Viable mycobacterium avium subsp. paratuberculosis in retail artisanal coalho cheese from northeastern Brazil.

2014

Características físicas de frutos do pequizeiro (Caryocar coriaceum Wittm.) nativos da Chapada do Araripe - CE.

O pequizeiro, planta nativa explorada de forma extrativista, típica da região do cerrado, pertence ao gênero Caryocar e à família Caryocaraceae (LORENZI, 1992). Esta planta de grande importância socioeconômica, envolve as famílias de agricultores na atividade de catação do fruto, cuja venda in natura, gera emprego e renda para o município. São diversas as suas aplicações na culinária local, no preparo de arroz, feijão e galinha, na elaboração de bolos, biscoitos, doces e fabricação artesanal de licor, considerado o mais nobre subproduto da fruta. Seu óleo (da polpa ou da amêndoa) tem aplicações na indústria de cosméticos para fabricação de cremes e sabonetes e grande importância na farmacopéia popular no combate de bronquites, gripes, resfriados e tumores (BRAGA, 2001; ALMEIDA; SILVA, 1994). Sendo o pequizeiro uma planta nativa, é natural que apresente certa variabilidade em suas características gerais conforme evidenciado por pesquisas realizadas em frutos de pequizeiros de ocorrência natural no Estado de Goiás (VERA et al., 2005, 2007). Este achados motivaram a realização deste trabalho para determinar as características físicas dos frutos da espécie Caryocar coriaceum Wittm., presente na Chapada do Araripe CE, visando verificar possíveis diferenças entre os genótipos e identificar materiais de interesse agroindustrial e para estudos de melhoramento da espécie.

Enraizamento de estacas de seis espécies nativas de mata de galeria: Bauhinia rufa (Bong.) Steud., Calophyllum brasiliense Camb., Copaifera langsdorffii Desf., Inga laurina (Sw.) Willd., Piper arboreum Aubl. e Tibouchina stenocarpa (DC.) Cogn.

