LEPTOSPIROSIS: A NEGLECTED DISEASE FROM NORTH INDIA

Introduction: Leptospirosis is a zoonotic disease caused by Leptospira interrogans and has been reported from various countries worldwide. As very few studies were conducted on leptospirosis from north India, this study was conducted to know the status of this disease in this region. This retrospective hospital Material & Methods: based study was conducted in the Department of Microbiology of a tertiary care super specialty teaching institute from north India for a period of two consecutive years. Blood specimens from acute febrile illness cases were tested for presence of IgM antibodies against Leptospira interrogans by rapid card (Leptocheck from TULIP) testing and ELISA (Leptospira IgM ELISA from PanBio). Out of total 216 samples Results: collected and included in this study, 40 were found to be positive for presence of IgM antibodies against Leptospira interrogans. Seropositivity for leptospirosis was observed to be 19%. Maximum number of patients were from economically productive age groups, 31-40 years of age group followed by 21-30 and 41-50 years of age groups. CONCLUSION: Leptospirosis was found to be a major cause of acute febrile illness from north India. It is neglected and under reported from most of the regions of India due to lack of clinician's suspicion. More studies with more samples are required on leptospirosis from this region to reach on final conclusion.

Hemolysin Sph2 from Leptospira interrogans amplified inflammatory response in murine macrophages J774A.1 through HMGB1 / 中华微生物学和免疫学杂志

Objective:To investigate the possible mechanism of high mobility group box-1 (HMGB1) in amplifing inflammatory responses in Leptospira interrogans hemolysin Sph2-treated J774A.1 macrophages. Methods:Recombinant Sph2 was incubated with J774A.1 macrophages. The damage of cell membrane was detected by lactate dehydrogenase(LDH) determination; the changes of cell structure were observed by cryo-electron microscope; ELISA was used to determine the expression of HMGB1. After the commercial recombinant HMGB1 was incubated with mouse J774A.1 macrophages, the phosphorylation of NF-κB, p38-MAPK and JNK signaling pathway wsa detected by Western blot, and the expression of IL-1β, IL-6, and KC (IL-8) was detected by ELISA.Results:Recombinant hemolysin rSph2 induced significant changes in the structures of J774A.1 cells, including nucleus disappearance, cell membrane structure damage, cell lysis and membrane swelling. The yields of LDH and HMGB1 also increased significantly. Phosphorylated-NF-κB, -p38-MAPK and -JNK were increased by HMGB1. The expression of IL-1β, IL-6 and KC in J774A.1 cells was up-regulated by HMGB1 and inhibited via inhibitors of NF-κB, p38-MAPK and JNK signal pathways.Conclusions:Hemolysin rSph2 damaged the membrane of J774A.1 cells, and induced the secretion of HMGB1. Secreted-HMGB1 might induce the expression of IL-1β, IL-6 and KC in J774A.1 cells via NF-κB, p38-MAPK and JNK signal pathways, thus amplifying the inflammatory responses caused by Sph2.

Eclipta alba Derived Phytochemicals against Jaundice

Phytochemicals otherwise known assecondary metabolites means the small organic molecules that are not essential for growth, development and reproduction but can protect from various diseases. Infact these phytochemicals are the key source of medicine. Secondary metabolites from Eclipta alba plant extract are traditionally used to cure Jaundice which is caused by Leptospira interrogans. The curative property of Eclipta alba against Leptospira interrogans was proved by molecular docking method in “Biovia Discovery Studio”. “High positive values of -CDOCKER energy and -CDOCKER interaction energy” recommended that pentadecan, heptadecane, 6,10,14-trimethyl-2-petadecanone can successfully deactivate the thioredoxin-disulfide reductase enzyme thereby disrupting the cellular function as well as the lifecycle of causative organism

High level of serovar copenhageni detection in dog sera from Patrocínio, MG, Brazil / Alto nível de detecção do sorovar copenhageni em soros de cães de Patrocínio, MG, Brasil

This study aimed to evaluate the occurrence of canine leptospirosis and the possible risk factors associated with the disease in the municipality of Patrocínio, MG, Brazil. A cross-sectional observational study was carried out from July through August 2017. The municipality was divided into four regions (north, south, east and west) and a predefined number of neighborhoods (25) were randomly sampled in each region. Samples of blood serum were collected from 241 domiciled male and female dogs of different breeds and ages. To investigate the risk factors for canine leptospirosis, the owners of the animals were asked to fill out an epidemiological questionnaire. The following factors were evaluated: breed, sex, age, presence of rodents, type of diet, access to the street, vaccination, presence of flooded areas, and educational level of the owners. Blood serum samples were evaluated by the Microscopic Agglutination Test (MAT), using a collection of 24 live antigens. Of the 241 dogs evaluated, 32 (13.2%) were reactive. The most frequent serovars were: Copenhageni (37.5%) and Canicola (21.8%), followed by Icterohaemorrhagiae and Grippotyphosa (12.5%), Pomona, Tarassovi and Butembo (9.3%) and Hardjo (6.2%). The presence of canine leptospirosis was associated with purebred dogs (OR=0.3059 [95% CI: 0.1430 ­ 0.6547]) and vaccination (OR=2.581 [95% CI: 1.198 ­ 5.563]). It was concluded that some dogs in the municipality of Patrocínio, MG have anti-Leptospiraspp. antibodies and that the serovars most frequently identified were Copenhageni (37.5%) and Canicola (21.8%). Pure breeds and vaccination were factors associated with the prevalence of infection.

