Rapid detection of bovine coronavirus by a semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

Perfil bioquímico, inclusive proteinograma, do soro lácteo de búfalas primíparas e pluríparas sadias ao longo da lactação

Resumo: Para avaliar o perfil bioquímico, inclusive proteínas, do soro lácteo de búfalas Murrah primíparas e pluríparas sadias foram analisadas amostras de leite de 30 fêmeas bubalinas durante uma lactação completa. Os animais foram distribuídos em três grupos: G1 - 10 búfalas primíparas, G2 - 10 búfalas pluríparas com duas a três lactações e G3 - 10 búfalas pluríparas com mais de três lactações. O período de lactação foi dividido em: fase inicial (I: primeiro ao terceiro mês de lactação), fase intermediária (T: quarto ao sexto mês de lactação) e fase final (F: sétimo ao nono mês de lactação). Antes da colheita das amostras de leite foram realizados o exame físico da glândula mamária, o teste da caneca de fundo escuro e o California Mastitis Test (CMT). Após a assepsia dos quartos mamários, foram colhidas mensalmente, durante uma lactação completa, amostras de 20mL de leite de cada quarto mamário, em frascos plásticos esterilizados e sem conservante, para a realização do isolamento microbiológico, determinação do perfil bioquímico e fracionamento proteico por meio de eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE), e amostras de 30mL de leite de cada quarto mamário, em frascos plásticos esterilizados contendo conservante bronopol, para contagem de células somáticas (CCS). Das 1.042 amostras de leite colhidas dos três grupos experimentais durante a lactação, 923 amostras de leite apresentaram reação negativa ao CMT e isolamento microbiológico negativo e foram selecionadas para as análises do perfil bioquímico e fracionamento proteico em SDS-PAGE. Notou-se influência da ordem de parto e da fase da lactação no perfil bioquímico e no proteinograma do soro lácteo de búfalas da raça Murrah sadias. As búfalas primíparas (G1) apresentaram maior atividade das enzimas gamaglutamiltransferase (GGT: 2.346U/L) e fosfatase alcalina (ALP: 181U/L) e maiores concentrações de fósforo (P: 56,6mg/dL), potássio (K: 32,0mg/dL) e α-lactoalbumina (458mg/dL). As fêmeas com duas a três lactações (G2) apresentaram maior CCS (70.700 células/mL) e maiores concentrações de proteína total (1,55g/dL), albumina (100mg/dL), magnésio (Mg: 8,80mg/dL), cloretos (Cl: 176mg/dL), ferro (Fe: 10,7μg/dL), sódio (Na: 178mMol/L) e lactoferrina (59,5mg/dL). As fêmeas com mais de três lactações (G3) apresentaram maiores concentrações de cálcio total (Ca: 41,8mg/dL), cálcio ionizado (Cai: 2,92mMol/L), imunoglobulina A (IgA: 1,32mg/dL), albumina sérica (99,1mg/dL), imunoglobulina G (IgG: 49,7mg/dL) e b-lactoglobulina (1.068mg/dL). Durante a lactação foi observado aumento da CCS, aumento das atividades das enzimas GGT e ALP, aumento das concentrações de proteína total, albumina, P, Mg, Cl, Na, lactoferrina, albumina sérica, IgG, α-lactoalbumina e redução das concentrações de Ca, Fe, Cai, K, IgA e b-lactoglobulina no soro lácteo das búfalas. Os resultados obtidos podem ser utilizados como referências para a espécie bubalina e auxiliar no diagnóstico e no prognóstico de doenças de ocorrência comum na fase de lactação.

