Microbiological quality of hamburgers sold in the streets of Cuiabá - MT, Brazil and vendor hygiene-awareness

This study evaluated the microbiological quality of hamburgers and the microbe community on the hands of vendors in Cuiabá, Mato Grosso, Brazil, in relation to vendors´ awareness as to what constitute acceptable food-handling practices as part of a broad-spectrum research programme on street foods in Brazil . Sale of the hamburger known as the 'baguncinha' is common and widespread in urban Cuiabá, Mato Grosso, Brazil. Food inspectors encounter various difficulties in carrying out inspections. One hundred and five hamburgers samples were evaluated using conventional methods including tests for facultative aerobic and/or anaerobic mesophytic bacteria, coliform counts at 45 °C, the coagulase test for Staphylococcus, Gram-staining for the presence of Bacillus cereus, Clostridium sulphite reductase and Salmonella spp. The hamburgers were categorized as unsuitable for human consumption in 31.4% of samples, with those testing positive for coliforms and Staphylococcus at unacceptably high levels by Brazilian standards. High levels of microbiological contamination were detected on the hands of the food handlers and mesophytic bacterial counts reached 1.8 × 10(4) CFU/hand. Interviews were carried out by means of questionnaires to evaluate levels of awareness as to acceptable food handling practices and it was found that 80,1% of vendors had never participated in any kind of training.

Ocorrência de Giardia, Cryptosporidium e microsporídios em animais silvestres em área de desmatamento no Estado de São Paulo, Brasil

A ocorrência de Giardia, Cryptosporidium e microsporídios foi investigada por meio da análise de 98 amostras fecais de animais silvestres capturados em uma área de desmatamento para a construção das barragens de Paraitinga e Biritiba, localizadas nos Municípios de Mogi das Cruzes, Salesópolis e Biritiba-Mirim, no Estado de São Paulo. As amostras foram obtidas de 46 roedores, 21 marsupiais, 16 sapos, nove morcegos, três primatas e três lagartos. As técnicas de centrífugo-flutuação com sulfato de zinco, de Kinyoun e a coloração de Gram-Chromotrope foram utilizadas, respectivamente, para a pesquisa de Giardia, de Cryptosporidium e de microsporídios. O total de animais parasitados por um dos protozoários investigados foi de 17,35% (17/98). Cistos de Giardia foram encontrados em amostras fecais de dois pequenos roedores da espécie Coendou villosus (ouriço-cacheiro). Os três animais positivos para Cryptosporidium foram roedores das espécies Akodon montensis, Thaptomys nigrita (ambos conhecidos como ratos do mato) e Sciurus aestuans (serelepe ou caxinguelê). Esporos de microsporídios foram encontrados nas fezes de 12 animais, sendo seis roedores das espécies Oligoryzomys sp.(um), Akodon montensis (três) e Coendou villosus (dois), três marsupiais pertencentes às espécies Didelphis aurita (dois) e Marmosops incanus (um) e três morcegos da espécie Diphylla ecaudata. Este é o primeiro relato de microsporidiose em animais silvestres no Brasil. A presente investigação enfatiza a importância de animais silvestres, particularmente pequenos mamíferos, como potenciais fontes de infecção desses protozoários para outras populações animais, incluindo o homem, em áreas de desmatamento.

Morphological aspects of buffaloes (Bubalus bubalis) umbilical cord

Buffalo is an important livestock resource, with a great participation in agricultural systems, providing milk, meat, and work power. Umbilical cord is responsible for maternal-fetal nutrients exchange during pregnancy, and its alterations can compromise the fetal development. We investigated ten pregnant uteruses collected from cross-bread buffaloes in different stages of gestation. Pregnancy and fetal age was determined by measuring the apex sacral length and development period was calculated by previously published formula. Umbilical cords were measured for length determination. Umbilical cord vascular net and anastomosis were observed by injection of Neoprene latex. Histological sections of the umbilical cord were studied after stain with HE, picrossirius, toluidine blue, orceine, and PAS reaction. Buffaloes' umbilical cord was formed by two central arteries, an allantois duct and two peripheral veins. The artery wall was composed by large quantity of collagen, elastic fibers, fibroblasts and large number of vasa vasorum. The allantois duct was located between the arteries and presented a great number of small nourishing vessels. Small nourishing vessels should be carefully considered to avoid to be mistaken to the arterials and veins vasa vasorum. Medium length of umbilical cord from buffalos was 11.8cm (minimum of 6.8cm and maximum of 17.4cm).