O objetivo deste estudo foi avaliar a ocorrência da leptospirose canina e os possíveis fatores de riscos associados à doença no município de Patrocínio ­ MG. Foi realizado um estudo observacional transversal, durante os meses de Julho à Agosto de 2017. O município foi divido em quatro regiões (norte, sul, leste e oeste) e um número predefinido de bairros (25) foi amostrado aleatoriamente em cada região. Foram colhidas 241 amostras de soro sanguíneo de cães domiciliados de ambos os sexos e de diferentes raças e idades. Para investigação dos fatores de risco da leptospirose canina foi aplicado um questionário epidemiológico aos tutores dos animais, foram avaliados os fatores: raça, sexo, idade, presença de roedores, tipo de dieta, acesso à rua, vacinação, presença de áreas alagadas, terrenos baldios e grau de escolaridade dos tutores. As amostras de soro sanguíneo foram avaliadas pelo exame de Soroaglutinação Microscópica (SAM), com uma coleção de vinte e quatro antígenos vivos. Dos 241 cães avaliados, 32 (13,2%) apresentaram-se reagentes. Os sorovares de maior frequência foram: Copenhageni (37,5%) e Canicola (21,8%), seguido por Icterohaemorrhagiae e Grippotyphosa (12,5%), Pomona, Tarassovi e Butembo (9,3%) e Hardjo (6,2%). A presença de leptospirose canina foi associada em cães com raça definida (OR = 0,3059 [IC 95%: 0,1430 ­ 0,6547]) e vacinação (OR = 2,581 [IC 95%: 1.198 ­ 5.563]). Concluiu-se que existem cães que apresentam anticorpos anti-Leptospira spp., no município de Patrocínio-MG e que os sorovares Copenhageni (37,5%) e Canicola (21,8%) foram os de maior ocorrência. Apresentar raça definida e a vacinação foram fatores associados à prevalência da infecção.

DNA nanovaccines prepared using LemA antigen protect Golden Syrian hamsters against Leptospira lethal infection

BACKGROUND Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.

In-vitro antileptospiral activity of Canarium odontophyllum Miq. (Dabai) leaves extract

Aims@#To evaluate the anti-leptospiral activity of Canarium odontophyllum leaves against Leptospira interrogans serovar Bataviae and Leptospira borgpetersenii serovar Javanica. @*Methodology and results@#The extracts (hexane, acetone, methanol and aqueous) used in this study were tested at concentration ranging from 0.049 mg/mL to 25 mg/mL using broth microdilution method. Percentage inhibition (%) was obtained through OD reading at 400 nm. Only methanol extract was incubated with Leptospira to observe population changes under dark field microscope prior to subjected for DNA damaging studies through gel electrophoresis of genomic DNA. Methanol extract showed the highest percentage inhibition of 66% against L.interrogans serovar Bataviae and 74% against L. borgpetersenii serovar Javanica. The IC50 value of methanol extract was 4.60 mg/mL and 2.25 mg/mL against serovar Bataviae and serovar Javanica, respectively. Both Leptospira culture which was treated with IC50 value of methanol extract showed drastic decrease in population compared to untreated Leptospira for both serovar. There was no DNA damage towards serovar Bataviae. However, serovar Javanica exhibited DNA damage as observed from the presence of DNA fragmentation on the gel electrophoresis. @*Conclusion, significance and impact of study@#These findings confirmed that methanol leaves extract from of Canarium odontophyllum has a potential to control leptospirosis.