Tecnicas de propagacao por meio de enraizamento de estacas tem sido amplamente empregadas na fruticultura, floricultura e silvicultura. No entanto, no Brasil, poucos estudos tem sido feitos com especies nativas. Neste estudo foi testado o enraizamento de estacas caulinares de seis especies nativas de Mata de Galeria do bioma Cerrado: Copaifera langsdorffii Desf. (copaiba), Tibouchina stenocarpa (DC.) Cogn. (quaresmeira), Piper arboreum Aubl. (pimenta-de-macaco), Inga laurina (Sw.) Willd. (inga), Calophyllum brasiliense Camb. (landim) e Bauhinia rufa (Bong.) Steud. (unha-de-vaca). Foi estudada a influencia de diferentes concentracoes de acido indolbutirico, AIB (0, 1000, 2000 e 4000 ppm), em talco, e tratamento com agua sob gotejamento no enraizamento de estacas basais e/ou apicais, conforme a especie, em duas epocas do ano, final das chuvas (marco-maio/98) e inicio da seca (junho/98). Com P. arboreum tambem foi feito um tratamento onde as estacas foram enraizadas usando agua como substrato. Os resultados mostraram que as especies apresentaram diferentes habilidades para formar raizes adventicias em estacas. As estacas de C. langsdorffii e T. stenocarpa nao formaram raizes em qualquer das epocas realizadas, enquanto P. arboreum e I. laurina formaram raizes nas estacas apicais coletadas nas duas epocas (chuvosa e seca). Porem, as estacas basais de P. arboreum formaram menos raizes do que as apicais e as estacas basais de I. laurina nao enraizaram. Ja em C. brasiliense observaram-se altas taxas de sobrevivencia das estacas coletadas nas duas epocas (final das chuvas e inicio da seca), mas nao houve enraizamento. Por ultimo, as estacas de B. rufa enraizaram somente nas coletas realizadas na estacao chuvosa. Os tratamentos auxinicos (AIB) nao tiveram efeitos sobre a percentagem final de enraizamento das estacas de P. arboreum (apicais e basais), de I. laurina (apicais) e de B. rufa, bem como sobre a porcentagem final de sobrevivencia das estacas de C. brasiliense. Entre as especies estudadas, as estacas apicais de P. arboreum apresentaram os percentuais de enraizamento mais elevados: de 63% a 83% no periodo chuvoso e de 63% a 90% no periodo seco. Nas estacas basais o enraizamento foi menor, variando de 7% a 20%, no periodo chuvoso, e de 0 a 13%, no periodo seco. A epoca de coleta afetou a sobrevivencia e o peso seco das estacas apicais de P. arboreum, mas nao o numero de estacas enraizadas. Ja nas estacas basais, onde a capacidade de enraizamento foi menor, a epoca de coleta afetou o enraizamento das estacas, mas nao a sobrevivencia. Nas estacas basais houve uma grande mortalidade das estacas nas duas epocas estudadas (chuva e seca). Em geral, os melhores resultados de enraizamento ocorreram na epoca seca para as estacas apicais e na epoca chuvosa para as estacas basais. A utilizacao de agua de torneira como substrato proporcionou resultados satisfatorios no enraizamento das estacas basais de P. arboreum tanto em copos (200 ml) com agua colocados na casa de vegetacao (87%) quanto no tanque com agua corrente (77%). Em I. laurina a epoca de coleta influenciou a sobrevivencia das estacas apicais. As estacas coletadas na estacao chuvosa apresentaram melhores resultados sobrevivencia do que aquelas coletadas na estacao seca. Nas coletas feitas no final da epoca chuvosa obteve-se uma media de 15% de enraizamento e uma variacao de 3 a 23% de estacas enraizadas conforme os tratamentos. Na seca a media foi de 7% e a variacao foi de 0 a 13%. No periodo seco as medidas de peso seco das raizes foram mais inferiores do que na epoca chuvosa. A maioria das estacas vivas de I. laurina que nao enraizaram formaram calos, sugerindo que um periodo maior de observacao pode levar a maiores percentuais de enraizamento. Em C. brasiliense a epoca de coleta influenciou a sobrevivencia das estacas, de forma que no periodo chuvoso a porcentagem media de sobrevivencia (81%) foi maior do que na epoca seca (71%). Bauhinia rufa teve uma baixa capacidade de enraizamento por meio de estacas coletadas nas duas epocas do ano: apenas 3% das estacas enraizaram quando tratadas com 1000 e 4000 ppm de AIB; nos outros tratamentos nao ocorreu enraizamento. Pelos resultados, sugere-se que P. arboreum e uma especie de facil enraizamento por meio de estacas apicais, nas duas datas estudadas (no final das chuvas e inicio da seca), mas o uso de estacas basais nestas datas e inviavel. Sugere-se tambem que, devido aos baixos percentuais de enraizamento, nas duas epocas (chuva e seca), I. laurina e B. rufa sao especies de dificil enraizamento por meio de estacas apicais e basais com folhas. Ja C. Brasiliense, C. langsdorffii e T. stenocarpa nao enraizaram e podem ser consideradas especies de dificil enraizamento, nestas duas epocas estudadas (chuvosa e seca).

In vitro seed germination and multiplication of Calophyllum brasiliense.

