Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira that infects humans and a wide range of animals. By combining computational prediction and high-accuracy tandem m...Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira that infects humans and a wide range of animals. By combining computational prediction and high-accuracy tandem mass spectra, we revised the genome annotation of Leptospira interrogans serovar Lai, a free-living pathogenic spirochete responsible for leptospirosis, providing substantial peptide evidence for novel genes and new gene boundaries. Subsequently, we presented a high-coverage proteome analysis of protein expression and multiple posttranslational modifications （PTMs）. Approximately 64.3% of the predicted L. interrogans proteins were cataloged by detecting 2 540 proteins. Meanwhile, a profile of multiple PTMs was concurrently established, containing in total 32 phosphorylated, 46 acetylated and 155 methylated proteins. The PTM systems in the serovar Lai show unique features. Unique eukaryotic-like features of L. interrogans protein modifications were demonstrated in both phosphorylation and arginine methylation. This systematic analysis provides not only comprehensive information of high-coverage protein expression and multiple modifications in prokaryotes but also a view suggesting that the evolutionarily primitive L. interrogans shares significant similarities in protein modification systems with eukaryotes.展开更多
BACKGROUND Leptospira is an uncommon pathogen for adult severe community-acquired pneumonia and its nonspecific manifestations and limited diagnostic tests make it difficult to identify.Although conventional penicilli...BACKGROUND Leptospira is an uncommon pathogen for adult severe community-acquired pneumonia and its nonspecific manifestations and limited diagnostic tests make it difficult to identify.Although conventional penicillin remains efficacious to treat leptospirosis,failure in early diagnosis and treatment can lead to progression into a deadly syndrome with multiple organ dysfunction.Next generation sequencing is of great value to understand cases with infection of unknown cause,which could help in the diagnosis of uncertain Leptospira infection.CASE SUMMARY We recently managed a patient with fever,cough and dyspnea on admission that progressed into persistent adult respiratory distress syndrome,hemoptysis and hematuria after admission.In this case,the rare Leptospira infection was clouded by the positive influenza tests at admission,delaying early Leptospira-targeted antibiotics administration.Next generation sequencing,a novel molecular diagnostic tool,provided a key hint to uncover the crucial pathogen,Leptospira interrogans,further supported by the possible occupational exposure history.Subsequent conventional penicillin and mechanical respiratory support were administrated to cure the patient successfully without any sequela.CONCLUSION Clinicians must pay attention to possible exposure history and keep uncommon Leptospira in mind when managing pneumonia with unknown causes.展开更多
Objective.To construct recombinant BCG again st leptospirosis.Methods.We amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain 017.Two recombin ant plasmids pBQ...Objective.To construct recombinant BCG again st leptospirosis.Methods.We amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain 017.Two recombin ant plasmids pBQ1and pBQ2were constructed by oriented ligation based on the E.coli-BCG shuttle plasmids pMV261and pMV361respectively.The recombinant plasmids were transformed into BCG by electroporation.The rBCGs bearing pBQ1and pBQ2were induced by high temperature of 45℃.Results.The expressed product,a 35kD prote in was detected by SDS-PAGE.The resu lt indicates that pBQ1and pBQ2can express OmpL1in rBCG.Conclusion.The technical methods in this study may help detect the immunogenicity a nd immunoprotection of OmpL1and develop more safe,highl y effective rBCG bearing leptospira l antigen with long-lasting protection.展开更多
Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutin...Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.展开更多
AIM： To evaluate the production of reactive oxygen species （ROS） and the expression of inducible nitric oxide synthase （iNOS） in rat isolated Kupffer cells （KCs） stimulated by Leptospira interrogans and Borreli...AIM： To evaluate the production of reactive oxygen species （ROS） and the expression of inducible nitric oxide synthase （iNOS） in rat isolated Kupffer cells （KCs） stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS： Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Pudfied Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS： B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION： Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.展开更多
