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.

[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.

[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.

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.

[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.