Serological and molecular characteristics of pathogenic Leptospira in rodent populations in Fujian Province, China, 2018-2020.

BACKGROUND: Leptospirosis is a significant emerging infectious disease worldwide. Rodents are considered to be the most critical hosts of Leptospira spp. Fujian Province is a region highly endemic for leptospirosis in China. However, the genetic diversity of leptospires circulating among rodents in Fujian is limited. RESULTS: The carrier status of rodents for Leptospira spp. was investigated by culture and serological detection in Fujian during 2018-2020. A total of 710 rodents, including 11 species, were trapped, with Rattus losea being the dominant trapped species (50.56%). Fourteen pathogenic Leptospira strains were obtained. Seven L. borgpetersenii serogroup Javanica strains belonging to ST143, 4 L. interrogans serogroup Icterohaemorrhagiae strains belonging to ST1 and ST17, 2 L. interrogans serogroup Bataviae strains belonging to ST96 and ST333, and 1 L. interrogans serogroup Pyrogenes strains belonging to ST332 were identified using 16S rDNA gene sequencing, microscopic agglutination test (MAT) and Multilocus sequence typing (MLST). L. borgpetersenii serogroup Javanica belonging to ST143 was the dominant type (50.00%). A total of 387 rodent serum samples were tested by MAT. Serum were considered positive for seroreactivity at a titer ≥ 1:160 against at least one serovar. A total of 90 (23.26%) serum samples tested positive, and four serogroups were identified, with Javanica being the dominant serogroup (87.78%), which was similar to the dominant serogroup isolated from rodents. This study demonstrates a high prevalence of leptospirosis in rodents and public health education among high-risk workers is highly recommended. CONCLUSIONS: R. losea was the dominant trapped rodent, and L. borgpetersenii serogroup Javanica ST143 was widely distributed among rodents in Fujian from 2018 to 2020. Despite the low number of isolates obtained from rodents, this study suggests that continuous epidemiological surveillance of the aetiological characteristics of pathogenic Leptospira in wild animal reservoirs may help reduce the possible risk of disease transmission.

Genetic characteristics of pathogenic Leptospira in wild small animals and livestock in Jiangxi Province, China, 2002-2015.

BACKGROUND: Leptospirosis is one of the most important neglected tropical bacterial diseases worldwide. However, there is limited information on the genetic diversity and host selectivity of pathogenic Leptospira in wild small mammal populations. METHODOLOGY/PRINCIPAL FINDINGS: Jiangxi Province, located in southern China, is a region highly endemic for leptospirosis. In this study, among a total of 3,531 trapped rodents dominated by Apodemus agrarius (59.7%), 330 Leptospira strains were successfully isolated from six different sites in Jiangxi between 2002 and 2015. Adding 71 local strains from humans, various kinds of livestock and wild animals in Jiangxi, a total of 401 epidemic strains were characterized using 16S rRNA gene senquencing, multilocus sequence typing (MLST) and the microscopic agglutination test (MAT). Among them, the most prevalent serogroup was Icterohaemorrhagiae (61.10%), followed by Javanica (19.20%) and Australis (9.73%); the remaining five serogroups, Canicola, Autumnalis, Grippotyphosa, Hebdomadis and Pomona, accounted for 9.97%. Species identification revealed that 325 were L. interrogans and 76 were L. borgpetersenii. Moreover, L. interrogans was the only pathogenic species in Fuliang and Shanggao and was predominant in Shangrao (95.0%); L. borgpetersenii was the most common in the remaining three sites. Twenty-one sequence types (STs) were identified. Similarly, ST1 and serogroup Icterohaemorrhagiae were most prevalent in Shangrao (86.0% and 86.4%) and Fuliang (90.4% and 90.4%), ST143 and serogroup Javanica in Shangyou (88.5% and 90.4%) and Longnan (73.1% and 73.1%), and ST105 and serogroup Australis in Shanggao (46.3% and 56.1%). Serogroup Icterohaemorhagiae primarily linked to A. agrarius (86.9%), serogroup Canicola to dogs (83.3%). There were significant differences in the distribution of leptospiral species/serogroups/STs prevalence across host species/collected locations among the 394 animal-associated strains (Fisher's exact test, p<0.001). CONCLUSIONS/SIGNIFICANCE: Our study demonstrated high genetic diversity of pathogenic Leptospira strains from wild small animals in Jiangxi from 2002 to 2015. A. agrarius was the most abundantly trapped animal reservoir, and serogroup Icterohaemorrhagiae and ST1 were the most dominant in Jiangxi. Significant geographic variation and host diversity in the distribution of dominant species, STs and serogroups were observed. Moreover, rat-to-human transmission might play a crucial role in the circulation of Leptospirosis in Jiangxi. Details of the serological and molecular characteristics circulating in this region will be essential in implementing prevention and intervention measures to reduce the risk of disease transmission in China. However, phylogenetic analysis of more Leptospira isolates should explore the impact of ecological change on leptospirosis transmission dynamics and investigate how such new knowledge might better impact environmental monitoring for disease control and prevention at a public health level.

