Non-pathogenic Serovars Research Articles

Molecular Detection of lsa21 Gene Encoding an Adhesion Protein Among Pathogenic Leptospiral Serovars

Background: Leptospirosis, as an emerging global public health problem, is a widespread zoonosis and substantial infectious disease caused by pathogenic Leptospira spp. The diagnosis of the disease remains a challenge due to its non-specific clinical symptoms. The Lsa21 protein is one of the leptospiral proteins that has extracellular matrix-binding properties that are expressed during the infection of pathogenic Leptospira serovars. Objectives: The study aimed to assess the presence of the lsa21 gene in Leptospira serovars. Materials and Methods: The study was conducted on 22 pathogenic Leptospira serovars and non-pathogenic Leptospira serovars, obtained from the Reference Laboratory for Leptospira, Razi Vaccine, and Serum Research Institute, Karaj, Iran. DNA Leptospira serovars were extracted and amplified by the polymerase chain reaction (PCR) using specific primers. Results: An approximately 540-bp DNA fragment from the DNA of all pathogenic Leptospira serovars was amplified by the PCR not found in non-pathogenic serovars. Conclusion: Molecular detection of pathogenic leptospires based on the lsa21 gene can be utilized for laboratory diagnosis and could be a good candidate for developing vaccines against leptospirosis.

Biochemical analysis of leptospiral LPS explained the difference between pathogenic and non-pathogenic serogroups

Lipopolysaccharide (LPS) is the major surface antigen of Leptospira. In this study, the genes involved in the LPS biosynthesis were analyzed and compared by bioinformatics tools. Also, the chemical composition analysis of leptospiral lipopolysaccharides (LPS) extracted from 5 pathogenic serovars like Autumnalis, Australis, Ballum, Grippotyphosa, Pomona, and the nonpathogenic serovar Andamana was performed. Methods used were Limulus amebocyte lysate assay (LAL), gas chromatography-mass spectrometry (GC-MS), fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance spectroscopy (NMR). LAL assay showed a significantly higher level of endotoxicity among pathogenic serovars (~0.490 EU/mL) than that of nonpathogenic Andamana (~0.102 EU/mL). FAMES analysis showed the presence of palmitic acid (C16:0), hydroxy lauric acid (3-OH-C12:0), and oleic acid (C18:0). Palmitoleic acid (C16: 1), and 3- hydroxy palmitate (3-OH-C16:0) was detected only in pathogenic serovars. In contrast myristoleic acid (C14:1) and stearic acid (C18:0) were present in Andamana. FTIR analysis revealed C–O–C stretch of esters, 3°ROH functional groups and carbohydrate vibration range were similar among pathogenic serovars. The NMR analysis reveals similarity for 6 deoxy sugars and methyl groups of Autumnalis, Australis, and Ballum. Further, the presence of palmitoleic acid and 3-hydroxy palmitate may be the significant pathogen-associated predisposing factor. This mediates high osmolarity glycerol (HOG) mediated stress response in leptospiral LPS mediated pathogenesis.

Cloning and sequencing of the ompL37 gene present in Leptospira interrogans, a surface protein in pathogenic leptospires

Leptospirosis, an infection caused by pathogenic leptospires, is associated with insufficient sanitation and poverty. Leptospira is transmitted through contact with contaminated urine of reservoir animals. The primary objective of this study was to clone and sequence the ompL37 gene present in local and vaccine serovars. A total of 16 Leptospira interrogans serovars were cultured in EMJH liquid medium. After growing, genomic DNA was extracted using phenol-chloroform method. Primer pair was synthesized to amplify the 996 bp ompL37 sequence. The amplified ompL37 gene was cloned into pTZ57R/T vector. The sequences obtained from this study were compared with an only recorded sequence in the Genbank by the Meg Align software. PCR products showed an amplified 996bp ompL37 gene product belonging to pathogenic serovars, while no ompL37 products were amplified in non-pathogenic serovars. Sequences comparison tests from 16 native serotypes examined in this study displayed a similarity range of 84% to 99.5% among serovars used. The results showed that two serotypes of L. interrogans including Serjoehardjo (RTCC2810 and RTCC2821) had the highest identity up to 95.5%. Two serovars of L. interrogans including Pomona (RTCC2822) and Icterohaemorrhagiae (RTCC2823) had the lowest identity about 84%. As the results showed, ompL37, present on the surface of such bacteria, showed a conserved sequence. ompL37, as a key role in cell adhesion and pathogenicity, can be used for designing diagnostic tests and vaccines. Furthermore, sequencing of various sites in ompL37 gene, including binding sites and immunogenic epitopes, can be valuable alternatives for future studies.