Calophyllum brasiliense é uma espécie arbórea com sistema de propagação natural limitada. A germinação in vitro pode ser uma alternativa para obtenção de plântulas de qualidade. Sementes foram mantidas em água antes da desinfestação e comparadas com sementes controle (não imersas), sem diferença entre os tratamentos. HgCl2 usado durante a desinfestação reduziu a contaminação das culturas. A contaminação fúngica foi reduzida com fungicida adicionado ao meio (23 para 6,4%), mas a porcentagem de bactérias foi aumentada (24 para 36%). Em outro experimento, as sementes foram imersas em plant preservative mixture (PPM?) antes da desinfestação. Combinando a imersão por 48 h e 2 mL L-1 no meio de cultura, a contaminação foi de 6%. A imersão das sementes em GA3 antes da desinfestação reduziu a formação de raízes conforme a concentração foi aumentada. A germinação e o IVG foram reduzidos, respectivamente, de 72% e 0,129 (24 h) para 60% e 0,092 (48 h), de acordo com o tempo de exposição a GA3. Após 90 dias em meio de multiplicação contendo benzilaminopurina, o número médio de brotações por segmento nodal foi 3,4. A germinação in vitro de C. brasiliense é viável em meio WPM sem sacarose, com até 93,3% de sobrevivência.

Métodos para detecção de Clavibacter michiganense subsp. michiganense e Xanthomonas campestris pv. vesicatoria em sementes de tomate.

Visando detectar Clavibacter michiganense subsp. michiganense (Cm) e Xanthomonas campestris pv. vesicatoria (Xcv) em sementes de tomate, duas técnicas foram comparadas: meio semi-seletivo e planta indicadora. Os seguintes parâmetros foram avaliados: soluções extratoras de Cm e Xcv de sementes inteiras e moídas, especificidade e sensibilidade. Os resultados mostraram que os meios semi-seletivos MB1M (MB1 + telurito de potássio, ácido borico e benomil) e TAM (peptona, brometo de potássio, cloreto de cálcio, agar + Tween 80, cefalexina e clorotalonil), foram mais eficientes para detecção de Cm e Xcv, a partir de sementes moídas em tampão fosfato do que os meios disponíveis e, apresentaram maior especificidade e sensibilidade, detectando 10(2) - 10(3) ufc/ml de Cme Xcv em comparacao a 10(3) - 10(4) ufc/ml da inoculação em plântulas de tomateiro (cvs. Angela Gigante e Santa Cruz).

Morfologia de plântulas de cinco leguminosas genistóides arbóreas do Brasil (Leguminosae-Papilionoideae)

This work aims to describe, illustrate and compare the seedling morphology of five tree species of the genera Bowdichia, Cyclolobium, Diplotropis, Ormosia, and Poecilanthe, which belong to the genistoid clade (Leguminosae Papilionoideae). Phanero-epigeal-foliaceous seedlings are found in Bowdichia virgilioides Kunth, Cyclolobium brasiliense Benth. has phanero-epigeal-reserve seedlings, while Ormosia arborea (Vell.) Harms, Diplotropis martiusii Benth., and Poecilanthe parviflora Benth. possess crypto-hypogeal-reserve seedlings. Some other relevant seedling morphological characters are discussed and compared with those of previously studied species in these genera.

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.

Detection of the bacterium, Xylella fastidiosa, in saliva of glassy-winged sharpshooter, Homalodisca vitripennis

Homalodisca vitripennis ( Germar) ( Hemiptera: Cicadellidae), the glassy- winged sharpshooter, is one of the most important vectors of the bacterium, Xylella fastidiosa subsp. piercei ( Xanthomonadales: Xanthomonadaceae) that causes Pierce's Disease in grapevines in California. In the present study we report a new method for studying pathogen transmission or probing behavior of H. vitripennis. When confined, H. vitripennis attempt to probe the surface of sterile containers 48 hours post- acquisition of X. f. piercei. The saliva deposited during attempted feeding probes was found to contain X. f. piercei. We observed no correlation between X. f. piercei titers in the foregut of H. vitripennis that fed on Xylella- infected grapevines and the presence of this bacterium in the deposited saliva. The infection rate after a 48 h post- acquisition feeding on healthy citrus and grapevines was observed to be 77% for H. vitripennis that fed on grapevines and 81% for H. vitripennis that fed on citrus, with no difference in the number of positive probing sites from H. vitripennis that fed on either grapevine or citrus. This method is amenable for individual assessment of X. f. piercei- infectivity, with samples less likely to be affected by tissue contamination that is usually present in whole body extracts.