Ossificação endocondral em embriões e fetos de bovinos

Estudou-se a ossificação endocontral de 18 embriões e 12 fetos de até três meses de gestação, os quais foram coletados de úteros gestantes em frigoríficos e abatedouros. Os úteros foram dissecados e, em seguida, realizou-se uma incisão dorsal até o cérvix para avaliações macroscópicas dos embriões e fetos. Para o estudo microscópico foram realizadas técnicas de inclusão, seguidas de marcação dos depósitos de cálcio e fósforo, responsável pela ossificação dos moldes de cartilagem. Foram identificados hipertrofia da cartilagem e morte dos condrócitos e aumento da área de depósito de cálcio e fósforo, por volta da 10ª semana gestacional (74 dias). Durante a 11ª semana de gestação (81 dias), os grupamentos de carbonato de cálcio e fósforo espalharam-se por todo o osso, sendo mais intenso na diáfise.

Detection of anti-Toxoplasma gondii antibodies in experimentally and naturally infected non-human primates by Indirect Fluorescence Assay (IFA) and indirect ELISA

The Indirect Fluorescence Assay (IFA) and the indirect ELISA were comparatively used to detect IgG and IgM antibodies for Toxoplasma gondii in experimentally and naturally infected primates. In the experimentally infected group, antibodies of diagnostic value were detected at day 9 post-infection (PI) with the IFA (IgG and IgM) and with IgG-ELISA. IgM-ELISA detected antibodies for T. gondii starting at day 3 PI until the end of the experiment (102 days PI). Of the 209 naturally infected sera tested, from many zoos of State of Sao Paulo, 64.59 and 67.94% were positive in the IgG-IFA test and IgG-ELISA respectively. IgM-ELISA test detected seropositivity in 52.63% of the sera although IgM-IFA test detected it in only in 0.96% of the samples. The differential toxoplasmosis diagnosis was accomplished with Neospora caninum by IFA, observing 61 (29.2%) seropositive animals for this parasite and 149 (70.8%) negative. Sixty animals were positive for both T. gondii and N. caninum. Pneumonia, splenomegaly, and intestinal ulcers were macroscopically observed. Unremarkable interstitial pneumonia, enteritis, colitis, splenitis, and glomerulitis were microscopically observed. The immunohistochemical stain could not detect the presence of T. gondii in the tissues of the animals infected experimentally.

Análise das metodologias diretas e indiretas para a contagem de células somáticas no leite de cabras hígidas

A particularidade da secreção láctea caprina, do tipo apócrina, diferente da secreção merócrina da vaca, leva a erros de interpretação durante a realização de técnicas de avaliação da celularidade do leite de fêmeas desta espécie. Portanto, o presente trabalho teve o objetivo de determinar a contagem de células somáticas pelo método indireto California Mastitis Test (CMT), e por métodos diretos, incluindo a contagem por citometria de fluxo e a contagem microscópica direta, através da coloração de verde de metil e pironina-Y, além de comparar os métodos de contagem celular. Foram analisadas 102 amostras de 51 fêmeas caprinas, das raças Saanen, Parda Alpina e Toggenburg, criadas no Estado de São Paulo. Os animais foram categorizados segundo a fase da lactação, exame físico da glândula mamária e exame do leite. As amostras foram colhidas, após a realização do exame Califórnia Mastitis Test, em duas alíquotas, uma destinada à contagem celular automática e a outra, a contagem microscópica direta, utilizando-se o corante verde de metil e pironina- Y. De acordo com os diferentes escores do CMT, observou- se 74,5% de amostras negativas, 8,8% de amostras com escore traços, 8,8% de amostras ligeiramente positivas (+), 6,8% de amostras fracamente positivas (++) e 0,9% de amostras fortemente positivas (+++). Os valores medianos das contagens de células somáticas presentes no leite de cabras, avaliadas através de contador automático e microscopia direta, e analisadas de acordo com os diferentes escores do CMT, foram, respectivamente, 181.000, 578.000, 628.000, 1.421.500 e 5.542.000 células/mL de leite e 74.991, 271.396, 71.420, 640.995 e 5.049.394 células/ mL de leite, nos escores negativo, traços, +, ++ e +++. Os valores medianos obtidos através da contagem de células somáticas pelo método automático e microscópico direto, de acordo com as fases de lactação foram de 159.500, 508.000 e 277.500 células/mL de leite, e 62.493, 89.275 e 146.411. A correlação obtida entre a contagem celular automática e microscópica direta foi de 88%. A partir dos resultados observados pode-se concluir que existe diferença na contagem celular determinada através do método automático e microscópico sendo este último o mais adequado para a determinação da celularidade no leite de cabras.

