A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.     

Belangrike ongewingsfaktore wat die natuurlike verspreiding van schmidtia kalahariensis stent en enneapogon cenchroides (licht) c. e. hubbard (e. mollis lehm) in Suidwes‐Afrika beïnvloed 1

Die resultate van ‘n studie van die reaksie van twee belangrike eenjarige weidingsgrasse van Suidwes‐Afrika, Schmidtia kalahariensis en Enneapogon cenchroides, teenoor verskillende omgewingsfaktore word gerapporteer. Verskille tussen die twee spesies ten opsigte van ligenergievereistes, lengte van die lewensiklus, voedingsbehoeftes, smaaklikheid (veroorsaak deur suurafskeidmg) en ontkiemingseienskappe is uitgewys. Dit is moontlik om hierdie verskille in verband te bring met die natuurlike verspreiding van die twee spesies.     

Production of irrigated Trifolium repens L. under a cutting regime in the subtropical region of Gauteng,South Africa

Abstract

The productivity of Trifolium repens L. cv. Ladino, under irrigation in the subtropical region of Gauteng, South Africa, is unknown. The performance of T. repens was determined over five years (1981/1982 to 1985/1986), at two defoliation frequencies, four levels of nitrogen (0, 400, 800 and 1 600 kg N ha^?1 ‐ total applied over five years), and either the removal or recycling of harvested material. Frequent, compared to infrequent defoliation, resulted in higher dry matter (DM) yields from the second year onwards. Dry matter yields were positively affected by the recycling of material, compared to no recycling. A mean DM yield of 191 ha^?1 in the first year and 12 t ha^?1 in the final year, was obtained. The highest DM yields occurred during October for all years and lowest DM yields from January to March. The first and final year showed similar peaks (late October) and troughs (March till April). Crude protein (CP) content of the herbage was influenced more by frequency of defoliation than recycling of material, where the highest CP content in any year was obtained from frequent defoliation. Nitrogen fertiliser applied up to a total of 1 600 kg N ha^?1 over five years had no effect on either DM yield or crude protein content. This study has shown that T. repens could be a productive pasture for at least five years in the subtropical region of Gauteng, South Africa. Productivity and quality of T. repens were enhanced by a defoliation interval of less than five weeks and when soil high fertility was maintained by recycled plant material.     

Insights into the Effect of a Natural Arsenate Dose on Growth, Nodulation and Redox Metabolism of Soybean Plants

<正>Dear Editor,Arsenic(As) is a harmful metalloid that occurs in soil and water; its concentration varies considerably among geographic regions, with groundwater being the principal source of human contamination(Smedley and Kinniburgh, 2002). Besides the direct contamination effect of drinking water that contains high As concentration, human poisoning may also occur after inges-     

Genome sequencing analysis of Brazilian chicken anemia virus isolates that lack MSB-1 cell culture tropism

Specific amino acid (aa) substitutions in VP1, VP2 and VP3 genes were reported as a distinctive feature of the American CIA-1 strain, characterized as having a variable rate of growth and tropism for different MSB-1 cell sublines [Renshaw RW, Soiné C, Weinkle T, O'Connell PH, Ohashi K, Watson S, et al. A hypervariable region in VP1 of chicken anemia virus mediates rate of spread and cell tropism in tissue culture. J Virol 1996;70(12):8872-8]. DNA sequencing of 878 nucleotides from twelve Brazilian CAV, eight of which tested for in vitro isolation in three different sources of MDCC-MSB1 cell line and identified as lacking capacity to propagate in any of these cells, were compared to sequence data available for CAV strains propagated or not in cell culture. Alignment of the deduced aa resulted in a lack of singled out amino acid substitutions in the partial genomic sequences of Brazilian isolates that would entirely contrast them to viruses propagated in MSB-1 cells, indicating that the combined VP1, VP2 and VP3 substitutions observed may not entirely account as sole determinants of CAV isolation and propagation in MDCC-MSB-1 cells.     

Cloning and expression of chicken anemia virus VP3 protein in Escherichia coli

Purification of chicken anemia virus (CAV) VP3 protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study. CAV particle was obtained from infected liver of chicken and DNA was extracted. The VP3 protein gene was amplified from the extracted DNA by polymerase chain reaction (PCR) and cloned. The recombinant expression construct (pTrc-VP3) was identified by PCR and sequencing analysis. Expression of VP3 protein with a molecular mass of approximately 21kDa was confirmed by Western blotting analysis with CAV-specific antibodies. The in vitro expressed VP3 protein was purified to near homogeneity by elution from the gel, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP3 protein was recognized by CAV antibodies in a Western blotting assay. This finding indicates that recombinant VP3 expressed in the pTrcHis2 vector system can be used as antigen to detect anti-CAV antibodies.     