Behavior and viability of spontaneous oxidative stress-resistant Lactococcus lactis mutants in experimental fermented milk processing

Previously, we isolated two strains of spontaneous oxidative (SpOx2 and SpOx3) stress mutants of Lactococcus lactis subsp cremoris. Herein, we compared these mutants to a parental wild- type strain (J60011) and a commercial starter in experimental fermented milk production. Total solid contents of milk and fermentation temperature both affected the acidification profile of the spontaneous oxidative stress- resistant L. lactis mutants during fermented milk production. Fermentation times to pH 4.7 ranged from 6.40 h (J60011) to 9.36 h (SpOx2); V(max) values were inversely proportional to fermentation time. Bacterial counts increased to above 8.50 log(10) cfu/mL. The counts of viable SpOx3 mutants were higher than those of the parental wild strain in all treatments. All fermented milk products showed post-fermentation acidification after 24 h of storage at 4 degrees C; they remained stable after one week of storage.

Increased CLA content in organic milk fermented by bifidobacteria or yoghurt cultures

This study investigates the kinetics of acidification, fatty acid (FA) profile and conjugated linoleic acid (CLA, C18:2 c9, t11) content in fermented milks prepared from organic and conventional milk. Fermented milks were manufactured with five mixed cultures: four different strains of Bifidobacterium animalis subsp. lactis (BL04, B94, BB12 and HN019) and Lactobacillus delbrueckii subsp. bulgaricus LB340, in co-culture with Streptococcus thermophilus TA040. The composition of milk was evaluated, and the kinetics of acidification was followed by continuous pH measurement using the Cinac system. The profile of FA, including CLA, was analyzed by gas chromatography. The chemical composition of conventional and organic milk was similar, with the exception of protein and Fe, the concentrations of which were higher in the organic milk. The rate of acidification was significantly influenced by the type of milk and the bacterial strain used. Co-cultures St-HN019 and St-BB12 showed higher maximal acidification rates in both milks. Final counts of S. thermophilus (9.0-10.1 log(10) colony forming units (CFU) . mL(-1), L)actobacillus bulgaricus (8.2-8.5 log(10) CFU . mL(-1)) and B. animalis subsp. lactis strains (8.3-9.3 log(10) CFU . mL(-1)) did not differ significantly in either milk. Unexpectedly, all fermented organic milks contained significantly higher amounts of CLA than the same milk before fermentation, whereas CLA amounts did not change during fermentation of conventional milk. Regardless of the type of milk, CLA was found to be significantly positively correlated with trans-vaccenic acid and negatively correlated with linoleic acid. Moreover, the CLA contents were significantly higher in fermented milks showing shorter fermentation times.

Characterization of new IS elements and studies of their dispersion in two subspecies of Leifsonia xyli

Background: Leifsonia xyli is a xylem-inhabiting bacterial species comprised of two subspecies: L. xyli subsp. xyli (Lxx) and L. xyli subsp. cynodontis (Lxc). Lxx is the causal agent of ratoon stunting disease in sugarcane commercial fields and Lxc colonizes the xylem of several grasses causing either mild or no symptoms of disease. The completely sequenced genome of Lxx provided insights into its biology and pathogenicity. Since IS elements are largely reported as an important source of bacterial genome diversification and nothing is known about their role in chromosome architecture of L. xyli, a comparative analysis of Lxc and Lxx elements was performed. Results: Sample sequencing of Lxc genome and comparative analysis with Lxx complete DNA sequence revealed a variable number of IS transposable elements acting upon genomic diversity. A detailed characterization of Lxc IS elements and a comparative review with IS elements of Lxx are presented. Each genome showed a unique set of elements although related to same IS families when considering features such as similarity among transposases, inverted and direct repeats, and element size. Most of the Lxc and Lxx IS families assigned were reported to maintain transposition at low levels using translation regulatory mechanisms, consistent with our in silico analysis. Some of the IS elements were found associated with rearrangements and specific regions of each genome. Differences were also found in the effect of IS elements upon insertion, although none of the elements were preferentially associated with gene disruption. A survey of transposases among genomes of Actinobacteria showed no correlation between phylogenetic relatedness and distribution of IS families. By using Southern hybridization, we suggested that diversification of Lxc isolates is also mediated by insertion sequences in probably recent events. Conclusion: Collectively our data indicate that transposable elements are involved in genome diversification of Lxc and Lxx. The IS elements were probably acquired after the divergence of the two subspecies and are associated with genome organization and gene contents. In addition to enhancing understanding of IS element dynamics in general, these data will contribute to our ongoing comparative analyses aimed at understanding the biological differences of the Lxc and Lxx.

