Non‐invasive fast real‐time PCR assay for detection of the enteric parasite Enteromyxum scophthalmi in cultured turbot (Scophthalmus maximus L.)

Enteromyxum scophthalmi is a myxozoan parasite that causes severe parasitic diseases in cultured turbot affecting mainly the intestine of the host. It is characterized by producing acute enteritis, starvation and eventually death. Current diagnosis of E. scopthalmi use traditional techniques, based on the identification of the morphology of the parasite. These techniques take extended time to be carried out and do not favour the adoption of control measure at turbot farms and require the sacrifice of fish. This study develops a fast real‐time PCR molecular tool for the detection of E. scophthalmi in infected farmed turbot. This methodology is applicable for routine controls on the farm at every stage of the parasite infection. Results of the study demonstrate the robustness, specificity, efficiency and reliability of the technique. In addition, this study also provides a non‐invasive procedure of sampling through swaps. This allows control, prevention and diagnosis of the parasite infection at turbot farms while maintaining the welfare of the cultivated fish and avoiding sacrifice of the fish sampled.     

Application of isotope dilution analysis for the evaluation of extraction conditions in the determination of total selenium and selenomethionine in yeast-based nutritional supplements

Isotope dilution analysis (IDA) has been used to quantify total selenium, total solubilized selenium, and the selenomethionine (SeMet) amount in yeast and yeast-based nutritional supplements after acid microwave digestion and different enzymatic extraction procedures. For this purpose, both a (77)Se-enriched SeMet spike, previously synthesized and characterized in our laboratory, and a (77)Se(VI) spike were used. In the analysis of the nutritional supplements, the SeMet spike was added to the sample and extracted under different conditions, and the (78)Se/(77)Se and (80)Se/(77)Se isotope ratios were measured as peak area ratios after high-performance liquid chromatography (HPLC) separation and inductively coupled plasma mass spectrometry (ICP-MS) detection. The formation of SeH(+) and mass discrimination were corrected using a natural SeMet standard injected every three samples. Similarly, total solubilized selenium was measured in the extracts after enzymatic hydrolysis using the (77)Se-enriched SeMet as a spike by direct nebulization without a chromatographic separation. To establish a mass balance, total selenium was also determined by IDA-ICP-MS on the yeast tablets after microwave digestion using (77)Se(VI) as a spike. Results showed that all enzymatic procedures tested were able to solubilize total selenium quantitatively from the solid. However, the recovery for the species SeMet, the major selenium compound detected, was seriously affected by the enzymatic procedure employed and also by the matrix composition of the supplement evaluated. For the yeast sample, SeMet recovery increased from 68 to 76% by the combined use of driselase and protease. For the nutritional supplements, the two most effective procedures appeared to be protease and driselase/protease, with a SeMet recovery ranging from 49 to 63%, depending upon the supplement evaluated. In the case of in vitro gastrointestinal enzymolysis, the results obtained showed 26-37% SeMet recovery, while the rest of selenium was solubilized as other unknown compounds (probably Se-containing peptides).     

Trypanocidal Action of (?)-Elatol Involves an Oxidative Stress Triggered by Mitochondria Dysfunction

Natural compounds have shown good potential for the discovery of new chemotherapeutics for the treatment of Chagas’ disease. Recently, our group reported the effective trypanocidal activity of (−)-elatol, extracted from the red macroalgae Laurencia dendroidea present in the Brazilian coast against Trypanosoma cruzi. However, the mechanism of action of this compound has remained unclear. There are only hypotheses concerning its action on mitochondrial function. Here, we further investigated the mechanisms of action of (−)-elatol on trypomastigotes of T. cruzi. For this, we evaluated some biochemical alterations in trypomastigotes treated with (−)-elatol. Our results show that (−)-elatol induced depolarization of the mitochondrial membrane, an increase in the formation of mitochondrial superoxide anion and loss of cell membrane and DNA integrity. Additionally, (−)-elatol induced formation of autophagic vacuoles and a decrease in cell volume. All together, these results suggest that the trypanocidal action of (−)-elatol involves multiple events and mitochondria might be the initial target organelle. Our hypothesis is that the mitochondrial dysfunction leads to an increase of ROS production through the electron transport chain, which affects cell membrane and DNA integrity leading to different types of parasite death.     

Comparison of lignin and cellulose solubilities in ionic liquids by COSMO-RS analysis and experimental validation

In this work, the solubilities of lignin and cellulose in ionic liquids (ILs) have been analyzed and compared by COSMO-RS simulation. The excess enthalpy of the IL + lignin/cellulose mixtures is evaluated as reference property to describe the affinity of lignin and cellulose for different ionic liquids, in terms of the intermolecular interactions contributing to their mixing properties. Thus, the anion plays the main role in the dissolution process of both lignin and cellulose, in which the hydrogen bonding forces are the major interactions. The most promising anions for the cellulose and lignin dissolution are acetate, formate and chloride, whereas different cations can be employed. Computational results indicated that a rational combination of the anions and cations allows designing ILs able to dissolve with different selectivity lignin and cellulose. Some of these solutions are tested in the laboratory to validate the model employed and the regenerated solids are analyzed by FTIR spectroscopy.     

