Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions

Background: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (K-D) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a K-D of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

OCCURRENCE OF ANTIBODIES TO TOXOPLASMA GONDII AND LEPSTOSPIRA SPP. IN MANATEES (TRICHECHUS INUNGUIS) OF THE BRAZILIAN AMAZON

The presence of Toxoplasma gondii and Leptospira spp. antibodies was investigated in 74 manatees (Trichechus inunguis [Mammalia: Sirenia]) kept in captivity in two rescue units in the northern region of Brazil. Antibodies to T gondii were detected in 29 (39.2%) of 74 animals by using the modified agglutination test (titer, 1:25). For antibodies against Leptospira spp., sera were diluted 1:50 and tested against 24 strains of leptospires by microscopic agglutination microtechnique, and positive samples were end titrated. Twenty-three (31.1%) of 74 animals were reactive to four serovars (Patoc 21/23, Castellonis 2/23, Icterohaemorrhagiae 1/23, and Butembo 1/23), with titers ranging from 100 to 1,600. This is the first report of antibodies against T gondii and Leptospira spp. in T. inunguis from the Brazilian Amazon.

Leptospiral Immunoglobulin-like Proteins Interact With Human Complement Regulators Factor H, FHL-1, FHR-1, and C4BP

Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular matrix components, coagulation cascade proteins, and human complement regulators. Here we demonstrate that acquisition of factor H (FH) on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes, besides interacting with FH, FH related-1, and C4b binding protein (C4BP), also acquire FH like-1 from human serum. We also demonstrate that binding to these complement regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins, previously shown to interact with fibronectin, laminin, collagen, elastin, tropoelastin, and fibrinogen. Factor H binds to Lig proteins via short consensus repeat domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins. Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by factor I. In conclusion, Lig proteins are multifunctional molecules, contributing to leptospiral adhesion and immune evasion.

Differences in early biomechanical properties after myopic and hyperopic lasik using mechanical microkeratome Sub-Bowman's Keratomileusis (SBK)

Purpose: To evaluate biomechanical changes measured with the ORA (Ocular Response Analyzer (R); Reichert Ophthalmic Instruments, Buffalo, New York, USA) after Lasik with the Moria One Use Plus and to compare the biomechanics changes after myopic and hyperopic ablations. Methods: Fourteeneyes for hyperopia (H) and 19 eyes for myopia (M) were evaluated with the ORA preoperatively and 1 month after Lasik with thin flap (100 microns) using SBK-OUP (Sub-Bowman Keratomileusis-One Use Plus, Moria (R)). CH (Corneal Hysteresis), CRF (Corneal Resistance Factor), IOPg (gold-standard, Goldmann correlated Intraocular pressure), IOPcc (Corneal compensated Intraocular pressure) and more 38 variables derived from the corneal biomechanical response signal of the ORA were analyzed. The Wilcoxon test was used to assess differences between the variables before and after surgery for each group and the differences between the pre and postoperative (1 month) myopic eyes were compared with those obtained in hyperopic eyes, using the Mann-Whitney test. Results: There was a significant difference before and after Lasik in myopic and hyperopic eyes in IOPg (Wilcoxon, p<0.05), but not in IOPcc. Only myopic eyes showed a significant difference in CH and CRF measurements before and after LASIK, as well as 9 other biomechanical parameters (aspect1, h1, dive1, path1, p1area1, W11, H11, and w2 path11; Wilcoxon, p<0, 05), 8 of these being related to the first sign of flattening. Five parameters related to the sign of the second applanation showed significant variation only in the eyes before and after hyperopic Lasik (aspect2, h2, dive2, mslew2 and H21; Wilcoxon, p<0,05). There was a difference in both myopic and hyperopic on three parameters related to the applanation signal areas (p1area, and p2area p2area1; Wilcoxon, p<0.05). Differences in IOPg and p1area, before and after surgery were significantly higher in myopic eyes than in hyperopic eyes (Mann-Whitey, p<0.05). Conclusion: There are several significant differences in biomechanical parameters after Lasik with Moria OUP_SBK. Overall, the impact of myopic LASIK on corneal biomechanics is higher than of hyperopic Lasik. The parameters derived from the first sign of the ORA are more affected in myopic LASIK, whereas parameters derived from the second applanation are more affected in hyperopic LASIK.

