ISOLATION OF Rickettsia bellii FROM Amblyomma ovale AND Amblyomma incisum TICKS FROM SOUTHERN BRAZIL

Objective. To isolate and characterize rickettsiae from the ticks Amblyomma ovale and Amblyomma incisum collected in the state of Sao Paulo. Materials and methods. Adult, free-living A. ovale and A. incisum were collected in an Atlantic rainforest area in the state of Sao Paulo, Brazil. Each tick was tested using the hemolymph assay; samples from positive ticks were placed in shell vials in order to isolate rickettsiae and subsequently grown in Vero cells. Amplification of three rickettsial genes ( gltA, htrA and ompA) was attempted using polymerase chain reaction (PCR) for each isolate obtained. Amplicons were subsequently sequenced. Results. A total of 388 A. incisum and 50 A. ovale were collected. Only one A. incisum and one A. ovale were hemolymph-test positive. Rickettsiae were successfully isolated from these ticks; however establishment in Vero cell culture was successful only for the isolate from A. ovale. Bacterial contamination in the first cell passage of the A. incisum isolate precluded successful isolation of the organism. PCR products were obtained with the gltA and htrA primers for the two isolates, but no product was obtained with the ompA primers. By BLAST analysis, partial gltA and htrA sequences of isolates from A. ovale and A. incisum were similar to the corresponding sequences of R. bellii. Conclusions. This is the first report of R. bellii infecting A. incisum and the first successful isolation from A. ovale.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Detection and molecular characterization of a canine piroplasm from Brazil

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name ""nambiuvu"", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvu. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp.sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of ""nambiuvu"" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia. (C) 2011 Elsevier B.V. All rights reserved.

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63 kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. (C) 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Rickettsia monteiroi sp nov., Infecting the Tick Amblyomma incisum in Brazil

Free-living adult Amblyomma incisum ticks were collected in an Atlantic rainforest area at Intervales State Park, State of Sao Paulo, Brazil. From an A. incisum specimen, rickettsiae were successfully isolated in Vero cell culture by the shell vial technique. Rickettsial isolation was confirmed by optical microscopy, transmission electron microscopy, and PCRs targeting portions of the rickettsial genes gltA, htrA, rrs, and sca1 on infected cells. Fragments of 1,089, 457, 1,362, and 443 nucleotides of the gltA, htrA, rrs, and sca1 genes, respectively, were sequenced. By BLAST analysis, the partial sequence of rrs of the A. incisum rickettsial isolate was closest to the corresponding sequence of Rickettsia bellii (99.1% similarity). The gltA partial sequence was closest to the corresponding sequences of ""Candidatus Rickettsia tarasevichiae"" (96.1% similarity) and Rickettsia canadensis (95.8% similarity). The htrA partial sequence was closest to the corresponding sequence of R. canadensis (89.8% similarity). The sca1 partial sequence was closest to the corresponding sequence of R. canadensis (95.2% similarity). Since our rickettsial isolate was genetically distinct from other Rickettsia species, we propose a new species designated Rickettsia monteiroi sp. nov. Phylogenetic analyses indicated that R. monteiroi belongs to the canadensis group within the genus Rickettsia, together with the species R. canadensis and ""Candidatus R. tarasevichiae"". Little or no antibody cross-reaction was observed between sera of R. monteiroi-inoculated guinea pigs and R. bellii-, Rickettsia rickettsii-, or R. canadensis-inoculated guinea pigs.

Short-term experimental data of drying shrinkage of ground granulated blast-furnace slag cement concrete

This paper presents results of laboratory testing of unrestrained drying shrinkage during a period of 154 days of different concrete mixtures from the Brazilian production line that utilize ground granulated blast-furnace slag in their compositions. Three concrete mixtures with water/cement ratio of 0.78(M1), 0.41(M2), and 0.37(M3) were studied. The obtained experimental data were compared with the analytical results from prediction models available in the literature: the ACI 209 model (ACI), the B3 model (B3), the Eurocode 2 model (EC2), the GL 2000 model (GL), and the Brazilian NBR 6118 model (NBR), and an analysis of the efficacy of these models was conducted utilizing these experimental data. In addition, the development of the mechanical properties (compressive strength and modulus of elasticity) of the studied concrete mixtures was also measured in the laboratory until 126 days. From this study, it could be concluded that the ACI and the GL were the models that most approximated the experimental drying shrinkage data measured during the analyzed period of time.