Leptospirosis is a potential threat to public health. An increasing number of people infected with Leptospira were reported in Bantul District, Yogyakarta special region with a case fatality rate (CFR) of 7.8%. Infect...Leptospirosis is a potential threat to public health. An increasing number of people infected with Leptospira were reported in Bantul District, Yogyakarta special region with a case fatality rate (CFR) of 7.8%. Infected areas in the district have increased from 2 to 15 sub districts. Leptospirosis is caused by Leptospira bacteria and spread by direct contact with infected rodents and indirect contact through contaminated water or soil. Leptospira in rats, water and soil were detected using real-time quantitative polymerase chain reaction (qPCR). The sites of sampled materials were geocoded using Global Positioning System (GPS). Spatial analysis was used to predict the spread of Spira. This study aims to perform the mapping, clustering, and predicting the spread of Leptospira in Bantul Yogyakarta Indonesia. Data were collected from three sub-districts: Sedayu, Sewon and Bantul. The result showed that 38.04% from 368 samples were Spira positive. There were four significant clusters of infection spread source. Spira is predicted to spread in, and out from, Bantul District.展开更多
Objective:To compare the efficiency of routine diagnostic PCR assays in detecting pathogenic Leptospira isolated from water and soils.Methods:Seven routine assays targeting six genes(lip L32,fla B,gyr B,lfb1,sec Y and...Objective:To compare the efficiency of routine diagnostic PCR assays in detecting pathogenic Leptospira isolated from water and soils.Methods:Seven routine assays targeting six genes(lip L32,fla B,gyr B,lfb1,sec Y and lig B)were evaluated and compared on the cultures of two groups of pathogenic Leptospira from different sources.One group included 19 described reference strains recovered from infected human or animals,and another group included 22 environmental isolates from recreational and residential sites in Malaysia.The latter have been confirmed for presence of pathogenic Leptospira DNA.PCR positivity or detection sensitivity of each assay was determined and compared between the two groups.Results:Validation on reference strains showed 100.0%PCR sensitivity for all assays except lig B-PCR(95.0%)that failed to amplify Leptospira interrogans serovar Pomona.In marked contrast,there was a notable decline in sensitivity in the environmental isolates(lip L32-PCR,95.5%;fla B-PCR,90.9%;gyr B-PCR,77.3%;lfb1-PCR,59.1%;sec Y-PCRs,40.9%G1/G2-PCR,36.4%;lig B-PCR,13.6%),implying a large genetic distance between the two groups,as well as nucleotide polymorphism among environmental isolates.Conclusions:High proportion of false-negative PCR results suggests a need of prudent selection of primers in detecting environmental pathogenic Leptospira.These findings offer valuable insights on the extensive biodiversity of genus Leptospira and its impact on the efficacy and development of molecular detection tool.展开更多
Objective: To detect genetic variations among pathogenic Leptospira isolated from rats using 16 S r RNA gen as chronometer. Methods: This is an observational study with cross sectional design. Rats saples were taken i...Objective: To detect genetic variations among pathogenic Leptospira isolated from rats using 16 S r RNA gen as chronometer. Methods: This is an observational study with cross sectional design. Rats saples were taken in Yogyakarta Special Region of Indonesia. Leptospira in the rats was detected by two methods ie. real time PCR(q PCR) by using primers correspond to16 S r RNA gene of Leptospira, and standard PCR by using dif erent set of primer correspond to the 16 S r RNA gene of Leptospira. The standard PCR amplicon then subjected for DNA sequencing. Analysis genetic variation was performed using MEGA 6.2. Software. Results:There were 99 DNA samples from rats included in this study. Detection of Leptospira by using q PCR revealed 25 samples positive for pathogenic Leptospira, while only 6 samples were able to be detected using standard PCR. The new primer set correspond to 16 S r RNA gene was able to detect specii cally pathogenic Leptospira in the rats. Sequencing analysis of 6 PCR amplicons showed that the Leptospira which infect the rats catched in Yogyakarta genetically close related with pathogenic Leptospira which were isolated from human, animal, rodents, and environment. Conclusions: It can be considered that rats are the most important vector and reservoir of Leptospira.展开更多