Associação de variáveis ambientais à ocorrência de leptospirose canina e humana na cidade de São Paulo

Foi realizada uma análise espacial da ocorrência de leptospirose humana e canina na Supervisão de Vigilância em Saúde do Butantã, situada no município de São Paulo, no ano de 2007, associada a variáveis ambientais de risco, tais como: focos de enchente e áreas de desratização. Foram encontrados aglomerados espaciais de pontos de alagamentos em 12 setores censitários e de casos de leptospirose humana em quatro setores censitários, sem correlação entre ambos. Não foram encontrados agrupamentos de casos em cães, possivelmente devido à subnotificação. As proporções casos humanos de leptospirose : população humana dentro e fora da área de desratização foram 7:199.600 e 9:257.980, respectivamente. Conclui-se que medidas de controle de roedores como a desratização foram responsáveis pela minimização dos efeitos dos fatores de risco para a transmissão de leptospirose para humanos.

Molecular and serological characterization of Leptospira interrogans serovar Canicola isolated from dogs, swine, and bovine in Brazil

The identification of Leptospira clinical isolates through genotyping and serotyping, besides the recognition of its reservoirs, are important tools for understanding the epidemiology of leptospirosis, and they are also keys for identifying new species and serovars. Fourteen clinical isolates from animals were characterized by means of single enzyme amplified length polymorphism, variable number of tandem repeat analysis, pulsed field gel electrophoresis, and serotyping. All isolates were identified as Leptospira interrogans, serovar Canicola. Infections by this serovar occur in urban regions, where dogs represent the main maintenance hosts, whereas bovine and swine may act as reservoirs of serovar Canicola in rural areas. Both urban and rural aspects of leptospirosis, and the role of domestic animals as maintenance hosts, cannot be neglected in developing and developed countries.

Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+

Abstract Background Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. Results The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. Conclusion LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

“Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions”

Abstract Background Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Prevalência de leptospirose em caprinos leiteiros do semiárido paraibano

Este trabalho teve como objetivos determinar a prevalência de propriedades de caprinos leiteiros positivas e de animais soropositivos para leptospirose no semiárido paraibano. A amostragem foi delineada para a determinação da prevalência de propriedades positivas (focos) e de animais soropositivos para a infecção por Leptospira spp. No total, foram colhidas amostras de sangue de 975 animais procedentes de 110 propriedades leiteiras localizadas no Munícipio de Monteiro, microrregião do Cariri Ocidental, Estado da Paraíba. Para o diagnóstico da infecção por Leptospira spp., foi utilizado o teste de soroaglutinação microscópica (SAM), utilizando como antígenos 24 sorovares. Uma propriedade foi considerada foco quando apresentou pelo menos um animal soropositivo. As prevalências de propriedades positivas e de animais soropositivos foram de 43,6% (IC 95% = 34,2-53,4%) e de 8,7% (IC 95% = 5,7-12,9%), respectivamente. Nos animais, os sorovares mais frequentes foram Autumnalis (1,74%; IC 95% = 0,97-3,09%), Sentot (1,71%; IC 95% = 0,82-3,52%) e Whitcomb (1,39%; IC 95% = 0,65-2,93%), e nas propriedades, os sorovares Autumnalis (10,9%; IC 95% = 5,8-18,3%), Whitcomb (8,2%; IC 95% = 3,8-15,0%) e Sentot e Patoc (7,3%; IC 95% = 3,2-13,8%) foram os mais frequentes. Sugere-se que a infecção está distribuída em caprinos da região, e que há necessidade de implantação de medidas de controle e prevenção, com o intuito de reduzir a ocorrência da infecção e, consequentemente, diminuir perdas econômicas ocasionadas e bloquear a possível transmissão do agente aos seres humanos.

Prevalência e fatores de risco de leptospirose bovina no Estado do Maranhão

Foram investigados a prevalência e os fatores de risco da leptospirose bovina no Estado do Maranhão. O Estado foi dividido em quatro circuitos amostrais com base em parâmetros de produção distintos que variam conforme os diferentes sistemas de produção, as práticas de manejo, a finalidade de exploração, o tamanho médio dos rebanhos e os sistemas de comercialização. Objetivou-se estudar as características epidemiológicas da leptospirose bovina no Estado do Maranhão, de modo a determinar a prevalência em bovinos e em rebanhos, detectar as sorovariedades de Leptospira spp. presentes, identificar os fatores de risco eventualmente associados à leptospirose em bovinos e diferenciar os circuitos pecuários entre si no que se refere à prevalência de leptospirose. A pesquisa foi realizada em 136 propriedades rurais pertencentes ao circuito I, no qual 841 fêmeas bovinas com idade igual ou superior a 24 meses foram analisadas; 238 do circuito II, com 2.582 fêmeas analisadas; 122 do circuito III, com 869 fêmeas analisadas; e 77 do circuito IV, com 540 fêmeas analisadas; no total, 573 propriedades e 4.832 fêmeas foram estudadas. A presença de anticorpos contra Leptospira spp. foi verificada pela técnica de soroaglutinação microscópica (SAM). Das 4.832 fêmeas bovinas analisadas, 1.904 (35,94%; IC 95% = 33,01% - 38,98%) foram reagentes. Das 573 propriedades analisadas, 380 (64,81%; IC 95% = 61,10% - 68,35%) foram consideradas positivas. As sorovariedades Hardjo e Wolffi foram as mais frequentes em todo o Estado. O circuito III foi o que apresentou menor prevalência de leptospirose em todas as comparações. As variáveis identificadas como fatores de risco de leptospirose foram: presença de equinos (p = 0,000), presença de capivaras (p = 0,034) e rebanhos bovinos com 32 ou mais fêmeas adultas (p = 0,002).