Study of gastrointestinal parasites in water buffalo (Bubalus bubalis) reared under Mexican humid tropical conditions

Tropical Animal Health and Production - The objective was to determine the frequency of gastrointestinal parasites (GP) genera affecting water buffalo (Bubalus bubalis) reared under humid tropical...     

A novel application of the ecological field theory to the definition of physiographic and climatic potential areas of forest species

A new approach to the definition of physiographic and climatic potential areas for forest species, based on the ecological field theory, is outlined in this paper. The proposed formulation is tested on the Spanish juniper (Juniperus thurifera L.), using data from 883 permanent and temporary plots throughout its distribution area in the Spanish autonomous region of Castilla y León. The suitability of the territory for the species is assessed by previously studying its habitat, which in turn is analyzed through physiographic and climatic parameters. This new method is rooted in an additive index that depends on the Mahalanobis distance in the parametric space that evaluates the ecological resemblance between the studied site and each of the points defining the parametric habitat. Thereby the ecological potential of any site within the territory can be established, integrated in a geographical information systems and accordingly charted. The results are compared with those obtained with the methodology traditionally used by Spanish foresters (factorial index), showing that the overall potential area is similar in size but quite different in its distribution.     

Shiga toxin-producing Escherichia coli O157:H7 from extensive cattle of the fighting bulls breed

Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with cattle recognized as their main natural reservoir. The aim of this work was to determine the prevalence and the pheno-genotypic characteristics of STEC O157:H7 in a herd with 268 cattle of the fighting bulls breed (De Lidia breed) managed under extensive conditions in the South-West of Spain. Rectal-anal swabs of all animals were collected and examined for STEC O157:H7 by performing an immunomagnetic concentration and separation procedure combined with PCR, and the resulting isolates were characterized by both phenotypic and genotypic methods. Overall, STEC O157:H7 was isolated from seven animals (2.6%) in the herd. The PCR procedure indicated that all seven isolates displayed stx₂, eae-γ1, ehxA, O157 rfbE, and fliCh7 genes. They belonged to phage types 4 (one isolate) and 42 (two isolates), and four isolates reacted with typing phages but did not conform to a recognized pattern. Among the seven isolates there were five indistinguishable PFGE patterns and other two which differed only in ?2 restriction fragments, supporting the existence of horizontal transmission among animals in the herd. The present study demonstrates that cattle managed under extensive conditions in Spain can excrete STEC O157:H7 with their faeces. To our knowledge this is the first isolation of this pathogen from De Lidia cattle.     

Artificial neural network implementation in single low-cost chip for the detection of insecticides by modeling of screen-printed enzymatic sensors response

This work presents a hardware implementation of artificial neural networks (ANNs) using a dsPIC^® microcontroller to resolve mixtures of pesticides measured by amperometric acetylcholinesterase (AChE) biosensors. The response of three biosensors with different concentrations of Chlorpyrifos Oxon (CPO) and Chlorfenvinfos (CFV) was modeled by two ANNs, which were implemented on the dsPIC^®. The performance of the ANNs was good, the prediction ability was better than 0.986 when the obtained values were compared with those expected for a set of eight external test samples, which were not used for training. This implementation is proposed to develop low-cost analytical chemical specialized tools.     

Manufacture and properties of bifidogenic saccharides derived from wood mannan

Pinus pinaster wood samples were subjected to double hydrothermal processing. The liquors coming from the second stage, containing soluble saccharides of polymeric or oligomeric nature from hemicelluloses (POHs), were subjected to membrane processing (operating in discontinuous diafiltration) for refining and fractionation. Refined POH fractions were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry and chromatographic techniques. The most complex POH component was made up of 14 hexoses and contained 4 acetyl groups. The fermentability of purified POHs by human fecal inocula was assessed by measuring both carbon source consumption and formation of short-chain fatty acids. The bifidogenic ability of POHs was confirmed by fluorescence in situ hybridization. The stimulatory effects on the bifidobacterial population reached by POHs were of the same order as those obtained with commercial fructooligosaccharides.     

Avian CD154 enhances humoral and cellular immune responses induced by an adenovirus vector-based vaccine in chickens

Recombinant adenoviral vectors have emerged as an attractive system for veterinary vaccines development. However, for poultry vaccination a very important criterion for an ideal vaccine is its low cost. The objective of this study was to test the ability of chicken CD154 to enhance the immunogenicity of an adenoviral vector-based vaccine against avian influenza virus in order to reduce the amount of antigen required to induce an effective immune response in avian. Chickens were vaccinated with three different doses of adenoviral vectors encoding either HA (AdHA), or HA fused to extracellular domain chicken's CD154 (AdHACD). Hemagglutination inhibition (HI) assay and relative quantification of IFN-γ showed that the adenoviral vector encoding for the chimeric antigen is able to elicit an improved humoral and cellular immune response, which demonstrated that CD154 can be used as a molecular adjuvant allowing to reduce in about 50-fold the amount of adenoviral vector vaccine required to induce an effective immune response.