Discovery of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones as a new class of pathogen specific anti-leptospiral agents

A simple and efficient method for the synthesis of a series of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one derivatives starting from 5-carboalkoxy-2,3-dihydropyranone (5-CDHPs) has been developed. Pyranoisoxazolones 10a-j, dihydronaphthopyran-4-one (DHNPs) class of natural product 12b and 12c and its analogues 12a and 13a-c were preliminarily screened against pathogenic leptospiral serovar Autumnalis strain N2 at various concentrations. Six pyranoisoxazolones, 10b, 10d, 10f, 10g, 10i and 10j which displayed very good anti-leptospiral activity was taken for secondary screening against twelve strains of pathogenic and one non-pathogenic leptospiral serovars. While all the compounds displayed significant anti-leptospiral activity against the pathogenic serovars at MIC of 62.5–500 μg/mL. Compounds 10d, 10g and 10j did not show any significant effect on non-pathogenic serovar. Inhibition of leptospires at a significant level by pyranoisoxazolone 10g was confirmed using RT-qPCR assay. In vivo treatment of BALB/c mice with compound 10g revealed that, it has 95% survivability against the pathogenic strain Canicola and also showed inhibition of renal colonization of leptospires. Compound 10g was found to show cytotoxicity against THP-1 cells only at higher concentration (≥75 μg/mL). Effective binding of compound 10g with leptospiral outer membrane protein LipL32 observed via in silico molecular docking provided a suitable explanation for pathogen specificity of compound 10g. Antibiotics acting against leptospirosis in human are very few. The results obtained from in vitro, in vivo and in silico study reveals that 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones class of compounds are lead molecules for further development as pathogen specific anti-leptospiral agents.

Evaluation of polymerase chain reaction assay for pathogenic Leptospira detection in stored urine

Pathogenic Leptospira spp. is the etiological agent of leptospirosis, a worldwide zoonotic infectious disease that causes jaundice, hemorrhages, renal failure and abortion in susceptible species. Urine is one of the preferred clinical samples for the detection of the agent. However due to its reliability, detection of leptospires in stored samples is challenged. Here we evaluated the capability of a polymerase chain reaction (PCR) assay for detecting pathogenic leptospira DNA in a non-sterile collected urine, stored at different times and temperatures. Our results indicate that the PCR protocol used detect pathogenic leptospira DNA but not non-pathogenic serovars or other non-leptospiral microorganisms. The sensitivity of the assay was of 100 Leptospira interrogans in 10 mL refrigerated neutralized urine within 72 h post collection. This protocol could be of considerable interest for public health workers, field veterinarians and laboratory scientists, in sampling and processing urine for the detection of pathogenic Leptospira spp.

Analysis of differential proteomes in pathogenic and non‐pathogenic Leptospira: Potential pathogenic and virulence factors

Leptospirosis is a bacterial zoonotic disease caused by spirochetes in the genus Leptospira. To date, factors determining the pathogenicity and virulence of leptospires remain unclear. We performed a gel-based proteomic analysis to evaluate differential leptospiral proteomes in the pathogenic L. interrogans (serovars Australis, Bratislava, Autumnalis, and Icterohaemorrhagiae) and the non-pathogenic L. biflexa (serovar Patoc). Quantitative proteome analysis and MS protein identification revealed 42 forms of 33 unique proteins whose levels were significantly greater in the pathogenic serovars compared with the non-pathogenic serovar. Among the four pathogenic serovars, the more virulent serovar Icterohaemorrhagiae (which is most commonly associated with severe leptospirosis in patients) had significantly greater levels of 14 forms of 12 unique proteins, when compared with the other three pathogenic serovars. Some of these identified proteins may serve as the pathogenic and/or virulence factors of leptospirosis.