Differences in engulfing ability and phagolysosome formation between mononuclear-macrophages and neutrophils during Leptospira interrogans infection / 中华微生物学和免疫学杂志

Objective To understand the differences in engulfing ability and phagolysosome for-mation between mononuclear-macrophages and neutrophils during Leptospira interrogans infection. Methods Human THP-1 monocytes and HL-60 cells were pretreated with PMA ( phorbol-12-myristate-13-acetate) and ATRA ( all-trans retinoic acid) to differentiate them into mononuclear-macrophages and neutrophils, respec-tively. The phagocytosis of Leptospira interrogans in THP-1-PMA mononuclear-macrophages and HL-60-AT-RA neutrophils was detected by confocal microscopy. The morphology of intracellular Leptospira was deter-mined by transmission electron microscopy. The viability of phagocytized Leptospira and the percentages of dead Leptospira were analyzed by confocal microscopy and spectrofluorimetry, respectively. Confocal micros-copy was used to measure the formation of phagolysosomes in different phagocytes. Results Both THP-1-PMA mononuclear-macrophages and HL-60-ATRA neutrophils could phagocytize Leptospira interrogans, but the phagocytic ability of the former was notably stronger than that of the latter (P<0. 05). Intracellular Lep-tospira were surrounded by phagocytic vesicles in both types of phagocytes. THP-1-PMA mononuclear-mac-rophages were better than HL-60-ATRA neutrophils in killing intracellular Leptospira (P<0. 05). More phagolysosomes were formed in THP-1-PMA mononuclear-macrophages than in HL-60-ATRA neutrophils ( P<0. 05). Conclusions Human mononuclear-macrophages but not neutrophils act as major phagocytes that play an important role in phagocytizing and killing Leptospira during infection. Less fusion of the phagosomes with lysosomes may be responsible for the lower Leptospira-killing ability of neutrophils.

PAF-AH and PLA2 activity of Leptospira interrogans LA_2144 gene product / 中华微生物学和免疫学杂志

Objective To analyze the enzymatic activity of Leptospira interrogans ( L. interrogans) LA_2144 gene product to hydrolyze platelet activating factor acetylhydrolase ( PAF-AH) and phosphatidase A2(PLA2). Methods Bioinformatic softwares were used to predict transmembrane regions, signal peptides and domains of the LA_2144 gene of L. interrogans strain Lai. A prokaryotic expression system for signal peptide-free LA_2144 gene was established. The expressed target recombinant protein rLep2144 was extrac-ted by Ni-NTA affinity chromatography and then renatured. Spectrometry was used to detect the activity of rLep2144 to hydrolyze PAF-AH substrate 2-thio PAF and the Km and Kcat values as well as the activity to hy-drolyze PLA2 substrate arachidonoyl 2-thio PC. Real-time fluorescence quantitative RT-PCR and Western blot were performed to detect the transcription, protein expression and secretion of LA_2144 gene during infection of human and mouse vascular endothelial cells ( HUVEC and EOMA) with L. interrogans. Results L. interrogans LA_2144 gene contained a signal peptide and a domain belonging to SGNH hydrolase super-family, but no transmembrane regions. The established prokaryotic expression system for signal peptide-free LA_2144 gene could efficiently express rLep2144. The extracted rLep2144 was shown as a single protein fragment in separation gel and then successfully renatured. rLep2144 had a stronger PAF-AH activity with the Km and Kcat values of 688. 235 μmol/L and 0. 976/s, but its PLA2 activity was relatively weak. Expres-sion of the LA_2144 gene at mRNA and protein levels in HUVEC and EOMA was rapidly increased after the cells were infected with L. interrogans (P<0. 05) and the secretion of LA_2144 gene product could be detec-ted. Conclusions L. interrogans LA_2144 gene product had a stronger PAF-AH and a certain PLA2 activi-ty, which might involve in the hemorrhage and inflammatory response in leptospirosis.

Comparison of the phagocytosis and inflammatory response of murine macrophages infected by Leptospira strains 56606v and 56606a / 上海交通大学学报（医学版）

Objective : To compare the response and consequence of mouse macrophages infected by Leptospira interrogans 56606v and 56606a, and explore the mechanisms of pathogenic Leptospira to cause disease. Methods: Peritoneal macrophages of C57BL/6 mice were infected by pathogenic Leptospira 56606v and non-pathogenic Leptospira 56606a respectively. Immunofluorescence staining was performed to observe phagocytosis and clearance of Leptospira after infection, and realtime-PCR was used to determine cytokine production of macrophages. Results: After 72-hour infection, strain 56606v exhibited a lower phagocytic rate but survived after incubation with peritoneal macrophages compared with strain 56606a, which showed a higher phagocytic rate but was cleared at that time point. Additionally, cytokine production of macrophages incubated with 56606v was lower than that with 56606a. Conclusion: Leptospira interrogans strain 56606v can survive in macrophages, which may contribute to evasion from phagocytic clearance and lead to disease, while strain 56606a can be cleared, which implicates a lower pathogenicity.

