Etiologic diagnosis of bovine infectious abortion by PCR

Infectious abortion is a significant cause of reproductive failure and economic losses in cattle. The goal of this study was to detect nucleic acids of several infectious agents known to cause abortion including Arcanobacterium pyogenes, Bovine Herpesvirus 1, Brucella abortus, Campylobacter fetus subsp. venerealis, Chlamydophila abortus, Leptospira sp., Listeria monocytogenes, Salmonella sp., Mycoplasma bovis, Mycoplasma bovigenitalium, Neospora caninum, and Tritrichomonas foetus. Tissue homogenates from 42 fetuses and paraffin-embedded tissues from 28 fetuses and 14 placentas/endometrium were included in this study. Brucella abortus was detected in 14.2% (12/84) of the samples. Salmonella sp. DNA was amplified from 2 fetuses, and there was one positive for Neospora caninum, and another for Listeria monocytogenes. This PCR-based approach resulted in identification of the etiology in 19% of samples, or 20% if considered fetal tissues only.

EMJH medium with 5-fluorouracil and nalidixic acid associated with serial dilution technique used to recover Leptospira spp from experimentally contaminated bovine semen

Bovine semen experimentally contaminated with Leptospira santarosai serovar Guaricura was submitted to the modified EMJH medium with 5-fluorouracil (300mg/L) and nalidixic acid (20mg/L), named as "selective medium" and using the serial dilution technique, in order to evaluate the percentage of recovery of the added microorganism. The selective EMJH medium was found with higher percentage of recovery of leptospiras and minor losses of samples due to contamination with opportunistic microorganisms than the non-selective EMJH medium: 151/376 (40.0%) of positive growth; and 38/376 (10.0%) contamination and 58/376 (15%) and 129/376 (34.0%), respectively. These results were statistically significant (p<0. 0001; Fisher). Differences were found when the frequencies of positive leptospires recovery have been compared in the serial dilution technique (10-1 to 10-4) between the selective and non-selective media at different dilution factors. At 1/10th dilution the percentages found were (0%, 0/80) and (38%, 30/80), at 1/100th dilution, (3%, 2/80) and (49%, 39/80) and at 1/1,000th dilution, (25%, 20/80) and (50%, 40/80), respectively. The percentage of recovery of leptospires was found to be directly proportional to the dilution used. The methodology of the serial dilution technique (setting at least three dilutions) and the use of selective EMJH medium have been found to be efficient for the isolation of leptospires from the bovine semen samples.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

Damage assessment of the equine sperm membranes by fluorimetric technique

To validate a practical technique of simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in equine spermatozoa three fluorescent probes (PI, FITC-PSA and MITO) were associated. Four ejaculates from three stallions (n=12) were diluted in TALP medium and split into 2 aliquots, 1 aliquot was flash frozen in liquid nitrogen to induce damage in cellular membranes. Three treatments were prepared with the following fixed ratios of fresh semen: flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen of each treatment was added of 2 µL of PI, 2 µL of MITO and 80 µL of FITC-PSA; incubated at 38.5ºC/8 min, and sperm cells were evaluated by epifluorescent microscopy. Based in regression analysis, this could be an efficient and practical technique to assess damage in equine spermatozoa, as it was able to determine the sperm percentage more representative of the potential to fertilize the oocyte.

Protective effects of mate tea (Ilex paraguariensis) on H2O2-induced DNA damage and DNA repair in mice

Yerba mate (Ilex paraguariensis) is rich in several bioactive compounds that can act as free radical scavengers. Since oxidative DNA damage is involved in various pathological states such as cancer, the aim of this study was to evaluate the antioxidant activity of mate tea as well as the ability to influence DNA repair in male Swiss mice. Forty animals were randomly assigned to four groups. The animals received three different doses of mate tea aqueous extract, 0.5, 1.0 or 2.0 g/kg, for 60 days. After intervention, the liver, kidney and bladder cells were isolated and the DNA damage induced by H2O2 was investigated by the comet assay. The DNA repair process was also investigated for its potential to protect the cells from damage by the same methodology. The data presented here show that mate tea is not genotoxic in liver, kidney and bladder cells. The regular ingestion of mate tea increased the resistance of DNA to H2O2-induced DNA strand breaks and improved the DNA repair after H2O2 challenge in liver cells, irrespective of the dose ingested. These results suggest that mate tea could protect against DNA damage and enhance the DNA repair activity. Protection may be afforded by the antioxidant activity of the mate tea's bioactive compounds

Spectral Region Optimization for Raman-Based Optical Biopsy of Inflammatory Lesions

