Molecular typing of Leptospira interrogans strains isolated from Rattus tanezumi in Guizhou Province, Southwest of China.

OBJECTIVE: To identify and type three leptospires isolated from Rattus tanezumi in Guizhou Province by using three molecular techniques (PFGE, MLVA, and MLST), reveal the molecular characteristic of causative agents of local leptospirosis and evaluate these three molecular methods based on their detection resolution and efficiency. METHODS: Three Leptospira strains were isolated from the kidney of Rattus tanezumi and cultured with EMJH medium. PFGE, MLVA, and MLST assays were applied to type the three strains isolated from Rattus tanezumi in Guizhou Province. RESULTS: PFGE, MLVA, and MLST typing showed that the three leptospiral isolates matched with leptospiral serogroup Icterohaemorrhagiae serovar Lai. The findings of the genotyping methods were consistent. MLVA and MLST defined genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes, and the findings of molecular typing were also consistent with those of traditional techniques. CONCLUSION: Three leptospiral isolates from Guizhou Province matched with leptospiral serogroup Icterohaemorrhagiae serovar Lai, and PFGE, MLVA, and MLST, as reliable molecular techniques for identifying and typing of Leptospira interrogans, would contribute to the active surveillance, outbreak investigation and source tracking for leptospirosis in Guizhou Province.

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.

[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.

[Surveillance on severe acute respiratory syndrome associated coronavirus in animals at a live animal market of Guangzhou in 2004].

OBJECTIVE: To study the prevalence of severe acute respiratory syndrome coronavirus (SARS-CoV) like virus in animals at a live animal market of Guanzhou in 2004 before and after culling of wild animal action taken by the local authority, in order to predict the re-emerging of SARS from animal originals in this region. METHODS: Animals at live animal market were sampled for rectal and throat swabs in triplicate. A single step realtime reverse transcription-polymerase chain reaction (RT-PCR) diagnostic kit was performed for screening SARS-CoV like virus, the manual nested RT- PCR and DNA sequencing were performed for confirmation. Only specimens which tested positive for both of the N and P genes by nested RT-PCR were scored as positive. RESULTS: In 31 animals sampled in January 5 2004 before culling of wild animals at Guangdong Province, including 20 cats (Felis catus), 5 red fox (Vulpes vulpes) and 6 Lesser rice field rats (Rattus losea), 8 (25.8%) animals were tested positive for SARS-CoV like virus by RT-PCR methods, of which 4 cats, 3 red fox and one Lesser rice field rats were included. However, two weeks after culling of animals and disinfection of the market were implemented, in 119 animals sampled in January 20 2004, including 6 rabbits (Oryctolagus cuniculus), 13 cats, 46 red jungle fowl (Gallus gallus), 13 spotbill duck (Anas platyrhynchos), 10 greylag goose (Anser anser), 31 Chinese francolin (Franclinus pintadeanus), only rectal swab from one greylag goose was tested positive for SARS-CoV like virus. Furthermore, in 102 animals that including 14 greylag gooses, 3 cats, 5 rabbits, 9 spotbill duck (Anaspoecilorhyncha), 2 Chinese francolin (Franclinus pintadeanus), 8 common pheasant (Phasianus colchicus), 6 pigeons, 9 Chinese muntjac (Muntiacus reevesi), 19 wild boar (Sus scrofa), 16 Lesser rice field rats, 5 dogs, 1 mink (Mustela vison), 3 goats, 2 green peafowl (Pavo muticus) sampled in April, May, June, July, August and November, only rectal swab from one pig was tested positive. However, of 12 and 10 palm civets sampled in November and December including five of which had been at the live animals market for 2 days, none of them was tested positive. CONCLUSION: This findings revealed that animals being sampled in April, May, June, July, August and November of 2004, only one rectal swab from a pig was tested positive as SARS-CoV like virus, much lower than the results from the previous year, suggesting that the possibility of re-emerging of human infection from animal origins is low for the winter of 2004-2005.

Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing.

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum and Borrelia burgdorferi, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.

[Distribution of virulence associated genes among strains of Leptospira].

OBJECTIVE: To analyze factors related to the virulence associated genes of Leptospires. METHODS: Twelve putative virulence associated genes were detected by polymerase chain reaction (PCR) method in 38 reference strains, 81 field strains of Leptospira interrogans isolated from patients or animals, and 12 avirulent strains of Leptospira biflexa. RESULTS: These putative virulent genes were widely distributed among the strains of Leptospira interrogans, but only few of them were detected in Leptospira biflexa. Gene lipL32 was detected in all strains of Leptospira interrogans. Distribution of gene lipL36 was varied significantly with detected rates from 0 to 90.91%. Gene la1608 had a positive rate of 87.50% for strains of serogroup Icterohaemorrhagiae, but was only detected in few strains of other serogroups with a range from 0 to 25.00%. Rate of detection on gene sphA was 17.65% in Leptospira interrogans, and was absent in serovar hardjo reference strain. CONCLUSION: Results indicated that these genes might be of importance for the virulence and pathogenicity of Leptospira interrogans, while gene lipL32 might be one of the common antigens. Gene lipL36 might be involved in serogroup specificity with genetic diversity, but gene la1608 was as one of the genes with specificity for serogroup Icterohaemorrhagiae. However, serovar hadjo might hold quite different genetic characteristics when compared with the other serovars of Leptospires.