Objective:Pulmonary haemorrhage is an increasing cause of death in leptospirosis patients.However,molecular mechanism underlying pathologies in this organ is not clearly understood.It has been shown that sodium transp...Objective:Pulmonary haemorrhage is an increasing cause of death in leptospirosis patients.However,molecular mechanism underlying pathologies in this organ is not clearly understood.It has been shown that sodium transport was disturbed following Leptospira infection.LipL21 is the second abundant outer membrane protein found only in pathogenic Leptospira.Its expression in vivo has been shown which suggests that this protein may be involved in survival in hosts or pathogenesis.However,the expression of this protein in host organs and its role in lung pathology has not been demonstrated.In this study we demonstrated the expression of LipL21 in lungs of hamsters infected with pathogenic Leptospira.Methods:Lung tissues were collected from Golden Syrian hamsters injected with Leptospira interrogans serovar Pyrogenes at days 3,5 and 7 post-infection.Four hamsters were used for each time point.Lungs from non-infected hamsters were collected as a control group.LipL21 mRNA expression in lung tissues was investigated by reverse transcription and nested PCR.Results:LipL21 mRNA expression was detected in all lung tissues from hamsters infected with pathogenic Leptospira.No PCR product was detected when tissues from non-infected hamsters were investigated.Conclusion:Our data demonstrated that LipL21 is expressed in lungs of hamsters infected with pathogenic Leptospira.Additional experiments such as quantitation and localization of LipL21 expression in lungs will provide further information whether this protein is involved in pathogenesis.展开更多
To explore the closing mechanism of the substrate pocket,we perform a 16,000 ps molecular dynamics simulation separately on the ligand-free and actinonin-bound peptide deformylase from Leptospira interrogans.Our resul...To explore the closing mechanism of the substrate pocket,we perform a 16,000 ps molecular dynamics simulation separately on the ligand-free and actinonin-bound peptide deformylase from Leptospira interrogans.Our results show that the CD-loop, hydrophilic inhibitor and hydrophobic cluster are necessary for the formation of semi-open conformation,and Tyr71 plays an important role in mediating the movements of CD-loop.The average MD structure of the actinonin-bound LiPDF complex approaches to the crystal structure.These are consistent with experiment very well.展开更多
Objective:To report the renal histological lesions in synanthropic rodents,Mus musculus and Rattus rattus,naturally infected with Leptospira spp.,captured in a rural community in Yucatan,Mexico.Methods:Kidney samples ...Objective:To report the renal histological lesions in synanthropic rodents,Mus musculus and Rattus rattus,naturally infected with Leptospira spp.,captured in a rural community in Yucatan,Mexico.Methods:Kidney samples of synanthropic rodents were collected from a rural community in Yucatan,Mexico.Polymerase chain reaction was used to detect Leptospira spp.infection.Tissue kidney was fixed in 10%buffered formalin,processed according to the usual techniques for paraffin inclusion,cut and stained with hematoxylin and eosin,and examined using a conventional electronic microscope.Results:A total of 187 rodents were captured.Nine individuals(4.8%)were positive for Leptospira spp.in the molecular analysis.All renal lesions observed in the histopathological study had been reported previously for Leptospira spp.infection.Conclusions:The histopathological lesions are present in the kidneys,plus the results of the polymerase chain reaction confirm that these rodents are true carriers of Leptospira spp.展开更多
Objective: To establish a DNA detection platform based on a tapered optical fiber to detect Leptospira DNA by targeting the leptospiral secY gene.Methods: The biosensor works on the principle of light propagating in t...Objective: To establish a DNA detection platform based on a tapered optical fiber to detect Leptospira DNA by targeting the leptospiral secY gene.Methods: The biosensor works on the principle of light propagating in the special geometry of the optical fiber tapered from a waist diameter of 125 to 12 μm. The fiber surface was functionalized through a cascade of chemical treatments and the immobilization of a DNA capture probe targeting the secY gene. The presence of the target DNA was determined from the wavelength shift in the optical transmission spectrum.Results: The biosensor demonstrated good sensitivity, detecting Leptospira DNA at 0.001 ng/μL, and was selective for Leptospira DNA without cross-reactivity with non-leptospiral microorganisms. The biosensor specifically detected DNA that was specifically amplified through the loop-mediated isothermal amplification approach.Conclusions: These findings warrant the potential of this platform to be developed as a novel alternative approach to diagnose leptospirosis.展开更多