Whole-genome sequencing of Leptospira interrogans from southern Brazil: genetic features of a highly virulent strain

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

Primer informe de Leptospira interrogans en el roedor sigmodontino Scapteromys aquaticus / First report on Leptospira interrogans in the sigmodontine rodent Scapteromys aquaticus / Primeiro relato da presença de Leptospira interrogans em roedores sigmodontíneos Scapteromys aquaticus

RESUMEN La leptospirosis es una enfermedad zoonótica de distribución mundial que puede transmitirse por contacto directo o indirecto con orina o tejidos de animales infectados. En Argentina, la leptospirosis es endémica en la provincia de Santa Fe y presenta brotes epidémicos durante las inundaciones. Sin embargo, se sabe muy poco sobre el papel que cumplen los roedores silvestres en la diseminación de la enfermedad en el país. El objetivo de este estudio fue identificar las especies hospederas de leptospiras patógenas entre los roedores presentes en un asentamiento ribereño de la provincia de Santa Fe. Se realizó un muestreo de roedores durante octubre de 2015. Los riñones de los animales capturados se analizaron por real-time PCR para el gen LipL32 de leptospiras patógenas. En los animales que resultaron positivos, se realizó test de microaglutinación (MAT) y tipificación molecular por amplificación del gen 16S rRNA y dos esquemas de MLST. Se capturaron 37 roedores de las especies Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus rattus y Scapteromys aquaticus. En el análisis por real-time PCR resultó positivo un macho de Scapteromys aquaticus. El suero de este individuo y del resto de los S. aquaticus capturados (n = 18) se analizaron por test de microaglutinación (MAT), y fueron no reactivos para los 10 serovares probados. La amplificación del gen 16S rRNA identificó la especie infectante como Leptospira interrogans, mientras que no se obtuvo amplificación para los dos esquemas de MLST. El hallazgo de este estudio aporta nueva información acerca de presencia de leptospiras patógenas en roedores silvestres, que es relevante para la zona por tratarse de una especie ampliamente distribuida en ambientes pantanosos e inundables de América del Sur.(AU)

ABSTRACT Leptospirosis is a globally distributed zoonosis that can be transmitted through direct or indirect contact with the urine or tissues of infected animals. In Argentina, leptospirosis is endemic in the province of Santa Fe and epidemic outbreaks occur during floods. However, very little is known about the role that wild rodents play in the spread of the disease in Argentina. The objective of this study was to identify the host species of pathogenic Leptospira among rodents in a riverine settlement in the province of Santa Fe. We conducted a trapping session in October 2015. Kidneys of the captured animals were analyzed by real-time PCR for the LipL32 gene of pathogenic Leptospira. Animals that were positive were subjected to microscopic agglutination test (MAT) and molecular typing by amplification of the 16S rRNA gene and two multilocus sequence typing (MLST) schemes. A total of 37 rodents of the species Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus rattus, and Scapteromys aquaticus were captured. Real-time PCR found one male Scapteromys aquaticus that was positive. The serum of this individual and of the rest of the S. aquaticus captured (n = 18) were analyzed by MAT and were non-reactive for the 10 serovars tested. Amplification of the 16S rRNA gene identified the infective species as Leptospira interrogans, while amplification could not be obtained for the two MLST schemes. The findings of this study contribute new information concerning the presence of pathogenic Leptospira in wild rodents, which is relevant in this region because the species is widely distributed in swampy and flood-prone environments of South America.(AU)

RESUMO A leptospirose é uma doença zoonótica de distribuição mundial transmitida pelo contato direto ou indireto com a urina ou os tecidos de animais infectados. Na Argentina, a leptospirose é endêmica na Província de Santa Fé com surtos epidêmicos ocorrendo com as enchentes. Sabe-se pouco sobre o papel dos roedores silvestres na propagação da doença no país. O objetivo deste estudo foi identificar as espécies hospedeiras de leptospiras patogênicas em roedores encontrados em um núcleo de povoamento ribeirinho na Província de Santa Fé. A amostragem dos roedores foi feita no mês de outubro de 2015. Os tecidos dos rins dos animais capturados foram analisados com a técnica de reação em cadeia da polimerase em tempo real (PCR-RT) quanto à presença do gene LipL32 de leptospiras patógenas. Para os animais com resultados positivos, foi realizado o teste de microaglutinação (MAT) e tipagem molecular baseada na amplificação do gene 16S rRNA e dois esquemas de tipagem por sequenciamento de locos múltiplos (MLST). Ao todo, foram capturados 37 roedores das espécies Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus e Scapteromys aquaticus. O ensaio de PCR-RT foi positivo em um roedor macho da espécie Scapteromys aquaticus. Os soros deste animal e dos outros S. aquaticus capturados (n = 18) foram analisados com o MAT e os resultados foram não reagentes para os 10 sorovares testados. A amplificação do gene 16S rRNA permitiu identificar a espécie infetante como sendo Leptospira interrogans e não houve amplificação nos dois esquemas de MLST. O achado deste estudo fornece um novo dado quanto à presença de leptospiras patogênicas em roedores silvestres, importante para esta área por se tratar de uma espécie de ampla distribuição em terras pantanosas e inundáveis da América do Sul.(AU)

Influences of Leptospira interrogans infection on the expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 / 中华微生物学和免疫学杂志

