Cytokine gene expression and molecular detection of Mycobacterium avium subspecies paratuberculosisin organs of experimentally infected mice

AbstractMycobacterium avium subspecies paratuberculosis (MAP) can infect ruminants and remain subclinical for long periods within herds. The identification of organs that are more susceptible to infection and the evaluation of cytokine expression at the site of infection are important to understand the pathogenesis of MAP. In this study, the probability of detection of MAP-DNA and the expression of cytokines in organs of C57BL/6 mice infected intraperitoneally for 120 days were evaluated. Among the evaluated organs, the spleen (85%), colon (75%) and liver (60%) had the highest frequency of positivity. When compared these frequencies between organs, it has been found that the spleen had 1.54 times as likely to be positive in relation to the ileum, and 2.0 times more likely in relation to the Peyer's patches. In addition, at 60 days post-infection, the spleen and the liver were responsible for upregulation of IFN-γ , and the ileum by TNF-α and IL-4. The results indicate that the spleen is the best organ for evaluating an experimental infection by MAP, especially in the initial stages of the infection. Moreover, it showed that the spleen, liver and ileum have a direct role in the inflammatory response in experimental models.

Detection of virulence factors and antimicrobial resistance patterns in shiga toxin-producing Escherichia coli isolates from sheep

Abstract: In order to detect virulence factors in Shiga toxin-producing Escherichia coli (STEC) isolates and investigate the antimicrobial resistance profile, rectal swabs were collected from healthy sheep of the races Santa Inês and Dorper. Of the 115 E. coli isolates obtained, 78.3% (90/115) were characterized as STEC, of which 52.2% (47/90) carried stx1 gene, 33.3% (30/90) stx2 and 14.5% (13/90) both genes. In search of virulence factors, 47.7% and 32.2% of the isolates carried the genes saa and cnf1. According to the analysis of the antimicrobial resistance profile, 83.3% (75/90) were resistant to at least one of the antibiotics tested. In phylogenetic classification grouped 24.4% (22/90) in group D (pathogenic), 32.2% (29/90) in group B1 (commensal) and 43.3% (39/90) in group A (commensal). The presence of several virulence factors as well as the high number of multiresistant isolates found in this study support the statement that sheep are potential carriers of pathogens threatening public health.

Covalidation of Integral Transforms and Method of Lines in Nonlinear Convection-Diffusion with Mathematica

The Mathematica system (version 4.0) is employed in the solution of nonlinear difusion and convection-difusion problems, formulated as transient one-dimensional partial diferential equations with potential dependent equation coefficients. The Generalized Integral Transform Technique (GITT) is first implemented for the hybrid numerical-analytical solution of such classes of problems, through the symbolic integral transformation and elimination of the space variable, followed by the utilization of the built-in Mathematica function NDSolve for handling the resulting transformed ODE system. This approach ofers an error-controlled final numerical solution, through the simultaneous control of local errors in this reliable ODE's solver and of the proposed eigenfunction expansion truncation order. For covalidation purposes, the same built-in function NDSolve is employed in the direct solution of these partial diferential equations, as made possible by the algorithms implemented in Mathematica (versions 3.0 and up), based on application of the method of lines. Various numerical experiments are performed and relative merits of each approach are critically pointed out.

Detection of Horizontal Two-Phase Flow Patterns Through a Neural Network Model

One of the main problems related to the transport and manipulation of multiphase fluids concerns the existence of characteristic flow patterns and its strong influence on important operation parameters. A good example of this occurs in gas-liquid chemical reactors in which maximum efficiencies can be achieved by maintaining a finely dispersed bubbly flow to maximize the total interfacial area. Thus, the ability to automatically detect flow patterns is of crucial importance, especially for the adequate operation of multiphase systems. This work describes the application of a neural model to process the signals delivered by a direct imaging probe to produce a diagnostic of the corresponding flow pattern. The neural model is constituted of six independent neural modules, each of which trained to detect one of the main horizontal flow patterns, and a last winner-take-all layer responsible for resolving when two or more patterns are simultaneously detected. Experimental signals representing different bubbly, intermittent, annular and stratified flow patterns were used to validate the neural model.