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.

Whole genome sequencing revealed host adaptation-focused genomic plasticity of pathogenic Leptospira.

Leptospirosis, caused by pathogenic Leptospira spp., has recently been recognized as an emerging infectious disease worldwide. Despite its severity and global importance, knowledge about the molecular pathogenesis and virulence evolution of Leptospira spp. remains limited. Here we sequenced and analyzed 102 isolates representing global sources. A high genomic variability were observed among different Leptospira species, which was attributed to massive gene gain and loss events allowing for adaptation to specific niche conditions and changing host environments. Horizontal gene transfer and gene duplication allowed the stepwise acquisition of virulence factors in pathogenic Leptospira evolved from a recent common ancestor. More importantly, the abundant expansion of specific virulence-related protein families, such as metalloproteases-associated paralogs, were exclusively identified in pathogenic species, reflecting the importance of these protein families in the pathogenesis of leptospirosis. Our observations also indicated that positive selection played a crucial role on this bacteria adaptation to hosts. These novel findings may lead to greater understanding of the global diversity and virulence evolution of Leptospira spp.

Molecular Typing of Pathogenic Leptospira Serogroup Icterohaemorrhagiae Strains Circulating in China during the Past 50 Years.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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 L. interrogans, L. borgpetersenii, and L. kirschneri, 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 Leptospira 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. CONCLUSIONS/SIGNIFICANCE: 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 Leptospira serotyping.

A novel approach for differentiating pathogenic and non-pathogenic Leptospira based on molecular fingerprinting.

Leptospirosis is a worldwide, deadly zoonotic disease. Pathogenic Leptospira causes leptospirosis. The rapid and accurate identification of pathogenic and non-pathogenic Leptospira strains is essential for appropriate therapeutic management and timely intervention for infection control. The molecular fingerprint is a simple and rapid alternative tool for microorganisms identification, which is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this study, molecular fingerprint was performed to identify pathogenic strains of Leptospira. Phylogenetic analysis based on 16S rRNA gene sequences was used as the reference method. In addition, a label-free technique was used to reveal the different proteins of pathogenic or non-pathogenic Leptospira. A reference database was constructed using 30 Leptospira strains, including 16 pathogenic strains and 14 non-pathogenic strains. Two super reference spectra that were associated with pathogenicity were established. Overall, 33 Leptospira strains were used for validation, and 32 of 33 Leptospira strains could be identified on the species level and all the 33 could be classified as pathogenic or non-pathogenic. The super reference spectra and the major spectra projection (MSP) dendrogram correctly categorized the Leptospira strains into pathogenic and non-pathogenic groups, which was consistent with the 16S rRNA reference methods. Between the pathogenic and non-pathogenic strains, 108 proteins were differentially expressed. molecular fingerprint is an alternative to conventional molecular identification and can rapidly distinguish between pathogenic and non-pathogenic Leptospira strains. Therefore, molecular fingerprint may play an important role in the clinical diagnosis, treatment, surveillance, and tracking of epidemic outbreaks of leptospirosis. BIOLOGICAL SIGNIFICANCE: Leptospirosis is a worldwide zoonosis that is caused by spirochetes of the genus Leptospira. Leptospirosis is a serious zoonotic disease that has become an important public health problem. Traditional serological methods are the gold standard for the detection of pathogenic strains of Leptospira. However, serological procedures are cumbersome, require more complex experimental techniques, and are based on a large number of international and domestic reference strains. Additionally, these experiments involve the immunization of animals with antigens from different serotypes to produce immune serum, and improper techniques may result in a rapid decrease in antibody titer, which would affect the final results. It is difficult to perform cumbersome detection procedures in a basic laboratory. Therefore, the use of conventional serological methods is limited, which significantly impacts daily leptospirosis epidemic surveillance, prevention, and control. Molecular biology methods, such as 16S rRNA and PCR-based methods, can be used to identify the pathogenic Leptospira. However, DNA extraction and gene sequencing methods are laborious and time consuming. Therefore, more rapid and reliable high-throughput identification methods are urgently needed for the clinical diagnosis of leptospirosis to improve epidemic control. Here, molecular fingerprinting technique was use to identify the pathogenicity. We constructed the reference spectra database and the super reference spectra of pathogenic and non-pathogenic Leptospira, which can rapidly identified Leptospira at the species level and the pathogenicity of these isolates can be simultaneously confirmed. Furthermore, the protein components of Leptospira pathogenicity were revealed. These findings thus provide a new way for Leptospira pathogenicity identification.