Chronic kidney disease is commonly diagnosed in dogs,and clinical signs may be aggravated when infected agents are involved.In this case report,33 dogs with chronic kidney disease were clinically evaluated and serolog...Chronic kidney disease is commonly diagnosed in dogs,and clinical signs may be aggravated when infected agents are involved.In this case report,33 dogs with chronic kidney disease were clinically evaluated and serologically tested for Leptospira spp.,Ehrlichia canis,and Anaplasma phagocytophilum.The seroprevalence for Leptospira spp.was 39.4%.The most frequent serovars found were Pyrogenes,Canicola,Bratislava and Australis,with serological titers between 1:100 to 1:800.Clinical signs included fever,depression,decreased body condition,vomiting and hema‑turia.Signifcant laboratory fndings were anemia,leukocytosis,thrombocytopenia,increased liver enzymes,urea and creatinine,hyperbilirubinemia and hyperphosphatemia.All leptospira seronegative dogs were positive for one or both monitored homoparasites(i.e.,E.canis and A.phagocytophilum);only three leptospira seropositive dogs were positive for one or both hemoparasites.Findings also suggest that endemic hemoparasites of dogs should be moni‑tored in dogs with a kidney condition for a better clinical picture of the patients and therapeutic approach.展开更多
To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira b...To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.展开更多
To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemor- rhagiae serovar l...To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemor- rhagiae serovar lai stain 56601 were cloned and their prokaryotic expression systems were constructed. It was demonstrated that the cloned flhA and flhB2 genes had 2118 bp in length and showed 100% and 99.9% of homologies in their nucleotide sequences and 100% and 98.8% of homologies in their putative amino acid sequences respectively, in comparison with those of previously reported. The prokaryotic expression systems under the induction with IPTG could efficiently express the target proteins rFlhA and rFlhB2 with the outputs of approximate 10% of the total bacterial proteins. Based on the sequences of the cloned genes flhA and flhB2, the structural features in associated with pathogenesis and the functions of the target proteins were analyzed with bioinformatics softwares, in which the FlhA was found to have 7 major transmembrane helices, while the FlhB2 had 5 ones. The conserved domains in the FlhA showed high similarity to those of the FHIPEP of the other bacterial FlhA and EscV families, but the conserved domains in the FlhB2 were similar to those of bac-export-2 and EscU families, EscV and EscU families being the protein products of the type IR secretion system in association with pathogenesis. The FlhA and FlhB2 also contained protein kinase C （PKC） and protein tyrosine kinase （PTK） phosphorylation sites, indicating that PKC and PTK of host cells were involved in the internalization and intracellular proliferation in the pathogenesis of microorganisms. All these data leads to a conclusion that the flhA and flhB2 genes of L. interrogans are relatively conserved and their gene products have great potential in the pathogenesis of this organism.展开更多
The life-threatening infections caused by Leptospira serovars demand the need for designing anti-leptospirosis drugs. The present study encompasses exploring inhibitors against phosphoheptose isomerase （GmhA） of Lep...The life-threatening infections caused by Leptospira serovars demand the need for designing anti-leptospirosis drugs. The present study encompasses exploring inhibitors against phosphoheptose isomerase （GmhA） of Leptospira, which is vital for lipopolysaccharide （LPS） biosynthesis and is identified as a common drug target through the subtractive genomic approach. GmhA model was built in Modeller 9v7. Structural refinement and energy minimization of the predicted model was carried out using Maestro 9.0. The refined model reliability was assessed through Procheck, ProSA, ProQ and Profile 3D. The substrate-based virtual high-throughput screening （VHTS） in Ligand.Info Meta-Database tool generated an in-house library of 354 substrate structural analogs. Furthermore, structure-based VHTS from the in-house library with different conformations of each ligand provided 14 novel competitive inhibitors. The model together with insight gained from the VHTS would be a promising starting point for developing anti-leptospirosis competitive inhibitors targeting LPS biosynthesis pathway.展开更多