DETECTION OF GLYPHOSATE-RESISTANT PALMER AMARANTH (Amaranthus palmeri) IN AGRICULTURAL AREAS OF MATO GROSSO, BRAZIL

ABSTRACT The recent introduction of Palmer amaranth (Amaranthus palmeri) in Brazilian agricultural areas may promote several changes on weed management, especially in no-till systems and in glyphosate-resistant crops, since glyphosate-resistant biotypes of A. palmerihave been frequently selected in other countries. Therefore, this research was developed in order to evaluate the glyphosate susceptibility of a Palmer amaranth biotype recently identified in the State of Mato Grosso, Brazil. For this purpose, glyphosate susceptibility of three Amaranthusbiotypes was compared: A.hybridus var. patulus, collected in the State of Rio Grande do Sul - Brazil; A.hybridus var. patulus, collected in the State of São Paulo - Brazil; and A.palmeri, collected in the State of Mato Grosso - Brazil. Dose-response curves were generated for all biotypes, considering eight rates of glyphosate and six replicates. All the experiments were repeated twice. Both A.hybridus biotypes were satisfactorily controlled by glyphosate, demanding rates equal to or lower than 541.15 g a.e. ha-1 for 80% control (LD80). The A.palmeri biotype was not controlled by glyphosate in any of the assessments and required rates greater than 4,500 g a.e. ha-1 to reach LD80, which are economically and environmentally unacceptable. Comparison of the Brazilian A.palmeri biotype to the A. hybridus biotypes, as well as, to the results available in scientific international literature, led to the conclusion that the Brazilian Palmer amaranth biotype is resistant to glyphosate.

Cyanobacterial occurrence and detection of microcystin by planar chromatography in surface water of Billings and Guarapiranga Reservoirs, SP, Brazil

Billings and Guarapiranga Reservoirs were deeply affected by environmental disturbances, which more evident consequence are the cyanobacterial blooms. Microcystins are the most common cyanotoxin in freshwaters and more than 70 types are known. Different methods for microcystins analysis in water can be used, among which ELISA and HPLC are the most frequently employed. However, less sophisticated and more economic methods can also be used. This is the case of planar chromatography (thin-layer chromatography) method previously used in cyanotoxins purification but gradually replaced by others. Posterior optimization of the microcystin chromatography conditions and because of its simplicity, rapidity, efficiency and low cost, this method is again considered an option for the analysis of microcystins and nodularins. Considering the importance of Billings and Guarapiranga Reservoirs for drinking water supplies and the few scientific data about cyanobacteria and cyanotoxins in these water bodies, the aims of this work are to analyze the biodiversity of cyanobacteria in the Billings and Guarapiranga Reservoirs and the detection of dissolved microcystins in the water. It was possible to identify 17 species of cyanobacteria, 9 of them being potentially toxic. In Billings Reservoir Microcystis aeruginosa (Kützing) Kützing and Cylindrospermopsis raciborskii (Woloszynska) Seenayya & Subba Raju are the most common species, while in Guarapiranga Reservoir only M. aeruginosa was considered as a common species. Microcystins were detected in all Billings Reservoir samples and in only one sample from Guarapiranga Reservoir.

Detection of anthraquinones and identification of 1,4-naphthohydroquinone in cell suspension cultures of Rudgea jasminoides (Cham.) Müll. Arg. (Rubiaceae)

In Rubiaceae, anthraquinones and naphthoquinones are secondary metabolites characteristic of the subfamily Rubioideae, in which Rudgea jasminoides is included. Thin-layer chromatography using specific solvent systems and spray reagents indicated the presence of anthraquinones constitutively produced by cell suspension cultures of R. jasminoides. GC/MS analysis detected 1,4-naphthohydroquinone as a product of biosynthesis only after elicitation of the cells with yeast extract (Saccharomyces cerevisiae). The latter compound is probably a phytoalexin produced by suspension cultures of R. jasminoides.

Inhibitory effects in the detection of 1 cpd jo targets superimposed to angular frequency stimuli or sinewave gratings

Independence among channels processing different aspects of spatial information, including orthogonal stimuli, has been generally assumed in the literature. We tested independence between the processing of jo targets and the processing of either vertical sinusoidal gratings or angular frequency stimuli with suprathreshold summation. We found the detection of a jo target at 1 cpd to be affected in an inhibitory fashion by either background angular frequencies in the range of 3-96 cycles or sinewave gratings in the range of 0.8-3.0 cpd. These results demonstrate interactions both among orthogonal stimuli and among channels processing vertical sinewave gratings and jo target stimuli. Our discussion focuses on the hypothesis of frequency decomposition in polar coordinates

Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni

Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS) before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 µg/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula) are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite

Detection of cytotoxic activity on Vero cells in clinical isolates of Serratia marcescens

Cytotoxin production was studied in 60 Serratia marcescens strains isolated from hospitalized patients. Association of cytotoxic activity with serotype, source of isolation and presence of plasmids was also evaluated. Thirteen of the 60 S. marcescens strains produced a cytotoxic effect on Vero cells. These strains were isolated from distinct clinical sources and classified into seven different serotypes (O1:H7; O4:NM; O10:NT; O19:NM; O6,14:H4; O6,14:NM and O6,14:H1). No relationship was observed between cytotoxic activity and clinical source or serotypes of the strains. Plasmids from five cytotoxin-producing S. marcescens strains were transferred to E. coli K12/711. The transconjugants did not exhibit cytotoxicity, indicating that the cytotoxic effect is not plasmid-mediated among these strains. Although a cytotoxic activity was demonstrated in filtrates of some S. marcescens strains, further studies should be performed to assess the role of this toxin in pathogenesis

Patch-clamp detection of macromolecular translocation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus

Mouse inoculation for the detection of non-cultivable gastric tightly spiralled bacteria