Objective: The biochemical alterations between inflammatory fibrous hyperplasia (IFH) and normal tissues of buccal mucosa were probed by using the FT-Raman spectroscopy technique. The aim was to find the minimal set of Raman bands that would furnish the best discrimination. Background: Raman-based optical biopsy is a widely recognized potential technique for noninvasive real-time diagnosis. However, few studies had been devoted to the discrimination of very common subtle or early pathologic states as inflammatory processes that are always present on, for example, cancer lesion borders. Methods: Seventy spectra of IFH from 14 patients were compared with 30 spectra of normal tissues from six patients. The statistical analysis was performed with principal components analysis and soft independent modeling class analogy cross-validated, leave-one-out methods. Results: Bands close to 574, 1,100, 1,250 to 1,350, and 1,500 cm(-1) (mainly amino acids and collagen bands) showed the main intragroup variations that are due to the acanthosis process in the IFH epithelium. The 1,200 (C-C aromatic/DNA), 1,350 (CH(2) bending/collagen 1), and 1,730 cm(-1) (collagen III) regions presented the main intergroup variations. This finding was interpreted as originating in an extracellular matrix-degeneration process occurring in the inflammatory tissues. The statistical analysis results indicated that the best discrimination capability (sensitivity of 95% and specificity of 100%) was found by using the 530-580 cm(-1) spectral region. Conclusions: The existence of this narrow spectral window enabling normal and inflammatory diagnosis also had useful implications for an in vivo dispersive Raman setup for clinical applications.

Evaluation technique for determining wheel performance in the grinding of aerospace materials

Nickel-based super alloys are used in a variety of applications in which high-temperature strength and resistance to creep, corrosion, and oxidation are required, such as in aircraft gas turbines, combustion chambers, and automotive engine valves. The properties that make these materials suitable for these applications also make them difficult to grind. Grinding systems for such materials are often built around vitrified cBN (cubic boron nitride) wheels to realize maximum productivity and minimum cost per part. Conditions that yield the most economical combination of stock removal rate and wheel wear are key to the successful implementation of the grinding system. Identifying the transition point for excessive wheel wear is important. The aim of this study is to compare the performance of different cBN wheels when grinding difficult-to-grind (DTG) materials by determining the 'wheel wear characteristic curve', which correlates the G-ratio to the calculated tangential force per abrasive grain. With the proposed methodology, a threshold force per grit above which the wheel wear rate increases rapidly can be quickly identified. A comparison of performance for two abrasive product formulations in the grinding of three materials is presented. The obtained results can be applied for the development of grinding applications for DTG materials.

Computational adjustment technique for digital mammographic images based on the digitizer characteristic curve

We evaluated the performance of a novel procedure for segmenting mammograms and detecting clustered microcalcifications in two types of image sets obtained from digitization of mammograms using either a laser scanner, or a conventional ""optical"" scanner. Specific regions forming the digital mammograms were identified and selected, in which clustered microcalcifications appeared or not. A remarkable increase in image intensity was noticed in the images from the optical scanner compared with the original mammograms. A procedure based on a polynomial correction was developed to compensate the changes in the characteristic curves from the scanners, relative to the curves from the films. The processing scheme was applied to both sets, before and after the polynomial correction. The results indicated clearly the influence of the mammogram digitization on the performance of processing schemes intended to detect microcalcifications. The image processing techniques applied to mammograms digitized by both scanners, without the polynomial intensity correction, resulted in a better sensibility in detecting microcalcifications in the images from the laser scanner. However, when the polynomial correction was applied to the images from the optical scanner, no differences in performance were observed for both types of images. (C) 2008 SPIE and IS&T [DOI: 10.1117/1.3013544]

DEVELOPMENT OF MICROSATELLITE MARKERS FOR AULONEMIA ARISTULATA (POACEAE) AND CROSS-AMPLIFICATION IN OTHER BAMBOO SPECIES

Premise of the study: Microsatellite primers were developed for Aulonemia aristulata, an endangered species of economic interest, to further describe its genetic variability and population structure. We also tested cross-amplification in 18 other bamboo species. Methods and Results: Using an enrichment genomic library, 13 microsatellite loci were isolated and characterized in A. aristulata. Seven of these loci were polymorphic. Twelve markers were cross-amplified in at least ten of the tested bamboo species. Conclusions: These markers will be useful for studies on the genetic diversity and structure of A. aristulata, which are important for future conservation, management and breeding programs of this species.

Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis

We evaluated the genetic and physiological variability of Moniliophthora perniciosa obtained from healthy and diseased branches of cacao (Theobroma cacao) plants. The diversity of the isolates was evaluated by RAPD technique and by studies of virulence and exoenzyme production. The genetic variability of endophytic and pathogenic M. perniciosa was evaluated in association with pathogenicity assays. RAPD analysis showed eight genetic groups, which were not related to plant disease status (healthy versus diseased branches). Isolates from cacao were included in three groups, excluding isolates from other host plants. Pathogenicity and enzyme analysis showed that the virulence of the isolates is not related to exoenzyme production. This is the first evidence that M. perniciosa colonizes healthy parenchymatic tissues, showing that endophytic behavior may occur in this species.