Mechanical properties, drying and autogenous shrinkage of blast furnace slag activated with hydrated lime and gypsum

This article reports the characteristics of blast furnace slag (BFS) pastes activated with hydrated lime (5%) and hydrated lime (2%) plus gypsum (6%) in relation to compressive strength, shrinkage (autogenous and drying) and microstructure (porosity, hydrated products). The paste mixtures were characterized using powder X-ray diffraction (XRD), mercury intrusion porosimetry (MIP) and thermogravimetric analysis (TG/DTG). BSF activated with lime and gypsum (LG) results in larger amounts of ettringite when compared with BFS activated with lime (L). Although the porosities of the L and LG mixtures were about the same, there was a greater pore refinement for the BFS activated with lime, with an increase in mesopores volume with age. The presence of ettringite and the higher volumes of macropores cause the compressive strength of BSF activated with hydrated lime plus gypsum to be smaller than that of BFS activated with lime. For both chemical activators, compressive strength developed slowly at early ages. Autogenous and drying shrinkage were greater for the BFS activated with lime, believed to result from the more refined porous structure in comparison with the mixture activated with gypsum plus lime. (c) 2010 Elsevier Ltd. All rights reserved.

Digital subtraction radiographic analysis of the combination of bioabsorbable membrane and bovine morphogenetic protein pool in human periodontal infrabony defects

OBJECTIVES: This study assessed the bone density gain and its relationship with the periodontal clinical parameters in a case series of a regenerative therapy procedure. MATERIAL AND METHODS: Using a split-mouth study design, 10 pairs of infrabony defects from 15 patients were treated with a pool of bovine bone morphogenetic proteins associated with collagen membrane (test sites) or collagen membrane only (control sites). The periodontal healing was clinically and radiographically monitored for six months. Standardized pre-surgical and 6-month postoperative radiographs were digitized for digital subtraction analysis, which showed relative bone density gain in both groups of 0.034 ± 0.423 and 0.105 ± 0.423 in the test and control group, respectively (p>0.05). RESULTS: As regards the area size of bone density change, the influence of the therapy was detected in 2.5 mm² in the test group and 2 mm² in the control group (p>0.05). Additionally, no correlation was observed between the favorable clinical results and the bone density gain measured by digital subtraction radiography (p>0.05). CONCLUSIONS: The findings of this study suggest that the clinical benefit of the regenerative therapy observed did not come with significant bone density gains. Long-term evaluation may lead to a different conclusions.

Isolation of Pseudomonas aeruginosa strains from dental office environments and units in Barretos, state of São Paulo, Brazil, and analysis of their susceptibility to antimicrobial drugs

A wide variety of opportunistic pathogens has been detected in the tubing supplying water to odontological equipment, in special in the biofilm lining of these tubes. Among these pathogens, Pseudomonas aeruginosa, one of the leading causes of nosocomial infections, is frequently found in water lines supplying dental units. In the present work, 160 samples of water, and 200 fomite samples from forty dental units were collected in the city of Barretos, State of São Paulo, Brazil and evaluated between January and July, 2005. Seventy-six P. aeruginosa strains, isolated from the dental environment (5 strains) and water system (71 strains), were tested for susceptibility to six antimicrobial drugs most frequently used against P. aeruginosa infections. Susceptibility to ciprofloxacin, followed by meropenem was the predominant profile. The need for effective means of reducing the microbial burden within dental unit water lines is emphasized, and the risk of exposure and cross-infection in dental practice, in special when caused by opportunistic pathogens like P. aeruginosa, are highlighted.

