The structures of the honeydew oligosaccharides synthesized by Claviceps africana

The structures of the principal oligosaccharides in the honeydew exudate of the sorghum ergot pathogen Claviceps africana, which has become epidemic in the Americas, have been characterized through linkage analysis using FAB-MS and GC-MS techniques, as 1,6-di-b-D-fructofuranosyl-D-mannitol and 1,5-di-b-D-fructofuranosyl-D-arabitol trisaccharides, 1-b-D-fructofuranosyl-D-mannitol and 5-b-D-fructofuranosyl-D-arabitol disaccharides and other minor disaccharides and trisaccharides. Their structural diversity is explained according to perceived biosynthetic interrelationships in pathways that appear to be unique amongst ergot fungi, particularly concerning intra-molecular reduction of fructose. The oligosaccharide, 1,6-di-b-D-fructofuranosyl-D-mannitol, which inhibits C. africana macrospore germination at a concentration in water of 1 g/mL or more, forms together with other slightly less bioactive oligosaccharides, the basis of a novel strategy to limit ergot disease losses in hybrid sorghum seed production.

Progress of Asian soybean rust and airborne urediniospores of Phakopsora pachyrhizi in southern Brazil

Asian soybean rust, caused by the fungus Phakopsora pachyrhizi, was reported at epidemic levels in 2003/2004 and is the main soybean disease in Brazil. The aim of this study was to investigate the spread of Asian soybean rust and to quantify airborne urediniospores in the region of Campo Mourão, Paraná State, Brazil. Three experiments were conducted under field conditions during the 2007/08 and 2008/09 crop seasons. Using the disease gradient method, provided by the application of increasing levels of the fungicide tebuconazole, four Asian soybean rust epidemics at different intensities were obtained in each experiment. To quantify the urediniospores, weathercock-type spore collectors were installed during and between the two crop seasons. Disease progress curves were plotted for each epidemic, and maximum severity was estimated. The curves were fit to the logistic model, which provided higher coefficients of determination and more randomly distributed residuals plotted over time. Analyses of the area under the disease progress curve showed that the largest epidemics occurred in the 2007/2008 crop season and that the progress rates were higher for severity, even among plants protected with the fungicide. The number of urediniospores collected in the air was related to the presence of soybean plants in the cultivated crops. The quantity of urediniospores was also positively correlated to the disease severity and incidence, as well as to cumulative rainfall and favorable days for P. Pachyrhizi infection.

Diagrammatic scale for the assessment of blast on wheat spikes

The correct quantification of blast caused by the fungus Magnaporthe oryzae on wheat (Triticum aestivum) spikes is an important component to understand the development of this disease aimed at its control. Visual quantification based on a diagrammatic scale can be a practical and efficient strategy that has already proven to be useful against several plant pathosystems, including diseases affecting wheat spikes like glume blotch and fusarium head blight. Spikes showing different disease severity values were collected from a wheat field with the aim of elaborating a diagrammatic scale to quantify blast severity on wheat spikes. The spikes were photographed and blast severity was determined by using resources of the software ImageJ. A diagrammatic scale was developed with the following disease severity values: 3.7, 7.5, 21.4, 30.5, 43.8, 57.3, 68.1, 86.0, and 100.0%. An asymptomatic spike was added to the scale. Scale validation was performed by eight people who estimated blast severity by using digitalized images of 40 wheat spikes. The precision and the accuracy of the evaluations varied according to the rater (0.82

Use of pyrosequencing to identify streptococci and to detect mutations causing antimicrobial resistance

