Background and Objective: Leptospirosis is a disease caused by pathogenic Leptospira prevalent in tropical countries like the Philippines. Some studies have shown that the role of currently used antibiotics for leptospirosis is unclear since trials have found no significant benefit to patient outcomes compared to placebo. This signals the need for alternative therapies, such as herbal medicines, which may provide effective therapeutic regimens in treating this infection. In this study, we characterized the antibacterial potential of three Philippine herbal medicines against Leptospira interrogans. Methods: Crude methanolic extracts of Momordica charantia, Cassia alata, and Allium sativum were subjected to an optimized broth microdilution assay against L. interrogans, utilizing the resazurin-resorufin reaction as a cell proliferation and viability indicator. Results: The respective minimum inhibitory concentrations of the plants were found to be as follows: 1.25 mg/mL (M. charantia), 2.5 mg/mL (C. alata), and >5 mg/mL (A. sativum). Conclusions Among the three herbal medicines, M. charantia and C. alata proved to have antibacterial activity against L. interrogans. Given the promising potential of two of these plant extracts, exploring the use of other solvents to extract natural compounds from these plants, and discovering possible synergistic effects between these plants and conventional antibiotics may be worthwhile.
Leptospira interrogans ; Herbal Medicine

BACKGROUND AND OBJECTIVE

Leptospirosis is a disease caused by pathogenic Leptospira prevalent in tropical countries like the Philippines. Some studies have shown that the role of currently used antibiotics for leptospirosis is unclear since trials have found no significant benefit to patient outcomes compared to placebo. This signals the need for alternative therapies, such as herbal medicines, which may provide effective therapeutic regimens in treating this infection. In this study, we characterized the antibacterial potential of three Philippine herbal medicines against Leptospira interrogans.

METHODS

Crude methanolic extracts of Momordica charantia, Cassia alata, and Allium sativum were subjected to an optimized broth microdilution assay against L. interrogans, utilizing the resazurin-resorufin reaction as a cell proliferation and viability indicator.

RESULTS

The respective minimum inhibitory concentrations of the plants were found to be as follows: 1.25 mg/mL (M. charantia), 2.5 mg/mL (C. alata), and >5 mg/mL (A. sativum).

CONCLUSIONS

Among the three herbal medicines, M. charantia and C. alata proved to have antibacterial activity against L. interrogans. Given the promising potential of two of these plant extracts, exploring the use of other solvents to extract natural compounds from these plants, and discovering possible synergistic effects between these plants and conventional antibiotics may be worthwhile.

Leptospira Interrogans ; Momordica Charantia ; Herbal Medicine ; Herbal

Rodents are well-known reservoirs and vectors of many emerging and re-emerging infectious diseases, but little is known about their role in zoonotic disease transmission in Bhutan. In this study, a cross-sectional investigation of zoonotic disease pathogens in rodents was performed in Chukha district, Bhutan, where a high incidence of scrub typhus and cases of acute undifferentiated febrile illness had been reported in people during the preceding 4–6 months. Twelve rodents were trapped alive using wire-mesh traps. Following euthanasia, liver and kidney tissues were removed and tested using PCR for Orientia tsutsugamushi and other bacterial and rickettsial pathogens causing bartonellosis, borreliosis, human monocytic ehrlichiosis, human granulocytic anaplasmosis, leptospirosis, and rickettsiosis. A phylogenetic analysis was performed on all rodent species captured and pathogens detected. Four out of the 12 rodents (33.3%) tested positive by PCR for zoonotic pathogens. Anaplasma phagocytophilum, Bartonella grahamii, and B. queenslandensis were identified for the first time in Bhutan. Leptospira interrogans was also detected for the first time from rodents in Bhutan. The findings demonstrate the presence of these zoonotic pathogens in rodents in Bhutan, which may pose a risk of disease transmission to humans.
Anaplasma ; Anaplasma phagocytophilum ; Anaplasmosis ; Animals ; Bartonella ; Bartonella Infections ; Bhutan ; Communicable Diseases, Emerging ; Ehrlichiosis ; Euthanasia ; Humans ; Incidence ; Kidney ; Leptospira ; Leptospira interrogans ; Leptospirosis ; Liver ; Orientia tsutsugamushi ; Polymerase Chain Reaction ; Rodentia ; Scrub Typhus ; Zoonoses

Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.
Antigens, Bacterial/*immunology ; Argentina ; Bulgaria ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Leptospira/isolation & purification/metabolism ; Leptospira interrogans/isolation & purification/metabolism ; Leptospirosis/*diagnosis/microbiology ; Male ; Polysaccharides/*immunology ; Reagent Kits, Diagnostic/*standards ; Republic of Korea ; Sensitivity and Specificity

Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.
Antigens, Bacterial/*immunology ; Argentina ; Bulgaria ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Leptospira/isolation & purification/metabolism ; Leptospira interrogans/isolation & purification/metabolism ; Leptospirosis/*diagnosis/microbiology ; Male ; Polysaccharides/*immunology ; Reagent Kits, Diagnostic/*standards ; Republic of Korea ; Sensitivity and Specificity

In this study, we selected only serologically identified 15 Leptosira interrogans isolates in the past and analyzed and identified them by using molecular method. The partial 16S rDNA and LipL32 genes were amplified from the bacteria by polymerase chain reaction (PCR) and sequenced. Sizes of the PCR products were 529 bp and 819 bp respectively and analysis of the nucleotide sequence of 16S rDNA and LipL32 genes showed that 14 out the 15 Leptospira showed 99.4~100% and 99.2~99.9% similarity respectively to those of L. interrogans lai and one isolate named HS-7 showed 100% and 100% similarity to L. interrogans canicola. The phylogenetic tree based on the 16S rDNA and LipL32 genes obtained the study revealed that 14 of the Leptospira composed a cluster distinct to that of L. interrogans lai and HS-7 composed to L. interrogans canicola.
Bacteria ; Base Sequence ; DNA, Ribosomal* ; Leptospira interrogans ; Leptospira* ; Polymerase Chain Reaction

OBJECTIVETo construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters.

METHODSThe groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT.

RESULTSThe nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels.

CONCLUSIONThe GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

Agglutination Tests ; Animals ; Antigens, Bacterial ; immunology ; Chaperonin 60 ; genetics ; immunology ; Cricetinae ; Gene Expression ; Leptospira interrogans ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

OBJECTIVETo determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism.

METHODSOmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays.

RESULTSThe bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01).

CONCLUSIONExpression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cell Line ; Chaperonin 60 ; genetics ; metabolism ; Humans ; Leptospira interrogans ; genetics ; immunology ; pathogenicity ; Lipoproteins ; genetics ; metabolism ; Macrophages ; microbiology

Leptospirosis is an important public health problem in the Republic of Korea (ROK), occurring sporadically or in outbreaks during the autumn season. Wild rodents, Apodemus agrarius, have been mainly involved in human leptospirosis. The majority of carrier animals are infected with Leptospira interrogans serovar lai. The characteristic pulmonary involvement or hemorrhage may increase the clinical severity or result in fatal outcomes, and these aspects continue to be a threat to people in endemic areas. While the disease incidence has been relatively low in recent years, there have been newer findings of livestock (zoo animals and racing horses) and rats (Rattus norvegicus) captured in urban environments as potential animal carriers. Many avenues of research are still open to define current changes in ecology, epidemiology, and the disease burden in both humans and animals in the ROK, together with global warming and climate change issues. In addition, national policy regarding the weighted wildlife monitoring system and the enhanced disease surveillance program is required to facilitate better monitoring and understanding of this disease.
Animals ; Climate Change ; Continental Population Groups ; Disease Outbreaks ; Ecology ; Fatal Outcome ; Global Warming ; Hemorrhage ; Humans ; Incidence ; Leptospira ; Leptospira interrogans ; Leptospirosis ; Livestock ; Murinae ; Public Health ; Rats ; Republic of Korea ; Rodentia ; Seasons

A 21-kDa leptospiral lipoprotein (LipL21) was evaluated for its diagnostic potential to detect bovine leptospirosis by ELISA. Both native LipL21 (nLipL21) and recombinant LipL21 (rLipL21) proteins were tested and compared regarding diagnostic efficiency, and no statistically significant difference was observed. The sensitivity of rLipL21 ELISA for 62 microscopic agglutination test (MAT) positive sera was 100% and the specificity with 378 MAT negative sera was 97.09%. Thus, rLipL21 protein-based ELISA could be used as an alternative to MAT for the diagnosis of bovine leptospirosis.
Animals ; Antibodies, Bacterial/blood ; Antigens, Bacterial/biosynthesis/*chemistry/genetics ; Bacterial Outer Membrane Proteins/biosynthesis/*chemistry/genetics ; Cattle ; Cattle Diseases/blood/*microbiology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Leptospira interrogans/*isolation & purification ; Leptospirosis/blood/microbiology/*veterinary ; Lipoproteins/biosynthesis/*chemistry/genetics ; Recombinant Proteins/biosynthesis/chemistry/genetics ; Sensitivity and Specificity