Primeiro relato de isolamento de Histophilus somni em ovino com quadro de endometrite no Brasil

Este trabalho tem como objetivo relatar um caso de isolamento em cultura pura de Histophilus somni em secreção vaginal de uma fêmea ovina da raça Texel, em propriedade localizada no estado de São Paulo, a qual apresentou histórico de endometrite e infertilidade. No exame clínico específico do aparelho genital, foi observada na vagina secreção de coloração esbranquiçada, com aspecto mucoso e odor fétido. Os exames sorológicos para Brucella ovis, Leptospirose e Neospora caninum resultaram negativos, enquanto o exame para Toxoplasma gondii foi positivo.

LH surge in response to the treatment with GnRH analog or estradiol in ovariectomized buffaloes with or without progesterone pre-exposition

The aim of the present study was to evaluate the LH surge after EB (estradiol benzoate) or GnRH administration with or without P4 (progesterone) pre-exposure in ovariectomized (OVX) buffalo cows. Females were randomly assigned to receive an intravaginal P4 device (D0–D9). They were then given EB 24 h or GnRH 36 h post-P4 device removal (factorial 2×2, n=6 per group). Blood collection for LH measurement began 36 h after the P4 device removal and continued at 3 h intervals. The area under the LH curve (AUC; 30.2 ng2 and 13.41 ng2; P=0.007) and the area of the LH peak (AP; 19.0 ng2 and 8.9 ng2; P=0.009) were greater for EB than GnRH. We did not observe an effect of P4 pre-exposure on the AUC and AP. Furthermore, there was no interaction between P4 pre-exposure and EB or GnRH treatment on the AUC and AP. However, there was an interaction (P<0.01) between P4 pre-exposure and the type of inducer (EB or GnRH) to release a preovulatory-like LH surge at the beginning (BP), final (FP) and time (TP) of the LH peak. The P4 pre-exposure anticipated the BP (2.5 and 7.4 h), TP (6.0 and 12.0 h) and FP (11.5 and 17.1 h) when EB was used to induce a preovulatory-like LH surge (P<0.01). However, there was no effect of P4 pre-exposure on BP (0.4 and 0.4 h), TP (3.0 and 3.0 h) and FP (5.9 and 6.1 h) with GnRH treatment. There was also no effect of the pre-exposure to P4, type of inducer or interaction on the amplitude of the LH peak. We concluded that EB therefore led to greater LH release than GnRH, and pre-exposure to P4 before EB administration anticipated the preovulatory-like LH surge in buffalo cows.

Longitudinal bar buckling behavior

Reinforced concrete columns might fail because of buckling of the longitudinal reinforcing bar when exposed to earthquake motions. Depending on the hoop stiffness and the length-over-diameter ratio, the instability can be local (in between two subsequent hoops) or global (the buckling length comprises several hoop spacings). To get insight into the topic, an extensive literary research of 19 existing models has been carried out including different approaches and assumptions which yield different results. Finite element fiberanalysis was carried out to study the local buckling behavior with varying length-over-diameter and initial imperfection-over-diameter ratios. The comparison of the analytical results with some experimental results shows good agreement before the post buckling behavior undergoes large deformation. Furthermore, different global buckling analysis cases were run considering the influence of different parameters; for certain hoop stiffnesses and length-over-diameter ratios local buckling was encountered. A parametric study yields an adimensional critical stress in function of a stiffness ratio characterized by the reinforcement configuration. Colonne in cemento armato possono collassare per via dell’instabilità dell’armatura longitudinale se sottoposte all’azione di un sisma. In funzione della rigidezza dei ferri trasversali e del rapporto lunghezza d’inflessione-diametro, l’instabilità può essere locale (fra due staffe adiacenti) o globale (la lunghezza d’instabilità comprende alcune staffe). Per introdurre alla materia, è proposta un’esauriente ricerca bibliografica di 19 modelli esistenti che include approcci e ipotesi differenti che portano a risultati distinti. Tramite un’analisi a fibre e elementi finiti si è studiata l’instabilità locale con vari rapporti lunghezza d’inflessione-diametro e imperfezione iniziale-diametro. Il confronto dei risultati analitici con quelli sperimentali mostra una buona coincidenza fino al raggiungimento di grandi spostamenti. Inoltre, il caso d’instabilità globale è stato simulato valutando l’influenza di vari parametri; per certe configurazioni di rigidezza delle staffe e lunghezza d’inflessione-diametro si hanno ottenuto casi di instabilità locale. Uno studio parametrico ha permesso di ottenere un carico critico adimensionale in funzione del rapporto di rigidezza dato dalle caratteristiche dell’armatura.