Perfil mineral de vacas mestiças Girolanda no período de transição em sistema semi-intensivo em duas estações do ano

Neste estudo foi avaliado o perfil metabólico de vacas leiteiras no período de transição durante o verão e o inverno. Foram utilizados 31 animais pluríparos mestiços girolando em cada estação, totalizando 62 vacas. No verão permaneciam em pasto com suplementação de silagem de milho e concentrado. No inverno, a exigência nutricional era suprida apenas com silagem de milho e concentrado. Foram feitas um total de 11 coletas de sangue de cada animal segundo o seguinte protocolo: quatro coletas pré-parto espaçadas semanalmente, no dia do parto e com 2, 5, 10, 15, 21 e 30 dias pós-parto. Foram avaliadas as concentrações de cálcio, fósforo e magnésio. Todos os analitos variaram em função do estatus fisiológico. As concentrações médias de cálcio e magnésio foram maiores no verão do que no inverno. Em ambas as estações, as menores concentrações médias de cálcio ocorreram próximas ao parto, sendo que 75% dos animais no inverno e 35,48% dos animais no verão estavam hipocalcêmicos. Apenas no décimo dia as concentrações de cálcio voltaram aos níveis do pré-parto, demonstrando que esse tempo é necessário para a adaptação da nova condição de lactante. As concentrações de magnésio foram menores no pós-parto do que no pré-parto nas duas estações, demonstrando a necessidade desse mineral para a produção de leite. As concentrações médias de magnésio permaneceram sempre dentro dos valores de referência, porém 19% dos animais aos dois dias pós-parto no inverno e 7% dos animais aos 10 dias pós-parto no verão tinham concentrações inferiores a esses limites. As concentrações de fósforo sofreram grande variação ao longo do peri-parto, porém sempre em níveis elevados, sendo que 49,8% dos animais no inverno e 37,3% no verão tinham níveis de fósforo acima dos valores de referência

Modeling of the Fluid-Structure Interaction in Inelastic Piping Systems

In many engineering applications, compliant piping systems conveying liquids are subjected to inelastic deformations due to severe pressure surges such as plastic tubes in modern water supply transmission lines and metallic pipings in nuclear power plants. In these cases the design of such systems may require an adequate modeling of the interactions between the fluid dynamics and the inelastic structural pipe motions. The reliability of the prediction of fluid-pipe behavior depends mainly on the adequacy of the constitutive equations employed in the analysis. In this paper it is proposed a systematic and general approach to consistently incorporate different kinds of inelastic behaviors of the pipe material in a fluid-structure interaction analysis. The main feature of the constitutive equations considered in this work is that a very simple numerical technique can be used for solving the coupled equations describing the dynamics of the fluid and pipe wall. Numerical examples concerning the analysis of polyethylene and stainless steel pipe networks are presented to illustrate the versatility of the proposed approach.

Weed community and growth under the canopy of trees adapted to the brazilian semi-arid region

The objectives of this work were to evaluate the floristic composition and dry biomass of weeds under the canopy of seven perennial species adapted to the Semi-Arid region of Brazil, and correlate these characteristics with growth traits of the perennial species. The following perennial species were evaluated in two experiments (E1 and E2): mesquite (Prosopis juliflora), jucá (Caesalpinia ferrea), white popinac (Leucaena leucocephala), mofumbo (Combretum leprosum), neem (Azadirachata indica), sabiá (Mimosa caesalpiniaefolia) and tamarind (Tamarindus indica). In E1, the seven species were evaluated in a random block design with four replicates and nine plants per plot. In E2, evaluation comprised four species (mesquite, jucá, white popinac, and tamarind) in a random block design with eight replicates and nine plants per plot. A circle with an area of 1.77 m² was established around the trunk of each plant, two years after they were transplanted to the permanent location. The weeds collected within this circle were cut even with the ground, classified and weighed. At this time, plant height, and crown and stem diameters were evaluated in all trees of each plot. In E1 there were no differences between tree species as to weed frequency under their canopies; however, weed growth was smaller under the canopy of sabiá trees. Mesquite and sabiá had the greatest plant height and crown diameter means, but only sabiá had the greatest stem diameter. In E2, the perennial species were not different with regard to weed frequency and growth under their canopies, but mesquite had the greatest growth, as measured by plant height (with significant results for jucá as well) and crown and stem diameter.