Analysis of TMJ vibration sounds before and after use of two types of occlusal splints

Temporomandibular joint (TMJ) sounds are important and common physical signs of temporomandibular disorders (TMD). The aim of this study was to evaluate the influence of the effect of the use of occlusal bite splints (stabilizing and repositioning) on the sounds produced in the TMJ, by means of the electrovibratography (EVG). Thirty-one patients with TMD from the Dental School of Ribeirão Preto, University of São Paulo, Brazil were selected for this study. Group 1 (n=23) wore stabilizing bite splints and Group 2 (n=8) used anterior repositioning splints. Before and after treatment with occlusal splints both groups were analyzed using the SonoPAK Q/S recording system (BioResearch System, Inc.). The treatments with stabilizing bite splints were satisfactory, since they reduced the total amount of the sound energies (p<0.05), but the use of anterior repositioning splints for no more than 4 weeks produced significantly better results (p<0.01). The total amount of vibration energy involved in the vibrating movements of the TMJ showed significant improvement using anterior repositioning splints.

Adhesion of an endodontic sealer to dentin and gutta-percha: shear and push-out bond strength measurements and SEM analysis

The use of an adequate method for evaluation of the adhesion of root canal filling materials provides more reliable results to allow comparison of the materials and substantiate their clinical choice. The aims of this study were to compare the shear bond strength (SBS) test and push-out test for evaluation of the adhesion of an epoxy-based endodontic sealer (AH Plus) to dentin and gutta-percha, and to assess the failure modes on the debonded surfaces by means of scanning electron microscopy (SEM). Three groups were established (n=7): in group 1, root cylinders obtained from human canines were embedded in acrylic resin and had their canals prepared and filled with sealer; in group 2, longitudinal sections of dentin cylinders were embedded in resin with the canal surface smoothed and turned upwards; in group 3, gutta-percha cylinders were embedded in resin. Polyethylene tubes filled with sealer were positioned on the polished surface of the specimens (groups 2 and 3). The push-out test (group 1) and the SBS test (groups 2 and 3) were performed in an Instron universal testing machine running at crosshead speed of 1 mm/min. Means (±SD) in MPa were: G1 (8.8±1.13), G2 (5.9±1.05) and G3 (3.8±0.55). Statistical analysis by ANOVA and Student's t-test (a=0.05) revealed statistically significant differences (p<0.01) among the groups. SEM analysis showed a predominance of adhesive and mixed failures of AH Plus sealer. The tested surface affected significantly the results with the sealer reaching higher bond strength to dentin than to gutta-percha with the SBS test. The comparison of the employed methodologies showed that the SBS test produced significantly lower bond strength values than the push-out test, was skilful in determining the adhesion of AH Plus sealer to dentin and gutta-percha, and required specimens that could be easily prepared for SEM, presenting as a viable alternative for further experiments.

Morphological analysis of glass, carbon and glass/carbon fiber posts and bonding to self or dual-cured resin luting agents

OBJECTIVE: The aim of this study was to evaluate the morphology of glass (GF), carbon (CF) and glass/carbon (G/CF) fiber posts and their bond strength to self or dual-cured resin luting agents. MATERIAL AND METHODS: Morphological analysis of each post type was conducted under scanning electron microscopy (SEM). Bond strength was evaluated by microtensile test after bisecting the posts and re-bonding the two halves with the luting agents. Data were subjected to two-way ANOVA and Tukey's test (α=0.05). Failure modes were evaluated under optical microscopy and SEM. RESULTS: GF presented wider fibers and higher amount of matrix than CF, and G/CF presented carbon fibers surrounded by glass fibers, and both involved by matrix. For CF and GF, the dual-cured material presented significantly higher (p<0.05) bond strength than the self-cured agent. For the dual agent, CF presented similar bond strength to GF (p>0.05), but higher than that of G/CF (p<0.05). For the self-cured agent, no significant differences (p>0.05) were detected, irrespective of the post type. For GF and G/CF, all failures were considered mixed, while a predominance of adhesive failures was detected for CF. CONCLUSION: The bonding between fiber posts and luting agents was affected by the type of fibers and polymerization mode of the cement. When no surface treatment of the post is performed, the bonding between glass fiber post and dual-cured agent seems to be more reliable.