In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1（＋）. The guinea pigs were immuni...In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1（＋）. The guinea pigs were immunized with pcDNA3.1（＋）-lipL21, pcDNA3.1（＋） or PBS. Six weeks after the second immunization, the splenocytes were isolated to detect their proliferative ability by lymphocyte transformation experiments. In addition, microscopic agglutination test was used for quantitative detection of specific antibodies. The rest guinea pigs were challenged intraperitoneally with L. interogans sorevar Lai. Then, protective effect was evaluated on the basis of survival and histopathological lesions in the kidneys, lungs, and liver. The lipL21 gene was successfully expressed in COS-7 cells through recombinant pcDNA3.1（＋）-lipL21. The titer of specific antibodies substantially increased, and the stimulation index of splenocytes increased significantly. Hence, the pcDNA3.1（＋）-lipL21 could protect the immunized guinea pigs from homotypic Leptospira infection. Furthermore, no obvious pathologic changes were observed in the pcDNA3.1（＋）-lipL21 immunized guinea pigs. The results showed that the protective effect with pathogenic strains of Leptospira was shared by LipL21 mediated through a plasmid vector. Consequently, these results indicated that the lipL21 DNA vaccine was a promising candidate for the prevention of leptospirosis.展开更多
文摘Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira that infects humans and a wide range of animals. By combining computational prediction and high-accuracy tandem mass spectra, we revised the genome annotation of Leptospira interrogans serovar Lai, a free-living pathogenic spirochete responsible for leptospirosis, providing substantial peptide evidence for novel genes and new gene boundaries. Subsequently, we presented a high-coverage proteome analysis of protein expression and multiple posttranslational modifications （PTMs）. Approximately 64.3% of the predicted L. interrogans proteins were cataloged by detecting 2 540 proteins. Meanwhile, a profile of multiple PTMs was concurrently established, containing in total 32 phosphorylated, 46 acetylated and 155 methylated proteins. The PTM systems in the serovar Lai show unique features. Unique eukaryotic-like features of L. interrogans protein modifications were demonstrated in both phosphorylation and arginine methylation. This systematic analysis provides not only comprehensive information of high-coverage protein expression and multiple modifications in prokaryotes but also a view suggesting that the evolutionarily primitive L. interrogans shares significant similarities in protein modification systems with eukaryotes.
基金Basic Public Welfare Research Scheme of Zhejiang Province,No.GF19H030001.
文摘BACKGROUND Leptospira is an uncommon pathogen for adult severe community-acquired pneumonia and its nonspecific manifestations and limited diagnostic tests make it difficult to identify.Although conventional penicillin remains efficacious to treat leptospirosis,failure in early diagnosis and treatment can lead to progression into a deadly syndrome with multiple organ dysfunction.Next generation sequencing is of great value to understand cases with infection of unknown cause,which could help in the diagnosis of uncertain Leptospira infection.CASE SUMMARY We recently managed a patient with fever,cough and dyspnea on admission that progressed into persistent adult respiratory distress syndrome,hemoptysis and hematuria after admission.In this case,the rare Leptospira infection was clouded by the positive influenza tests at admission,delaying early Leptospira-targeted antibiotics administration.Next generation sequencing,a novel molecular diagnostic tool,provided a key hint to uncover the crucial pathogen,Leptospira interrogans,further supported by the possible occupational exposure history.Subsequent conventional penicillin and mechanical respiratory support were administrated to cure the patient successfully without any sequela.CONCLUSION Clinicians must pay attention to possible exposure history and keep uncommon Leptospira in mind when managing pneumonia with unknown causes.
文摘Objective.To construct recombinant BCG again st leptospirosis.Methods.We amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain 017.Two recombin ant plasmids pBQ1and pBQ2were constructed by oriented ligation based on the E.coli-BCG shuttle plasmids pMV261and pMV361respectively.The recombinant plasmids were transformed into BCG by electroporation.The rBCGs bearing pBQ1and pBQ2were induced by high temperature of 45℃.Results.The expressed product,a 35kD prote in was detected by SDS-PAGE.The resu lt indicates that pBQ1and pBQ2can express OmpL1in rBCG.Conclusion.The technical methods in this study may help detect the immunogenicity a nd immunoprotection of OmpL1and develop more safe,highl y effective rBCG bearing leptospira l antigen with long-lasting protection.
文摘Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.
文摘AIM： To evaluate the production of reactive oxygen species （ROS） and the expression of inducible nitric oxide synthase （iNOS） in rat isolated Kupffer cells （KCs） stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS： Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Pudfied Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS： B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION： Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.