Structure and dynamics of a woody plant community of a tropical semi-deciduous seasonal forest in the "Estação Ecológica do Panga", municipality of Uberlândia, Minas Gerais, Brazil

Studies of plant community dynamics are essential in understanding the demographic patterns of species since changes in demographic rates can affect the floristic composition and future structure. The purpose of the present study was to analyze the changes in the community structure and floristic composition of woody plants in a tropical semi-deciduous forest in Uberlândia in central Brazil, during a 10-years period. The data were collected in 1989 and in 2000 in 50 quadrats (10 m x 10 m) where all trees with a minimum circumference at breast height of 10 cm were sampled. In 1989, 93 species and 1103 individuals were registered. Over a period of 10 years, seven new species were added to the community, although eight disappeared. The main change that occurred during this period in the floristic composition was the replacement of savannah species occurring in forest gaps by those from the forest understory.

Comportamento fenológico de três espécies de Jatropha (Euphorbiaceae) da Caatinga, semi-árido do Brasil

O comportamento fenológico vegetativo e reprodutivo de Jatropha mollissima (Pohl) Baill., Jatropha mutabilis (Pohl) Baill. e Jatropha ribifolia (Pohl) Baill. foi comparado em uma área de caatinga hiperxerófila arbustiva-arbórea do nordeste brasileiro, no período de julho de 2005 a junho de 2007. Adicionalmente, investigou-se a correlação entre as fenofases e as variáveis abióticas (temperatura, umidade relativa, precipitação e fotoperíodo), a capacidade de armazenamento de água e o padrão de distribuição espacial. Todas as espécies apresentaram alta capacidade de armazenamento de água e comportamento fenológico contínuo e irregular com ausência de flores, frutos, queda foliar e brotamento nos meses mais frios e úmidos do ano. Apenas a floração de J. ribifolia apresentou correlação significativa com a precipitação. As três espécies apresentaram populações com distribuição agregada, favorecida principalmente devido ao processo de dispersão de sementes por autocoria. Os resultados obtidos indicam que a alta capacidade de armazenamento de água pelas espécies garante a ocorrência dos eventos fenológicos mesmo na ausência de chuvas.

The genus Stachybotrys (anamorphic fungi) in the semi-arid region of Brazil

(The genus Stachybotrys (anamorphic fungi) in the semi-arid region of Brazil). Stachybotrys is characterized by macronematous, mononematous, unbranched or branched conidiophores, with discrete terminal and phialidic conidiogenous cells, and aseptate, reniform, ellipsoidal to spherical, smooth or verrucose conidia, which are produced in a slimy mass. Eight species have been reported from Brazil, occurring in the soil, air and leaf litter. During investigation of conidial fungi on decaying leaf litter in semi-arid areas of Brazil nine species were found: S. bisbyi (Sriniv.) G.L. Barron, S. chartarum (Ehrenb.) S. Hughes, S. globosa P. C. Misra & S. K. Srivast., S. kampalensis Hansf., S. longispora Matsush., S. nephrospora Hansf., S. nilagirica Subram., Stachybotrys parvispora S. Hughes and S. verrucispora Matsush. Stachybotrys nilagirica is a new record from Brazil. Descriptions, comments, geographic distribution and illustrations are presented for above mentioned species. A key for all species recorded in semi-arid region of Brazil is presented.

Photosynthetic responses of four tropical tree species grown under gap and understorey conditions in a semi-deciduous forest

Leaf CO2 assimilation (A) as a function of photosynthetic photon flux density (Q) or intercellular CO2 concentration (Ci) and chlorophyll fluorescence measurements were carried out on four tropical woody species growing in forest gap and understorey (Bauhinia forficata Link. and Guazuma ulmifolia Lam. as pioneers, and Hymenaea courbaril L. and Esenbeckia leiocarpa Engl. as non-pioneers). Chlorophyll fluorescence indicated similar acclimation capacities of photochemical apparatus to contrasting light environments irrespective to plant species. Maximum CO2 assimilation and quantum yield derived from A/Q curves indicated higher photosynthetic capacity in pioneer than in non-pioneer species in forest gap. However, the differences among species did not show a straightforward relation with their successional status regarding data derived from A/Q curves under understorey conditions. Both successional groups are able to sustain positive carbon balance under contrasting natural light availabilities, modifying photochemical and biochemical photosynthetic traits with similar phenotypic plasticity capacity.