Objective To investigate the influences of Leptospira interrogans (L.interrogans) in-fection on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion mole-cule-1 (VCAM-1) on endothelial cells. Methods Expression of ICAM-1 and VCAM-1 at mRNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR) after infecting human umbilical vein endothelial cells (HUVEC) with L.interrogans strain Lai. Silver staining was used to detect leptospires in lung,liver and kidney tissues of L.interrogans-infected C3H/HeJ mice. Expression of ICAM-1 and VCAM-1 in lung,liver and kidney tissues of L.interrogans-infected mice was measured with immunohistochemistry. Results L.interrogans infection increased the expression of ICAM-1 and VCAM-1 on HUVEC(P<0.05). Moreover,the expression of VCAM-1 at mRNA level was significantly higher than that of ICAM-1 (P<0.05). Silver-stained leptospires could be found in lung,liver and kidney tissues of L.interrogans-infected C3H/HeJ mice. Results of the immunohistochemical examination showed that increased expression of both ICAM-1 and VCAM-1 could be detected in ling,liver and kidney tissues of L.interrogans-infected mice,and the VCAM-1 level was significantly higher than that of ICAM-1 in every tissue sample(P<0.05). Conclu-sion L.interrogans infection could induce the expression of ICAM-1 and VCAM-1 on endothelial cells and increase the expression of VCAM-1 to a level significantly higher than that of ICAM-1, which mediated the infiltration of specific inflammatory cells to the site of infection.

Role of alkyl hydroperoxide reductase subunit C (AhpC) as a peroxiredoxin in Leptospira interro-gans against oxidative stress / 中华微生物学和免疫学杂志

Objective To analyze the enzymatic properties of alkyl hydroperoxide reductase sub-unit C (AhpC) from Leptospira interrogans (L.interrogans) and to elucidate its physiological roles in host-pathogen interactions in macrophages during Leptospira infection. Methods A prokaryotic expression system for ahpC gene of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was established to ex-press the recombinant AhpC(rAhpC). After purified by Ni-NTA affinity chromatography,the enzymatic ac-tivity of the rAhpC and its role in protecting DNA from oxidation were analyzed. The importance of each cys-teine in its molecule was evaluated through site-directed mutation. L.interrogans strains were pretreated with or without Conoidin A, a covalent inhibitor of peroxiredoxin, and then were used to infect macrophages. Changes in oxidative status in leptospires and survival rates of L.interrogans strains were analyzed by fluores-cence-activated cell sorting and colony counting method. Results The rAhpC was successfully expressed in the established prokaryotic expression system. It had peroxiredoxin activity that was able to catalyze the re-duction of hydrogen peroxide. Its ability of reducing hydrogen peroxide depended on the thioredoxin/thiore-doxin reductase system. Cys47 (a peroxidatic cysteine) and Cys167 (a resolving cysteine) were critical to maintaining the enzymatic activity of AhpC. AhpC could protect DNA from hydrogen peroxide induced-oxida-tive damage. When L.interrogans strains were pretreated with Conoidin A,the oxidative status in leptospires was elevated and the survival of L.interrogans in macrophages was significantly reduced in a dose-dependent manner. Conclusion The AhpC of L.interrogans is a thioredoxin-dependent peroxiredoxin that plays an im-portant role in protecting L.interrogans against oxidative stress in macrophages.

Differences between infiltration of mononuclear-macrophages and neutrophils during Leptospira inter-rogans infection and underlying mechanisms / 中华微生物学和免疫学杂志

Objective To understand the differences between infiltration of mononuclear-macro-phages and neutrophils during Leptospira interrogans (L.interrogans) infection and the underlying mecha-nisms. Methods Histological changes in mouse lung, liver and kidney tissues were detected using hema-toxylin and eosin (HE) staining following infection of C3H/HeJ mice with L.interrogans serovar Lai strain 56601. Infiltration of peripheral blood-derived CD11b+mononuclear-macrophages and Ly6G+neutrophils in lung,liver and kidney tissues collected form L.interrogans-infected C3H/HeJ mice was detected with immu-nohistochemistry. Levels of mononuclear-macrophage chemokines and neutrophil chemokines in serum sam-ples of L.interrogans-infected mice were detected with chemokine detection microarray. Results Lung,liv-er and kidney tissue samples collected from L. interrogans-infected C3H/HeJ mice presented typical his-topathological changes of leptospirosis, such as inflammatory cell infiltration in these tissues, pulmonary hemorrhage,extensive hepatocyte necrosis and serious nephrohemia. Results of immunohistochemical stai-ning showed that a large number of peripheral blood-derived CD11b+mononuclear-macrophages were presen-ted in lung,liver and kidney tissues of L.interrogans-infected mice, but few neutrophils could be found in these tissues. The mouse chemokine detection microarray confirmed that the levels of mononuclear-macro-phage chemokines (I-309,MCP-1,MCP-5,MIP-1α and RANTES) in serum samples of L.interrogans-in-fected C3H/HeJ mice were significantly increased during infection (P<0.05), but the neutrophil chemokines(KC,LIX and MIP-2) analyzed in this study were not notably increased (P>0.05). Conclu-sion Mononuclear-macrophages rather than neutrophils are the major infiltrating phagocytes during L.inter-rogans infection and play a crucial role in the elimination of Leptospira invasion.