文摘Leptospirosis is a potential threat to public health. An increasing number of people infected with Leptospira were reported in Bantul District, Yogyakarta special region with a case fatality rate (CFR) of 7.8%. Infected areas in the district have increased from 2 to 15 sub districts. Leptospirosis is caused by Leptospira bacteria and spread by direct contact with infected rodents and indirect contact through contaminated water or soil. Leptospira in rats, water and soil were detected using real-time quantitative polymerase chain reaction (qPCR). The sites of sampled materials were geocoded using Global Positioning System (GPS). Spatial analysis was used to predict the spread of Spira. This study aims to perform the mapping, clustering, and predicting the spread of Leptospira in Bantul Yogyakarta Indonesia. Data were collected from three sub-districts: Sedayu, Sewon and Bantul. The result showed that 38.04% from 368 samples were Spira positive. There were four significant clusters of infection spread source. Spira is predicted to spread in, and out from, Bantul District.
文摘Objective:To compare the efficiency of routine diagnostic PCR assays in detecting pathogenic Leptospira isolated from water and soils.Methods:Seven routine assays targeting six genes(lip L32,fla B,gyr B,lfb1,sec Y and lig B)were evaluated and compared on the cultures of two groups of pathogenic Leptospira from different sources.One group included 19 described reference strains recovered from infected human or animals,and another group included 22 environmental isolates from recreational and residential sites in Malaysia.The latter have been confirmed for presence of pathogenic Leptospira DNA.PCR positivity or detection sensitivity of each assay was determined and compared between the two groups.Results:Validation on reference strains showed 100.0%PCR sensitivity for all assays except lig B-PCR(95.0%)that failed to amplify Leptospira interrogans serovar Pomona.In marked contrast,there was a notable decline in sensitivity in the environmental isolates(lip L32-PCR,95.5%;fla B-PCR,90.9%;gyr B-PCR,77.3%;lfb1-PCR,59.1%;sec Y-PCRs,40.9%G1/G2-PCR,36.4%;lig B-PCR,13.6%),implying a large genetic distance between the two groups,as well as nucleotide polymorphism among environmental isolates.Conclusions:High proportion of false-negative PCR results suggests a need of prudent selection of primers in detecting environmental pathogenic Leptospira.These findings offer valuable insights on the extensive biodiversity of genus Leptospira and its impact on the efficacy and development of molecular detection tool.
文摘Objective: To detect genetic variations among pathogenic Leptospira isolated from rats using 16 S r RNA gen as chronometer. Methods: This is an observational study with cross sectional design. Rats saples were taken in Yogyakarta Special Region of Indonesia. Leptospira in the rats was detected by two methods ie. real time PCR(q PCR) by using primers correspond to16 S r RNA gene of Leptospira, and standard PCR by using dif erent set of primer correspond to the 16 S r RNA gene of Leptospira. The standard PCR amplicon then subjected for DNA sequencing. Analysis genetic variation was performed using MEGA 6.2. Software. Results:There were 99 DNA samples from rats included in this study. Detection of Leptospira by using q PCR revealed 25 samples positive for pathogenic Leptospira, while only 6 samples were able to be detected using standard PCR. The new primer set correspond to 16 S r RNA gene was able to detect specii cally pathogenic Leptospira in the rats. Sequencing analysis of 6 PCR amplicons showed that the Leptospira which infect the rats catched in Yogyakarta genetically close related with pathogenic Leptospira which were isolated from human, animal, rodents, and environment. Conclusions: It can be considered that rats are the most important vector and reservoir of Leptospira.
基金supported by National Research Council of Thailand and Rachadapiseksompoj Grant, Faculty of Medicine,Chulalongkorn University, Thailand.
文摘Objective:Pulmonary haemorrhage is an increasing cause of death in leptospirosis patients.However,molecular mechanism underlying pathologies in this organ is not clearly understood.It has been shown that sodium transport was disturbed following Leptospira infection.LipL21 is the second abundant outer membrane protein found only in pathogenic Leptospira.Its expression in vivo has been shown which suggests that this protein may be involved in survival in hosts or pathogenesis.However,the expression of this protein in host organs and its role in lung pathology has not been demonstrated.In this study we demonstrated the expression of LipL21 in lungs of hamsters infected with pathogenic Leptospira.Methods:Lung tissues were collected from Golden Syrian hamsters injected with Leptospira interrogans serovar Pyrogenes at days 3,5 and 7 post-infection.Four hamsters were used for each time point.Lungs from non-infected hamsters were collected as a control group.LipL21 mRNA expression in lung tissues was investigated by reverse transcription and nested PCR.Results:LipL21 mRNA expression was detected in all lung tissues from hamsters infected with pathogenic Leptospira.No PCR product was detected when tissues from non-infected hamsters were investigated.Conclusion:Our data demonstrated that LipL21 is expressed in lungs of hamsters infected with pathogenic Leptospira.Additional experiments such as quantitation and localization of LipL21 expression in lungs will provide further information whether this protein is involved in pathogenesis.