The construction and evaluation of reference spectra for the identification of human pathogenic microorganisms by MALDI-TOF MS.

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.

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.

A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species.

BACKGROUND: The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. METHODOLOGY AND FINDINGS: We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. CONCLUSION: The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.

Comparative proteogenomic analysis of the Leptospira interrogans virulence-attenuated strain IPAV against the pathogenic strain 56601.

The virulence-attenuated Leptospira interrogans serovar Lai strain IPAV was derived by prolonged laboratory passage from a highly virulent ancestral strain isolated in China. We studied the genetic variations of IPAV that render it avirulent via comparative analysis against the pathogenic L. interrogans serovar Lai strain 56601. The complete genome sequence of the IPAV strain was determined and used to compare with, and then rectify and reannotate the genome sequence of strain 56601. Aside from their highly similar genomic structure and gene order, a total of 33 insertions, 53 deletions and 301 single-nucleotide variations (SNVs) were detected throughout the genome of IPAV directly affecting 101 genes, either in their 5' upstream region or within their coding region. Among them, the majority of the 44 functional genes are involved in signal transduction, stress response, transmembrane transport and nitrogen metabolism. Comparative proteomic analysis based on quantitative liquid chromatography (LC)-MS/MS data revealed that among 1 627 selected pairs of orthologs, 174 genes in the IPAV strain were upregulated, with enrichment mainly in classes of energy production and lipid metabolism. In contrast, 228 genes in strain 56601 were upregulated, with the majority enriched in the categories of protein translation and DNA replication/repair. The combination of genomic and proteomic approaches illustrated that altered expression or mutations in critical genes, such as those encoding a Ser/Thr kinase, carbon-starvation protein CstA, glutamine synthetase, GTP-binding protein BipA, ribonucleotide-diphosphate reductase and phosphate transporter, and alterations in the translational profile of lipoproteins or outer membrane proteins are likely to account for the virulence attenuation in strain IPAV.

[Establishment and application of TaqMan Real-time PCR for the detection of pathogenic Leptospira species].