Biological activity and pathogenicity of M23 superfamily metalloendopeptidases of Leptospira interro-gans / 中华微生物学和免疫学杂志

Objective To understand and determine the biological activity and pathogenicity of metalloendopeptidases encoded by LA2582 and LA2901 genes of Leptospira interrogans(L.interrogans) sero-group Icterohaemorrhagiaeserovar Lai strain Lai. Methods Structures and functions of LA2582 and LA2901 genes were analyzed by using bioinformatic software. Prokaryotic expression systems for expressing the extra-cellular regions of LA2582 and LA2901 genes were generated. The target recombinant expression products, rLA2582 and rLA2901,were extracted by Ni-NTA affinity chromatography. The Azo-casein-hydrolyzingactiv-ity of rLA2582 and rLA2901 was detected by spectrophotometry. Activities of rLA2582 and rLA2901 in the hydrolysis of Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans, a fluorescence-labeling pentapeptide substrate, were de-tected by fluorospectrophotometry,and then the Km and Kcat values were determined. SDS-PAGE and spec-trophotometry were performed to detect the activities of rLA2582 and rLA2901 in hydrolyzing extracellular matrix molecules such as collagen type-Ⅰ (COL1), fibronectin (FN) and Congo red-labeling elastin (ELN). Real-time fluorescent quantitative RT-PCR(qRT-PCR) and Western blot were respectively used to measure the expression of LA2582 and LA2901 genes at mRNA and protein levels after infecting human um-bilical vein endothelial cells(HUVEC) with L. interrogans strain Lai. Results The gene products of LA2582 and LA2901 genes were identified as the signal peptide and matrix metalloproteinase motif HXH-containing Zn2+-dependent Gly-Gly metalloendopeptidases belonging to the M23 superfamily. rLA2582 and rLA2901 did not hydrolyze Azo-casein (Km=126.54 μmol/L, Kcat=4.67/s), but could hydrolyze the pentapeptide substrate (Km=190. 25 μmol/L, Kcat 4. 86/s). rLA2582 and rLA2901 could hydrolyze COL1, FN and ELN. Expression of LA2582 and LA2901 genes at both mRNA and protein levels was signifi-cantly increased after infection of HUVEC with L.interrogans strain Lai(P<0.05). Conclusion The prod-ucts of LA2582 and LA2901 genes of L.interrogans strain Lai are Zn2+-dependent M23 metalloendopeptidas-es, which can hydrolyze multiple ECM molecules and are closely associated with the leptospiral invasiveness.

Human mononuclear macrophages and neutrophils: differences in killing ability and mechanism against Leptospira interrogans / 中华微生物学和免疫学杂志

Objective To understand the differences in killing ability and mechanism of human mononuclear macrophages and neutrophils against Leptospira interrogans.Methods Human THP-1 and HL-60 cell lines were respectively pretreated with PMA (phorbol 12-myristate 13-acetate) and ATRA (all-trans retinoic acid) to induce their differentiation into macrophages and neutrophils.Confocal microscopy was used to detect the changes in total ROS (reactive oxygen species) and NO (nitric oxide) levels as well as free Ca2+ concentration ([Ca2+]i) in THP-1 monocyte-derived macrophages and HL-60 cell-derived neutrophils after infection with Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai.Fluorospectrophotometry was applied to analyze the differences in killing ability between THP-1 monocyte-derived macrophages and HL-60 cell-derived neutrophils against intracellular leptospires before and after treatment with total ROS and NO inhibitors and intracellular free Ca2+ chelator.Results The total ROS and NO levels and [Ca2+]i in THP-1 monocyte-derived macrophages and HL-60 cell-derived neutrophils were significantly increased after infection with the spirochete (P<0.05).Moreover,the total ROS and NO levels and [Ca2+]i in the former were significantly higher than those in the latter (P<0.05).The THP-1 monocyte-derived macrophages had stronger killing ability against intracellular leptospires than the HL-60 cell-derived neutrophils (P<0.05).Inhibiting total intracellular ROS,NO or free Ca2+ could result in decreased killing ability of THP-1 monocyte-derived macrophages against intracellular leptospires,but not affect the killing ability of HL-60 cell-derived neutrophils.Conclusion Mononuclear macrophages rather than neutrophils act as the main phagocytes eliminating Leptospira interrogans.High levels of total intracellular ROS,NO and free Ca2+ are closely associated with the ability of mononuclear macrophages to kill Leptospira interrogans.