基金supports of the National Key Basic Research Priorities Program(No.2003CCA027)the National Natural Science Foundation of China(No.20573064).
文摘To explore the closing mechanism of the substrate pocket,we perform a 16,000 ps molecular dynamics simulation separately on the ligand-free and actinonin-bound peptide deformylase from Leptospira interrogans.Our results show that the CD-loop, hydrophilic inhibitor and hydrophobic cluster are necessary for the formation of semi-open conformation,and Tyr71 plays an important role in mediating the movements of CD-loop.The average MD structure of the actinonin-bound LiPDF complex approaches to the crystal structure.These are consistent with experiment very well.
基金supported by PROMEP-México-Proyecto 103.5/09/1258(Red Epidemiológica de Enfermedades Zoonóticas y Transmitidas por Vector de Importancia en Salud Pública)Consejo Nacional de Ciencia y Tecnología de México(No.grant320021)
文摘Objective:To report the renal histological lesions in synanthropic rodents,Mus musculus and Rattus rattus,naturally infected with Leptospira spp.,captured in a rural community in Yucatan,Mexico.Methods:Kidney samples of synanthropic rodents were collected from a rural community in Yucatan,Mexico.Polymerase chain reaction was used to detect Leptospira spp.infection.Tissue kidney was fixed in 10%buffered formalin,processed according to the usual techniques for paraffin inclusion,cut and stained with hematoxylin and eosin,and examined using a conventional electronic microscope.Results:A total of 187 rodents were captured.Nine individuals(4.8%)were positive for Leptospira spp.in the molecular analysis.All renal lesions observed in the histopathological study had been reported previously for Leptospira spp.infection.Conclusions:The histopathological lesions are present in the kidneys,plus the results of the polymerase chain reaction confirm that these rodents are true carriers of Leptospira spp.
基金funded by Universiti Putra Malaysia through the Geran Inisiatif Putra Siswazah (GP-IPS/2019/9678200)。
文摘Objective: To establish a DNA detection platform based on a tapered optical fiber to detect Leptospira DNA by targeting the leptospiral secY gene.Methods: The biosensor works on the principle of light propagating in the special geometry of the optical fiber tapered from a waist diameter of 125 to 12 μm. The fiber surface was functionalized through a cascade of chemical treatments and the immobilization of a DNA capture probe targeting the secY gene. The presence of the target DNA was determined from the wavelength shift in the optical transmission spectrum.Results: The biosensor demonstrated good sensitivity, detecting Leptospira DNA at 0.001 ng/μL, and was selective for Leptospira DNA without cross-reactivity with non-leptospiral microorganisms. The biosensor specifically detected DNA that was specifically amplified through the loop-mediated isothermal amplification approach.Conclusions: These findings warrant the potential of this platform to be developed as a novel alternative approach to diagnose leptospirosis.
文摘Chronic kidney disease is commonly diagnosed in dogs,and clinical signs may be aggravated when infected agents are involved.In this case report,33 dogs with chronic kidney disease were clinically evaluated and serologically tested for Leptospira spp.,Ehrlichia canis,and Anaplasma phagocytophilum.The seroprevalence for Leptospira spp.was 39.4%.The most frequent serovars found were Pyrogenes,Canicola,Bratislava and Australis,with serological titers between 1:100 to 1:800.Clinical signs included fever,depression,decreased body condition,vomiting and hema‑turia.Signifcant laboratory fndings were anemia,leukocytosis,thrombocytopenia,increased liver enzymes,urea and creatinine,hyperbilirubinemia and hyperphosphatemia.All leptospira seronegative dogs were positive for one or both monitored homoparasites(i.e.,E.canis and A.phagocytophilum);only three leptospira seropositive dogs were positive for one or both hemoparasites.Findings also suggest that endemic hemoparasites of dogs should be moni‑tored in dogs with a kidney condition for a better clinical picture of the patients and therapeutic approach.