Syphilis at the Crossroad of Phylogenetics and Paleopathology

The origin of syphilis is still controversial. Different research avenues explore its fascinating history. Here we employed a new integrative approach, where paleopathology and molecular analyses are combined. As an exercise to test the validity of this approach we examined different hypotheses on the origin of syphilis and other human diseases caused by treponemes (treponematoses). Initially, we constructed a worldwide map containing all accessible reports on palaeopathological evidences of treponematoses before Columbus's return to Europe. Then, we selected the oldest ones to calibrate the time of the most recent common ancestor of Treponema pallidum subsp. pallidum, T. pallidum subsp. endemicum and T. pallidum subsp. pertenue in phylogenetic analyses with 21 genetic regions of different T. pallidum strains previously reported. Finally, we estimated the treponemes' evolutionary rate to test three scenarios: A) if treponematoses accompanied human evolution since Homo erectus; B) if venereal syphilis arose very recently from less virulent strains caught in the New World about 500 years ago, and C) if it emerged in the Americas between 16,500 and 5,000 years ago. Two of the resulting evolutionary rates were unlikely and do not explain the existent osseous evidence. Thus, treponematoses, as we know them today, did not emerge with H. erectus, nor did venereal syphilis appear only five centuries ago. However, considering 16,500 years before present (yBP) as the time of the first colonization of the Americas, and approximately 5,000 yBP as the oldest probable evidence of venereal syphilis in the world, we could not entirely reject hypothesis C. We confirm that syphilis seems to have emerged in this time span, since the resulting evolutionary rate is compatible with those observed in other bacteria. In contrast, if the claims of precolumbian venereal syphilis outside the Americas are taken into account, the place of origin remains unsolved. Finally, the endeavor of joining paleopathology and phylogenetics proved to be a fruitful and promising approach for the study of infectious diseases.

Optical fiber laser induced fluorescence spectroscopy as a citrus canker diagnostic

Citrus canker is a serious disease caused by Xanthomonas citri subsp. citri bacteria, which infects citrus plants (Citrus spp.) leading to large economic losses in citrus production worldwide. In this work, laser induced fluorescence spectroscopy (LIF) was investigated as a diagnostic technique for citrus canker disease in citrus trees at an orchard using a portable optical fiber based spectrometer. For comparison we have applied LIF to leaves contaminated with citrus canker, citrus scab, citrus variegates chlorosis, and Huanglongbing (HLB, Greening). In order to reduce the noise in the data, we collected spectra from ten leaves with visual symptoms of diseases and from five healthy leaves per plant. This procedure is carried out in order to minimize the environmental effect on the spectrum (water and nutrient supply) of each plant. Our results show that this method presents a high sensitivity (similar to 90%), however it does present a low specificity (similar to 70%) for citrus canker diagnostic. We believe that such poor performance is due to the fact that the optical fiber collects light from only a small part of the leaf. Such results may be improved using the fluorescence imaging technique on the whole leaf. (C) 2010 Optical Society of America

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcU(AAAH)) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the Delta hrcU mutant with HrcU(AAAH) produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the Delta hrcU mutant complemented with HrcU(AAAH), suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the Delta hrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.