OBJECTIVE: To develop and evaluate a TaqMan Real-time PCR method for the detection of pathogenic Leptospira species. METHODS: rrs gene of part fragment on 16S rRNA was used to design primers and TaqMan probe. The target gene was cloned into vector pMD19-T in order to make the standard curve and be used for quality control. To determine the specificity and specificity, DNA from Chinese Leptospira strains belonging to 15 pathogenic reference strains, 21 non-pathogenic reference strains, and 50 different serotypes of pathogenic isolates as well as 27 other micro-organisms were included in this study. Eight serial DNA dilutions from pathogenic Leptospira and DNA from 25 kidney tissues were detected by Real-time PCR and conventional PCR simultaneously. RESULTS: A Real-time PCR methodology was developed and optimised. All the pathogenic Leptospira gave a positive amplification. Non-pathogenic Leptospira and all the other micro-organisms were not amplified. The plasmid sensitivity of Real-time PCR and conventional PCR were 10 copy/µl and 10(4)copy/µl respectively. The DNA sensitivity of Real-time PCR and conventional PCR were 100 fg/µl and 1 ng/µl respectively. The kidney tissue detection of the two methods appeared to be exactly the same. CONCLUSION: This research project successfully developed a Real-time PCR methodology with better sensitivity and specificity for the identification of pathogenic Leptospira, using the rrs gene.

[Molecular typing on Leptospira interrogans isolates from Jiangxi province, by pulsed-field gel electrophoresis].

OBJECTIVE: To perform a molecular epidemiological investigation on the types of Leptospira interrogans isolates from leptospirosis patients and animal hosts in Jiangxi province, using a pulsed-field gel electrophoresis (PFGE). METHODS: The extracted chromosomal DNA from leptospiral isolates were digested with restriction endonuclease Not I and the DNA segments were separated by using PFGE. By BiOnurerics V4.0 software and 75% similarity as the standard, the obtained PFGE images from leptospiral isolates were managed to establish a digitization database and then the PFGE maps of leptospiral isolates were compared with those of reference standard strains belonging to 15 serovars in 15 serogroups of L. interrogans, for cluster analysis. RESULTS: 139 strains of L. interrogans isolated from different areas of Jiangxi province were classified into 46 PFGE types. Among the PFGE types, LepNot I.0071, LepNot I.0072 and LepNot I.0043 were the predominant types that accounting for 28.06%, 15.11% and 7.19% of all the leptospiral isolates, respectively. The PFGE maps from 84.89% (118/139) of the 139 leptospiral isolates were found to basically match those of 6 reference standard strains belonging to 6 serovar in 6 serogroups of L. interrogans. In the 118 matched leptospiral isolates, 32.37% (45 strains), 15.83% (22 strains) and 15.11% (21 strains) belonged to sero-groups Icterohaemorrhagiae serovar Lai, sero-groups Australis serovar Australis and sero-group Javanica serovar Javanica, respectively. CONCLUSION: PFGE seemed a fast, accurate and effective method for typing of L. interrogans isolates. Serogroup Icterohaemorrhagiae serovar Lai and followed by serogroup Australis serovar Australis as well as serogroup Javanica serovar Javanica were the predominant L. interrogans species in humans and animal hosts in Jiangxi province.

Development of O-antigen gene cluster-specific PCRs for rapid typing six epidemic serogroups of Leptospira in China.

BACKGROUND: Leptospira is the causative agent of leptospirosis. The O-antigen is the distal part of the lipopolysaccharide, which is a key component of outer membrane of Gram-negative bacteria and confers serological specificity. The epidemiology and clinical characteristics of leptospirosis are relative to the serology based taxonomic unit. Identification of Leptospira strains by serotyping is laborious and has several drawbacks. RESULTS: In this study, the O-antigen gene clusters of four epidemic Leptospira serogroups (serogroup Canicola, Autumnalis, Grippotyphosa and Hebdomadis) in China were sequenced and all genes were predicted in silico. Adding published sequences of two serogroups, Icterohaemorrhagiae (strain Lai and Fiocruz L1-130) and Sejroe (strain JB197 and L550), we identified six O-antigen-specific genes for six epidemic serogroups in China. PCR assays using these genes were developed and tested on 75 reference strains and 40 clinical isolates. CONCLUSION: The results show that the PCR-based assays can be reliable and alternative means for rapid typing of these six serogroups of Leptospira.

Nosocomial transmission of human granulocytic anaplasmosis in China.