文摘To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.
文摘To determine the pathogenic potential of the leptospiral flagella-associated proteins, the genes flhA and flhB2 encoding the biosynthesis of flagella of leptospira interrogans serogroup icterohaemor- rhagiae serovar lai stain 56601 were cloned and their prokaryotic expression systems were constructed. It was demonstrated that the cloned flhA and flhB2 genes had 2118 bp in length and showed 100% and 99.9% of homologies in their nucleotide sequences and 100% and 98.8% of homologies in their putative amino acid sequences respectively, in comparison with those of previously reported. The prokaryotic expression systems under the induction with IPTG could efficiently express the target proteins rFlhA and rFlhB2 with the outputs of approximate 10% of the total bacterial proteins. Based on the sequences of the cloned genes flhA and flhB2, the structural features in associated with pathogenesis and the functions of the target proteins were analyzed with bioinformatics softwares, in which the FlhA was found to have 7 major transmembrane helices, while the FlhB2 had 5 ones. The conserved domains in the FlhA showed high similarity to those of the FHIPEP of the other bacterial FlhA and EscV families, but the conserved domains in the FlhB2 were similar to those of bac-export-2 and EscU families, EscV and EscU families being the protein products of the type IR secretion system in association with pathogenesis. The FlhA and FlhB2 also contained protein kinase C （PKC） and protein tyrosine kinase （PTK） phosphorylation sites, indicating that PKC and PTK of host cells were involved in the internalization and intracellular proliferation in the pathogenesis of microorganisms. All these data leads to a conclusion that the flhA and flhB2 genes of L. interrogans are relatively conserved and their gene products have great potential in the pathogenesis of this organism.
文摘The life-threatening infections caused by Leptospira serovars demand the need for designing anti-leptospirosis drugs. The present study encompasses exploring inhibitors against phosphoheptose isomerase （GmhA） of Leptospira, which is vital for lipopolysaccharide （LPS） biosynthesis and is identified as a common drug target through the subtractive genomic approach. GmhA model was built in Modeller 9v7. Structural refinement and energy minimization of the predicted model was carried out using Maestro 9.0. The refined model reliability was assessed through Procheck, ProSA, ProQ and Profile 3D. The substrate-based virtual high-throughput screening （VHTS） in Ligand.Info Meta-Database tool generated an in-house library of 354 substrate structural analogs. Furthermore, structure-based VHTS from the in-house library with different conformations of each ligand provided 14 novel competitive inhibitors. The model together with insight gained from the VHTS would be a promising starting point for developing anti-leptospirosis competitive inhibitors targeting LPS biosynthesis pathway.
基金grants from the Hunan Provincial Education Department (06B088)the Hunan Provincial Health Department (2004B165)
文摘In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1（＋）. The guinea pigs were immunized with pcDNA3.1（＋）-lipL21, pcDNA3.1（＋） or PBS. Six weeks after the second immunization, the splenocytes were isolated to detect their proliferative ability by lymphocyte transformation experiments. In addition, microscopic agglutination test was used for quantitative detection of specific antibodies. The rest guinea pigs were challenged intraperitoneally with L. interogans sorevar Lai. Then, protective effect was evaluated on the basis of survival and histopathological lesions in the kidneys, lungs, and liver. The lipL21 gene was successfully expressed in COS-7 cells through recombinant pcDNA3.1（＋）-lipL21. The titer of specific antibodies substantially increased, and the stimulation index of splenocytes increased significantly. Hence, the pcDNA3.1（＋）-lipL21 could protect the immunized guinea pigs from homotypic Leptospira infection. Furthermore, no obvious pathologic changes were observed in the pcDNA3.1（＋）-lipL21 immunized guinea pigs. The results showed that the protective effect with pathogenic strains of Leptospira was shared by LipL21 mediated through a plasmid vector. Consequently, these results indicated that the lipL21 DNA vaccine was a promising candidate for the prevention of leptospirosis.