Influence of cervical preflaring on determinationof apical file size in mandibular molars: SEM analysis

This study investigated the influence of cervical preflaring with different rotary instruments on determination of the initial apical file (IAF) in mesiobuccal roots of mandibular molars. Fifty human mandibular molars whose mesial roots presented two clearly separated apical foramens (mesiobuccal and mesiolingual) were used. After standard access opening and removal of pulp tissue, the working length (WL) was determined at 1 mm short of the root apex. Five groups (n=10) were formed at random, according to the type of instrument used for cervical preflaring. In group 1, the size of the IAF was determined without preflaring of the cervical and middle root canal thirds. In groups 2 to 5, preflaring was performed with Gates-Glidden drills, ProTaper instruments, EndoFlare instruments and LA Axxes burs, respectively. Canals were sized manually with K-files, starting with size 08 K-files, inserted passively up to the WL. File sizes were increased until a binding sensation was felt at the WL and the size of the file was recorded. The instrument corresponding to the IAF was fixed into the canal at the WL with methylcyanoacrylate. The teeth were then sectioned transversally 1 mm short of the apex, with the IAF in position. Cross-sections of the WL region were examined under scanning electron microscopy and the discrepancies between canal diameter and the diameter of IAF were calculated using the tool "rule" (FEG) of the microscope's proprietary software. The measurements (µm) were analyzed statistically by Kruskal-Wallis and Dunn's tests at 5% significance level. There were statistically significant differences among the groups (p<0.05). The non-flared group had the greatest discrepancy (125.30 ± 51.54) and differed significantly from all flared groups (p<0.05). Cervical preflaring with LA Axxess burs produced the least discrepancies (55.10 ± 48.31), followed by EndoFlare instruments (68.20 ± 42.44), Gattes Glidden drills (68.90 ± 42.46) and ProTaper files (77.40 ± 73.19). However, no significant differences (p>0.05) were found among the rotary instruments. In conclusion, cervical preflaring improved IAF fitting to the canals at the WL in mesiobuccal roots of maxillary first molars. The rotary instruments evaluated in this study did not differ from each other regarding the discrepancies produced between the IAF size and canal diameter at the WL.

Ex vivo analysis of root canal cleaning using Endo-PTC associated to NaOCl and different irrigant solutions

The aim of this study was to assess qualitatively, by means of SEM images, the cleaning of the dentin walls of root canals after chemical-surgical preparation using Endo-PTC cream with 0.5% and 1% sodium hypochlorite and different final irrigating solutions. Seventy-two single-rooted human teeth were divided into eight groups and prepared using Endo-PTC cream with sodium hypochlorite (NaOCl) at different concentrations, and irrigated with NaOCl at different concentrations. Final irrigation was performed with either EDTA-T or EDTA-C. The best results were obtained with Group 1, followed by Groups 5, 2, 7, 8, 3, 6 and 4. We can conclude that the use of 0.5% NaOCl during instrumentation and final flush of the root canals was more efficient in cleaning than was 1% sodium hypochlorite. EDTA-T was more efficient in removing smear layer than EDTA-C, and the cervical third presented better cleaning of the root canal walls than did the middle third, which showed cleaner dentin walls than the apical third.

Coronal and apical leakage analysis of two different root canal obturation systems

This study compared the coronal and apical leakage of AH Plus with gutta-percha to that of Epiphany with Resilon. Twenty-four single rooted teeth were instrumented and divided into 2 groups according to the solutions for smear layer removal and the obturation materials employed: Group A - 17% EDTA-T and AH Plus with gutta-percha; Group B - primer and Epiphany with Resilon. The Group B specimens were light-cured in the coronal area for 20 s. The external root surfaces were covered with a double layer of ethyl cyanoacrylate, except for the apical foramen and the cavity access. The teeth were immersed in 0.5% methylene blue for 48 h. The specimens were rinsed, dried and axially split for dye penetration measurement with the ImageLab 2.3 software. The t-test showed no significant differences for coronal leakage between the groups, but there were significant differences for apical leakage between the groups (P < 0.05). AH Plus with gutta-percha and Epiphany with Resilon provided the same coronal seal, whereas Epiphany with Resilon provided the best apical seal.