1H‐NMR metabolomic profiling of the crayfish Astacus leptodactylus subjected to polyphenol‐enriched diets

¹H‐NMR analysis of the hepatopancreas, muscle and haemolymph of Astacus leptodactylus after feeding with polyphenol‐enriched diet is reported. ¹H‐NMR spectra of lipophilic extracts showed the presence of cholesterol, fatty acid residues, phospholipids and triglycerides. ¹H‐NMR spectra of aqueous extracts identified 35 metabolites in the hepatopancreas, 31 in the muscle and 22 in the haemolymph. A total of 20 metabolites (amino acids and their derivatives) were present in the hepatopancreas, the muscle and the haemolymph. A total of 10 metabolites were present in both the hepatopancreas and the muscle (five amino acids, 2‐hydroxybutyrate, choline, myo‐inositol, glycogen and uracil). 2‐Hydroxyisobutyrate and creatine were present in both the hepatopancreas and the haemolymph. Phosphorylethanolamine, phosphocholine and fumarate were present only in the hepatopancreas and isoleucine only in the muscle. Statistical analysis showed that the percentage of weight gain was statistically higher in polyphenol‐enriched diet groups compared to the control and that polyphenols had a stimulating effect on the general metabolism. No stress‐related metabolites were higher in crayfish fed with polyphenol‐enriched diet. Conversely, phosphatidylcholine, cholesterol and DHA, linked to resistance to environmental stress and diseases, were higher compared to the control diet. This study indicates that ¹H‐NMR is a useful tool to study the metabolomics in relation to diet differences.     

Influence of the Processing Parameters on the Characteristics of Spherical Bacterial Cellulose

Bacterial Cellulose (BC) biosynthesized under agitated conditions can produce hydrogels with spherical shapes and the processing parameters govern this formation of BC hydrogels. The variations of these parameters cause different results in the formation of spherical BC and it is relevant to evaluate these effects. Carbon source, inoculum concentration, volume of the culture medium, stirring speed and incubation temperature were the processing parameters evaluated in this work. Therefore, the results obtained were characterized in terms of dry mass production, water uptake and sphericity of the spherical BC. To do so, Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were employed as tools for morphological and chemical evaluations. Among the parameters analyzed, the carbon source had the greatest effect on the production of dry mass and water uptake. Glycerol had the greatest growing rate, generating a higher dry mass production with spherical and homogeneous bodies with excellent water absorption indexes. Furthermore, the morphological analysis of BC revealed a typical structure, with high crystallinity index and the presence of typical functional groups. These results indicate a path for the optimized and controlled production of spherical BC, allowing it to be used in many applications, such as enzyme immobilization, water treatment, bone regeneration, spherical cancer model, as adsorption agent and drug delivery.     

Phytodecontamination of the Endocrine Disruptor 4-Nonylphenol in Water Also in the Presence of Two Natural Organic Fractions

The objective of this study was to assess the removal of the endocrine disruptor 4-nonylphenol (NP) at a concentration of 1?mg?L^?1 by ryegrass and radish during germination and growth. The decontamination process was evaluated in water only or in water containing two organic fractions, a soil humic acid (HA) and a river natural organic matter (NOM) at concentrations of 10 and 200?mg?L^?1. The addition of these fractions aimed to simulate the organic content of real aqueous systems. At the end of germination and growth, residual NP was measured by chromatographic analysis. Also, NP phytotoxicity was evaluated during germination. In germination experiments, NP in water only was not toxic for ryegrass and radish which removed, respectively, 37 and 51?% of the initial NP added. In water added with HA or NOM at both doses, in general, NP did not influence or inhibited seed germination. Both doses of HA in water promoted the removal of NP by germinating seeds, whereas NOM exerted differentiated results in the two species as a function of the dose applied. After 2?days of growth, in all treatments both species almost completely removed NP and accumulated a very little fraction of product. This study demonstrated that both ryegrass and radish possess a relevant capacity to remove the endocrine disruptor NP from water also in the presence of different organic fractions, thus suggesting their use in the decontamination of real aquatic systems.