Leptospira DNA detection for the diagnosis of human leptospirosis

This study aims to analyse PCR applicability to the diagnosis of human leptospirosis and to compare the sensitivity of two primer pairs in urine and blood samples. PCR with G1/G2 and LP1/LP2 primers was specific and able to detect 10pg of DNA by agarose gel and 1pg by hybridization. Twenty-one serovars, representing 20 serogroups of pathogenic leptospires, were amplified with G1/G2 primers. DNA from two non-pathogenic serovars, Andamana and Patoc, was not amplified. For hybridization, one probe employing DNA from most prevalent leptospires (serovars Icterohaemorrhagiae, Copenhageni, and Autumnalis) was chosen in accordance with the microagglutination titres in patient samples. It was observed that not all serovars hybridized with the PCR products of G1/G2 and LP1/LP2 primer amplification, suggesting heterogeneity in the sequence amplified by these primers. G1/G2-primed amplifications of blood and/or urine samples were shown to be significantly more sensitive (57.6%) than the LP1/LP2 primers (33.3%), P=0.04, when positivity of patients is considered. When each primer pair and only urine samples were considered, PCR positivity was higher for G1/G2 primers than for LP1/LP2 (P=0.007). G1/G2 presented greater sensitivity in urine than in blood, and LP1/LP2 presented greater sensitivity in blood than in urine, although these differences were not statistically significant. The positivity of PCR per patient using both primers in blood and/or urine samples was 63.6%, with 84.4% efficiency. PCR was useful for patients without microagglutination detectable antibodies, for whom it was able to diagnose nine out of 11 patients (81.8%).

Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira

Two hybridomas secreting monoclonal antibodies (MAbs) that react with a lipoprotein (LipL32) of the outermembrane of pathogenic Leptospira were obtained. For hybridoma production, spleen cells from BALB/cmice imunized with recombinant LipL32 (rLipL32) were fused to SP2/O-Ag14 cells, selected in HAT mediumand screened in an indirect ELISA. One MAb produced was of the IgG2b isotype and the other was an IgM.MAbs specificity was confirmed by indirect ELISA and immunoblotting using purified rLipL32 and whole-cellantigen preparations from Escherichia coli (E. coli) expressing LipL32 and from pathogenic and non-pathogenic serovars. Both Mabs reacted with most of the pathogenic serovars tested and none reacted withnon-pathogenic Leptospira. The MAbs described have potential for use in diagnostic tests for leptospirosis.

Pulsed-field gel electrophoretic analysis of leptospiral DNA

The genomic structures of spirochete species are not well characterized, and genetic studies on these organisms have been hampered by lack of a genetic exchange mechanism in these bacteria. In view of these observations, pulsed-field gel electrophoresis was used to examine the genomes of Leptospira species. Live cells, prepared in agarose plugs, were lysed in situ, and the DNA was analyzed under different electrophoretic conditions. Pulsed-field gel electrophoresis of DNA digested with infrequently cutting restriction enzymes showed that the genome of Leptospira interrogans serovar canicola is approximately 3.1 Mb, while that of the saprophytic L. biflexa serovar patoc I is 3.5 Mb. DNA forms of approximately 2,000 and 350 kb which were present in samples from L. interrogans serovars were not readily detected in nonpathogenic serovars. Three distinct populations, designated type alpha, beta, and gamma, of L. interrogans DNA molecules were further analyzed with two-dimensional gel electrophoresis. Evidence suggested that two of these DNA forms, type alpha and gamma, were linear structures. Pulsed-field gel electrophoresis has proven to be a valuable tool with which to size bacterial genomes and to take the first steps toward characterization of a form of leptospiral DNA which behaves as a linear molecule and which may be related to the virulence of L. interrogans.

Prevalence of leptospirosis in man. Pilot survey.

A survey on the prevalence of leptospirosis was performed on the population living in an area of central Italy. The size of the sample was calculated in order to provide significant results in the case of a prevalence of infection in not less than 1% of the population. Results demonstrated an unexpectedly wide circulation of leptospirosis in the surveyed area, showing a prevalence rate of infection of 11.34% for people living in rural areas and 3.08% for people living in the main town. The highest prevalence of infection (17.44%) was found in people between 30 and 44 years of age, living in rural areas. Such a wide circulation of undiagnosed past leptospiral infections was attributed both to the prevalence of mild clinical cases of leptospirosis in humans and the lack of microbiological tests performed to differentiate current leptospirosis from other infectious illnesses. An unexpected persistence in sera of co-agglutinins towards non-pathogenic serovars of L. biflexa was also noticed in healthy people. Criteria were established for the extension of the survey on the prevalence of leptospirosis to cover larger areas by limiting sampling to the more exposed age groups and to areas representative of a larger land belt.