CONTEXT: Human granulocytic anaplasmosis (HGA) is an emerging tick-borne disease in China. A cluster of cases among health care workers and family members following exposure to a patient with fulminant disease consistent with HGA prompted investigation. OBJECTIVE: To investigate the origin and transmission of apparent nosocomial cases of febrile illness in the Anhui Province. DESIGN, SETTING, AND PATIENTS: After exposure to an index patient whose fatal illness was characterized by fever and hemorrhage at a primary care hospital and regional tertiary care hospital's isolation ward, secondary cases with febrile illness who were suspected of being exposed were tested for antibodies against Anaplasma phagocytophilum and by polymerase chain reaction (PCR) and DNA sequencing for A. phagocytophilum DNA. Potential sources of exposure were investigated. MAIN OUTCOME MEASURE: Cases with serological or PCR evidence of HGA were compared with uninfected contacts to define the attack rate, relative risk of illness, and potential risks for exposure during the provision of care to the index patient. RESULTS: In a regional hospital of Anhui Province, China, between November 9 and 17, 2006, a cluster of 9 febrile patients with leukopenia, thrombocytopenia, and elevated serum aminotransferase levels were diagnosed with HGA by PCR for A. phagocytophilum DNA in peripheral blood and by seroconversion to A. phagocytophilum. No patients had tick bites. All 9 patients had contact with the index patient within 12 hours of her death from suspected fatal HGA while she experienced extensive hemorrhage and underwent endotracheal intubation. The attack rate was 32.1% vs 0% (P = .04) among contacts exposed at 50 cm or closer, 45% vs 0% (P = .001) among those exposed for more than 2 hours, 75% vs 0% (P < .001) among those reporting contact with blood secretions, and 87.5% vs 0% (P = .004) among those reporting contact with respiratory secretions from the index patient. CONCLUSION: We report the identification of HGA in China and likely nosocomial transmission of HGA from direct contact with blood or respiratory secretions.

[Establishment of standardized method for pulsed-field gel electrophoresis on Leptospira interrogans and analysis on their patterns].

OBJECTIVE: To establish a standardized operation procedure for pulsed-field gel electrophoresis (PFGE) on Leptospira interrogans as well as a figure digital database to develop the Chinese representative reference strains. METHODS: Under the characteristics of strains and referring to the other SOPs of PFGE on pathogens provided by CDC and PulseNet Asia Pacific, genomic chromosome DNA purification, restriction endonuclease digestion and the parameters for running PFGE were optimized. RESULTS: Not I digestion patterns of leptospiral genome for the Chinese representative strains were established and partial isolates of serogroup icterohaemorrhagiae from the leptospirosis surveillance in Sichuan and Anhui provinces were analyzed by PFGE. Results showed that each of all the 15 Chinese representative strains had a unique pattern. 91.67% (22/24) of the 24 isolates identified as serogroup icterohaemorrhagiae matched to the map of the reference strain 56601 (serogroup icterohaemorrhagiae serovar lai). CONCLUSION: The PFGE figures were clear with high resolution and the fragments were equally distributed by this standardized operating procedure so as to reveal the molecular-genetic characteristics of Leptospira interrogans. The patterns had high relativity with the serological identification and seemed to be very important for genetic analysis of strains in studying the outbreak of leptospirosis.

Genetic diversity among major endemic strains of Leptospira interrogans in China.