Helminth Coinfection Does Not Affect Therapeutic Effect of a DNA Vaccine in Mice Harboring Tuberculosis

Background: Helminthiasis and tuberculosis (TB) coincide geographically and there is much interest in exploring how concurrent worm infections might alter immune responses against bacilli and might necessitate altered therapeutic approaches. A DNA vaccine that codifies heat shock protein Hsp65 from M. leprae (DNAhsp65) has been used in therapy during experimental tuberculosis. This study focused on the impact of the co-existence of worms and TB on the therapeutic effects of DNAhsp65. Methodology/Principal Findings: Mice were infected with Toxocara canis or with Schistosoma mansoni, followed by coinfection with M. tuberculosis and treatment with DNAhsp65. While T. canis infection did not increase vulnerability to pulmonary TB, S. mansoni enhanced susceptibility to TB as shown by higher numbers of bacteria in the lungs and spleen, which was associated with an increase in Th2 and regulatory cytokines. However, in coinfected mice, the therapeutic effect of DNAhsp65 was not abrogated, as indicated by colony forming units and analysis of histopathological changes. In vitro studies indicated that Hsp65-specific IFN-gamma production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-beta and IL-10 production, which could be associated with long-term protection. Conclusions/Significance: We have demonstrated that the therapeutic effects of DNAhsp65 in experimental TB infection are persistent in the presence of an unrelated Th2 immune response induced by helminth infections.

Influence of memory in deterministic walks in random media: Analytical calculation within a mean-field approximation

Consider a random medium consisting of N points randomly distributed so that there is no correlation among the distances separating them. This is the random link model, which is the high dimensionality limit (mean-field approximation) for the Euclidean random point structure. In the random link model, at discrete time steps, a walker moves to the nearest point, which has not been visited in the last mu steps (memory), producing a deterministic partially self-avoiding walk (the tourist walk). We have analytically obtained the distribution of the number n of points explored by the walker with memory mu=2, as well as the transient and period joint distribution. This result enables us to explain the abrupt change in the exploratory behavior between the cases mu=1 (memoryless walker, driven by extreme value statistics) and mu=2 (walker with memory, driven by combinatorial statistics). In the mu=1 case, the mean newly visited points in the thermodynamic limit (N >> 1) is just < n >=e=2.72... while in the mu=2 case, the mean number < n > of visited points grows proportionally to N(1/2). Also, this result allows us to establish an equivalence between the random link model with mu=2 and random map (uncorrelated back and forth distances) with mu=0 and the abrupt change between the probabilities for null transient time and subsequent ones.

Pulse train fluorescence technique for measuring triplet state dynamics

We report on a method to study the dynamics of triplet formation based on the fluorescence signal produced by a pulse train. Basically, the pulse train acts as sequential pump-probe pulses that precisely map the excited-state dynamics in the long time scale. This allows characterizing those processes that affect the population evolution of the first excited singlet state, whose decay gives rise to the fluorescence. The technique was proven to be valuable to measure parameters of triplet formation in organic molecules. Additionally, this single beam technique has the advantages of simplicity, low noise and background-free signal detection. (C) 2011 Optical Society of America

Gene Expression Profiles in Radiation Workers Occupationally Exposed to Ionizing Radiation

Ionizing radiation OR) imposes risks to human health and the environment. IR at low doses and low (lose rates has the potency to initiate carcinogenesis. Genotoxic environmental agents such as IR trigger a cascade of signal transduction pathways for cellular protection. In this study, using cDNA microarray technique, we monitored the gene expression profiles in lymphocytes derived from radiation-ex posed individuals (radiation workers). Physical dosimetry records on these patients indicated that the absorbed dose ranged from 0.696 to 39.088 mSv. Gene expression analysis revealed statistically significant transcriptional changes in a total of 78 genes (21 up-regulated and 57 clown-regulated) involved in several biological processes such as ubiquitin cycle (UHRF2 and PIAS1), DNA repair (LIG3, XPA, ERCC5, RAD52, DCLRE1C), cell cycle regulation/proliferation (RHOA, CABLES2, TGFB2, IL16), and stress response (GSTP1, PPP2R5A, DUSP22). Some of the genes that showed altered expression profiles in this study call be used as biomarkers for monitoring the chronic low level exposure in humans. Additionally, alterations in gene expression patterns observed in chronically exposed radiation workers reinforces the need for defining the effective radiation dose that causes immediate genetic damage as well as the long-term effects on genomic instability, including cancer.

Characterization of Hepatitis B virus (HBV) genotypes in patients from Rondonia, Brazil

Background: Hepatitis B virus (HBV) can be classified into nine genotypes (A-I) defined by sequence divergence of more than 8% based on the complete genome. This study aims to identify the genotypic distribution of HBV in 40 HBsAg-positive patients from Rondonia, Brazil. A fragment of 1306 bp partially comprising surface and polymerase overlapping genes was amplified by PCR. Amplified DNA was purified and sequenced. Amplified DNA was purified and sequenced on an ABI PRISM (R) 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were aligned with reference sequences obtained from the GenBank using Clustal X software and then edited with Se-Al software. Phylogenetic analyses were conducted by the Markov Chain Monte Carlo (MCMC) approach using BEAST v.1.5.3. Results: The subgenotypes distribution was A1 (37.1%), D3 (22.8%), F2a (20.0%), D4 (17.1%) and D2 (2.8%). Conclusions: These results for the first HBV genotypic characterization in Rondonia state are consistent with other studies in Brazil, showing the presence of several HBV genotypes that reflects the mixed origin of the population, involving descendants from Native Americans, Europeans, and Africans.