Search for Borrelia sp. in ticks collected from potential reservoirs in an urban forest reserve in the State of Mato Grosso do Sul, Brazil: a short report

A total of 128 ticks of the genus Amblyomma were recovered from 5 marsupials (Didelphis albiventris) - with 4 recaptures - and 17 rodents (16 Bolomys lasiurus and 1 Rattus norvegicus) captured in an urban forest reserve in Campo Grande, State of Mato Grosso do Sul, Brazil. Of the ticks collected, 95 (78.9%) were in larval form and 22 (21.1%) were nymphs; the only adult (0.8%) was identified as A. cajennense. Viewed under dark-field microscopy in the fourth month after seeding, 9 cultures prepared from spleens and livers of the rodents, blood of the marsupials, and macerates of Amblyomma sp. nymphs revealed spiral-shaped, spirochete-like structures resembling those of Borrelia sp. Some of them showed little motility, while others were non-motile. No such structures could be found either in positive Giemsa-stained culture smears or under electron microscopy. No PCR amplification of DNA from those cultures could be obtained by employing Leptospira sp., B. burgdorferi, and Borrelia sp. primers. These aspects suggest that the spirochete-like structures found in this study do not fit into the genera Borrelia or Leptospira, requiring instead to be isolated for proper identification.

Unique genome replication mechanism of the archaeal virus AFV1.

The exceptional genomic content and genome organization of the Acidianus filamentous virus 1 (AFV1) that infects the hyperthermophilic archaeon Acidianus hospitalis suggest that this virus might exploit an unusual mechanism of genome replication. An analysis of replicative intermediates of the viral genome by two-dimensional (2D) agarose gel electrophoresis revealed that viral genome replication starts by the formation of a D-loop and proceeds via strand displacement replication. Characterization of replicative intermediates using dark-field electron microscopy, in combination with the 2D agarose gel electrophoresis data, suggests that recombination plays a key role in the termination of AFV1 genome replication through the formation of terminal loops. A terminal protein was found to be attached to the ends of the viral genome. The results allow us to postulate a model of genome replication that relies on recombination events for initiation and termination.

Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far?

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench. [Authors]

Influence of average size and interface passivation on the spectral emission of Si nanocrystals embedded in SiO2

The correlation between the structural (average size and density) and optoelectronic properties [band gap and photoluminescence (PL)] of Si nanocrystals embedded in SiO2 is among the essential factors in understanding their emission mechanism. This correlation has been difficult to establish in the past due to the lack of reliable methods for measuring the size distribution of nanocrystals from electron microscopy, mainly because of the insufficient contrast between Si and SiO2. With this aim, we have recently developed a successful method for imaging Si nanocrystals in SiO2 matrices. This is done by using high-resolution electron microscopy in conjunction with conventional electron microscopy in dark field conditions. Then, by varying the time of annealing in a large time scale we have been able to track the nucleation, pure growth, and ripening stages of the nanocrystal population. The nucleation and pure growth stages are almost completed after a few minutes of annealing time at 1100°C in N2 and afterward the ensemble undergoes an asymptotic ripening process. In contrast, the PL intensity steadily increases and reaches saturation after 3-4 h of annealing at 1100°C. Forming gas postannealing considerably enhances the PL intensity but only for samples annealed previously in less time than that needed for PL saturation. The effects of forming gas are reversible and do not modify the spectral shape of the PL emission. The PL intensity shows at all times an inverse correlation with the amount of Pb paramagnetic centers at the Si-SiO2 nanocrystal-matrix interfaces, which have been measured by electron spin resonance. Consequently, the Pb centers or other centers associated with them are interfacial nonradiative channels for recombination and the emission yield largely depends on the interface passivation. We have correlated as well the average size of the nanocrystals with their optical band gap and PL emission energy. The band gap and emission energy shift to the blue as the nanocrystal size shrinks, in agreement with models based on quantum confinement. As a main result, we have found that the Stokes shift is independent of the average size of nanocrystals and has a constant value of 0.26±0.03 eV, which is almost twice the energy of the Si¿O vibration. This finding suggests that among the possible channels for radiative recombination, the dominant one for Si nanocrystals embedded in SiO2 is a fundamental transition spatially located at the Si¿SiO2 interface with the assistance of a local Si-O vibration.

Imaging the ultrasmall-angle x-ray scattering distribution with grating interferometry.

X-ray imaging with grating interferometry has previously been regarded as a technique providing information only in direct space. It delivers absorption, phase, and dark-field contrast, which can be viewed as parameters of the underlying but unresolved scattering distribution. Here, we present a method that provides the ultrasmall-angle x-ray scattering distribution and, thus, allows simultaneous access to direct and reciprocal space information.