In the present study we compared the inoculation of swine gastric mucus into the stomach of mice, the urease test and carbolfuchsin-stained smears for the diagnosis of the infection with "Gastrospirillum suis" ("Helicobacter heilmannii" type 1), an uncultivated tightly spiralled gastric bacterium. Fragments obtained from the antral and oxyntic mucosa of the stomach of 50 slaughtered pigs were used for urease test, for carbolfuchsin-stained smears and for obtaining scrapings of mucus for mouse inoculation. The mice were killed by spinal dislocation 10 days after inoculation and fragments of the antral and oxyntic mucosa were used for spiral bacterium identification (urease test and carbolfuchsin-stained smears). Among the methods employed for the diagnosis of "H. heilmannii" infection, the inoculation of gastric mucus into the stomach of mice was the most sensitive and demonstrated bacterial positivity in 31 (62.0%) swine. Direct examination showed tightly spiralled bacteria in the gastric mucosa of only 4 (8.0%) of the 50 pigs studied. Among them, 3 (6.0%) presented a positive preformed urease test. Spiral bacteria were not seen in the gastric mucosa of any control mice. These results show that the use of the mouse inoculation method improved the detection of "H. heilmannii" in swine

Western blot detection of infectious bursal disease virus infection

In order to evaluate the use of a Western blot methodology for the diagnosis of infectious bursal disease virus (IBDV) infection, chickens were experimentally infected with IBDV strains and tested for the presence of viral antigens and antibodies by a blocking Western blot test (bWB). The viral proteins obtained from the bursa of Fabricius (BF) were transferred to a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the chicken sera obtained by heart puncture were used for the detection of these proteins. In order to eliminate nonspecific reactions, we used a rabbit anti-chicken serum (blocking tool). By the use of the bWB test, two distinct viral proteins of 43-kDa (VP2) and 32-kDa (VP3) were detected. We suggest the use of this methodology for the detection of IBDV infection in animals suspected of having IBDV reinfection and a chronic subclinical form of the disease. With the use of the rabbit anti-chicken sera for blocking, this method is practical, sensitive and less time consuming

Improvement of the indirect hemagglutination test for the detection of antibodies to Streptococcus pyogenes

An indirect hemagglutination test for a seroepidemiological survey of Streptococcus pyogenes infection was standardized. This is an improved modification of the indirect hemagglutination test which utilizes an unstable reagent prepared with fresh blood cells. Two types of bacterial antigens represented by extracellular products and purified streptolysin O were assayed, but only the former antigen gave good results. Pretreatment of the bacterial antigen with 0.15 M NaOH and neutralization to pH 5.5, as well as postfixation of sensitized red cells with 0.1% glutaraldehyde at 56oC for 30 min were found to be essential to give long stability to the reagent in liquid suspension, at least 9 months at 4oC. A total of 564 serum samples with high, moderate and low anti-streptolysin O antibodies as determined by the neutralization assay were studied by the indirect hemagglutination test using the new reagent. The sensitivity, specificity, efficiency, positive predictive value and negative predictive value of the test in relation to the neutralization assay were 0.950, 0.975, 0.963, 0.973, and 0.955, respectively. The kappa agreement index between the two techniques was high (0.926) and ranked as "almost perfect". Antibody levels detected by both techniques also presented a high positive correlation (rs = 0.726). Five reagent batches successively produced proved to be reproducible. Thus, the improved indirect hemagglutination test seems to be useful for public health laboratories.

Detection of adrenocortical autoantibodies in Addison's disease with a peroxidase-labelled protein A technique

Adrenocortical autoantibodies (ACA), present in 60-80% of patients with idiopathic Addison's disease, are conventionally detected by indirect immunofluorescence (IIF) on frozen sections of adrenal glands. The large-scale use of IIF is limited in part by the need for a fluorescence microscope and the fact that histological sections cannot be stored for long periods of time. To circumvent these restrictions we developed a novel peroxidase-labelled protein A (PLPA) technique for the detection of ACA in patients with Addison's disease and compared the results with those obtained with the classical IIF assay. We studied serum samples from 90 healthy control subjects and 22 patients with Addison's disease, who had been clinically classified into two groups: idiopathic (N = 13) and granulomatous (N = 9). ACA-PLPA were detected in 10/22 (45%) patients: 9/13 (69%) with the idiopathic form and 1/9 (11%) with the granulomatous form, whereas ACA-IIF were detected in 11/22 patients (50%): 10/13 (77%) with the idiopathic form and 1/9 (11%) with the granulomatous form. Twelve of the 13 idiopathic addisonians (92%) were positive for either ACA-PLPA or ACA-IIF, but only 7 were positive by both methods. In contrast, none of 90 healthy subjects was found to be positive for ACA. Thus, our study shows that the PLPA-based technique is useful, has technical advantages over the IIF method (by not requiring the use of a fluorescence microscope and by permitting section storage for long periods of time). However, since it is only 60% concordant with the ACA-IIF method, it should be considered complementary instead of an alternative method to IIF for the detection of ACA in human sera.