[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.

[Study on the location of membrane and detection of antibody in the sera of genus-specific antigen LipL41s in patients with Leptospira interrogans].

OBJECTIVE: To determine the location on outer envelope and natural antibody response and types of genus-specific lipoprotein antigen LipL41s in patients with Leptospira interrogans. METHODS: Microscope agglutination test (MAT) was used to examine leptospirosis patients' serum samples from Sichuan area, China. Ni-NTA affinity chromatography was performed to extract the target recombinant rLipL41/1 and rLipL41/2 products that expressed under inducement of IPTG. Western blot assay was performed to detect the immunoreactivity between the sera from the patients infected with different serogroups of L. interrogans and rLipL41s. Immune aurosol electron microscopy was selected to locate the position of LipL41s on leptospiral envelope. ELISA based on rLipL41s was established to confirm the level and types of specific antibody. RESULTS: L. interrogans serogroup icterohaemorrhagiae remained to be the most dominant leptospiral serogroup in Sichuan area. All the sera from patients infected with different serogroups of L. interrogans could efficiently recognize the LipL41s which were the protein molecular that located on the external surface of leptospiral envelope. In the 156 serum samples from MAT positive leptospirosis patients, the positive rates for rLipL41/1 or rLipL41/2 specific IgM appeared to be 84.6%-87.8% and 78.2%-83.3%, respectively, while for rLipL41/1 or rLipL41/2 specific IgG they were 69.2%-81.4% and 75.0%-80.1%, respectively. CONCLUSION: LipL41s were the leptospiral superficial protein antigen of L. interrogans. Both the LipL41/1 and LipL41/2 could induce serum antibodies IgM and IgG with extensive antigenic-cross reaction during natural infection of L. interrogans in general populations. Hence, rLipL41/1 or rLipL41/2 could be used as the antigen candidate for developing universal genetic engineering vaccine and detection kit.

[Membrane location and naturally antibody responses of genus-specific antigen OmpL1s of Leptospira interrogans].

The major aim of this study is to determine the location on outer envelope of the genus-specific antigen OmpL1s of Leptospira interrogans, and the inducement of naturally antibody response and types of the antigen, which will offer the evidences to use OmpL1s as the antigen candidate for developing universal genetic engineering vaccine and detection kit. The serum samples from 156 leptospirosis patients in Sichuan area were detected using microscope agglutination test (MAT). By using PCR plus nucleotide sequence analysis, the genotypes of the dominant L. interrogans serogroups in China were demonstrated. Routine genetic engineering technique was applied to construct the prokaryotic expression systems of genotypes ompL1/1 and ompL1/2, and Ni-NTA affinity chromatography was performed to extract the target recombinant products rOmpL1/1 and rOmpL1/2. Immune aurosol electron microscopy was selected to locate the position of OmpL1s on leptospiral envelope. ELISAs based on rOmpL1s were established to confirm the level and types of specific antibody. The results indicated that L. interrogans serogroup Icterohaemorrhagiae remains to be the most important dominant leptospiral serogroup in Sichuan area. There are two ompL1 genotypes of ompL1/1 and ompL1/2 in the dominant leptospiral serogroup in China. And remarkable differences of the nucleotide and putative amino acid sequence similarities between the two genotypes are present. OmpL1s are the protein molecular that located on the external surface of leptospiral envelope. In the 156 cases of leptospirosis patients' serum samples using different dilutions, the positive rates for rOmpL1/1 or rOmpL1/2 specific IgM are 67.9%-79.5% and 75.0%-75.6%, while for for rOmpL1/1 or rOmpL1/2 specific IgG are 71.8%-79.5% and 75.0%-76.9%, respectively. All the results mentioned above lead to the conclusions that OmpL1s is the leptospiral genus-specific superficial protein antigen of L. interrogans and both rOmpL1/1 and rOmpL1/2 can induce humoral response in individuals naturally infected with L. interrogans as well as produce two serum antibodies IgM and IgG with extensive antigenic-cross reaction. Therefore, rOmpL1/1 and rOmpL1/2 can be used as the antigen candidate for developing universal genetic engineering vaccine and detection kit.