BACKGROUND: Leptospirosis is a world-widely distributed zoonosis. Humans become infected via exposure to pathogenic Leptospira spp. from contaminated water or soil. The availability of genomic sequences of Leptospira interrogans serovar Lai and serovar Copenhageni opened up opportunities to identify genetic diversity among different pathogenic strains of L. interrogans representing various kinds of serotypes (serogroups and serovars). RESULTS: Comparative genomic hybridization (CGH) analysis was used to compare the gene content of L. interrogans serovar Lai strain Lai with that of other 10 L. interrogans strains prevailed in China and one identified from Brazil using a microarray spotted with 3,528 protein coding sequences (CDSs) of strain Lai. The cutoff ratio of sample/reference (S/R) hybridization for detecting the absence of genes from one tested strain was set by comparing the ratio of S/R hybridization and the in silico sequence similarities of strain Lai and serovar Copenhageni strain Fiocruz L1-130. Among the 11 strains tested, 275 CDSs were found absent from at least one strain. The common backbone of the L. interrogans genome was estimated to contain about 2,917 CDSs. The genes encoding fundamental cellular functions such as translation, energy production and conversion were conserved. While strain-specific genes include those that encode proteins related to either cell surface structures or carbohydrate transport and metabolism. We also found two genomic islands (GIs) in strain Lai containing genes divergently absent in other strains. Because genes encoding proteins with potential pathogenic functions are located within GIs, these elements might contribute to the variations in disease manifestation. Differences in genes involved in O-antigen biosynthesis were also identified for strains belonging to different serogroups, which offers an opportunity for future development of genomic typing tools for serological classification. CONCLUSION: CGH analyses for pathogenic leptospiral strains prevailed in China against the L. interrogans serovar Lai strain Lai CDS-spotted microarrays revealed 2,917 common backbone CDSs and strain specific genes encoding proteins mainly related to cell surface structures and carbohydrated transport/metabolism. Of the 275 CDSs considered absent from at least one of the L. interrogans strains tested, most of them were clustered in the rfb gene cluster and two putative genomic islands (GI A and B) in strain Lai. The strain-specific genes detected via this work will provide a knowledge base for further investigating the pathogenesis of L interrogans and/or for the development of effective vaccines and/or diagnostic tools.

Expression and comparative analysis of genes encoding outer membrane proteins LipL21, LipL32 and OmpL1 in epidemic leptospires.

Leptospiral outer membrane proteins (OMPs) are highly conserved in different species, and play an essential role in the development of new immunoprotection and serodiagnosis strategies. The genes encoding LipL21, LipL32 and OmpL1 were cloned from the complete genome sequence of Leptospira interrogans serovar lai strain Lai and expressed in vitro. Sequence comparison analysis revealed that the three genes were highly conserved among distinct epidemic leptospires, including three major epidemic species Leptospira interrogans, Leptospira borgpetersenii and Leptospira weilii, in China. Immunoblot analysis was further performed to scrutinize 15 epidemic Leptospira reference strains using the antisera of the recombinant OMPs. Both immunoblot assay and reverse transcription-polymerase chain reaction demonstrated that these three OMPs were conservatively expressed in pathogenic L. interrogans strains and other pathogenic leptospires. Additionally, the use of these recombinant OMPs as antigens in enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of leptospirosis was evaluated. The recombinant LipL32 and OmpL1 proteins showed a high degree of ELISA reactivity with sera from patients infected with L. interrogans strain Lai and other pathogenic leptospires. These results may contribute to the identification of candidates for broad-range vaccines and immunodiagnostic antigens in further research.

Molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms.

Massive numbers of palm civets were culled to remove sources for the reemergence of severe acute respiratory syndrome (SARS) in Guangdong Province, China, in January 2004, following SARS coronavirus detection in market animals. The virus was identified in all 91 palm civets and 15 raccoon dogs of animal market origin sampled prior to culling, but not in 1,107 palm civets later sampled at 25 farms, spread over 12 provinces, which were claimed to be the source of traded animals. Twenty-seven novel signature variation residues (SNVs) were identified on the spike gene and were analyzed for their phylogenetic relationships, based on 17 sequences obtained from animals in our study and from other published studies. Analysis indicated that the virus in palm civets at the live-animal market had evolved to infect humans. The evolutionary starting point was a prototype group consisting of three viral sequences of animal origin. Initially, seven SNV sites caused six amino acid changes, at positions 147, 228, 240, 479, 821, and 1080 of the spike protein, to generate low-pathogenicity viruses. One of these was linked to the first SARS patient in the 2003-2004 period. A further 14 SNVs caused 11 amino acid residue changes, at positions 360, 462, 472, 480, 487, 609, 613, 665, 743, 765, and 1163. The resulting high-pathogenicity groups were responsible for infections during the so-called early-phase epidemic of 2003. Finally, the remaining six SNVs caused four amino acid changes, at positions 227, 244, 344, and 778, which resulted in the group of viruses responsible for the global epidemic.