 128 <i>Leptospira</i> strains belonged to three major clusters corresponding to 3 <i>Leptospira</i> species in different colors: Red: <i>L</i>. <i>int</i>; Yellow: <i>L</i>. <i>kri</i>; Blue: <i>L</i>. <i>bor</i>.</p

Isolated location and STs of 128 pathogenic <i>Leptospira</i> strains.

<p>Isolated location and STs of 128 pathogenic <i>Leptospira</i> strains.</p

Geographic distribution and STs of the 244 Chinese and international <i>Leptospira</i> isolates.

<p>Geographic distribution and STs of the 244 Chinese and international <i>Leptospira</i> isolates.</p

Minimum spanning tree analysis of the 22 STs representing 244 global <i>Leptospira</i> strains.

<p>In the Minimum Spanning Tree (MST) analysis, each circle represents a different sequence type (ST) as labeled, its size is proportional to the number of strains and the color indicates the country of origin. The width of the lines reflects the genetic difference between two STs, where in dark/heavy lines connect single locus variants (SLVs) or double locus variants (DLVs) and dotted lines indicate the most likely connection between two STs differing by three or more loci.</p