Rapid identification and resistance determination of pathogens in clinical specimens is vital for accurate treatment and monitoring of infectious diseases. Antimicrobial drug resistance is increasing globally and healthcare settings are facing this cost-intensive and even life-threatening problem. The incidence of resistant pathogens in Finland has remained relatively steady and manageable at least for the time being. DNA sequencing is the gold standard method for genotyping, mutation analysis, and identification of bacteria. Due to significant cost decrease in recent years, this technique is available to many research and clinical laboratories. Pyrosequencing technique, a rapid real-time DNA sequencing method especially suitable for analyzing fairly short stretches of DNA, was used in this study. Due to its robustness and versatility, pyrosequencing was applied in this study for identification of streptococci and detection of certain mutations causing antimicrobial resistance in different bacteria. Certain streptococcal species such as S. pneumoniae and S. pyogenes are significantly important clinical pathogens. S. pneumoniae causes e.g. pneumonia and otitis media and is one of the most important community-acquired pathogens. S. pyogenes, also known as group A streptococcus, causes e.g. angina and erysipelas. In contrast, the socalled alpha-haemolytic streptococci, such as S. mitis and S. oralis, belong to the normal microbiota, which are regarded to be non-pathogenic and are nearly impossible to identify by phenotypic methods. In this thesis, a pyrosequencing method was developed for identification of streptococcal species based on the 16S rRNA sequences. Almost all streptococcal species could be differentiated from one another by the developed method, including S. pneumoniae from its close relatives S. mitis and S. oralis . New resistance genes and their variants are constantly discovered and reported. In this study, new methods for detecting certain mutations causing macrolide resistance or extended spectrum beta-lactamase (ESBL) phenotype were developed. These resistance detection approaches are not only suitable for surveillance of mechanisms causing antimicrobial resistance but also for routine analysis of clinical samples particularly in epidemic settings. In conclusion, pyrosequencing was found to be an accurate, versatile, cost-effective, and rapid DNA sequencing method that is especially suitable for mutation analysis of short DNA fragments and identification of certain bacteria.

Esikaupunkien ruoka-aavikoilta urbaaniin terveysajatteluun

Kirjallisuusarvostelu

Mecanismos cirúrgicos de controle do diabetes mellitus tipo 2 após cirurgia bariátrica

Type 2 diabetes mellitus is an epidemic health problem. Approximately, 90% of diabetic patients are overweight or are obese. The current increase in the prevalence of obesity has been associated with an increase in the prevalence of type 2 diabetes. Bariatric surgery is the most effective treatment for morbid obese patients in terms of controlling weight and co-morbidities. Sustained normal plasma concentration of glucose has been reported in most diabetic morbid obese patients, which has been managed surgically. Available data show a significant alteration in the production of some gastrointestinal hormones, which might explain the improvement of glucose metabolism following these procedures. Diabetic patient improvements following some bariatric surgeries seems to be an independent factor unrelated to the amount of weight loss. The authors reviewed data published on the effects of bariatric surgery in diabetic patient improvements and the possible mechanisms responsible for this control.

Infecções por micobactérias de crescimento rápido resistentes a desinfetantes: uma problemática nacional?

Rapidly growing mycobacteria (RGM) are opportunistic microorganisms and widely distributed into aqueous environment and soil. Human RGM infections are usually associated with contaminated solutions or medical instruments used during invasive procedures. RGM postsurgical infections have recently emerged in Brazil and have caused national alert, considering the risk factors and epidemiological aspects. This study aimed at analysing the main factors linked to the recent RGM outbreaks, with focus on the national epidemic of Mycobacterium massiliense infections related to the BRA100 strains resistant to 2% glutaraldehyde commercial solutions commonly used for preoperative high-level disinfection. Based on previous studies and laboratorial results of assays and colaborations, it has been observed that the cases have been associated with videolaparoscopy for different applications and elective esthetic procedures, such as lipoaspiration and mammary prosthesis implant. Furthermore, outbreaks between 2004 and 2008 and the epidemic in Rio de Janeiro state may be considered particular Brazilian events. Although there are a few epidemiological published studies, some hypotheses based on common aspects related to most national nosocomial occurrences are possible, such as lack of protocols for cleaning and high-level disinfection, use of 2% glutaraldehyde as high-level disinfectant for surgical instruments, and dissemination of M. massiliense BRA100 by unknown mechanisms.

Two-Photon Excited Fluorescence Detection Technology in Screening for Methicillin-Resistant Staphylococcus aureus

Control of the world-wide spread of methicillin-resistant Staphylococcus aureus (MRSA) has been unsuccessful in most developed countries. A few countries have been able to maintain a low MRSA prevalence, plausibly due to their strict MRSA control policies. Such policies require wide-scale screening of patients with suspected MRSA colonization, in order to nurse the MRSA-positive patients in contact isolation. The aim of this study was to develop and introduce a 2-photon excited fluorescence detection (TPX) technique for screening of MRSA directly from clinical samples. The assay principle involves specific online immunometric monitoring of S. aureus growth under selective antibiotic pressure. After the novel TPX approach had been set up, its applicability for the detection of MRSA was evaluated using a large MRSA collection including practically all epidemic MRSA strains identified in Finland between 1991 and 2009. The TPX assay was found both sensitive (97.9%) and specific (94.1%) in this epidemiological setting, illustrating that the method is tolerant to wide biological variation as well as to environments with rapidly emerging MRSA strains. When MRSA was screened directly from colonization samples, all patients positive for MRSA by conventional methods were positive also by the TPX assay. The assay capacity was 48 samples per a test run, and the median time required for confirmation of a true-positive screening test result was 3 h 26 min. Collectively, the findings presented in this thesis suggest that the TPX MRSA screening assay could be applicable for direct screening of MRSA colonization samples without any prior steps of isolation. This can potentially mean that contact isolation of suspected carriers testing negative could be discontinued earlier, thereby reducing the costs and burden associated with the containment of MRSA. In case of infection, a positive test result would ensure an early onset of effective therapy.