Further ultrastructural evidence that spirochaetes may play a role in the aetiology of Alzheimer's disease

Recently it was reported that, at autopsy, in neuropathologically confirmed cases of Alzheimer's disease spirochaetes were found in blood and cerebrospinal fluid using dark-field microscopy. Moreover, the spirochaetes were isolated and cultured from brain tissue. We now show, using scanning electron microscopy and atomic force microscopy that the helically shaped microorganisms isolated and cultured from the Alzheimer brains possess axial filaments. This indicates that these microorganisms taxonomically indeed belong to the order Spirochaetales. A morphometric analysis reinforces this notion.

Persisting atypical and cystic forms of Borrelia burgdorferi and local inflammation in Lyme neuroborreliosis

BACKGROUND: The long latent stage seen in syphilis, followed by chronic central nervous system infection and inflammation, can be explained by the persistence of atypical cystic and granular forms of Treponema pallidum. We investigated whether a similar situation may occur in Lyme neuroborreliosis. METHOD: Atypical forms of Borrelia burgdorferi spirochetes were induced exposing cultures of Borrelia burgdorferi (strains B31 and ADB1) to such unfavorable conditions as osmotic and heat shock, and exposure to the binding agents Thioflavin S and Congo red. We also analyzed whether these forms may be induced in vitro, following infection of primary chicken and rat neurons, as well as rat and human astrocytes. We further analyzed whether atypical forms similar to those induced in vitro may also occur in vivo, in brains of three patients with Lyme neuroborreliosis. We used immunohistochemical methods to detect evidence of neuroinflammation in the form of reactive microglia and astrocytes. RESULTS: Under these conditions we observed atypical cystic, rolled and granular forms of these spirochetes. We characterized these abnormal forms by histochemical, immunohistochemical, dark field and atomic force microscopy (AFM) methods. The atypical and cystic forms found in the brains of three patients with neuropathologically confirmed Lyme neuroborreliosis were identical to those induced in vitro. We also observed nuclear fragmentation of the infected astrocytes using the TUNEL method. Abundant HLA-DR positive microglia and GFAP positive reactive astrocytes were present in the cerebral cortex. CONCLUSION: The results indicate that atypical extra- and intracellular pleomorphic and cystic forms of Borrelia burgdorferi and local neuroinflammation occur in the brain in chronic Lyme neuroborreliosis. The persistence of these more resistant spirochete forms, and their intracellular location in neurons and glial cells, may explain the long latent stage and persistence of Borrelia infection. The results also suggest that Borrelia burgdorferi may induce cellular dysfunction and apoptosis. The detection and recognition of atypical, cystic and granular forms in infected tissues is essential for the diagnosis and the treatment as they can occur in the absence of the typical spiral Borrelia form.

Description of Lyme disease-like syndrome in Brazil: is it a new tick borne disease or Lyme disease variation?

An emerging clinical entity that reproduces clinical manifestations similar to those observed in Lyme disease (LD) has been recently under discussion in Brazil. Due to etiological and laboratory particularities it is named LD-like syndrome or LD imitator syndrome. The condition is considered to be a zoonosis transmitted by ticks of the genus Amblyomma, possibly caused by interaction of multiple fastidious microorganisms originating a protean clinical picture, including neurological, osteoarticular and erythema migrans-like lesions. When peripheral blood of patients with LD-like syndrome is viewed under a dark-field microscope, mobile uncultivable spirochete-like bacteria are observed. PCR carried out with specific or conservative primers to recognize Borrelia burgdorferi sensu stricto or the genus Borrelia has been negative in ticks and in biological samples. Two different procedures, respectively involving hematoxylin and eosin staining of cerebrospinal fluid and electron microscopy analysis of blood, have revealed spirochetes not belonging to the genera Borrelia, Leptospira or Treponema. Surprisingly, co-infection with microorganisms resembling Mycoplasma and Chlamydia was observed on one occasion by electron microscopy analysis. We discuss here the possible existence of a new tick-borne disease in Brazil imitating LD, except for a higher frequency of recurrence episodes observed along prolonged clinical follow-up.

Chemical sensing with microbent optical fiber

We propose and demonstrate the possibility of using a permanently microbent bare optical fiber for detecting chemical species. Two detection schemes, viz., a bright-field detection scheme (for the core modes), and a dark-field detection scheme (for the cladding modes) have been employed to produce a fiber-optic sensor. The sensor described here is sensitive enough to detect concentrations as low as nanomoles per liter of a chemical species, with a dynamic range of more than 6 orders of magnitude.

A microbent fiber optic pH sensor

Optical fiber sensors developed for measuring pH values usually employ an unclad and unstrained section of the fiber. In this paper, we describe the design and fabrication of a microbent fiber optic sensor that can be used for pH sensing. In order to obtain the desired performance, a permanently microbent portion of a plastic optic fiber is coated with a thin film of dye impregnated sol–gel material. The measurements are simultaneously carried out in two independent detection schemes viz., the bright field detection configuration for detecting the core modes and dark field detection configuration, for detecting the cladding modes. The results of measurements of core mode-power and cladding mode-power variation with change in pH of a solution surrounding the coated portion of the fiber is presented. This paper thus demonstrates how a bare plastic fiber can be modified for pH sensing in a simple and cost effective manner.