The use of remote sensing imagery for monitoring recovery in the aftermath of a natural disaster: the Hurricane Katrina Case

Although Recovery is often defined as the less studied and documented phase of the Emergency Management Cycle, a wide literature is available for describing characteristics and sub-phases of this process. Previous works do not allow to gain an overall perspective because of a lack of systematic consistent monitoring of recovery utilizing advanced technologies such as remote sensing and GIS technologies. Taking into consideration the key role of Remote Sensing in Response and Damage Assessment, this thesis is aimed to verify the appropriateness of such advanced monitoring techniques to detect recovery advancements over time, with close attention to the main characteristics of the study event: Hurricane Katrina storm surge. Based on multi-source, multi-sensor and multi-temporal data, the post-Katrina recovery was analysed using both a qualitative and a quantitative approach. The first phase was dedicated to the investigation of the relation between urban types, damage and recovery state, referring to geographical and technological parameters. Damage and recovery scales were proposed to review critical observations on remarkable surge- induced effects on various typologies of structures, analyzed at a per-building level. This wide-ranging investigation allowed a new understanding of the distinctive features of the recovery process. A quantitative analysis was employed to develop methodological procedures suited to recognize and monitor distribution, timing and characteristics of recovery activities in the study area. Promising results, gained by applying supervised classification algorithms to detect localization and distribution of blue tarp, have proved that this methodology may help the analyst in the detection and monitoring of recovery activities in areas that have been affected by medium damage. The study found that Mahalanobis Distance was the classifier which provided the most accurate results, in localising blue roofs with 93.7% of blue roof classified correctly and a producer accuracy of 70%. It was seen to be the classifier least sensitive to spectral signature alteration. The application of the dissimilarity textural classification to satellite imagery has demonstrated the suitability of this technique for the detection of debris distribution and for the monitoring of demolition and reconstruction activities in the study area. Linking these geographically extensive techniques with expert per-building interpretation of advanced-technology ground surveys provides a multi-faceted view of the physical recovery process. Remote sensing and GIS technologies combined to advanced ground survey approach provides extremely valuable capability in Recovery activities monitoring and may constitute a technical basis to lead aid organization and local government in the Recovery management.

Valorization of pomaces from the mechanical extraction of virgin olive oils in dairy animal feeding

This research work is aimed at the valorization of two types of pomace deriving from the extra virgin olive oil mechanical extraction process, such as olive pomace and a new by-product named “paté”, in the livestock sector as important sources of antioxidants and unsaturated fatty acids. In the first research the suitability of dried stoned olive pomace as a dietary supplement for dairy buffaloes was evaluated. The effectiveness of this utilization in modifying fatty acid composition and improving the oxidative stability of buffalo milk and mozzarella cheese have been proven by means of the analysis of qualitative and quantitative parameters. In the second research the use of paté as a new by-product in dietary feed supplementation for dairy ewes, already fed with a source of unsaturated fatty acids such as extruded linseed, was studied in order to assess the effect of this combination on the dairy products obtained. The characterization of paté as a new by-product was also carried out, studying the optimal conditions of its stabilization and preservation at the same time. The main results, common to both researches, have been the detection and the characterization of hydrophilic phenols in the milk. The analytical detection of hydroxytyrosol and tyrosol in the ewes’ milk fed with the paté and hydroxytyrosol in buffalo fed with pomace showed for the first time the presence in the milk of hydroxytyrosol, which is one of the most important bioactive compounds of the oil industry products; the transfer of these antioxidants and the proven improvement of the quality of milk fat could positively interact in the prevention of some human cardiovascular diseases and some tumours, increasing in this manner the quality of dairy products, also improving their shelf-life. These results also provide important information on the bioavailability of these phenolic compounds.