Outbreak of caprine abortion by Toxoplasma gondii in Midwest Brazil

An outbreak of abortion by Toxoplasma gondii in goats on a farm in the Brazilian Midwest is reported. Gross lesions were not observed in seven aborted fetuses submitted to the Veterinary Pathology Laboratory, Federal University of Mato Grosso, for necropsy investigation. The main histologic lesions were mononuclear cell pneumonia and necrotizing encephalitis in varying degrees of intensity. PCR for Brucella abortus and Neospora caninum and aerobic cultures were negative in all cases. Antibody titles against T. gondii varying from 1:1024 to 1:32.768 were detected in serum samples from four aborted goats. Nested-PCR assay for T. gondii were positive in brain samples of all cases submitted. These findings indicate that T. gondii infection should be considered in the diagnosis of abortion in goats in Midwest Brazil.

Receptors and endocytosis of coxsackievirus A9

Coxsackievirus A9 (CV-A9) belongs to human enteroviruses within family Picornaviridae, which are the main cause of aseptic meningitis. In addition, CV-A9 causes a wide range of other clinical manifestations of acute disease including respiratory infections, myocarditis, encephalitis and severe generalized infections in newborns. In this study, the functions of integrins αVβ6 and αVβ3 in the attachment and cellular entry of CV-A9 were analyzed. Further, virus and cell surface interactions and endocytosis of CV-A9 were studied in specific cell lines. Also, a method for production of GFP-expressing CV-A9 particles by long PCR-mediated mutagenesis and in vivo transcription was developed. The results indicated that RGD-motif (arginine-glycine-asparagine) that resides in the viral capsid is important for CV-A9 infection particularly in cell lines expressing integrin αVβ6 and that this integrin serves as a high affinity attachment receptor for the virus. CV-A9 is also capable of infecting certain cell lines independently of αV-integrins by binding to the cell surface HSPA5 protein. Regardless of the attachment stage, the internalization of the virus occurs via the same entry pathway and is dependent on β2M, dynamin, and Arf6 but independent of clathrin and caveolin-1. Furthermore, the virus internalization occurs within Arf6-containing vesicles suggesting that Arf6 is central mediator of CV-A9 endocytosis. While in this study the results of CV-A9 endocytosis were based on microscopical visualization within individual fixed cells, a rapid method for generation of a virus for real-time imaging was also described.

Concomitant infection of Neospora caninum and Bovine Herpesvirus type 5 in spontaneous bovine abortions

Bovine Herpesvirus type 5 (BoHV-5) has not been conclusively demonstrated to cause bovine abortion. Brain lesions produced by Neospora caninum and Bovine Herpesvirus type 1 (BoHV-1) exhibit common features. Therefore, careful microscopic evaluation and additional diagnostic procedures are required to achieve an accurate final etiological diagnosis. The aim of the present work was to investigate the occurrence of infections due to BoHV-1, BoHV-5 and N. caninum in 68 cases of spontaneous bovine abortions which showed microscopic lesions in the fetal central nervous system. This study allowed the identification of 4 (5.9%) fetuses with dual infection by BoHV-5 and N. caninum and 33 (48.5%) cases in which N. caninum was the sole pathogen identified. All cases were negative to BoHV-1. The results of this study provide evidence that dual infection by BoHV-5 and N. caninum occur during pregnancy in cattle; however, the role of BoHV-5 as a primary cause of bovine abortion needs further research. Molecular diagnosis of BoHV-5 and N. caninum confirmed the importance of applying complementary assays to improve the sensitivity of diagnosing bovine abortion.