The right to the screen: from media education to educommunication in Brazil

Nowadays in Brazil, some social organizations, governments and mass media are discussing the need to establish an oversight committee to guarantee the quality of television programmes, as well as the need to set a system to determine what kind of pro, gram is appropriate for every television time slot. Across Brazil, a representative body of children and young people have come to the conclusion that the right to receive quality television programmes is not enough. The children of the new generations think they have the right to access new technologies and the production of their own messages, in accordance with their own creativity, interests and lifestyle projects within society.

Origins of the Xylella fastidiosa Prophage-Like Regions and Their Impact in Genome Differentiation

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes.

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

Background: In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties. Results: The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e. g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity. Conclusions: Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.

The role of disulfide bridges in the 3-D structures of the antimicrobial peptides gomesin and protegrin-1: a molecular dynamics study

Some antimicrobial peptides have a broad spectrum of action against many different kinds of microorganisms. Gomesin and protegrin-1 are examples of such antimicrobial peptides, and they were studied by molecular dynamics in this research. Both have a beta-hairpin conformation stabilized by two disulfide bridges and are active against Gram-positive and Gram-negative bacteria, as well as fungi. In this study, the role of the disulfide bridge in the maintenance of the tertiary peptide structure of protegrin-1 and gomesin is analyzed by the structural characteristics of these peptides and two of their respective variants, gomy4 and proty4, in which the four cysteines are replaced by four tyrosine residues. The absence of disulfide bridges in gomy4 and proty4 is compensated by overall reinforcement of the original hydrogen bonds and extra attractive interactions between the aromatic rings of the tyrosine residues. The net effects on the variants with respect to the corresponding natural peptides are: i) maintenance of the original beta-hairpin conformation, with great structural similarities between the mutant and the corresponding natural peptide; ii) combination of positive F and. Ramachandran angles within the hairpin head region with a qualitative change to a combination of positive (F) and negative (.) angles, and iii) significant increase in structural flexibility. Experimental facts about the antimicrobial activity of the gomesin and protegrin-1 variants have also been established here, in the hope that the detailed data provided in the present study may be useful for understanding the mechanism of action of these peptides.

Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection

Background: It has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of L. interrogans allowed identification of a repertoire of putative leptospiral surface proteins. Results: Here, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis. Conclusion: Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the sigma(54) regulon

Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a sigma(54)-dependent manner. A more complete picture of the sigma(54) regulon was achieved by combining the transcriptome data with an in silico search for potential sigma(54)-dependent promoters, using a position weight matrix approach. One of these sigma(54)-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a sigma(54)-dependent promoter. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the sigma(54) regulon.

Renal hypothermia: Experience in pigs and clinical trial

Purpose: This study was designed to compare the effectiveness of two methods of inducing renal hypothermia through laparoscopy in pigs and humans. Materials and Methods: Twelve pigs were divided into four groups of three animals each. Both kidneys of the animals in Groups A, B, and C were submitted to pelvic irrigation with cold saline (4 degrees C) for 20 minutes, with flow rates of 5 mL/min, 10 mL/min, and 15 mL/min, respectively. In Group D renal hypothermia was induced by intracorporeal ice slush applied to the surface for 20 minutes. All maneuvers were performed laparoscopically and renal cortex temperature was measured by a thermocouple needle. Five human patients also underwent laparoscopic partial nephrectomy due to renal cell carcinoma. In one case renoprotection was induced by retrograde endoscopic cold saline perfusion at a flow rate of 10 mL/min. In the remaining four patients we induced renal hypothermia via laparoscopic application of ice slush. The renal temperature of the human patients was also monitored using a thermocouple needle. Results: In the pigs, at 20 minutes of renal pelvis perfusion the mean renal temperature, the temperature drop, and saline flow per gram of kidney were: Group A, -29.5 degrees C +/- 1.1 (-6.3 degrees C; 0.10 mL); Group B, -22.8 degrees C +/- 1.1 (-13.1 degrees C; 0.22 mL); and Group C, -21.1 degrees C +/- 0.9 (-14.9 degrees C; 0.31 mL). In Group D the mean renal cortex temperature at 20 minutes was 13.6 degrees C +/- 1.2, a drop of -22.5 degrees C. There were striking differences among the groups (P < 0.0001). The laparoscopic partial nephrectomy was uneventful in all five human patients. The lowest renal cortex temperature was 32.5 degrees C, seen in the patient who submitted to pelvic irrigation with cold saline, and the mean temperature drop was 19.1 degrees C +/- 2.5 degrees C in the patients who submitted to ice slush-induced renal hypothermia. Conclusions: Induction of renal hypothermia using intracorporeal ice slush confers lower kidney temperatures than endoscopically-induced cold saline perfusion.

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. Methodology/Principal Findings: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. Conclusions/Significance: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.