Cyclic diguanylate (c-di-GMP) regulates the motility of Leptospira interrogans via chemotaxis regulatory proteins CheB/CheR / 中华微生物学和免疫学杂志

Objective To investigate the regulatory effects of cyclic diguanylate (c-di-GMP) signaling on CheB and CheR, which were chemotaxis regulatory proteins relating to the motility of Leptospira interrogans.Methods Real-time PCR was used to determine the expression of cheB1, cheB2, cheB3, cheR1 and cheR2 genes at mRNA level during Leptospira interrogans infection.Fragments of these genes were amplified and cloned into the expression vector pET-28a, respectively, to construct the prokaryotic expression system for them.Colony morphologies of Escherichia coli (E.coli) strains that overexpressed the target genes were observed to determine the regulatory effects of c-di-GMP on CheB and CheR.Results The expression of cheB1 gene at mRNA level increased 60 min after infection and reached the peak at 90 min.Compared with the control group, the expression of cheB3 gene at mRNA level were up-regulated, while no significant difference in the expression of cheB2 and cheR genes was observed 60 min after infection.The prokaryotic expression system for the five genes was successfully constructed and the purified proteins were obtained.CheB1, CheB3 and CheR2 improved the motility of E.coli, but that was inhibited by the inhibitor of diguanylate cyclase (DGC) or phosphodiesterase (PDE).Conclusion CheB and CheR regulate the swarming motility of E.coli and are affected by intracellular c-di-GMP.

Discriminación genotípica de serovariedades del serogrupo Icterohaemorrhagiae pertenecientes a Leptospira interrogans (Spirochaetales: Leptospiraceae) y el análisis de distancias génicas mediante coordenadas principales

La leptospirosis es una zoonosis de amplia distribución mundial y de importancia en salud pública y veterinaria. El serogrupo Icterohaemorrhagiae toma importancia, ya que ha sido aislado con frecuencia en roedores y en casos clínicos de humanos, caninos, bovinos y porcinos; y se ha logrado aislar a partir de agua del rio Reconquista, provincia de Buenos Aires, Argentina. El objetivo de este estudio fue discriminar molecularmente entre las serovariedades Copenhageni e Icterohaemorrhagiae del serogrupo Icterohaemorrhagiae de importancia en Argentina, utilizando la técnica del análisis de repeticiones en tándem en múltiples locus (MLVA) y analizar las distancias génicas entre los genotipos determinados a partir de cepas aisladas de animales. Para ello, genotipificamos las 4 cepas referenciales del serogrupo Icterohaemorrhagiae de la especie Leptospira interrogans serovariedad Copenhageni cepa M20 y cepa Ictero I y de la serovariedad Copenhageni cepa RGA y cepa Fiocruz L1-130. Para este estudio se incluyeron 24 cepas aisladas de animales domésticos como silvestres. Logramos mediante esta técnica determinar un código numérico para cada serovariedad y así pudimos discriminar entre las serovariedades de este serogrupo. El análisis de coordenadas principales (PCoA) demostró la variabilidad génica existente entre las 4 cepas referenciales pertenecientes al serogrupo Icterohaemorrhagiae, el genotipo con mayor representación fue el serovar Copenhageni cepa Fiocruz L1-130 con un total de 13 cepas aisladas con ese perfil genético. Discriminar las serovariedades de este importante serogrupo nos permitirá en un futuro investigar si existen diferencias entre la sintomatología clínica y/o respuesta al tratamiento en los casos clínicos producidos por las distintas serovariedades del serogrupo Icterohaemorrhagiae discriminadas en este trabajo.

Leptospirosis is the world`s most widely distributed zoonosis and of great importance in animal health and in public health. The relevance of the serogroup Icterohaemorrhagiae relies on the frequent isolation from rodents and from clinical cases of humans, canines, bovines and swine; and this serogroup was also isolated from water samples of the Reconquista river, Buenos Aires province, Argentina. The objective of this study was to discriminate molecularly between serovars Copenhageni and Icterohaemorrhagiae being of importance in Argentina, using Multiple Locus Variable number tandem repeats analysis (MLVA) and analyzing the genetic distances among genotypes obtained from animal isolates in this study. We genotyped the four referential strains of the serogroup Icterohaemorrhagiae belonging to the species Leptospira interrogans serovar Copenhageni strain M20 and strain Ictero I and of the serovar Copenhageni strain RGA and strain Fiocruz L1-130. In this study we included 24 strains isolated from animals domestic and wildlife. Using this technique we could determine numeric codes for each serovar and in this way, discriminate between serovars of this serogroup. The principal coordinates analysis (PCoA) showed the genetic variability of the four referential strains from the serogroup Icterohaemorrhagiae, the genotype with better representation was serovar Copenhageni strain Fiocruz L1-130 with 13 isolated strains with this genetic profile. Discrimination of the serovars from this important serogroup allows to establish differences between clinical signs and/or response to treatment in the clinical cases produced by different serovars of the serogroup Icterohaemorrhagiae successfully distinguished in this study.