Floristic diversity of two crystalline rocky outcrops in the Brazilian northeast semi-arid region

Floristic composition and structure of vegetation were studied in two rocky outcrop areas in the semi-arid region of northeastern Brazil. From April 2007 to September 2008, 18 monthly field trips were carried out. Vascular plants were randomly collected throughout the outcrop areas. For structural analysis, 30 plots of 1 × 1 m were set in the vegetation islands. The checklist presented combines 211 species (69 families and 168 genera), although only 56 species were collected in the plots. Fabaceae (18 spp.; 8.5%), Asteraceae (17 spp.; 8%), Orchidaceae (13 spp.; 6.1%), Euphorbiaceae (13 spp.; 6.1%), Bromeliaceae (10 spp.; 4.7%), and Poaceae (eight spp.; 3.8%) are the richest families. Overall, 1,792 shrub and herbaceous specimens were counted in the plots. The Shannon-Wiener (H) diversity index values were 2.572 and 2.547 nats individual-1. The species that presented the highest absolute abundance values (number of plants) had low frequencies in the plots and vice-versa. The biological spectrum had a high proportion of phanerophytes and therophytes, followed by cryptophytes, chamaephytes, and hemicryptophytes. The studied flora shares floristic components similar to other rocky outcrop areas of the semi-arid region in northeastern Brazil, including in relation to dominant groups in the vegetation structure.

Sistemas de polinização e de reprodução de três espécies de Jatropha (Euphorbiaceae) na Caatinga, semi-árido do Brasil

São apresentados a biologia floral, a dinâmica de produção de néctar, visitantes florais e sistemas reprodutivos de Jatropha ribifolia (Pohl) Baill. (Euphorbiaceae) e uma comparação dos dados obtidos para J. mollissima (Pohl) Baill. e J. mutabilis (Pohl) Baill. O estudo foi desenvolvido em uma área de caatinga hiperxerófila, na Estação Biológica de Canudos, Bahia, Brasil, de maio de 2005 a junho de 2007. As flores das três espécies estão organizadas em dicásios protogínicos. Em J. ribifolia as flores estaminadas e pistiladas duram cerca de 48 horas e a abertura é diurna, enquanto em J. mollissima e J. mutabilis as estaminadas duram entre 12 e 15 horas e as pistiladas entre 36 e 48 horas e a abertura é crepuscular. A produção de néctar, a viabilidade polínica e a receptividade estigmática iniciaram-se logo após a abertura total das flores e se sobrepuseram até sua senescência. A produção de néctar variou ao longo do dia e as flores pistiladas produziram maiores volumes que as estaminadas. A viabilidade polínica e a receptividade estigmática foram observadas nas três espécies durante toda a vida da flor. Houve diferença significativa entre os tratamentos dos sistemas reprodutivos para J. mollissima (KW = 59,796), J. mutabilis (KW = 59,058) e J. ribifolia (KW = 63,660). As três espécies produziram frutos por geitonogamia manual e xenogamia manual e apenas J. ribifolia produziu frutos por geitonogamia espontânea. As abelhas Apis mellifera, Xylocopa frontalis e X. grisescens e os beija-flores Chlorostilbon lucidus e Anopetia gounellei são os polinizadores potenciais de J. mollissima e J. mutabilis. Já para J. ribifolia, os potenciais polinizadores são A. mellifera e X. grisescens. A utilização de uma gama variada de vetores de pólen permite o fluxo polínico e a formação de frutos nas três espécies. Por sua vez, o sequenciamento no período da floração e diferenças nas dimensões das flores e nos horários da antese ajudam a manter o isolamento reprodutivo das três espécies e evitam a perda de pólen interespecífico, que poderia ser alta devido à partilha de alguns dos polinizadores.