Long period gratings in multimode optical fibers: application in chemical sensing

We propose and demonstrate a new technique for evanescent wave chemical sensing by writing long period gratings in a bare multimode plastic clad silica fiber. The sensing length of the present sensor is only 10 mm, but is as sensitive as a conventional unclad evanescent wave sensor having about 100 mm sensing length. The minimum measurable concentration of the sensor reported here is 10 nmol/l and the operating range is more than 4 orders of magnitude. Moreover, the detection is carried out in two independent detection configurations viz., bright field detection scheme that detects the core-mode power and dark field detection scheme that detects the cladding mode power. The use of such a double detection scheme definitely enhances the reliability and accuracy of the results. Furthermore, the cladding of the present fiber need not be removed as done in conventional evanescent wave fiber sensors.

Microbent optical fibers as evanescent wave sensors

Microbent optical fibers are potential candidates for evanescent wave sensing. We investigate the behavior of a permanently microbent fiber optic sensor when it is immersed in an absorbing medium. Two distinct detection schemes, namely, bright-field and dark-field detection configuration, are employed for the measurements. The optical power propagating through the sensor is found to vary in a logarithmic fashion with the concentration of the absorbing species in the surrounding medium. We observe that the sensitivity of the setup is dependent on the bending amplitude and length of the microbend region for the bright-field detection scheme, while it is relatively independent of both for the dark-field detection configuration. This feature can be exploited in compact sensor designs where reduction of the sensing region length is possible without sacrificing sensitivity.

Bilayer graphene formed by passage of current through graphite: evidence for a three-dimensional structure

The passage of an electric current through graphite or few-layer graphene can result in a striking structural transformation, but there is disagreement about the precise nature of this process. Some workers have interpreted the phenomenon in terms of the sublimation and edge reconstruction of essentially flat graphitic structures. An alternative explanation is that the transformation actually involves a change from a flat to a three-dimensional structure. Here we describe detailed studies of carbon produced by the passage of a current through graphite which provide strong evidence that the transformed carbon is indeed three-dimensional. The evidence comes primarily from images obtained in the scanning transmission electron microscope using the technique of high-angle annular dark-field imaging, and from a detailed analysis of electron energy loss spectra. We discuss the possible mechanism of the transformation, and consider potential applications of “three-dimensional bilayer graphene”.

Effects of calcining conditions on the microstructure of sugar cane waste ashes (SCWA): Influence in the pozzolanic activation

in this paper a study of calcining conditions on the microstructural features of sugar cane waste ash (SCWA) is carried out. For this purpose, some microparticles (< 90 mu m) of sugar cane straw ash and sugar cane bagasse ash of samples calcined at 800 degrees C and 1000 are studied by combining the bright field and the dark field images with the electron diffraction patterns in the transmission electron microscopy (TEM). It is appreciated that the morphology and texture of these microparticles change when silicon or calcium are present. Furthermore, it is observed that iron oxide (magnetite Fe(3)O(4)) is located in the calcium-rich particles. The microstructural information is correlated with the results of a kinetic-diffusive model that allows the computing of the kinetic parameters of the pozzolanic reaction (mainly the reaction rate constant). The results show that the sugar cane wastes ash calcined at 800 and 1000 degrees C have properties indicative of high pozzolanic activity. The X-ray diffraction patterns, the TEM images and the pozzolanic activity tests show the influence of different factors on the activation of these ashes. (c) 2008 Elsevier Ltd. All rights reserved.

Detection of Leptospira spp. in the semen and urine of bulls serologically reactive to Leptospira interrogans serovar hardjo

O presente trabalho avaliou a PCR na detecção de leptospiras em sêmen e urina de dez touros sorologicamente reagentes, comparando seus resultados com aqueles obtidos por outras técnicas de diagnóstico. Foram realizadas duas colheitas de materiais em dias alternados. As amostras de sêmen e de urina foram separadas em alíquotas para visualização direta em microscopia de campo escuro, inoculação em hamsters (apenas para o sêmen), isolamento em meio de cultura e PCR. Nenhum hamster apresentou positividade na prova de soroaglutinação microscópica (SAM); fragmentos de rins e fígado desses animais foram utilizados para a tentativa de isolamento em meio de cultura, sendo positivo o cultivo a partir do rim de hamster inoculado com semen de um touro, e do fígado de hamsters inoculados com o semen de três touros. O isolamento em meio de cultura foi negativo para todas as amostras de sêmen, mas foi positivo para cinco amostras de urina. Na PCR não houve resultado positivo para as amostras de sêmen, e apenas uma amostra de urina apresentou resultado positivo, sendo coincidente com uma das culturas positivas. Não foi possível visualizar leptospiras em nenhuma das amostras por exame direto em microscopia de campo escuro.