Effects of Leptospira interrogans infection on the activation of NLRP3 in THP-1 and J774A. 1 cells / 中华微生物学和免疫学杂志

Objective To analyze the effects of Leptospira interrogans ( L. interrogans) infection on the activation of NLRP3 in THP-1 and J774A. 1 cells and to further understand the mechanism of inflam-mation caused by L. interrogans in different hosts. Methods Human mononuclear macrophage cell line (THP-1) and murine mononuclear macrophage cell line (J774A. 1) were infected with L. interrogans strain 56601. The expression of NLRP3 at mRNA and protein levels were measured by using real-time RT-PCR and flow cytometry analysis, respectively. The NLRP3-mediated secretion of IL-1β, IL-18 and IL-33 was detec-ted by ELISA combined with the NLRP3 inhibitory test. Results Compared with the normal cells, the ex-pression of NLRP3 at mRNA level in L. interrogans-infected THP-1 cells was respectively increased by 4. 05, 0. 34, 0. 33, 0. 06 and 1. 66 times at the time points of 1 h, 2 h, 4 h, 12 h and 24 h after infection ( P0. 05). Results of the inhibitory test showed that the up-regulation of IL-1β , IL-18 and IL-33 in THP-1 and J774A. 1 cells were effectively inhibited by the specific inhibitor of NLRP3. Conclusion NL-RP3 inflammasome was activated and involved in the production of specific inflammatory cytokines IL-1βand IL-18 in both THP-1 and J774A. 1 cells after L. interrogans infection, but the inflammatory cytokines induced by L. interrogans infection varied in different cells. L. interrogans induced earlier and higher level of IL-1βand IL-18 production in human macrophages than in murine macrophages.

Effects of Leptospira interrogans infection on the activities of NADPH oxidase and the levels of ROS in THP-1 and J774A. 1 cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of Leptospira interrogans ( L. interrogans) infec-tion on the activities of NADPH oxidase ( nicotinamide adenine dinucleotide phosphate-oxidase) and the lev-els of reactive oxygen species (ROS) in THP-1 and J774A. 1 cells and to understand the bactericidal mecha-nisms of macrophages in different hosts against L. interrogans. Methods Human mononuclear macrophage cell line (THP-1 cells) and murine mononuclear macrophage cell line (J774A. 1 cells) were infected with L. interrogans strain 56601. The activities of NADPH oxidase and the levels of superoxide ion ( O-2 ) were measured with spectrophotography. Changes in the levels of ROS were detected with immunofluorescence as-say. Results Compared with the normal cells, the activities of NADPH oxidase in L. interrogans-infected J774A. 1 cells changed from 0. 619 0 μmol · min-1 · mg-1 to 0. 305 5 μmol · min-1 · mg-1 , 6. 141 5μmol·min-1 ·mg-1 , 1. 487 1μmol·min-1 ·mg-1 and 0. 964 6μmol·min-1 ·mg-1 after 2, 4, 12 and 24 hours of infection, respectively (P<0. 05), while the activities of NADPH oxidase in L. interrogans-infected THP-1 cells were up-regulated from 0. 723 5μmol·min-1 ·mg-1 to 0. 884 2μmol·min-1 ·mg-1 , 1. 897 1μmol·min-1 ·mg-1 , 1. 125 4 μmol·min-1 ·mg-1 and 0. 562 7 μmol·min-1 ·mg-1 , respectively ( P<0. 05). The levels of O-2 in L. interrogans-infected J774A. 1 cells at the time points of 2 h, 4 h, 12 h and 24 h after infection increased from 0. 189 0μmol/L to 0. 236 3μmol/L, 0. 297 7μmol/L, 0. 324 0μmol/L and 0. 305 7 μmol/L, respectively (P<0. 05), while the levels of O-2 in L. interrogans-infected THP-1 cells rose from 0. 123 7 μmol/L to 0. 149 3 μmol/ L, 0. 249 0 μmol/ L, 0. 270 0 μmol/ L and 0. 272 7μmol/L, respectively (P<0. 05). The fluorescence intensity of ROS in THP-1 and J774A. 1 cells increased gradually after infection with L. interrogans for 2 h and decreased after reaching the peak at 24 h. Conclu-sion Both the activities of NADPH oxidase and the levels of O-2 in J774A. 1 and THP-1 cells were signifi-cantly upregulated after infected with L. interrogans, especially in J774A. 1 cells. The results of this study provided references for further elucidating the bactericidal mechanisms of macrophages in different hosts against L. interrogans.