A semi-automatic computerized method to measure baroreflex-mediated heart rate responses that reduces interobserver variability

Arterial baroreflex sensitivity estimated by pharmacological impulse stimuli depends on intrinsic signal variability and usually a subjective choice of blood pressure (BP) and heart rate (HR) values. We propose a semi-automatic method to estimate cardiovascular reflex sensitivity to bolus infusions of phenylephrine and nitroprusside. Beat-to-beat BP and HR time series for male Wistar rats (N = 13) were obtained from the digitized signal (sample frequency = 2 kHz) and analyzed by the proposed method (PRM) developed in Matlab language. In the PRM, time series were low-pass filtered with zero-phase distortion (3rd order Butterworth used in the forward and reverse direction) and presented graphically, and parameters were selected interactively. Differences between basal mean values and peak BP (deltaBP) and HR (deltaHR) values after drug infusions were used to calculate baroreflex sensitivity indexes, defined as the deltaHR/deltaBP ratio. The PRM was compared to the method traditionally (TDM) employed by seven independent observers using files for reflex bradycardia (N = 43) and tachycardia (N = 61). Agreement was assessed by Bland and Altman plots. Dispersion among users, measured as the standard deviation, was higher for TDM for reflex bradycardia (0.60 ± 0.46 vs 0.21 ± 0.26 bpm/mmHg for PRM, P < 0.001) and tachycardia (0.83 ± 0.62 vs 0.28 ± 0.28 bpm/mmHg for PRM, P < 0.001). The advantage of the present method is related to its objectivity, since the routine automatically calculates the desired parameters according to previous software instructions. This is an objective, robust and easy-to-use tool for cardiovascular reflex studies.

The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99%) samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99%) samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.

Manual and semi-automatic quantification of in vivo ¹H-MRS data for the classification of human primary brain tumors

In vivo proton magnetic resonance spectroscopy (¹H-MRS) is a technique capable of assessing biochemical content and pathways in normal and pathological tissue. In the brain, ¹H-MRS complements the information given by magnetic resonance images. The main goal of the present study was to assess the accuracy of ¹H-MRS for the classification of brain tumors in a pilot study comparing results obtained by manual and semi-automatic quantification of metabolites. In vivo single-voxel ¹H-MRS was performed in 24 control subjects and 26 patients with brain neoplasms that included meningiomas, high-grade neuroglial tumors and pilocytic astrocytomas. Seven metabolite groups (lactate, lipids, N-acetyl-aspartate, glutamate and glutamine group, total creatine, total choline, myo-inositol) were evaluated in all spectra by two methods: a manual one consisting of integration of manually defined peak areas, and the advanced method for accurate, robust and efficient spectral fitting (AMARES), a semi-automatic quantification method implemented in the jMRUI software. Statistical methods included discriminant analysis and the leave-one-out cross-validation method. Both manual and semi-automatic analyses detected differences in metabolite content between tumor groups and controls (P < 0.005). The classification accuracy obtained with the manual method was 75% for high-grade neuroglial tumors, 55% for meningiomas and 56% for pilocytic astrocytomas, while for the semi-automatic method it was 78, 70, and 98%, respectively. Both methods classified all control subjects correctly. The study demonstrated that ¹H-MRS accurately differentiated normal from tumoral brain tissue and confirmed the superiority of the semi-automatic quantification method.

Semi-automatic measurement of visual verticality perception in humans reveals a new category of visual field dependency

Previous assessment of verticality by means of rod and rod and frame tests indicated that human subjects can be more (field dependent) or less (field independent) influenced by a frame placed around a tilted rod. In the present study we propose a new approach to these tests. The judgment of visual verticality (rod test) was evaluated in 50 young subjects (28 males, ranging in age from 20 to 27 years) by randomly projecting a luminous rod tilted between -18 and +18° (negative values indicating left tilts) onto a tangent screen. In the rod and frame test the rod was displayed within a luminous fixed frame tilted at +18 or -18°. Subjects were instructed to verbally indicate the rod’s inclination direction (forced choice). Visual dependency was estimated by means of a Visual Index calculated from rod and rod and frame test values. Based on this index, volunteers were classified as field dependent, intermediate and field independent. A fourth category was created within the field-independent subjects for whom the amount of correct guesses in the rod and frame test exceeded that of the rod test, thus indicating improved performance when a surrounding frame was present. In conclusion, the combined use of subjective visual vertical and the rod and frame test provides a specific and reliable form of evaluation of verticality in healthy subjects and might be of use to probe changes in brain function after central or peripheral lesions.