Fatal Human herpesvirus 1 (HHV-1) infection in captive marmosets (Callithrix jacchus and Callithrix penicillata) in Brazil: clinical and pathological characterization

Fatal Human herpesvirus 1 (HHV-1) was diagnosed in 12 captive marmosets (Callithrix jacchus and Callithrix penicillata) from metropolitan region of São Paulo, São Paulo State. Clinical signs were variable among the cases, but most affected marmosets presented signs associated with viral epithelial replication: oral, lingual and facial skin ulcers and hypersalivation, and viral replication in the central nervous system: prostration, seizure and aggressive behavior. Consistent microscopic findings were diffuse mild to severe nonsuppurative necrotizing meningoencephalitis with gliosis, vasculitis and neuronal necrosis. Additionally, in the brain, oral cavity, skin, adrenal gland and myoenteric plexus intranuclear inclusion bodies were present. Immunohistochemistry confirmed the presence of the HHV-1 antigen in association with lesions in the brain, oral and lingual mucosa, facial skin, adrenal gland and myoenteric plexus. HHV-1-specific polymerase chain reaction (PCR) analysis of the brain was carried out and the virus was detected in 7/8 infected marmosets. It is concluded that HHV-1 causes widespread fatal infection in marmosets.

Numerical Simulation of Stochastic Di erential Equations

Stochastic differential equation (SDE) is a differential equation in which some of the terms and its solution are stochastic processes. SDEs play a central role in modeling physical systems like finance, Biology, Engineering, to mention some. In modeling process, the computation of the trajectories (sample paths) of solutions to SDEs is very important. However, the exact solution to a SDE is generally difficult to obtain due to non-differentiability character of realizations of the Brownian motion. There exist approximation methods of solutions of SDE. The solutions will be continuous stochastic processes that represent diffusive dynamics, a common modeling assumption for financial, Biology, physical, environmental systems. This Masters' thesis is an introduction and survey of numerical solution methods for stochastic differential equations. Standard numerical methods, local linearization methods and filtering methods are well described. We compute the root mean square errors for each method from which we propose a better numerical scheme. Stochastic differential equations can be formulated from a given ordinary differential equations. In this thesis, we describe two kind of formulations: parametric and non-parametric techniques. The formulation is based on epidemiological SEIR model. This methods have a tendency of increasing parameters in the constructed SDEs, hence, it requires more data. We compare the two techniques numerically.

Two variants of HIV-1 B serotype are transmitted heterosexually in São Paulo, Brazil

HIV-1 variability may have an important impact on transmission and pathogenicity. Better characterization of the HIV epidemic in Brazil is necessary for the development of vaccine trials in this country. We analyzed sera from 108 HIV-1-infected volunteers from São Paulo City to determine serotype and reactivity for V3 motifs of HIV in this population, and the relationship to transmission mode. We concluded that the HIV-1 B serotype is frequent among heterosexually infected women, even in the absence of anal sex, and that two major V3 motifs, GPGR and GWGR, had similar prevalence among women (48% and 52%, respectively) and men (56% and 44%, respectively). We also observed an equal distribution of these strains regardless of their CD4+ T cell counts, clinical status, and mode of transmission. Even though V3 serology for HIV-1 subtyping is an inexpensive tool for use in developing countries, additional methods, such as heteroduplex mobility assay and direct DNA sequencing, should be included to determine HIV-1 genetic diversity.

Expression of extracellular matrix components and their receptors in the central nervous system during experimental Toxoplasma gondii and Trypanosoma cruzi infection

Alterations in extracellular matrix (ECM) expression in the central nervous system (CNS) usually associated with inflammatory lesions have been described in several pathological situations including neuroblastoma and demyelinating diseases. The participation of fibronectin (FN) and its receptor, the VLA-4 molecule, in the migration of inflammatory cells into the CNS has been proposed. In Trypanosoma cruzi infection encephalitis occurs during the acute phase, whereas in Toxoplasma infection encephalitis is a chronic persisting process. In immunocompromised individuals such as AIDS patients, T. cruzi or T. gondii infection can lead to severe CNS damage. At the moment, there are no data available regarding the molecules involved in the entrance of inflammatory cells into the CNS during parasitic encephalitis. Herein, we characterized the expression of the ECM components FN and laminin (LN) and their receptors in the CNS of T. gondii- and T. cruzi-infected mice. An increased expression of FN and LN was detected in the meninges, leptomeninges, choroid plexus and basal lamina of blood vessels. A fine FN network was observed involving T. gondii-free and T. gondii-containing inflammatory infiltrates. Moreover, perivascular spaces presenting a FN-containing filamentous network filled with a4+ and a5+ cells were observed. Although an increased expression of LN was detected in the basal lamina of blood vessels, the CNS inflammatory cells were a6-negative. Taken together, our results suggest that FN and its receptors VLA-4 and VLA-5 might be involved in the entrance, migration and retention of inflammatory cells into the CNS during parasitic infections.